Dimeric antigen receptor (DAR)

Abstract

The present invention provides a dimeric antigen receptor (DAR) construct comprising a heavy chain binding region on one polypeptide chain and a light chain binding region on another polypeptide chain. The two polypeptide chains comprising the dimeric antigen receptor can dimerize to form an antigen-binding domain. The dimeric antigen receptor has properties similar to antibodies, such as specific binding to a target antigen. The dimeric antigen receptor can be used for targeted cell therapy.

Description

[0001] References to Related Applications

[0002] This application claims priority under 35 U.S.C. § 119 to U.S. Provisional Application No. 62 / 640,775, filed on March 9, 2018, entitled “Dimeric Antigen Receptors and Bivalent Antigen Receptors,” the entire contents of which are incorporated herein by reference.

[0003] Throughout this application, various publications, patents, and / or patent applications are referenced, the disclosures of which are hereby incorporated by reference in their entireties in order to more fully describe the state of the art to which this disclosure pertains. Field of the Invention

[0004] The present disclosure provides a dimeric antigen receptor (DAR) protein construct that specifically binds to a target antigen, a nucleic acid encoding the dimeric antigen receptor, a vector comprising the nucleic acid, and a host cell carrying the vector. Background Art

[0005] Chimeric antigen receptors (CARs) have been used to develop antibodies targeting antigens particularly associated with cancer. The first generation of CARs was designed to include a signaling domain (TCRζ) that only transmits activation stimuli (signal 1) (Geiger et al., J. 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. 20 (1): 70-75, 2002). Due to suboptimal activation, T cells transplanted with first-generation CARs showed only limited anti-tumor efficacy (Beecham et al., J. Immunother. 23(6):631-642, 2000). The second-generation CAR immunoglobulin CD28-T cell receptor (IgCD28TCR) integrates the co-stimulatory CD28 (signal 2) into the first-generation receptor (Gerstmayer et al., J. Immunol. 158(10):4584-4590, 1997; Emtage et al., Clin. Cancer Res. 14(24):8112-8122, 2008; Lo, Ma et al., Clin. Cancer Res. 16(10):2769-2780, 2010), giving CAR-T cells greater anti-tumor ability (Finney et al., J. Immunol. 161(6):2791-2797, 1998; Hombach et al., Cancer Res. 61(5):1976-1982, 2001, Maher et al., Cancer Res. 61(5):1976-1982, 2001). al., Nat. Biotechnol. 20(1):70-75, 2002). Various CAR variants have been developed by replacing the signaling domains of TCRζ or CD28 with molecules with similar functions (e.g., FcRγ, 4-1BB, and OX40) (Eshhar et al., Proc. Natl. Acad. Sci. USA 90(2):720-724, 1993).TCR CAR-T cells targeting various tumor antigens have been developed (Ma et al., Cancer Gene Ther. 11(4):297-306, 2004; Ma et al., Prostate 61(1):12-25, 2004; Lo et al., Clin. Cancer Res. 16(10):2769-2780, 2010; Kong et al., Clin. Cancer Res. 18(21):5949-5960, 2012; Ma et al., Prostate 74(3):286-296, 2014; Katz et al., Clin. Cancer Res. 21(14):3149-3159, 2015; Junghans et al., 2016 The Prostate, 76(14):1257-1270).

[0006] Adoptive immunotherapy, through the infusion of T cells engineered with chimeric antigen receptors (CARs) that redirect tumor-killing activity, represents a potentially highly specific modality for treating metastatic cancer. CAR-T cells targeting CD19, a molecule expressed on B cells, have shown success in treating B-cell malignancies and have received FDA approval, with some trials demonstrating efficacy rates as high as 70%, including sustained complete responses.

[0007] Therefore, there is still a need in the art for antibody-directed cell therapy with antibodies having heavy chain binding regions and light chain binding regions in separate polypeptide chains, thereby utilizing the powerful efficacy of CAR therapy. The present disclosure is intended to meet this need in the art. Summary of the Invention

[0008] The present disclosure provides a dimeric antigen receptor (DAR) precursor polypeptide comprising ten regions, in order from amino terminus to 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.

[0009] The present disclosure provides a dimeric antigen receptor (DAR) precursor polypeptide comprising ten regions, in order from amino terminus to 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.

[0010] In one embodiment, the precursor polypeptide optionally comprises a hinge sequence of an antibody selected from the group consisting of IgG, IgA, IgM, IgE, and IgD. In one embodiment, the precursor polypeptide comprises a hinge sequence from the hinge region of CD8α or CD28. In one embodiment, the precursor polypeptide comprises a hinge region comprising the amino acid sequence of CPPC or SPPC.

[0011] In one embodiment, the precursor polypeptide comprises a transmembrane region from CD8α, CD8β, 4-1BB / CD137, CD28, CD34, CD4, FcεRIγ, CD16, OX40 / CD134, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRζ, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD33, CD37, CD64, CD80, CD86, CD137, CD154, LFA-1 T cell co-receptor, CD2 T cell co-receptor / adhesion molecule, CD40, CD4OL / CD154, VEGFR2, FAS, or FGFR2B.

[0012] In one embodiment, the precursor polypeptide comprises an intracellular signaling region comprising an intracellular signaling sequence from two to five signaling sequences selected from the group consisting of 4-1BB, CD3ζ, CD28, CD27, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, GITR (TNFRSF18), DR3 (TNFRSF25), TNFR2, and / or CD226, in any combination, in any order.

[0013] In one embodiment, the precursor polypeptide comprises the amino acid sequence of SEQ ID NO: 15 or 18.

[0014] The present disclosure provides a dimeric antigen receptor (DAR) comprising: (a) a first polypeptide chain comprising, in sequence from amino terminus to carboxyl terminus, five regions: (i) an antibody heavy chain variable region (VH), (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, in sequence from amino terminus to carboxyl terminus, two regions: (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.

[0015] The present disclosure provides a dimeric antigen receptor (DAR) comprising: (a) a first polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, five regions: (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, sequentially from amino terminus to carboxyl terminus, two regions: (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.

[0016] In one embodiment, the dimeric antigen receptor (DAR) comprises an antibody heavy chain constant region and an antibody light chain constant region dimerized via one or two disulfide bonds.

[0017] In one embodiment, the dimeric antigen receptor (DAR) optionally comprises a hinge region, which can be derived from an antibody selected from the group consisting of IgG, IgA, IgM, IgE, and IgD. In one embodiment, the hinge comprises a CD8α or CD28 hinge region. In one embodiment, the hinge region comprises a CPPC or SPPC amino acid sequence.

[0018] In one embodiment, the dimeric antigen receptor (DAR) comprises a transmembrane region from CD8α, CD8β, 4-1BB / CD137, CD28, CD34, CD4, FcεRIγ, CD16, OX40 / CD134, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRζ, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD33, CD37, CD64, CD80, CD86, CD137, CD154, LFA-1 T cell co-receptor, CD2 T cell co-receptor / adhesion molecule, CD40, CD4OL / CD154, VEGFR2, FAS, and FGFR2B.

[0019] In one embodiment, the dimeric antigen receptor (DAR) comprises an intracellular signaling region comprising an intracellular signaling sequence from two to five of the following signaling sequences, in any combination in any order: 4-1BB, CD3ζ, CD28, CD27, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, GITR (TNFRSF18), DR3 (TNFRSF25), TNFR2, and / or CD226.

[0020] The present disclosure provides a dimeric antigen receptor (DAR) comprising: (a) a first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, five regions: (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: 21, (iv) a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO: 6, (v) an intracellular signaling region comprising a 4-1BB intracellular signaling sequence having the amino acid sequence of SEQ ID NO: 7 and a CD3ζ intracellular signaling sequence having the amino acid sequence of SEQ ID NO: 9 or 20; and (b) a second polypeptide chain comprising, in order from amino terminus to carboxyl terminus, two regions: (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 CD38 antibody light chain constant region having the amino acid sequence of NO: 4, 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 that binds to the CD38 protein. In one embodiment, the hinge region is optional.

[0021] In one embodiment, the dimeric antigen receptor (DAR) 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.

[0022] In one embodiment, the dimeric antigen receptor (DAR) 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.

[0023] The present disclosure provides a nucleic acid encoding a precursor polypeptide, the 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;

[0024] (4) an optional hinge 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 signal sequence or any combination of two or more signal sequences selected from the group consisting of a 4-1BB signaling sequence comprising the amino acid sequence of SEQ ID NO: 7, a CD28 signaling sequence comprising the amino acid sequence of SEQ ID NO: 8, a CD3ζ (long) signaling sequence comprising the amino acid sequence of SEQ ID NO: 9, and / or a CD3ζ (short) signaling sequence having an ITAM 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.

[0025] The present disclosure provides nucleic acids encoding a precursor polypeptide comprising the amino acid sequence of SEQ ID NO: 15 or 18.

[0026] The present disclosure provides a nucleic acid encoding (a) a first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, five regions: (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: 21, (iv) a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO: 6, (v) an intracellular signaling region comprising a 4-1BB intracellular signaling sequence having the amino acid sequence of SEQ ID NO: 7 and a CD3 zeta intracellular signaling sequence having the amino acid sequence of SEQ ID NO: 9 or 20; and the same nucleic acid or a second nucleic acid encoding (b) a second polypeptide chain comprising, in order from amino terminus to carboxyl terminus, two regions: (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 antibody light chain constant region of the amino acid sequence of NO: 4, 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 that binds to the CD38 protein. In one embodiment, the hinge region is optional.

[0027] In one embodiment, 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.

[0028] In one embodiment, 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.

[0029] The present disclosure provides a vector operably linked to a nucleic acid encoding a precursor polypeptide, the precursor polypeptide comprising:

[0030] (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 hinge 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 signal sequence or any combination of two or more signal sequences selected from the group consisting of a 4-1BB signaling sequence comprising the amino acid sequence of SEQ ID NO: 7, a CD28 signaling sequence comprising the amino acid sequence of SEQ ID NO: 8, a CD3ζ (long) signaling sequence comprising the amino acid sequence of SEQ ID NO: 9, and / or a CD3ζ (short) signaling sequence having an ITAM 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: 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; (10) an antibody light chain constant region comprising the amino acid sequence of SEQ ID NO: 4.

[0031] The present disclosure provides a vector operably linked to a nucleic acid encoding a precursor polypeptide comprising the amino acid sequence of SEQ ID NO: 15 or 18.

[0032] In one embodiment, the vector comprises an expression vector operably linked to a nucleic acid encoding a precursor polypeptide comprising the amino acid sequence of SEQ ID NO: 15 or 18.

[0033] In one embodiment, the expression vector in a host cell or population of host cells directs the transient introduction of a nucleic acid encoding a precursor polypeptide into the host cell or population of host cells.

[0034] In one embodiment, the expression vector in the host cell or population of host cells stably inserts the nucleic acid encoding the precursor polypeptide into the genome of the host cell.

[0035] In one embodiment, the expression vector in the host cell or host cell group directs the transcription and / or translation of the nucleic acid encoding the precursor polypeptide in the host cell or host cell group. For example, the expression vector may include one or more regulatory sequences, such as inducible and / or constitutive promoters and enhancers, which direct the transcription and / or translation (e.g., expression) of the nucleic acid encoding the precursor polypeptide in the host cell or host cell group.

[0036] The present disclosure provides a vector operably linked to a nucleic acid encoding (a) a first polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, five regions: (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: 21, (iv) a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO: 6, (v) an intracellular signaling region comprising a 4-1BB intracellular signaling sequence having the amino acid sequence of SEQ ID NO: 7 and a CD3 zeta intracellular signaling sequence having the amino acid sequence of SEQ ID NO: 9 or 20; and the same vector or a second vector is operably linked to a nucleic acid encoding (b) a second polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, two regions: (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: 21, (iv) a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO: 6, (v) an intracellular signaling region comprising a 4-1BB intracellular signaling sequence having the amino acid sequence of SEQ ID NO: 7 and a CD3 zeta intracellular signaling sequence having the amino acid sequence of SEQ ID NO: 9 or 20; NO:3 and (ii) an antibody light chain variable region comprising the amino acid sequence of CD38 SEQ ID NO:4, 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 that binds to the CD38 protein. In one embodiment, the vector comprises an expression vector. In one embodiment, the hinge region is optional.

[0037] In one embodiment, the expression vector is operably linked to nucleic acids encoding 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.

[0038] In one embodiment, the first expression vector is operably linked to a nucleic acid encoding a first polypeptide comprising the amino acid sequence of SEQ ID NO: 13, and the second vector is operably linked to a nucleic acid encoding a second polypeptide comprising the amino acid sequence of SEQ ID NO: 14.

[0039] In one embodiment, the expression vector is operably linked to nucleic acids encoding 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.

[0040] In one embodiment, the first expression vector is operably linked to a nucleic acid encoding a first polypeptide comprising the amino acid sequence of SEQ ID NO: 16, and the second vector is operably linked to a nucleic acid encoding a second polypeptide comprising the amino acid sequence of SEQ ID NO: 17.

[0041] In one embodiment, the expression vector in the host cell or population of host cells directs the transient introduction of the nucleic acid encoding the first and / or second polypeptide into the host cell or population of host cells.

[0042] In one embodiment, the expression vector in the host cell or population of host cells stably inserts the nucleic acid encoding the first and / or second polypeptide into the genome of the host cell.

[0043] In one embodiment, the expression vector in the host cell or host cell population directs transcription and / or translation of the nucleic acid encoding the first and / or second polypeptide in the host cell or host cell population. For example, the expression vector may comprise 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 host cell population.

[0044] The present disclosure provides a host cell or a population of host cells carrying a vector operably linked to a nucleic acid encoding a precursor polypeptide, wherein the precursor polypeptide 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: 1; (3) an antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 2; (4) an optional hinge 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 signal sequence or any combination of two or more signal sequences selected from the group consisting of a 4-1BB signaling sequence comprising the amino acid sequence of SEQ ID NO: 7, a CD28 signaling sequence comprising the amino acid sequence of SEQ ID NO: 8, a CD3ζ (long) signaling sequence comprising the amino acid sequence of SEQ ID NO: 9, and / or a sequence having an ITAM 3 motif and comprising the amino acid sequence of SEQ ID NO: NO: 20; (7) a CD3ζ (short) signaling 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.

[0045] In one embodiment, the host cell or host cell population carries a vector operably linked to a nucleic acid encoding a precursor polypeptide comprising the amino acid sequence of SEQ ID NO: 15 or 18.

[0046] In one embodiment, the vector carried by the host cell or host cell population comprises an expression vector, which directs the transient introduction of a nucleic acid encoding a precursor polypeptide into the host cell or host cell population.

[0047] In one embodiment, the expression vector in the host cell or population of host cells stably inserts the nucleic acid encoding the precursor polypeptide into the genome of the host cell.

[0048] In one embodiment, the expression vector in the host cell or host cell group directs the transcription and / or translation of the nucleic acid encoding the precursor polypeptide in the host cell or host cell group. For example, the expression vector may include one or more regulatory sequences, such as inducible and / or constitutive promoters and enhancers, which direct the transcription and / or translation (e.g., expression) of the nucleic acid encoding the precursor polypeptide in the host cell or host cell group.

[0049] The present disclosure provides a host cell or a host cell population carrying a vector operably linked to a nucleic acid encoding (a) a first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, five regions: (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: 21, (iv) a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO: 6, (v) an intracellular signaling region comprising a 4-1BB intracellular signaling sequence having the amino acid sequence of SEQ ID NO: 7 and a CD3 zeta intracellular signaling sequence having the amino acid sequence of SEQ ID NO: 9 or 20; and (b) a second polypeptide chain comprising, in order from amino terminus to carboxyl terminus, two regions: (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: 21, (iv) a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO: 6, (v) an intracellular signaling region comprising a 4-1BB intracellular signaling sequence having the amino acid sequence of SEQ ID NO: 7 and a CD3 zeta intracellular signaling sequence having the amino acid sequence of SEQ ID NO: 9 or 20; NO:3, and (ii) a CD38 antibody light chain variable region comprising the amino acid sequence of SEQ ID NO:4, wherein the antibody heavy chain constant region and the antibody light chain constant region form an antigen binding domain that binds to the CD38 protein. In one embodiment, the hinge region is optional. In one embodiment, the host cell or host cell population carries an expression vector that directs the transcription and / or translation (e.g., expression) of the first and / or second polypeptide chain. In one embodiment, the host cell expresses the first and / or second polypeptide chain.

[0050] In one embodiment, the host cell or host cell population carries a vector operably linked to a nucleic acid encoding 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.

[0051] In one embodiment, the host cell or host cell population carries a first vector operably linked to a nucleic acid encoding a first polypeptide comprising the amino acid sequence of SEQ ID NO: 13, and a second vector operably linked to a nucleic acid encoding a second polypeptide comprising the amino acid sequence of SEQ ID NO: 14.

[0052] In one embodiment, the host cell or host cell population carries the vector operably linked to a nucleic acid encoding 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.

[0053] In one embodiment, the host cell or host cell population carries a first vector operably linked to a nucleic acid encoding a first polypeptide comprising the amino acid sequence of SEQ ID NO: 16, and a second vector operably linked to a nucleic acid encoding a second polypeptide comprising the amino acid sequence of SEQ ID NO: 17.

[0054] The present disclosure provides a method for treating a subject suffering from a disease, disorder or condition associated with the deleterious expression of a tumor antigen, comprising: administering to the subject a host cell or a population of host cells carrying an expression vector operably linked to a nucleic acid encoding a precursor polypeptide, wherein the precursor polypeptide comprises: ten regions, in order from amino terminus to 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 comprising 2 to 5 intracellular signal transduction sequences, (7) a T2A cleavage sequence region, (8) a light chain leader sequence, (9) an antibody light chain variable region, and (10) an antibody light chain constant region.

[0055] In one embodiment, the host cell or host cell population carries an expression vector operably linked to a nucleic acid encoding a precursor polypeptide, the 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 hinge 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 signal sequence or any combination of two or more signal sequences selected from the group consisting of a 4-1BB signaling sequence comprising the amino acid sequence of SEQ ID NO: 7, a CD28 signaling sequence comprising the amino acid sequence of SEQ ID NO: 8, a CD3ζ (long) signaling sequence comprising the amino acid sequence of SEQ ID NO: 9, and / or a sequence having an ITAM 3 motif and comprising the amino acid sequence of SEQ ID NO: NO: 20; (7) a CD3ζ (short) signaling 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.

[0056] In one embodiment, the host cell or host cell population carries an expression vector operably linked to a nucleic acid encoding a precursor polypeptide comprising the amino acid sequence of SEQ ID NO: 15 or 18.

[0057] The present disclosure provides a method for treating a subject suffering from a disease, disorder, or condition associated with deleterious expression of a tumor antigen, comprising: administering to the subject a host cell or a population of host cells carrying an expression vector operably linked to a nucleic acid encoding (a) a first polypeptide chain comprising, from amino terminus to carboxyl terminus, five regions: (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: 21, (iv) a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO: 6, (v) an intracellular signaling region comprising a 4-1BB intracellular signaling sequence having the amino acid sequence of SEQ ID NO: 7, and (v) a CD38 antibody heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 8. NO: 9 or 20; and (b) a second polypeptide chain comprising, from amino terminus to carboxyl terminus, two regions: (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. In one embodiment, 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 to the CD38 protein. In one embodiment, the hinge region is optional.

[0058] In one embodiment, the host cell or host cell population is selected from the group consisting of T lymphocytes (e.g., T cells, regulatory T cells, γδ T cells, or cytotoxic T cells), NK (natural killer) cells, macrophages, dendritic cells, mast cells, eosinophils, B lymphocytes, or monocytes.

[0059] In one embodiment, the host cell comprises a NK cell, which comprises a cord blood-derived NK cell or a placenta-derived NK cell.

[0060] In one embodiment, the expression vector in the host cell or population of host cells directs the transient introduction of the nucleic acid encoding the first and / or second polypeptide into the host cell or population of host cells.

[0061] In one embodiment, the expression vector in the host cell or population of host cells directs the stable insertion of the nucleic acid encoding the first and / or second polypeptide into the genome of the host cell.

[0062] In one embodiment, the expression vector in the host cell or host cell population directs the transcription and / or translation of the nucleic acid encoding the first and / or second polypeptide in the host cell or host cell population. For example, the expression vector can comprise one or more regulatory sequences, such as inducible and / or constitutive promoters and enhancers, which direct the transcription and / or translation (e.g., expression) of the nucleic acid encoding the first and / or second polypeptide in the host cell or host cell population.

[0063] In one embodiment, the disease, disorder or condition associated with deleterious expression of a tumor antigen is cancer, including but not limited to hematological breast cancer, ovarian cancer, prostate cancer, head and neck cancer, lung cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, liver cancer, kidney cancer, esophageal cancer, leiomyoma, leiomyosarcoma, glioma and glioblastoma.

[0064] In one embodiment, the disease is a blood cancer selected from the group consisting of non-Hodgkin lymphoma (NHL), Burkitt lymphoma (BL), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphoblastic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin lymphoma (HL), chronic myeloid leukemia (CML) and multiple myeloma (MM).

[0065] Illustration

[0066] Figure 1 is a schematic diagram showing an exemplary dimeric antigen receptor comprising two intracellular signaling sequences.

[0067] Figure 2 is a schematic diagram showing an exemplary dimeric antigen receptor comprising three intracellular signaling sequences.

[0068] Figure 3 is a schematic diagram showing an exemplary dimeric antigen receptor comprising two intracellular signaling sequences.

[0069] Figure 4 is a schematic diagram showing an exemplary dimeric antigen receptor comprising three intracellular signaling sequences.

[0070] Figure 5 is a schematic diagram showing an exemplary precursor polypeptide molecule comprising a T2A cleavage sequence and three intracellular signaling sequences.

[0071] Figure 6 is a schematic diagram showing an exemplary precursor polypeptide molecule comprising a T2A cleavage sequence and two intracellular signaling sequences.

[0072] Figure 7 is a schematic diagram showing an exemplary precursor polypeptide molecule comprising a T2A cleavage sequence and three intracellular signaling sequences.

[0073] Figure 8 is a schematic diagram showing an exemplary precursor polypeptide molecule comprising a T2A cleavage sequence and two intracellular signaling sequences.

[0074] Figure 9 The results of flow cytometry studies are shown, which compare the T cells expressing CD38 chimeric antigen receptor (CAR) constructs with the T cells expressing CD38 dimer antigen receptor (DAR) constructs. The negative control is a cell line carrying a knockout TRAC (T cell receptor α constant) gene. The CD38 DAR (V1) construct includes a long hinge sequence comprising CD8 and CD28 hinge sequences and a signaling region comprising CD28 and long CD3 ζ intracellular signaling sequences.

