DAP10 / DAP12 fusion polypeptides

Abstract

To provide: fusion polypeptides comprising a DNAX-activating protein 10 (DAP10) polypeptide and a DNAX-activating protein 12 (DAP12) polypeptide; cells comprising such fusion proteins; and their use in treating cancer.SOLUTION: A fusion polypeptide is provided which comprises (i) a DAP10 polypeptide or a functional variant thereof and (ii) a DAP12 polypeptide or a functional variant thereof. The DAP10 polypeptide (or functional variant thereof) and the DAP12 polypeptide (or functional variant thereof) may be directly fused together, or may be joined by a linker. The fusion polypeptide may further comprise N-terminal or C-terminal amino acid sequences, e.g., to assist with detection or purification, to improve expression, or to increase the half-life.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a fusion polypeptide comprising a DNAX - activating protein 10 (DAP10) polypeptide and a DNAX - activating protein 12 (DAP12) polypeptide. The present disclosure also relates to cells comprising such fusion polypeptides and their use in the treatment of cancer.

Background Art

[0002] Immunotherapy using chimeric antigen receptor (CAR) - modified T cells has proven to bring about a revolution in the management of B - cell malignancies and multiple myeloma. However, applying this technology to solid - tumor immunotherapy is hampered by the lack of tumor - selective targets. Most tumor antigens are intracellular and thus cannot be easily recognized by CAR T cells. As a result, most of the CARs currently under development for solid tumors bind to targets that are up - regulated in tumor cells but are seen at lower levels in normal tissues.

[0003] One of several target groups showing high tumor selectivity is the NKG2D ligands. In men, these include a group of eight stress-inducible proteins (MICA, MICB, ULBP1-6) that are abnormally expressed on virtually all types of tumor cells. Additionally, NKG2D ligands are also found on tumor-associated stromal elements such as endothelium, regulatory T cells, and myeloid-derived suppressor cells (Parihar, R., et al., 2019, Cancer Immunol. Res. 7(3):363-375; Schmiedel & Mandelboim, 2018, Front. Immunol. (9)2040). Mice genetically deficient in NKG2D have impaired immune surveillance for both epithelial and lymphoid malignancies. Evidence that NKG2D ligands are safe therapeutic targets is supported by the fact that they are not found in healthy tissues. Furthermore, ongoing clinical trials involving NKG2D-targeted CARs have not resulted in significant safety issues even when combined with lympho depleting chemotherapy with fludarabine / cyclophosphamide (see https: / / www.celyad.com / en / news / celyad-presents-update-on-autologous-allogeneic-nkg2d-based-car-t-therapies-in-solid-tumors).

[0004] The NKG2D receptor is naturally expressed by natural killer (NK) and some T cell populations. Each NKG2D homodimer associates with two homodimeric DAP10 adapter molecules via complementary charged amino acids within the cell membrane. This interaction is required for cell surface expression and function of NKG2D. DAP10 has an ability similar to CD28 to provide co-stimulation via phosphatidylinositol 3-kinase, but importantly, it lacks the p56lck-binding motif that promotes unwanted mobilization of regulatory T cells (Kofler, et al., 2011, Mol. Ther. 19:760-767). The efficacy of DAP10 co-stimulation is emphasized by its persistent ability to signal after internalization. However, since DAP10 lacks an immunoreceptor tyrosine-based activation motif (ITAM), binding of NKG2D does not result in complete T cell activation.

[0005] In addition to costimulation (also known as signal 2), various CARs using different methods have been developed to provide ITAM-dependent signal 1 (since both signal types are required for complete T cell activation). The first NKG2D-targeted CAR was developed by Sentman et al. and consists of the fusion of NKG2D to CD3ζ (Zhang et al, 2005, Blood 106:1544-1551). Nominally a first-generation CAR, it associates with endogenous DAP10 in T cells, meaning that both signal 1 and signal 2 are provided. This CAR is currently in clinical development as Cyad-01 by Celyad S.A. Recently, Chang et al (2013, Cancer Res. 73:1777-1786) modified NK cells to co-express the same CAR in addition to exogenous DAP10. Two additional NKG2D CARs incorporating 4-1BB (Song et al., 2013, Hum. Gene Ther. 24:295-305) or CD28 (Lehner et al., 2012, PLoS One 7:e31210) have also been described to provide alternative forms of costimulation instead of that provided by DAP10. All of these CARs enabled T cell-mediated tumor cell killing with cytokine production, and the CAR described by Chang et al. also showed transient in vivo anti-tumor activity.

Summary of the Invention

Problems to be Solved by the Invention

[0006] One isoform of murine NKG2D found in NK cells can associate in trans with both DAP10 and DAP12 (which contains a single ITAM), thereby potentially delivering both signal 1 and signal 2 for T cell activation. Based on this observation, the inventors modified a fusion polypeptide containing both DAP10 and DAP12. When co-expressed with NKG2D, the fusion polypeptide and NKG2D co-associate at the cell membrane via complementary charged amino acid residues, forming a compact adapter-based CAR that results in complete T cell activation.

Means for Solving the Problem

[0007] Accordingly, the present disclosure provides a fusion polypeptide comprising (i) a DNAX-activating protein 10 (DAP10) polypeptide, or a functional variant thereof, and (ii) a DNAX-activating protein 12 (DAP12) polypeptide, or a functional variant thereof. The DAP10 polypeptide (or a functional variant thereof) and the DAP12 polypeptide (or a functional variant thereof) may be directly fused or may be linked by a linker. The fusion polypeptide may further comprise an N-terminal or C-terminal amino acid sequence, for example, to assist in detection or purification, to improve expression, or to increase the half-life.

[0008] In one embodiment, the present disclosure provides a fusion polypeptide having, from the N-terminus to the C-terminus, the formula: A-B-C-D-E where A = an optional N-terminal sequence that may be present B = a DAP10 polypeptide or a functional variant thereof C = an optional linker sequence that may be present D = a DAP12 polypeptide or a functional variant thereof E = an optional C-terminal sequence that may be present.

[0009] ​In one embodiment, the fusion polypeptide does not include, or does not consist of, SEQ ID NO: 1 of WO2019 / 182425 (referred to herein as SEQ ID NO: 84). In some embodiments, the fusion polypeptide does not include an anti-EpCAM peptide. In some embodiments, the fusion polypeptide does not include an EpCAM-specific antigen receptor. In some embodiments, the fusion polypeptide does not include, or does not consist of, SEQ ID NO: 9 of WO2019 / 182425 (referred to herein as SEQ ID NO: 85). In some embodiments, the DAP10 polypeptide or a functional variant thereof does not include, or does not consist of, SEQ ID NO: 85. In some embodiments, the fusion polypeptide does not include, or does not consist of, SEQ ID NO: 11 of WO2019 / 182425 (referred to herein as SEQ ID NO: 86). In some embodiments, the DAP12 polypeptide or a functional variant thereof does not include, or does not consist of, SEQ ID NO: 86. In some embodiments, the fusion polypeptide does not include, or does not consist of, both SEQ ID NO: 85 and SEQ ID NO: 86.

[0010] In other aspects, the disclosure relates to nucleic acid molecules (e.g., isolated nucleic acid molecules) comprising DNA and RNA molecules encoding the fusion polypeptides described herein. Such nucleic acid molecules may encode an NKG2D polypeptide or a functional variant thereof.

[0011] Vectors, particularly expression vectors, comprising the nucleic acid molecules of the disclosure are also disclosed.

[0012] The disclosure also provides host cells comprising nucleic acids and / or vectors encoding the fusion polypeptides described herein. Host cells comprising the fusion polypeptides of the invention are also provided herein.

[0013] The disclosure also provides a method of making the fusion polypeptides described herein, the method comprising maintaining a host cell of the disclosure under conditions suitable for expression of the nucleic acid, whereby the recombinant nucleic acid is expressed and the fusion polypeptide is produced.

[0014] The present disclosure also provides a pharmaceutical composition comprising a fusion polypeptide, nucleic acid molecule, vector, or host cell of the present disclosure, which may further comprise a pharmaceutically or physiologically acceptable carrier.

