CN117545493A

CN117545493A - Dimeric Antigen Receptor (DAR) binding to GD2

Info

Publication number: CN117545493A
Application number: CN202280044868.4A
Authority: CN
Inventors: 季红俊; 郭文忠; 张延良; 丁蓓蓓; 王旸; G·F·考夫曼
Original assignee: Sorento Pharmaceutical Co ltd
Current assignee: Sorento Pharmaceutical Co ltd
Priority date: 2021-04-23
Filing date: 2022-04-22
Publication date: 2024-02-09

Abstract

The present disclosure provides transgenic T cells that express an engineered Dimeric Antigen Receptor (DAR) that binds to GD2, wherein the DAR comprises a heavy chain binding region on one polypeptide chain and a light chain binding region on a separate polypeptide chain. The two polypeptide chains that make up the dimeric antigen receptor may dimerize to form an antigen binding domain. The transgenic T cells may be used for targeted cell therapy.

Description

Dimeric Antigen Receptor (DAR) binding to GD2

The present application claims priority from U.S. provisional application No. 63/179,147 filed on month 4 and 23 of 2021 and U.S. provisional application No. 63/188,284 filed on month 5 and 13 of 2021, the entire contents of which are incorporated herein by reference.

Technical Field

The present disclosure provides Dimeric Antigen Receptor (DAR) protein constructs that specifically bind to a target antigen, nucleic acids encoding the dimeric antigen receptor, vectors comprising the nucleic acids, and host cells harboring the vectors.

Background

GD2 is a tumor of neuroectodermal origin, including human neuroblastoma and melanoma, over-expressed bisialoganglioside. GD2 expression is limited to cerebellum and peripheral nerves in normal human tissue. A murine anti-GD 2 monoclonal antibody 14.18 was used to engineer a chimeric monoclonal antibody comprising the variable region of murine antibody 14.18 and the constant region of human IgG-K (ch 14.18, herein referred to as 14.18) (Gillies, 1989 J.Immunol. Method (Journal of Immunological Methods) 125:191-202). The humanized version of murine antibody 14.18 (hu 14.18) included variable region framework mutations designed to reduce the immunogenicity of the antibody in humans (U.S. patent No. 7,169,904).

Adoptive immunotherapy with T cells engineered with Chimeric Antigen Receptors (CARs) by infusion for redirecting tumoricidal activity represents a potentially highly specific form for the treatment of metastatic cancers. Different versions of CARs have been developed for target antigens associated with cancer. The first generation of CARs were engineered to contain a signaling domain (TCR ζ) that delivers only activating 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 research (Cancer Res.)) (61 (5): 1976-1982,2001; hombach et al, J. Immunol.) (167 (11): 6123-6131,2001; maher et al, nature Biotechnology (Nat. Biotechnol.)) (20 (1): 70-75,2002). T cells transplanted with first generation CAR alone showed limited anti-tumor efficacy due to suboptimal activation (Beecham et al J. Immunothether.) (23 (6): 631-642, 2000). The second generation of CARs, immunoglobulin-CD 28-T cell receptor (IgCD 28 TCR), incorporates the co-stimulatory CD28 domain (Signal 2) into the first generation of receptor (Gerstmayer et al, J.Immunol.158 (10): 4584-4590,1997; emtage et al, clinical cancer research (Clin. Cancer Res.) 14 (24): 8112-8122,2008; lo, ma et al, clinical cancer research 16 (10): 2769-2780, 2010), which confers greater antitumor capacity on CAR-T cells (Finney et al, J.Immunol.161 (6): 2791-2797,1998; hombach et al, cancer research 61 (5): 1976-1982,2001, maher et al, nature Biotechnology 20 (1): 70-75,2002). Various CAR variants have been developed by substituting signal domains of tcrζ or CD28 with molecules having similar functions, such as fcrγ, 4-1BB and OX40 (Eshhar et al, proc. Natl. Acad. U S A), 90 (2): 720-724, 1993).

TCR CAR-T cells have been developed against various tumor antigens (Ma et al, cancer Gene therapy (Cancer Gene Ther.) 11 (4): 297-306,2004; ma et al, prostate (Prostate) 61 (1): 12-25,2004; lo et al, clinical Cancer research 16 (10): 2769-2780,2010; kong et al, clinical Cancer research 18 (21): 5949-5960,2012; ma et al, prostate 74 (3): 286-296 (4); zhao et al, clinical Cancer research 21 (14): 3149-3159,2015; junghans et al, 2016 Prostate (14): 1257-1270). CD 19-targeting CAR-T cells (i.e., molecules expressed on B cells) have been successful in treating B cell malignancies and have been FDA approved, with some assays showing response rates up to 70%. However, CAR-T cells may exhibit non-specific activation, which may lead to potentially serious adverse events due to inappropriate immune activity.

Disclosure of Invention

Provided herein are engineered dimeric antigen receptors having a first polypeptide chain and a second polypeptide chain that associate with each other to form an antigen binding domain that binds to a GD2 molecule. The antigen binding domain consists of an antibody heavy chain variable region located on a first polypeptide and an antibody light chain variable region located on a second polypeptide, or alternatively, consists of an antibody light chain variable region located on the first polypeptide and an antibody heavy chain variable region located on the second polypeptide. The antibody heavy and light chain variable regions are followed by antibody constant regions (CH 1 and CL, respectively), and the first polypeptide further comprises a transmembrane domain and at least one intracellular signaling domain. When the first and second polypeptides of the DAR are expressed by a host cell, the first and second polypeptides associate with each other outside the cell through disulfide bonds located between antibody constant regions, allowing the heavy and light chain variable regions of the two polypeptides to associate to form an antigen binding domain outside the cell. The DAR is assembled outside the cell by the antibody constant region thereby producing a Fab fragment linked to the transmembrane domain and the intracellular signaling domain of the first polypeptide. DAR expressing host cells, such as T cells, may be used in cell-based therapies, for example, in the treatment of cancer.

In a first aspect, provided herein is a Dimeric Antigen Receptor (DAR) that binds GD2, wherein the DAR comprises: a) A first polypeptide comprising, in order from amino terminus to carboxy terminus, a plurality of polypeptide regions; (i) an antibody heavy chain variable region; (ii) an antibody heavy chain constant region (CH 1); (iii) optionally a hinge region; (iv) a transmembrane region; and (v) an intracellular region; and (b) a second polypeptide comprising, in order from amino terminus to carboxy terminus, a plurality of polypeptide regions: (i) an antibody light chain variable region; and (ii) an antibody light chain constant region (CL). Alternatively, provided herein are DARs in combination with GD2, which can include: a first polypeptide chain comprising, in order from amino terminus to carboxy terminus, a plurality of polypeptide regions: (i) an antibody light chain variable region; (ii) an antibody light chain constant region (CL); (iii) optionally a hinge region; (iv) a transmembrane region; and (v) an intracellular region; and (b) a second polypeptide chain comprising, in order from amino terminus to carboxy terminus, a plurality of polypeptide regions: (i) an antibody heavy chain variable region; and (ii) an antibody heavy chain constant region (CH 1). The second polypeptide of DAR as provided herein does not include a transmembrane region. The antibody heavy chain constant region and the antibody light chain constant region form a dimerization domain to associate the first polypeptide and the second polypeptide to form the DAR, and the antibody heavy chain variable region and the antibody light chain variable region form an antigen binding domain that binds GD 2.

In some implementations, GD2 DAR as provided herein includes: a) A first polypeptide consisting essentially of: (i) an antibody heavy chain variable region; (ii) an antibody heavy chain constant region (CH 1); (iii) optionally a hinge region; (iv) a transmembrane region; and (v) an intracellular region; and (b) a second polypeptide, the second polypeptide chain consisting essentially of: (i) an antibody light chain variable region; and (ii) an antibody light chain constant region (CL). Alternatively, provided herein are DARs in combination with GD2, wherein the DARs may comprise: a) A first polypeptide consisting essentially of: (i) an antibody light chain variable region; (ii) an antibody light chain constant region (CL); (iii) optionally a hinge region; (iv) a transmembrane region; and (v) an intracellular region; and b) a second polypeptide consisting essentially of: (i) an antibody heavy chain variable region; and (ii) an antibody heavy chain constant region. The first and second polypeptides of the DAR dimerize via their antibody heavy chain constant regions and antibody light chain constant regions, which form one or more disulfide bonds. Two mature polypeptides, a transmembrane (first) polypeptide and a secreted (second) polypeptide, when produced by a host cell genetically modified to express genes encoding the first and second polypeptides, can be assembled outside the cell by a cysteine bridge in their antibody constant domains, thereby forming a GD2 binding domain.

The antibody heavy chain variable region and the antibody light chain variable region of a GD2DAR polypeptide may be derived from a GD2 antibody, which may be a 14.18 antibody or a humanized version (hu 14.18), as non-limiting examples. In some embodiments, the first polypeptide of GD2DAR comprises a heavy chain variable region of 14.18 (SEQ ID NO: 2) or a heavy chain variable region having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:2, and comprises a light chain variable region of 14.18 (SEQ ID NO: 5) or a light chain variable region having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 5. Alternatively, in some embodiments, the first polypeptide of GD2DAR comprises a light chain variable region of 14.18 (SEQ ID NO: 5) or a light chain variable region having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and comprises a heavy chain variable region of 14.18 (SEQ ID NO: 2) or a heavy chain variable region having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In various embodiments, the heavy chain variable region having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO. 2 comprises the heavy chain variable region CDR sequence of SEQ ID NO. 2. In various embodiments, the light chain variable region having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO. 5 comprises the light chain variable region CDR sequence of SEQ ID NO. 5.

In further embodiments, the first polypeptide of GD2 DAR comprises the heavy chain variable region of hu14.18 (SEQ ID NO: 3) or a heavy chain variable region having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:3, and comprises the light chain variable region of hu14.18 (SEQ ID NO: 6) or a light chain variable region having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 6. Alternatively, in some embodiments, the first polypeptide of GD2 DAR comprises the light chain variable region of hu14.18 (SEQ ID NO: 6) or a light chain variable region having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and comprises the heavy chain variable region of hu14.18 (SEQ ID NO: 3) or a heavy chain variable region having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 3. In various embodiments, the heavy chain variable region having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO. 3 comprises the heavy chain variable region CDR sequence of SEQ ID NO. 3. In various embodiments, the light chain variable region having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO. 6 comprises the light chain variable region CDR sequence of SEQ ID NO. 6.

The heavy chain variable region of the first DAR polypeptide or the second DAR polypeptide is followed by a heavy chain constant region (CH 1), e.g., SEQ ID No. 4 or a constant region having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID No. 4. The light chain variable region of the first DAR polypeptide or the second DAR polypeptide is followed by a light chain constant region, which may be a light chain kappa constant region (SEQ ID NO:7 or a constant region having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto) or a light chain lambda constant region (SEQ ID NO:8 or a constant region having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto).

The hinge region of the first polypeptide is optional and may comprise a hinge sequence from an antibody or other immune molecule or a portion thereof, if present, for example, may be selected from a hinge region of IgG, igA, igM, igE or IgD or a portion of any of them. Hinge regions including, but not limited to, at least a portion of a hinge region derived from one or more naturally occurring proteins include three, four, five, six, seven, eight, nine, or ten or more amino acids, for example, about three to about twenty amino acids, about ten to about thirty amino acids, about twenty to about fifty amino acids, about thirty to about sixty amino acids, about forty to about eighty amino acids, about fifty to about one hundred amino acids, about sixty to about 120 amino acids, or about eighty to about 150 amino acids or more. In further non-limiting examples, the hinge region of the first polypeptide of the DAR may comprise a CD8 a hinge region (SEQ ID NO: 10), a CD28 hinge region (SEQ ID NO: 9), or a CD8 a/CD 28 hinge region (SEQ ID NO: 11), or may comprise a hinge region having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to either.

As non-limiting examples, the transmembrane region of the first polypeptide may include a transmembrane region of CD8 (SEQ ID NO: 13), CD28 (SEQ ID NO: 12), 4-1BB (SEQ ID NO: 14), or CD3 ζ (SEQ ID NO: 15), or a transmembrane region having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of them.

The intracellular region of the first polypeptide may comprise one or more intracellular amino acid sequences selected from the group consisting of: 4-1BB intracellular region (SEQ ID NO: 16), CD3 zeta (SEQ ID NO: 19) with ITAM 1, 2 and 3, CD3 zeta (SEQ ID NO: 20) with ITAM 1, CD3 zeta (SEQ ID NO: 22) with ITAM 3, or CD28 (SEQ ID NO: 17), CD27, OX40 (SEQ ID NO: 18), CD30, CD40, PD-1, ICOS, lymphocyte function associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B-H3, GITR (TNFRSF 18), DR3 (TNFRSF 25), TNFR2 and/or CD226, or an intracellular amino acid sequence having at least 95% identity to any of them. In some embodiments, the intracellular region comprises a CD3 zeta intracellular region (SEQ ID NO: 19) having ITAMs 1, 2, and 3 or an intracellular region having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 19. In some embodiments, the intracellular region comprises a 4-1BB intracellular region (SEQ ID NO: 16) or an intracellular region having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 16. In some embodiments, the intracellular domain comprises or consists essentially of: 4-1BB intracellular region (SEQ ID NO: 16) and CD3 ζ intracellular region (SEQ ID NO: 19) having ITAMs 1, 2 and 3.

In some embodiments of GD2 DAR, the first polypeptide comprises the amino acid sequence of SEQ ID NO. 32 or an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and the second polypeptide comprises the amino acid sequence of SEQ ID NO. 33 or an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments of GD2 DAR, the first polypeptide comprises the amino acid sequence of SEQ ID NO. 36 or an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto, and the second polypeptide comprises the amino acid sequence of SEQ ID NO. 37 or an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto.

In a further aspect, a nucleic acid molecule encoding GD2 DAR is provided, as any of the nucleic acid molecules described herein. For example, one or more nucleic acid molecules can encode one or more precursor polypeptides that can be expressed by a host cell to produce a DAR having a first polypeptide and a second polypeptide as described herein. In some embodiments, one or more nucleic acid molecules encoding a DAR encode one or more precursor polypeptides, which may include, for example, one or more leader sequences (signal peptides) for membrane insertion or secretion of the first or second polypeptide produced. Further, the nucleic acid molecule encoding the first polypeptide and the second polypeptide of DAR may include a peptide "self-cleaving" or "2A" sequence located between the sequences encoding the first polypeptide and the second polypeptide such that the nucleic acid molecule encodes a single open reading frame that includes both the mature first polypeptide and the mature second polypeptide, wherein both polypeptide-encoding sequences are preceded by a sequence encoding a signal peptide (i.e., encoded as a precursor polypeptide) and the first polypeptide-encoding sequence and the second polypeptide-encoding sequence are separated by a sequence encoding a 2A peptide (e.g., SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO: 29) or other sequences that result in the production of two separate polypeptides. As non-limiting examples, a nucleic acid molecule encoding a DAR as provided herein can encode a precursor polypeptide of SEQ ID:30 or a precursor polypeptide having 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID:30, wherein a host cell expressing a DAR coding sequence produces the first polypeptide and the second polypeptide, or can encode a precursor polypeptide of SEQ ID NO:34 or a precursor polypeptide having 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:34, wherein a host cell expressing a DAR coding sequence produces the first polypeptide and the second polypeptide.

Other configurations of nucleic acid molecules that can encode a DAR as provided herein include, but are not limited to, open reading frames encoding a first precursor polypeptide and a second precursor polypeptide of the DAR, wherein the open reading frames are separated by an Internal Ribosome Entry Site (IRES) that allows translation of two polypeptides from the same promoter. Also contemplated is a nucleic acid molecule encoding a first polypeptide and a second polypeptide as separate open reading frames, each having its own promoter. Two vectors are further contemplated, which together encode two polypeptides of DAR. In these embodiments, a signal peptide will typically be included at the N-terminus of each polypeptide to ensure membrane integration of the first polypeptide and secretion of the second polypeptide. Non-limiting examples of signal peptides are provided as SEQ ID NOs 23, 24 and 25.

The nucleic acid molecule encoding a DAR precursor polypeptide can include a promoter operably linked to one or more sequences encoding a first polypeptide and a second polypeptide of DAR. Provided herein are expression cassettes comprising a promoter operably linked to a nucleic acid sequence encoding a DAR precursor polypeptide, wherein the DAR precursor polypeptide can encode one or both of the DAR polypeptides. The promoter may be, for example, a promoter active in eukaryotic cells, e.g., a promoter active in mammalian cells. As non-limiting examples, the promoter may be a JeT promoter (SEQ ID NO: 39), a CMV promoter, an HTLV promoter, an EF1 alpha promoter or an EF1 alpha/HTLV mixed promoter.

In some embodiments, provided herein are nucleic acid expression cassettes encoding at least one polypeptide of GD2 DAR, wherein the expression cassettes flank sequences of a human locus, e.g., sequences of the human TRAC gene.

In some exemplary embodiments, a nucleic acid as provided herein may encode a precursor polypeptide of SEQ ID NO. 30 that encodes a first polypeptide and a second polypeptide of GD2 DAR and may have at least 65%, at least 70%, at least 75%, at least 80% or at least 85%, at least 90% or at least 95% identity with SEQ ID NO. 31. In some exemplary embodiments, a nucleic acid as provided herein may encode a precursor polypeptide of SEQ ID NO. 34 that encodes a first polypeptide and a second polypeptide of GD2 DAR and may have at least 65%, at least 70%, at least 75%, at least 80% or at least 85%, at least 90% or at least 95% identity with SEQ ID NO. 35.

Also provided herein is the use of a nucleic acid molecule encoding GD2 DAR, any GD2 DAR as disclosed herein, for the preparation of a medicament for the treatment of cancer. The drug may be, for example, a genetically engineered cell, such as a T cell or NK cell, that expresses GD2 DAR.

In another aspect, a genetically engineered host cell is provided that includes one or more nucleic acid molecules as provided herein for expressing GD2 DAR, any of the nucleic acid molecules disclosed herein. As non-limiting examples, the cells may be T cells or NK cells. In some embodiments, one or more nucleic acid molecules encoding at least one DAR polypeptide are integrated into the genome of the engineered host cell. For example, one or more nucleic acid molecules encoding at least one DAR polypeptide may be integrated into the TRAC locus of an engineered host cell. In various embodiments provided herein, a genetically engineered host cell expresses GD2 DAR as provided herein and does not express a T cell receptor. Further provided herein is a population of T cells in which at least 20%, at least 30%, at least 40%, or at least 50% of the cells express GD2 DAR and do not express a T cell receptor. Further provided herein is a population of T cells in which at least 90% or at least 95% of cells expressing GD2 DAR do not express a T cell receptor.

In some embodiments, the genetically engineered cell expressing GD2 DAR is a T cell (DAR-T cell), e.g., a human T cell. In some embodiments, the DAR-T cells are primary T cells. In alternative embodiments, the genetically engineered cells expressing GD2 DAR are Natural Killer (NK) cells (DAR-NK cells), e.g., human NK cells. In some embodiments, the DAR-NK cells are primary NK cells.

In a further aspect, provided herein are methods of treating cancer by administering anti-GD 2 DAR-T cells to a subject having cancer. The cells may be administered in a single dose or in multiple doses, e.g., about 10 may be administered ⁵ From about 10 to about ⁹ Individual cells. The cell can be a cell of a population in which at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% of the cells express a DAR construct and less than 10%, less than 5%, or less than 1% of the cells express an endogenous T cell receptor (e.g., less than 1% of the cells are CD3 positive).

Further provided are genetically engineered cells that express GD2 DAR as disclosed herein and/or include a nucleic acid molecule encoding GD2 DAR, any of the nucleic acid molecules described herein, for use in a method of treating cancer by administering the genetically engineered GD2 DAR-expressing cells to a subject having cancer.

Further provided is the use of a genetically engineered cell expressing GD2 DAR as provided herein, any GD2 DAR as disclosed herein, for the preparation of a medicament for the treatment of cancer.

Further embodiments according to the present disclosure are set forth in the claims and the detailed description.

Drawings

Figures 1A-D provide diagrams illustrating tissues and domains of four exemplary configurations of dimeric antigen receptors. A) Is a diagram of a DAR showing a disulfide bond between constant antibody regions of a first polypeptide and a second polypeptide, wherein the first polypeptide comprising a transmembrane domain and two intracellular signaling regions comprises a heavy chain variable region of an antibody and the second polypeptide comprising no transmembrane domain or any intracellular domain comprises a light chain variable region of an antibody. B) Is a diagram of a DAR showing a disulfide bond between constant antibody regions of a first polypeptide and a second polypeptide, wherein the first polypeptide comprising a transmembrane domain and three intracellular signaling regions comprises a heavy chain variable region of an antibody and the second polypeptide comprising no transmembrane domain or any intracellular domain comprises a light chain variable region of an antibody. C) Is a diagram of a DAR showing a disulfide bond between constant antibody regions of a first polypeptide and a second polypeptide, wherein the first polypeptide comprising a transmembrane domain and two intracellular signaling regions comprises a light chain variable region of an antibody and the second polypeptide comprising no transmembrane domain or any intracellular domain comprises a heavy chain variable region of an antibody. D) Is a diagram of a DAR showing a disulfide bond between constant antibody regions of a first polypeptide and a second polypeptide, wherein the first polypeptide comprising a transmembrane domain and three intracellular signaling regions comprises a light chain variable region of an antibody and the second polypeptide comprising no transmembrane domain or any intracellular domain comprises a heavy chain variable region of an antibody.

Figures 2A-D provide diagrams illustrating tissues and domains of four exemplary configurations of precursors of dimeric antigen receptors, which in some embodiments may be encoded by nucleic acid molecules that may be transfected into host cells to express DAR. A and B) provide diagrams for expressing a precursor polypeptide of a DAR shown in fig. 1B and 1A, respectively, wherein the precursor polypeptide comprises a leader sequence for directing a first polypeptide to a membrane, a "self-cleaving sequence" for allowing expression of the precursor polypeptide as a first polypeptide and a second polypeptide, and a second leader sequence for secreting the second polypeptide. C) And D) provides a map for expressing the precursor polypeptides of DARs shown in FIGS. 1D and 1C, respectively, wherein the precursor polypeptides include a leader sequence for directing the first polypeptide to the membrane, a "self-cleaving sequence" for allowing the precursor polypeptides to be expressed as the first polypeptide and the second polypeptide, and a second leader sequence for secreting the second polypeptide.

FIG. 3 shows the results of flow cytometry performed on several cell lines including NCI-H524, SK-MEL-5, K562, and H460 using the labeled antibodies against GD2 (x-axis).

FIG. 4 shows the results of flow cytometry detecting expression of GD2 (14.18) CAR (anti-GD 2 (14.18) -scFv-28 z), GD2 (hu 14.18) CAR (anti-GD 2 (hu 14.18) -scFv-28 z) or GD2 (14.18) DAR (anti-GD 2 (14.18) -DAR-BBz) in transgenic T cells (x-axis) fourteen days after transfection. Very few transgenic CAR-T and DAR-T expressing transgenic cells expressed CD3 (y axis).