[0075] Figure 10 It is a figure showing the cytotoxicity percentage of T cells expressing CD38 CAR or CD38 DAR to RPMI 8226 target cells. The negative control (line A) is a cell line carrying a knockout TRAC gene. The CD38DAR (V1) construct (dashed line B) includes a long hinge sequence containing CD8 and CD28 hinge sequences and a signal transduction region containing CD28 and long CD3 ζ intracellular signal transduction sequences. CD38 CAR constructs (line C) are indicated.

[0076] Figure 11 Figure 5 shows the results of flow cytometry studies comparing T cells expressing CD38 CAR V1 or V2 constructs. Negative controls were Figure 1 The cell line used was the same as that described in (TRAC KO). The CD38 DAR (V1) construct contained a long hinge sequence containing CD8 and CD28 hinge sequences and a signaling region containing CD28 and a long CD3ζ intracellular signaling sequence. The CD38 DAR (V2b) construct contained a short hinge sequence and a signaling region containing CD28 and a long CD3ζ intracellular signaling sequence.

[0077] Figure 121 is a graph showing the percentage of cytotoxicity of T cells expressing CD38 CAR, CD38 DAR (V1) or CD38 DAR (V2b) constructs to RPMI 8226 target cells. Negative control (line A) is a cell line carrying a knockout TRAC gene. CD38 DAR (V1) construct (line B) includes a long hinge sequence containing CD8 and CD28 hinge sequences and a signaling region containing CD28 and long CD3 ζ intracellular signaling sequences. CD38 DAR (V2b) construct (dashed line C) includes a short hinge sequence, and the signaling region includes CD28 and long CD3 ζ intracellular signaling sequences. CD38 CAR (line D) is indicated.

[0078] Figure 13 : It is a bar graph showing the level of IFN-γ released from T cells expressing CD38 CAR, CD38 DAR (V1) or CD38 DAR (V2b) constructs. Each data set shows RPMI 8226 cells, K562 cells and culture medium from left to right. The negative control is a cell line carrying a knockout TRAC (T cell receptor alpha constant) gene. The CD38 DAR (V1) construct comprises a long hinge sequence containing CD8 and CD28 hinge sequences and a signaling region comprising CD28 and a long CD3 ζ intracellular signaling sequence. The CD38 DAR (V2b) construct comprises a short hinge sequence and a signaling region comprising CD28 and a long CD3 ζ intracellular signaling sequence.

[0079] Figure 14 It is a bar graph showing the level of TNF-α released from T cells expressing CD38 CAR, CD38 DAR (V1) or CD38 DAR (V2b) constructs. Each data set shows RPMI 8226 cells, K562 cells and culture medium from left to right. The negative control is a cell line carrying a knockout TRAC (T cell receptor α constant) gene. The CD38 DAR (V1) construct includes a long hinge sequence containing CD8 and CD28 hinge sequences and a signaling region comprising CD28 and a long CD3 ζ cell intracellular signaling sequence. The CD38 DAR (V2b) construct includes a short hinge sequence, and the signaling region includes CD28 and a long CD3 ζ cell intracellular signaling sequence.

[0080] Figure 151 is a histogram showing the release of IL-2 levels from T cells expressing CD38 CAR, CD38 DAR (V1) or CD38 DAR (V2b) constructs. Each data set shows RPMI 8226 cells, K562 cells and culture medium from left to right. The negative control is a cell line carrying a knockout TRAC gene. The CD38 DAR (V1) construct comprises a long hinge sequence containing CD8 and CD28 hinge sequences and a signaling region comprising CD28 and a long CD3 ζ intracellular signaling sequence. The CD38 DAR (V2b) construct comprises a short hinge sequence, and the signaling region comprises CD28 and a long CD3 ζ intracellular signaling sequence.

[0081] Figure 16 Figure 1 is a flow cytometry study showing the results of flow cytometry studies comparing the proliferative capacity of T cells expressing CAR or DAR (V1) or (V2b) constructs when co-cultured with K562 or RPMI 8226 cells. The negative control was a cell line carrying a knockout of the TRAC (T cell receptor alpha constant) gene. The CD38 DAR (V1) construct comprises a long hinge sequence containing CD8 and CD28 hinge sequences and a signaling region comprising CD28 and a long CD3ζ intracellular signaling sequence. The CD38 DAR (V2b) construct comprises a short hinge sequence, and the signaling region comprises CD28 and a long CD3ζ intracellular signaling sequence.

[0082] Figure 17 It is a bar graph showing the proliferation fold change of T cells expressing CAR or DAR (V1) or (V2b) constructs when co-cultured with K562 or RPMI 8226 cells. The negative control is a cell line carrying a knockout TRAC (T cell receptor alpha constant) gene. The CD38 DAR (V1) construct includes a long hinge sequence containing CD8 and CD28 hinge sequences and a signaling region comprising CD28 and a long CD3 ζ intracellular signaling sequence. The CD38 DAR (V2b) construct includes a short hinge sequence, and the signaling region includes CD28 and a long CD3 ζ intracellular signaling sequence.

[0083] Figure 18 Figure 2 shows the evaluation of the tumor killing activity of CD38 DAR T cells in a xenograft animal model. Negative control mice were administered phosphate-buffered saline or a cell line carrying a knockout TRAC gene. Test mice were administered T cells expressing a CD38 DAR (V2b) construct containing a short hinge sequence and a signaling region including CD28 and a long CD3ζ intracellular signaling sequence.

[0084] Figure 19Figure 2 shows the evaluation of the tumor killing activity of CD38 DAR (V2a) T cells in a xenograft animal model. Negative control mice were administered phosphate-buffered saline or a cell line carrying a knockout TRAC gene. Test mice were administered T cells expressing a CD38 DAR (V2a) construct comprising a short hinge sequence and a signaling region including 4-1BB and a long CD3ζ intracellular signaling sequence.

[0085] Figure 20 Results of flow cytometry studies are shown comparing T cells expressing CD38 DAR (V2a) or (V3) constructs. Negative controls included activated T cells and a cell line carrying a knockout of the TRAC gene. The CD38 DAR (V2a) construct included a short hinge sequence, and the signaling region included 4-1BB and a long CD3ζ intracellular signaling sequence. The CD38 DAR (V3) construct included a short hinge sequence, and the signaling region included 4-1BB and CD3ζ with a shortened ITAM region.

[0086] Figure 21 Graph showing the percentage of cytotoxicity of T cells expressing CD38 DAR (V2a) against RPMI 8226 target cells compared to CD38 DAR (V3). The negative control is a cell line carrying a knockout TRAC gene (line A). The CD38 DAR (V2a) construct (line B) contains a short hinge sequence, and the signaling region contains 4-1BB and a long CD3ζ intracellular signaling sequence. The CD38 DAR (V3) construct (line C) contains a short hinge sequence, and the signaling region contains 4-1BB and CD3ζ with a shortened ITAM region.

[0087] Figure 22 A histogram of the levels of IFN-γ release in T cells expressing CD38 DAR (V2a) compared to the DAR (V3) construct is shown. Each data set shows RPMI 8226 cells, K562 cells, and culture medium from left to right. The negative control is a cell line carrying a knockout TRAC gene. The CD38 DAR (V2a) construct includes a short hinge sequence, and the signaling region includes 4-1BB and a long CD3ζ intracellular signaling sequence. The CD38 DAR (V3) construct includes a short hinge sequence, and the signaling region includes 4-1BB and CD3ζ with a shortened ITAM region.

[0088] Figure 23: It is a bar graph showing the level of TNF-α released by T cells expressing CD38 DAR (V2a) compared to DAR (V3) construct. Each data set shows RPMI 8226 cells, K562 cells and culture medium from left to right. The negative control is a cell line carrying a knockout TRAC gene. The CD38 DAR (V2a) construct includes a short hinge sequence, and the signaling region includes 4-1BB and a long CD3ζ intracellular signaling sequence. The CD38 DAR (V3) construct includes a short hinge sequence, and the signaling region includes 4-1BB and CD3ζ with a shortened ITAM region.

[0089] Figure 24 Figure 2 shows the results of flow cytometry studies measuring the proportion 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 region includes 4-1BB and a long CD3ζ intracellular signaling sequence. The CD38 DAR(V3) construct includes a short hinge sequence, and the signaling region includes 4-1BB and CD3ζ with a shortened ITAM region.

[0090] Figure 25 The results of flow cytometry studies are shown, which compare the T cells expressing CD38 CAR, second generation CD38 DAR and third generation CD38 DAR constructs. Negative control is a cell line carrying a knockout TRAC gene. Second generation 28Z constructs include short hinge sequences, and signaling region includes CD28 and CD3 ζ signaling sequences (for example: DAR (V2b) constructs). BBZ second generation constructs include short hinge sequences, and signaling region includes 4-1BB and CD3 ζ signaling sequences (for example: DAR (V2a) constructs). 28BBZ third generation constructs include short hinge sequences and signaling region includes CD28, 4-1BB and CD3 ζ signaling sequences (for example: DAR (V2c) constructs).

[0091] Figure 26It is a diagram showing the percentage of cytotoxicity of T cells expressing CD38 CAR, one of two different CD38 second-generation DARs or CD38 third-generation DAR constructs to RPMI 8226 target cells. Negative control (line A) is a cell line carrying a knockout TRAC gene. 28Z second-generation constructs (line E) include short hinge sequences and signaling regions include CD28 and CD3 ζ signaling sequences (e.g., DAR (V2b) constructs). BBZ second-generation constructs (line C) include short hinge sequences and signaling regions include 4-1BB and CD3 ζ signaling sequences (e.g., DAR (V2a) constructs). 28BBZ third-generation constructs (dashed line D) include short hinge sequences and signaling regions include CD28, 4-1BB and CD3 ζ signaling sequences (e.g., DAR (V2c) constructs). Cells expressing CD38 CAR (line B) are indicated.

[0092] Figure 27 It is a histogram showing the T cell secretion IL-2 level of expressing CD38 CAR, one of two different CD38 second generation DARs or CD38 third generation DARs.Each data set shows K562 cells, RPMI 8226 cells and only T cells from left to right.Negative control is a cell line carrying the knockout TRAC gene.Second generation 28Z construct includes short hinge sequence, and signaling region includes CD28 and CD3 ζ signaling sequence (for example: DAR (V2b) construct).BBZ second generation construct includes short hinge sequence, and signaling region includes 4-1BB and CD3 ζ signaling sequence (for example: DAR (V2a) construct).28BBZ third generation construct includes short hinge and signaling region includes CD28, 4-1BB and CD3 ζ signaling sequence (for example: DAR (V2c) construct).

[0093] Figure 28 The system shows a histogram of TNF-α secretion levels of T cells from expressing CD38 CAR, one of two different CD38 second-generation DARs or CD38 third-generation DARs. Each data set shows K562 cells, RPMI 8226 cells and only T cells from left to right. The negative control is a cell line carrying a knockout TRAC gene. The second-generation 28Z construct includes a short hinge sequence, and the signaling region includes CD28 and CD3 ζ signaling sequences (e.g., DAR (V2b) construct). The BBZ second-generation construct includes a short hinge sequence, and the signaling region includes 4-1BB and CD3 ζ signaling sequences (e.g., DAR (V2a) construct). The 28BBZ third-generation construct includes a short hinge and a signaling region includes CD28, 4-1BB and CD3 ζ signaling sequences (e.g., DAR (V2c) construct).

[0094] Figure 29 It is a histogram showing the IFN-γ secretion levels of T cells from expressing CD38 CAR, one of two different CD38 second generation DARs or CD38 third generation DARs. Each data set shows K562 cells, RPMI 8226 cells and only T cells from left to right. The negative control is a cell line carrying a knockout TRAC gene. The second generation 28Z construct includes a short hinge sequence and the signaling region includes CD28 and CD3 ζ signaling sequences (for example: DAR (V2b) construct). The BBZ second generation construct includes a short hinge sequence and the signaling region includes 4-1BB and CD3 ζ signaling sequences (for example: DAR (V2a) construct). The 28BBZ third generation construct includes a short hinge and the signaling region includes CD28, 4-1BB and CD3 ζ signaling sequences (for example: DAR (V2c) construct).

[0095] Figure 30 It is shown that the tumor killing activity of CD38 CAR T cells is assessed in a xenograft animal model. Negative control mice are administered phosphate-buffered saline, activated T cells or cell lines carrying knockout TRAC genes. Test mice are administered T cells expressing 28Z or BBZ second-generation constructs or third-generation 28BBZ DAR constructs. 28ZDAR second-generation constructs include short hinge sequences, and signaling regions include CD28 and CD3 ζ signaling sequences (e.g., DAR (V2b) constructs). BBZ second-generation constructs include short hinge sequences, and signaling regions include 4-1BB and CD3 ζ signaling sequences (e.g., DAR (V2a) constructs). 28BBZ third-generation constructs include short hinge sequences and the signaling regions include CD28, 4-1BB and CD3 ζ signaling sequences (e.g., DAR (V2c) constructs).

[0096] Figure 31The results of flow cytometry studies are shown, and the T cells expressing CD38 DAR V2, V3 or V4 constructs are compared. Negative controls include activated T cells and carry the cell line knocking out the TRAC gene. CD38 V2 DAR constructs include short hinges, and signaling regions include 4-1BB and CD3 ζ signaling sequences (e.g., DAR (V2a) constructs). CD38 V3 DAR constructs include short hinge sequences, and signaling regions include 4-1BB and CD3-ζ (only with ITAM 3 motifs) signaling sequences (e.g., DAR (V3) constructs). CD38 DAR V4 constructs lack hinge sequences, and signaling regions include 4-1BB and CD3-ζ (only with ITAM 3 motifs) signaling sequences (e.g., DAR (V4) constructs).

[0097] Figure 32 : It is a graph showing the percentage of cytotoxicity of T cells expressing CD38 DAR V2, V3 or V4 constructs to RPMI 8226 target cells. The negative control (line A) is a cell line carrying a knockout TRAC gene. The CD38 DAR V4 construct (line B) does not have a hinge sequence, and the signaling region includes 4-1BB and CD3-ζ (only with ITAM 3 motifs) signaling sequences (e.g., DAR (V4) construct). The CD38 V2 DAR construct (dashed line C) includes a short hinge sequence, and the signaling region includes 4-1BB and CD3ζ 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-ζ (only with ITAM 3 motifs) signaling sequences (e.g., DAR (V3) construct).

[0098] Figure 33 It is a bar graph showing the IFN-γ levels released from T cells expressing CD38 DAR V2, V3 or V4 constructs. Each data set shows RPMI 8226 cells, K562 cells and culture medium from left to right. The negative control is a cell line carrying a knockout TRAC gene. The CD38 V2 DAR construct comprises a short hinge, and the signaling region comprises 4-1BB and CD3 ζ signaling sequences (e.g., DAR (V2a) construct). The CD38 V3 DAR construct comprises a short hinge sequence, and the signaling region comprises 4-1BB and CD3-ζ (only with ITAM 3 motifs) signaling sequences (e.g., DAR (V3) construct). The CD38 DARV4 construct lacks a hinge sequence, and the signaling region includes 4-1BB and CD3-ζ (only with ITAM 3 motifs) signaling sequences (e.g., DAR (V4) construct).

[0099] Figure 34 It is a bar graph showing the TNF-α levels released from T cells expressing CD38 DAR V2, V3 or V4 constructs. Each data set shows RPMI 8226 cells, K562 cells and culture medium from left to right. The negative control is a cell line carrying a knockout TRAC gene. The CD38 V2 DAR construct comprises a short hinge, and the signaling region comprises 4-1BB and CD3 ζ signaling sequences (e.g., DAR (V2a) construct). The CD38 V3 DAR construct comprises a short hinge sequence, and the signaling region comprises 4-1BB and CD3-ζ (only with ITAM 3 motifs) 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-ζ (only with ITAM 3 motifs) signaling sequences (e.g., DAR (V4) construct).

[0100] Figure 35A The amino acid sequences of the different regions of the CD38 dimeric antigen receptor (DAR) construct are shown.

[0101] Figure 35B The amino acid sequences of the first polypeptide, second polypeptide and precursor polypeptide of the CD38 dimeric antigen receptor (DAR) construct V2a are shown.

[0102] Figure 35C The amino acid sequences of the first polypeptide, second polypeptide and precursor polypeptide of CD38 dimeric antigen receptor (DAR) construct V3 are shown.

[0103] Figure 35D The amino acid sequences of the long hinge sequence comprising the CD8 and CD28 hinge sequences, as well as the CD3ζ signaling region with only the ITAM 3 motif, the CD8 hinge sequence, and the anti-CD38 CAR construct are shown.

[0104] manual

[0105] Unless otherwise defined, technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art. Generally, terms related to cell and tissue culture techniques, molecular biology, immunology, microbiology, genetics, transgenic cell production, protein chemistry and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques provided herein are generally performed according to conventional procedures well known in the art and are described in the various general and more specific references cited and discussed herein. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). Many basic textbooks describe standard antibody production procedures, including: Borrebaeck (ed) Antibody Engineering, 2nd Edition Freeman and Company, NY, 1995; McCafferty et al. Antibody Engineering, A Practical Approach IRL at Oxford Press, Oxford, England, 1996; and Paul (1995) Antibody Engineering Protocols Humana Press, Towata, NJ, 1995; Paul (ed.), Fundamental Immunology, Raven Press, NY, 1993;

[0106] Coligan (1991) Current Protocols in Immunology Wiley / Greene, NY; Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY; Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, Calif., and references cited therein; Coding Monoclonal Antibodies: Principles and Practice (2nd ed.) Academic Press, New York, NY, 1986, and Kohler and Milstein Nature 256:495-497, 1975. All references cited herein are incorporated by reference in their entirety. Enzymatic reactions and enrichment / purification techniques are also well known and performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature, as well as the laboratory procedures and techniques, described herein, relating to analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry 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.

[0107] The headings provided herein are not limitations of the various aspects of the disclosure, which can be understood by reference to the specification as a whole.

[0108] Unless the context otherwise requires, singular terms shall include pluralities and plural terms shall include the singular. The singular forms "a," "an," and "the," as well as any word in the singular, shall include pluralities unless clearly and unequivocally limited to one object.

[0109] It should be understood that use of alternatives (eg, "or") herein is taken to mean one or both or any combination of the alternatives.

[0110] As used herein, the term "and / or" is to be understood as a specific disclosure of each particular feature or component with or without each other. For example, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to cover the following: 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).

[0111] As used herein, the terms "comprises," "including," "having," and "containing," and grammatical variations thereof, as used herein, are non-limiting, such that the inclusion of one or more items in a list does not exclude other items that can be substituted or added to the listed items. It should be understood that wherever aspects are described herein using the language "comprising," similar aspects described using "consisting of" and / or "consisting essentially of" are also provided.

[0112] As used herein, the term "approximately" refers to a value or composition that is within an acceptable error range for a 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, according to the practice in the art, "approximately" or "approximately" can mean within one or more standard deviations. Alternatively, depending on the limitations of the measurement system, "approximately" or "approximately" can refer to a range of up to 10% (i.e., ±10%) or greater. For example, approximately 5 mg may include any number between 4.5 mg and 5.5 mg. In addition, particularly with respect to biological systems or processes, these terms can represent up to an order of magnitude or 5 times a value. When a specific value or composition is provided in the present disclosure, unless otherwise stated, it should be assumed that the meaning of "approximately" or "approximately" is within an acceptable error range for that specific value or composition.

[0113] As used herein, the terms "peptide," "polypeptide," and "protein," and other related terms, are used interchangeably and refer to polymers 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 undergone post-translational modifications such as proteolytic cleavage, cleavage due to T2A ribosome skipping, hydroxylation, methylation, lipidation, acetylation, sumoylation, ubiquitination, glycosylation, phosphorylation, and / or disulfide bond formation. These terms encompass natural and artificial proteins, protein fragments, and polypeptide analogs (e.g., mutants, variants, chimeric proteins, and fusion proteins) of protein sequences, as well as proteins that have been post-translationally modified, either covalently or non-covalently. Described herein are dimeric antigen receptors comprising two polypeptide chains.

[0114] As used herein, the terms "nucleic acid," "polynucleotide," and "oligonucleotide," and other related terms are used interchangeably and refer 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 DNA or RNA produced using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. Nucleic acid molecules can be single-stranded or double-stranded. In one embodiment, the nucleic acid molecules of the present disclosure comprise a continuous open reading frame encoding an antibody or fragment thereof or scFv, derivative, mutein, or variant. In one embodiment, the nucleic acid comprises a polynucleotide of one type or a mixture of two or more different types of polynucleotides. Nucleic acids encoding dimeric antigen receptors are described herein.

[0115] Antigen binding proteins can have the structure of, for example, an immunoglobulin. In one embodiment, an "immunoglobulin" refers to a tetrameric molecule consisting of two pairs of identical polypeptide chains, each pair having a "light" (approximately 25 kDa) and a "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region that is primarily responsible for effector function. Human light chains are classified as kappa or lambda light chains. Heavy chains are classified as μ, δ, γ, α, or ε, and define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also includes a "D" region of approximately 10 or more amino acids. Reference is made generally to Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)) (the entire contents of which are incorporated herein by reference for all purposes). The variable region of each of the light / heavy chain pairs forms an antibody binding site, such that a complete immunoglobulin has two antigen binding sites. In one embodiment, the antigen binding protein can be a synthetic molecule having a structure that is different from a tetrameric immunoglobulin molecule but still binds to a target antigen or binds to two or more target antigens. For example, a synthetic antigen binding protein can comprise an antibody fragment, 1-6 or more polypeptide chains, an asymmetric assembly of polypeptides, or other synthetic molecules. Described herein are dimeric antigen receptors with immunoglobulin-like properties that specifically bind to a target antigen.

[0116] As used herein, "antibody" and "antibody" and related terms refer to an intact immunoglobulin or an antigen-binding portion thereof that specifically binds an antigen. The antigen-binding portion can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. Antigen-binding portions particularly include 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 comprising at least a portion of an immunoglobulin sufficient to bind a specific antigen to the polypeptide.

[0117] 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 specificity). Antibodies include tetrameric antibodies, light chain monomers, heavy chain monomers, light chain dimers, and 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 fragments (scFv), camelized antibodies, affibodies, disulfide-linked Fvs (sdFv), anti-idiotypic antibodies (anti-Id), and minibodies. Antibodies include monoclonal and polyclonal populations. This article describes dimeric antigen receptors that behave like antibodies.

[0118] As used herein, "antigen binding domain," "antigen binding region," or "antigen binding site" and other related terms refer to a portion of an antigen binding protein that comprises the amino acid residues (or other portions) that interact with an antigen and confer specificity and affinity to the antigen binding protein. For an antibody that specifically binds to its antigen, this will include at least a portion of at least one of its CDR domains. Described herein are dimeric antigen receptors having an antibody heavy chain variable region and an antibody light chain variable region that form an antigen binding domain.