[0015] The present disclosure also provides a method of treating a patient suffering from a medical condition, the method comprising administering to a subject in need thereof a fusion polypeptide, nucleic acid molecule, vector, or host cell comprising a fusion polypeptide of the present disclosure.

[0016] The present disclosure also provides a fusion polypeptide, nucleic acid molecule, vector, host cell, or pharmaceutical composition according to the present disclosure for (i) use in therapy and (ii) in the manufacture of a medicament for the treatment of a disease or condition disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0017]

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[0018] DAP10 polypeptide and its functional variants The DAP10 polypeptide used in the fusion polypeptides described herein may be mammalian, such as human. Wild-type human DAP10 is encoded by an amino acid sequence having UniProt accession number Q9UBK5 (SEQ ID NO: 1). This is a 93aa polypeptide. The first 18aa are thought to be a signal / leader sequence, and amino acids 19 - 48 are the extracellular domain, amino acids 49 - 69 are the transmembrane domain, and amino acids 70 - 93 are thought to be the cytoplasmic / intracellular domain.

[0019] In one embodiment, the DAP10 polypeptide used in the fusion polypeptides of the present disclosure comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% sequence identity to the DAP10 polypeptide of SEQ ID NO: 1. In some embodiments, the DAP10 polypeptide used in the fusion polypeptides of the present disclosure comprises the amino acid sequence of SEQ ID NO: 1.

[0020] In another embodiment, the functional mutant DAP10 polypeptide used in the fusion polypeptides of the present disclosure may contain one or more (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) point mutations that add, delete or substitute any amino acid of DAP10 (e.g., an amino acid of wild-type human DAP10 (SEQ ID NO: 1)).

[0021] Truncated forms of the DAP10 polypeptide can also be used in the fusion polypeptides of the present disclosure. For example, a truncated form of DAP10 containing only amino acids 19-93 of SEQ ID NO: 1 (i.e., amino acids 1-18 (signal / leader sequence) are deleted) may be used in the fusion polypeptides of the present disclosure. Such a sequence is referred to herein as SEQ ID NO: 2. Other truncated forms may include amino acids 19-69 of SEQ ID NO: 1, such sequences contain only the extracellular and transmembrane domains of DAP10 and are referred to herein as SEQ ID NO: 3. Further truncated forms of DAP10 used in the present invention may include amino acids 1-71 of SEQ ID NO: 1 (i.e., the signal / leader sequence, extracellular domain, transmembrane domain, and 2 amino acids of the cytoplasmic / intracellular domain), and are referred to herein as SEQ ID NO: 4. Further truncated forms of DAP10 used in the present invention may include amino acids 19-71 of SEQ ID NO: 1 (i.e., the extracellular domain, transmembrane domain, and 2 amino acids of the cytoplasmic / intracellular domain), and are referred to herein as SEQ ID NO: 5. Further truncated forms of DAP10 used in the present invention may include amino acids 70-93 of SEQ ID NO: 1 (i.e., the intracellular domain), and are referred to herein as SEQ ID NO: 6. Further truncated forms of DAP10 used in the present invention may include amino acids 49-93 of SEQ ID NO: 1 (i.e., the transmembrane and cytoplasmic / intracellular domains), and are referred to herein as SEQ ID NO: 7. Further truncated forms of DAP10 used in the present invention may include amino acids 49-69 of SEQ ID NO: 1 (i.e., the transmembrane domain), and are referred to herein as SEQ ID NO: 8.

[0022] Other mutant or truncated forms of the DAP10 polypeptide are also suitable for use in the present disclosure. In some embodiments, such mutant or truncated forms used as functional variants of the DAP10 polypeptide in the present disclosure maintain the activity of the wild-type polypeptide shown in SEQ ID NO: 1.

[0023] In one embodiment, a functional variant of the DAP10 polypeptide of the present disclosure maintains at least 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more) of the activity of the wild-type polypeptide shown in SEQ ID NO: 1. In one embodiment, the activity can be measured by evaluating tyrosine phosphorylation of DAP10 and / or recruitment and activation of the p85 subunit of phosphatidylinositol 3-kinase and downstream anti-apoptotic kinase (AKT).

[0024] DAP12 polypeptide and its functional variants The DAP12 polypeptide used in the fusion polypeptides described herein may be mammalian, such as human. Wild-type human DAP12 is encoded by the amino acid sequence having UniProt accession number O43914 (SEQ ID NO: 9). The first 21 amino acids are thought to be a signal / leader sequence, amino acids 22-40 are the extracellular domain, amino acids 41-61 are the transmembrane domain, and amino acids 62-113 are thought to be the cytoplasmic / intracellular domain.

[0025] In one embodiment, the DAP12 polypeptide used in the fusion polypeptides of the present disclosure comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% sequence identity to the DAP12 polypeptide of SEQ ID NO: 9. In some embodiments, the DAP12 polypeptide used in the fusion polypeptides of the present disclosure comprises the amino acid sequence of SEQ ID NO: 9.

[0026] In another embodiment, a functional variant DAP12 polypeptide used in the fusion polypeptides of the present disclosure may comprise one or more (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) point mutations that add, delete or substitute any amino acid of DAP12 (e.g., the amino acids of wild-type human DAP12 (SEQ ID NO: 9)).

[0027] Truncated forms of the DAP12 polypeptide can also be used in the fusion polypeptides of the present disclosure. For example, a truncated form of DAP12 that includes only amino acids 22 to 113 of SEQ ID NO: 9 (i.e., amino acids 1 to 21 (signal / leader sequence) are deleted) may be used in the fusion polypeptides of the present disclosure. Such a sequence is referred to herein as SEQ ID NO: 10. Other truncated forms may include amino acids 62 to 113 of SEQ ID NO: 9, and such sequences contain only the cytoplasmic / intracellular domain of DAP12 and are referred to herein as SEQ ID NO: 11. Other truncated forms may include amino acids 41 to 61 of SEQ ID NO: 9 (i.e., the transmembrane domain), and are referred to herein as SEQ ID NO: 12. Another truncated form may include amino acids 22 to 61 of SEQ ID NO: 9 (i.e., the extracellular and transmembrane domains), and is referred to herein as SEQ ID NO: 13.

[0028] Other mutant or truncated forms of the DAP12 polypeptide are also suitable for use in the present disclosure. Mutant or truncated forms used as functional variants of the DAP12 polypeptide in the present disclosure may maintain the activity of the wild-type polypeptide shown in SEQ ID NO: 9.

[0029] In one embodiment, the functional variant of the DAP12 polypeptide of the present disclosure maintains at least 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more) of the activity of the wild-type polypeptide shown in SEQ ID NO: 9. In one embodiment, the activity can be measured by measuring cytokine secretion by ELISA using a functional assay such as MTT.

[0030] Polypeptides that associate with the fusion polypeptides of the present invention The fusion polypeptides of the present invention can associate with other polypeptides. Such association can be due to electrostatic forces (e.g., provided by complementary charged amino acids).

[0031] An example of such a polypeptide that can associate with the fusion polypeptide of the present invention is the NKG2D polypeptide (Wu et al., 2000, J. Exp. Med., 192(7):1059-1067 and Rosen et al., 2004, J. Immunol., 173(4):2470-2478).

[0032] In one embodiment, such a polypeptide can be genetically encoded as part of a contiguous chimeric construct having a gene encoding the fusion polypeptide of the present disclosure. The fusion polypeptide and the other polypeptide can then be separated during translation (e.g., using a ribosomal skip peptide), or separated by post-translational cleavage (e.g., using a furin cleavage site). The fusion polypeptide and the other polypeptide can thus be linked by an optional linker that may be present. Such a linker may include a cleavage site to facilitate cleavage.

[0033] NKG2D polypeptide and functional variants thereof The NKG2D polypeptide used in the chimeric polypeptides described herein can be mammalian, such as human. Wild-type human NKG2D is encoded by an amino acid sequence having the UniProt accession number P26718 (SEQ ID NO: 14). The polypeptide is thought to include a cytoplasmic domain (amino acids 1-51), a transmembrane domain (amino acids 52-72) and an extracellular domain (amino acids 73-216).