FIG. 5 shows the results of flow cytometry monitoring for expansion over time of transgenic T cells expressing GD2 CAR or GD2-DAR co-cultured with cell lines expressing GD2 (GD2+) or very low to no expression of GD2 (expressed as GD 2-). The column labeled "T only" indicates CAR T cells or DAR T cells cultured alone. anti-GD 2 (14.18) -scFv-28z represents a CAR construct prepared with the scFv of the 14.18 (chimeric) anti-GD 2 antibody; anti-GD 2 (hu 14.18) -scFv-28z represents a CAR construct prepared with scFv of hu14.18 (humanized) anti-GD 2 antibody; anti-GD 2 (14.18) DAR-BBz represents DAR constructs made with the heavy and light chain sequences of the 14.18 (chimeric) anti-GD 2 antibody. GD2 construct expression along the x-axis; CD3 expression is along the y-axis.

FIG. 6A is a graph showing the percentage of in vitro cytotoxicity obtained using GD2 CAR or GD2 DAR expressing transgenic T cells as effectors, and SK-MEL-5 (GD2+) target cells. The population used in the assay resulting from isolated T cells transfected with CAR or DAR constructs is designated by the letter "T"; the population generated from PBMCs transfected with CAR or DAR constructs used in the assay is designated by the letter "P". anti-GD 2 (14.18) -scFv-28z represents a CAR construct prepared with the scFv of the 14.18 (chimeric) anti-GD 2 antibody; anti-GD 2 (hu 14.18) -scFv-28z represents a CAR construct prepared with scFv of hu14.18 (humanized) anti-GD 2 antibody; anti-GD 2 (14.18) DAR-BBz represents DAR constructs made with the heavy and light chain sequences of the 14.18 (chimeric) anti-GD 2 antibody. TRAC KO is a control T cell knocked out of T cell receptor but not transfected with CAR or DAR constructs. The percentage of populations of transfected T cells or PBMCs expressing GD2 CAR or DAR constructs used in the assay are also indicated in the legend. FIG. 6B is a graph showing the percentage of in vitro cytotoxicity obtained using transgenic T cells expressing GD2 CAR or GD2-DAR and NCI-H460 (GD 2-) target cells as effectors. Cell killing use Real-time cell analysis (RTCA) system (Agilent) measurements). The cell population is shown in figure 6A.

FIG. 7A is a bar graph showing IFN- γ release levels (48 hours post target stimulation) in T cells with TRAC knockdown alone (TRAC KO) or transgenic T cells expressing GD2 CAR or GD2-DAR constructs. Target cells include T cells only (no target control), NCI-H524 (GD2+) cells, or K562 (GD2-) cells. Each assay set includes (from left to right): TRAC KO; GD2 (14.18) CAR (isolated T cells); GD2 (hu 14.18) CAR (isolated T cells); GD2 (14.18) DAR (isolated T cells); GD2 (14.18) CAR (PBMC); GD2 (hu 14.18) CAR (PBMC); GD2 (14.18) DAR (PBMC). The arrow indicates the value of the GD2 (14.18) DAR population.

FIG. 7B is a bar graph showing GM-CSF release levels (48 hours post target stimulation) in T cells with TRAC knockdown alone (TRAC KO) or transgenic T cells expressing GD2 CAR or GD2-DAR constructs. Assays included no target control (T cells only), or NCI-H524 (GD2+) or K562 (GD2-) cells as target cells. Each assay set includes (from left to right): TRAC KO; GD2 (14.18) CAR (isolated T cells); GD2 (hu 14.18) CAR (isolated T cells); GD2 (14.18) DAR (isolated T cells); 14.18CAR (PBMC); hu14.18 CAR (PBMC); 14.18DAR (PBMC).

FIG. 8 provides in vivo images taken 7 weeks after treatment of mice vaccinated with SK-MEL-5 tumor cells and then treated with PBS only, TRAC knockout T cells, GD2 (hu 14.18) DAR-T cells (labeled anti-GD 2 hDAR T) in which the DAR includes hu14.18 heavy and light chain variable regions, and GD2 (14.18) DAR-T cells (labeled anti-GD 2 mDAR T) in which the DAR includes 14.18 heavy and light chain variable regions.

Fig. 9A is a graph of average tumor volume obtained by treating the group of mice shown in fig. 8 over time. Fig. 9B is a graph of average body weight obtained by treating the group of mice shown in fig. 8 for a certain period of time.

Fig. 10 provides a survival curve for the mice shown in fig. 8.

Detailed Description

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

The headings provided herein are for the convenience of the reader only and do not limit the various aspects of the disclosure, which can be had by reference to the specification in its entirety.

Definition of the definition

Unless defined otherwise, technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art. Generally, terms relating to cell and tissue culture, molecular biology, immunology, microbiology, genetics, transgenic cell production, protein chemistry and nucleic acid chemistry, and hybridization techniques described herein are 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 as described in various general and more specific references cited and discussed herein. See, e.g., sambrook et al, molecular cloning: laboratory Manual (Molecular Cloning: ALaboratory Manual), 2 nd edition, cold spring harbor laboratory Press (Cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y.) of Cold spring harbor, N.Y. (1989) Ausubel et al, molecular biology laboratory Manual (Current Protocols in Molecular Biology), green publication society (Greene Publishing Associates) (1992). Many basic texts describe standard antibody production procedures, including borreba eck (editions) & antibody engineering (Antibody Engineering), 2 nd edition new york frieman company (Freeman and Company, NY), 1995; mcCafferty et al, antibody engineering methods of practical use (Antibody Engineering, A Practical Approach), oxford press IRL of Oxford, england (IRL at Oxford Press, oxford, 1996; paul (1995) antibody engineering protocol (Antibody Engineering Protocols), new Jersey Totolva Ha Men Press (Humana Press, towata, N.J.), 1995; paul (eds.), "basic immunology (Fundamental Immunology)," New York Raven Press (Raven Press, N.Y), 1993; coligan (1991) current guidelines for immunology experiments (Current Protocols in Immunology), wiley/Greene, N.Y.; harlow and Lane (1989) antibodies: laboratory manuals (Antibodies: A Laboratory Manual), cold spring harbor laboratory Press, new York; stites et al, (editions) basic and clinical immunology (Basic and Clinical Immunology) (4 th edition) Langerhans medical publication (Lange Medical Publications, los Altos, calif.) to Los Aweiss, calif., and references cited therein; coding for monoclonal antibodies: principle and practice (Coding Monoclonal Antibodies: principles and Practice) (2 nd edition) Academic Press (Academic Press, new York, N.Y.), 1986, and Kohler and Milstein Nature, 256:495-497,1975. All references cited herein are incorporated herein in their entirety. Enzymatic reactions and enrichment/purification techniques are also well known and are performed according to manufacturer's instructions as commonly accomplished in the art or as described herein. The terminology used in connection with analytical chemistry, synthetic organic chemistry, and pharmaceutical chemistry described herein, as well as laboratory procedures and techniques, are well known and commonly used in the art. Standard techniques may be used for chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and treatment of patients.

Unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. The singular uses of the singular forms "a", "an" and "the" and any of the words include plural referents unless expressly and unequivocally limited to one referent.

It should be understood that the use of alternatives (e.g., "or") herein means one or both of the alternatives or any combination thereof.

The term "and/or" as used herein means that the specific disclosure of each particular feature or component is accompanied or not accompanied by other features or components. 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). Also, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following aspects: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

As used herein, the terms "include," "including," "having," "contains," and "containing," and grammatical variants thereof, are intended to be non-limiting such that one or more items in a list do not exclude other items that may be substituted or added to the listed items. It should be understood that where an aspect is described herein by the language "comprising," other similar aspects described in terms of "consisting of …" and/or "consisting essentially of …" are also contemplated. The phrase "consisting essentially of …" is intended to include the materials or steps specified as well as those materials or steps that do not materially affect the basic and novel characteristics of the described compositions or methods.

As used herein, the term "about" refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, in accordance with the practice in the art, "about" or "approximately" may mean within one or more than one standard deviation. Alternatively, "about" or "approximately" may mean a range of up to 10% (i.e., ±10%) or greater, depending on the limitations of the measurement system. For example, about 5mg may include any number between 4.5mg and 5.5 mg. Furthermore, with specific reference to a biological system or process, this term may mean at most one order of magnitude or at most 5 times the value. When a particular value or composition is provided in this disclosure, unless otherwise stated, the meaning of "about" or "approximately" should be assumed to be within an acceptable error range for the particular value or composition.

The terms "peptide," "polypeptide chain," and "protein," as well as other related terms used herein, are used interchangeably and refer to a polymer of amino acids, and are not limited to any particular length. The polypeptides may include natural amino acids and unnatural amino acids. Polypeptides include recombinant forms or chemically synthesized forms. Polypeptides also include precursor molecules and mature molecules. Precursor molecules include those that have not been subjected to cleavage, such as cleavage by a secreted signal peptide or by non-enzymatic cleavage at certain amino acid residues. Polypeptides include mature molecules that have undergone cleavage. These terms encompass natural proteins, recombinant proteins and artificial proteins, protein fragments and polypeptide analogs of protein sequences (e.g., muteins, variants, chimeric proteins and fusion proteins), post-translationally or otherwise covalently or non-covalently modified proteins. Two or more polypeptides (e.g., 2-6 or more polypeptide chains) may associate with each other by covalent and/or non-covalent association to form a polypeptide complex. Association of polypeptide chains may also include peptide folding. Thus, the polypeptide complex may be a dimer, trimer, tetramer or higher order complex, depending on the number of polypeptide chains forming the complex. Described herein are Dimeric Antigen Receptors (DARs) comprising two polypeptide chains.

The terms "nucleic acid," "polynucleotide," and "oligonucleotide," as well as other related terms used herein, are used interchangeably and refer to a polymer of nucleotides and are not limited to any particular length. Nucleic acids include recombinant forms 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. The nucleic acid molecule may be single-stranded or double-stranded. In one embodiment, the nucleic acid molecules of the present disclosure include a contiguous open reading frame encoding an antibody or fragment or scFv, derivative, mutein or variant thereof. In one embodiment, the nucleic acid comprises one type of polynucleotide or a mixture of two or more different types of polynucleotides. Nucleic acids encoding Dimeric Antigen Receptors (DARs) or antigen binding portions thereof are described herein.

The term "recovering" and other related terms refer to obtaining a protein (e.g., an antibody or antigen binding portion thereof) from a host cell culture medium or host cell lysate or host cell membrane. In one embodiment, the protein is expressed by the host cell as a recombinant protein fused to a secretion signal peptide (leader peptide sequence) sequence that mediates secretion of the expressed protein from the host cell (e.g., from a mammalian host cell). The secreted protein may be recovered from the host cell culture medium. In one embodiment, the protein is expressed by the host cell as a recombinant protein lacking the secretion signal peptide sequence, which can be recovered from the host cell lysate. In one embodiment, the protein is expressed by the host cell as a membrane-bound protein that can be recovered using a detergent to release the expressed protein from the host cell membrane. In one embodiment, regardless of the method used to recover the protein, the protein may be subjected to a procedure that removes cellular debris from the recovered protein. For example, the recovered protein may be subjected to chromatography, gel electrophoresis, and/or dialysis. In one embodiment, chromatography includes any one procedure or any combination of procedures or two or more procedures, including affinity chromatography, hydroxyapatite chromatography, ion exchange chromatography, reverse phase chromatography, and/or chromatography on silica gel. In one embodiment, the affinity chromatography comprises protein a or G (a cell wall component from staphylococcus aureus (Staphylococcus aureus)).

The term "isolated" refers to a protein (e.g., an antibody or antigen-binding portion thereof) or polynucleotide that is substantially free of other cellular material. Proteins may be substantially free of naturally associated components (or components associated with cellular expression systems or chemical synthesis methods for producing antibodies) by isolation using protein purification techniques well known in the art. In some embodiments, the term isolated also refers to proteins or polynucleotides that are substantially free of other molecules of the same species, e.g., other proteins or polynucleotides having different amino acid or nucleotide sequences, respectively. The purity or homogeneity of the desired molecule can be determined using techniques well known in the art, including low resolution methods such as gel electrophoresis and high resolution methods such as HPLC or mass spectrometry. In one embodiment, the isolated precursor polypeptide and the first and second polypeptide chains of the DAR or antigen binding portion thereof of the present disclosure are isolated.

Antibodies can be obtained from sources such as serum or plasma containing immunoglobulins with various antigen specificities. Such antibodies, if affinity purified, can be enriched for a particular antigen specificity. Such enriched antibody preparations typically consist of less than about 10% of antibodies having specific binding activity for a particular antigen. Several rounds of affinity purification of these formulations can increase the proportion of antibodies that have specific binding activity for the antigen. Antibodies prepared in this manner are commonly referred to as "monospecific". A monospecific antibody preparation may consist of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 99.9% of antibodies having specific binding activity for a particular antigen. Antibodies can be produced using recombinant nucleic acid techniques as described below.

The term "leader sequence" or "leader peptide" or "peptide signal sequence" or "signal peptide" or "secretion signal peptide" refers to a peptide sequence located at the N-terminus of a polypeptide. The leader sequence directs the polypeptide chain to the cell secretory pathway and may direct integration and anchoring of the polypeptide into the lipid bilayer of the cell membrane. Typically, the leader sequence is about 10-50 amino acids in length. The leader sequence may direct the transport of the precursor polypeptide from the cytosol to the endoplasmic reticulum. In one embodiment, the leader sequence comprises a signal sequence comprising a CD8 a, CD28 or CD16 leader sequence. In one embodiment, the signal sequence comprises a mammalian sequence comprising, for example, a mouse or human igy secretion signal peptide. In one embodiment, the leader sequence comprises a mouse Ig gamma leader peptide sequence MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 23).

As used herein, "antigen binding protein" and related terms refer to proteins that include a moiety that binds to an antigen and optionally a scaffold or framework portion that allows the antigen binding moiety to adopt a conformation that facilitates binding of the antigen binding protein to the antigen. Examples of antigen binding proteins include antibodies, antibody fragments (e.g., antigen binding portions of antibodies), antibody derivatives, and antibody analogs. The antigen binding proteins may include, for example, alternative protein scaffolds or artificial scaffolds with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds including, for example, mutations introduced to stabilize the three-dimensional structure of the antigen binding protein, and fully synthetic scaffolds including, for example, biocompatible polymers. See, e.g., korndorfer et al, 2003, protein: structure, function and bioinformatics (Proteins: structure, function, and Bioinformatics), volume 53, phase 1: 121-129; roque et al, 2004, biotechnology progress (Biotechnol. Prog.) 20:639-654. In addition, peptide antibody mimics ("PAM") and scaffolds based on antibody mimics that utilize a fibronectin component as a scaffold may be used. Antigen binding proteins that include a Dimeric Antigen Receptor (DAR) are described herein.

The antigen binding protein may have a structure such as an immunoglobulin. In one embodiment, an "immunoglobulin" refers to a tetrameric molecule composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region consisting of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as either kappa or lambda light chains. Heavy chains are classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, wherein the heavy chain further comprises a "D" region of about 10 or more amino acids. See generally basic immunology (Fundamental Immunology), chapter 7 (Paul, W., editorial, 2 nd edition, raven Press, N.Y.), new York (1989) (incorporated by reference in its entirety for all purposes). The heavy and/or light chain may or may not include a leader sequence for secretion. The variable region of each light/heavy chain pair forms an antibody binding site such that the intact immunoglobulin has two antigen binding sites. In one embodiment, the antigen binding protein may be a synthetic molecule having a structure that differs from a tetrameric immunoglobulin molecule but that still binds to a target antigen or to two or more target antigens. For example, a synthetic antigen binding protein may include an antibody fragment, 1-6 or more polypeptide chains, an asymmetric assembly of polypeptides, or other synthetic molecules. Described herein are antigen binding proteins that contain DAR structures that specifically bind to a target antigen (e.g., GD2 antigen) that have immunoglobulin-like properties.

The variable regions of an immunoglobulin chain exhibit the same general structure of relatively conserved Framework Regions (FR) connected by three hypervariable regions (also known as complementarity determining regions or CDRs). From N-terminal to C-terminal, both the light and heavy chains include domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

One or more CDRs can be incorporated covalently or non-covalently into a molecule to make it an antigen binding protein. The antigen binding protein may incorporate the CDR as part of a larger polypeptide chain, may covalently link the CDR to another polypeptide chain, or may non-covalently incorporate the CDR. CDRs allow the antigen binding proteins to specifically bind to a particular antigen of interest.

The amino acid assignment to each domain is performed according to the following definition: kabat et al, protein sequence of immunological significance (Sequences of Proteins of Immunological Interest), 5 th edition, public health agency (PHS) of the United states department of health and human services (USDept. Of Health and Human Services), national Institutes of Health (NIH), NIH publication No. 91-3242, 1991 ("Kabat numbering"). Other numbering systems for amino acids in immunoglobulin chains include IMGT.RTM (International immunogenetics information System (international ImMunoGeneTics information system); lefranc et al, development of competitive immunology (Dev. Comp. Immunol.)) (29:185-203; 2005) and AHo (Honyger and Pluckaphun, J. Mol. Biol.) (309 (3): 657-670; 2001); chothia (Al-Lazikani et Al, 1997 journal of molecular biology 273:927-948); contact (Maccallum et al, 1996 journal of molecular biology 262:732-745) and Aho (Honygger and Pluckaphun 2001 journal of molecular biology 309:657-670).

As used herein, "antibodies" (and related terms refer to intact immunoglobulins or antigen-binding portions thereof that specifically bind to an antigen). The antigen binding portion may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the intact antibody. Antigen binding portions include, inter alia, fab ', F (ab') ₂ Fv, domain antibodies (dabs) and Complementarity Determining Region (CDR) fragments, single chain antibodies (scFv), chimeric antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, and polypeptides comprising at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide.

Antibodies include recombinantly produced antibodies and antigen-binding portions. Antibodies include non-human antibodies, chimeric antibodies, humanized antibodies, 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 variable fragments (scFv), humped (camelized) antibodies, affibodies, disulfide-linked Fv (sdFv), anti-idiotype antibodies (anti-Id), minibodies. Antibodies include monoclonal populations and polyclonal populations. Antibody-like molecules that include Dimeric Antigen Receptor (DAR) are described herein.

As used herein, an "antigen binding domain," "antigen binding region," or "antigen binding site," and other related terms, refer to a portion of an antigen binding protein that contains amino acid residues (or other portions) that interact with an antigen and facilitate the specificity and affinity of the antigen binding protein for the antigen. For antibodies that specifically bind to their antigen, the term will include at least a portion of at least one of its CDR domains. Described herein are Dimeric Antigen Receptors (DARs) having an antibody heavy chain variable region and an antibody light chain variable region that form an antigen binding domain.

Antibodies or anti-antibodies as hereinThe term "specific binding" or "specific binding" specifically binds or specifically binding "and other related terms, as used in the context of a pro-binding protein or antibody fragment, refer to non-covalent or covalent preferential binding to an antigen relative to other molecules or moieties (e.g., specific binding of an antibody to a particular antigen relative to other available antigens). In one embodiment, if the antibody is at 10 ^-5 M or less, or 10 ^-6 M or less, or 10 ^-7 M or less, or 10 ^-8 M or less, or 10 ^-9 M or less, or 10 ^-10 M or less, or 10 ^-11 Dissociation constant K of M or less _D Binding to the antigen, the antibody specifically binds to the target antigen. In one embodiment, described herein is a Dimeric Antigen Receptor (DAR) that specifically binds to its target antigen (e.g., GD2 antigen).

In one embodiment, the binding specificity of an antibody or antigen binding protein or antibody fragment may be measured by ELISA, radioimmunoassay (RIA), electrochemiluminescence assay (ECL), immunoradiometric assay (IRMA) or Enzyme Immunoassay (EIA).

In one embodiment, the dissociation constant (K _D ) The measurements may be made using BIACORE Surface Plasmon Resonance (SPR) assays. Surface plasmon resonance refers to an optical phenomenon that allows analysis of real-time interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIACORE system (GE healthcare institute of life sciences, new jersey (Biacore Life Sciences division of GE Healthcare, piscataway, NJ)).

An "epitope" and related terms as used herein refer to a portion of an antigen that is bound by an antigen binding protein (e.g., by an antibody or antigen binding portion thereof). An epitope may comprise a portion of two or more antigens bound by an antigen binding protein. An epitope may include one antigen or two or more discrete portions of an antigen (e.g., amino acid residues that are discontinuous in the primary sequence of an antigen but sufficiently close to each other in the context of the tertiary and quaternary structure of an antigen to bind through an antigen binding protein). In general, the variable regions of antibodies, specifically CDRs, interact with an epitope. In one embodiment, described herein is a Dimeric Antigen Receptor (DAR) or antigen binding portion thereof that binds to an epitope of GD2 antigen.

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 a molecule that includes, in addition to an intact antibody, a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') ₂ The method comprises the steps of carrying out a first treatment on the surface of the Fd; and Fv fragments, as well as dabs; a bifunctional antibody; a linear antibody; single chain antibody molecules (e.g., scFv); a polypeptide comprising at least a portion of an antibody sufficient to confer specific antigen binding to the polypeptide. The antigen binding portion of an antibody may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the intact antibody. Antigen binding portions include, inter alia, fab ', F (ab') 2, fv, domain antibodies (dAb) and Complementarity Determining Region (CDR) fragments, chimeric antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, and polypeptides comprising at least a portion of an immunoglobulin sufficient to confer antigen binding properties to the antibody fragment. In one embodiment, described herein are dimeric antigen receptors comprising Fab fragments conjugated to a hinge, a transmembrane region, and an intracellular signaling region.

The terms "Fab", "Fab fragment" and other related terms are meant to include variable light chain regions (V _L ) Constant light chain region (C) _L ) Variable heavy chain region (V) _H ) And a first constant region (C _H1 ) Monovalent fragments of (a). Fab is capable of binding to antigen. F (ab') ₂ Fragments are bivalent fragments comprising two Fab fragments linked at the hinge region by a disulfide bridge. F (Ab') ₂ Has antigen binding ability. Fd fragment includes V _H Region and C _H1 A zone. Fv fragments comprising V _L Region and V _H A zone. Fv can bind antigen. dAb fragment has V _H Domain, V _L Domain or V _H Or an antigen binding fragment of a VL domain (U.S. Pat. Nos. 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). In one embodiment, described herein are dimeric antigen receptors comprising Fab fragments conjugated to a hinge, a transmembrane region, and an intracellular signaling region.

Single chain antibody (scFv) is V _L And V _H The regions are joined by a linker (e.g., a synthetic sequence of amino acid residues) to form an antibody of a continuous protein chain. In one embodiment, the linker is long enough to allow the protein chain to fold upon itself and form a monovalent antigen binding site (see, e.g., bird et al, 1988, science 242:423-26 and Huston et al, 1988, proc. Natl. Acad. Sci. USA 85:5879-83).