[0119] As used herein in the context of an antibody or antigen binding protein or antibody fragment, the term "specifically binds" and other related terms refer to the 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). In one embodiment, if the antibody binds to an antigen at 10 -5 M or smaller, or 10 -6 M or smaller, or 10 -7 M or smaller, or 10 -8 M or smaller, or 10 -9 M or smaller or 10 -10 Dissociation constant K of M or less DThe antibody specifically binds to the target antigen. This article describes a dimeric antigen receptor that specifically binds to the target antigen.

[0120] In one embodiment, the dissociation constant (K) can be measured using the BIACORE surface plasmon resonance (SPR) assay. D Surface plasmon resonance is an optical phenomenon that can be used to analyze real-time interactions by detecting changes in protein levels in a biosensor matrix, for example using the BIACORE system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).

[0121] As used herein, "epitope" and related terms refer to a portion of an antigen that is bound by an antigen binding protein (e.g., an antibody or its antigen binding portion). An epitope may comprise portions of two or more antigens that are bound by an antigen binding protein. An epitope may comprise a discontinuous portion of an antigen, or discontinuous portions of two or more antigens (e.g., amino acid residues that are discontinuous in the primary sequence of an antigen, but are close enough to each other to be bound by an antigen binding protein in the context of the tertiary and quaternary structures of the antigen). Typically, the variable regions of an antibody, particularly CDRs, interact with an epitope. Described herein are dimeric antigen receptors that bind to an epitope of a target antigen (e.g., CD38).

[0122] As used herein, "antibody fragment," "antibody portion," "antigen-binding fragment of an antibody," or "antigen-binding portion of an antibody" and other related terms refer to molecules other than intact antibodies that comprise a portion of an intact antibody that binds to an antigen that binds to the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; Fd; Fv fragments and dAbs; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); polypeptides comprising at least a portion of an antibody sufficient to bind a specific antigen to the polypeptide. Antigen-binding portions of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include, among others, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs) and complementarity determining region (CDR) fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides comprising at least a portion of an immunoglobulin sufficient to bind the antigen to the antibody fragment. Described herein are dimeric antigen receptors comprising Fab fragments connected to hinge, transmembrane, and intracellular signaling regions.

[0123] The terms "Fab", "Fab fragment" and other related terms refer to a monovalent fragment comprising the variable light chain region (V L ), constant light chain region (C L), variable heavy chain region (V H ) and the first constant region (C H1 ). Fab is able to bind to antigen. The F(ab')2 fragment is a bivalent fragment containing two Fab fragments connected by a disulfide bond in the hinge region. F(Ab')2 has antigen binding ability. The Fd fragment contains V H and C H1 The Fv fragment contains V L and V H Fv can bind to antigen. dAb fragment has V H domain, V L domain or V H or V L Antigen-binding fragments of the leukemia / antigen binding 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).

[0124] The term "human antibody" refers to an antibody having one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all variable and constant domains are derived from human immunoglobulin sequences (e.g., fully human antibodies). These antibodies can be prepared in a variety of ways, examples of which are described below, including by recombinant methods or by immunizing mice with the antigen of interest, which mice are genetically modified to express antibodies derived from human heavy and / or light chain encoding genes. Described herein are dimeric antigen receptors comprising fully human antibody heavy chain variable regions and antibody light chain variable regions.

[0125] The term "hinge" refers to an amino acid segment that is typically between two domains of a protein and that allows for flexibility of the overall construct and movement of one or both domains relative to each other. Structurally, the hinge region comprises about 10 to about 100 amino acids, for example, about 15 to about 75 amino acids, about 20 to about 50 amino acids, or about 30 to about 60 amino acids. In one embodiment, 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 may be derived from the hinge region of a naturally occurring protein, such as a CD8 hinge region or a fragment thereof, a CD8α hinge region or a fragment thereof, an antibody (e.g., IgG, IgA, IgM, IgE, or IgD antibody), or a hinge region connecting the constant domains CH1 and CH2 of an antibody. The hinge region may be derived from an antibody and may or may not include one or more constant regions of an antibody, or the hinge region may include the hinge region of an antibody and the CH3 constant region of an antibody, or the hinge region may include the hinge region of an antibody and the CH2 and CH3 constant regions of an antibody, or the hinge region may be a non-naturally occurring peptide, or the hinge region may be located between the C-terminus of the scFv and the N-terminus of the transmembrane domain.

[0126] The term "leader sequence," "leader peptide," "peptide signal sequence," or "signal peptide" refers to a peptide sequence located at the N-terminus of a polypeptide. The leader sequence directs the polypeptide chain into the cellular secretory pathway and can direct the integration and anchoring of the polypeptide into the lipid bilayer of the cell membrane. Typically, leader sequences are approximately 10-50 amino acids in length. The leader sequence can direct the transport of the precursor polypeptide from the cytoplasm to the endoplasmic reticulum. Leader sequences include signal sequences comprising the CD8α, CD28, or CD16 leader sequences.

[0127] The term "chimeric antigen receptor" or "CAR" refers to a single-chain fusion protein comprising an extracellular antigen binding protein fused to an intracellular signaling domain. The CAR extracellular binding domain is a single-chain variable fragment (scFv or sFv) derived from a variable heavy region and a variable light region of a fusion monoclonal antibody (e.g., a human monoclonal antibody). The disclosed construct is a DARs different from CARs because DARs do not use single-chain antibodies for targeting, but rather use separated heavy and light chain variable domain regions.

[0128] As used herein, "vector" and related terms refer to nucleic acid molecules (e.g., DNA or RNA) that can be operably linked to foreign genetic material (e.g., nucleic acid transgenes). A vector can be used as a vector for introducing foreign genetic material into a cell (e.g., a host cell). The vector may include at least one restriction endonuclease recognition sequence for inserting the transgene into the vector. The vector may include at least one gene sequence that confers antibiotic resistance or selectable characteristics to help select host cells carrying transgenic vector constructs. The vector can be a single-stranded or double-stranded nucleic acid molecule. The vector can be a linear or circular nucleic acid molecule. The donor nucleic acid for the gene editing method using zinc finger nucleases, TALENs, or CRISPR / Cas can be a type of vector. One type of vector is a "plasmid," which refers to a linear or circular double-stranded extrachromosomal DNA molecule that can be connected to a transgene and can replicate, transcribe, and / or translate the transgene in a host cell. Viral vectors typically contain a viral RNA or DNA backbone sequence that can be connected to a transgene. The viral backbone sequence can be modified to render the infection ineffective, but the viral backbone and the co-connected transgene are retained to be inserted into the host cell genome. Examples of viral vectors include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, baculoviruses, pasteurian viruses, vaccinia viruses, herpes simplex viruses, and Epstein-Barr virus vectors. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors containing bacterial replication origins and episomal mammalian vectors). After introduction into the host cell, other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell, thereby replicating along with the host genome.

[0129] An "expression vector" is a vector that may contain one or more regulatory sequences, such as inducible and / or constitutive promoters and enhancers. An expression vector may include a ribosome binding site and / or a polyadenylation site. Regulatory sequences direct the transcription or transcription and translation of a transgene linked to the expression vector that is transduced into a host cell. The regulatory sequences can control the level, timing, and / or location of transgene expression. The regulatory sequences can, for example, act directly on the transgene or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequences and / or nucleic acids). The regulatory sequence can be part of the vector. Other examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif., and Baron et al., 1995, Nucleic Acids Res. 23:3605-3606. Expression vectors comprising a nucleic acid encoding a dimeric antigen receptor are described herein.

[0130] A transgene is "operably linked" to a vector when the linkage between the transgene and the vector allows for the function or expression of the transgene sequences contained in the vector. In one embodiment, a transgene is "operably linked" to a regulatory sequence when the regulatory sequence affects the expression of the transgene (e.g., the level, timing, or location of expression).

[0131] As used herein, the term "transfected" or "transformed" or "transduced" or other related terms refer to the process of transferring or introducing an exogenous nucleic acid (e.g., a transgene) into a host cell. A "transfected" or "transformed" or "transduced" host cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid (transgene). The host cell includes primary subject cells and their progeny. Exogenous nucleic acids encoding at least a portion of any dimeric antigen receptor are described herein and can be introduced into a host cell. An expression vector comprising at least a portion of any dimeric antigen receptor described herein can be introduced into a host cell, and the host cell can express a first and a second polypeptide that dimerizes to form a dimeric antigen receptor described herein.

[0132] As used herein, the term "host cell" or "or host cell group" or related terms refer to a cell (or its cell group) into which an exogenous (exogenous or transgenic) nucleic acid has been introduced. Exogenous nucleic acid can include an expression vector operably linked to a transgene, and the host cell can be used to express nucleic acids and / or polypeptides (transgenes) encoded by the exogenous nucleic acid. The host cell (or its cell group) can be a cultured cell or can be extracted from a subject. The host cell (or its cell group) includes primary subject cells and their progeny (without considering the number of passages). The host cell (or its cell group) includes an immortal cell line. Compared to the parent cell, the progeny cells may or may not carry the same genetic material. Host cells include daughter cells. In one embodiment, as disclosed herein, 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. In one example, as described herein, an expression vector can be introduced into a host cell (or its cell group) that is operably linked to a nucleic acid encoding a desired antibody or its antigen-binding portion thereof as described herein. Host cells and cell populations thereof can carry expression vectors that are stably integrated into the host genome, or can carry extrachromosomal expression vectors. In one embodiment, host cells and cell populations thereof can carry extrachromosomal vectors that exist after several cell divisions, or exist transiently and are lost after several cell divisions.

[0133] The host cell can be a prokaryotic organism, such as Escherichia coli, or a eukaryotic organism, such as a unicellular eukaryotic organism (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), a mammalian cell (e.g., human cell, monkey cell, hamster cell, rat cell, mouse cell, or insect cell), or a hybridoma cell. In one embodiment, an expression vector operably linked to a nucleic acid encoding a desired antibody can be introduced into the host cell to produce a transfected / transformed host cell, the host cell is cultured under conditions suitable for antibody expression by the transfected / transformed host cell, and the antibody is optionally recovered from the transfected / transformed host cell (e.g., from a host cell lysate) or from the culture medium. In one embodiment, the host cell comprises a non-human cell, including CHO, BHK, NS0, SP2 / 0, and YB2 / 0. In one embodiment, the host cell comprises a human cell, including HEK293, HT-1080, Huh-7, and PER.C6. Examples of host cells include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or derivatives thereof, such as Veggie CHO and related cell lines grown in serum-free medium (see Rasmussen et al., 1998, Cytotechnology 28:31) or the DHFR-deficient CHO strain DX-B 11 (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20), HeLa cells, the BHK (ATCC CRL10) cell line, the CV1 / EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) (see McMahan et al., 1991, EMBO J.10:2821), human embryonic kidney cells such as 293, 293EBNA or MSR 293, human epidermal A431 cells, human Colo 205 cells, other transformed primate cell lines, normal diploid cells, cells derived from primary tissues, in vitro cultured cell lines, primary explants, HL-60, U937, HaK or Jurkat cells. In one embodiment, the host cell comprises a lymphoid cell, such as Y0, NS0 or Sp20. In one embodiment, the host cell is a mammalian host cell, but not a human host cell. Generally, the host cell is a cultured cell that can be transformed or transfected with a nucleic acid encoding a polypeptide, which can then be expressed in the host cell.The phrases "transgenic host cell" or "recombinant host cell" may be used to refer to a host cell into which a nucleic acid to be expressed has been introduced (e.g., transformed or transfected). A host cell may also be a cell that contains the nucleic acid but does not express it at the desired level unless a regulatory sequence is introduced into the host cell to operably link it to the nucleic acid. It should be understood that the term host cell refers not only to the specific subject cell, but also to the progeny or potential progeny of that cell. Because certain modifications may occur in subsequent generations due to, for example, mutations or environmental influences, such progeny may not actually be the same as the parent cell, but are still included within the scope of the term as used herein.

[0134] The host cell or host cell population comprises T lymphocytes (e.g., T cells, regulatory T cells, γ-δ T cells, and cytotoxic T cells), NK (natural killer) cells, macrophages, dendritic cells, mast cells, eosinophils, B lymphocytes, monocytes. In one embodiment, the NK cells comprise umbilical cord blood-derived NK cells or placental-derived NK cells.

[0135] Transgenic host cells can be prepared using non-viral methods, including well-known designer nucleases, including zinc finger nucleases, TALENS or CRISPR / Cas. Genome editing technology, such as zinc finger nucleases, can be used to introduce transgenes into the genome of host cells. Zinc finger nucleases include a pair of chimeric proteins, each of which comprises a non-specific endonuclease domain of a restriction endonuclease (e.g., FokI) fused to the DNA binding domain of an engineered zinc finger motif. The DNA binding domain can be designed to bind to a specific sequence in the host genome, and the endonuclease domain performs double-stranded cleavage. The donor DNA carries a transgene, such as any of the nucleic acids encoding the CAR or DAR constructs described herein, and flanking sequences homologous to either side of the region of the expected insertion site in the host cell genome. The DNA repair mechanism of the host cell can accurately insert transgenes by homologous DNA repair. 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). Transgenic host cells can be prepared using TALEN (transcription activator-like effector nuclease), which is similar to zinc finger nucleases in that they include a non-specific nuclease domain fused to a DNA binding domain that can deliver precise transgene insertion. Like zinc finger nucleases, TALEN also introduces a double-stranded nick in the host's DNA. Transgenic host cells can be prepared using CRISPR (clustered regularly interspaced short palindromic repeats). CRISPR couples Cas nucleases to guide RNAs for target-specific donor DNA integration. The guide RNA includes a conserved polynucleotide (the conserved polynucleotide includes a pre-spacer 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 nucleases and TALEN, the CRISPR / Cas system can be used to introduce site-specific insertion of donor DNA with flanking sequences homologous to the insertion site. Examples of CRISPR / Cas systems for modifying genomes are described in, for example, U.S. Patent Nos. 8,697,359, 10,000,772, 9,790,490 and U.S. Patent Application Publication No. US2018 / 0346927. In one embodiment, zinc finger nucleases, TALEN or CRISPR / Cas systems can be used to prepare transgenic host cells, and the host target site can be a TRAC gene (T Cell Receptor AlphaConstant). The donor DNA may include, for example, any nucleic acid encoding a CAR or DAR construct as described herein.Electroporation, nucleofection, or lipofection can be used to deliver the donor DNA together with zinc finger nucleases, TALENs, or CRISPR / Cas systems into host cells.

[0136] Transgenic host cells can be prepared by transducing T cells with a retroviral vector carrying a CAR or DAR construct. The transduction can be substantially as described below: Ma et al., 2004 The Prostate 61: 12-25; and Ma et al., The Prostate 74 (3): 286-296, 2014 (all disclosures of which are incorporated herein by reference). FuGene reagent (Promega, Madison, WI) can be used to transfect the anti-CD38 CAR or DAR MFG retroviral vector plasmid DNA into the Phoenix-Eco cell line (ATCC) to produce ecotropic (Ecotropic) retrovirus, and then the transient viral supernatant (ecotropic virus) can be collected and used to transduce PG13 packaging cells with a Gal-V envelope to produce retrovirus to infect human cells. The viral supernatant of 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 as follows: Normal healthy donor peripheral blood mononuclear cells (PBMCs) are activated with 100 ng / ml mouse anti-human CD3 antibody OKT3 (Orth Biotech, Rartian, NJ) or anti-CD3, anti-CD28 TransAct (Miltenyi Biotech, Germany) according to the manufacturer's instructions and grown for two days in AIM-V growth medium (GIBCO-Thermo Fisher scientific, Waltham, MA) supplemented with 300-1000 U / ml IL-2 and 5% FBS. Approximately 5 × 10 cells can be transduced with 3 ml of viral supernatant in 6-well plates pre-coated with 10 μg / ml retronectin (Takara Bio USA). 6 Activated human T cells are centrifuged at 1000 g for approximately 1 hour at approximately 32° C. Following transduction, transduced T cells can be expanded in AIM-V growth medium supplemented with 5% FBS and 300-1000 U / ml IL2.

[0137] The polypeptides disclosed herein (e.g., antibodies and antigen-binding proteins) can be produced using any method known in the art. In one example, the polypeptides are produced by recombinant nucleic acid methods, where a nucleic acid sequence (e.g., DNA) encoding the polypeptide is inserted into a recombinant expression vector, which is introduced into a host cell and expressed by the host cell under conditions that promote expression.

[0138] The general techniques of recombinant nucleic acid manipulation are described in, for example, Sambrook et al., in Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or F. Ausubel et al., in Current Protocols in Molecular Biology (Green Publishing and Wiley-Interscience: New York, 1987) and regularly updated, the entire contents of which are incorporated herein by reference. The nucleic acid encoding the polypeptide (e.g., DNA) is operably linked to an expression vector carrying one or more suitable transcription or translation regulatory elements derived from mammalian, viral or insect genes. Such regulatory elements include a transcription promoter, an optional operator sequence for controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for controlling transcription and translation termination. The expression vector may include an origin of replication that imparts replication capability in the host cell. The expression vector may include a gene that imparts selectivity to aid recognition of a transgenic host cell (e.g., a transformant).

[0139] The recombinant DNA may also encode any type of protein tag sequence that can be used to purify the protein. Examples of protein tags include, but are not limited to, histidine tags, FLAG tags, myc tags, HA tags, or GST tags. Suitable cloning and expression vectors for bacterial, fungal, yeast, and mammalian cell hosts can be found in Cloning Vectors: A Laboratory Manual, (Elsevier, NY, 1985).

[0140] The expression vector construct can be introduced into the host cell using a method appropriate to the host cell. Various methods for introducing nucleic acids into host cells are known in the art, including but not limited to electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; viral transfection; non-viral transfection; microprojectile bombardment; lipofection; and infection (e.g., the vector is an infectious agent). Suitable host cells include prokaryotes, yeast, mammalian cells, or bacterial cells.

[0141] Suitable bacteria include Gram-negative or Gram-positive bacteria, such as Escherichia coli or Bacillus. Yeast, for example, from the genus Saccharomyces, such as Saccharomyces cerevisiae, can also be used to produce polypeptides. Various mammalian or insect cell culture systems can also be used to express recombinant proteins. Luckow and Summers, (Bio / Technology, 6:47, 1988) reviewed baculovirus systems for producing heterologous proteins in insect cells. Examples of suitable mammalian host cell lines include endothelial cells, COS-7 monkey kidney cells, CV-1, 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 a suitable host / vector system to express the recombinant protein. The protein is then purified from culture medium or cell extracts. Either the first or second polypeptide that forms the dimeric antigen receptor can be expressed by a transgenic host cell.

[0142] Antibodies and antigen-binding proteins disclosed herein can also be produced using a cell-based translation system. For this purpose, the nucleic acid encoding the polypeptide must be modified to allow in vitro transcription to produce mRNA and to allow the mRNA to be translated in the specific cell-free system used (eukaryotic, e.g., mammalian or yeast cell-free translation systems, or prokaryotic, e.g., bacterial cell-free translation systems).

[0143] Nucleic acids encoding any of the various polypeptides disclosed herein can be chemically synthesized. Codon usage can be selected to improve expression in cells. Such codon usage will depend on the cell type selected. Specific codon usage patterns have been developed for E. coli and other bacteria, as well as mammalian cells, plant cells, yeast cells, and insect cells. See, for example: Mayfield et al., Proc. Natl. Acad. Sci. USA. 2003 100(2):438-42; Sinclair et al. Protein Expr. Purif. 2002(1):96-105; Connell ND. Curr. Opin.

[0144] Biotechnol.2001 12(5):446-9; Makrides et al.Microbiol.Rev.1996 60(3):512-38; and Sharp etal.Yeast.1991 7(7):657-78.

[0145] The antibodies and antigen-binding proteins described herein can also be chemically synthesized (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, 111.) Modifications to proteins can also be produced by chemical synthesis.

[0146] The antibodies and antigen-binding proteins described herein can be purified by separation / purification methods of 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, reverse phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution, or any combination of these. After purification, the polypeptide can be exchanged into different buffers and / or concentrated by any of a variety of methods known in the art (including but not limited to filtration and dialysis).

[0147] 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 value of purity, the polypeptide is sufficiently pure for use as a pharmaceutical product. Any dimeric antigen receptor described herein can be expressed by a transgenic host cell and then purified to a purity of about 65-98% or higher using any method known in the art.

[0148] In certain embodiments, the antibodies and antigen-binding proteins herein may further comprise post-translational modifications. Exemplary post-translational protein modifications include phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, sulfonylation, biotinylation, or the addition of polypeptide side chains or hydrophobic groups. Thus, the modified polypeptide may comprise non-amino acid components, such as lipids, polysaccharides or monosaccharides, and phosphates. In one embodiment, glycosylation may be sialylation, which conjugates one or more sialic acid moieties to the polypeptide. The sialic acid moiety improves solubility and serum half-life while also reducing the potential immunogenicity of the protein. See Raju et al. Biochemistry. 2001 31; 40(30): 8868-76.

[0149] In one embodiment, the antibodies and antigen-binding proteins described herein can be modified into soluble polypeptides by linking the antibodies and antigen-binding proteins to a non-protein polymer. In one embodiment, the non-protein polymer comprises polyethylene glycol ("PEG"), polypropylene glycol, or a polyoxyalkylene in the manner specified in U.S. Patent Nos. 640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

[0150] The present disclosure provides therapeutic compositions comprising any dimeric antigen receptor described herein mixed with a pharmaceutically acceptable excipient. Excipients include carriers, stabilizers, and excipients. Pharmaceutically acceptable excipients include, for example, inert diluents or fillers (e.g., sucrose and sorbitol), lubricants, glidants, and anti-adherents (e.g., magnesium stearate, zinc stearate, stearic acid, silicon dioxide, hydrogenated vegetable oil, or talc). Other examples include buffers, stabilizers, preservatives, nonionic detergents, antioxidants, and isotonic agents.

[0151] Therapeutic compositions and methods for their preparation are well known in the art and are described, 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 and can include, for example, excipients, sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of plant origin, or hydrogenated naphthalene. Biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the antibodies (or their antigen-binding proteins) described herein. Nanoparticle formulations (e.g., biodegradable nanoparticles, solid lipid nanoparticles, liposomes) can be used to control the biodistribution of the antibodies (or their antigen-binding proteins). Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The level of the antibody (or antigen binding protein thereof) in the formulation will depend on a number of factors, including the dose of the drug used and the route of administration.

[0152] Any of the dimeric antigen receptors described herein can optionally be administered in the form of a pharmaceutically acceptable salt, such as a non-toxic acid addition salt or metal complex commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic acid, lactic acid, pamoic acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzoic acid, palmitic acid, suberic acid, salicylic acid, tartaric acid, methanesulfonic acid, toluenesulfonic acid, or trifluoroacetic acid; polymeric acids such as tannic acid, carboxymethyl cellulose; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like. Metal complexes include zinc, iron, and the like. In one example, the antibody (or its antigen-binding portion) is formulated in the presence of sodium acetate to increase thermal stability.