[0034] In one embodiment, the NKG2D polypeptide used in the present disclosure includes an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% sequence identity to the NKG2D polypeptide of SEQ ID NO: 14. In some embodiments, the NKG2D polypeptide used in the present disclosure includes the amino acid sequence of SEQ ID NO: 14.

[0035] In another embodiment, the functional variant NKG2D polypeptide used in the present disclosure may contain one or more (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) point mutations that add, delete or substitute any amino acid of NKG2D (e.g., the amino acids of wild-type human NKG2D (SEQ ID NO: 14)).

[0036] Truncated forms of the NKG2D polypeptide may also be used in the polypeptides of the present disclosure. For example, a truncated form of NKG2D containing only amino acids 73-216 of SEQ ID NO: 14 (i.e., the extracellular domain) may be used in the present disclosure. Such a sequence is referred to herein as SEQ ID NO: 15. Other truncated forms may include amino acids 82-216 of SEQ ID NO: 14, and such sequences, which contain a portion of the extracellular domain of NKG2D, are referred to herein as SEQ ID NO: 16. A further truncated form includes amino acids 52-216 of SEQ ID NO: 14 (i.e., the transmembrane and extracellular domains), which is referred to herein as SEQ ID NO: 17.

[0037] Other mutant or truncated forms of the NKG2D polypeptide are also useful for use in the present disclosure. Of course, any such mutant or truncated form used as a functional variant of the NKG2D polypeptide in the present disclosure should preferably maintain the activity of the wild-type polypeptide shown in SEQ ID NO: 14.

[0038] In one embodiment, the functional variant of the NKG2D polypeptide of the present disclosure maintains at least 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more) of the activity of the wild-type polypeptide shown in SEQ ID NO: 14. In one embodiment, the activity can be measured by various cell culture assays (e.g., MTT and ELISA) aimed at confirming target cell lysis, cytokine secretion and co-stimulation, using flow cytometry to confirm continuous binding to the NKG2D ligand.

[0039] Linker The DAP10 and DAP12 moieties described in the present disclosure may be directly linked to each other in a continuous polypeptide chain or may be indirectly linked to each other via a suitable linker. The linker may be a peptide linker. Peptide linkers are commonly used in fusion polypeptides, and methods for selecting or designing linkers are well known (see, for example, Chen X et al., 2013, Adv. Drug Deliv. Rev. 65(10):1357 - 01369 and Wriggers W et al., 2005, Biopolymers 80:736 - 746). The linker may be used to link the fusion polypeptide of the present disclosure to another polypeptide (such as an NKG2D polypeptide) within the chimeric construct described above.

[0040] Peptide linkers are generally classified into i) flexible linkers, ii) helix - forming linkers, and iii) cleavable linkers, and examples of each type are known in the art. In one example, a flexible linker is included in the fusion polypeptide described herein. A flexible linker may contain a majority of sterically unhindered amino acids, such as glycine and alanine. The hydrophilic amino acid Ser is also conventionally used in flexible linkers. Examples of flexible linkers include, but are not limited to: polyglycine (e.g., (Gly)4 and (Gly)5), polyalanine, poly(Gly - Ala), and poly(Gly - Ser) (e.g., (Gly n -Ser n ) n or (Ser n -Gly n ) n where each n is independently an integer of 1 or more).

[0041] The peptide linker may be of an appropriate length. The peptide linker sequence may be of a length of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or more amino acid residues. For example, the peptide linker may be about 5 to about 50 amino acids in length; about 10 to about 40 amino acids in length; about 15 to about 30 amino acids in length; or about 15 to about 20 amino acids in length. Variations in the length of the peptide linker can maintain or enhance activity and result in excellent efficacy in activity tests. The peptide linker sequence may consist of natural or non-natural origin amino acids, or may consist of a mixture of both natural and non-natural origin amino acids.

[0042] In some embodiments, the amino acids glycine and serine are included in the amino acids within the linker sequence. In certain embodiments, the linker region comprises a set of glycine repeats (GSG3) n (SEQ ID NO: 18), wherein n is a positive integer of 1 or more (e.g., 1 to about 20). More specifically, the linker sequence may be GSGGG (SEQ ID NO: 19). The linker sequence may be GSGG (SEQ ID NO: 20). In certain other embodiments, the orientation of the linker region comprises a set of glycine repeats (SerGly3) n wherein n is a positive integer of 1 or more (e.g., 1 to about 20) (SEQ ID NO: 21).

[0043] In other embodiments, the linker may contain glycine (G) and serine (S) in a random or repeating pattern. For example, the linker may be (GGGG S) n (SEQ ID NO: 22), wherein n is an integer in the range of 1 to 20, for example 1 to 4. In a specific example, n is 4 and the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 23). In another specific example, n is 3 and the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 24).

[0044] In other embodiments, the linker may include glycine (G), serine (S), and proline (P) in a random or repeating pattern. For example, the linker may be (GPPGS) n wherein n is an integer in the range of 1 to 20, such as 1 to 4. In one specific example, n is 1 and the linker is GPPGS (SEQ ID NO: 25).

[0045] Generally, the linker is not immunogenic when administered to a patient such as a human. Thus, the linker can be selected to be of low immunogenicity or to be considered to have low immunogenicity.

[0046] The linkers described herein are exemplary, and the linker can optionally include other amino acids such as Glu and Lys. The peptide linker can optionally include multiple repeats of, for example, (G3S) (SEQ ID NO: 26), (G4S) (SEQ ID NO: 27), (GYS) (SEQ ID NO: 28), and / or (GlySer) (SEQ ID NO: 29). In certain embodiments, the peptide linker can optionally include multiple repeats of, for example, (SG4) (SEQ ID NO: 30), (SG3) (SEQ ID NO: 31), (SG2) (SEQ ID NO: 32), (SG)2 (SEQ ID NO: 33), or (SerGly) (SEQ ID NO: 34).

[0047] In other embodiments, the peptide linker can include combinations and multiple repeating amino acid sequence units, for example, (G3S)+(G4S)+(GlySer) (SEQ ID NO: 26+SEQ ID NO: 27+SEQ ID NO: 29). In other embodiments, Ser can be replaced with Ala, for example, (G4A) (SEQ ID NO: 35) or (G3A) (SEQ ID NO: 36). In yet other embodiments, the linker includes the motif (EAAAK) n wherein n is a positive integer of 1 or more, such as 1 to about 20 (SEQ ID NO: 37). In certain embodiments, the peptide linker may include a cleavable linker.

[0048] The linker may further comprise additional domains and / or features, such as a furin cleavage site (RRKR) (SEQ ID NO: 38), a P2A ribosome skipping peptide (ATNFSLLKQAGDVEENPGP) (SEQ ID NO: 39) and / or a T2A ribosome skipping peptide (EGRGSLLTCGDVEENPGP) (SEQ ID NO: 40). Examples of linkers containing these domains include the SGSG+P2A ribosome skipping peptide (SGSGATNFSLLKQAGDVEENPGP) (SEQ ID NO: 41), the SGSG+T2A ribosome skipping peptide (SGSGEGRGSLLTCGDVEENPGP) (SEQ ID NO: 42), and versions that also include the furin cleavage site, namely the furin cleavage site+SGSG+P2A ribosome skipping peptide (RRKRSGSGATNFSLLKQAGDVEENPGP) (SEQ ID NO: 43) and the furin cleavage site+SGSG+T2A ribosome skipping peptide (RRKRSGSGEGRGSLLTCGDVEENPGP) (SEQ ID NO: 44). Alternative ribosome skipping peptides that can be used in the present invention include F2A (VKQTLNFDLLKLAGDVESNPGP) (SEQ ID NO: 45) and E2A (QCTNYALLKLAGDVESNPGP) (SEQ ID NO: 46).