A bifunctional antibody is a bivalent antibody comprising two polypeptide chains, wherein each polypeptide chain comprises a V linked by a linker that is too short to pair between two domains on the same chain, thus allowing each domain to pair with a complementary domain on the other polypeptide chain _H And V _L Domains (see, e.g., holliger et al, 1993 Proc. Natl. Acad. Sci. USA 90:6444-48 and Poljak et al, 1994, structure 2:1121-23). If the two polypeptide chains of a bifunctional antibody are identical, then the bifunctional antibody produced by its pairing will have two identical antigen binding sites. Polypeptide chains having different sequences can be used to prepare bifunctional antibodies having two different antigen binding sites. Similarly, a trifunctional antibody and a tetrafunctional antibody are antibodies that include three and four polypeptide chains, respectively, and form three and four antigen binding sites, respectively, which may be the same or different. Bifunctional, trifunctional, and tetrafunctional antibody constructs may be prepared using antigen binding moieties from any of the Dimeric Antigen Receptors (DARs) described herein.

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 of the variable domains 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 immunization with a mouse antigen of interest genetically modified to express antibodies derived from human heavy and/or light chain encoding genes. Described herein are Dimeric Antigen Receptors (DARs) comprising a fully human antibody heavy chain variable region and a fully human antibody light chain variable region.

By "humanized" antibody is meant an antibody having a sequence that differs from the sequence of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and/or additions, such that the humanized antibody is less likely to induce an immune response and/or induce a less severe immune response when administered to a human subject than an antibody of a non-human species. For example, certain amino acids in the framework domains and constant domains of the heavy and/or light chains of a non-human species antibody may be mutated to produce a humanized antibody. In other examples, the constant domain from a human antibody may be fused to a variable domain of a non-human species. In further embodiments, CDR sequences of non-human antibodies may replace CDRs in human antibodies or otherwise be engineered into the framework of human antibodies. In other examples, one or more amino acid residues in one or more CDR sequences of a non-human antibody are altered to reduce the potential immunogenicity of the non-human antibody upon administration of the non-human antibody to a human subject, wherein the altered amino acid residues are not critical for immunospecific binding of the antibody to its antigen or the alteration made to the amino acid sequence is a conservative alteration such that binding of the humanized antibody to the antigen is not significantly worse than binding of the non-human antibody to the antigen. Examples of how to prepare humanized antibodies can be found in U.S. Pat. nos. 6,054,297, 5,886,152 and 5,877,293.

The term "chimeric antibody" and related terms as used herein refer to an antibody that contains one or more regions from a first antibody and one or more regions from one or more other antibodies. In one embodiment, one or more of the CDRs are derived from a non-human antibody. In another embodiment, all CDRs are derived from a human antibody. In another embodiment, CDRs from more than one non-human antibody are mixed and matched in a chimeric antibody. For example, a chimeric antibody may include CDR1 from the light chain of a first non-human antibody, CDR2 and CDR3 from the light chain of a second non-human antibody, and CDR from the heavy chain of a third functional antibody. For example, the framework sequences other than CDR sequences may be human antibody sequences. In other examples, the CDRs may be derived from different species such as human and mouse, or human and rabbit, or human and goat. Those skilled in the art will appreciate that other combinations are possible. In some embodiments, including some embodiments disclosed herein, a chimeric GD2 antibody, such as a chimeric 14.18 antibody, comprises a variable domain of a GD2 antibody of a non-human species (e.g., mouse) fused to a constant domain of a human antibody. For example, a chimeric GD2 heavy chain or portion thereof for a recombinant receptor disclosed herein may comprise the heavy chain variable region of a mouse 14.18 antibody fused to a human antibody constant region, and a chimeric GD2 light chain or portion thereof for a recombinant receptor disclosed herein may comprise the light chain variable region of a mouse 14.18 antibody fused to a human antibody constant region.

Further, the framework regions may be derived from one of the same antibodies, from one or more different antibodies such as a human antibody, or from a humanized antibody. In one example of a chimeric antibody, a portion of the heavy and/or light chain is identical to, homologous to, or derived from an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to, homologous to, or derived from an antibody from another species or belonging to another antibody class or subclass. Fragments of such antibodies that exhibit the desired biological activity (i.e., the ability to specifically bind to a target antigen) are also included.

As used herein, the term "variant" polypeptides and "variants" of polypeptides refer to polypeptides that include amino acid sequences having one or more amino acid residues inserted into, deleted from, and/or substituted into the amino acid sequence relative to a reference polypeptide sequence. Polypeptide variants include fusion proteins. In the same manner, variant polynucleotides include nucleotide sequences having one or more nucleotides inserted into, deleted from and/or substituted into the nucleotide sequence relative to another polynucleotide sequence. Polynucleotide variants include fusion polynucleotides.

As used herein, the term "derivative" of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified, e.g., by conjugation, phosphorylation, and glycosylation with another chemical moiety, such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), and the like. Unless otherwise indicated, the term "antibody" includes derivatives, variants, fragments and muteins thereof, except for antibodies that include full length heavy and full length light chains, examples of which are described below.

The term "hinge" refers to an amino acid segment that is typically present between two domains of a protein and can allow flexibility in the overall construction and movement of one or both domains relative to each other. Structurally, the hinge region comprises from about 10 to about 100 amino acids, for example, from about 15 to about 75 amino acids, from about 20 to about 50 amino acids, or from about 30 to about 60 amino acids. 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 a naturally occurring protein hinge region (e.g., a CD8 hinge region or fragment thereof, a CD8 a hinge region or fragment thereof), an antibody (e.g., igG, igA, igM, igE or IgD antibody) hinge region, or a hinge region that joins 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 includes a hinge region of an antibody and a CH3 constant region of an antibody, or the hinge region includes a hinge region of an antibody and CH2 and CH3 constant regions of an antibody, or the hinge region is a non-naturally occurring peptide, or the hinge region is disposed between the C-terminus of the scFv and the N-terminus of the transmembrane domain. In one embodiment, the hinge region comprises any one region or any combination of two or more regions comprising an immunoglobulin from IgG1, igG2, igG3 or IgG4 An upper hinge sequence, a core hinge sequence, or a lower hinge sequence of the molecule. In one embodiment, the hinge region comprises an IgG1 upper hinge sequence EPKSCDKTHT (SEQ ID NO: 47). In one embodiment, the hinge region comprises an IgG1 core hinge sequence CPXC, whereinXP, R or S (SEQ ID NO: 48). In one embodiment, the hinge region includes a lower hinge/CH 2 sequence PAPELLGGP (SEQ ID NO: 49). In one embodiment, the hinge is joined to an Fc region (CH 2) having the amino acid sequence SVFLFPPKPKDT (SEQ ID NO: 50). In one embodiment, the hinge region comprises the amino acid sequence of the upper hinge, core hinge, or lower hinge and comprises EPKSCDKTHTCPPCPAP ELLGGP (SEQ ID NO: 51). In one embodiment, the hinge region includes one, two, three or more cysteines that may form at least one, two, three or more interchain disulfide bonds.

As used herein, the term "Fc" or "Fc region" refers to the portion of an antibody heavy chain constant region that begins in or after the hinge region and ends at the C-terminus of the heavy chain. The Fc region includes at least a portion of the CH2 and CH3 regions, and may or may not include a portion of the hinge region. In one embodiment, two polypeptide chains each carrying a half-Fc region may dimerize to form an Fc region. The Fc region may bind to Fc cell surface receptors and some proteins of the immune complement system. The Fc region exhibits effector functions including any one or any combination of two or more activities including Complement Dependent Cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), antibody Dependent Phagocytosis (ADP), opsonization, and/or cell binding. In one embodiment, the Fc region may include mutations that increase or decrease any one of these functions or any combination of these functions. The Fc region may bind to Fc receptors including fcyri (e.g., CD 64), fcyrii (e.g., CD 32), and/or fcyriii (e.g., CD16 a). The Fc region can bind to complement component C1 q. In one embodiment, the Fc domain comprises a LALA-PG mutation that reduces effector function (e.g., equivalent to L234A, L235A, P G). In one embodiment, the Fc domain mediates the serum half-life of the protein complex, and mutations in the Fc domain may increase or decrease the serum half-life of the protein complex. In one embodiment, the Fc domain affects the thermal stability of the protein complex, and mutations in the Fc domain may increase or decrease the thermal stability of the protein complex.

The term "labeled antibody" or related terms as used herein refer to antibodies and antigen-binding portions thereof that are unlabeled or conjugated to a detectable label or moiety for detection, wherein the detectable label or moiety is radioactive, colorimetric, antigenic, enzymatic, detectable beads (e.g., magnetic or electron dense (e.g., gold) beads), biotin, streptavidin, or protein a. A variety of labels may be employed including, but not limited to, radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, and ligands (e.g., biotin, hapten).

As used herein, "percent identity" or "percent homology" and related terms refer to a quantitative measurement of similarity between two polypeptides or between two polynucleotide sequences. The percent identity between two polypeptide sequences is a function of the number of identical amino acids shared between the two polypeptide sequences at the aligned position, taking into account the number of gaps and the length of each gap that may need to be introduced to optimize the alignment of the two polypeptide sequences. In a similar manner, the percent identity between two polynucleotide sequences is a function of the number of identical nucleotides in common between the two polynucleotide sequences at the aligned positions, taking into account the number of gaps and the length of each gap that may need to be introduced to optimize the alignment of the two polynucleotide sequences. Sequence comparison and determination of the percent identity between two polypeptide sequences or two polynucleotide sequences can be accomplished using mathematical algorithms. For example, the "percent identity" or "percent homology" of two polypeptides or two polynucleotide sequences can be determined by comparing the sequences using their default parameters using the GAP computer program (GCG Wisconsin Package, version 10.3 (Accelrys, san Diego, calif.) the test sequences include residues at least X% identical to residues of Y when aligned to sequence Y as described above.

In one embodiment, the amino acid sequence of an antibody may be similar to, but not identical to, any of the amino acid sequences of the antigen binding portions of DARs and/or the antibody constant regions described herein. The similarity between an antibody and a polypeptide may be at least 95%, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to the sequence of any polypeptide comprising a DAR or antigen-derived portion thereof described herein. In one embodiment, the similar polypeptide may contain amino acid substitutions within the heavy and/or light chain. In one embodiment, the amino acid substitutions comprise one or more conservative amino acid substitutions. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is substituted with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. In the case where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upward to correct the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, e.g., pearson, (1994) Methods of molecular biology (Methods mol. Biol.) 24:307-331, which is incorporated herein by reference in its entirety. Examples of amino acid groups having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chain: lysine, arginine, and histidine; (6) acidic side chain: aspartic acid and glutamic acid; and (7) the sulfur-containing side chains are cysteine and methionine.

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) obtained by conjugation to the variable heavy and light regions of a monoclonal antibody, such as a human monoclonal antibody. In one embodiment, the CAR comprises: (i) An antigen binding protein comprising a heavy chain Variable (VH) domain and a light chain Variable (VL) domain, wherein the VH and VL domains are joined together by a peptide linker; (ii) a hinge domain; (iii) a transmembrane domain; and (iv) an intracellular domain comprising an expanded intracellular signaling sequence. The disclosed constructs are DARs, which differ from CARs in that the DARs do not use single chain antibody targeting but use separate heavy and light chain variable domain regions.

As used herein, the term "dimeric antigen receptor" (DAR) refers to an engineered receptor comprising two polypeptide chains: a first polypeptide comprising an antigen binding region (heavy chain variable region or light chain variable region) of an antibody, followed by an antibody constant region, a transmembrane region, and an intracellular signaling region; and a second polypeptide comprising an antigen binding region (light chain variable region or heavy chain variable region), followed by an antibody constant region. Wherein the first polypeptide comprises a heavy chain variable region and the second polypeptide comprises a light chain variable region, and vice versa, such that the DAR produces an Fab fragment outside of the cell linked to the transmembrane domain and intracellular signaling domain of the first polypeptide via the antibody constant region. The first polypeptide is anchored to the cell membrane by a transmembrane domain, while the second polypeptide lacks a transmembrane domain and is secreted outside the cell, where it is assembled with the second polypeptide. The two polypeptide chains that make up the dimeric antigen receptor can dimerize to form a protein complex upon specific binding thereof to a target antigen and have antibody-like properties. The dimeric antigen receptors may be used in directed cell therapies.

The present disclosure provides transgenic T cells engineered to express anti-GD 2 constructs having an antigen-binding extracellular portion, optionally a hinge portion, a transmembrane portion, and an intracellular portion having a costimulatory and/or intracellular signaling region. Fab fragments which bind to the transmembrane and intracellular regions. In one embodiment, the DAR construct comprises an optional hinge region between the Fab fragment and the transmembrane region. In some embodiments, the presently disclosed DAR constructs provide unexpected and surprising results, for example, based on comparing a DAR structure with Fab format antibodies to a scFv format CAR structure with the same antibodies. Furthermore, the DAR and CAR formats can be directly compared because the hinge, transmembrane, and two intracellular regions can be identical. However, the DAR format can provide superior results relative to the corresponding CAR format in terms of cell binding to the expressed target antigen, antigen-induced cytokine release, and/or antigen-induced cytotoxicity.

The present disclosure provides DAR constructs that include a heavy chain binding region on one polypeptide chain and a light chain binding region on a separate polypeptide chain. The two polypeptide chains that make up the dimeric antigen receptor may dimerize to form a protein complex. The dimeric antigen receptor has antibody-like properties when it specifically binds to a target antigen. The dimeric antigen receptors may be used in directed cell therapies.

Dimeric Antigen Receptors (DARs) and their various configurations and domains, constructs encoding DARs and cells expressing DARs, and their use in cell therapies are also disclosed in WO 2019/173837 and WO 2021/046445, which are incorporated by reference in their entirety.

As used herein, "vector" and related terms refer to a nucleic acid molecule (e.g., DNA or RNA) that can be operably linked to foreign genetic material (e.g., a nucleic acid transgene). The vector may be used as a vehicle to introduce foreign genetic material into a cell (e.g., a host cell). The vector may include at least one restriction endonuclease recognition sequence for inserting a transgene into the vector. The vector may include at least one gene sequence that confers antibiotic resistance or selectable properties to aid in the selection of host cells carrying the vector-transgene construct. The vector may be a single-stranded or double-stranded nucleic acid molecule. The vector may be a linear or circular nucleic acid molecule. The donor nucleic acid for gene editing methods employing zinc finger nucleases, TALENs or CRISPR/Cas may be one 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 ligated to a transgene, and is capable of replication in a host cell, and transcription and/or translation of the transgene. Viral vectors typically contain viral RNA or DNA backbone sequences that can be linked to a transgene. Viral backbone sequences can be modified to stop infection but retain insertion of the viral backbone and the co-linked transgene into the host cell genome. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, baculovirus vectors, papova vectors, vaccinia vectors, herpes simplex virus vectors, and epstein barr virus (Epstein Barr viral) vectors. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors including a bacterial origin of replication as well as episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

An "expression vector" is a type of vector that may contain one or more regulatory sequences, such as inducible and/or constitutive promoters and enhancers. The expression vector may include a ribosome binding site and/or a polyadenylation site. The expression vector may include one or more origin of replication sequences. Regulatory sequences direct the transcription or transcription and translation of a transgene linked to an expression vector that is transduced into a host cell. Regulatory sequences may control the expression level, timing and/or location of the transgene. Regulatory sequences may exert their effect on a transgene, for example, directly or by the action of one or more other molecules (e.g., polypeptides that bind to regulatory sequences and/or nucleic acids). The regulatory sequence may be part of the vector. Further examples of regulatory sequences are described below: for example, goeddel,1990, gene expression techniques: enzymatic methods (Gene Expression Technology: methods in Enzymology) 185, academic Press (Academic Press, san Diego, calif.), baron et al, 1995, nucleic Acids Res 23:3605-3606. The expression vector may include a nucleic acid encoding at least a portion of any of the Dimeric Antigen Receptors (DARs) described herein or antigen binding portions thereof.

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

The term "transfected" or "transformed" or "transduced" or other related terms as used herein refer to the process of transferring or introducing an exogenous nucleic acid (e.g., transgene) into a host cell. A "transfected" or "transformed" or "transduced" host cell is one that has been introduced with an exogenous nucleic acid (transgene). Host cells include primary subject cells and their progeny. Exogenous nucleic acids encoding at least a portion of any of the Dimeric Antigen Receptors (DARs) described herein or antigen binding portions thereof may be introduced into a host cell. An expression vector comprising at least a portion of any Dimeric Antigen Receptor (DAR) or antigen binding portion thereof described herein may be introduced into a host cell, and the host cell may express a polypeptide comprising at least a portion of a DAR or antigen binding portion thereof described herein.

The term "host cell" or population of host cells "or related terms as used herein refer to a cell (or population thereof) into which exogenous (exogenous or transgenic) nucleic acid has been introduced. The foreign nucleic acid may comprise an expression vector operably linked to the transgene, and the host cell may be used to express the nucleic acid and/or polypeptide encoded by the foreign nucleic acid (transgene). The host cell (or population thereof) may be a cultured cell or may be extracted from the subject. Host cells (or populations thereof) include primary subject cells and their progeny, regardless of the number of passages. Host cells (or populations thereof) include immortalized cell lines. The progeny cells may or may not carry the same genetic material as the parent cells. Host cells encompass offspring cells. In one embodiment, a host cell describes any cell (including progeny thereof) that has been modified, transfected, transduced, transformed and/or manipulated in any manner to express an antibody as disclosed herein. In one example, a host cell (or population thereof) can be introduced with an expression vector operably linked to a nucleic acid encoding a desired antibody or antigen-binding portion thereof described herein. The host cells and populations thereof can carry expression vectors stably integrated into the genome of the host or can carry extrachromosomal expression vectors. In one embodiment, the host cells and populations thereof may carry extrachromosomal vectors that exist after several cell divisions, or that are temporarily present and lost after several cell divisions.

Transgenic host cells can be prepared using non-viral methods including well-known designer nucleases, including zinc finger nucleases, TALENS, meganucleases, or by gene editing using CRISPR/Cas. The transgene may be introduced into the genome of the host cell using a zinc finger nuclease. Zinc finger nucleases include chimeric protein pairs, each containing a non-specific endonuclease domain of a restriction endonuclease (e.g., fokl) fused to a DNA binding domain from an engineered zinc finger motif. The DNA binding domain may be engineered to bind to a specific sequence in the host genome, and the endonuclease domain double-stranded cleaves. 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 regions on either side of the intended insertion site in the host cell genome. The DNA repair mechanism of the host cell enables precise insertion of transgenes through homologous DNA repair. Transgenic mammalian host cells have been prepared using zinc finger nucleases (U.S. Pat. nos. 9,597,357, 9,616,090, 9,816,074 and 8,945,868). Transgenic host cells can be made using TALENs (transcription activator-like effector nucleases) similar to zinc finger nucleases, in that the zinc finger nucleases include a non-specific endonuclease domain fused to a DNA binding domain that can precisely deliver a transgene insertion. Like zinc finger nucleases, TALENs also introduce double-stranded cleavage into host DNA. Transgenic host cells can be prepared using meganucleases that function as site-specific, rare-cutting endonucleases that recognize recognition sites on double-stranded DNA of about 12-40 base pairs in length. Meganucleases include the LAGLIDADG (SEQ ID NO: 52) family that is commonly found in most mitochondria and chloroplasts of eukaryotic unicellular organisms. Examples of meganuclease systems for modifying genomes are described, for example, in U.S. patent No. 9,889,160. Transgenic host cells can be made using CRISPR (clustered regularly interspaced short palindromic repeats, clustered Regularly Interspaced Short Palindromic Repeat).

CRISPR employs Cas endonuclease coupled to guide RNA for target-specific donor DNA integration. The guide RNA includes a conserved polynucleotide comprising a Protospacer Adjacent Motif (PAM) sequence upstream of the gRNA binding region in the target DNA and hybridizes to a host cell target site in which the Cas endonuclease cleaves double-stranded target DNA. The guide RNA may be designed to hybridize to a specific target site. Similar to zinc finger nucleases and TALENs, CRISPR/Cas systems can be used to introduce site-specific insertion of donor DNA with flanking sequences homologous to the insertion site. Non-limiting examples of Cas endonucleases that may be employed include Cas9, cas12a, casX, and related enzymes including variants of Cas9, cas12a, and CasX, as well as enzymes such as MAD7 and variants thereof, ceCpf1, prCpf1, lb2Cpf1, and Lb2Cpf 1-KY. Examples of CRISPR/Cas systems for modifying genomes are described, for example, in U.S. patent nos. 8,697,359; 10,000,772; 9,790,490; and 10,570,415, and U.S. patent application Ser. Nos. 2020/0109382, 2021/0309981, and 2021/0155911, all of which are incorporated herein by reference in their entirety. CRISPR RNA directed use of endonucleases can also be used to disrupt genes whose expression may be optional or undesirable, and in some embodiments, insertion of a CAR or DAR construct (e.g., GD2 DAR construct) can be targeted to a gene encoding a TCR chain, e.g., a TRAC or TRBC gene, such that the TCR is not expressed in cells expressing the CAR or DAR. CRISPR/Cas systems and methods for construct integration, gene knockout and simultaneous knock-in/knockout at targeted sites are described, for example, in US2020/0224160; in WO 2020/176740 and WO 2020/185867, all of these documents are incorporated herein by reference in their entirety.

In various embodiments, the transgenic host cell can be prepared using zinc finger nucleases, TALENs or CRISPR/Cas systems, and the host target site can be the TRAC gene (T cell receptor alpha constant). The donor DNA can include, for example, any nucleic acid encoding a CAR or DAR construct described herein. Electroporation, nuclear transfection or lipofection may be used to co-deliver donor DNA, zinc finger nucleases, TALENs or CRISPR/Cas systems into host cells.

Transgenic host cells can also be prepared by transducing a host cell (e.g., a T cell) with a retroviral vector carrying a nucleic acid encoding a CAR or DAR construct. Transduction may be substantially as described in Ma et al, 2004, prostate 61:12-25; and Ma et al, prostate 74 (3): 286-296,2014 (the disclosure of which is incorporated herein by reference in its entirety). Retroviral vectors can be transfected into the Phoenix-Eco cell line (ATCC) using FuGene reagent (Promega, madison, wis.) to produce a eosinophil retrovirus, and then the transient virus supernatant (the nucleophile) can be used to transduce PG13 packaging cells with Gal-V envelope to produce a retrovirus, thereby infecting human cells. Viral supernatants from PG13 cells can be used to transduce activated T cells (or PBMC) two to three days after activation of CD3 or CD3/CD 28. Activated human T cells can be prepared by activating normal healthy donor Peripheral Blood Mononuclear Cells (PBMC) with 100ng/ml mouse anti-human CD3 antibody OKT3 (Orth Biotech, rartian, NJ) or anti-CD 3, anti-CD 28 TransAct (Miltenyi Biotech, germany) according to manufacturer's manual and AIM-V growth medium (Wolmer Ji Boke, inc. of Simmer Fei technology (GIBCO-Thermo Fisher scientific, waltham, mass)) containing 300-1000U/ml IL2 supplemented with 5% FBS for two days. About 5X 10 ⁶ The individual activated human T cells can be transduced in 10ug/ml fibronectin (Takara Bio Inc. (Takara Bio USA)) and precoated with 3ml viral supernatantThe 6-well plate was centrifuged at 1000g for about 1 hour at about 32 ℃. After transduction, transduced T cells can be expanded in AIM-V growth medium supplemented with 5% FBS and 300-1000U/ml IL 2.