[0153] Any of the dimeric antigen receptors described herein can be formulated for oral use, including tablets containing the active ingredient mixed with a non-toxic pharmaceutically acceptable excipient. Oral formulations can also be provided in the form of chewable tablets, or in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, or in the form of soft gelatin capsules in which the active ingredient is mixed with water or an oil medium.

[0154] The present disclosure provides dimeric antigen receptors (DARs) comprising a Fab fragment connected to a transmembrane region and an intracellular signaling region. In one embodiment, the DAR construct comprises an optional hinge region between the Fab fragment and the transmembrane region. The DAR structure disclosed herein provides unexpected and surprising results, which compares the DAR structure of an antibody in the form of a Fab with the CAR structure in the form of an scFv with the same antibody. In addition, the DAR and CAR structures are directly compared because the hinge region, the transmembrane region and the two intracellular signaling regions are identical. However, the activity of the DAR structure is superior to its corresponding CAR structure.

[0155] The present disclosure provides a dimeric antigen receptor (DAR) construct comprising a heavy chain binding region on one polypeptide chain and a light chain binding region on another polypeptide chain. The two polypeptide chains comprising the dimeric antigen receptor can dimerize to form a protein complex. Dimeric antigen receptors have properties similar to antibodies, such as their specific binding to target antigens. The dimeric antigen receptors can be used for targeted cell therapy.

[0156] The present disclosure provides a structure of a DAR (dimeric antigen receptor) construct having a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an antibody heavy chain variable region, and the second polypeptide chain comprises an antibody light chain variable region, wherein when the first polypeptide chain and the second polypeptide chain are expressed by the same cell, the first polypeptide chain is connected to the second polypeptide chain via one or more disulfide bonds on the external region of the transduced cell. More specifically, the DAR construct comprises a first polypeptide chain, which in turn comprises an antibody heavy chain (or light chain) having a variable domain region and a CH1 region (κ (K) or λ (L)) with a corresponding CL / CK region, a hinge region, a transmembrane region, and one or two signaling domains, and a second polypeptide chain, wherein the second polypeptide chain comprises an antibody light chain (or heavy chain) variable domain region (κ (K) or λ (L)) with a corresponding CL / CK region, wherein the CH region in each of the first and second polypeptide chains is connected by one or two disulfide bonds on the hinge sequence.

[0157] The present disclosure provides a structure of a DAR (dimeric antigen receptor) construct having a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an antibody heavy chain variable region, and the second polypeptide chain comprises an antibody light chain variable region, wherein when the first polypeptide chain and the second polypeptide chain are expressed by the same cell, the first polypeptide chain is connected to the second polypeptide chain by one or more disulfide bonds on the external region of the transduced cell. More specifically, the DAR construct comprises a first polypeptide chain, which in turn comprises an antibody heavy chain having a variable domain region and a CH1 region (κ (K) or λ (L)) having a corresponding CL / CK region, a hinge region, a transmembrane region, one or two signaling domains, and a second polypeptide chain, wherein the second polypeptide chain comprises an antibody light chain variable domain region (κ or λ) having a corresponding CL / CK region, wherein the CH region in each of the first and second polypeptide chains is connected by one or two disulfide bonds on the hinge sequence.

[0158] In one embodiment, 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.

[0159] In one embodiment, the hinge region is about 10 to about 100 amino acids in length. In one embodiment, the hinge region is independently selected from the group consisting of a CD8 hinge region or a fragment thereof, a CD8α hinge region or a fragment thereof, a hinge region of an antibody (IgG, IgA, IgM, IgE or IgD) connecting the constant domains CH1 and CH2 of an antibody. The hinge region may be derived from an antibody and may or may not include one or more constant regions of an antibody.

[0160] In one embodiment, the transmembrane domain can be derived from a membrane protein sequence region selected from the group consisting of CD8α, CD8β, 4-1BB / CD137, CD28, CD34, CD4, FcεRIγ, CD16, OX40 / CD134, CD3

[0161] ζ, CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRζ, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD33, CD37, CD64, CD80, CD86, CD137, CD154, LFA-1, T cell co-receptor, CD2 T cell co-receptor / adhesion molecule, CD40, CD4OL / CD154, VEGFR2, FAS, and FGFR2B.

[0162] In one embodiment, the signaling region is selected from the group consisting of CD3-zeta chain, 4-1BB, CD28, CD27, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, GITR (TNFRSF18), DR3 (TNFRSF25), TNFR2, CD226, and combinations thereof.

[0163] In one embodiment, the overall design of the dimeric antigen receptor comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an antigen binding region, which is connected to a dimerization region, to a hinge region, to a transmembrane region, to one or more intracellular signaling sequence regions, and wherein the second polypeptide chain comprises an antigen binding domain and a dimerization domain. In one embodiment, the antigen binding domain of one or both of the first and 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 a combination thereof. In one embodiment, the dimerization domain of 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, a myc-max component, and a combination thereof. Figure 1-4 In the present invention, the "SS" represents any chemical bond or association that leads to dimerization of the first and second polypeptide chains, including a disulfide bond, a leucine zipper, or a myc-max component.

[0164] The present disclosure provides dimeric antigen receptor (DAR) constructs, wherein the first polypeptide chain carries a heavy chain variable region (VH) and a heavy chain constant region (CH), and the second polypeptide chain carries a light chain variable region (VL) and a light chain constant region (CL) (e.g., Figure 1 and 2In one embodiment, the dimeric antigen receptor (DAR) construct comprises: (a) a first polypeptide chain comprising, from amino terminus to carboxyl terminus, five regions: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) an optional hinge region, (iv) a transmembrane region (TM), and (v) an intracellular signaling region; and (b) a second polypeptide chain comprising, from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region (VL) (e.g., κ or λ) and (ii) an antibody light chain constant region (CL).

[0165] The present disclosure provides dimeric antigen receptor (DAR) constructs, wherein the first polypeptide chain carries a light chain variable region (VL) and a light chain constant region (CL), and the second polypeptide chain carries a heavy chain variable region (VH) and a heavy chain constant region (CH) (e.g., Figure 3 and 4 In one embodiment, the dimeric antigen receptor (DAR) construct comprises (a) a first polypeptide chain comprising, from amino terminus to carboxyl terminus, five regions: (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; and (b) a second polypeptide chain comprising, from amino terminus to carboxyl terminus, two regions: (i) an antibody heavy chain variable region (VH) and (ii) an antibody heavy chain constant region (CH).

[0166] In one embodiment, for Figure 1-4 In the dimer antigen receptor shown, the antibody heavy chain constant region (CH) and the antibody light chain constant region (CL) can dimerize to form a dimerization domain. In one embodiment, the antibody heavy chain constant region and the antibody light chain constant region dimerize through one or two disulfide bonds.

[0167] In one embodiment, for Figure 1-4 In the dimer antigen receptor shown, 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. For example, when the antibody heavy chain constant region and the antibody light chain constant region dimerize, the antibody heavy chain variable region and the antibody light chain variable region associate with each other.

[0168] In one embodiment, for Figure 1-4 The dimeric antigen receptor shown, the antigen binding domain formed by the antibody heavy chain variable region and the antibody light chain variable region binds to the target antigen.

[0169] In one embodiment, for Figure 1-4 The dimeric antigen receptor shown, the antibody heavy chain variable region and the antibody light chain variable region are fully human antibody regions.

[0170] In one embodiment, for Figure 1-4 The dimeric antigen receptor shown in FIG, the hinge region is about 10 to about 100 amino acids in length. In one embodiment, the hinge region comprises a hinge region or a fragment thereof from an antibody (e.g., IgG, IgA, IgM, IgE, or IgD). In one embodiment, the hinge region comprises a CD8 (e.g., CD8α) or CD28 hinge region or a fragment thereof. In one embodiment, the hinge region comprises a CPPC or SPPC amino acid sequence. In one embodiment, the hinge region comprises both CD8 and CD28 hinge sequences (e.g., a long hinge region), only a CD8 sequence (short hinge), or only a CD28 hinge sequence (e.g., a short hinge region).

[0171] In one embodiment, for Figure 1-4 The dimeric antigen receptor shown, the transmembrane regions of the first and second polypeptide chains can be independently derived from CD8α, CD8β, 4-1BB / CD137, CD28, CD34, CD4, FcεRI

[0172] γ, CD16, OX40 / CD134, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCR

[0173] ζ, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD33, CD37, CD64, CD80, CD86, CD137, CD154, LFA-1 T cell co-receptor, CD2 T cell co-receptor / adhesion molecule, CD40, CD4OL / CD154, VEGFR2, FAS, and FGFR2B.

[0174] In one embodiment, for Figure 1-4The dimeric antigen receptor shown, the intracellular signaling region of the first polypeptide comprises an intracellular signaling sequence selected from the following two to five signaling sequences in any order in any combination: 4-1BB, CD3ζ, CD28, CD27, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, GITR (TNFRSF18), DR3 (TNFRSF25), TNFR2, CD226 and combinations thereof. In one embodiment, the intracellular signaling region comprises an intracellular signaling sequence from any one or two or more of CD28, 4-1BB and / or CD3-ζ. In one embodiment, the intracellular signaling region comprises CD28 and CD3-ζ intracellular signaling sequences, or 4-1BB and CD3-ζ intracellular signaling sequences. In one embodiment, the CD3-ζ portion of the intracellular signaling region comprises ITAM (immunoreceptor tyrosine-based activation motif) motifs 1, 2, and 3 (e.g., long CD3-ζ). In one embodiment, the CD3-ζ portion of the intracellular signaling region comprises only one of the ITAM motifs, e.g., only ITAM 1, 2, or 3 (e.g., short CD3-ζ).

[0175] The present disclosure provides a dimeric antigen receptor (DAR) construct having a first and a second polypeptide chain that bind to each other to form an antigen binding domain that binds to a CD38 protein antigen. In one embodiment, the CD38 protein is derived from humans, cynomolgus monkeys, and / or mice. In one embodiment, the CD38 protein comprises a wild-type or mutant CD38 protein.

[0176] In one embodiment, 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. In one embodiment, the antibody heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 2. In one embodiment, 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: 21, or a hinge region comprising the CD28 and CD8 hinge sequences (e.g., long hinge) of SEQ ID NO: 19. In one embodiment, the transmembrane region is a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO: 6. In one embodiment, the intracellular signaling region comprises a signaling sequence selected from any one or any combination of two or more of the following groups: a 4-1BB signaling sequence comprising the amino acid sequence of SEQ ID NO: 7; a CD28 signaling sequence comprising the amino acid sequence of SEQ ID NO: 8; a CD3 zeta (long) signaling sequence comprising the amino acid sequence of SEQ ID NO: 9 and / or a CD3 zeta (short) signaling sequence having an ITAM 3 motif and comprising the amino acid sequence of SEQ ID NO: 20. In one embodiment, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 13 (e.g., with or without Figure 35B In one embodiment, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 13 and the CD28 signaling sequence (SEQ ID NO: 8) between the 4-1BB and CD3ζ signaling sequences. In one embodiment, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16 (e.g., with or without the amino acid sequence in Figure 35C In one embodiment, 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 CD3ζ signaling sequences.

[0177] In one embodiment, 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. In one embodiment, the antibody light chain constant region comprises the amino acid sequence of SEQ ID NO: 4. In one embodiment, the full length of the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 14 (e.g., with or without Figure 35BIn one embodiment, the full length of the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 17 (e.g., with or without Figure 35C The leader sequence is underlined in Figure 5).

[0178] The present disclosure provides a dimeric antigen receptor (DAR) construct comprising: (a) a first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, five regions: (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 having the amino acid sequence of SEQ ID NO: 7 and a CD3ζ intracellular signaling sequence having the amino acid sequence of SEQ ID NO: 9, and (b) a second polypeptide chain comprising, in order from amino terminus to carboxyl terminus, two regions: (i) a CD38 antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 3, and (ii) a CD28 hinge region comprising the amino acid sequence of SEQ ID NO: 5. NO: 4 amino acid sequence of the CD38 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 a binding CD38 protein (for example: Figure 1 )'s antigen-binding domain.

[0179] The present disclosure provides a dimeric antigen receptor (DAR) construct comprising: (a) a first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, five regions: (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 having the amino acid sequence of SEQ ID NO: 7 and a CD3ζ intracellular signaling sequence comprising an ITAM 3 motif and having the amino acid sequence of SEQ ID NO: 20, and (b) a second polypeptide chain comprising, in order from amino terminus to carboxyl terminus, two regions: (i) a CD38 antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 3, and (ii) a CD28 hinge region comprising the amino acid sequence of SEQ ID NO: 5. NO: 4 amino acid sequence of the CD38 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 a binding CD38 protein (for example: Figure 1 )'s antigen-binding domain.

[0180] The present disclosure provides a version 1 (e.g., V1) dimeric antigen receptor (DAR) construct comprising a first polypeptide chain carrying a heavy chain variable region (VH) and a heavy chain constant region (CH), and a second polypeptide chain carrying a light chain variable region (VL) and a light chain constant region (CL) (e.g., V2). Figure 1), wherein (a) the first polypeptide chain comprises, from amino terminus to carboxyl terminus, five regions: (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 a CD28 transmembrane sequence (e.g., SEQ ID NO: 6), and (v) an intracellular signaling region comprising a CD28 signaling sequence (e.g., SEQ ID NO: 8) and a CD3-ζ signaling sequence having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 9); and (b) a second polypeptide chain comprises, from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region (VL) (e.g., κ or λ) and (ii) an antibody light chain constant region (CL). In one embodiment, the antibody heavy chain variable region (VH) 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).

[0181] The present disclosure provides a version 2 (e.g., V2) dimeric antigen receptor (DAR) construct comprising a first polypeptide chain carrying a heavy chain variable region (VH) and a heavy chain constant region (CH), and a second polypeptide chain carrying a light chain variable region (VL) and a light chain constant region (CL) (e.g., Figure 1 and 2 ), wherein (a) a first polypeptide chain comprises, in order from amino terminus to carboxyl terminus, five regions: (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 a CD28 transmembrane sequence (e.g., SEQ ID NO: 6), and (v) an intracellular signaling region comprising (1) a 4-1BB signaling sequence (e.g., SEQ ID NO: 7) and a CD3-ζ having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 9), or (2) a CD28 (e.g., SEQ ID NO: 8) signaling sequence and a CD3-ζ having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 9), or (3) a 4-1BB (e.g., SEQ ID NO: 7) signaling sequence and a CD28 (e.g., SEQ ID NO: 8) signaling sequence. NO: 8) signaling sequence and CD3-ζ having ITAM motifs 1, 2 and 3 (e.g., SEQ ID NO: 9); (b) a second polypeptide chain comprising, from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region (VL) (e.g., κ or λ) and (ii) an antibody light chain constant region (CL).

[0182] In one embodiment, the version 2a (V2a) DAR construct comprises an intracellular signaling domain having a 4-1BB signaling sequence (eg, SEQ ID NO: 7) and CD3-ζ having ITAM motifs 1, 2, and 3 (eg, SEQ ID NO: 9).

[0183] In one embodiment, the version 2b (V2b) DAR construct comprises an intracellular signaling domain having a CD28 signaling sequence (eg, SEQ ID NO: 8) and CD3-zeta having ITAM motifs 1, 2, and 3 (eg, SEQ ID NO: 9).

[0184] In one embodiment, the version 2c (V2c) DAR construct comprises an intracellular signaling region having a 4-1BB signaling sequence (e.g., SEQ ID NO: 7) and a CD28 signaling sequence (e.g., SEQ ID NO: 8) and CD3-ζ having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 9).

[0185] In one embodiment, DAR V2a and V2b are second generation DAR constructs, and DAR V2c is a third generation DAR construct. In one embodiment, the antibody heavy chain variable region (VH) 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).

[0186] The present disclosure provides a version 3 (e.g., V3) dimeric antigen receptor (DAR) construct carrying a first polypeptide chain having a heavy chain variable region (VH) and a heavy chain constant region (CH), and a second polypeptide chain (e.g., VL) carrying a light chain variable region (VL) and a light chain constant region (CL). Figure 1), wherein (a) a first polypeptide chain comprises, from amino terminus to carboxyl terminus, five regions: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a short chain region comprising a CD28 hinge sequence (e.g., SEQ ID NO: 5), (iv) a transmembrane region (TM) comprising a CD28 transmembrane sequence (e.g., SEQ ID NO: 6), and (v) an intracellular signaling region comprising a 4-1BB signaling sequence (e.g., SEQ ID NO: 7) and a CD3-ζ signaling sequence having only ITAM motif 3 (e.g., SEQ ID NO: 20); and (b) a second polypeptide chain comprises, from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region (VL) (e.g., κ or λ) and (ii) an antibody light chain constant region (CL). In one embodiment, the antibody heavy chain variable region (VH) 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).

[0187] The present disclosure provides a version 4 (e.g., V4) dimeric antigen receptor (DAR) construct comprising a first polypeptide chain carrying a heavy chain variable region (VH) and a heavy chain constant region (CH), and a second polypeptide chain carrying a light chain variable region (VL) and a light chain constant region (CL), wherein (a) the first polypeptide chain comprises, in order from amino terminus to carboxyl terminus, four regions: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a transmembrane region (TM) comprising a CD28 transmembrane sequence (e.g., SEQ ID NO: 6), and (iv) an intracellular signaling region comprising a 4-1BB signaling sequence (e.g., SEQ ID NO: 7) and a CD3-ζ signaling sequence having only ITAM motif 3 (e.g., SEQ ID NO: 20); and (b) the second polypeptide chain comprises, in order from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region (VL) (e.g., κ or λ) and (ii) an antibody light chain constant region (CL). The DAR V4 construct lacks a hinge sequence. In one embodiment, the antibody heavy chain variable region (VH) 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).

[0188] The present disclosure provides precursor polypeptides. In one embodiment, the precursor polypeptide can be processed into first and second polypeptide chains that associate / assemble to form a dimeric antigen receptor (DAR) construct.

[0189] The present disclosure provides a precursor polypeptide comprising ten regions, in order 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. Figure 5 and 6 In a non-limiting example, the intracellular signaling region comprises an intracellular signaling sequence of any combination of at least two of 4-1BB, CD3ζ, and / or CD28 ( Figure 5 and 6 ). The skilled artisan will appreciate that other combinations of intracellular signaling sequences are possible. The T2A cleavage sequence is an amino acid sequence that promotes ribosome skipping and de novo protein translation, thereby producing two independent polypeptides. In one embodiment, the precursor polypeptide population includes a mixture of polypeptides that have or have not been cleaved at the T2A cleavage sequence, and / or a mixture of polypeptides that have been cleaved at the heavy chain and / or light chain leader sequence.

[0190] The present disclosure provides a precursor polypeptide comprising ten regions, in order 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. Figure 7 and 8 In a non-limiting example, the intracellular signaling region comprises an intracellular signaling sequence of any combination of at least two of 4-1BB, CD3ζ, and / or CD28 ( Figure 7 and 8 ). The skilled artisan will appreciate that other combinations of intracellular signaling sequences are possible. The T2A cleavage sequence is an amino acid sequence that promotes ribosome skipping and de novo protein translation, thereby producing two independent polypeptides. In one embodiment, the precursor polypeptide population includes a mixture of polypeptides that have or have not been cleaved at the T2A cleavage sequence and / or a mixture of polypeptides that have been cleaved at the heavy chain and / or light chain leader sequence.

[0191] In one embodiment, for Figure 5-8The precursor polypeptide shown, the heavy chain and light chain leader sequences comprise peptide signal sequences that target the polypeptide chains (e.g., the first and second polypeptide chains) to the secretory pathway of the cell and will allow the polypeptide to be integrated and anchored into the lipid bilayer of the cell membrane. The heavy chain and light chain leader sequences can direct the transport of the precursor polypeptide from the cytosol to the endoplasmic reticulum of the host cell. The heavy chain and light chain leader sequences comprise signal sequences containing CD8α, CD28, or CD16 leader sequences.

[0192] In one embodiment, for Figure 5-8 The precursor polypeptide shown includes a first peptide signal sequence (eg, a heavy chain or light chain leader sequence) at its N-terminus.

[0193] In one embodiment, for Figure 5-8 The precursor polypeptide shown may include a second peptide signal sequence (eg, a heavy chain or light chain leader sequence) located after the cleavage sequence.

[0194] In one embodiment, the precursor polypeptide can be cleaved at the cleavage sequence, thereby producing a first and a second polypeptide chain, each having a peptide signal sequence at its N-terminus.

[0195] In one embodiment, for Figure 5-8 The precursor polypeptide shown, wherein processing of the precursor polypeptide includes cleavage of the precursor into first and second polypeptide chains, secretion of the precursor, and / or anchoring the precursor in the cell membrane.

[0196] In one embodiment, for Figure 5-8 The precursor polypeptide shown, after the precursor polypeptide chain is cut to produce the first and second polypeptide chains, the antibody heavy chain constant region (CH) (in one polypeptide chain) and the antibody light chain constant region (CL) (of the other polypeptide chain) can dimerize to form a dimerization domain. In one embodiment, the antibody heavy chain constant region and the antibody light chain constant region dimerize through one or two disulfide bonds.

[0197] In one embodiment, for Figure 5-8 In the precursor polypeptide shown, after the precursor polypeptide chain is cleaved to produce the first and second polypeptide chains, the antibody heavy chain variable region (VH) (in one polypeptide chain) and the antibody light chain variable region (VL) (in the other polypeptide chain) bind to each other to form an antigen-binding domain. For example, when the antibody heavy chain constant region and the antibody light chain constant region dimerize, the antibody heavy chain variable region and the antibody light chain variable region bind to each other.

[0198] In one embodiment, for Figure 5-8 The precursor polypeptide shown, the antigen binding domain formed by the antibody heavy chain variable region and the antibody light chain variable region binds to the target antigen.

[0199] In one embodiment, for Figure 5-8 The precursor polypeptide, antibody heavy chain variable region and antibody light chain variable region shown are fully human antibody regions.

[0200] In one embodiment, for Figure 5-8 The precursor polypeptide shown in FIG, wherein the hinge region is about 10 to about 100 amino acids in length. In one embodiment, the hinge region comprises a hinge region or a fragment thereof from an antibody (e.g., IgG, IgA, IgM, IgE, or IgD). In one embodiment, the hinge region comprises a CD8 (e.g., CD8α) or CD28 hinge region or a fragment thereof. In one embodiment, the hinge region comprises a CPPC or SPPC amino acid sequence. In one embodiment, the hinge region comprises a CD8 and CD28 hinge sequence (e.g., a long hinge region), only a CD8 sequence (short hinge), or only a CD28 hinge sequence (e.g., a short hinge region).