[0049] N-terminal sequence and C-terminal sequence Various sequences may be attached to the N-terminus or C-terminus of the fusion polypeptides of the present disclosure, or to the NKG2D polypeptides disclosed herein. These may be functional, such as signal peptides, purification tags / sequences, or half-life extension moieties, or may simply comprise spacer sequences. Alternatively, they may comprise functions such as T cell stimulating functions.

[0050] Purification Tags and Markers To aid purification, various tags or markers may be attached to the N-terminus or C-terminus of the fusion polypeptides of the present disclosure. Any affinity tag may be combined with the fusion polypeptides of the present disclosure to aid purification. Examples of such affinity tags are His tag, FLAG tag, Arg tag, T7 tag, Strep tag, S tag, aptamer tag, V5 tag, AviTag™, myc epitope tag or any combination of these tags. In one embodiment, the affinity tag is a His tag (usually containing 5 to 10 histidine residues), for example, a 6His tag (i.e., HHHHHH) (SEQ ID NO: 47). In another embodiment, the affinity tag is a FLAG tag (i.e., DYKDDDDK) (SEQ ID NO: 48). In another embodiment, the affinity tag is an AviTag™ (i.e., GLNDIFEAQKIEWHE) (SEQ ID NO: 49). In another embodiment, the affinity tag is a V5 tag (GKPIPNPLLGLDST) (SEQ ID NO: 50) or (IPNPLLGLD) (SEQ ID NO: 51). In another embodiment, the affinity tag is a myc epitope tag recognized by the 9e10 antibody (EQKLISEEDL) (SEQ ID NO: 52). Various other tags for use in the present disclosure are well known in the art.

[0051] Combinations of affinity tags containing one or more tags at the N-terminus, one or more tags at the C-terminus, or one or more tags at each of the N-terminus and C-terminus may be used. Examples of such combinations include a His tag (H) combined with an AviTag (A), or a His tag (H) combined with both an AviTag (A) and a FLAG tag (F). The tags may be in either orientation, thus, the AviTag / His tag may have the orientation of N-AH-C or N-HA-C, and the Avi / His / FLAG tag may have the orientation of N-AHF-C, N-FHA-C, etc.

[0052] In one embodiment, the fusion polypeptide according to the present disclosure includes an "AHF" tag having the sequence "GLNDIFEAQKIEWHEGGHHHHHHDYKDDDDK" (SEQ ID NO: 53). In another embodiment, the fusion polypeptide according to the present disclosure includes an "FHA" tag having the sequence "DYKDDDDKHHHHHHGGGLNDIFEAQKIEWHE" (SEQ ID NO: 54).

[0053] The CD8α leader sequence (amino acids 1 - 21 of UniProt: P01732, or a truncated derivative including amino acids 1 - 18) is a commonly used T cell sequence, which is referred to as SEQ ID NO: 55 herein.

[0054] Costimulatory sequence Various T cell costimulatory activation sequences are known from previous studies for engineering CAR - T cells. These can also be added to the fusion polypeptides of the present disclosure.

[0055] The 4 - 1BB endodomain (amino acids 214 - 255 of UniProt: Q07011) can also be used as an N - terminal or C - terminal sequence. The 4 - 1BB endodomain is referred to as SEQ ID NO: 56 herein. The 4 - 1BB endodomain can act as a costimulatory domain.

[0056] The CD27 endodomain (amino acids 213 - 260 of UniProt: P26842) can also be used as an N - terminal or C - terminal sequence. The CD27 endodomain is referred to as SEQ ID NO: 57 herein. The CD27 endodomain can act as a costimulatory domain.

[0057] The human IgG1 hinge (amino acids 218 - 229 of UniProt: P0DOX5) can also be used as an N - terminal or C - terminal sequence. The human IgG1 hinge is referred to as SEQ ID NO: 58.

[0058] The truncated CD8α hinge (amino acids 138 - 182 of Uniprot:P01732) can also be used as an N-terminal or C-terminal sequence. The truncated CD8a hinge is referred to as SEQ ID NO:59.

[0059] Exemplary constructs The present disclosure provides the following exemplary fusion polypeptide constructs in Table 1.

[0060] JPEG2025108507000001.jpg169166

[0061] Furthermore, as described above, the fusion polypeptides of the present disclosure can be expressed as a single chimeric construct having the NKG2D polypeptide for translational or post-translational cleavage. In such constructs, after expression, the translated polypeptide is cleaved to yield separate polypeptides, which then self-associate to form the CAR. In one embodiment, the fusion polypeptides of the present disclosure are cleaved from the NKG2D polypeptide. Examples of such constructs are shown in Table 2.

[0062] JPEG2025108507000002.jpg208166JPEG2025108507000003.jpg221166

[0063] Nucleic acid molecules encoding the fusion polypeptides of the present disclosure Another aspect of the disclosure relates to a nucleic acid molecule encoding a fusion polypeptide or chimeric construct of the disclosure. This may be as DNA or RNA. Unless otherwise specifically limited herein, the term encompasses nucleic acids having similar properties as the reference nucleic acid and including known analogs of natural nucleotides that are metabolized in a manner similar to natural origin nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly recited sequences. In particular, as detailed below, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues (Batzer et al., 1991, Nucleic Acid Res. 19:5081; Ohtsuka et al., 1985, J. Biol. Chem. 260:2605-2608; and Rossolini et al., 1994, Mol. Cell. Probes 8:91-98).

[0064] Accordingly, the disclosure also provides a nucleic acid comprising a nucleotide sequence encoding any one or more of the polypeptide sequences of SEQ ID NOs: 60-69.

[0065] The disclosure further provides a nucleic acid comprising a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% sequence identity to a nucleic acid encoding any of SEQ ID NOs: 60-69. Sequence identity is typically measured along the full length of the reference sequence.

[0066] The present disclosure further provides a nucleic acid comprising a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% sequence identity to any one of SEQ ID NOs: 70-79.

[0067] The present disclosure also provides a nucleic acid comprising a nucleotide sequence of any one of SEQ ID NOs: 70-79. The present disclosure also provides a nucleic acid consisting of a nucleotide sequence of any one of SEQ ID NOs: 70-79.

[0068] The polynucleotide sequence can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence (such as the sequences described in the following examples). Direct chemical synthesis of nucleic acids can be achieved by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method of Brown et al., 1979, Meth. Enzymol. 68:109; the diethylphosphoramidite method of Beaucage et al., 1981, Tetra. Lett., 22:1859; and the solid support method of U.S. Patent No. 4,458,066. Introducing mutations into a polynucleotide sequence by PCR can be performed as described, for example, in PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, N.Y., 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, Calif, 1990; Mattila et al., 1991, Nucleic Acids Res. 19:967; and Eckert et al., 1991, PCR Methods and Applications 1:17.

[0069] Vector The present disclosure also provides vectors comprising one or more nucleic acid molecules of the present disclosure.

[0070] For expression in a host cell, the nucleic acid encoding the fusion polypeptide can be present in a suitable vector and, after introduction into a suitable host, the sequence can be expressed and the encoded fusion polypeptide can be produced according to standard cloning and expression techniques known in the art (e.g., as described in Sambrook, J., Fritsh, E.F., and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). The present disclosure also relates to vectors comprising the nucleic acid sequences according to the present disclosure.

[0071] Using various expression vectors, the polynucleotide encoding the fusion polypeptide of the present disclosure can be expressed. Both viral and non-viral expression vectors can be used to produce the fusion polypeptide in host cells, such as mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors (typically containing an expression cassette for expressing protein or RNA), and human artificial chromosomes (see, for example, Harrington et al., 1997, Nat Genet. 15:345). For example, non-viral vectors useful for the expression of the polynucleotide and polypeptide of the fusion polypeptide of the present disclosure in mammalian (e.g., human) cells include pThioHis A, B, and C, pcDNA3.1 / His, pEBVHis A, B, and C, (Invitrogen, San Diego, Calif.), MPS V vector, and a very many other vectors known in the art for expressing other proteins. Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, SV40, papillomaviruses, HBP Epstein-Barr virus, vaccinia virus vectors, and Semliki Forest virus (SFV). See Brent et al., supra; Smith, 1995, Annu. Rev. Microbiol. 49:807; and Rosenfeld et al., 1992, Cell 68:143. In particular, retroviral, lentiviral, adenoviral, or adeno-associated viral vectors are commonly used for expression in T cells. Examples of such vectors include the SFG retroviral expression vector (see Riviere et al., 1995, Proc. Natl. Acad. Sci. (USA) 92:6733-6737). In one embodiment, lentiviral vectors are used, including self-inactivating lentiviral vectors (so-called SIN vectors).