The host cell may be a prokaryote, such as e.coli (e.coli), or it may be a eukaryote, such as a single-cell eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cells), a mammalian cell (e.g., human, monkey, hamster, rat, mouse, or insect cells), or a hybridoma cell. In one embodiment, the host cell may be introduced with an expression vector operably linked to nucleic acid encoding the desired antibody, thereby producing a transfected/transformed host cell that is cultured under conditions suitable for expression of the antibody by the transfected/transformed host cell, and optionally recovering the antibody from the transfected/transformed host cell (e.g., from a host cell lysate) or from the culture medium. In one embodiment, the host cell comprises a non-human cell comprising CHO, BHK, NS0, SP2/0 and YB2/0. In one embodiment, the host cell comprises a human cell comprising HEK293, HT-1080, huh-7 and PER.C6. Examples of host cells include COS-7 lines 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 grown in serum-free medium and related Cell lines (see Ramussen et al, 1998, cytotechnologic 28:31) or CHO strain DX-B11 lacking DHFR (see Urlaub et al, 1980, proc. Natl. Acad. Sci. USA 77:4216-20), heLa cells, BHK (ATCC CRL 10) Cell lines, CV1/EBNA Cell lines derived from the African green monkey kidney Cell line CV1 (ATCC CCL 70) (see McMahan et al, 1991, J. European molecular biology (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, cell strains derived from primary tissue cultured in vitro, primary explants, HL-60, U937, haK or Jurkat cells. In one embodiment, the host cell comprises a lymphocyte, such as Y0, NS0, or Sp20. In one embodiment, the host cell is a mammalian host cell, but not a human host cell. Typically, a host cell is a cultured cell that can be transformed or transfected with a polypeptide-encoding nucleic acid, which can then be expressed in the host cell. The phrase "transgenic host cell" or "recombinant host cell" may be used to refer to a host cell that has been introduced (e.g., transduced, transformed or transfected) with an exogenous nucleic acid that is or is not to be expressed. The host cell may also be a cell that includes a nucleic acid but does not express the nucleic acid at a desired level until a regulatory sequence is introduced into the host cell such that the regulatory sequence is operably linked to the nucleic acid. It is to be understood that the term host cell refers not only to a particular subject cell, but also to the progeny or potential progeny of such a cell. Because certain modifications may exist in succeeding passages, e.g., due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. Described herein are host cells or populations of host cells that carry a vector (e.g., an expression vector) operably linked to at least one nucleic acid encoding one or more polypeptides comprising a DAR or antigen-binding portion thereof.

The host cell or population of host cells includes T lymphocytes (e.g., T cells, regulatory T cells, gamma delta T cells, and/or cytotoxic T cells), NK (natural killer) cells, macrophages, dendritic cells, mast cells, eosinophils, B lymphocytes, monocytes. In some embodiments, the host cell is a T cell, and the cell expressing GD2 DAR may be referred to as a GD2 DAR-T cell. In some embodiments, the host cell is an NK cell, and the cell expressing GD2 DAR may be referred to as a GD2 DAR-NK cell. In some embodiments, the NK cells comprise umbilical cord blood-derived NK cells and/or placenta-derived NK cells.

The polypeptides of the disclosure (e.g., dimeric Antigen Receptor (DAR)) may be produced using any method known in the art. In one example, the polypeptide is produced by recombinant nucleic acid methods by inserting a nucleic acid sequence (e.g., DNA) encoding the polypeptide into a recombinant expression vector that is introduced into a host cell and expressed by the host cell under conditions that allow expression.

General techniques for recombinant nucleic acid manipulation are described, for example, in Sambrook et al, molecular cloning: in the handbook, volumes 1-3, cold spring harbor laboratory Press, 2 nd edition, 1989 or F.Ausubel et al, guidelines for molecular biology experiments (Green publication and Wili's intersection science press (Green Publishing and Wiley-Interscience): new York, 1987), which are incorporated herein by reference in their entirety, and updated periodically. The nucleic acid (e.g., DNA) encoding the polypeptide is operably linked to an expression vector carrying one or more suitable transcriptional or translational regulatory elements derived from mammalian, viral, or insect genes. Such regulatory elements include transcriptional promoters, optional operator sequences for controlling transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. Expression vectors may include an origin of replication that confers replication in a host cell. Expression vectors can include genes that confer selection to facilitate recognition of a transgenic host cell (e.g., a transformant).

The recombinant DNA may also encode any type of protein tag sequence that can be used to purify a 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: laboratory Manual (Cloning Vectors: ALaboratory Manual) (New York Esculer (Elsevier, N.Y.), 1985).

The expression vector construct may be introduced into a host cell using methods suitable for the host cell. Various methods for introducing nucleic acids into host cells are known in the art, including but not limited to electroporation; transfection is carried out by adopting calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; virus transfection; non-viral transfection; bombarding the micro-shell; lipofection; and infection (e.g., when the vector is an infectious agent). Suitable host cells include prokaryotes, yeast, mammalian cells, or bacterial cells.

Suitable bacteria include gram negative or gram positive organisms, for example, E.coli or Bacillus spp yeasts, for example, yeasts from Saccharomyces species (Saccharomyces species), such as Saccharomyces cerevisiae (S.cerevisiae), may also be used to produce the polypeptides. Various mammalian or insect cell culture systems can also be used to express recombinant proteins. Baculovirus systems for the production of foreign proteins in insect cells are described by Luckow and Summers (Bio/Technology, 6:47, 1988). 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, 293T, and BHK cell lines. The purified polypeptide is prepared by culturing a suitable host/vector system to express the recombinant protein. The protein is then purified from the culture medium or cell extract. Any polypeptide chain including a Dimeric Antigen Receptor (DAR) or antigen binding portion thereof may be expressed by the transgenic host cell.

Antibodies and antigen binding proteins disclosed herein can also be produced using a cellular translation system. For such purposes, the nucleic acid encoding the polypeptide must be modified to allow in vitro transcription to produce mRNA and to allow cell-free translation of the mRNA in the particular cell-free system utilized (a translation system that does not contain eukaryotic cells such as mammalian cells or yeast cells or a translation system that does not contain prokaryotic cells such as bacterial cells).

Nucleic acids encoding any of the various polypeptides disclosed herein can be chemically synthesized. Codon usage can be selected to enhance expression in a cell. 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 expression and purification (Protein expr. Purif.) (2002 (1): 96-105); connell N D. (Curr. Opin. Biotechnol.) 2001 12 (5): 446-9; makrides et al, microbiology review (Microbiol. Rev.) 1996 60 (3): 512-38; and Sharp et al, (Yeast.) (1991) 7 (7): 657-78).

Antibodies and antigen binding proteins described herein may also be produced by chemical synthesis (e.g., by methods described in pierce chemistry (The Pierce Chemical co., rockford, ill.) of rocford, il.) (solid phase peptide synthesis (Solid Phase Peptide Synthesis), 2 nd edition, 1984). Modification of proteins may also be produced by chemical synthesis.

Antibodies and antigen binding proteins described herein may be purified by methods of protein isolation/purification generally known in the art of protein chemistry. Non-limiting examples include extraction, recrystallization, salting out (e.g., using 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 may be exchanged into a different buffer and/or concentrated by any of a variety of methods known in the art, including, but not limited to, filtration and dialysis.

Purified antibodies and antigen binding proteins described herein are at least 65% pure, at least 75% pure, at least 85% pure, at least 95% pure, or at least 98% pure. Regardless of the exact numerical value of purity, the polypeptide is sufficiently pure for use as a pharmaceutical product. Any Dimeric Antigen Receptor (DAR) described herein, or antigen binding portion thereof, may be expressed by the transgenic host cell and then purified to a purity of about 65% -98% or high level purity using any method known in the art.

In certain embodiments, the antibodies and antigen binding proteins described herein (e.g., DARs) can further comprise post-translational modifications. Exemplary post-translational protein modifications include phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, defucosylation, carbonylation, threonization, biotinylation, or addition of polypeptide side chains or hydrophobic groups. Thus, the modified polypeptides may contain non-amino acid elements such as lipids, polysaccharides or monosaccharides, as well as phosphates. In one embodiment, glycosylation can be sialylation that conjugates one or more sialic acid moieties with a polypeptide. Sialic acid moieties improve 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.

In one embodiment, the Dimeric Antigen Receptor (DAR) described herein may be modified to include soluble polypeptides that link antibodies and antigen binding proteins to non-proteinaceous polymers. In one embodiment, the non-proteinaceous polymer comprises polyethylene glycol ("PEG"), polypropylene glycol, or polyoxyalkylene in a manner such as described in U.S. Pat. nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The present disclosure provides therapeutic compositions comprising any DAR expressing cell described herein in admixture with a pharmaceutically acceptable excipient. Excipients encompass carriers, stabilizers, and excipients. Excipients of pharmaceutically acceptable excipients include, for example, inert diluents or fillers (e.g., sucrose and sorbitol), salts, buffers, stabilizers, preservatives, cryoprotectants, non-ionic detergents, antioxidants and isotonic agents.

Therapeutic compositions and methods for preparing the therapeutic compositions are well known in the art and are described, for example, in "leimington: pharmaceutical science and practice (Remington: the Science and Practice of Pharmacy), (20 th edition, a.r. gennaro ar. Edit, 2000, philadelphia Wilkins publishing company (Lippincott Williams & Wilkins, philiadelphia, pa.)) in Philadelphia. The therapeutic compositions may be formulated for parenteral administration, possibly and may for example contain excipients, sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymers, lactide/glycolide copolymers or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the antibodies (or antigen binding proteins thereof) described herein. Nanoparticle formulations (e.g., biodegradable nanoparticles, solid lipid nanoparticles, liposomes) can be used to control the biodistribution of antibodies (or antigen binding proteins thereof). Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The concentration of antibody (or antigen binding protein thereof) in the formulation varies depending on a variety of factors, including the dosage of drug to be administered and the route of administration.

Any GD2 DAR expressing cells described herein can be administered in the form of a solution of a pharmaceutically acceptable salt, such as a non-toxic acid addition salt. Examples of the 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 the like; inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, and the like. DAR expressing cells can be provided in the form of a composition comprising, for example, cell culture medium, PBS, HBSS, ringer's solution, or Tyrode's solution, and can include growth factors or other proteins that maintain the activity or stability of a cell population or allow the spread of DAR expressing cells.

As used herein, the term "subject" refers to human and non-human animals, including vertebrates, mammals, and non-mammals. In one embodiment, the subject can be a human, a non-human primate, a simian, a mouse (e.g., mouse and rat), a cow, a pig, an horse, a dog, a cat, a goat, a wolf, a frog, or a fish.

The term "administering" and grammatical variations refer to the physical introduction of an agent into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Exemplary routes of administration of the formulations disclosed herein that include GD2 DAR-expressing cells include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" means modes of administration other than enteral and topical administration (typically by injection) and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. In one embodiment, the formulation is administered by a parenteral route (e.g., orally). Other parenteral routes include topical, epidermal or mucosal routes of administration, e.g., intranasal, vaginal, rectal, sublingual or topical. Administration may also be performed, for example, one, multiple times, and/or over one or more extended periods of time. Any GD2 DAR expressing cells described herein can be administered to a subject using methods and delivery routes known in the art.

The terms "effective amount," "therapeutically effective amount," or "effective dose" or related terms are used interchangeably and refer to an amount of any DAR expressing cell described herein sufficient to affect a measurable improvement or prevention of a disease or disorder associated with tumor or cancer antigen expression when administered to a subject. The therapeutically effective amounts of the DAR-T or DAR-NK cells provided herein, when used alone or in combination, will vary depending on the relative activity of the antibody and combination (e.g., in inhibiting cell growth) and on the subject and disease condition being treated, the weight, age and sex of the subject, the severity of the subject's disease condition, the manner of administration, and the like, which can be readily determined by one of ordinary skill in the art.

In one embodiment, the therapeutically effective amount will depend on the subject being treated and on certain aspects of the condition being treated and can be determined by one of skill in the art using known techniques. Typically, DAR-expressing cells (e.g., GD2DAR-T cells or GD2 DAR-NK cells) are with or without lymphocyte deletionIs about 1x 10 ³ -1x 10 ¹² The amount of individual cells/kg is administered to a human subject. DAR-T cells may be administered daily (e.g., once, twice, three times, or four times per day) or less frequently (e.g., weekly, biweekly, tricyclically, monthly, or quarterly). In addition, as known in the art, it may be desirable to adjust age and weight, general health, sex, diet, time of administration, drug interactions, and severity of the disease.

In one embodiment, the therapeutically effective amount comprises about 10 administered to the subject ³ -10 ¹² Dosage of individual DAR-T cells/kg body weight. In one embodiment, the transgenic host cell carries one or more expression vectors expressing a polypeptide chain comprising any of the DARs described herein. A therapeutically effective amount may be determined by considering the subject to be subjected to the therapeutically effective amount and the disease/condition to be treated, which may be determined by one of skill in the art using known techniques. The therapeutically effective amount may take into account factors related to the subject, such as age, weight, general health, sex, diet, time of administration, drug interactions, and severity of the disease. A therapeutically effective amount may consider the purity of the transgenic host cell, which may be about 65% -98% or higher levels of purity. A therapeutically effective amount of the transgenic host cell may be administered to the subject at least once or twice, three times, 4 times, 5 times or more over a period of time. The time period may be daily, weekly, monthly or yearly. The therapeutically effective amount of transgenic cells administered to a subject may be the same each time or may be increased or decreased at each administration event. A therapeutically effective amount of the transgenic cells can be administered to the subject until the tumor size or number of cancer cells is reduced by 5% -90% or more compared to the tumor size or number of cancer cells prior to administration of the transgenic host cells.

The present disclosure provides methods for treating a subject suffering from a disease/disorder associated with expression or overexpression of one or more tumor-associated antigens. The disease includes cancers or tumor cells that express tumor-associated antigens, e.g., GD2 antigen. In one embodiment, the cancer or tumor comprises neuroblastoma, melanoma, small cell lung cancer, medulloblastoma, astrocytoma, osteosarcoma, and other soft tissue sarcomas.

GD2 dimer antigen receptor

The present disclosure provides Dimeric Antigen Receptors (DARs) comprising two polypeptides that together comprise a Fab fragment that binds to GD2, wherein the Fab fragment is linked to a single transmembrane region and an intracellular region as a component of the second polypeptide. DAR as provided herein includes an antibody heavy chain variable region and an antibody light chain variable region on separate polypeptide chains, wherein the heavy chain variable region and the light chain variable region form an antigen binding domain. The present disclosure provides Dimeric Antigen Receptors (DARs) having a first polypeptide chain and a second polypeptide chain that associate with each other to form an antigen binding domain that binds to a GD2 molecule (e.g., a target antigen). In one embodiment, GD2 is overexpressed on tumors, such as tumors of neuroectodermal origin, including human neuroblastomas and melanomas.

In some embodiments, the GD2 DAR comprises an optional hinge region located between the Fab fragment and the transmembrane region, wherein the hinge region is located N-terminal to the transmembrane region of the first polypeptide. In some embodiments, host cells expressing GD2 DAR provided herein may exhibit greater specificity in response to GD 2-positive target cells, e.g., in terms of clonal expansion, cytokine release, and/or cytotoxicity, as compared to host cells expressing scFv format CARs with the same anti-GD 2 antibody.

The present disclosure provides GD2 DAR comprising an antibody heavy chain variable region comprising a transmembrane domain on one polypeptide chain and an antibody light chain variable region comprising no transmembrane domain on a separate polypeptide chain, or a light chain variable region comprising a transmembrane domain on one polypeptide chain and a heavy chain variable region comprising no transmembrane domain on a separate polypeptide chain. The two polypeptide chains that make up the DAR can dimerize outside the cell, e.g., through their antibody constant regions, to form a protein complex. DAR has antibody-like properties because it specifically binds to a target antigen. The dimeric antigen receptor may be expressed by cells used for targeted cell therapy.

The present disclosure provides transgenic T cells engineered to express GD2 DAR constructs having an antigen-binding extracellular portion, an optional hinge portion, a transmembrane portion, and an intracellular portion having a costimulatory and/or intracellular signaling region. Extracellular moieties exhibit high affinity and avidity to bind to GD 2-expressing diseased cells, resulting in T cell activation and diseased cell killing while protecting normal cells from injury. The intracellular portion of GD2 DAR includes costimulatory and/or signaling regions that mediate T cell activation upon antigen binding, which may result in enhanced T cell expansion, formation of memory T cells, and/or reduced T cell depletion. Various configurations of GD2 DARs are described herein that differ in the type and number of intracellular co-stimulatory and signaling regions, providing flexibility in designing GD2 DARs to produce a strong and rapid effector response and/or to produce a more durable memory T cell population (e.g., GD2 DAR that includes an intracellular 4-1BB co-stimulatory region).

The present disclosure provides GD2 DAR having a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a heavy chain variable region of an antibody and the second polypeptide chain comprises a light chain variable region of an antibody, wherein when both the first polypeptide chain and the second polypeptide chain are expressed by a cell, the first polypeptide chain is linked to the second polypeptide chain at a region outside the genetically modified cell by one or more disulfide bonds. In some embodiments, the GD2 DAR comprises a first polypeptide chain comprising, in order, an antibody heavy chain variable domain region and a heavy chain CH1 region, optionally a hinge region, a transmembrane region, and an intracellular region having 2-5 signaling domains, and a second polypeptide chain comprising an antibody light chain variable domain region having a corresponding light chain constant region (CL), which may be a kappa or lambda light chain constant region, wherein the CH1 region and CL region in each of the first and second polypeptide chains may be linked to one or two disulfide bonds (see, e.g., fig. 1A and 1B).

The present disclosure also provides GD2 DAR having a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a light chain variable region of an antibody and the second polypeptide chain comprises a heavy chain variable region of an antibody, wherein when both the first polypeptide chain and the second polypeptide chain are expressed by the same cell, the first polypeptide chain is linked to the second polypeptide chain at a region outside the transduced cell by one or more disulfide bonds. In some embodiments, a GD2 DAR construct comprises a first polypeptide chain comprising, in order, an antibody light chain variable domain region followed by a hinge region, a transmembrane region, and an intracellular region having 2-5 signaling domains with a corresponding light chain Constant (CL) region, which may be a kappa or lambda light chain constant region, and a second polypeptide chain comprising an antibody heavy chain variable domain region and a CH1 region, wherein the CL and CH1 regions in the first and second polypeptide chains may be linked by one or two disulfide bonds (see, e.g., fig. 1C and 1D). In some embodiments, a GD2 DAR construct consists essentially of a first polypeptide chain comprising, in order, an antibody light chain variable domain region having a corresponding light chain Constant (CL) region, which may be a kappa or lambda light chain constant region, followed by a hinge region, a transmembrane region, and an intracellular region having 2-5 signaling domains, and a second polypeptide chain comprising an antibody heavy chain variable domain region and a CH1 region, wherein the CL and CH1 regions in the first and second polypeptide chains may be linked by one or two disulfide bonds.

The first polypeptide chain of GD2 DAR may include, for example, an antibody heavy chain variable region comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of seq id no: a mouse 14.18 (14.18) heavy chain variable region according to SEQ ID NO. 2; a humanised 14.18 (hu 14.18) heavy chain variable region according to SEQ ID NO. 3; chimeric 3F8 (ch 3F 8) heavy chain variable region; or a humanized 3F8 (hu 3F 8) heavy chain variable region. The antibody heavy chain constant region includes sequences derived from a human antibody heavy chain constant region, e.g., a human CH1 domain (e.g., SEQ ID NO: 4). In various embodiments, the antibody heavy chain constant region may be derived from IgM, igA, igG, igE or IgD antibodies. The second polypeptide chain of GD2 DAR may include, for example, an antibody light chain variable region comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of seq id no: a mouse 14.18 (14.18) light chain variable region according to SEQ ID No. 5; a humanised 14.18 (hu 14.18) light chain variable region according to SEQ ID NO. 6; chimeric 3F8 (ch 3F 8) light chain variable region; or a humanized 3F8 (hu 3F 8) light chain variable region. The antibody light chain constant region may include sequences derived from a human antibody light chain constant region, e.g., a human CL domain, which may be a kappa or lambda CL domain (e.g., SEQ ID NO:7 or SEQ ID NO: 8).

Alternatively, the first polypeptide chain of GD2 DAR may comprise, for example, an antibody light chain variable region comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of seq id no: a mouse 14.18 (14.18) light chain variable region according to SEQ ID No. 5; a humanised 14.18 (hu 14.18) light chain variable region according to SEQ ID NO. 6; chimeric 3F8 (ch 3F 8) light chain variable region; or a humanized 3F8 (hu 3F 8) light chain variable region. The antibody light chain constant region may include sequences derived from a human antibody light chain constant region, e.g., a human CL domain, which may be a kappa or lambda CL domain (e.g., SEQ ID NO:7 or SEQ ID NO: 8). The second polypeptide chain of GD2 DAR may include, for example, an antibody heavy chain variable region comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of seq id no: a mouse 14.18 (14.18) heavy chain variable region according to SEQ ID NO. 2; a humanised 14.18 (hu 14.18) heavy chain variable region according to SEQ ID NO. 3; chimeric 3F8 (ch 3F 8) heavy chain variable region; or a humanized 3F8 (hu 3F 8) heavy chain variable region. The antibody heavy chain constant region includes sequences derived from a human antibody heavy chain constant region, e.g., a human CH1 domain (e.g., SEQ ID NO: 4). In one embodiment, the antibody heavy chain constant region may be derived from IgM, igA, igG, igE or IgD antibodies.

GD2 DAR as provided herein may or may not have a hinge region. In one embodiment, the GD2 DAR comprises a hinge region, wherein the hinge region is about 10 to about 120 amino acids in length. In some non-limiting examples, the hinge region can be a CD28 hinge region or fragment thereof (e.g., SEQ ID NO: 9), a CD8 alpha hinge region or fragment thereof (SEQ ID NO: 10), a hinge region that binds to the hinge regions of CD28 and CD8 (SEQ ID NO: 11), or a hinge region of an antibody (IgG, igAIgM, igE or IgD) that links constant domains CH1 and CH2 of an antibody, or a hinge region derived from any of these having at least 95%, 96%, 97%, 98%, 99% of any of them. The hinge region derived from an antibody may or may not include one or more constant regions of the antibody or the amino acid sequence thereof.