[0201] In one embodiment, for Figure 5-8 The precursor polypeptide shown, the transmembrane region of the precursor polypeptide chain can be derived from CD8α, CD8β, 4-1BB / CD137, CD28, CD34, CD4, FcεRIγ, CD16, OX40 / CD134, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRζ, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD33, CD37, CD64, CD80, CD86, CD137, CD154, LFA-1 T cell co-receptor, CD2 T cell co-receptor / adhesion molecule, CD40, CD4OL / CD154, VEGFR2, FAS, and FGFR2B.

[0202] In one embodiment, for Figure 5-8The dimeric antigen receptor shown, the intracellular signaling region of the first polypeptide comprises an intracellular signaling sequence selected from the following two to five signaling sequences in any combination in any order: 4-1BB, CD3ζ, CD28, CD27, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, GITR (TNFRSF18), DR3 (TNFRSF25), TNFR2, CD226. In one embodiment, the intracellular signaling region comprises an intracellular signaling sequence from any one or two or more of CD28, 4-1BB and / or CD3-ζ. In one embodiment, the intracellular signaling region comprises CD28 and CD3-ζ intracellular signaling sequences, or 4-1BB and CD3-ζ intracellular signaling sequences. In one embodiment, the CD3-ζ portion of the intracellular signaling region comprises ITAM (immunoreceptor tyrosine-based activation motif) motifs 1, 2, and 3 (e.g., long CD3-ζ). In one embodiment, the CD3-ζ portion of the intracellular signaling region comprises only one of the ITAM motifs, e.g., only ITAM 1, 2, or 3 (e.g., short CD3-ζ).

[0203] 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 region comprising the amino acid sequence of SEQ ID NO: 21; (5) a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO: 6; (6) an intracellular signaling region comprising any one or a combination of any three or more signaling sequences selected from the group consisting of a 4-1BB signaling sequence comprising the amino acid sequence of SEQ ID NO: 7, a CD28 signaling sequence comprising the amino acid sequence of SEQ ID NO: 8, a CD3ζ (long) signaling sequence comprising the amino acid sequence of SEQ ID NO: 9, and / or a CD3ζ (long) signaling sequence having an ITAM 3 motif and comprising the amino acid sequence of SEQ ID NO: NO: 20; (7) a CD3ζ (short) signaling 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) a CD38 antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 3; and (10) a CD38 antibody light chain constant region comprising the amino acid sequence of SEQ ID NO: 4. In one embodiment, 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 CD3ζ signaling sequences. In one embodiment, the precursor polypeptide can be cleaved at the T2A cleavable sequence to release the first and second polypeptide chains and secrete the precursor, and / or anchor the precursor to the cell 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 to the CD38 antigen. In one embodiment, the hinge region is optional.

[0204] 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 a CD3ζ (short) signaling sequence having an ITAM 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) a light chain leader sequence comprising the amino acid sequence of SEQ ID NO: 11; (9) a CD28 cleavage sequence comprising the amino acid sequence of SEQ ID NO: 13; NO: 3 amino acid sequence; (10) a CD38 antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 4; (11) a CD38 antibody light chain constant region comprising the amino acid sequence of SEQ ID NO: 5. In one embodiment, the full-length precursor polypeptide comprises the amino acid sequence of SEQ ID NO: 18. In one embodiment, the precursor polypeptide can be cleaved at the T2A cleavable sequence to release the first and second polypeptide chains and secrete the precursor, and / or anchor the precursor to the cell membrane. In one embodiment, after releasing the first and second polypeptide chains, 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 through 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 to the CD38 antigen.

[0205] The present disclosure provides nucleic acids encoding any of the first polypeptide chain, the second polypeptide chain, the first and second polypeptide chains, the dimeric antigen receptor, or the precursor polypeptide described herein.

[0206] The present disclosure provides a nucleic acid encoding a first polypeptide chain comprising: five regions, in order from amino terminus to 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.

[0207] The present disclosure provides a nucleic acid encoding a second polypeptide chain comprising: two regions, sequentially from amino terminus to carboxyl terminus: (i) an antibody light chain variable region and (ii) an antibody light chain constant region.

[0208] In one embodiment, the nucleic acid encodes a first and a second polypeptide chain, said first and second polypeptide chains comprising:

[0209] (a) a first polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, five regions: (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, sequentially from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region and (ii) an antibody light chain constant region (e.g., Figure 1 and 2 ).

[0210] The present disclosure provides a nucleic acid encoding a first polypeptide chain comprising: five regions, in order from amino terminus to 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.

[0211] The present disclosure provides a nucleic acid encoding a second polypeptide chain comprising: two regions, sequentially from amino terminus to carboxyl terminus: (i) an antibody heavy chain variable region and (ii) an antibody heavy chain constant region.

[0212] In one embodiment, the nucleic acid encodes a first and a second polypeptide chain, said first and second polypeptide chains comprising:

[0213] (a) a first polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, five regions: (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, sequentially from amino terminus to carboxyl terminus, two regions: (i) an antibody heavy chain variable region and (ii) an antibody heavy chain constant region (e.g., Figure 3 and 4 ).

[0214] The present disclosure provides a nucleic acid encoding a first polypeptide chain comprising: six regions, in order from amino terminus to 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.

[0215] The present disclosure provides a nucleic acid encoding a second polypeptide chain comprising: three regions, sequentially from amino terminus to carboxyl terminus: (i) a light chain leader region, (ii) an antibody light chain variable region, and (iii) an antibody light chain constant region.

[0216] In one embodiment, the nucleic acid encodes a first and a second polypeptide chain, said first and second polypeptide chains comprising:

[0217] (a) a first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, six regions: (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, in order from amino terminus to carboxyl terminus: (i) an antibody light chain variable region and (ii) an antibody light chain constant region.

[0218] The present disclosure provides a nucleic acid encoding a first polypeptide chain comprising: six regions, in order from amino terminus to 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.

[0219] The present disclosure provides a nucleic acid encoding a second polypeptide chain, wherein the first polypeptide chain comprises: three regions, sequentially from amino terminus to carboxyl terminus: (i) a heavy chain leader region, (ii) an antibody heavy chain variable region, and (iii) an antibody light chain constant region.

[0220] In one embodiment, the nucleic acid encodes a first and a second polypeptide chain, said first and second polypeptide chains comprising:

[0221] (a) a first polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, six regions: (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, sequentially from amino terminus to carboxyl terminus, two regions: (i) an antibody heavy chain variable region and (ii) an antibody heavy chain constant region.

[0222] The present disclosure provides a nucleic acid encoding a precursor polypeptide, wherein the precursor polypeptide comprises: ten regions from amino terminus to 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) a T2A cleavage 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., Figure 5 and 6 ).

[0223] The present disclosure provides a nucleic acid encoding a precursor polypeptide, wherein the precursor polypeptide comprises: ten regions from amino terminus to 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) a T2A cleavage 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., Figure 7 and 8 ).

[0224] The present disclosure provides a nucleic acid encoding a first polypeptide chain comprising, from amino terminus to carboxyl terminus, five regions: (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: 21, (iv) a CD28 transmembrane region comprising the amino acid sequence of SEQ ID NO: 6, and (v) an intracellular signaling region comprising a signaling sequence selected from any one or any combination of two or more of the following groups: a 4-1BB signaling sequence comprising the amino acid sequence of SEQ ID NO: 7; a CD28 signaling sequence comprising the amino acid sequence of SEQ ID NO: 8; a CD3 zeta (long) signaling sequence comprising the amino acid sequence of SEQ ID NO: 9 and / or a CD3 zeta (short) signaling sequence having an ITAM 3 motif and comprising the amino acid sequence of SEQ ID NO: 20. In one embodiment, the nucleic acid encodes a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 13 or 16 (eg, with or without Figure 35Bor the underlined leader sequence in 35C). In one embodiment, the nucleic acid encodes a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 13 and the CD28 signaling sequence (SEQ ID NO: 8) between the 4-1BB and CD3ζ signaling sequences. In one embodiment, the nucleic acid encodes a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 16 (e.g., with or without the amino acid sequence in Figure 35C In one embodiment, the nucleic acid encodes a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 16 and a CD28 signaling sequence (SEQ ID NO: 8) between the 4-1BB and CD3ζ signaling sequences. In one embodiment, the hinge region is optional.

[0225] In one embodiment, the nucleic acid encodes a second polypeptide chain of a dimeric antigen receptor comprising an antibody light chain variable region having the amino acid sequence of SEQ ID NO: 3. In one embodiment, the nucleic acid encodes a second polypeptide chain of a dimeric antigen receptor comprising an antibody light chain constant region having 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 Figure 35B or the underlined leader sequence in 35C).

[0226] The present disclosure provides a nucleic acid encoding a second polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, two regions: (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.

[0227] In one embodiment, the nucleic acid encodes a first polypeptide chain (SEQ ID NO: 13 or 16), a T2A cleavage sequence (SEQ ID NO: 12), and a second polypeptide chain (SEQ ID NO: 14 or 17). In one embodiment, the first polypeptide chain (SEQ ID NO: 13 or 16) comprises or lacks Figure 35B and the underlined leader sequence in C. In one embodiment, the second polypeptide chain (SEQ ID NO: 14 or 17) comprises or lacks Figure 35B or the underlined leader sequence in C.

[0228] The present disclosure provides a nucleic acid encoding a precursor polypeptide comprising the amino acid sequence of SEQ ID NO: 15 or 18,

[0229] The present disclosure provides nucleic acids encoding version 1 (e.g., V1) dimeric antigen receptor (DAR) constructs comprising a first polypeptide chain carrying a heavy chain variable region (VH) and a heavy chain constant region (CH), and a second polypeptide chain carrying a light chain variable region (VL) and a light chain constant region (CL) (e.g., V2). Figure 1 ), wherein (a) the first nucleic acid encodes a first polypeptide chain comprising five regions, sequentially 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 hinge region comprising CD8 and CD28 (e.g.,

[0230] The invention relates to a novel scaffold comprising a long hinge region comprising a CD28 transmembrane sequence (e.g., SEQ ID NO: 19), (iv) a transmembrane region (TM) comprising a CD28 transmembrane sequence (e.g., SEQ ID NO: 6), and (v) an intracellular signaling region comprising a CD28 signaling sequence (e.g., SEQ ID NO: 8) and a CD3-ζ signaling sequence having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO:

[0231] 9); (b) a second nucleic acid encoding a second polypeptide chain comprising two regions, sequentially from the amino terminus to the carboxyl terminus:

[0232] (i) an antibody light chain variable region (VL) (e.g., κ or λ) and (ii) an antibody light chain constant region (CL). In one embodiment, the antibody heavy chain variable region (VH) 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).

[0233] The present disclosure provides nucleic acids encoding version 2 (e.g., V2) dimeric antigen receptor (DAR) constructs comprising a first polypeptide chain carrying a heavy chain variable region (VH) and a heavy chain constant region (CH), and a second polypeptide chain carrying a light chain variable region (VL) and a light chain constant region (CL) (e.g., Figure 1 and 2), wherein (a) the first nucleic acid encodes a first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, five regions: (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 a CD28 transmembrane sequence (e.g., SEQ ID NO: 6), and (v) an intracellular signaling region comprising (1) a 4-1BB signaling sequence (e.g., SEQ ID NO: 7) and a CD3-ζ having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 9), or (2) a CD28 (e.g., SEQ ID NO: 8) signaling sequence and a CD3-ζ having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 9), or (3) a 4-1BB (e.g., SEQ ID NO: 7) signaling sequence and a CD28 (e.g., SEQ ID NO: 8) signaling sequence. NO: 8) signaling sequence and CD3-ζ having ITAM motifs 1, 2 and 3 (e.g., SEQ ID NO: 9); (b) a second nucleic acid encoding a second polypeptide chain comprising, from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region (VL) (e.g., κ or λ) and (ii) an antibody light chain constant region (CL).

[0234] In one embodiment, the nucleic acid encodes a version 2a (V2a) DAR construct comprising an intracellular signaling region having a 4-1BB signaling sequence (e.g., SEQ ID NO: 7) and CD3-ζ having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 9).

[0235] In one embodiment, the nucleic acid encodes a version 2b (V2b) DAR construct comprising an intracellular signaling region having a CD28 (e.g., SEQ ID NO: 8) signaling sequence and CD3-ζ having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 9).

[0236] In one embodiment, the nucleic acid encodes a version 2c (V2c) DAR construct comprising an intracellular signaling region having a 4-1BB (e.g., SEQ ID NO: 7) signaling sequence and a CD28 (e.g., SEQ ID NO: 8) signaling sequence and CD3-ζ (e.g., SEQ ID NO: 9) having ITAM motifs 1, 2, and 3. In one embodiment, DAR V2a and V2b are second generation DAR constructs, while DAR V2c is a third generation DAR construct. In one embodiment, the antibody heavy chain variable region (VH) 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).

[0237] The present disclosure provides nucleic acids encoding version 3 (e.g., V3) dimeric antigen receptor (DAR) constructs comprising a first polypeptide chain carrying a heavy chain variable region (VH) and a heavy chain constant region (CH), and a second polypeptide chain carrying a light chain variable region (VL) and a light chain constant region (CL) (e.g., Figure 1 ), wherein (a) the first nucleic acid encodes a first polypeptide chain comprising five regions, sequentially from amino terminus to 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 a CD28 transmembrane sequence (e.g., SEQ ID NO: 6), and (v) an intracellular signaling region comprising a 4-1BB signaling sequence (e.g., SEQ ID NO:

[0238] 7) and a CD3-ζ signaling sequence having only ITAM motif 3 (e.g., SEQ ID NO: 20); (b) a second nucleic acid encoding a second polypeptide chain comprising, from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region (VL) (e.g., κ or λ) and (ii) an antibody light chain constant region (CL). In one embodiment, the antibody heavy chain variable region (VH) 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).

[0239] The present disclosure provides nucleic acids encoding version 4 (e.g., V4) dimeric antigen receptor (DAR) constructs, the construct comprising a first polypeptide chain carrying a heavy chain variable region (VH) and a heavy chain constant region (CH), and a second polypeptide chain carrying a light chain variable region (VL) and a light chain constant region (CL), wherein (a) the first nucleic acid encodes a first polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, four regions: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a transmembrane region (TM) comprising a CD28 transmembrane sequence (e.g., SEQ ID NO: 6), and (iv) an intracellular signaling region comprising a 4-1BB signaling sequence (e.g., SEQ ID NO: 7) and a CD3-ζ signaling sequence having only ITAM motif 3 (e.g., SEQ ID NO: 20);

[0240] (b) The second nucleic acid encodes a second polypeptide chain comprising, from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region (VL) (e.g., kappa or lambda) and (ii) an antibody light chain constant region (CL). The DAR V4 construct lacks a hinge sequence. In one embodiment, the antibody heavy chain variable region (VH) 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).

[0241] The present disclosure provides vectors operably linked to a nucleic acid encoding any of the first polypeptide chain, the second polypeptide chain, the first and second polypeptide chains, the dimeric antigen receptor, or the precursor polypeptide described herein.

[0242] The present disclosure provides a vector operably linked to a nucleic acid encoding a first polypeptide chain, the first polypeptide chain comprising: five regions, in order from amino terminus to 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.

[0243] The present disclosure provides a vector operably linked to a nucleic acid encoding a second polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region and (ii) an antibody light chain constant region.

[0244] In one embodiment, the vector is operably linked to nucleic acids encoding first and second polypeptide chains, the first and second polypeptide chains comprising: (a) a first polypeptide chain comprising: five regions, sequentially from amino terminus to carboxyl terminus: (i) a heavy antibody 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, sequentially from amino terminus to carboxyl terminus: (i) an antibody light chain variable region and (ii) an antibody light chain constant region.

[0245] The present disclosure provides a vector operably linked to a nucleic acid encoding a first polypeptide chain, the first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, five regions: (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.

[0246] The present disclosure provides a vector operably linked to a nucleic acid encoding a second polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, two regions: (i) an antibody heavy chain variable region and (ii) an antibody heavy chain constant region.

[0247] In one embodiment, the vector is operably linked to nucleic acids encoding first and second polypeptide chains, the first and second polypeptide chains comprising: (a) a first polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, five regions: (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, sequentially from amino terminus to carboxyl terminus: (i) an antibody heavy chain variable region and (ii) an antibody heavy chain constant region.

[0248] The present disclosure provides a vector operably linked to a nucleic acid encoding a first polypeptide chain, the first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, six regions: (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.

[0249] The present disclosure provides a vector operably linked to a nucleic acid encoding a second polypeptide chain, the second polypeptide chain comprising: three regions, sequentially from amino terminus to carboxyl terminus: (i) a light chain leader region, (ii) an antibody light chain variable region, and (iii) an antibody light chain constant region.

[0250] In one embodiment, the vector is operably linked to nucleic acids encoding first and second polypeptide chains, the first and second polypeptide chains comprising: (a) a first polypeptide chain comprising, in sequence from amino terminus to carboxyl terminus, six regions: (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: in sequence from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region and (ii) an antibody light chain constant region.

[0251] The present disclosure provides a vector operably linked to a nucleic acid encoding a first polypeptide chain, the first polypeptide chain comprising: six regions, in order from amino terminus to 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.

[0252] The present disclosure provides a vector operably linked to a nucleic acid encoding a second polypeptide chain, the second polypeptide chain comprising: three regions, sequentially from amino terminus to carboxyl terminus: (i) a heavy chain leader region, (ii) an antibody heavy chain variable region, and (iii) an antibody light chain constant region.

[0253] In one embodiment, the vector is operably linked to nucleic acids encoding first and second polypeptide chains, the first and second polypeptide chains comprising: (a) a first polypeptide chain comprising, in sequence from amino terminus to carboxyl terminus, six regions: (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: in sequence from amino terminus to carboxyl terminus, two regions: (i) an antibody heavy chain variable region and (ii) an antibody heavy chain constant region.

[0254] The present disclosure provides a vector operably linked to a nucleic acid encoding a precursor polypeptide, wherein the precursor polypeptide includes: ten regions from amino terminus to 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) a T2A cleavage sequence region, (8) a light chain leader region, (9) an antibody light chain variable region, and (10) an antibody light chain constant region.

[0255] The present disclosure provides a vector operably linked to a nucleic acid encoding a precursor polypeptide, wherein the precursor polypeptide includes: ten regions from amino terminus to 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) a T2A cleavage sequence region, (8) a heavy chain leader region, (9) an antibody heavy chain variable region, and (10) an antibody heavy chain constant region.

[0256] The present disclosure provides a vector operably linked to a nucleic acid encoding a first polypeptide chain comprising, in order from amino terminus to 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 CD3ζ intracellular signaling sequence comprising the amino acid sequence of SEQ ID NO: 9.

[0257] The present disclosure provides a vector operably linked to a nucleic acid encoding a second polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, two regions: (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.

[0258] In one embodiment, the vector is operably linked to a nucleic acid encoding a first polypeptide chain (SEQ ID NO: 13 or 16) and a second polypeptide chain (SEQ ID NO: 14 or 17).

[0259] In one embodiment, the vector is operably linked to a nucleic acid encoding a first polypeptide chain (SEQ ID NO: 13 or 16), a T2A cleavage sequence (SEQ ID NO: 12), and a second polypeptide chain (SEQ ID NO: 140 or 17). In one embodiment, the first polypeptide chain (SEQ ID NO: 13 or 16) includes or lacks Figure 35B and the underlined leader sequence in C. In one embodiment, the second polypeptide chain (SEQ ID NO: 14 or 17) comprises or lacks Figure 35Band the leader sequence underlined in C. The present disclosure provides a vector operably linked to a nucleic acid encoding a precursor polypeptide comprising the amino acid sequence of SEQ ID NO: 15 or 18.

[0260] The present disclosure provides vectors operably linked to nucleic acids encoding version 1 (e.g., V1) of a dimeric antigen receptor (DAR) construct comprising a first polypeptide chain carrying a heavy chain variable region (VH) and a heavy chain constant region (CH), and a second polypeptide chain carrying a light chain variable region (VL) and a light chain constant region (CL) (e.g., V2). Figure 1 ), wherein (a) a first vector is operably linked to a first nucleic acid encoding a first polypeptide chain, the first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, five regions: (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 a CD28 transmembrane sequence (e.g., SEQ ID NO: 6), and (v) an intracellular signaling region comprising a CD28 signaling sequence (e.g., SEQ ID NO: 8) and a CD3-ζ signaling sequence having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 9); and (b) the first vector is operably linked to a second nucleic acid encoding a second polypeptide chain, the second polypeptide chain comprising, in order from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region (VL) (e.g., κ or λ) and (ii) an antibody light chain constant region (CL). In one embodiment, the antibody heavy chain variable region (VH) 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).

[0261] The present disclosure provides vectors operably linked to nucleic acids encoding version 2 (e.g., V2) dimeric antigen receptor (DAR) constructs comprising a first polypeptide chain carrying a heavy chain variable region (VH) and a heavy chain constant region (CH), and a second polypeptide chain carrying a light chain variable region (VL) and a light chain constant region (CL) (e.g., V3). Figure 1 and 2), wherein (a) a first vector is operably linked to a first nucleic acid encoding a first polypeptide chain, the first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, five regions: (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 a CD28 transmembrane sequence (e.g., SEQ ID NO: 6), and (v) an intracellular signaling region comprising (1) a 4-1BB signaling sequence (e.g., SEQ ID NO: 7) and a CD3-ζ having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 9), or (2) a CD28 (e.g., SEQ ID NO: 8) signaling sequence and a CD3-ζ having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 9), or (3) a 4-1BB (e.g., SEQ ID NO: 7) signaling sequence and a CD28 (e.g., SEQ ID NO: 8) signaling sequence. NO: 8) signaling sequence and CD3-ζ having ITAM motifs 1, 2 and 3 (e.g., SEQ ID NO: 9); (b) the first vector is operably linked to a second nucleic acid encoding a second polypeptide chain, the second polypeptide chain comprising two regions, sequentially from amino terminus to carboxyl terminus: (i) an antibody light chain variable region (VL) (e.g., κ or λ) and (ii) an antibody light chain constant region (CL).

[0262] In one embodiment, the vector is operably linked to a nucleic acid encoding a version 2a (V2a) DAR construct comprising an intracellular signaling region having a 4-1BB signaling sequence (e.g., SEQ ID NO: 7) and CD3-ζ having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 9).

[0263] In one embodiment, the vector is operably linked to a nucleic acid encoding a version 2b (V2b) DAR construct comprising an intracellular signaling region having a CD28 (e.g., SEQ ID NO: 8) signaling sequence and a CD3-ζ (e.g., SEQ ID NO: 9) having ITAM motifs 1, 2, and 3.