[0072] The selection of an expression vector depends on the intended host cell in which the vector is to be expressed. Expression vectors for mammalian host cells can include expression control sequences such as origins of replication, promoters, and enhancers (see, e.g., Queen, et al., 1986, Immunol. Rev. 89:49-68), necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination factor sequences. These expression vectors usually contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific, and / or regulatable or inducible. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPS V promoter, the tetracycline-inducible CMV promoter (e.g., the human immediate-early CMV promoter), the constitutive CMV promoter, the EF1α promoter, the phosphoglycerate kinase (PGK) promoter, and promoter-enhancer combinations known in the art.

[0073] The culture of the transformed organism can be expanded under non-inducing conditions without biasing the population towards a particular coding sequence for which the expression product is better tolerated by the host cell. In addition to the promoter, other regulatory elements may also be necessary or desirable for the efficient expression of the antibodies or fragments thereof of the present disclosure. These elements typically include the ATG start codon and adjacent ribosome binding sites or other sequences. Furthermore, the expression efficiency can be enhanced by including enhancers appropriate for the cell line used (see, e.g., Scharf et al., 1994, Results Probl. Cell Differ. 20:125; and Bittner et al., 1987, Meth. Enzymol., 153:516). For example, the SV40 enhancer or the CMV enhancer can be used to increase expression in mammalian host cells.

[0074] The present disclosure provides a cloning vector or an expression vector comprising a nucleic acid having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% sequence identity with a nucleic acid encoding any one of SEQ ID NOs: 60 to 69. Further, the present disclosure provides a cloning vector or an expression vector comprising a nucleic acid encoding one or more of SEQ ID NOs: 60 to 69. The present disclosure provides a cloning vector or an expression vector comprising any one nucleic acid sequence of SEQ ID NOs: 70 to 79.

[0075] Host cell There is provided a host cell comprising the polypeptide of the present disclosure, the nucleic acid of the present disclosure, the vector of the present disclosure, or any combination of one or both thereof. Such cells are generally used for the expression of the fusion polypeptide according to the present disclosure.

[0076] The nucleic acid or vector can be transfected into a host cell by standard techniques.

[0077] The various forms of the term "transfection" are intended to include a wide variety of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like.

[0078] Alternatively, the nucleic acid or vector can be delivered into a host cell by transduction. For example, the viral vectors disclosed above can be used for the delivery of the nucleic acid or vector.

[0079] The fusion polypeptides of the present disclosure can be expressed in prokaryotic or eukaryotic host cells. Representative host cells include many strains of E. coli, mammalian cell lines such as CHO, CHO-K1, and HEK293; insect cells such as Sf9 cells; and yeast cells such as S. cerevisiae and P. pastoris. In one embodiment, the host cell is an immunoresponsive cell, such as an NK cell (primary NK cell, or NK cell line) or a T cell (primary T cell, or T cell line). Other types of host cells include macrophages, induced pluripotent stem cells (iPSCs), neutrophils, and invariant NKT (iNKT) cells. The T cell can be a CD4 + or CD8 + T cell. In one embodiment, the host cell is a human cell. In one embodiment, the host cell is a human T cell. In another embodiment, the host cell is a primary human T cell. Cell lines that can be used include the NK cell line NK-92.

[0080] Mammalian host cells for expressing the fusion polypeptides of the present disclosure include Chinese hamster ovary (CHO cells) (dhfr-CHO cells used with a DHFR selectable marker (e.g., as described in R.J. Kaufman and P.A. Sharp, 1982, Mol. Biol. 159:601-621), as described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220), NSO myeloma cells, COS cells, and SP2 cells. In one embodiment, the host cell is a CHO K1PD cell. In another embodiment, the host cell is an NSO1 cell. In particular, for use with NSO myeloma cells, another expression system is the GS gene expression system shown in WO87 / 04462, WO89 / 01036, and EP338,841. When a recombinant expression vector encoding a fusion polypeptide is introduced into a mammalian host cell, the fusion polypeptide can be produced by culturing the host cell for a period sufficient to allow expression of the fusion polypeptide in the host cell or secretion of the fusion polypeptide into the culture medium in which the host cell has been grown. The fusion polypeptide can be recovered from the culture medium using standard protein purification methods.

[0081] Production method The present disclosure also provides a method of producing immunoreactive cells comprising the fusion polypeptides of the present disclosure. Such a method may include the step of transducing cells with a nucleic acid or vector encoding the fusion polypeptides of the present disclosure. The method may further include the step of culturing the cells such that the fusion polypeptide is expressed and associates with the NKG2D polypeptide to form a CAR.

[0082] In one embodiment, the present disclosure provides a method of preparing immunoreactive cells comprising: (i) the step of transducing an immunoreactive cell with a nucleic acid or vector encoding the fusion polypeptides of the present disclosure, and (ii) the step of culturing the immunoreactive cell such that the fusion polypeptide is expressed and associates with the NKG2D polypeptide to form a CAR.

[0083] In yet another embodiment, the present disclosure provides a method comprising: (i) obtaining T cells and / or NK cells from a patient; (ii) transducing a nucleic acid or vector encoding a fusion polypeptide of the present disclosure into the T cells and / or NK cells; and (iii) culturing the T cells and / or NK cells such that the fusion polypeptide is expressed and associates with an NKG2D polypeptide to form a CAR.

[0084] A variety of methods for culturing immunoreactive cells are well known in the art. See, for example, Parente-Pereira AC et al. 2014, J. Biol. Methods 1(2):e7, Ghassemi S et al. 2018, Cancer Immunol Res 6(9):1100-1109, and Denman CJ et al. 2012, PLoS One 7(1):e30264.

[0085] Composition The present disclosure also provides a pharmaceutical composition comprising a fusion polypeptide, nucleic acid, vector, or host cell described herein. Such pharmaceutical compositions can include a pharmaceutically or physiologically acceptable diluent and / or carrier. The carrier is generally selected to be appropriate for the intended mode of administration and can include agents for modifying, maintaining, or preserving the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or permeability of the composition. Typically, these carriers include aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including saline and / or buffered media.

[0086] Suitable agents for inclusion within the pharmaceutical composition include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antibacterial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (e.g., boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids), fillers (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin), proteins (e.g., free serum albumin, gelatin, or immunoglobulins), coloring agents, flavoring agents and diluents, emulsifying agents, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (e.g., sodium), preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (e.g., glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (e.g., mannitol or sorbitol), suspending agents, surfactants or wetting agents (e.g., pluronic; PEG; sorbitan esters; polysorbates, e.g., polysorbate 20 or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancers (e.g., sucrose or sorbitol), osmotic pressure enhancers (e.g., alkali metal halides, e.g., sodium chloride or potassium chloride, or mannitol sorbitol), delivery vehicles, diluents, excipients and / or formulation adjuvants.

[0087] Parenteral vehicles include saline, Ringer's glucose, glucose and sodium chloride, and lactated Ringer's. Appropriate physiologically acceptable thickeners, such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and alginates, may be included. Intravenous vehicles include those based on fluids and nutritional and electrolyte replenishment, such as Ringer's glucose. Optionally, agents for adjusting the osmotic pressure of the composition, such as saccharides, polyalcohols, such as mannitol, sorbitol, or sodium chloride, may be included in the pharmaceutical composition. For example, in many cases, it is desirable for the composition to be substantially isotonic. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, and inert gases, may be present. The exact formulation depends on the route of administration. Further relevant principles, methods, and components related to pharmaceutical formulations are well known (see, for example, Allen, Loyd V. Ed, (2012) Remington’s Pharmaceutical Sciences, 22 nd Edition).