In various embodiments, the transmembrane domain may be derived from a region of a membrane protein sequence selected from the group consisting of: CD8 a (e.g., SEQ ID NO: 13), CD8 beta, 4-1BB/CD137, CD28 (e.g., SEQ ID NO: 12), CD34, CD4, fepsilon RIgamma, CD16, OX40/CD134, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, TCR alpha, TCR beta, TCR zeta, CD32, CD64, CD45, CD5, CD9, CD22, CD33, CD37, CD64, CD80, CD86, CD137, CD154, LFA-1T cell co-receptor, CD 2T cell co-receptor/adhesion molecule, CD40, CD4OL/CD154, VEGFR2, FAS and FGFR2B.

In one embodiment, the signaling region may be selected from the group consisting of signaling regions from: CD 3-zeta chain, 4-1BB (e.g., SEQ ID NO: 16), CD28 (e.g., SEQ ID NO: 17), CD27, OX40 (e.g., SEQ ID NO: 18), CD30, CD40, PD-1, ICOS, lymphooocyte function-related antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B-H3, GITR (TNFRSF 18), DR3 (TNFRSF 25), TNFR2, CD226, and combinations thereof. For example, the signaling region of a DAR first polypeptide may have two or three signaling regions derived from any of these signaling regions. In some embodiments, GD2 DAR as provided herein has a signaling region comprising ITAM1, ITAM2, and/or ITAM3 signaling regions derived from CD3 zeta with a CD3 zeta signaling region (SEQ ID NO: 19), and at least one additional signaling region (or costimulatory signaling domain), which may be, as non-limiting examples, a 4-1BB (SEQ ID NO: 16), CD28 (SEQ ID NO: 17), or OX40 (SEQ ID NO: 18) costimulatory domain. In some embodiments, the intracellular region includes a CD28 costimulatory region (e.g., SEQ ID NO: 17) and a CD 3-zeta intracellular signaling sequence (e.g., SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO: 22) or a 4-1BB costimulatory sequence and a CD 3-zeta intracellular signaling sequence. In some embodiments, the CD3- ζ portion of the intracellular signaling region includes ITAM (immune receptor tyrosine based activation motif) motifs 1, 2, and 3 (e.g., long CD3- ζ). In some embodiments, the CD3- ζ portion of the intracellular signaling region includes only one of the ITAM motifs, such as ITAM1, 2, or 3 only (e.g., short CD3- ζ).

In one embodiment, the hinge region comprises a CD28 hinge comprising the amino acid sequence of SEQ ID NO. 9, or a CD8 hinge comprising the amino acid sequence of SEQ ID NO. 10, or a hinge region (e.g., a long hinge) comprising the CD28 and CD8 hinge sequences of SEQ ID NO. 11. In one embodiment, the first polypeptide lacks a hinge region. In one embodiment, the transmembrane region comprises the amino acid sequence: SEQ ID NO. 12 (from CD 28); SEQ ID NO. 13 (from CD 8); SEQ ID NO. 14 (from 4-1 BB); or SEQ ID NO. 15 (from CD3 ζ). In one embodiment, the intracellular region comprises an amino acid sequence from any one or any combination of two or more intracellular sequences selected from the group consisting of: SEQ ID NO. 16 (from 4-1 BB); SEQ ID NO. 17 (from CD 28); SEQ ID NO. 18 (from OX 40); SEQ ID NO. 19 (CD 3 ζITAM 1, 2 and 3); SEQ ID NO. 20 (CD 3 ζITAM 1); SEQ ID NO. 21 (CD 3 ζITAM 2); and/or SEQ ID NO. 22 (CD 3 zeta ITAM 3). In one embodiment, the first polypeptide chain comprises a leader sequence comprising the amino acid sequence of SEQ ID NO. 23, 24 or 25, or the first polypeptide lacks a leader sequence.

Also provided are nucleic acid molecules encoding GD2 DARs described herein, including a precursor polypeptide of one or both of the first DAR polypeptide and the second DAR polypeptide (e.g., fig. 2A-D). In various embodiments, the GD2 DAR coding sequence may be operably linked to a promoter in an expression cassette, such as a promoter that functions in a eukaryotic cell.

Further included are host cells genetically engineered to include a nucleic acid sequence encoding a GD2DAR provided herein. The cells in various embodiments may be knocked out for T cell receptor expression and may be, as non-limiting examples, T cells or NK cells, such as human T cells or NK cells. The genetically engineered cell may be a primary cell. A population of cells, such as T cells or NK cells, that have been genetically engineered to express GD2DAR as described herein may be a population in which at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% of the cells in the population express GD2 DAR. A population of cells, such as T cells or NK cells, that have been genetically engineered to express GD2DAR as described herein may be a population in which at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% of the cells in the population express GD2DAR and do not express a T cell receptor, e.g., do not express an endogenous T cell receptor.

Host cells provided herein that express GD2DAR can be provided as pharmaceutical compositions in buffers, saline solutions, or cell culture media formulations including, for example, PBS, HBSS, ringer's solution, or a table's solution. The compositions comprising host cells expressing GD2DAR provided herein may further comprise a cryoprotectant, such as DMSO, glycerol, or a sugar alcohol, and the compositions may be provided as frozen compositions. Compositions comprising host cells expressing GD2DAR provided herein may optionally include proteins, peptides, sugars, lipids, polymers, antioxidants, enzymes, small molecules, or other compounds that may contribute to the stability, viability, or function of the cells and/or may include one or more compounds that may provide therapeutic benefits, including, but not limited to, antibodies (including engineered polypeptides with antibody domains), cytokines, growth factors, or small molecules.

Host cells expressing GD2 DAR as provided herein can be used in a method of treating a subject having cancer, e.g., neuroblastoma, melanoma, small cell lung cancer, medulloblastoma, astrocytoma, or osteosarcoma. In some embodiments, T cells that may lack expression of endogenous T cell receptors may be allogeneic with respect to the subject being treated. The method may comprise administering to a subject an effective amount of a population of host cells comprising at least one nucleic acid sequence encoding GD2 DAR as provided herein. Administration may be by any practical route. In some examples, the administration is intravenous administration. In some embodiments, administration may include injection, such as intratumoral or peri-cancerous injection. In some embodiments, one or more doses of the GD2 DAR T cell population may be administered after the onset of cancer or detection of cancer, and optionally for the length of time required to treat the disease.

Examples

The following examples are intended to be illustrative and may be used to further understand embodiments of the present disclosure. The described embodiments should not be construed as limiting the scope of the present teachings in any way.

Example 1: GD2 is expressed in tumor cell lines.

GD2 on the following cell lines was detected by flow cytometry using anti-GD 2 antibodies conjugated to Allophycocyanin (APC): NCI-H524 (gd2+) (small cell lung cancer); SK-MEL-5 (gd2+) (melanoma); k562 (GD 2-) (erythroleukemia (CML)); and H460 (gd2+) (non-small cell lung cancer). In a control experiment, cells were incubated with IgG conjugated to APC. FIG. 3 shows that H524 and SK-MEL-5 have detectable GD2 expression, while K562 and H460 have low or no detectable GD2 on the cell surface.

Example 2: isolation of human PBMC cells and primary T cells.

Primary human T cells were isolated from healthy human donors from buffy coat (san diego blood bank (San Diego blood bank)), fresh blood or white blood cell isolation products (stem cell technology company of vancomer, canada (StemCell Technologies, vancouver, CA)). Peripheral Blood Mononuclear Cells (PBMCs) were isolated by density gradient centrifugation. In some experiments, PBMCs were transfected with GD2 DAR and CAR constructs instead of isolated T cells.

T cells were isolated from PBMCs by magnetic negative selection using EASYSEP human T cell isolation kit (stem cell technologies) or positive selection and activation by DYNABEADS human T amplicon CD3/CD28 (walthamer feichi technologies, thermo Fisher Scientific, waltham, MA, USA) according to manufacturer's instructions.

In some transfection, T cells were expanded from PBMCs by implantation of PBMCs in coated cell culture flasks for one to two hours after single cell removal according to manufacturer's instructions, after which non-adherent lymphocytes were washed from the flasks and then activated with T cells tranact (maytansino biotechnology company (Miltenyi Biotec, bergisch Gladbach, germany) of Bei Jishi gladbach, germany) in new flasks.

Isolated T cells or T cell populations expanded from PBMCs from which monocytes are removed are freshly isolated or thawed from frozen stock for transfection with CAR or DAR constructs. CTS OPTMIZER T cell expansion SFM supplemented with 5% CTS immune cell SR (Semer Feishmanikin Co.) and 300U/mL IL-2 (Proleukin) at 10 ⁶ Cells were cultured at a density of individual cells/mL. Cells were treated with 3uL/10 ⁶ Individual cells/mL T cell tranact (containing CD3 and CD28 agonists, meitian and biotech) were activated for two to three days, followed by transfection with nucleic acid molecules encoding the precursor anti-GD 2CAR or the precursor anti-GD 2 DAR.

Example 3: t cell receptor knockdown/DAR-T cells were prepared.

Transfection of activated T cells with nucleic acids encoding GD2 DAR or GD2CAR (approximately 9X 10 ⁶ (9e6) Individual cells) or PBMCs (in some cases). CRISPR techniques are used to insert CARs or DAR constructs directly into the TRAC (T cell receptor alpha constant) gene (also known as TRA gene, NCBI gene ID: 6955) such that the T cell receptor is knocked out in cells where the engineered GD2 receptor is stably integrated into the genome and expressed on the cell surface.

The two GD2 DAR constructs introduced into primary T cells or PBMCs differ only in the sequence of the antibody domains of the first and second polypeptide chains of the DAR. GD2-14.18DAR includes the heavy and light chain variable regions of anti-GD 2 antibody 14.18 (Mujoo et al (1989) cancer research 49:2857-2861). The GD2-14.18DAR first polypeptide included an anti-GD 2 monoclonal antibody 14.18 heavy chain variable region (SEQ ID NO: 2) linked to a human heavy chain constant region (SEQ ID NO: 4), and the GD2-14.18DAR second polypeptide included an anti-GD 2 monoclonal antibody 14.18 light chain variable region (SEQ ID NO: 5) linked to a human kappa light chain constant region (SEQ ID NO: 7). GD2-hu14.18DAR includes the heavy and light chain variable regions of humanized anti-GD 2 antibody hu14.18 (U.S. patent No. 7,169,904). The GD2-hu14.18DAR first polypeptide included an anti-GD 2 humanized antibody hu14.18 heavy chain variable region (SEQ ID NO: 3) linked to a human heavy chain constant region (SEQ ID NO: 4), and the GD2-hu14.18DAR second polypeptide included an anti-GD 2 humanized antibody hu14.18 light chain variable region (SEQ ID NO: 6) linked to a human kappa light chain constant region (SEQ ID NO: 7).

Both GD2 DAR have the same configuration (fig. 1A) and the same hinge, transmembrane and intracellular regions.

GD2-14.18DAR first polypeptide included, from N-terminus to C-terminus, the 14.18 antibody heavy chain variable region of SEQ ID NO. 2 followed by the heavy chain CH1 region (SEQ ID NO. 4), the hinge region of CD28 (SEQ ID NO. 9), the transmembrane region of CD28 (SEQ ID NO. 12), the co-stimulatory intracellular domain of 4-1BB (SEQ ID NO. 16), and the internal signaling domain of CD3 zeta including ITAMs 1, 2, and 3 (SEQ ID NO. 19). The second polypeptide of GD2-14.18DAR includes, from N-terminus to C-terminus, the 14.18 antibody light chain variable region of SEQ ID NO. 5 followed by the light chain CL kappa region (SEQ ID NO: 7). The nucleic acid construct encoding the first polypeptide and the second polypeptide comprises sequences encoding signal peptides at the N-terminus of the first polypeptide and the second polypeptide for synthesis of the precursor polypeptides for integration into the membrane of the host cell and secretion from the host cell, respectively: the sequence encoding SEQ ID NO. 23 precedes the sequence encoding the VH domain of the first polypeptide and the sequence encoding SEQ ID NO. 24 precedes the sequence encoding the VL domain of the second polypeptide. The two precursor polypeptides comprising the signal peptide are encoded by a single transcriptional unit in which a first polypeptide-encoding sequence and a second polypeptide-encoding sequence are linked by a sequence encoding a T2A amino acid sequence (SEQ ID NO: 26) such that the two polypeptides encoded by a single transcript will be translated and processed into two mature polypeptides (SEQ ID NO:32 and SEQ ID NO: 33) that will assemble outside the cell into DAR through disulfide bonds through their antibody constant regions. The nucleic acid sequence encoding the two precursor polypeptides linked by the T2A sequence (SEQ ID NO: 30) is provided as SEQ ID NO:31. The nucleic acid sequence of SEQ ID NO. 31 is operably linked to a JeT promoter (SEQ ID NO. 39) to provide an expression cassette for transfection into T cells.

The second GD2 DAR construct encoding GD2-hu14.18 DAR comprises the heavy and light chain variable regions of anti-GD 2 antibody hu 14.18. The GD2-hu14.18 DAR first polypeptide included, from N-terminus to C-terminus, the hu14.18 heavy chain variable region of SEQ ID NO. 3 followed by the heavy chain CH1 region (SEQ ID NO. 4), the hinge region of CD28 (SEQ ID NO. 9), the transmembrane region of CD28 (SEQ ID NO. 12), the co-stimulatory intracellular domain of 4-1BB (SEQ ID NO. 16), and the internal signaling domain of CD3 zeta including ITAMs 1, 2, and 3 (SEQ ID NO. 19). The second polypeptide of GD2-hu14.18 DAR includes, from N-terminus to C-terminus, the hu14.18 light chain variable region of SEQ ID NO. 6 followed by the light chain CL kappa region (SEQ ID NO: 7). The nucleic acid construct encoding the first polypeptide and the second polypeptide comprises sequences encoding signal peptides at the N-terminus of the first polypeptide and the second polypeptide for synthesis of the precursor polypeptides for integration into the membrane of the host cell and secretion from the host cell, respectively: the sequence encoding SEQ ID NO. 23 precedes the sequence encoding the VH domain of the first polypeptide and SEQ ID NO. 24 precedes the sequence encoding the VH domain of the second polypeptide. The two precursor polypeptides comprising the signal peptide are encoded by a single transcriptional unit in which a first polypeptide-encoding sequence and a second polypeptide-encoding sequence are linked by a sequence encoding a T2A amino acid (SEQ ID NO: 26) such that the two polypeptides encoded by a single transcript will be translated and processed into two mature polypeptides (SEQ ID NO:36 and SEQ ID NO: 37) that will assemble outside the cell into DAR through disulfide bonds through their antibody constant regions. The nucleic acid sequence encoding the two polypeptides of GD2 hu14.18 DAR linked by a T2A sequence is provided as SEQ ID NO. 35. The nucleic acid sequence of SEQ ID NO. 35 is operably linked to a JeT promoter (SEQ ID NO. 39) to provide an expression cassette for transfection into T cells.

A GD2 CAR construct was also produced in which scFv based on anti-GD 2.18 antibody heavy and light chain sequences joined by a GS linker (SEQ ID NO: 38) to SEQ ID NO:2 and SEQ ID NO:5 was linked to the CD28 hinge region (SEQ ID NO: 9), CD28 transmembrane region (SEQ ID NO: 12) and CD28 co-stimulatory domain (SEQ ID NO: 17) and CD3 zeta intracellular signaling domain (SEQ ID NO: 19). Another construct included the hu14.18 antibody heavy and light chain sequences of SEQ ID NO. 3 and SEQ ID NO. 6 joined by the same GS linker (SEQ ID NO. 38) and also linked to the CD28 hinge region (SEQ ID NO. 9), the CD28 transmembrane region (SEQ ID NO. 12), the CD28 co-stimulatory domain (SEQ ID NO. 17) and the CD3 zeta intracellular signaling domain (SEQ ID NO. 19). Constructs encoding these CARs were also ligated to the JeT promoter (SEQ ID NO: 39) in the expression cassette for transfection of primary T cells.

Cas9 RNA-guided endonucleases are used to generate GD2 CAR-T cells and GD2DAR-T cells that knock out the TRAC gene simultaneously. The GD2DAR and CAR constructs cloned downstream of the JeT promoter (SEQ ID NO: 39) described above were cloned into a vector between the 5 'and 3' homologous regions (SEQ ID NO:40 and SEQ ID NO:41, respectively) of the T cell receptor alpha constant (TRAC) gene (Entrez gene ID: 28755) located at the target site (SEQ ID NO: 42) flanking the AAV vector pAAV-MCS for Cas 9-mediated integration. Bacterial clones containing GD2DAR or CAR constructs operably linked to the JeT promoter and flanked by TRAC gene homology regions were confirmed by sequencing.

For CAR or DAR knock-in/TCR knock-out using Cas9, the target sequence comprising SEQ ID NO. 42 is first combinedCRISPR-Cas9 crRNA and +.>The RNP complex was prepared by CRISPR-Cas9 tracrRNA (both from algav Hua Zhouke ralvier IDT company) and heating the mixture at 95 ℃ for 5 minutes. The mixture was then cooled to room temperature (18-25 ℃) at the top of the bench for about 20 minutes to make crRNA: tracrRNA duplex. For each transfection, 10 μg of wild-type SpCas9 protein including a nuclear localization sequence (IDT) was mixed with 200pmol crRNA:tracrRNA duplex and the mixture was incubated at 4 ℃ for 30 min to form RNP.

Cas 9-mediated insertion of donor DNA for the GD2DAR construct was generated from pAAV plasmids including the GD2-14.18DAR construct of SEQ ID NO:31 or the GD2-hu14.18 DAR construct of SEQ ID NO:35 flanked by 5 'and 3' homologous sequences from the TRAC gene (SEQ ID NO:40 and SEQ ID NO:41, respectively). The donor fragment with the shorter homology arm of SEQ ID NO. 45 (171 bp) and SEQ ID NO. 46 (161 bp) was generated using a forward primer having the sequence: tmC mA mcggagcagctggtttct (SEQ ID NO: 43), the reverse primer having the sequence: GACCTCATGTCTAGCACAGTTTTG (SEQ ID NO: 44). The forward primer (SEQ ID NO: 43) includes a phosphorothioate bond (represented by an asterisk) between the first nucleotide and the second nucleotide, the third nucleotide and the fourth nucleotide, and the fourth nucleotide and the fifth nucleotide starting from the 5' end. Nucleotides at the third, fourth and fifth positions starting from the 5 'end of the forward oligonucleotide primer were modified with 2' -O-methyl groups (designated mC, mA and mC). The reverse primer (SEQ ID NO: 44) does not have these modifications but includes a 5' -terminal phosphate. PCR was performed essentially as described above to generate a double stranded donor DNA molecule with a GD2DAR expression cassette flanked by TRAC locus homology arms of 171bp and 161bp (SEQ ID NO:45 and SEQ ID NO: 46). The resulting double stranded GD2DAR donor DNA fragment is used as a double stranded molecule with Cas9 RNP in independent transfection of activated T cells.

Donor DNA comprising GD2 CAR construct was designed and synthesized in the same way as the donor comprising GD2DAR construct. The same primers (SEQ ID NO:43 and SEQ ID NO: 44) were used to generate double stranded donor fragments with homology arms of 171bp and 161 bp.

Cells transfected with GD2 CAR and GD2DAR donor DNA plus Cas9 RNP targeting exon 1 of the TRAC locus were essentially as described in US2020/0224160; this is done as described in WO 2020/176740 and WO 2020/185867, all of which are incorporated herein by reference in their entirety. After electroporation with Cas9 RNP and donor fragment, the cells were diluted into medium and at 37 ℃ with 5% CO ₂ The cells were incubated in an Optmizer MT cell expansion SFM supplemented with 5% serum replacement and 300U/ml IL2 at 37 ℃. Once the cells are in expansion culture, cell counts are obtained every 2 or 3 days and cell concentration is maintained at 5X 10 ⁵ (5e5) Up to 1X 10 ⁶ (1e6)/mL。

For use as control cells, isolated T cells were transfected with TRAC-targeted RNP in the absence of donor fragment. Such cells have an disrupted TRAC gene, but do not insert GD2DAR or CAR constructs, and are thus TCR knockout (TRAC KO) cells.

Figure 4 provides the results of flow cytometry after fourteen days of culture using a labeled antibody directed against CD3 for detection of T cell receptors and an anti-idiotype antibody 1A7 that mimics GD2 for detection of GD2DAR or CAR. Cells transfected with two versions of GD2 CAR had approximately 36% of the population expressing GD2 CAR (x-axis) in the absence of T cell receptor expression (y-axis). The proportion of GD2DAR expressing cells of the GD2 (14.18) DAR transfected cell population in the absence of T cell receptor expression was somewhat lower (about 30%). Almost all cells expressing GD2DAR did not express T cell receptor (i.e., were CD3 negative) (bottom panel).

Example 3: in vitro expansion of CAR-T and DAR-T cells.

Clonal expansion of GD2 CAR-T and DAR-T cells was tested on cells expressing GD2 (gd2+) and on cells with detectable expression of GD2 (GD 2-) as a control without GD2 (fig. 3). Cell populations transfected with GD2 CAR and DAR were co-cultured with NCI-H542 GD2+ cells, SK-MEL-5GD2+ cells, K562 GD 2-cells and H460GD 2-cells alone. After 7 days of co-culture (fig. 5), the level of cell expansion was measured using flow cytometry when the percentage of T cell populations expressing GD2 DAR or CAR in the absence of T cell receptor expression was evaluated substantially as described in example 2. Figure 5 shows that when compared to the percentage of a population expressing GD2 DAR or CAR that was cultured for the same amount of time in the absence of co-cultured cells (leftmost panel), the percentage of DAR-T cells increased with co-culture with gd2+ cells, whereas CAR-T cells did not, the fraction in the T cell population did not increase after 7 days co-culture with gd+ cells.

Example 4: in vitro cytotoxicity assay.

For in vitro cytotoxicity assays, lentiviral transduced cell lines NCI-H524 (small cell lung cancer), SK-MEL-5 (melanoma) and H460 (non-small cell lung cancer) carrying luciferase and GFP genes were used. Single cell clones with luciferase and GFP expression (NCI-H524-Fluc-GFP/puro and SK-MEL-5-Fluc-GFP/puro) were selected and amplified for assay use.

anti-GD 2 CAR-T or DAR-T cells were co-cultured with GD2+ NCI-H524-Fluc/GFP/puro or SK-MEL-5-Fluc-GFP/puro cells or GD2-H460-Fluc-GFP/puro cells. Using H524 cells as targets, the ratio of effector to target cells ranges from 0.3:1 to 3:1; and SK-MEL-5 cells and H460 cells were used as targets, the ratio ranging from 10:1 to 1.25:1. After overnight incubation, the cells were subjected to flow cytometry to measure GFP-expressing cell populations to determine specific target cell killing against GD2 CAR or DAR T cells.