[0264] In one embodiment, the vector is operably linked to a nucleic acid encoding a version 2c (V2c) DAR construct comprising a 4-1BB (e.g., SEQ ID NO: 7) signaling sequence and a CD28 (e.g., SEQ ID NO: 8) signaling sequence and a CD3-ζ (e.g., SEQ ID NO: 9) signaling sequence having ITAM motifs 1, 2, and 3.

[0265] 9). In one embodiment, DAR V2a and V2b are second-generation DAR constructs, while DAR V2c is a third-generation DAR construct. In one embodiment, the antibody heavy chain variable region (VH) 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).

[0266] The present disclosure provides vectors operably linked to nucleic acids encoding version 3 (e.g., V3) dimeric antigen receptor (DAR) constructs comprising a first polypeptide chain carrying a heavy chain variable region (VH) and a heavy chain constant region (CH), and a second polypeptide chain carrying a light chain variable region (VL) and a light chain constant region (CL) (e.g., V3). Figure 1 ), wherein (a) the first vector is operably linked to a first nucleic acid encoding a first polypeptide chain, the first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, five regions: (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 a CD28 transmembrane sequence (e.g., SEQ ID NO: 6), and (v) an intracellular signaling region comprising a 4-1BB signaling sequence (e.g., SEQ ID NO: 7) and a CD3-ζ signaling sequence having only ITAM motif 3 (e.g., SEQ ID NO: 20); and (b) the first vector is operably linked to a second nucleic acid encoding a second polypeptide chain, the second polypeptide chain comprising, in order from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region (VL) (e.g., κ or λ) and (ii) an antibody light chain constant region (CL). In one embodiment, the antibody heavy chain variable region (VH) 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).

[0267] The present disclosure provides vectors operably linked to a nucleic acid encoding a version 4 (e.g., V4) dimeric antigen receptor (DAR) construct, the construct comprising a first polypeptide chain carrying a heavy chain variable region (VH) and a heavy chain constant region (CH), and a second polypeptide chain carrying a light chain variable region (VL) and a light chain constant region (CL), wherein (a) the first vector is operably linked to a first nucleic acid encoding the first polypeptide chain, the first polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, four regions: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a transmembrane region (TM) comprising a CD28 transmembrane sequence (e.g., SEQ ID NO: 6), and (iv) a CD3-ζ signaling sequence comprising a 4-1BB signaling sequence (e.g., SEQ ID NO: 7) and a CD3-ζ signaling sequence having only ITAM motif 3 (e.g., SEQ ID NO: 8). NO: 20) intracellular signaling region; (b) the first vector is operably linked to a second nucleic acid encoding a second polypeptide chain, the second polypeptide chain comprising two regions, sequentially from amino terminus to carboxyl terminus: (i) an antibody light chain variable region (VL) (e.g., κ or λ) and (ii) an antibody light chain constant region (CL). The DAR V4 construct lacks a hinge sequence. In one embodiment, the antibody heavy chain variable region (VH) 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).

[0268] The present disclosure provides host cells or host cell populations carrying one or more expression vectors operably linked to a nucleic acid transgene encoding any of the first polypeptide chain, second polypeptide chain, first and second polypeptide chains, dimeric antigen receptor, or precursor polypeptide described herein.

[0269] In one embodiment, host cells or host cell groups are introduced with one or more expression vectors, wherein the vector is operably linked to a nucleic acid transgene encoding any dimeric antigen receptor (DAR) construct as described herein. The host cell or host cell group include T lymphocytes (for example: 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. In one embodiment, NK cells include umbilical cord blood-derived NK cells or placental-derived NK cells.

[0270] In one embodiment, the host cell or host cell population carries one or more expression vectors that can guide the transient introduction of a transgene into the host cell or the stable insertion of a transgene into the genome of the host cell. The expression vector can guide the transcription and / or translation of the transgene in the host cell. The expression vector can include a nucleic acid backbone sequence derived from a retrovirus, a lentivirus, or an adenovirus. The expression vector can include one or more regulatory sequences, such as inducible and / or constitutive promoters and enhancers. The expression vector can include a ribosome binding site and / or a polyadenylation site.

[0271] In one embodiment, an expression vector operably linked to a nucleic acid encoding a dimeric antigen receptor (DAR) construct can direct the production of the dimeric antigen receptor (DAR) construct, which can be displayed on the surface of a transgenic host cell or secreted into the cell culture medium.

[0272] In one embodiment, host cells can carry one or more expression vectors operably linked to a nucleic acid transgene encoding any dimeric antigen receptor, and the host cells can be cultured in an appropriate culture medium to transiently or stably express the dimeric antigen receptor construct.

[0273] The present disclosure provides a host cell or a population of host cells carrying an expression vector operably linked to a nucleic acid encoding a first polypeptide chain, the first polypeptide chain comprising: five regions, in order from amino terminus to 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.

[0274] The present disclosure provides a host cell or a population of host cells carrying an expression vector operably linked to a nucleic acid encoding a second polypeptide chain, the second polypeptide chain comprising: two regions, sequentially from amino terminus to carboxyl terminus: (i) an antibody light chain variable region and (ii) an antibody light chain constant region.

[0275] In one embodiment, the host cell or host cell population carries a first expression vector operably linked to a nucleic acid encoding a first polypeptide chain, and a second expression vector operably linked to a nucleic acid encoding a second polypeptide chain, wherein (a) the first polypeptide chain comprises, in order from amino terminus to carboxyl terminus, five regions: (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 comprises: in order from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region and (ii) an antibody light chain constant region.

[0276] In one embodiment, the host cell or host cell population carries an expression vector operably linked to a nucleic acid encoding a first and a second polypeptide chain, which comprises: (a) a first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, five regions: (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: in order from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region and (ii) an antibody light chain constant region.

[0277] In one embodiment, the host cell or population of host cells expresses the first and second polypeptide chains.

[0278] The present disclosure provides a host cell or a population of host cells carrying an expression vector operably linked to a nucleic acid encoding a first polypeptide chain, the first polypeptide chain comprising: five regions, in order from amino terminus to 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.

[0279] The present disclosure provides a host cell or a population of host cells carrying an expression vector operably linked to a nucleic acid encoding a second polypeptide chain, the second polypeptide chain comprising: two regions, sequentially from amino terminus to carboxyl terminus: (i) an antibody heavy chain variable region and (ii) an antibody heavy chain constant region.

[0280] In one embodiment, the host cell or host cell population carries a first expression vector operably linked to a nucleic acid encoding a first polypeptide chain, and a second expression vector operably linked to a nucleic acid encoding a second polypeptide chain, wherein (a) the first polypeptide chain comprises, in order from amino terminus to carboxyl terminus, five regions: (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) the second polypeptide chain comprises: in order from amino terminus to carboxyl terminus, two regions: (i) an antibody heavy chain variable region and (ii) an antibody heavy chain constant region.

[0281] In one embodiment, the host cell or host cell population carries an expression vector operably linked to a nucleic acid encoding a first and a second polypeptide chain, the first and the second polypeptide chain comprising: (a) a first polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, five regions: (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, sequentially from amino terminus to carboxyl terminus:

[0282] (i) an antibody heavy chain variable region and (ii) an antibody heavy chain constant region.

[0283] In one embodiment, the host cell or population of host cells expresses the first and second polypeptide chains.

[0284] The present disclosure provides a host cell or a population of host cells carrying an expression vector operably linked to a nucleic acid encoding a first polypeptide chain, the first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, six regions: (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.

[0285] The present disclosure provides a host cell or a host cell population carrying an expression vector operably linked to a nucleic acid encoding a second polypeptide chain, wherein the second polypeptide chain comprises: three regions, from amino terminus to carboxyl terminus: (i) a light chain leader region, (ii) an antibody light chain variable region, and (iii) an antibody light chain constant region.

[0286] In one embodiment, the host cell or host cell population carries a first expression vector operably linked to a nucleic acid encoding a first polypeptide chain, and a second expression vector operably linked to a nucleic acid encoding a second polypeptide chain, wherein (a) the first polypeptide chain comprises, in order from amino terminus to carboxyl terminus, six regions: (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 comprises: two regions, in order from amino terminus to carboxyl terminus: (i) an antibody light chain variable region and (ii) an antibody light chain constant region.

[0287] In one embodiment, the host cell or host cell population carries an expression vector operably linked to a nucleic acid encoding a first and a second polypeptide chain, the first and the second polypeptide chain comprising: (a) a first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, six regions: (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: in order from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region and (ii) an antibody light chain constant region.

[0288] In one embodiment, the host cell or population of host cells expresses the first and second polypeptide chains.

[0289] The present disclosure provides a host cell or a population of host cells carrying an expression vector operably linked to a nucleic acid encoding a first polypeptide chain, the first polypeptide chain comprising: six regions, in order from amino terminus to 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.

[0290] The present disclosure provides a host cell or a population of host cells carrying an expression vector operably linked to a nucleic acid encoding a second polypeptide chain, wherein the second polypeptide chain comprises, in order from amino terminus to carboxyl terminus, three regions: (i) a heavy chain leader region, (ii) an antibody heavy chain variable region, and (iii) an antibody light chain constant region.

[0291] In one embodiment, the host cell or host cell population carries a first expression vector operably linked to a nucleic acid encoding a first polypeptide chain, and a second expression vector operably linked to a nucleic acid encoding a second polypeptide chain, wherein (a) the first polypeptide chain comprises, in order from amino terminus to carboxyl terminus, six regions: (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 comprises: two regions, in order from amino terminus to carboxyl terminus: (i) an antibody heavy chain variable region and (ii) an antibody heavy chain constant region.

[0292] In one embodiment, the host cell or host cell population carries an expression vector operably linked to a nucleic acid encoding a first and a second polypeptide chain, the first and the second polypeptide chain comprising: (a) a first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, six regions: (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: in order from amino terminus to carboxyl terminus, two regions: (i) an antibody heavy chain variable region and (ii) an antibody heavy chain constant region.

[0293] In one embodiment, the host cell or population of host cells expresses the first and second polypeptide chains.

[0294] The present disclosure provides a host cell or a host cell population carrying an expression vector operably linked to a nucleic acid encoding a precursor polypeptide, wherein the precursor polypeptide includes: ten regions from amino terminus to 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) a T2A cleavage sequence region, (8) a light chain leader region, (9) an antibody light chain variable region, and (10) an antibody light chain constant region.

[0295] In one embodiment, the host cell or population of host cells expresses a precursor polypeptide.

[0296] The present disclosure provides a host cell or a host cell population carrying an expression vector operably linked to a nucleic acid encoding a precursor polypeptide, wherein the precursor polypeptide includes: ten regions from amino terminus to 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) a T2A cleavage sequence region, (8) a heavy chain leader region, (9) an antibody heavy chain variable region, and (10) an antibody heavy chain constant region.

[0297] In one embodiment, the host cell or population of host cells expresses a precursor polypeptide.

[0298] The present disclosure provides a host cell or a population of host cells carrying an expression vector of a nucleic acid operably linked to a first polypeptide chain, the first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, five regions: (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 having the amino acid sequence of SEQ ID NO: 7 and a CD3 zeta intracellular signaling sequence having the amino acid sequence of SEQ ID NO: 9.

[0299] The present disclosure provides a host cell or a population of host cells carrying an expression vector operably linked to a nucleic acid encoding a second polypeptide chain, the second polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, two regions: (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.

[0300] In one embodiment, the vector is operably linked to nucleic acids encoding the first and second polypeptide chains (SEQ ID NOs: 6 and 7).

[0301] In one embodiment, the vector is operably linked to a nucleic acid encoding a first polypeptide chain (SEQ ID NO: 6), a T2A cleavage sequence (SEQ ID NO: 12), and a second polypeptide chain (SEQ ID NO: 7).

[0302] The present disclosure provides a vector operably linked to a nucleic acid encoding a precursor polypeptide comprising the amino acid sequence of SEQ ID NO:15.

[0303] In one embodiment, the host cell expresses the first polypeptide chain (SEQ ID NO: 13 or 16) and the second polypeptide chain (SEQ ID NO: 14 or 17). In one embodiment, the host cell expresses the precursor polypeptide of SEQ ID NO: 15 or 18.

[0304] In one embodiment, the first polypeptide chain (SEQ ID NO: 13 or 16) comprises or lacks Figure 35B Or the underlined leader sequence in C. In one embodiment, the second polypeptide chain (SEQ ID NO: 14 or 17) comprises or lacks Figure 35B or the underlined leader sequence in C.

[0305] The present disclosure provides a host cell or a population of host cells carrying an expression vector operably linked to a nucleic acid encoding a version 1 (e.g., V1) dimeric antigen receptor (DAR) construct comprising a first polypeptide chain carrying a heavy chain variable region (VH) and a heavy chain constant region (CH), and a second polypeptide chain carrying a light chain variable region (VL) and a light chain constant region (CL) (e.g., V2). Figure 1 ), wherein the host cell or host cells carry (a) a first vector operably linked to a first nucleic acid encoding a first polypeptide chain, the first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, five regions: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a long chain region comprising CD8 and CD28 hinge sequences (e.g., SEQ ID NO: 19), (iv) a transmembrane region (TM) comprising a CD28 transmembrane sequence (e.g., SEQ ID NO: 6), and (v) an intracellular signaling region comprising a CD28 signaling sequence (e.g., SEQ ID NO: 8) and a CD3-ζ signaling sequence having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 9); and (b) the first vector operably linked to a second nucleic acid encoding a second peptide chain, the second peptide chain comprising, in order from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region (VL) (e.g., κ or λ) and (ii) an antibody light chain constant region (CL). In one embodiment, the antibody heavy chain variable region (VH) 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).

[0306] The present disclosure provides a vector operably linked to a nucleic acid encoding a version 2 (e.g., V2) dimeric antigen receptor (DAR) construct comprising a first polypeptide chain carrying a heavy chain variable region (VH) and a heavy chain constant region (CH), and a second polypeptide chain carrying a light chain variable region (VL) and a light chain constant region (CL) (e.g., V2). Figure 1 and 2), wherein the host cell or host cell population carries (a) a first vector operably linked to a first nucleic acid encoding a first polypeptide chain, the first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, five regions: (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 a CD28 transmembrane sequence (e.g., SEQ ID NO: 6), and (v) an intracellular signaling region comprising (1) a 4-1BB signaling sequence (e.g., SEQ ID NO: 7) and a CD3-ζ having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 9), or (2) a CD28 (e.g., SEQ ID NO: 8) signaling sequence and a CD3-ζ having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 9), or (3) a 4-1BB (e.g., SEQ ID NO: 10) signaling sequence and a CD3-ζ having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 11). NO: 7) signaling sequence and CD28 (e.g., SEQ ID NO: 8) signaling sequence and CD3-ζ having ITAM motifs 1, 2 and 3 (e.g., SEQ ID NO: 9); (b) the first vector is operably linked to a second nucleic acid encoding a second polypeptide chain, the second polypeptide chain comprising two regions, sequentially from amino terminus to carboxyl terminus: (i) an antibody light chain variable region (VL) (e.g., κ or λ) and (ii) an antibody light chain constant region (CL).

[0307] In one embodiment, the vector is operably linked to a nucleic acid encoding a version 2a (V2a) DAR construct comprising an intracellular signaling region having a 4-1BB signaling sequence (e.g., SEQ ID NO: 7) and CD3-ζ having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 9).

[0308] In one embodiment, the vector is operably linked to a nucleic acid encoding a version 2b (V2b) DAR construct comprising an intracellular signaling region having a CD28 (e.g., SEQ ID NO: 8) signaling sequence and a CD3-ζ (e.g., SEQ ID NO: 9) having ITAM motifs 1, 2, and 3.

[0309] In one embodiment, the vector is operably linked to a nucleic acid encoding a version 2c (V2c) DAR construct comprising a 4-1BB (e.g., SEQ ID NO: 7) signaling sequence and a CD28 (e.g., SEQ ID NO: 8) signaling sequence and CD3-ζ (e.g., SEQ ID NO: 9) having ITAM motifs 1, 2, and 3. In one embodiment, DAR V2a and V2b are second generation DAR constructs, while DAR V2c is a third generation DAR construct. In one embodiment, the antibody heavy chain variable region (VH) 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).

[0310] The present disclosure provides a host cell or a population of host cells carrying an expression vector operably linked to a nucleic acid encoding a version 3 (e.g., V3) dimeric antigen receptor (DAR) construct, the construct comprising a first polypeptide chain carrying a heavy chain variable region (VH) and a heavy chain constant region (CH), and a second polypeptide chain carrying a light chain variable region (VL) and a light chain constant region (CL) (e.g., Figure 1 ), wherein the host cell or host cell carries (a) a first vector operably linked to a first nucleic acid encoding a first polypeptide chain, the first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, five regions: (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 a CD28 transmembrane sequence (e.g., SEQ ID NO: 6), and (v) an intracellular signaling region comprising a 4-1BB signaling sequence (e.g., SEQ ID NO: 7) and a CD3-ζ signaling sequence having only ITAM motif 3 (e.g., SEQ ID NO: 20); and (b) the first vector operably linked to a second nucleic acid encoding a second polypeptide chain, the second polypeptide chain comprising, in order from amino terminus to carboxyl terminus, (i) an antibody light chain variable region (VL) (e.g., κ or λ), and (ii) an antibody light chain constant region (CL). In one embodiment, the antibody heavy chain variable region (VH) 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).

[0311] The present disclosure provides a host cell or a population of host cells carrying a gene operably linked to a coding version 4 (e.g.,

[0312] V4) a dimeric antigen receptor (DAR) construct comprising a first polypeptide chain comprising a heavy chain variable region (VH) and a heavy chain constant region (CH), and a second polypeptide chain comprising a light chain variable region (VL) and a light chain constant region (CL), wherein the host cell or host cell population carries (a) a first vector operably linked to a first nucleic acid encoding the first polypeptide chain, the first polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, four regions: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a transmembrane region (TM) comprising a CD28 transmembrane sequence (e.g., SEQ ID NO: 6), and (iv) an intracellular signaling region comprising a 4-1BB signaling sequence (e.g., SEQ ID NO: 7) and a CD3-ζ signaling sequence comprising only ITAM motif 3 (e.g., SEQ ID NO: 8). NO: 20); (b) the first vector is operably linked to a second nucleic acid encoding a second polypeptide chain, the second polypeptide chain comprising, from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region (VL) (e.g., κ or λ), and (ii) an antibody light chain constant region (CL). The DAR V4 construct lacks a hinge sequence. In one embodiment, the antibody heavy chain variable region (VH) 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).

[0313] The present disclosure further provides methods of adoptive cell therapy by administering to a subject transgenic host cells that have been engineered to express a dimeric antigen receptor construct.

[0314] The present disclosure further provides a method for treating a subject having a disease, disorder or condition associated with deleterious expression (e.g., elevated expression) of a tumor antigen. The method includes, for example, administering to the subject a host cell carrying an expression vector operably linked to a nucleic acid encoding any first polypeptide chain or second polypeptide chain, or any first and second polypeptide chain, or any precursor polypeptide chain as described herein. In one embodiment, the host cell or host cell population expresses any first and second polypeptide chain, or any precursor polypeptide chain as described herein.

[0315] In one embodiment, one or more expression vectors are introduced together into a host cell or host cell group for treating a subject, wherein the vector is operably connected to a nucleic acid transgene encoding any dimeric antigen receptor (DAR) construct as described herein. The host cell or host cell group include T lymphocytes (for example: 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. In one embodiment, NK cells include umbilical cord blood-derived NK cells or placental-derived NK cells.

[0316] In one embodiment, the host cell or host cell population used to treat the subject carries one or more expression vectors that can guide the transient introduction of a transgene into the host cell or the stable insertion of a transgene into the genome of the host cell. The expression vector can guide the transcription and / or translation of the transgene in the host cell. In one embodiment, the transgene comprises a nucleic acid encoding any first polypeptide chain or second polypeptide chain, or any first and second polypeptide chain, or any precursor polypeptide chain as described herein. The expression vector can comprise a nucleic acid backbone sequence derived from a retrovirus, a lentivirus, or an adenovirus. The expression vector can comprise one or more regulatory sequences, such as inducible and / or constitutive promoters and enhancers. The expression vector can comprise a ribosome binding site and / or a polyadenylation site.

[0317] In one embodiment, the expression vector operably linked to the nucleic acid encoding the dimeric antigen receptor (DAR) construct can direct the production of the dimeric antigen receptor (DAR) construct, which can be displayed on the surface of the transgenic host cell or secreted into the cell culture medium.

[0318] In one embodiment, host cells can carry one or more expression vectors operably linked to a nucleic acid transgene encoding any dimeric antigen receptor, and the host cells can be cultured in an appropriate culture medium to transiently or stably express the dimeric antigen receptor construct.

[0319] In one embodiment, the host cell or host cell population used to treat a subject is autologous and is derived from the subject being treated. In one embodiment, 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.

[0320] In one embodiment, the host cell or host cell population used to treat a subject is allogeneic and derived from different subjects. Allogeneic cells can be obtained from the whole blood of different subjects in the same manner as autologous cells. In one embodiment, allogeneic cells are derived from placenta or umbilical cord (chord) tissue after pregnancy.

[0321] In one embodiment, the desired cells are obtained from the subject being treated or from a different subject and are engineered to carry one or more expression vectors that can direct the expression of any of the first or second polypeptides or precursor polypeptides, thereby producing a transgenic host cell. The transgenic host cell can express the first or second polypeptide, or a precursor polypeptide. The host cell can express a first and a second polypeptide chain that dimerizes to form a dimeric antigen receptor that specifically binds to a tumor antigen in the subject. The host cell can express a precursor polypeptide chain that can be cleaved to form a first and a second polypeptide chain that dimerizes to form a dimeric antigen receptor that specifically binds to a tumor antigen in the subject. The transgenic host cell (e.g., carrying the expression vector or expressing the polypeptide chain) can be administered to a subject to treat a disease, disorder, or condition associated with the harmful expression of a tumor antigen.

[0322] 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 hematological breast cancer, ovarian cancer, prostate cancer, head and neck cancer, lung cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, liver cancer, kidney cancer, esophageal cancer, leiomyoma, leiomyosarcoma, glioma, and glioblastoma.

[0323] In one embodiment, the cancer is a blood cancer selected from the group consisting of non-Hodgkin lymphoma (NHL), Burkitt lymphoma (BL), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin lymphoma (HL), chronic myeloid leukemia (CML) and multiple myeloma (MM).

[0324] The present disclosure further provides a method for treating a subject suffering from 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 carrying an expression vector operably linked to a nucleic acid encoding a first polypeptide chain, the first polypeptide chain comprising: from amino terminus to carboxyl terminus, five regions: (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., Figure 1 and 2 ).

[0325] The present disclosure further provides a method for treating a subject suffering from 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 carrying an expression vector operably linked to a nucleic acid encoding a second polypeptide chain, the second polypeptide chain comprising: two regions, sequentially from amino terminus to carboxyl terminus: (i) an antibody light chain variable region and (ii) an antibody light chain constant region (e.g., Figure 1 and 2 ).