[0088] The pharmaceutical compositions of the present disclosure can be administered by one or more routes of administration using one or more of the various methods known in the art. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired result. Routes of administration for the pharmaceutical compositions of the present disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, intraspinal or other parenteral routes of administration, such as by injection or infusion. As used herein, the phrase "parenteral administration" means a mode of administration other than enteral and topical administration, and is usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subdural, intrathecal, epidural and intrasternal injections and infusions. In one embodiment, the pharmaceutical composition is administered intratumorally. When attempting parenteral administration, the pharmaceutical composition is usually in the form of a sterile pyrogen-free, parenterally acceptable composition. A vehicle particularly suitable for parenteral injection is a properly preserved sterile isotonic solution. The pharmaceutical composition may be in the form of a lyophilizate, such as a lyophilized cake.

[0089] Alternatively, the pharmaceutical compositions described herein can be administered by non-parenteral routes, such as by topical, epithelial or mucosal routes of administration, such as intranasal, oral, vaginal, rectal, sublingual or topical administration.

[0090] In certain embodiments, the pharmaceutical composition is for subcutaneous administration. Components and methods of suitable formulations for subcutaneous administration of polypeptide therapeutics (e.g., antibodies, fusion polypeptides, etc.) are known in the art, see, for example, US2011 / 0044977, US8465739 and US8476239. Typically, pharmaceutical compositions for subcutaneous administration include suitable stabilizers (e.g., amino acids such as methionine, and / or saccharides such as sucrose), buffers and tonicity agents.

[0091] Typically, in cell therapy, a composition containing host cells is administered to a patient by intravenous infusion.

[0092] Use and method The fusion polypeptide, nucleic acid, vector, host cell or pharmaceutical composition of the present disclosure can be administered to a subject and can be used for treating a disease, preventing and / or delaying the onset of disease symptoms.

[0093] Accordingly, the present disclosure provides the fusion polypeptide, nucleic acid, vector, host cell or pharmaceutical composition of the present disclosure for use in therapy or as a medicament. The present disclosure further provides the fusion polypeptide, nucleic acid, vector, host cell or pharmaceutical composition of the present disclosure for use in treating a pathological disorder. The present disclosure also provides the use of the fusion polypeptide, nucleic acid, vector, host cell or pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating a pathological disorder. The present disclosure further provides a method of treating a patient suffering from a pathological disorder, the method comprising administering to the patient a therapeutically effective amount of the fusion polypeptide, nucleic acid, vector, host cell or pharmaceutical composition of the present disclosure.

[0094] As used herein, the term "pathological disorder" includes, but is not limited to, cancers including solid tumor cancers, soft tissue tumors, metastatic lesions and hematological cancers. For example, cancers include liver cancer, lung cancer, breast cancer, prostate cancer, lymphatic cancer, colon cancer, kidney cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia including: acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, pediatric solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis carcinoma, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, squamous cell carcinoma, squamous cell carcinoma, T cell lymphoma, myelodysplastic syndrome (MDS), chronic myeloid leukemia-chronic phase (CMLCP), diffuse large B cell lymphoma (DLBCL), cutaneous T cell lymphoma (CTCL), peripheral T cell lymphoma (PTCL), hepatocellular carcinoma (HCC), gastrointestinal stromal tumor (GIST), non-small cell lung carcinoma (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), environmentally induced cancers including those induced by asbestos, and combinations of the above cancers. In particular, the cancer may be breast cancer, for example estrogen receptor positive (ERpos) breast cancer and / or metastatic forms of breast cancer.

[0095] In one embodiment, the cancer is a solid tumor cancer. In one embodiment, the treatment of the pathological disorder includes targeting non-tumor cells, such as tumor-associated stromal cells. Exemplary types of such tumor-associated stromal cells include pancreatic stromal cells. Other types of non-tumor cells that can be targeted include macrophages, regulatory T cells and myeloid-derived suppressor cells.

[0096] In one embodiment, the patient has been pre-treated with a chemotherapeutic agent.

[0097] In one embodiment, administration of host cells to a patient results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% reduction in tumor size compared to the untreated tumor.

[0098] The amount of host cells administered to a patient (patent) should take into account the route of administration, the cancer being treated, the patient's weight and / or the patient's age. Generally, about 1×10 6 ~ about 1×10 11 cells are administered to the patient. In one embodiment, about 1×10 7 ~ about 1×10 10 cells, or about 1×10 8 ~ about 1×10 9 cells are administered to the patient.

[0099] General Sequence identity can be determined by standard methods commonly used to compare the similarity at the amino acid positions of two polypeptides. Using a computer program such as BLAST, FASTA or Clustal Omega, the two polypeptides are aligned for optimal matching of their respective amino acids (along the entire length of one or both sequences, or along a predetermined portion of one or both sequences). The program gives a default opening penalty and a default gap penalty, and a scoring matrix such as PAM250 [standard scoring matrix; see Dayhoff et al. (Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978))] can be used with the computer program. For example, the identity (%) can be calculated as follows: multiply the total number of matching matches by 100 and divide it by the length of the longer sequence within the alignment range and the total number of gaps introduced into the longer sequence to align the two sequences.

[0100] Throughout the description and claims of this specification, the words "comprise", "contain" and variations of those words, such as "comprising" and "comprises", are to be interpreted as "including but not limited to", and do not exclude other components, integers or steps. Further, unless the context otherwise requires, the singular form includes the plural: in particular, when an indefinite article is used, the description is to be understood to include the plural as well as the singular, unless the context otherwise requires.

[0101] The term "about" with respect to a numerical value x means, for example, x ± 5%.

[0102] The features of each aspect of the present disclosure may be described in relation to any other aspect. It is clearly intended that, within the scope of this application, the various aspects, embodiments, examples, and alternatives described in the preceding paragraphs, claims and / or the following description and drawings, and in particular their individual features, may be taken independently or in any combination. That is, all embodiments and / or features of any embodiment may be combined in any manner and / or combination, provided such features are not inconsistent.

Examples

[0103] General methods Isolation and retroviral transduction of T cells Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples from healthy donors by density gradient centrifugation. T cells were activated for 48 hours using paramagnetic beads coated with anti-CD3 and anti-CD28 (1:2 T cell:bead ratio). 1×10 6Individual T cells were plated on plates coated with Retronectin that had been pre-treated with 3 mL of retroviral supernatant. Subsequently, each well was treated with 3 mL of fresh viral supernatant and 100 IU / mL IL-2. Retroviral transduction was performed using virus particles produced by stable gibbon ape leukemia virus (GALV)-pseudotyped 293T Vec stable packaging cells. Thereafter, 100 IU / mL + 5% normal human AB serum in RPMI 1640 medium was added to the T cells, and fresh medium and IL-2 (100 IU / mL) were provided thrice a week.

[0104] Flow cytometry T cell transduction and 293T cell transfection were evaluated by flow cytometry and compared to appropriate isotype controls when indicated. To evaluate the expression of NKG2D-based constructs, cells were stained with mouse anti-human CD4-FITC, mouse anti-human NKG2D-PE, and mouse anti-human CD8-APC and appropriately corrected. CD8 + Due to high levels of endogenous NKG2D expression in T cells, transduction efficiency was compared to untransduced CD4 + T cells for NKG2D expression in T cells. Expression of panErbB-specific T4 and TMY CARs was evaluated using biotinylated goat anti-human EGF followed by PE-conjugated streptavidin. Transduction efficiency was calculated by comparison to N1012 + T cells stained with the same reagent. Prior to use, transduction efficiency between constructs was normalized by spiking untransduced T cells at the required ratio. This ensured that all conditions were identical with respect to the total number of CAR + T cells and the overall T cell concentration.