Luciferase-based assays were performed in 96-well plates. Mu.l firefly luciferase-tagged NCI-H524 cells were treated with 5X 10 ⁵ Individual cells/ml were added and 100 μ l T cells were added to obtain E:T ratios of 3:1, 1:1 and 0.3:1. For tumor only control, 100 μl of medium was added, and for complete lysis control, 100 μl of medium containing 0.5% Triton X-100 was added. The plates were incubated 24 hours after T-cell addition, at which time 75. Mu.l of assay culture was transferred to wells of a black wall 96-well plate (Corning) and mixed with 75. Mu.l of ONE-Glo luciferase reagent (Promega). Five minutes after incubation, luminosity was measured using a microplate reader (BioTek) and cytotoxicity was calculated after normalization relative to tumor only control and complete lysis control.

Assays using SK-MEL-5 and H460 cells as targets were performed using an xcelligent real-time cell analyzer assay. In E-Plate View 96 (Eisen Biosciences), at 1X 10 per well ⁴ The individual cells were seeded with SK-MEL-5 and H460 cells and the plates were returned to the xCELLigence RTCA MP instrument (Essen Bioty). After overnight incubation, the medium was changed to 100 μl fresh medium and 100 μ l T cells were added to give E:T ratios of 10:1, 5:1, 2.5:1 and 1.25:1. For tumor only control, 100 μl of medium was added, and for complete lysis control, 100 μl of medium containing 0.5% Triton X-100 was added. The plate was returned to the xCELLigence RTCAMP instrument (elsen biosciences) for real-time impedance monitoring. Using RTCA softPart pro (elsen biosystems) calculated cytotoxicity.

Both GD2 DAR-T cells prepared by transfecting isolated T cells and those prepared by transfecting PBMCs killed H524 (gd2+) target cells, and also killed SK-MEL-5 (gd2+) target cells in a dose dependent manner (fig. 6A), while showing no cytotoxicity to GD2-H460 cells (fig. 6B). However, most CAR-T cell preparations showed cytotoxicity to GD2-H460 cells (fig. 6B).

Example 5: in vitro cytokine secretion assay.

GD2 CAR-T cells, GD2 DAR-T cells, and control T cells were nutrient starved overnight with IL-2, and then co-cultured with NCI-H524 (gd2+) cells or K562 (GD 2-) cells. Following incubation for 40 hours according to the manufacturer's instructions, the cells were centrifuged to collect the supernatant for detection of the cytokines IFN-gamma (ELISA MAX Delux kit, hundred-Ind (BioLegend)) or GM-CSF (human GM-CSF uncoated ELISA kit from England Ind (Invitrogen)/Siemens Feishmanic technologies).

Mu.l of NCI-H524 or K-562 cells were plated in a 96-well plate at 5X 10 ⁵ Each cell/ml was added, and for T cells, 100. Mu.l of medium was added. 100 μl of 1.5X10 were added ⁶ T cells per ml to give a 3:1 E:T ratio. The plates were returned to the incubator for incubation. After 48 hours of incubation, supernatants were collected and levels of IFN-. Gamma.and GM-SCF were measured by ELISA.

FIG. 7A shows the amount of IFN-gamma secreted by cells transfected with GD2 DAR and GD2 CAR constructs, either alone or in combination with NCI-H524 cells or K562 cells. Both T cells and PBMCs transfected with GD2 (14.18) DAR constructs expressed significant amounts of IFN- γ when cultured with NCI-H524 (gd2+) cells, but had negligible IFN- γ when cultured with K562 (GD 2-) cells. While one GD2 (hu 14.18) CAR-T cell population showed significant amounts of IFN- γ production on NCI-H524 (gd2+) cells, these CAR-T cells produced even greater amounts of IFN- γ when cultured with K562 (GD 2-) cells and also produced significant amounts of IFN- γ when cultured alone.

FIG. 7B shows the amount of GM-CSF secreted by cells transfected with GD2 DAR and GD2 CAR constructs, either alone or in combination with NCI-H524 cells or K562 cells. Both T cells and PBMCs transfected with GD2 (14.18) DAR constructs expressed IFN- γ when cultured with NCI-H524 (gd2+) cells, but produced undetectable amounts of GM-CSF when cultured with K562 (GD 2-) cells. The GD2 (hu 14.18) CAR-T cell population again showed significant amounts of GM-CSF production on NCI-H524 (GD2+) cells, but for IFN-gamma production these CAR-T cells produced even greater amounts of IFN-gamma when cultured with K562 (GD 2-) cells and also produced significant amounts of IFN-gamma when cultured alone.

Example 6: in vivo tumor killing in a mouse model.

In xenograft mouse models, SK-MEL-5 (gd2+) tumor cell lines (tumor-Fluc) expressing luciferase were used to test for tumoricidal activity against GD2 CAR or DAR T cells. Will total about 3 x 10 ⁶ Individual tumor-Fluc cells were suspended in 100 μl PBS and then subcutaneously injected to the right of each mouse.

After 15 days, 5X 10 ⁷ Personal (5X 10) ⁶ Individual GD2 DAR positive) anti-GD 2-hu14.18 DAR T cells or 5×10 ⁷ Personal (1.25X10) ⁷ Individual GD2 DAR positive) anti-GD 2-14.18DAR T cells were administered by tail vein in 200 μl PBS. The control group was given the same amount of TRAC knockout T cells intravenously, and the fourth group received only 200. Mu.L of PBS.

Tumor burden was monitored for each animal using bioluminescence from IVIS imaging, as shown in fig. 8. Tumor volumes and animal body weights over time are provided in fig. 9, which shows that GD2 DAR treated mice have little tumor establishment compared to the control group, wherein the tumors grew steadily during the experiment and did not experience the loss of body weight exhibited by the control group. Figure 10 shows that all mice in the GD2-hu14.18 DAR-T cell treated group survived to the end of the study (approximately 8 weeks) with higher survival than any other group. At the end of the study, the survival rate was 60% for the GD2-14.18DAR-T cell treated group, while PBS treatment and TRAC knockout treatment resulted in only 10% and 20% survival rates, respectively.

Sequence(s)

beta-1,4N-acetylgalactosamine transferase 1 isoform 1 precursor

-UniProtKB Q00973(B4GN1_HUMAN)SEQ ID NO:1

MWLGRRALCALVLLLACASLGLLYASTRDAPGLRLPLAPWAPPQSPRRPELPDLAPEPRYAHIPVRIKEQVVGLLAWNNCSCESSGGGLPLPFQKQVRAIDLTKAFDPAELRAASATREQEFQAFLSRSQSPADQLLIAPANSPLQYPLQGVEVQPLRSILVPGLSLQAASGQEVYQVNLTASLGTWDVAGEVTGVTLTGEGQADLTLVSPGLDQLNRQLQLVTYSSRSYQTNTADTVRFSTEGHEAAFTIRIRHPPNPRLYPPGSLPQGAQYNISALVTIATKTFLRYDRLRALITSIRRFYPTVTVVIADDSDKPERVSGPYVEHYLMPFGKGWFAGRNLAVSQVTTKYVLWVDDDFVFTARTRLERLVDVLERTPLDLVGGAVREISGFATTYRQLLSVEPGAPGLGNCLRQRRGFHHELVGFPGCVVTDGVVNFFLARTDKVREVGFDPRLSRVAHLEFFLDGLGSLRVGSCSDVVVDHASKLKLPWTSRDAGAETYARYRYPGSLDESQMAKHRLLFFKHRLQCMTSQ

GD2 resistance 14.18VH SEQ ID NO:2

EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATL TVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSS

anti-GD2hu14.18 VH SEQ ID NO:3

EVQLVQSGAEVEKPGASVKISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSTSTAYMHLKSLRSEDTAVYYCVSGMEYWGQGTSVTVSS

anti-GD 2CH1 SEQ ID NO:4

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT

anti-GD 214.18 VL SEQ ID NO:5

EIVMTQSPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELK

anti-GD2hu14.18VL SEQ ID NO:6

DVVMTQTPLSLPVTPGEPASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELK

Anti GD214.18CL (kappa) SEQ ID NO:7

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

anti-GD2ch14.18CL (lambda) SEQ ID NO. 8

GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

CD28 hinge SEQ ID NO:9

PRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

CD8 hinge SEQ ID NO:10

AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPR

CD8 hinge+CD 28 hinge (long hinge) SEQ ID NO:11

AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

CD28 transmembrane SEQ ID NO:12

FWVLVVVGGVLACYSLLVTVAFIIFWV

CD8 transmembrane SEQ ID NO:13

IYIWAPLAGTCGVLLLSLVITLY

4-1BB transmembrane SEQ ID NO:14

IISFFLALTSTALLFLLFFLTLRFSVV

CD3 zeta transmembrane SEQ ID NO:15

LCYLLDGILFIYGVILTALFL

4-1BB costimulatory sequence SEQ ID NO:16

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

CD28 costimulatory sequence SEQ ID NO:17

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

OX40 costimulatory sequence SEQ ID NO. 18

ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

CD3ζITAM 1、2、3 SEQ ID NO: 19

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD3ζITAM 1 SEQ ID NO:20

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKR

CD3ζ ITAM 2SEQ ID NO:21

RVKFSRSADRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM

CD3ζITAM 3 SEQ ID NO:22

RVKFSRSADKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Leader sequence SEQ ID NO:23

MEWSWVFLFFLSVTTGVHS

Light chain leader SEQ ID NO. 24

MSVPTQVLGLLLLWLTDARC

Alternative leader sequence SEQ ID NO:25

MALPVTALLLPLALLLHAARP

T2A self-cleavage sequence SEQ ID NO:26

GSGEGRGSLLTCGDVEENPGP

P2A self-assemblyCleavage sequence SEQ ID NO:27

GSGATNFSLLKQAGDVEENPGP

E2A self-cleaving sequence SEQ ID NO. 28

GSGQCTNYALLKLAGDVESNPGP

F2A self-cleavage sequence SEQ ID NO:29

GSGVKQTLNFDLLKLAGDVESNPGP

Precursor: GD2-14.18DAR (V2 a version): SEQ (SEQ) ID NO:30

MEWSWVFLFFLSVTTGVHSEVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGEGRGSLLTCGDVEENPGPMSVPTQVLGLLLLWLTDARCEIVMTQSPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Nucleic acid sequence, GD2-14.18DAR precursor, SEQ ID NO:31

ATGGAATGGAGCTGGGTGTTCCTCTTCTTCCTCAGCGTGACCACCGGCGTGCACTCCGAGGTGCAATTATTACAGTCCGGACCCGAGCTGGAGAAGCCCGGCGCCTCCGTGATGATCAGCTGTAAAGCCTCCGGCAGCTCCTTTACCGGCTACAACATGAACTGGGTGAGGCAGAACATTGGAAAGTCTTTAGAGTGGATCGGCGCCATCGATCCCTACTACGGCGGCACCTCCTATAACCAGAAGTTCAAGGGTCGTGCCACCCTCACCGTCGACAAGTCCTCCAGCACAGCTTATATGCATTTAAAGTCCCTCACCAGCGAGGACTCCGCCGTGTATTATTGTGTGAGCGGCATGGAATATTGGGGCCAAGGAACCAGCGTCACCGTGTCCAGCGCTTCCACAAAGGGACCCAGCGTCTTCCCTCTGGCCCCCAGCAGCAAAAGCACCAGCGGCGGAACCGCTGCTTTAGGATGTTTAGTGAAGGACTACTTTCCCGAACCCGTGACCGTGAGCTGGAATTCCGGCGCTCTCACCAGCGGCGTGCACACCTTTCCCGCTGTCCTCCAGTCCAGCGGCCTCTACTCTTTATCCTCCGTGGTGACAGTGCCTAGCTCCTCCCTCGGCACCCAGACCTACATTTGTAACGTGAACCACAAGCCTTCCAATACAAAGGTGGACAAGAGGGTGGAGCCCAAAAGCTGCGATAAGACCCACACTCCTCGTAAGATTGAGGTCATGTACCCCCCCCCCTATCTGGACAACGAGAAGAGCAACGGCACAATCATCCACGTGAAGGGCAAGCATCTGTGCCCCTCCCCTTTATTTCCCGGACCTTCCAAACCCTTTTGGGTTTTAGTGGTCGTGGGAGGCGTGCTGGCTTGTTACTCTTTACTGGTGACAGTCGCCTTCATCATCTTTTGGGTGAAGAGGGGTCGTAAGAAACTGCTGTACATCTTCAAACAGCCCTTCATGAGGCCCGTGCAGACAACCCAAGAAGAGGATGGATGTTCTTGTCGTTTCCCCGAAGAGGAGGAGGGAGGCTGTGAACTGAGGGTGAAGTTCTCTCGTAGCGCTGACGCCCCCGCCTACCAACAAGGTCAAAACCAGTTATACAACGAGCTGAATTTAGGAAGGAGAGAGGAGTATGACGTTTTAGACAAGAGAAGGGGAAGAGACCCCGAAATGGGCGGCAAGCCCAGAAGGAAGAACCCCCAAGAAGGTTTATACAATGAGCTCCAGAAGGACAAGATGGCCGAGGCCTACAGCGAAATTGGCATGAAAGGCGAGAGGAGGAGGGGAAAGGGCCATGACGGCCTCTATCAAGGTTTAAGCACCGCCACCAAGGACACCTACGACGCTTTACATATGCAAGCTTTACCTCCTAGGGGAAGCGGAGAAGGAAGGGGATCTTTACTGACTTGTGGCGACGTGGAGGAGAACCCCGGCCCTATGTCCGTGCCTACCCAAGTTCTGGGTTTACTGCTGCTCTGGCTGACAGACGCTCGTTGCGAGATCGTGATGACCCAATCCCCCGCTACACTCTCCGTGAGCCCCGGAGAGAGGGCTACTTTAAGCTGTCGTTCCTCCCAGAGCCTCGTCCACAGAAACGGCAATACCTATTTACACTGGTATTTACAGAAGCCCGGTCAGAGCCCCAAGTTATTAATTCACAAAGTCAGCAACAGATTCAGCGGCGTGCCCGATCGTTTCAGCGGCTCCGGCAGCGGAACCGACTTTACACTCAAGATCAGCAGAGTGGAGGCCGAGGATCTGGGCGTGTACTTCTGCTCCCAGTCCACCCATGTGCCCCCTCTCACATTCGGAGCCGGAACCAAGCTGGAGCTGAAAAGAACAGTCGCCGCCCCCTCCGTCTTCATTTTTCCCCCTTCCGATGAGCAGCTGAAGAGCGGCACAGCCAGCGTGGTCTGTCTGCTCAACAATTTTTACCCTCGTGAGGCCAAGGTCCAGTGGAAGGTGGATAATGCTCTGCAGTCCGGCAACTCCCAAGAATCCGTGACAGAGCAAGACTCCAAGGATTCCACCTATTCTTTATCCTCCACCCTCACTTTAAGCAAGGCTGACTACGAGAAGCATAAGGTGTACGCTTGTGAAGTGACACACCAAGGTTTATCCAGCCCCGTGACAAAGAGCTTCAATAGGGGAGAGTGC

Polypeptide 1, GD2-14.18DAR, SEQ ID NO:32

EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTPRKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Polypeptide No. 2, GD2-14.18DAR, SEQ ID NO:33

EIVMTQSPATLSVSPGERATLSCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRTVAAPSVFIFPPSD

EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Precursor: GD2-hu14.18 DAR: SEQ ID NO. 34

MEWSWVFLFFLSVTTGVHSEVQLVQSGAEVEKPGASVKISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSTSTAYMHLKSLRSEDTAVYYCVSGMEYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGEGRGSLLTCGDVEENPGPMSVPTQVLGLLLLWLTDARCDVVMTQTPLSLPVTPGEPASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Nucleic acid sequence, GD2-hu14.18DAR precursor, SEQ ID NO:35

atggaatggagctgggtctttctcttcttcctgtcagtaaccaccggtgtccactccGAAGTGCAACTCGTTCAAAGCGGAGCCGAGGTCGAAAAACCCGGAGCCTCAGTCAAGATTAGCTGTAAAGCTAGTGGCAGCAGCTTTACAGGGTACAACATGAACTGGGTCCGACAGAACATTGGGAAGTCACTCGAGTGGATTGGGGCTATTGACCCGTACTACGGTGGAACATCATACAATCAGAAATTTAAAGGACGAGCAACCCTCACGGTAGACAAAAGTACTTCCACGGCGTATATGCATCTCAAGAGTCTCAGAAGTGAAGACACAGCGGTCTATTATTGTGTATCTGGTATGGAGTATTGGGGCCAAGGTACGTCCGTCACTGTTTCATCTGCTAGCACAAAAGGCCCCTCCGTCTTTCCTTTAGCCCCTAGCTCCAAATCCACCTCCGGCGGAACAGCCGCCCTCGGATGTCTCGTCAAAGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAATTCCGGCGCTTTAACCTCCGGAGTGCACACCTTTCCCGCTGTTTTACAGTCCAGCGGACTGTATTCTTTAAGCTCCGTCGTCACCGTGCCTAGCAGCTCTTTAGGCACCCAGACCTACATTTGCAACGTCAACCACAAGCCTAGCAACACCAAGGTGGACAAAAGGGTGGAGCCTAAGTCTTGTGACAAGACCCACACCAAGATCGAGGTCATGTACCCCCCCCCCTATTTAGACAACGAGAAAAGCAACGGCACAATTATCCATGTGAAAGGCAAGCATCTCTGCCCCTCCCCTCTGTTTCCCGGACCCTCCAAGCCTTTTTGGGTGCTCGTGGTGGTGGGCGGCGTGCTGGCTTGTTATTCTTTACTGGTGACCGTCGCCTTTATCATCTTCTGGGTGAAGAGGGGTCGTAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGGCCCGTTCAAACCACCCAAGAAGAGGACGGCTGCAGCTGTCGTTTCCCCGAGGAGGAAGAGGGAGGATGCGAGCTGAGGGTGAAGTTCTCTCGTAGCGCCGATGCCCCCGCTTATCAGCAAGGTCAGAACCAGTTATACAACGAGCTGAATTTAGGTCGTAGGGAGGAGTACGACGTGCTGGACAAGAGAAGAGGTCGTGACCCCGAGATGGGCGGCAAACCTCGTAGGAAGAACCCCCAAGAAGGTTTATACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGAGGAGAAGGGGCAAGGGCCACGATGGTTTATACCAAGGTTTAAGCACCGCCACCAAGGACACCTACGATGCTTTACACATGCAAGCTTTACCTCCTCGTGGAAGCGGCGAAGGAAGGGGCTCTTTACTGACATGTGGCGACGTGGAGGAAAACCCCGGCCCTATGTCCGTGCCCACACAAGTTCTCGGACTGTTATTACTGTGGCTGACAGATGCTCGTTGCGATGTCGTTATGACGCAAACCCCGCTTTCCTTGCCGGTTACGCCTGGAGAACCAGCTTCCATCTCATGTCGGTCTTCACAATCTCTGGTTCATCGCAACGGAAACACGTATCTTCATTGGTATCTTCAGAAGCCAGGCCAATCCCCAAAGCTCCTCATTCACAAGGTCTCTAACAGATTCAGTGGGGTTCCTGACAGATTTTCCGGGTCTGGATCAGGTACTGACTTCACATTGAAAATTAGCCGCGTAGAAGCTGAGGACCTGGGCGTATACTTTTGTTCTCAATCCACCCATGTACCACCTCTCACTTTCGGCGCAGGAACGAAACTGGAACTGAAGAGAACCGTGGCCGCCCCTAGCGTTTTCATTTTCCCCCCCAGCGACGAACAGCTGAAGTCCGGCACAGCCTCCGTGGTGTGTTTACTGAATAACTTCTACCCTCGTGAGGCTAAGGTCCAATGGAAGGTGGATAATGCTTTACAGTCCGGAAATTCCCAAGAAAGCGTGACCGAGCAAGATAGCAAAGATAGCACCTACAGCTTAAGCTCCACACTGACACTGTCCAAAGCCGACTACGAGAAACACAAGGTGTACGCTTGTGAAGTGACCCATCAAGGTTTAAGCTCCCCCGTCACAAAGAGCTTTAACAGAGGAGAATGC

Polypeptide 1, GD2-hu14.18DAR, SEQ ID NO. 36

EVQLVQSGAEVEKPGASVKISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSTSTAYMHLKSLRSEDTAVYYCVSGMEYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Polypeptide No. 2, GD2-hu14.18DAR, SEQ ID NO. 37

DVVMTQTPLSLPVTPGEPASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

G4S linker SEQ ID NO:38

GGGGSGGGGSGGGGS

JeT promoterSEQ ID NO:39

GAATTCGGGCGGAGTTAGGGCGGAGCCAATCAGCGTGCGCCGTTCCGAAAGTTGCCTTTTATGGCTGGGCGGAGAATGGGCGGTGAACGCCGATGATTATATAAGGACGCGCCGGGTGTGGCACAGCTAGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTCTTGTTTGTGGATCCCTGTGATCGTCAGTTGACA

SEQ ID NO:40

DNA

Intellectual figure

5' homology arm from exon 1 of TRAC gene, cas9 target site, 660nt

GGCACCATATTCATTTTGCAGGTGAAATTCCTGAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTAAACGGTAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTTCAAAACCTCTATCAATGAGAGAGCAATCTCCTGGTAATGTGATAGATTTCCCAACTTAATGCCAACAT

ACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGA

TGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGC

TGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATT

TCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTG

GCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAG

ATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTC

CATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAAC

CCTGATCCTCTTGTCCCACA

SEQ ID NO:41

DNA

Intellectual figure

3' homology arm of exon 1 from TRAC gene, cas9 target site, 650nt

GATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTG

TCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTA

TATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCC

TGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAGGACA

CCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGG

AATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTC

GGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAGT

CCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAG

CCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTTGCCCCTTACTGC

TCTTCTAGGCCTCATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCA

AAAAATCTTT

SEQ ID NO:42

DNA

Intellectual figure

Cas9 target site, TRAC locus

CAGGGTTCTGGATATCTGT

SEQ ID NO:43

DNA

Manual work

Forward primer

A*TmC*mA*mCGAGCAGCTGGTTTCT

( * Represents a phosphorothioate bond; mA represents 2' -O-methyladenosine; mC represents 2' -O-methylcytidine )

SEQ ID NO:44

DNA

Manual work

Reverse primer

GACCTCATGTCTAGCACAGTTTTG

SEQ ID NO:45

DNA

Intellectual figure

171bp 5' homologous region, cas9 target site

ATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACA

SEQ ID NO:46

DNA

Intellectual figure

161bp 3' homologous region, cas9 target site

GATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTC

SEQ ID NO:47

IgG1 upper hinge sequence

EPKSCDKTHT

SEQ ID NO:48

IgG1 core hinge sequences

CPXC, whereinXP, R or S

SEQ ID NO:49

Lower hinge/CH 2 sequence

PAPELLGGP

SEQ ID NO:50

Fc region segment (CH 2)

SVFLFPPKPKDT

SEQ ID NO:51

Hinge sequence comprising an upper hinge, a core hinge and a lower hinge

EPKSCDKTHTCPPCPAPELLGGP

SEQ ID NO:52

LAGLIDADG

Sequence listing

<110> Sorrento pharmaceutical Co., ltd (SORRENTO THERAPEUTICS, INC.)