[0326] 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, the method comprising: administering to the subject a host cell or a population of host cells, the host cell or population of host cells carrying a first and a second expression vector, wherein the first expression vector is operably linked to a nucleic acid encoding a first polypeptide chain, and the second expression vector is operably linked to a nucleic acid encoding a second polypeptide chain, wherein (a) the first polypeptide chain comprises, sequentially from amino terminus to carboxyl terminus, five regions: (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 comprises, sequentially from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region, and (ii) an antibody light chain constant region (e.g., Figure 1 and 2 ).

[0327] The present disclosure further provides a method for treating a subject suffering from 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 carrying an expression vector operably linked to a nucleic acid encoding a first and a second polypeptide chain, the first and the second polypeptide chain comprising: (a) a first polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, five regions: (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, sequentially from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region and (ii) an antibody light chain constant region (e.g., Figure 1 and 2 ).

[0328] In one embodiment, 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 gene expressing a first polypeptide chain, a second polypeptide chain, or both a first and a second polypeptide chain (e.g., Figure 1 and 2 In one embodiment, the first and second polypeptide chains dimerize to form a dimeric antigen receptor that specifically binds to a tumor antigen in the subject.

[0329] The present disclosure further provides a method for treating a subject suffering from 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 carrying an expression vector operably linked to a nucleic acid encoding a first polypeptide chain, the first polypeptide chain comprising: from amino terminus to carboxyl terminus, five regions: (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., Figure 3 and 4 ).

[0330] The present disclosure further provides a method for treating a subject suffering from 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 carrying an expression vector operably linked to a nucleic acid encoding a second polypeptide chain, the second polypeptide chain comprising: two regions, sequentially from amino terminus to carboxyl terminus: (i) an antibody heavy chain variable region and (ii) an antibody heavy chain constant region (e.g., Figure 3 and 4 ).

[0331] The present disclosure further provides a method for treating a subject suffering from 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, the host cell or population of host cells carrying a first and a second expression vector, wherein the first expression vector is operably linked to a nucleic acid encoding a first polypeptide chain, and the second expression vector is operably linked to a nucleic acid encoding a second polypeptide chain, wherein (a) the first polypeptide chain comprises, sequentially from amino terminus to carboxyl terminus, five regions: (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 comprises, sequentially from amino terminus to carboxyl terminus, two regions: (i) an antibody heavy chain variable region and (ii) an antibody heavy chain constant region (e.g., Figure 3 and 4 ).

[0332] The present disclosure further provides a method for treating a subject suffering from 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 carrying an expression vector operably linked to a nucleic acid encoding a first and a second polypeptide chain, the first and the second polypeptide chain comprising: (a) a first polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, five regions: (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, sequentially from amino terminus to carboxyl terminus, two regions: (i) an antibody heavy chain variable region and (ii) an antibody heavy chain constant region (e.g., Figure 3 and Figure 4 ).

[0333] In one embodiment, 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 vaccine expressing a first polypeptide chain, a second polypeptide chain, or a first and a second polypeptide chain (e.g., Figure 3 and 4 In one embodiment, the first and second polypeptide chains dimerize to form a dimeric antigen receptor that specifically binds to a tumor antigen in a subject.

[0334] The present disclosure further provides a method for treating a subject suffering from 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 carrying an expression vector operably linked to a nucleic acid encoding a first polypeptide chain, the first polypeptide chain comprising: six regions, in order from amino terminus to 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., Figure 1 and 2 ).

[0335] The present disclosure further provides a method for treating a subject suffering from 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 carrying an expression vector operably linked to a nucleic acid encoding a second polypeptide chain, the second polypeptide chain comprising: three regions, sequentially from amino terminus to 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., Figure 1 and 2 ).

[0336] The present disclosure further provides a method for treating a subject suffering from 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 carrying a first and a second expression vector, wherein the first expression vector is operably linked to a nucleic acid encoding a first polypeptide chain, and the second expression vector is operably linked to a nucleic acid encoding a second polypeptide chain, wherein (a) the first polypeptide chain comprises, sequentially from amino terminus to carboxyl terminus, six regions: (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 comprises, sequentially from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region and (ii) an antibody light chain constant region (e.g., Figure 1 and 2 ).

[0337] The present disclosure further provides a method for treating a subject suffering from 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 carrying an expression vector operably linked to a nucleic acid encoding a first and a second polypeptide chain, the first and the second polypeptide chain comprising: (a) a first polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, six regions: (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, sequentially from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region and (ii) an antibody light chain constant region (e.g., Figure 1 and 2 ).

[0338] In one embodiment, 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 gene expressing a first polypeptide chain, a second polypeptide chain, or both a first and a second polypeptide chain (e.g., Figure 1 and 2 In one embodiment, the first and second polypeptide chains dimerize to form a dimeric antigen receptor that specifically binds to a tumor antigen in the subject.

[0339] The present disclosure further provides a method for treating a subject suffering from a disease, disorder or condition associated with deleterious expression of a tumor antigen, the method comprising: administering to the subject a host cell or a population of host cells carrying an expression vector operably linked to a nucleic acid encoding a first polypeptide chain, the first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, six regions: (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 ( Figure 3 and 4 ).

[0340] The present disclosure further provides a method for treating a subject suffering from a disease, disorder, or condition associated with deleterious expression of a tumor antigen, the method comprising: administering to the subject a host cell or a population of host cells carrying an expression vector operably linked to a nucleic acid encoding a second polypeptide chain, the second polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, three regions: (i) a heavy chain leader region, (ii) an antibody heavy chain variable region, and (iii) an antibody light chain constant region. Figure 3 and 4 ).

[0341] The present disclosure further provides a method for treating a subject suffering from a disease, disorder or condition associated with deleterious expression of a tumor antigen, the method comprising: administering to the subject a host cell or a population of host cells carrying a first and a second expression vector, wherein the first expression vector is operably linked to a nucleic acid encoding a first polypeptide chain, and the second expression vector is operably linked to a nucleic acid encoding a second polypeptide chain, wherein (a) the first polypeptide chain comprises, sequentially from amino terminus to carboxyl terminus, six regions: (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 comprises, sequentially from amino terminus to carboxyl terminus, two regions: (i) an antibody heavy chain variable region and (ii) an antibody heavy chain constant region ( Figure 3 and 4 ).

[0342] The present invention further provides a method for treating a subject suffering from a disease, disorder or condition associated with deleterious expression of a tumor antigen, the method comprising: administering to the subject a host cell or a population of host cells carrying an expression vector operably linked to a nucleic acid encoding a first and a second polypeptide chain, the first and the second polypeptide chain comprising: (a) a first polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, six regions: (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, sequentially from amino terminus to carboxyl terminus, two regions: (i) an antibody heavy chain variable region and (ii) an antibody heavy chain constant region. Figure 3 and 4 ).

[0343] In one embodiment, 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 gene expressing a first polypeptide chain, a second polypeptide chain, or both a first and a second polypeptide chain ( Figure 3 and 4 In one embodiment, the first and second polypeptide chains dimerize to form a dimeric antigen receptor that specifically binds to a tumor antigen in the subject.

[0344] The present disclosure further provides a method for treating a subject suffering from a disease, disorder, or condition associated with deleterious expression of a tumor antigen, the method comprising administering to the subject a host cell or a population of host cells carrying an expression vector operably linked to a nucleic acid encoding a precursor polypeptide, the precursor polypeptide comprising: ten regions, in order from amino terminus to carboxyl terminus: (1) a heavy chain leader, (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) a T2A cleavage sequence region, (8) a light chain leader, (9) an antibody light chain variable region, and (10) an antibody light chain constant region (e.g., Figure 5 and 6 In one embodiment, the precursor polypeptide is cleaved to form a first and a second polypeptide chain, which dimerize to form a dimeric antigen receptor that specifically binds to a tumor antigen in a subject.

[0345] The present disclosure further provides a method for treating a subject suffering from a disease, disorder or condition associated with deleterious expression of a tumor antigen, the method comprising: administering to the subject a host cell or a population of host cells carrying an expression vector operably linked to a nucleic acid encoding a precursor polypeptide, the precursor polypeptide comprising: ten regions, in order from amino terminus to 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) a T2A cleavage sequence region, (8) a heavy chain leader region, (9) an antibody heavy chain variable region, and (10) an antibody heavy chain constant region ( Figure 7 and 8 In one embodiment, the precursor polypeptide is cleaved to form a first and a second polypeptide chain, which dimerize to form a dimeric antigen receptor that specifically binds to a tumor antigen in a subject.

[0346] In one embodiment, 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 carrying an expression vector operably linked to a nucleic acid encoding a first polypeptide chain comprising, from amino terminus to carboxyl terminus, five regions: (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 CD3 zeta intracellular signaling sequence comprising the amino acid sequence of SEQ ID NO: 9.

[0347] In one embodiment, a method for treating a subject suffering from 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 carrying an expression vector operably linked to a nucleic acid encoding a second polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, two regions: (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.

[0348] In one embodiment, 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 carrying a first and a second expression vector, wherein the first expression vector is operably linked to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 13 or 16, and the second expression vector is operably linked to a nucleic acid encoding the amino acid sequence of SEQ ID NO: 14 or 17.

[0349] In one embodiment, 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 carrying a vector operably linked to a nucleic acid encoding a first and a second polypeptide chain, wherein the vector comprises the amino acid sequence of SEQ ID NOS: 13 and 14 or SEQ ID NOS: 16 and 17.

[0350] In one embodiment, a method for treating a subject suffering from 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 carrying an expression vector encoding a precursor polypeptide comprising the amino acid sequence of SEQ ID NO: 15 or 18 (e.g., CD38 precursor, generation 2).

[0351] In one embodiment, 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 expressing a first polypeptide chain (SEQ ID NO: 13 or 16), a second polypeptide chain (SEQ ID NO: 14 or 17), a first and a second polypeptide chain (SEQ ID NOS: 13 and 14 or SEQ ID NOS: 16 and 17), or a precursor polypeptide (SEQ ID NO: 15 or 18). In one embodiment, the first polypeptide chain (SEQ ID NO: 13 or 16) comprises or lacks Figure 35B Or the underlined leader sequence in C. In one embodiment, the second polypeptide chain (SEQ ID NO: 14 or 17) comprises or lacks Figure 35B Or the underlined leader sequence in C. In one embodiment, the first polypeptide chain and the second polypeptide chain dimerize to form a dimeric antigen receptor that specifically binds to a tumor antigen in a subject. In one embodiment, the precursor polypeptide is cleaved to form a first and a second polypeptide chain, which dimerize to form a dimeric antigen receptor that specifically binds to a tumor antigen (e.g., CD38) in a subject.

[0352] 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 hematological breast cancer, ovarian cancer, prostate cancer, head and neck cancer, lung cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, liver cancer, kidney cancer, esophageal cancer, leiomyoma, leiomyosarcoma, glioma, and glioblastoma.

[0353] In one embodiment, the cancer is a blood cancer selected from the group consisting of non-Hodgkin lymphoma (NHL), Burkitt lymphoma (BL), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin lymphoma (HL), chronic myeloid leukemia (CML) and multiple myeloma (MM).

[0354] The present disclosure provides a method for treating a subject suffering from 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 carrying an expression vector operably linked to a nucleic acid encoding a version 1 (e.g., V1) dimeric antigen receptor (DAR) construct comprising a first polypeptide chain carrying a heavy chain variable region (VH) and a heavy chain constant region (CH), and a second polypeptide chain carrying a light chain variable region (VL) and a light chain constant region (CL) (e.g., V2). Figure 1 ), wherein the host cell or host cell population carries (a) a first vector operably linked to a first nucleic acid encoding a first polypeptide chain, the first polypeptide chain comprising, from amino terminus to carboxyl terminus, five regions: (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 a CD28 transmembrane sequence (e.g., SEQ ID NO: 6), and (v) an intracellular signaling region comprising a CD28 signaling sequence (e.g., SEQ ID NO: 8) and a CD3-ζ signaling sequence having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 9); and (b) the first vector operably linked to a second nucleic acid encoding a second polypeptide chain, the second polypeptide chain comprising, from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region (VL) (e.g., κ or λ) and (ii) an antibody light chain constant region (CL). In one embodiment, the antibody heavy chain variable region (VH) 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).

[0355] 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 carrying an expression vector operably linked to a nucleic acid encoding a version 2 (e.g., V2) dimeric antigen receptor (DAR) construct, the construct comprising a first polypeptide chain carrying a heavy chain variable region (VH) and a heavy chain constant region (CH), and a second polypeptide chain carrying a light chain variable region (VL) and a light chain constant region (CL) (e.g., V2). Figure 1 and 2), wherein the host cell or host cell population carries (a) a first vector operably linked to a first nucleic acid encoding a first polypeptide chain, the first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, five regions: (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 a CD28 transmembrane sequence (e.g., SEQ ID NO: 6), and (v) an intracellular signaling region comprising (1) a 4-1BB signaling sequence (e.g., SEQ ID NO: 7) and a CD3-ζ having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 9), or (2) a CD28 (e.g., SEQ ID NO: 8) signaling sequence and a CD3-ζ having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 9), or (3) a 4-1BB (e.g., SEQ ID NO: 10) signaling sequence. NO: 7) signaling sequence and a CD28 (e.g., SEQ ID NO: 8) signaling sequence and a CD3-ζ having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 9); (b) the first vector is operably linked to a second nucleic acid encoding a second polypeptide chain, the second polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region (VL) (e.g., kappa or lambda) and (ii) an antibody light chain constant region (CL). In one embodiment, the version 2a (V2a) DAR construct comprises an intracellular signaling region having a 4-1BB signaling sequence (e.g., SEQ ID NO: 7) and a CD3-ζ having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 9). In one embodiment, the version 2b (V2b) DAR construct comprises an intracellular signaling region having a CD28 signaling sequence (e.g., SEQ ID NO: 8) and a CD3-ζ having ITAM motifs 1, 2, and 3 (e.g., SEQ ID NO: 9). In one embodiment, the version 2c (V2c) DAR construct comprises an intracellular signaling region having a 4-1BB signaling sequence (e.g., SEQ ID NO: 7) and a CD28 signaling sequence (e.g., SEQ ID NO: 8) and CD3-ζ (e.g., SEQ ID NO: 9) having ITAM motifs 1, 2, and 3. In one embodiment, DAR V2a and V2b are second generation DAR constructs, while DAR V2c is a third generation DAR construct. In one embodiment, the antibody heavy chain variable region (VH) 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).

[0356] 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 carrying an expression vector operably linked to a nucleic acid encoding a version 3 (e.g., V3) dimeric antigen receptor (DAR) construct, the construct comprising a first polypeptide chain carrying a heavy chain variable region (VH) and a heavy chain constant region (CH), and a second polypeptide chain carrying a light chain variable region (VL) and a light chain constant region (CL) (e.g., V3). Figure 1 ), wherein the host cell or host cell population carries (a) a first vector operably linked to a first nucleic acid encoding a first polypeptide chain, the first polypeptide chain comprising, in order from amino terminus to carboxyl terminus, five regions: (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 a CD28 transmembrane sequence (e.g., SEQ ID NO: 6), and (v) an intracellular signaling region comprising a 4-1BB signaling sequence (e.g., SEQ ID NO: 7) and a CD3-ζ signaling sequence having only ITAM motif 3 (e.g., SEQ ID NO: 20);

[0357] (b) The first vector is operably linked to a second nucleic acid encoding a second polypeptide chain, the second polypeptide chain comprising two regions, sequentially from amino terminus to carboxyl terminus: (i) an antibody light chain variable region (VL) (e.g., κ or λ) and (ii) an antibody light chain constant region (CL). In one embodiment, the antibody heavy chain variable region (VH) 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).

[0358] The present disclosure provides a method for treating a subject suffering from a disease, disorder, or condition associated with deleterious expression of a tumor antigen, the method comprising: administering to the subject a host cell or a population of host cells carrying an expression vector operably linked to a nucleic acid encoding a version 4 (e.g., V4) dimeric antigen receptor (DAR) construct comprising a first polypeptide chain comprising a heavy chain variable region (VH) and a heavy chain constant region (CH), and a second polypeptide chain comprising a light chain variable region (VL) and a light chain constant region (CL), wherein the host cell or population of host cells carries (a) a first vector operably linked to a first nucleic acid encoding a first polypeptide chain comprising, sequentially from amino terminus to carboxyl terminus, four regions: (i) an antibody heavy chain variable region (VH), (ii) an antibody heavy chain constant region (CH), (iii) a transmembrane region (TM) comprising a CD28 transmembrane sequence (e.g., SEQ ID NO: 6), and (iv) a transmembrane region (TM) comprising a 4-1BB signaling sequence (e.g., SEQ ID NO: 7). NO: 7) and an intracellular signaling region of a CD3-ζ signaling sequence having only ITAM motif 3 (e.g., SEQ ID NO: 20); (b) the first vector is operably linked to a second nucleic acid encoding a second polypeptide chain, the second polypeptide chain comprising, from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region (VL) (e.g., κ or λ) and (ii) an antibody light chain constant region (CL). The DAR V4 construct lacks a hinge sequence. In one embodiment, the antibody heavy chain variable region (VH) 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).

[0359] 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 hematological breast cancer, ovarian cancer, prostate cancer, head and neck cancer, lung cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, liver cancer, kidney cancer, esophageal cancer, leiomyoma, leiomyosarcoma, glioma, and glioblastoma.

[0360] In one embodiment, the cancer is a blood cancer selected from non-Hodgkin lymphoma (NHL), Burkitt lymphoma (BL), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin lymphoma (HL), chronic myeloid leukemia (CML) and multiple myeloma (MM). Example

[0361] The following examples are illustrative and may be used to further understand the embodiments of the present disclosure and should not be construed in any way as limiting the scope of this guidance.

[0362] Example 1: Isolation of human PBMC cells and primary T cells

[0363] Primary human T cells were isolated from healthy human donors using buffy coats (San Diego Blood Bank), fresh blood, or leukapheresis products (StemCell). Peripheral blood mononuclear cells were isolated by density gradient centrifugation. EasySep TM The Human T Cell Isolation Kit (STEMCELL) was used to isolate T cells from PBMCs by magnetic negative selection.

[0364] Example 2: Primary T cell culture

[0365] In supplementation with 5% CTS TM Immune Cell SR (Thermo Fisher Scientific), 300 U / mL IL-2 (Proleukin) CTS TM OpTmizer TM T cells were expanded in SFM at a density of 10 6 cells / mL to culture primary T cells. Stimulate fresh or frozen T cells. Use T cell TransAct (Miltenyi) 3uL / 10 6 cells / mL for two to three days. After transfection, T cells were cultured in medium containing 300 U / mL IL-2.

[0366] Example 3: Tumor cell lines

[0367] Multiple myeloma cell line RPMI 8226 is obtained from ATCC, and uses the slow virus carrying luciferase and GFP gene to be transduced.Selection has the single cell clone (RPMI8226-FLuc) of luciferase and GFP expression.By using the slow virus transduction K562 cell carrying RPE gene, similarly prepare K562 / RPE cell.Two kinds of cell lines are all cultivated in the RPMI1640 culture medium (ATCC) being supplemented with 10% fetal bovine serum (Sigma).

[0368] Example 4: Preparation of CAR and DAR T cells

[0369] Nucleic acid encoding the CAR or DAR construct is introduced into activated T cells.

[0370] The nucleic acid encoding the anti-CD38 CAR polypeptide comprises a heavy chain signal peptide (SEQ ID NO: 10), followed by two additional amino acid residues Asp and Ile, a myc tag EQKLISEEDL (SEQ ID NO: 24), an anti-CD38 heavy chain variable region (SEQ ID NO: 1), a 15 amino acid linker GGGGSGGGGSGGGGS (SEQ ID NO: 23), an anti-CD38 light chain variable region (SEQ ID NO: 3), a CD8 hinge region (SEQ ID NO: 21), a CD28 hinge region (SEQ ID NO: 5), a CD28 transmembrane region (SEQ ID NO: 6), a CD28 intracellular signaling region (SEQ ID NO: 8), and a CD3-ζ intracellular signaling region (SEQ ID NO: 9). The full-length anti-CD38 CAR construct has the amino acid sequence of SEQ ID NO: 22.

[0371] Examples of nucleic acids encoding anti-CD38 DAR precursor polypeptides (eg, V2a or V3) comprise the amino acid sequence of SEQ ID NO: 15 or 18.

[0372] Example 5: Cytotoxicity test

[0373] Two to three weeks after electroporation, the CAR, DAR, and control T cells were starved overnight with IL-2. The cells were co-cultured with a target cell mixture of CD38-positive RPMI-8226 / GFP cells or CD38-negative K562 / RPE cells. The effector to target cell ratio was 5:1 to 0.08:1. After overnight culture, the cells were subjected to flow cytometry to measure the GFP cell population to determine the killing effect of anti-CD38A2 CAR and DAR T cells on specific target cells.

[0374] Example 6: Cytokine secretion assay

[0375] Two to three weeks after electroporation, the CAR, DAR, and control T cells were nutrient restricted with IL-2 overnight. The cells were co-cultured with CD38-negative K562 or CD38-positive RPMI 8226 cells. The ratio of effector to target cells was 2:1. After overnight culture, the cells were centrifuged to collect the supernatant to detect cytokines IL-2, IFN-γ, and TNFα (Affymetrix eBioscience) according to the manufacturer's instructions.

[0376] Example 7: Detection of effector memory T cells

[0377] The DAR T cells were washed with 5% human serum albumin DPBS and then stained with anti-CD3-BV421 antibody (SK7, BioLegend) and PE or APC-conjugated CD38-Fc protein (Chimerigen Laboratories) at 4°C for 30-60 minutes. CD3 and CD38 were detected using iQue Screener Plus (Intellicyte Co). The markers used to identify effector memory T cells and central memory T cell fractions were CD45RO (BioLegend) and CCR7 (BioLegend).

[0378] Example 8: In vivo tumor killing

[0379] The tumor killing activity of anti-CD38A2 CART or DART cells was tested in the RPMI8226 xenograft mouse model. The multiple myeloma cell line RPMI8226 obtained from ATCC was transfected with a lentiviral vector with luciferase and GFP genes. A single clone (RPMI8226-FLuc) with luciferase and GFP expression was selected. A total of 7 x 10 6 RPMI8226-Fluc cells were suspended in 200 μL of PBS and then injected intravenously into the tail vein of each mouse. Based on bioluminescence imaging by IVIS, animals with very small or very large tumor burdens were excluded. The animals selected for the study were randomly divided into different groups.

[0380] Three weeks after tumor inoculation, a single treatment of approximately 10 million engineered CAR or DAR T cells in 200 μL of PBS was administered via the tail vein. The same amount of ATC or TRAC KO T cells or 200 μL of PBS was injected intravenously as a treatment control.