[0105] To perform intracellular staining, transfected 293T cells were fixed in 4% formaldehyde at room temperature for 10 minutes and then washed twice in permeabilization solution (PBS + 0.5% BSA + 0.1% saponin). Subsequently, cells were stained with 500 ng of PE-conjugated anti-human NKG2D or appropriate isotype control in the presence of 100 uL of permeabilization solution. After washing the cells two more times in permeabilization solution, they were analyzed by flow cytometry.

[0106] Dose-response assay 1×10 4 Individual tumor cells were plated at 1×10 per well (100 μL) in 96-well plates and incubated overnight at 37 °C and 5% CO2. After 24 hours, T cells were added at a log2 CAR T cell:tumor cell ratio ranging from 1:1 to 1:64. After 72 hours, the T cells were removed, 100 μL of MTT solution (500 μg / mL) was added, and the plates were incubated at 37 °C and 5% CO2 for approximately 1 hour. After removing the MTT solution, the resulting formazan crystals were solubilized in DMSO (100 μL / well), and the absorbance was measured at 560 nm. The viability of the tumor cells was calculated as follows: (absorbance of monolayer containing T cells / absorbance of monolayer without T cells) * 100.

[0107] Restimulation assay 1×10 5 Individual tumor cells were plated in triplicate wells of 24-well plates and incubated at 37 °C and 5% CO2 for 24 hours. After 24 hours, 1×10 5 individual CAR +1 mL containing T cells was given per well. After 72 hours, the T cells were gently removed and the wells were washed thoroughly with 1 mL of PBS. After removing the PBS, 1 mL of MTT (final concentration 500 μg / mL) was added to each well and the plate was incubated at 37 °C and 5% CO2 for approximately 1 hour. Absorbance was measured at 560 nm in the appropriate wells and the viability of the tumor cells was calculated as detailed in the “Dose response” section. Restimulation was considered successful if the viability of the tumor cells was measured to be less than 50%.

[0108] The T cells removed from the plate were centrifuged at 400 x g for 5 minutes to remove the supernatant. The pellet was resuspended in 3.2 mL of R5 medium and 1 mL was added in triplicate to each well of a fresh tumor monolayer (1 × 10 5 tumor cells per well of a 24-well plate). The total number of T cells was evaluated by trypan blue dye exclusion of the remaining 200 μL aliquot.

[0109] ELISA Secretion of IFN-γ and IL-2 by T cells was evaluated in supernatant aliquots taken 24 hours after the start of co-culture using Duo-set and Ready-Steady-Go ELISA kits, respectively.

[0110] Tumor spheroid formation To produce tumor spheroids, 1 × 10 3 tumor cells and 1 × 10 3 PS1-star cells were added per well of an ultra-low attachment 96-well plate and incubated at 37 °C and 5% CO2 for 72 hours. Spheroid formation was confirmed by visualization with a standard light microscope.

[0111] In vivo 1 × 10 5 individual firefly luciferase (ffLUC)-tagged BxPC3 cells were injected into the peritoneal cavity of NSG mice. Twelve days after tumor inoculation, the mice (n = 5 / group) were given PBS, 4 × 10 6 (N1012 +(N1012(lo)), T4 + or TMY + CAR T cells) or 1 × 10 7 individuals (N1012(hi) or NKG2D) CAR + T cells by intraperitoneal treatment. Alternatively, NSG mice were inoculated i.p. with 1 × 10 6 individuals of ffLUC-tagged H226 malignant mesothelioma cells. Eight days after tumor inoculation, the mice were treated with PBS or 4 × 10 6 individuals of N1012 + T cells. As a control, one group of mice was treated with 4 × 10 6 T cells expressing only NKG2D.

[0112] Tumor growth was monitored by BLI and all data were presented as total light flux (photons / second) or mean total light flux per treatment (photons / second). The mice were closely monitored and weighed three times a week for signs of ill health.

[0113] Example 1: Expression of NKG2D and DAP10 / 12 fusion proteins in 293T cells 293T cells were transfected with an SFG retroviral plasmid backbone containing either the DAP10 / 12 fusion protein N1012 or the NKG2D expression cassette. N1012 (SEQ ID NO: 64) contains a complex comprising an exogenous human NKG2D protein and a fusion exogenous DAP10 / 12 homodimer according to the present invention. The N1012 plasmid contains SEQ ID NO: 74, which encodes SEQ ID NO: 64. Surface expression of NKG2D was evaluated by flow cytometry 72 hours later. NKG2D expression was readily detected on the surface of 293T cells transfected with the N1012 plasmid, but not on the surface of those transfected with the plasmid encoding control NKG2D (Figure 2, upper panel). Considering that NKG2D surface expression is dependent on the expression of DAP10, the lack of NKG2D surface expression could be explained by the absence of DAP10 co-expression in the NKG2D plasmid. To confirm that the lack of surface NKG2D expression in 293T cells transfected with NKG2D was not due to low transfection, intracellular staining for the presence of NKG2D was performed. Importantly, intracellular expression of NKG2D was observed in 293T cells transfected with either the N1012 or NKG2D plasmid (Figure 2, lower panel). This indicates that expression of NKG2D from both constructs was successful and also confirms that DAP10 co-expression is required to achieve surface expression of NKG2D.

[0114] Example 2: Expression of N1012 and NKG2D in primary human T cells Primary human T cells were activated using paramagnetic beads coated with anti-human CD3 and anti-human CD28 antibodies. 48 hours after activation, the T cells were modified by retroviral transduction to express N1012 or NKG2D. Surface expression of NKG2D was evaluated by flow cytometry and co-stained for CD4 and CD8 expression. The percentage of NKG2D expression (Figure 3A) and the median fluorescence intensity (MFI, Figure 3B) were compared to non-transduced T cells. CD8 +Due to the endogenous expression of NKG2D in T cells, the data were gated on CD4 + T cells. As shown, both the NKG2D and N1012 constructs were reproducibly expressed at high levels on the surface of primary human T cells, in contrast to UT T cells or T cells expressing a control CAR (Figure 4).

[0115] Example 3: Evaluation of target cell destruction and recognition by N1012 T cells To evaluate cytotoxicity, N1012 + T cells were co-cultured with 11 different human tumor cell lines representing 5 different tumor types (mesothelioma, ovarian cancer, head and neck squamous cell carcinoma, pancreatic cancer and breast cancer), or tumor-associated stromal cells (PS1) at different E:T ratios. After 72 hours, the T cells were removed and an MTT assay was performed to evaluate tumor cell viability. A minimal decrease in tumor viability was observed when target cells were co-cultured with either UT T cells or T cells expressing NKG2D, but N1012 + T cells demonstrated potent lysis of all target cell lines, even at low E:T ratios (Figure 5 and Figures 7A and B). Analysis of co-culture supernatants by ELISA showed substantial secretion of both interferon-γ (IFN-γ) and interleukin-2 (IL-2) by N1012 + T cells, but not by UT T cells or T cells expressing a control NKG2D construct (Figure 6A and B). These data indicate that N1012 + T cells have the ability to recognize and lyse a wide variety of tumor types, including tumor-associated stromal cells.

[0116] Example 4: Demonstration of destruction of co-cultures of tumor cells and stromal cells by N1012 T cells N1012 + To determine whether N1012 4 T cells maintain the ability to lyse tumor cells when grown in the presence of stromal cells, they were co-cultured with a monolayer containing both tumor and stromal cells. To achieve this, 5×10 4Individual PS1 star cells were mixed and plated into triplicate wells of a 24-well plate. After 24 hours, 1×10 5 individual T cells were added and the plates were incubated for 72 hours. Subsequently, the T cells were removed and the viability of the monolayer of tumor cells and stromal cells was evaluated by MTT assay as detailed in Example 3. Potent lysis of both tumor and stromal cells was observed with N1012 + T cells, but minimal reduction in the viability of target cells was observed when co-cultured with either NKG2D or UT T cells (Figure 7C-D). N1012 + Both N1012

[0117] N1012 + T cells and T cells expressing A2028z CAR mediated lysis equivalent to that of isolated and grown BxPC3_LT cells. a2028zCAR is a pCAR that targets αvβ6 integrin. The targeting portion of this CAR consists of a 20-mer peptide of A20FMDV2 derived from VPI that binds to αvβ6 integrin (SEQ ID NO: 80). This was located downstream of the CD124 signal peptide (aa1-25 of Uniprot reference P24394, SEQ ID NO: 81). The targeting portion was fused via an AAA linker to the partial extracellular domain, transmembrane domain, and intracellular domain of CD28 (aa114-220, Uniprot reference P10747, SEQ ID NO: 82), and the B7-binding residues of CD28 (aa117-122) were replaced with aa410-419 of human c-myc (Uniprot reference P01106, SEQ ID NO: 52). This was fused in-frame with aa52-164 of CD247 (Uniprot reference P20963, SEQ ID NO: 83).