<120> Dimer Antigen Receptor (DAR) binding to GD2

<130> 01223-0095-00PCT

<150> US 63/179,147

<151> 2021-04-23

<150> US 63/188,284

<151> 2021-05-13

<160> 52

<170> patent In version 3.5

<210> 1

<211> 533

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 1

Met Trp Leu Gly Arg Arg Ala Leu Cys Ala Leu Val Leu Leu Leu Ala

1 5 10 15

Cys Ala Ser Leu Gly Leu Leu Tyr Ala Ser Thr Arg Asp Ala Pro Gly

20 25 30

Leu Arg Leu Pro Leu Ala Pro Trp Ala Pro Pro Gln Ser Pro Arg Arg

35 40 45

Pro Glu Leu Pro Asp Leu Ala Pro Glu Pro Arg Tyr Ala His Ile Pro

50 55 60

Val Arg Ile Lys Glu Gln Val Val Gly Leu Leu Ala Trp Asn Asn Cys

65 70 75 80

Ser Cys Glu Ser Ser Gly Gly Gly Leu Pro Leu Pro Phe Gln Lys Gln

85 90 95

Val Arg Ala Ile Asp Leu Thr Lys Ala Phe Asp Pro Ala Glu Leu Arg

100 105 110

Ala Ala Ser Ala Thr Arg Glu Gln Glu Phe Gln Ala Phe Leu Ser Arg

115 120 125

Ser Gln Ser Pro Ala Asp Gln Leu Leu Ile Ala Pro Ala Asn Ser Pro

130 135 140

Leu Gln Tyr Pro Leu Gln Gly Val Glu Val Gln Pro Leu Arg Ser Ile

145 150 155 160

Leu Val Pro Gly Leu Ser Leu Gln Ala Ala Ser Gly Gln Glu Val Tyr

165 170 175

Gln Val Asn Leu Thr Ala Ser Leu Gly Thr Trp Asp Val Ala Gly Glu

180 185 190

Val Thr Gly Val Thr Leu Thr Gly Glu Gly Gln Ala Asp Leu Thr Leu

195 200 205

Val Ser Pro Gly Leu Asp Gln Leu Asn Arg Gln Leu Gln Leu Val Thr

210 215 220

Tyr Ser Ser Arg Ser Tyr Gln Thr Asn Thr Ala Asp Thr Val Arg Phe

225 230 235 240

Ser Thr Glu Gly His Glu Ala Ala Phe Thr Ile Arg Ile Arg His Pro

245 250 255

Pro Asn Pro Arg Leu Tyr Pro Pro Gly Ser Leu Pro Gln Gly Ala Gln

260 265 270

Tyr Asn Ile Ser Ala Leu Val Thr Ile Ala Thr Lys Thr Phe Leu Arg

275 280 285

Tyr Asp Arg Leu Arg Ala Leu Ile Thr Ser Ile Arg Arg Phe Tyr Pro

290 295 300

Thr Val Thr Val Val Ile Ala Asp Asp Ser Asp Lys Pro Glu Arg Val

305 310 315 320

Ser Gly Pro Tyr Val Glu His Tyr Leu Met Pro Phe Gly Lys Gly Trp

325 330 335

Phe Ala Gly Arg Asn Leu Ala Val Ser Gln Val Thr Thr Lys Tyr Val

340 345 350

Leu Trp Val Asp Asp Asp Phe Val Phe Thr Ala Arg Thr Arg Leu Glu

355 360 365

Arg Leu Val Asp Val Leu Glu Arg Thr Pro Leu Asp Leu Val Gly Gly

370 375 380

Ala Val Arg Glu Ile Ser Gly Phe Ala Thr Thr Tyr Arg Gln Leu Leu

385 390 395 400

Ser Val Glu Pro Gly Ala Pro Gly Leu Gly Asn Cys Leu Arg Gln Arg

405 410 415

Arg Gly Phe His His Glu Leu Val Gly Phe Pro Gly Cys Val Val Thr

420 425 430

Asp Gly Val Val Asn Phe Phe Leu Ala Arg Thr Asp Lys Val Arg Glu

435 440 445

Val Gly Phe Asp Pro Arg Leu Ser Arg Val Ala His Leu Glu Phe Phe

450 455 460

Leu Asp Gly Leu Gly Ser Leu Arg Val Gly Ser Cys Ser Asp Val Val

465 470 475 480

Val Asp His Ala Ser Lys Leu Lys Leu Pro Trp Thr Ser Arg Asp Ala

485 490 495

Gly Ala Glu Thr Tyr Ala Arg Tyr Arg Tyr Pro Gly Ser Leu Asp Glu

500 505 510

Ser Gln Met Ala Lys His Arg Leu Leu Phe Phe Lys His Arg Leu Gln

515 520 525

Cys Met Thr Ser Gln

530

<210> 2

<211> 113

<212> PRT

<213> artificial sequence

<220>

<223> anti-GD 2.14.18 14.18 VH

<400> 2

Glu Val Gln Leu Leu Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala

1 5 10 15

Ser Val Met Ile Ser Cys Lys Ala Ser Gly Ser Ser Phe Thr Gly Tyr

20 25 30

Asn Met Asn Trp Val Arg Gln Asn Ile Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Ala Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met His Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Val Ser Gly Met Glu Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser

100 105 110

Ser

<210> 3

<211> 113

<212> PRT

<213> artificial sequence

<220>

<223> anti-GD 2 hu14.18 VH

<400> 3

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Glu Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Ser Ser Phe Thr Gly Tyr

20 25 30

Asn Met Asn Trp Val Arg Gln Asn Ile Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Ala Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met His Leu Lys Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Ser Gly Met Glu Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser

100 105 110

Ser

<210> 4

<211> 108

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 4

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

100 105

<210> 5

<211> 113

<212> PRT

<213> artificial sequence

<220>

<223> anti-GD 2.14.18 14.18 VL

<400> 5

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ser Ser Gln Ser Leu Val His Arg

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile His Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210> 6

<211> 113

<212> PRT

<213> artificial sequence

<220>

<223> anti-GD 2 hu14.18 VL

<400> 6

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Arg

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile His Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210> 7

<211> 107

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 7

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 8

<211> 106

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 8

Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser

1 5 10 15

Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp

20 25 30

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro

35 40 45

Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn

50 55 60

Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys

65 70 75 80

Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val

85 90 95

Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 9

<211> 42

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 9

Pro Arg Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu

1 5 10 15

Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro

20 25 30

Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro

35 40

<210> 10

<211> 48

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 10

Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro

1 5 10 15

Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro

20 25 30

Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Pro Arg

35 40 45

<210> 11

<211> 88

<212> PRT

<213> artificial sequence

<220>

<223> CD8 hinge+CD 28 hinge (Long hinge)

<400> 11

Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro

1 5 10 15

Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro

20 25 30

Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Pro Arg

35 40 45

Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser

50 55 60

Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro

65 70 75 80

Leu Phe Pro Gly Pro Ser Lys Pro

85

<210> 12

<211> 27

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 12

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 13

<211> 23

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 13

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr

20

<210> 14

<211> 27

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 14

Ile Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu

1 5 10 15

Leu Phe Phe Leu Thr Leu Arg Phe Ser Val Val

20 25

<210> 15

<211> 21

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 15

Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu

1 5 10 15

Thr Ala Leu Phe Leu

20

<210> 16

<211> 42

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 16

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 17

<211> 41

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 17

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 18

<211> 42

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 18

Ala Leu Tyr Leu Leu Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His

1 5 10 15

Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln

20 25 30

Ala Asp Ala His Ser Thr Leu Ala Lys Ile

35 40

<210> 19

<211> 112

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 19

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 20

<211> 38

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 20

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg

35

<210> 21

<211> 47

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 21

Arg Val Lys Phe Ser Arg Ser Ala Asp Arg Gly Arg Asp Pro Glu Met

1 5 10 15

Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu

20 25 30

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

35 40 45

<210> 22

<211> 45

<212> PRT

<213> Homo sapiens (Homo sapiens)

<400> 22

Arg Val Lys Phe Ser Arg Ser Ala Asp Lys Gly Glu Arg Arg Arg Gly

1 5 10 15

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

20 25 30

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

35 40 45

<210> 23

<211> 19

<212> PRT

<213> mice (Mus musculus)

<400> 23

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser

<210> 24

<211> 20

<212> PRT

<213> mice (Mus musculus)

<400> 24

Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr

1 5 10 15

Asp Ala Arg Cys

20

<210> 25

<211> 21

<212> PRT

<213> mice (Mus musculus)

<400> 25

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 26

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> T2A self-cleavage sequence

<400> 26

Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu

1 5 10 15

Glu Asn Pro Gly Pro

20

<210> 27

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> P2A self-cleavage sequence

<400> 27

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210> 28

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> E2A self-cleavage sequence

<400> 28

Gly Ser Gly Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp

1 5 10 15

Val Glu Ser Asn Pro Gly Pro

20

<210> 29

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> F2A self-cleaving sequence

<400> 29

Gly Ser Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala

1 5 10 15

Gly Asp Val Glu Ser Asn Pro Gly Pro

20 25

<210> 30

<211> 724

<212> PRT

<213> artificial sequence

<220>

<223> precursor: GD2-14.18 DAR (V2 a edition)

<400> 30

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Glu Val Gln Leu Leu Gln Ser Gly Pro Glu Leu Glu Lys

20 25 30

Pro Gly Ala Ser Val Met Ile Ser Cys Lys Ala Ser Gly Ser Ser Phe

35 40 45

Thr Gly Tyr Asn Met Asn Trp Val Arg Gln Asn Ile Gly Lys Ser Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn

65 70 75 80

Gln Lys Phe Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met His Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Val Ser Gly Met Glu Tyr Trp Gly Gln Gly Thr Ser Val

115 120 125

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

130 135 140

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

145 150 155 160

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

165 170 175

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

180 185 190

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

195 200 205

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

210 215 220

Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

225 230 235 240

Pro Arg Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu

245 250 255

Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro

260 265 270

Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val

275 280 285

Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe

290 295 300

Ile Ile Phe Trp Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

305 310 315 320

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

325 330 335

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

340 345 350

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

355 360 365

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

370 375 380

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

385 390 395 400

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

405 410 415

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

420 425 430

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

435 440 445

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly

450 455 460

Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu

465 470 475 480

Asn Pro Gly Pro Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu

485 490 495

Leu Trp Leu Thr Asp Ala Arg Cys Glu Ile Val Met Thr Gln Ser Pro

500 505 510

Ala Thr Leu Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg

515 520 525

Ser Ser Gln Ser Leu Val His Arg Asn Gly Asn Thr Tyr Leu His Trp

530 535 540

Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile His Lys Val

545 550 555 560

Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser

565 570 575

Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu

580 585 590

Gly Val Tyr Phe Cys Ser Gln Ser Thr His Val Pro Pro Leu Thr Phe

595 600 605

Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro Ser

610 615 620

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

625 630 635 640

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

645 650 655

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

660 665 670

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

675 680 685

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

690 695 700

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

705 710 715 720

Arg Gly Glu Cys

<210> 31

<211> 2172

<212> DNA

<213> artificial sequence

<220>

<223> GD2-14.18 DAR precursor

<400> 31

atggaatgga gctgggtgtt cctcttcttc ctcagcgtga ccaccggcgt gcactccgag 60

gtgcaattat tacagtccgg acccgagctg gagaagcccg gcgcctccgt gatgatcagc 120

tgtaaagcct ccggcagctc ctttaccggc tacaacatga actgggtgag gcagaacatt 180

ggaaagtctt tagagtggat cggcgccatc gatccctact acggcggcac ctcctataac 240

cagaagttca agggtcgtgc caccctcacc gtcgacaagt cctccagcac agcttatatg 300

catttaaagt ccctcaccag cgaggactcc gccgtgtatt attgtgtgag cggcatggaa 360

tattggggcc aaggaaccag cgtcaccgtg tccagcgctt ccacaaaggg acccagcgtc 420

ttccctctgg cccccagcag caaaagcacc agcggcggaa ccgctgcttt aggatgttta 480

gtgaaggact actttcccga acccgtgacc gtgagctgga attccggcgc tctcaccagc 540

ggcgtgcaca cctttcccgc tgtcctccag tccagcggcc tctactcttt atcctccgtg 600

gtgacagtgc ctagctcctc cctcggcacc cagacctaca tttgtaacgt gaaccacaag 660

ccttccaata caaaggtgga caagagggtg gagcccaaaa gctgcgataa gacccacact 720

cctcgtaaga ttgaggtcat gtaccccccc ccctatctgg acaacgagaa gagcaacggc 780

acaatcatcc acgtgaaggg caagcatctg tgcccctccc ctttatttcc cggaccttcc 840

aaaccctttt gggttttagt ggtcgtggga ggcgtgctgg cttgttactc tttactggtg 900

acagtcgcct tcatcatctt ttgggtgaag aggggtcgta agaaactgct gtacatcttc 960

aaacagccct tcatgaggcc cgtgcagaca acccaagaag aggatggatg ttcttgtcgt 1020

ttccccgaag aggaggaggg aggctgtgaa ctgagggtga agttctctcg tagcgctgac 1080

gcccccgcct accaacaagg tcaaaaccag ttatacaacg agctgaattt aggaaggaga 1140

gaggagtatg acgttttaga caagagaagg ggaagagacc ccgaaatggg cggcaagccc 1200

agaaggaaga acccccaaga aggtttatac aatgagctcc agaaggacaa gatggccgag 1260

gcctacagcg aaattggcat gaaaggcgag aggaggaggg gaaagggcca tgacggcctc 1320

tatcaaggtt taagcaccgc caccaaggac acctacgacg ctttacatat gcaagcttta 1380

cctcctaggg gaagcggaga aggaagggga tctttactga cttgtggcga cgtggaggag 1440

aaccccggcc ctatgtccgt gcctacccaa gttctgggtt tactgctgct ctggctgaca 1500

gacgctcgtt gcgagatcgt gatgacccaa tcccccgcta cactctccgt gagccccgga 1560

gagagggcta ctttaagctg tcgttcctcc cagagcctcg tccacagaaa cggcaatacc 1620

tatttacact ggtatttaca gaagcccggt cagagcccca agttattaat tcacaaagtc 1680

agcaacagat tcagcggcgt gcccgatcgt ttcagcggct ccggcagcgg aaccgacttt 1740

acactcaaga tcagcagagt ggaggccgag gatctgggcg tgtacttctg ctcccagtcc 1800

acccatgtgc cccctctcac attcggagcc ggaaccaagc tggagctgaa aagaacagtc 1860

gccgccccct ccgtcttcat ttttccccct tccgatgagc agctgaagag cggcacagcc 1920

agcgtggtct gtctgctcaa caatttttac cctcgtgagg ccaaggtcca gtggaaggtg 1980

gataatgctc tgcagtccgg caactcccaa gaatccgtga cagagcaaga ctccaaggat 2040

tccacctatt ctttatcctc caccctcact ttaagcaagg ctgactacga gaagcataag 2100

gtgtacgctt gtgaagtgac acaccaaggt ttatccagcc ccgtgacaaa gagcttcaat 2160

aggggagagt gc 2172

<210> 32

<211> 444

<212> PRT

<213> artificial sequence

<220>

<223> polypeptide 1, GD2-14.18 DAR

<400> 32

Glu Val Gln Leu Leu Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala

1 5 10 15

Ser Val Met Ile Ser Cys Lys Ala Ser Gly Ser Ser Phe Thr Gly Tyr

20 25 30

Asn Met Asn Trp Val Arg Gln Asn Ile Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Ala Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met His Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Val Ser Gly Met Glu Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser

100 105 110

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

115 120 125

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

180 185 190

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

195 200 205

Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Pro Arg Lys

210 215 220

Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn

225 230 235 240

Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu

245 250 255

Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly

260 265 270

Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe

275 280 285

Trp Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro

290 295 300

Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys

305 310 315 320

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe

325 330 335

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu

340 345 350

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

355 360 365

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

370 375 380

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

385 390 395 400

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

405 410 415

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

420 425 430

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

435 440

<210> 33

<211> 220

<212> PRT

<213> artificial sequence

<220>

<223> polypeptide No. 2, GD2-14.18 DAR

<400> 33

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ser Ser Gln Ser Leu Val His Arg

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile His Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 34

<211> 722

<212> PRT

<213> artificial sequence

<220>

<223> precursor: GD2-hu14.18 DAR

<400> 34

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Glu Lys

20 25 30

Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Ser Ser Phe

35 40 45

Thr Gly Tyr Asn Met Asn Trp Val Arg Gln Asn Ile Gly Lys Ser Leu

50 55 60

Glu Trp Ile Gly Ala Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn

65 70 75 80

Gln Lys Phe Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser

85 90 95

Thr Ala Tyr Met His Leu Lys Ser Leu Arg Ser Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Val Ser Gly Met Glu Tyr Trp Gly Gln Gly Thr Ser Val

115 120 125

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

130 135 140

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

145 150 155 160

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

165 170 175

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

180 185 190

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

195 200 205

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

210 215 220

Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

225 230 235 240

Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser

245 250 255

Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro

260 265 270

Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly

275 280 285

Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile

290 295 300

Phe Trp Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

305 310 315 320

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

325 330 335

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys

340 345 350

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln

355 360 365

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

370 375 380

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

385 390 395 400

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

405 410 415

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

420 425 430

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

435 440 445

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly

450 455 460

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

465 470 475 480

Gly Pro Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp

485 490 495

Leu Thr Asp Ala Arg Cys Asp Val Val Met Thr Gln Thr Pro Leu Ser

500 505 510

Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser

515 520 525

Gln Ser Leu Val His Arg Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu

530 535 540

Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile His Lys Val Ser Asn

545 550 555 560

Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

565 570 575

Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val

580 585 590

Tyr Phe Cys Ser Gln Ser Thr His Val Pro Pro Leu Thr Phe Gly Ala

595 600 605

Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe

610 615 620

Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val

625 630 635 640

Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp

645 650 655

Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr

660 665 670

Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

675 680 685

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

690 695 700

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly

705 710 715 720

Glu Cys

<210> 35

<211> 2166

<212> DNA

<213> artificial sequence

<220>

<223> GD2-hu14.18 DAR precursor

<400> 35

atggaatgga gctgggtctt tctcttcttc ctgtcagtaa ccaccggtgt ccactccgaa 60

gtgcaactcg ttcaaagcgg agccgaggtc gaaaaacccg gagcctcagt caagattagc 120

tgtaaagcta gtggcagcag ctttacaggg tacaacatga actgggtccg acagaacatt 180

gggaagtcac tcgagtggat tggggctatt gacccgtact acggtggaac atcatacaat 240

cagaaattta aaggacgagc aaccctcacg gtagacaaaa gtacttccac ggcgtatatg 300

catctcaaga gtctcagaag tgaagacaca gcggtctatt attgtgtatc tggtatggag 360

tattggggcc aaggtacgtc cgtcactgtt tcatctgcta gcacaaaagg cccctccgtc 420

tttcctttag cccctagctc caaatccacc tccggcggaa cagccgccct cggatgtctc 480

gtcaaagact acttccccga gcccgtgaca gtgtcttgga attccggcgc tttaacctcc 540

ggagtgcaca cctttcccgc tgttttacag tccagcggac tgtattcttt aagctccgtc 600

gtcaccgtgc ctagcagctc tttaggcacc cagacctaca tttgcaacgt caaccacaag 660

cctagcaaca ccaaggtgga caaaagggtg gagcctaagt cttgtgacaa gacccacacc 720

aagatcgagg tcatgtaccc ccccccctat ttagacaacg agaaaagcaa cggcacaatt 780

atccatgtga aaggcaagca tctctgcccc tcccctctgt ttcccggacc ctccaagcct 840

ttttgggtgc tcgtggtggt gggcggcgtg ctggcttgtt attctttact ggtgaccgtc 900

gcctttatca tcttctgggt gaagaggggt cgtaagaagc tgctgtacat cttcaagcag 960

cccttcatga ggcccgttca aaccacccaa gaagaggacg gctgcagctg tcgtttcccc 1020

gaggaggaag agggaggatg cgagctgagg gtgaagttct ctcgtagcgc cgatgccccc 1080

gcttatcagc aaggtcagaa ccagttatac aacgagctga atttaggtcg tagggaggag 1140

tacgacgtgc tggacaagag aagaggtcgt gaccccgaga tgggcggcaa acctcgtagg 1200

aagaaccccc aagaaggttt atacaatgag ctgcagaagg acaagatggc cgaggcctac 1260

agcgagatcg gcatgaaggg cgagaggaga aggggcaagg gccacgatgg tttataccaa 1320

ggtttaagca ccgccaccaa ggacacctac gatgctttac acatgcaagc tttacctcct 1380

cgtggaagcg gcgaaggaag gggctcttta ctgacatgtg gcgacgtgga ggaaaacccc 1440

ggccctatgt ccgtgcccac acaagttctc ggactgttat tactgtggct gacagatgct 1500

cgttgcgatg tcgttatgac gcaaaccccg ctttccttgc cggttacgcc tggagaacca 1560

gcttccatct catgtcggtc ttcacaatct ctggttcatc gcaacggaaa cacgtatctt 1620

cattggtatc ttcagaagcc aggccaatcc ccaaagctcc tcattcacaa ggtctctaac 1680

agattcagtg gggttcctga cagattttcc gggtctggat caggtactga cttcacattg 1740

aaaattagcc gcgtagaagc tgaggacctg ggcgtatact tttgttctca atccacccat 1800

gtaccacctc tcactttcgg cgcaggaacg aaactggaac tgaagagaac cgtggccgcc 1860

cctagcgttt tcattttccc ccccagcgac gaacagctga agtccggcac agcctccgtg 1920

gtgtgtttac tgaataactt ctaccctcgt gaggctaagg tccaatggaa ggtggataat 1980

gctttacagt ccggaaattc ccaagaaagc gtgaccgagc aagatagcaa agatagcacc 2040

tacagcttaa gctccacact gacactgtcc aaagccgact acgagaaaca caaggtgtac 2100

gcttgtgaag tgacccatca aggtttaagc tcccccgtca caaagagctt taacagagga 2160

gaatgc 2166

<210> 36

<211> 442

<212> PRT

<213> artificial sequence

<220>

<223> polypeptide 1, GD2-hu14.18 DAR

<400> 36

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Glu Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Ser Ser Phe Thr Gly Tyr

20 25 30

Asn Met Asn Trp Val Arg Gln Asn Ile Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Ala Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met His Leu Lys Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Ser Gly Met Glu Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser

100 105 110

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

115 120 125

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

180 185 190

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

195 200 205

Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Lys Ile Glu

210 215 220

Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr

225 230 235 240

Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro

245 250 255

Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu

260 265 270

Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

275 280 285

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

290 295 300

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

305 310 315 320

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

325 330 335

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

340 345 350

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

355 360 365

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

370 375 380

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

385 390 395 400

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

405 410 415

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

420 425 430

Ala Leu His Met Gln Ala Leu Pro Pro Arg

435 440

<210> 37

<211> 220

<212> PRT

<213> artificial sequence

<220>

<223> polypeptide No. 2, GD2-hu14.18 DAR

<400> 37

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Arg

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile His Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 38