[0381] Tumor growth was monitored weekly from the dorsal flank of each mouse until 4 or 5 weeks after tumor cell inoculation by measuring total photon flux using an IVIS Lumina III in vivo imaging system (Perkin Elmer Health Sciences, Inc.) Images were taken approximately 10 to 20 minutes after intraperitoneal administration of 150 mg / kg luciferin.

[0382] Example 9: Results of T cells expressing DAR V1 construct

[0383] Flow cytometry was used to compare the expression levels of transgenic T cells expressing either anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) version 1. Transgenic T cells expressed higher levels of version 1 DAR construct ( Figure 9 ).

[0384] The killing ability of T cells expressing anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) version 1 was compared in an in vitro cytotoxicity assay. Transgenic T cells expressing version 1 DAR construct (dashed line B) were comparable, but not superior, in in vitro cell killing compared to transgenic T cells expressing the CAR construct (line C). Figure 10 ).

[0385] The cytokine secretion capacity of T cells expressing either anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) version 1 was compared in an in vitro cytokine secretion assay. Transgenic T cells expressing version 1 DAR constructs exhibited higher levels of tumor necrosis factor alpha (TNFα) secretion compared to transgenic T cells expressing CAR constructs. Figure 14 ) and IL-2 secretion ( Figure 15 ), but lower levels of interferon-γ (IFNγ) ( Figure 13 ).

[0386] Flow cytometry was used to compare the in vitro clonogenic expansion capacity of T cells expressing anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) version 1 co-cultured with RPMI 8226 or K562 tumor target cells. Transgenic T cells expressing version 1 DAR constructs exhibited comparable fold-change expansion ( Figure 16 and 17 ).

[0387] Example 10: Results of T cells expressing DAR V2b construct

[0388] Flow cytometry was used to compare the expression levels of transgenic T cells expressing anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) V1 or V2b. Compared with transgenic T cells expressing DAR V1 or CAR constructs, transgenic T cells expressed higher levels of DAR V2b construct ( Figure 11 ).

[0389] The killing capacity of T cells expressing anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) V1 or V2b was compared in an in vitro cytotoxicity assay. Transgenic T cells expressing the DAR V2b construct (dashed line C) showed better in vitro cell killing than transgenic T cells expressing the DAR V1 construct (line B), and T cells expressing DAR V2b showed similar in vitro cell killing compared to transgenic T cells expressing the CAR construct (line D). Figure 12 ).

[0390] The killing capacity of T cells expressing anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) V2a, V2b, or V2c constructs was compared in an in vitro cytotoxicity assay. Transgenic T cells expressing the DAR V2b construct ("28Z" line E) showed significantly better in vitro cell killing than transgenic T cells expressing the CAR construct (line B) and T cells expressing DAR V2a ("BBZ" line C), and similar killing capacity compared to T cells expressing DAR V2c ("28BBZ" dashed line D) ( Figure 26 ).

[0391] The cytokine secretion capacity of T cells expressing anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) V1 or V2b was compared in an in vitro cytokine secretion assay. Transgenic T cells expressing the DAR V2b construct exhibited higher levels of IL-2 secretion compared to transgenic T cells expressing the CAR or DAR V1 construct ( Figure 15 ). Transgenic T cells expressing the DAR V2b construct showed slightly higher levels of interferon-γ (IFNγ) secretion compared to transgenic T cells expressing the CAR construct ( Figure 13 ), as well as higher levels of interferon-γ (IFNγ) secretion compared to transgenic T cells expressing the DAR V1 construct. Transgenic T cells expressing the DAR V2b construct exhibited lower levels of tumor necrosis factor α (TNFα) secretion compared to transgenic T cells expressing CAR or DAR V1 constructs ( Figure 14 ).

[0392] Flow cytometry was used to measure the in vitro clonal expansion capacity of T cells expressing anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) V1 or V2b, which were co-cultured with RPMI 8226 or K562 cells. When co-cultured with RPMI 8226 cells, transgenic T cells expressing DAR V2b constructs showed a higher level of fold change expansion ( ) compared to T cells expressing CAR or DAR V1 constructs. Figure 16 and 17 ).

[0393] An in vivo xenograft animal model was used to measure the tumor killing activity of CD38 DAR T cells in a xenograft animal model. Negative control mice were administered phosphate-buffered saline or a cell line carrying a knockout TRAC gene. Test mice were administered transgenic T cells expressing CD38 CAR or CD38 DAR (V2b) constructs. At four weeks, mice treated with T cells expressing the CD38 DAR (V2b) construct showed significantly less tumor burden ( ) compared to mice treated with T cells expressing the CD38 CAR construct. Figure 18 ).

[0394] Example 11: Results of T cells expressing DAR V2c construct

[0395] Flow cytometry was used to compare the expression levels of transgenic T cells expressing anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) V2a, V2b, or V2c constructs. Transgenic T cells expressed similar levels of DAR V2c (e.g., "BBZ") compared to transgenic T cells expressing DAR V2a (e.g., "BBZ").

[0396] "28BBZ") construct, and slightly higher levels of DARV2c("28BBZ") compared to T cells expressing the DAR V2b("28Z") construct ( Figure 25 ).

[0397] The killing capacity of T cells expressing anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) V2a, V2b, or V2c constructs was compared in an in vitro cytotoxicity assay. Transgenic T cells expressing the DAR V2c construct ("28BBZ" dashed line D) showed significantly higher levels of in vitro killing than transgenic T cells expressing the CAR construct (line B) and the DAR V2a construct (line C), but similar levels of cell killing compared to T cells expressing DAR V2b ("28Z" line E). Figure 26 ).

[0398] The cytokine secretion capacity of T cells expressing anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) V2a or V2b constructs was compared in an in vitro cytokine secretion assay. Transgenic T cells expressing DAR V2a ("BBZ") exhibited slightly higher levels of IL-2 secretion compared to T cells expressing DAR V2b ("28Z") and higher levels of IL-2 secretion compared to transgenic T cells expressing CAR constructs ( Figure 27 Compared with transgenic T cells expressing CAR or DAR V2b ("28Z") constructs, transgenic T cells expressing DAR V2a ("BBZ") constructs showed slightly higher levels of tumor necrosis factor alpha (TNFα) secretion ( Figure 28 Transgenic T cells expressing the DAR V2a ("BBZ") construct exhibited slightly higher levels of interferon-γ (IFNγ) secretion compared to transgenic T cells expressing the CAR construct, but cells expressing DAR V2a ("BBZ") exhibited slightly lower levels of IFNγ secretion compared to transgenic T cells expressing the DARV2b ("28Z") construct ( Figure 29 ).

[0399] An in vivo xenograft animal model was used to measure the tumor killing activity of CD38 DAR T cells in a xenograft animal model. Negative control mice were administered phosphate-buffered saline ("PBS") or a cell line carrying a knockout of the TRAC gene ("TRAC KO"). Test mice were administered transgenic T cells expressing either CD38 CAR or CD38 DAR(V2a) constructs. At four weeks, mice treated with T cells expressing the CD38 DAR(V2a) construct exhibited significantly less tumor burden ( ) compared to mice treated with T cells expressing the CD38 CAR construct. Figure 19 ).

[0400] In another in vivo xenograft animal model, the tumor killing activity of T cells expressing the CD38 DAR V2a construct was directly compared with that of T cells expressing DAR V2b or DAR V2c. Negative control mice were administered phosphate-buffered saline ("PBS") or activated T cells ("ATC") or a cell line carrying a knockout of the TRAC gene ("TRAC KO"). Test mice were administered transgenic T cells expressing CD38 DAR V2a, V2b, or V2c constructs. At week five, mice treated with T cells expressing the CD38 DAR V2a construct ("BBZ") exhibited measurably less tumor burden ( ) compared to mice treated with T cells expressing the CD38CAR V2b ("28Z") or V2c ("28BBZ") constructs. Figure 30).

[0401] Example 12: Results of T cells expressing DAR V2a constructs

[0402] Flow cytometry was used to compare the expression levels of transgenic T cells expressing anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) V2a or V2b constructs. Transgenic T cells expressed higher levels of DAR V2a (e.g., "BBZ") constructs ( Figure 25 ).

[0403] The cell killing ability of T cells expressing anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) V2a, V2b, or V2c constructs was compared in an in vitro cytotoxicity assay. Transgenic T cells expressing the DAR V2a construct ("BBZ" line C) exhibited better in vitro cell killing than transgenic T cells expressing the CAR construct (line B), but lower levels of cell killing than T cells expressing DAR V2b ("28Z" line E) or T cells expressing DAR V2c ("28BBZ" dashed line D) ( Figure 26 ).

[0404] The cytokine secretion capacity of T cells expressing anti-CD38 chimeric antigen receptor (CAR) or anti-CD38 dimeric antigen receptor (DAR) V2a or V2b constructs was compared in an in vitro cytokine secretion assay. Transgenic T cells expressing DAR V2a ("BBZ") exhibited slightly higher IL-2 secretion levels compared to T cells expressing DAR V2b ("28Z") and higher IL-2 secretion levels compared to transgenic T cells expressing CAR constructs ( Figure 27 Compared with transgenic T cells expressing CAR or DAR V2b ("28Z") constructs, transgenic T cells expressing DAR V2a ("BBZ") constructs showed slightly higher levels of tumor necrosis factor alpha (TNFα) secretion ( Figure 28 ). Transgenic T cells expressing the DARV2a ("BBZ") construct exhibited slightly higher levels of interferon-γ (IFNγ) secretion compared to transgenic T cells expressing the CAR construct, but cells expressing DAR V2a ("BBZ") exhibited slightly lower levels of IFNγ secretion compared to transgenic T cells expressing the DAR V2b ("28Z") construct ( Figure 29 ).

[0405] An in vivo xenograft animal model was used to measure the tumor killing activity of CD38 DAR T cells in a xenograft animal model. Negative control mice were administered phosphate-buffered saline ("PBS") or a cell line carrying a knockout of the TRAC gene ("TRAC KO"). Test mice were administered transgenic T cells expressing either CD38 CAR or CD38 DAR(V2a) constructs. At four weeks, mice treated with T cells expressing the CD38 DAR(V2a) construct exhibited significantly less tumor burden ( ) compared to mice treated with T cells expressing the CD38 CAR construct. Figure 19 ).

[0406] In another in vivo xenograft animal model, the tumor killing activity of T cells expressing the CD38 DAR V2a construct was directly compared with that of T cells expressing DAR V2b or DAR V2c. Negative control mice were administered phosphate-buffered saline ("PBS") or activated T cells ("ATC") or a cell line carrying a knockout of the TRAC gene ("TRAC KO"). Test mice were administered transgenic T cells expressing CD38 DAR V2a, V2B, or V2c constructs. At week five, mice treated with T cells expressing the CD38 DAR V2a construct ("BBZ") exhibited significantly less tumor burden ( ) compared to mice treated with T cells expressing the CD38CAR V2b ("28Z") or V2c ("28BBZ") constructs. Figure 30 ).

[0407] Example 13: Results of T cells expressing DAR V3 constructs

[0408] Flow cytometry was used to compare the expression levels of transgenic T cells expressing anti-CD38 dimeric antigen receptor (DAR) V2a or V3 constructs. Compared with transgenic T cells expressing DAR V2a, transgenic T cells expressed higher levels of DAR V3 construct ( Figure 20 and 31 ).

[0409] The killing capacity of T cells expressing anti-CD38 dimeric antigen receptor (DAR) V2a or V3 constructs was compared in an in vitro cytotoxicity assay. Transgenic T cells expressing DAR V3 (line C) exhibited a higher level of in vitro cell killing than transgenic T cells expressing DAR V2a construct (line B). Figure 21 ).

[0410] The cytokine secretion capacity of T cells expressing anti-CD38 dimeric antigen receptor (DAR) V2a or V3 constructs was compared in an in vitro cytokine secretion assay. Transgenic T cells expressing DAR V3 exhibited significantly higher levels of interferon-γ (IFNγ) secretion compared to T cells expressing DAR V2a ( Figure 22 Compared with transgenic T cells expressing the DAR V2a construct, transgenic T cells expressing the DAR V3 construct showed significantly higher levels of tumor necrosis factor alpha (TNFα) secretion ( Figure 23 ).

[0411] The cytokine secretion capacity of T cells expressing anti-CD38 dimeric antigen receptor (DAR) V2a or V3 was compared in an in vitro cytokine secretion assay. Transgenic T cells expressing the DAR V3 construct exhibited significantly higher levels of interferon-γ (IFNγ) secretion compared to transgenic T cells expressing the V2a construct. Figure 33 Compared with transgenic T cells expressing DAR V2a construct, transgenic T cells expressing DAR V3 construct showed lower tumor necrosis factor alpha (TNFα) secretion levels ( Figure 34 ).

[0412] Flow cytometry was used to detect the presence of central memory T cells (TCM) in T cell populations expressing DAR V2a or V3 constructs. T cells expressing DAR V3 constructs showed a higher percentage of central memory T cells (TCM) compared to T cells expressing DAR V2a ( Figure 24 ).

[0413] The cell killing capacity of T cells expressing anti-CD38 dimeric antigen receptor (DAR) V2a or V3 constructs was compared in an in vitro cytotoxicity assay. Transgenic T cells expressing DAR V2a construct (dashed line C) showed similar levels of in vitro cell killing (line D) compared to transgenic T cells expressing DAR V3 ( Figure 32 ).

[0414] Example 14: Results of T cells expressing DAR V4 constructs

[0415] Flow cytometry was used to compare the expression levels of transgenic T cells expressing anti-CD38 dimeric antigen receptor (DAR) V2a, V3, or V4 constructs. Transgenic T cells expressed higher levels of DAR V3 construct ( Figure 31 ).

[0416] The cell killing capacity of T cells expressing anti-CD38 dimeric antigen receptor (DAR) V2a, V3, or V4 constructs was compared in an in vitro cytotoxicity assay. Transgenic T cells expressing the DAR V4 construct (line B) exhibited similar levels of in vitro cell killing compared to transgenic T cells expressing the DAR V2a construct (dashed line C) or DAR V3 (dashed line D). Figure 32 The DAR V4 construct lacks the hinge region, and the cell killing results indicate that the DAR construct lacking the hinge region does not enhance the killing capacity of T cells expressing the DAR V4 construct compared to T cells expressing the V2a or V3 constructs, both of which contain a short hinge region (e.g., only the CD28 hinge sequence).

[0417] The cytokine secretion capacity of T cells expressing anti-CD38 dimeric antigen receptor (DAR) V2a, V3, or V4 constructs was compared in an in vitro cytokine secretion assay. Transgenic T cells expressing the DAR V4 construct showed significantly reduced interferon-γ (IFNγ) secretion levels compared to T cells expressing the DAR V3 construct ( Figure 33 Compared with transgenic T cells expressing DAR V3 construct, transgenic T cells expressing DAR V4 construct showed lower tumor necrosis factor alpha (TNFα) secretion level ( Figure 34 ).

Claims

1. A dimeric antigen receptor (DAR) construct comprising: an antigen binding domain comprising a first and a second polypeptide chain, wherein: a) the first polypeptide chain comprises five regions, sequentially from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH) comprising a CD38 antibody heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1, (ii) an antibody heavy chain constant region (CH), (iii) a hinge region or a hingeless region, (iv) a transmembrane region (TM), and (v) an intracellular signaling region; as well as b) the second polypeptide chain consists of two regions, sequentially from amino terminus to carboxyl terminus: (i) an antibody light chain variable region (VL) comprising the CD38 antibody light chain variable region consisting of the amino acid sequence of SEQ ID NO: 3 and (ii) an antibody light chain constant region (CL).

2. A dimeric antigen receptor (DAR) construct consisting of a Fab fragment connected to a transmembrane region and an intracellular signaling region, wherein the Fab fragment comprises (i) an antibody heavy chain variable region (VH), the antibody heavy chain variable region comprising a CD38 antibody heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1, and (ii) an antibody light chain variable region (VL), the antibody light chain variable region comprising a CD38 antibody light chain variable region consisting of the amino acid sequence of SEQ ID NO:

3.

3. The dimeric antigen receptor (DAR) construct of claim 2, wherein a hinge region is present between the Fab fragment and the transmembrane region.

4. A dimeric antigen receptor (DAR) construct comprising: an antigen binding domain comprising a first and a second polypeptide chain, wherein: a) The first polypeptide chain comprises five regions, sequentially from the amino terminus to the carboxyl terminus: (i) an antibody heavy chain variable region (VH), comprising a CD38 antibody heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1, (ii) an antibody heavy chain constant region (CH), comprising a CD38 antibody heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 2, (iii) a hinge region selected from the group consisting of: a CD38 antibody heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 3, NO:5, a CD8 hinge region consisting of the amino acid sequence of SEQ ID NO:21, a hinge region comprising the CD28 hinge region consisting of the amino acid sequence of SEQ ID NO:5 and the CD8 hinge region consisting of the amino acid sequence of SEQ ID NO:21, (iv) a transmembrane region (TM) comprising the CD28 transmembrane region consisting of the amino acid sequence of SEQ ID NO:6, and (v) an intracellular signaling region comprising a 4-1BB intracellular signaling sequence consisting of the amino acid sequence of SEQ ID NO:7 and a CD3ζ intracellular signaling sequence consisting of the amino acid sequence of SEQ ID NO:9 or 20; and b) the second polypeptide chain comprises, sequentially from amino terminus to carboxyl terminus, two regions: (i) an antibody light chain variable region (VL), comprising a CD38 antibody light chain variable region consisting of the amino acid sequence of SEQ ID NO: 3, and (ii) an antibody light chain constant region (CL), comprising a CD38 antibody light chain constant region consisting of the amino acid sequence of SEQ ID NO: 4, 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 that binds to the CD38 protein.

5. A nucleic acid encoding the dimeric antigen receptor (DAR) according to any one of claims 1 to 4.

6. A vector operably linked to the nucleic acid of claim 5.

7. A host cell or a host cell population carrying the vector according to claim 6.

8. The host cell or host cell population according to claim 7, wherein the vector is an expression vector, and wherein the expression vector directs the transcription and / or translation of the nucleic acid encoding the dimeric antigen receptor (DAR).

9. The host cell or host cell population according to claim 7 or 8, wherein the host cell or host cell population comprises T lymphocytes, NK (natural killer) cells, macrophages, dendritic cells, mast cells, eosinophils, B lymphocytes or monocytes.

10. Use of the host cell or host cell population according to claim 7 in the preparation of a medicament for treating a subject suffering from a disease associated with detrimental expression of a tumor antigen, wherein the disease is a blood cancer selected from the group consisting of non-Hodgkin lymphoma (NHL), Burkitt lymphoma (BL), B chronic lymphocytic leukemia (B- CLL), B and T acute lymphoblastic leukemia (ALL), T-cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin lymphoma (HL), chronic myeloid leukemia (CML), and multiple myeloma (MM).

11. Use of the host cell or host cell population according to claim 8 or 9 in the preparation of a medicament for treating a subject suffering from a disease associated with detrimental expression of a tumor antigen, wherein the disease is a blood cancer selected from the group consisting of non-Hodgkin lymphoma (NHL), Burkitt lymphoma (BL), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphoblastic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin lymphoma (HL), chronic myeloid leukemia (CML) and multiple myeloma (MM).

12. A precursor polypeptide comprising ten regions, in order from amino terminus to carboxyl terminus: (1) a heavy chain leader sequence, (2) an antibody heavy chain variable region comprising a CD38 antibody heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1, (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 comprising a CD38 antibody light chain variable region consisting of the amino acid sequence of SEQ ID NO: 3, and (10) an antibody light chain constant region.

13. The precursor polypeptide according to claim 12, wherein (1) the heavy chain leader sequence comprises the amino acid sequence of SEQ ID NO: 10; (3) the antibody heavy chain constant region comprises the CD38 antibody heavy chain constant region, and the CD38 antibody heavy chain constant region consists of the amino acid sequence of SEQ ID NO: 2; (4) the hinge region is selected from the group consisting of a CD28 hinge region consisting of the amino acid sequence of SEQ ID NO: 5, a CD8 hinge region consisting of the amino acid sequence of SEQ ID NO: 21, and a hinge region comprising a CD28 hinge region consisting of the amino acid sequence of SEQ ID NO: 5 and a CD8 hinge region consisting of the amino acid sequence of SEQ ID NO: 21; (5) the transmembrane region comprises a CD28 transmembrane region consisting of the amino acid sequence of SEQ ID NO: 6; (6) the intracellular signaling region comprises any one signal sequence or any combination of two or more signal sequences selected from the group consisting of a 4-1BB signaling sequence consisting of the amino acid sequence of SEQ ID NO: 7, a CD28 signaling sequence consisting of the amino acid sequence of SEQ ID NO: 8, a CD3ζ signaling sequence consisting of the amino acid sequence of SEQ ID NO: 9, and / or a CD3ζ signaling sequence having an ITAM 3 motif consisting of the amino acid sequence of SEQ ID NO: 20; (7) a T2A cleavage sequence consisting of the amino acid sequence of SEQ ID NO: 12; (8) a T2A cleavage sequence consisting of the amino acid sequence of SEQ ID NO: 13; NO: 11 amino acid sequence of the light chain leader sequence; and (10) the antibody light chain constant region comprises the CD38 antibody light chain constant region, the CD38 antibody light chain constant region is SEQ ID The amino acid sequence of NO:

4. The precursor polypeptide of claim 13 , comprising the amino acid sequence of SEQ ID NO: 15 or 18.

15. A nucleic acid encoding the precursor polypeptide according to claim 13 or 14.

16. A vector operably linked to the nucleic acid of claim 15.

17. A host cell or a host cell population carrying the vector according to claim 16.

18. The host cell or host cell population according to claim 17, wherein the vector is an expression vector, and wherein the expression vector directs the transcription and / or translation of the nucleic acid encoding the precursor polypeptide.

19. The host cell or host cell population according to claim 17 or 18, wherein the host cell or host cell population comprises T lymphocytes, NK (natural killer) cells, macrophages, dendritic cells, mast cells, eosinophils, B lymphocytes or monocytes.

20. Use of the host cell or host cell population according to claim 17 in the preparation of a medicament for treating a subject suffering from a disease associated with detrimental expression of a tumor antigen, wherein the disease is a blood cancer selected from the group consisting of non-Hodgkin lymphoma (NHL), Burkitt lymphoma (BL), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphoblastic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin lymphoma (HL), chronic myeloid leukemia (CML) and multiple myeloma (MM).

21. Use of the host cell or host cell population according to claim 18 or 19 in the preparation of a medicament for treating a subject suffering from a disease associated with detrimental expression of a tumor antigen, wherein the disease is a blood cancer selected from the group consisting of non-Hodgkin lymphoma (NHL), Burkitt lymphoma (BL), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphoblastic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin lymphoma (HL), chronic myeloid leukemia (CML) and multiple myeloma (MM).

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