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> G4S linker

<400> 38

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 39

<211> 195

<212> DNA

<213> artificial sequence

<220>

<223> JeT promoter

<400> 39

gaattcgggc ggagttaggg cggagccaat cagcgtgcgc cgttccgaaa gttgcctttt 60

atggctgggc ggagaatggg cggtgaacgc cgatgattat ataaggacgc gccgggtgtg 120

gcacagctag ttccgtcgca gccgggattt gggtcgcggt tcttgtttgt ggatccctgt 180

gatcgtcagt tgaca 195

<210> 40

<211> 660

<212> DNA

<213> Homo sapiens (Homo sapiens)

<400> 40

ggcaccatat tcattttgca ggtgaaattc ctgagatgta aggagctgct gtgacttgct 60

caaggcctta tatcgagtaa acggtagtgc tggggcttag acgcaggtgt tctgatttat 120

agttcaaaac ctctatcaat gagagagcaa tctcctggta atgtgataga tttcccaact 180

taatgccaac ataccataaa cctcccattc tgctaatgcc cagcctaagt tggggagacc 240

actccagatt ccaagatgta cagtttgctt tgctgggcct ttttcccatg cctgccttta 300

ctctgccaga gttatattgc tggggttttg aagaagatcc tattaaataa aagaataagc 360

agtattatta agtagccctg catttcaggt ttccttgagt ggcaggccag gcctggccgt 420

gaacgttcac tgaaatcatg gcctcttggc caagattgat agcttgtgcc tgtccctgag 480

tcccagtcca tcacgagcag ctggtttcta agatgctatt tcccgtataa agcatgagac 540

cgtgacttgc cagccccaca gagccccgcc cttgtccatc actggcatct ggactccagc 600

ctgggttggg gcaaagaggg aaatgagatc atgtcctaac cctgatcctc ttgtcccaca 660

<210> 41

<211> 650

<212> DNA

<213> Homo sapiens (Homo sapiens)

<400> 41

gatatccaga accctgaccc tgccgtgtac cagctgagag actctaaatc cagtgacaag 60

tctgtctgcc tattcaccga ttttgattct caaacaaatg tgtcacaaag taaggattct 120

gatgtgtata tcacagacaa aactgtgcta gacatgaggt ctatggactt caagagcaac 180

agtgctgtgg cctggagcaa caaatctgac tttgcatgtg caaacgcctt caacaacagc 240

attattccag aggacacctt cttccccagc ccaggtaagg gcagctttgg tgccttcgca 300

ggctgtttcc ttgcttcagg aatggccagg ttctgcccag agctctggtc aatgatgtct 360

aaaactcctc tgattggtgg tctcggcctt atccattgcc accaaaaccc tctttttact 420

aagaaacagt gagccttgtt ctggcagtcc agagaatgac acgggaaaaa agcagatgaa 480

gagaaggtgg caggagaggg cacgtggccc agcctcagtc tctccaactg agttcctgcc 540

tgcctgcctt tgctcagact gtttgcccct tactgctctt ctaggcctca ttctaagccc 600

cttctccaag ttgcctctcc ttatttctcc ctgtctgcca aaaaatcttt 650

<210> 42

<211> 19

<212> DNA

<213> Homo sapiens (Homo sapiens)

<400> 42

cagggttctg gatatctgt 19

<210> 43

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Forward primer

<220>

<221> misc_feature

<222> (1)..(2)

<223> phosphorothioate bond

<220>

<221> misc_feature

<222> (3)..(4)

<223> phosphorothioate bond

<220>

<221> modified_base

<222> (3)..(3)

<223> cm

<220>

<221> misc_feature

<222> (4)..(5)

<223> phosphorothioate bond

<220>

<221> modified_base

<222> (4)..(4)

<223> 2' -O-methyladenosine

<220>

<221> modified_base

<222> (5)..(5)

<223> cm

<400> 43

atcacgagca gctggtttct 20

<210> 44

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> reverse primer

<400> 44

gacctcatgt ctagcacagt tttg 24

<210> 45

<211> 171

<212> DNA

<213> Homo sapiens (Homo sapiens)

<400> 45

atcacgagca gctggtttct aagatgctat ttcccgtata aagcatgaga ccgtgacttg 60

ccagccccac agagccccgc ccttgtccat cactggcatc tggactccag cctgggttgg 120

ggcaaagagg gaaatgagat catgtcctaa ccctgatcct cttgtcccac a 171

<210> 46

<211> 161

<212> DNA

<213> Homo sapiens (Homo sapiens)

<400> 46

gatatccaga accctgaccc tgccgtgtac cagctgagag actctaaatc cagtgacaag 60

tctgtctgcc tattcaccga ttttgattct caaacaaatg tgtcacaaag taaggattct 120

gatgtgtata tcacagacaa aactgtgcta gacatgaggt c 161

<210> 47

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> IgG1 upper hinge region

<400> 47

Glu Pro Lys Ser Cys Asp Lys Thr His Thr

1 5 10

<210> 48

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> IgG1 core hinge region

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> X is P, R or S

<400> 48

Cys Pro Xaa Cys

1

<210> 49

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> lower hinge/CH 2 sequence

<400> 49

Pro Ala Pro Glu Leu Leu Gly Gly Pro

1 5

<210> 50

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> Fc region (CH 2)

<400> 50

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

1 5 10

<210> 51

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> hinge sequence including upper hinge, core hinge and lower hinge

<400> 51

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro

20

<210> 52

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> family name of site-specific DNA endonucleases

<400> 52

Leu Ala Gly Leu Ile Asp Ala Asp Gly

1 5

Claims

1. A genetically modified host cell or population of genetically modified host cells that express a Dimeric Antigen Receptor (DAR) that binds to GD2, wherein the DAR comprises:

a. a first polypeptide comprising, in order from amino terminus to carboxy terminus, a plurality of polypeptide regions:

(i) Antibody heavy chain variable regions; (ii) an antibody heavy chain constant region; (iii) a hinge region; (iv) a transmembrane region; and (vi) an intracellular region; and

b. a second polypeptide comprising, in order from amino terminus to carboxy terminus, a plurality of polypeptide regions:

(i) An antibody light chain variable region; and (ii) an antibody light chain constant region, wherein the second polypeptide does not comprise a transmembrane region;

Wherein the antibody heavy chain constant region and the antibody light chain constant region form a dimerization domain to form the DAR, and

wherein the antibody heavy chain variable region and the antibody light chain variable region form an antigen binding domain that binds GD 2.

2. The genetically modified host cell or population of genetically modified host cells of claim 1, wherein the DAR comprises:

a. a first polypeptide consisting essentially of: (i) an antibody heavy chain variable region; (ii) an antibody heavy chain constant region; (iii) a hinge region; (iv) a transmembrane region; and (vi) an intracellular region; and

b. a second polypeptide consisting essentially of: (i) an antibody light chain variable region; and (ii) an antibody light chain constant region.

3. A genetically modified host cell or population of genetically modified host cells that express a Dimeric Antigen Receptor (DAR) that binds to GD2, wherein the DAR comprises:

a) A first polypeptide chain comprising, in order from amino terminus to carboxy terminus, a plurality of polypeptide regions: (i) an antibody light chain variable region; (ii) an antibody light chain constant region; (iii) a hinge region; (iv) a transmembrane region; and

(v) An intracellular region; and

b) A second polypeptide chain comprising, in order from amino terminus to carboxy terminus, a plurality of polypeptide regions: (i) an antibody heavy chain variable region; and (ii) an antibody heavy chain constant region, wherein the second polypeptide does not comprise a transmembrane region;

wherein the antibody heavy chain constant region and the antibody light chain constant region form a dimerization domain to form the Dimeric Antigen Receptor (DAR), and

4. The genetically modified host cell or population of genetically modified host cells of claim 3, wherein the DAR comprises:

a. a first polypeptide consisting essentially of: (i) an antibody light chain variable region; (ii) an antibody light chain constant region; (iii) a hinge region; (iv) a transmembrane region; and (v) an intracellular region; and

b. a second polypeptide consisting of: (i) an antibody heavy chain variable region; and (ii) an antibody heavy chain constant region.

5. The genetically modified host cell or population of genetically modified host cells of any one of claims 1 to 4, wherein the antibody heavy chain constant region and the antibody light chain constant region dimerize via one or more disulfide bonds.

6. The genetically modified host cell or population of genetically modified host cells of any one of claims 1 to 4, wherein the hinge region comprises a hinge sequence from an antibody selected from the group consisting of: igG, igA, igM, igE and IgD.

7. The genetically modified host cell or population of genetically modified host cells of any one of claims 1 to 4, wherein the hinge region comprises a CD8 a hinge region, a CD28 hinge region, or a CD8 a/CD 28 hinge region, or a hinge region having at least 95% identity to any one thereof.

8. The genetically modified host cell or population of genetically modified host cells of claim 7, wherein the hinge comprises a CD28 hinge region (SEQ ID NO: 9) or a hinge region having at least 95% identity thereto.

9. The genetically modified host cell or population of genetically modified host cells of any one of claims 1 to 4, wherein the transmembrane region comprises a CD8, CD28, 4-1BB, or CD3 ζ transmembrane region or a transmembrane region having at least 95% identity to any one thereof.

10. The genetically modified host cell or population of genetically modified host cells of claim 9, wherein the transmembrane region comprises a transmembrane domain sequence from CD28 (SEQ ID NO: 12) or a sequence having at least 95% identity thereto.

11. The genetically modified host cell or population of genetically modified host cells of any one of claims 1 to 4, wherein the intracellular region comprises one or more intracellular amino acid sequences selected from the group consisting of seq id nos: 4-1BB intracellular region (SEQ ID NO: 16), CD3 zeta (SEQ ID NO: 19) with ITAM 1, 2 and 3, CD3 zeta (SEQ ID NO: 20) with ITAM 1, CD3 zeta (SEQ ID NO: 22) with ITAM 3, or CD28 (SEQ ID NO: 17), CD27, OX40 (SEQ ID NO: 18), CD30, CD40, PD-1, ICOS, lymphocyte function associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B-H3, GITR (TNFRSF 18), DR3 (TNFRSF 25), TNFR2 and/or CD226, or an intracellular amino acid sequence having at least 95% identity to any of them.

12. The genetically modified host cell or population of genetically modified host cells of claim 11, wherein the intracellular region comprises cd3ζ (SEQ ID NO: 19) having ITAMs 1, 2, and 3.

13. The genetically modified host cell or population of genetically modified host cells of claim 12, wherein the intracellular region further comprises a 4-1BB intracellular region (SEQ ID NO: 16).

14. The genetically modified host cell or population of genetically modified host cells of any one of claims 1 to 4, wherein the antibody heavy chain variable region comprises an amino acid sequence having at least 95% identity to SEQ ID No. 2.

15. The genetically modified host cell or population of genetically modified host cells of claim 14, wherein the antibody light chain constant region comprises an amino acid sequence having at least 95% identity to SEQ ID No. 5.

16. The genetically modified host cell or population of genetically modified host cells of any one of claims 1 to 4, wherein the antibody heavy chain variable region comprises an amino acid sequence having at least 95% identity to SEQ ID No. 3.

17. The genetically modified host cell or population of genetically modified host cells of claim 16, wherein the antibody light chain constant region comprises an amino acid sequence having at least 95% identity to SEQ ID No. 6.

18. The genetically modified host cell or population of genetically modified host cells of claim 1 or claim 2, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID No. 32.

19. The genetically modified host cell or population of genetically modified host cells of claim 18, wherein the second polypeptide chain comprises the amino acid sequence of SEQ ID No. 33.

20. The genetically modified host cell or population of genetically modified host cells of claim 1 or claim 2, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID No. 36.

21. The genetically modified host cell or population of genetically modified host cells of claim 20, wherein the second polypeptide chain comprises the amino acid sequence of SEQ ID No. 37.

22. The genetically modified host cell or population of genetically modified host cells of claim 1 or claim 2, wherein the DAR comprises:

a) A first polypeptide chain comprising or consisting essentially of, in order from amino terminus to carboxy terminus, a plurality of polypeptide regions: (i) A GD2 antibody heavy chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID No. 2; (ii) A GD2 antibody heavy chain constant region comprising an amino acid sequence having at least 95% identity to SEQ ID No. 4; (iii) The hinge region of CD28 (SEQ ID NO: 9) or a hinge region having at least 95% identity thereto; (iv) The transmembrane region of CD28 (SEQ ID NO: 12) or a transmembrane domain having at least 95% identity thereto; and (v) an intracellular region comprising the 4-1BB intracellular domain (SEQ ID NO: 16) and a CD3 zeta intracellular region comprising ITAM 1, 2 and 3 (SEQ ID NO: 19); and

b) A second polypeptide chain comprising or consisting essentially of, in order from amino terminus to carboxy terminus, a plurality of polypeptide regions: (i) A GD2 antibody light chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID No. 5; and (ii) a GD2 antibody light chain constant region comprising an amino acid sequence having at least 95% identity to SEQ ID No. 7, wherein the second polypeptide chain does not comprise a transmembrane domain.

23. The genetically modified host cell or population of genetically modified host cells of claim 1 or claim 2, wherein

a) The first polypeptide chain comprises or consists essentially of, in order from the amino terminus to the carboxy terminus, a plurality of polypeptide regions: (i) A GD2 antibody heavy chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID No. 3; (ii) A GD2 antibody heavy chain constant region comprising an amino acid sequence having at least 95% identity to SEQ ID No. 4; (iii) The hinge region of CD28 (SEQ ID NO: 9) or a hinge region having at least 95% identity thereto; (iv) The transmembrane region of CD28 (SEQ ID NO: 12) or a transmembrane domain having at least 95% identity thereto; and (v) an intracellular region comprising the 4-1BB intracellular domain (SEQ ID NO: 16) and a CD3 zeta intracellular region comprising ITAM 1, 2 and 3 (SEQ ID NO: 19); and is also provided with

b) The second polypeptide chain comprises or consists essentially of, in order from the amino terminus to the carboxy terminus, a plurality of polypeptide regions: (i) A GD2 antibody light chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID No. 6; and (ii) a GD2 antibody light chain constant region comprising an amino acid sequence having at least 95% identity to SEQ ID No. 7, wherein the second polypeptide chain does not comprise a transmembrane domain.

24. The genetically modified host cell or population of genetically modified host cells of claim 3 or claim 4, wherein the DAR comprises:

(a) A first polypeptide chain comprising or consisting essentially of, in order from amino terminus to carboxy terminus, a plurality of polypeptide regions: (i) A GD2 antibody light chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID No. 5; (ii) A GD2 antibody light chain constant region comprising an amino acid sequence having at least 95% identity to SEQ ID No. 7; (iii) The hinge region of CD28 (SEQ ID NO: 9) or a hinge region having at least 95% identity thereto; (iv) The transmembrane region of CD28 (SEQ ID NO: 12) or a transmembrane domain having at least 95% identity thereto; and (v) an intracellular region comprising the 4-1BB intracellular domain (SEQ ID NO: 16) and a CD3 zeta intracellular region comprising ITAM 1, 2 and 3 (SEQ ID NO: 19); and

(b) A second polypeptide chain comprising or consisting essentially of, in order from amino terminus to carboxy terminus, a plurality of polypeptide regions: (i) A GD2 antibody heavy chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID No. 2; and (ii) a GD2 antibody heavy chain constant region comprising an amino acid sequence having at least 95% identity to SEQ ID No. 4, wherein the second polypeptide chain does not comprise a transmembrane domain.

25. The genetically modified host cell or population of genetically modified host cells of claim 1 or claim 2, wherein

(a) The first polypeptide chain comprises or consists essentially of, in order from the amino terminus to the carboxy terminus, a plurality of polypeptide regions: (i) A GD2 antibody light chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID No. 6; (ii) A GD2 antibody light chain constant region comprising an amino acid sequence having at least 95% identity to SEQ ID No. 7; (iii) The hinge region of CD28 (SEQ ID NO: 9) or a hinge region having at least 95% identity thereto; (iv) The transmembrane region of CD28 (SEQ ID NO: 12) or a transmembrane domain having at least 95% identity thereto; and (v) an intracellular region comprising the 4-1BB intracellular domain (SEQ ID NO: 16) and a CD3 zeta intracellular region comprising ITAM 1, 2 and 3 (SEQ ID NO: 19); and is also provided with

(b) The second polypeptide chain comprises or consists essentially of, in order from the amino terminus to the carboxy terminus, a plurality of polypeptide regions: (i) A GD2 antibody heavy chain variable region comprising an amino acid sequence having at least 95% identity to SEQ ID No. 3; and (ii) a GD2 antibody heavy chain constant region comprising an amino acid sequence having at least 95% identity to SEQ ID No. 4, wherein the second polypeptide chain does not comprise a transmembrane domain.

26. The genetically modified host cell or population of genetically modified host cells of claim 22 or claim 23, wherein (a) the first polypeptide chain comprises (i) a GD2 antibody heavy chain variable region comprising the amino acid sequence of SEQ ID No. 2; and b) the second polypeptide chain comprises (i) a GD2 antibody light chain variable region comprising the amino acid sequence of SEQ ID No. 5.

27. The genetically modified host cell or population of genetically modified host cells of claim 22 or claim 23, wherein (a) the first polypeptide chain comprises (i) a GD2 antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO 3; and b) the second polypeptide chain comprises (i) a GD2 antibody light chain variable region comprising the amino acid sequence of SEQ ID No. 6.

28. The genetically modified host cell or population of genetically modified host cells of any one of claims 1 to 27, wherein the genetically modified host cell or population of genetically modified host cells comprises at least one nucleic acid sequence encoding the first polypeptide and the second polypeptide.

29. The genetically modified host cell or population of genetically modified host cells of claim 28, wherein at least one nucleic acid sequence encodes a polypeptide of SEQ ID No. 32 and a polypeptide of SEQ ID No. 33.

30. The genetically modified host cell or population of genetically modified host cells of claim 29, wherein at least one nucleic acid sequence encodes a precursor polypeptide of SEQ ID No. 30.

31. The genetically modified host cell or population of genetically modified host cells of claim 30, wherein at least one nucleic acid sequence comprises SEQ ID No. 31 or a sequence having at least 65% identity thereto.

32. The genetically modified host cell or population of genetically modified host cells of claim 28, wherein at least one nucleic acid sequence encodes a polypeptide of SEQ ID No. 36 and a polypeptide of SEQ ID No. 37.

33. The genetically modified host cell or population of genetically modified host cells of claim 32, wherein at least one nucleic acid sequence encodes a precursor polypeptide of SEQ ID No. 34.

34. The genetically modified host cell or population of genetically modified host cells of claim 33, wherein at least one nucleic acid sequence comprises SEQ ID No. 35 or a sequence having at least 65% identity thereto.

35. The genetically modified host cell or population of genetically modified host cells of any one of claims 1 to 34, comprising a T lymphocyte, NK (natural killer) cell, macrophage, dendritic cell, mast cell, eosinophil, B lymphocyte, or monocyte.

36. The population of host cells according to claim 35 wherein said cells are primary cells.

37. The population of host cells according to claim 35 wherein said cells are human cells.

38. The population of host cells according to claim 35 wherein said population comprises T cells.

39. The population of host cells according to claim 35 wherein at least 90% of said cells expressing said DAR do not express a T cell receptor.

40. The population of host cells according to claim 35 wherein at least 95% of said cells expressing said DAR do not express a T cell receptor.

41. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a population of host cells according to any one of claims 35 to 40.

42. The pharmaceutical composition of claim 41 provided in a bag, vial, tube or tray.

43. The pharmaceutical composition of claim 41, wherein the composition is frozen.

44. A method for treating a subject having cancer, the method comprising administering to the subject a population of host cells according to any one of claims 1 to 40 or a pharmaceutical composition according to any one of claims 41 to 43.

45. The method of claim 44, wherein the cancer is breast cancer, ovarian cancer, prostate cancer, head and neck cancer, lung cancer, bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer, liver cancer, renal cancer, esophageal cancer, leiomyoma, leiomyosarcoma, glioma, glioblastoma, neuroblastoma, small cell lung cancer, medulloblastoma, astrocytoma, or osteosarcoma.

46. The method of claim 44, wherein the cancer is melanoma or osteosarcoma.

47. The method of any one of claims 44 to 46, wherein the population of cells is administered multiple times over a period of days, weeks, or months.

48. At least one nucleic acid molecule encoding:

a) A first polypeptide comprising, in order from amino terminus to carboxy terminus, a plurality of polypeptide regions:

(i) Antibody heavy chain variable regions; (ii) an antibody heavy chain constant region; (iii) a transmembrane region; and (iv) an intracellular region; and

b) A second polypeptide comprising, in order from amino terminus to carboxy terminus, a plurality of polypeptide regions:

(i) An antibody light chain variable region; and (ii) an antibody light chain constant region, wherein the second polypeptide does not comprise a transmembrane domain;

49. At least one nucleic acid molecule encoding:

a) A first polypeptide chain comprising, in order from amino terminus to carboxy terminus, a plurality of polypeptide regions: (i) an antibody light chain variable region; (ii) an antibody light chain constant region; (iii) a transmembrane region; and (iv) an intracellular region; and

b) A second polypeptide chain comprising, in order from amino terminus to carboxy terminus, a plurality of polypeptide regions: (i) an antibody heavy chain variable region; and (ii) an antibody heavy chain constant region, wherein the second polypeptide does not comprise a transmembrane domain;

50. A nucleic acid molecule encoding a precursor polypeptide comprising, in order from amino terminus to carboxy terminus, a plurality of polypeptide regions: (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 region; (7) a self-cleaving sequence; (8) a light chain leader sequence; (9) an antibody light chain variable region; and (10) an antibody light chain constant region, wherein the self-cleaving sequence allows cleavage of the precursor polypeptide into a first polypeptide chain and a second polypeptide chain.

51. A nucleic acid molecule encoding a precursor polypeptide comprising, in order from amino terminus to carboxy terminus, a plurality of polypeptide regions: (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 region; (7) a self-cleaving sequence; (8) a heavy chain leader sequence; (9) an antibody heavy chain variable region; and (10) an antibody heavy chain constant region, wherein the self-cleaving sequence allows cleavage of the precursor polypeptide into a first polypeptide chain and a second polypeptide chain.

52. The one or more nucleic acid molecules of any one of claims 48 to 51, wherein the antibody heavy chain variable region comprises the amino acid sequence of SEQ ID No. 2.

53. The one or more nucleic acid molecules of any one of claims 48 to 52, wherein the antibody heavy chain constant region comprises the amino acid sequence of SEQ ID No. 3.

54. The one or more nucleic acid molecules of any one of claims 48 to 53, wherein the antibody light chain variable region comprises the amino acid sequence of SEQ ID No. 5.

55. The one or more nucleic acid molecules of any one of claims 48 to 54, wherein the antibody light chain constant region comprises the amino acid sequence of SEQ ID No. 6.

56. The nucleic acid molecule of claim 50 comprising the amino acid sequence of SEQ ID NO. 31 or EQ ID NO. 35.