[0118] Example 5: Demonstration of tumor spheroid destruction by N1012 T cells N1012 +To evaluate the ability of T cells to mediate target cell lysis within a 3D system, they were co-cultured with tumor spheroids. After spheroid generation, 6 × 10 3 CellTracker Violet-labeled T cells were added per well. The viability of tumor cells and astrocytes was evaluated by measuring GFP and RFP respectively using a fluorescence microscope at 72 hours and 192 hours. Quantification of GFP and RFP signals was performed using ImageJ software and represented as a percentage of fluorescence readings from spheroids grown in the absence of T cells. Potent lysis of spheroids was observed with N1012 + T cells but not with NKG2D or UT T cells (Figure 7I-J). Furthermore, only N1012 + T cells showed secretion of IFN-γ (Figure 7K).

[0119] Spheroid viability and T cell proliferation were evaluated using flow cytometry. To achieve this, spheroids were removed from plates at either 72 hours or 192 hours after addition of T cells and placed into flow cytometry tubes (up to a maximum of 5 spheroids treated with the same CAR T cells were added to the same tube). Disaggregation of spheroids was achieved using Accutase solution and resuspended thoroughly through a pipette tip. The resulting single cell suspension was washed in RPMI1640 medium + 5% normal human AB serum and then resuspended in PBS containing counting beads. The same number of counting beads was obtained per tube and the number of resulting tumor cells (evaluated by GFP and RFP fluorescence) and T cells (evaluated by CellTracker Violet fluorescence) was determined. Data are shown as a percentage of the total of GFP and RFP cells present in spheroids grown in the absence of T cells.

[0120] Example 6: Demonstration of continuous target recognition by N1012 T cells N1012 +To evaluate the ability of T cells to undergo serial lysis (“restimulation”) of target cells, they were co-cultured with fresh monolayers twice weekly until no single layer disruption was observed. UT or NKG2D + T cells mediated minimal target cell destruction and showed no evidence of proliferation, but N1012 + T cells mediated potent lysis through multiple rounds of restimulation (Figures 8A–B). Target cell destruction was associated with N1012 + substantial proliferation and expansion of T cells (Figures 8C–D).

[0121] Compared to CYAD-01 NKG2D CAR and control T cells, N1012 + T cells underwent significantly more rounds of restimulation against BxPC3_LT cells (Figure 14A). Furthermore, N1012 + T cells also showed substantially greater proliferation than CYAD-01 T cells or controls (Figure 14B).

[0122] Example 7: Efficacy of N1012 T cells in an in vivo model of pancreatic cancer N1012 + To determine the ability of N1012 T cells to target tumor cells in vivo, T cells expressing N1012, NKG2D or two different repeats of pan-ErbB targeting CAR (T4 and TMY) were produced. Expression of the various constructs in primary human T cells (Figure 9A), and the efficacy of the T cells against three cell lines after 72 hours of co-culture at a 1:1 ratio were shown in vitro (Figure 9B). To evaluate function in vivo, intraperitoneal firefly luciferase (ffLUC)-tagged BxPC3 tumors were established in NSG mice for 12 days. Tumor-bearing mice were intraperitoneally treated with PBS, 4×10 6 cells (N1012(lo), T4 or TMY), or 1×10 7 cells (N1012(hi) or NKG2D) of transduced T cells. Tumor growth was measured weekly by bioluminescence imaging and the mice were weighed three times a week. Data are shown as both mean total luminescence (photons / second) per treatment group (Figure 10A), or total luminescence (photons / second) per individual mouse (Figure 10B). NKG2D+ , T4 + or TMY + The tumor burden in mice administered with T cells was the same as that in the case of PBS administration, suggesting no efficacy. In contrast, the tumors were completely eradicated in 2 / 5 and 4 / 5 mice treated with N1012(lo) or N1012(hi), respectively. These mice maintained no tumors 76 days after T cell administration. No evidence of toxicity was observed when evaluated by measurement of body weight change (%) (Figure 10C).

[0123] N1012 + To determine whether T cells can engraft in NSG mice and provide immunological memory, mice with completely rejected tumors were given a second inoculation of 1 × 10 5 individual ffLUC-tagged BxPC3 cells into the peritoneal cavity 88 days after the first tumor inoculation (76 days after T cell infusion). An increase in luminescence was observed by BLI 24 hours after tumor rechallenge, but 4 / 5 of the mice then showed a substantial decrease in tumor size, thus suggesting + reactivation of N1012

[0124] N1012 against pancreatic cancer model + To further confirm the efficacy of N1012 7 T cells in vivo, repeated experiments were conducted. 1 × 10 + individual CAR 5 or control non-transduced T cells were injected i.p. into NSG mice 12 days after inoculation of 1 × 10 + individual ffLUC-tagged BxPC3 cells. Tumor growth was monitored weekly by bioluminescence imaging. The data are shown both as the mean total light intensity (photons / second) per treatment group (Figure 11A), and the total light intensity (photons / second) per individual mouse (Figure 11B). A significant and sustained tumor regression was again observed in mice treated with N1012 +It was completely eradicated in 5 / 6 of the mice treated with T cells. In contrast, the kinetics of tumor growth in mice treated with UT T cells were identical to those when PBS was administered. From these data, it was confirmed that tumor eradication was specific to N1012 + was confirmed.

[0125] To examine the potential formation of memory T cells, on day 41 (29 days after T cell infusion), tumor-free mice were rechallenged i.p. with a fresh bolus of 1×10 5 individual ffLUC-tagged BxPC3 cells. Mice were imaged on day 42 to confirm tumor take. Subsequent imaging showed that 5 / 5 of the rechallenged mice had reduced their tumor burden to below detection, and 3 / 5 of the mice showed long-term tumor control (Figure 11B). These data suggest that N1012 CAR T cells are capable of forming memory and of being reactivated in response to target reappearance.

[0126] Example 8: Efficacy of N1012 T cells in an in vivo model of malignant mesothelioma To confirm the efficacy of N1012 in another in vivo model, NSG mice were inoculated i.p. with 1×10 6 individual ffLUC-tagged H226 malignant mesothelioma cells. Eight days after tumor inoculation, the mice were treated with PBS or 4×10 6 individual N1012 + T cells. As a control, one group of mice was treated with 4×10 6 individual T cells expressing NKG2D only. Tumor growth was monitored weekly by bioluminescence imaging. The data are shown as the mean total light intensity per treatment (photons / second) (Figure 12A) and the total light intensity per individual mouse (photons / second) (Figure 12B). Consistent tumor growth was observed in mice administered PBS, but 100% tumor eradication was observed in mice administered N1012 + T cells.

[0127] To confirm T cell persistence and function maintenance, in all tumor-free mice, 91 days after the first tumor inoculation, an additional 1×10 6 individual ffLUC-tagged H226 cells were inoculated i.p. Tumor uptake was confirmed in all mice 24 hours later by bioluminescence imaging. All rechallenge mice rejected the tumors, and the persistence of N1012 + T cells and the ability of these T cells to mediate long-term tumor control were confirmed.

[0128] Example 9: Comparison of N1012 T cells with CYAD-01 T cells The potential for restimulation and proliferation of N1012 T cells was compared to CYAD-01 T cells. As previously described, the CYAD-01 CAR consists of the fusion of NKG2D to CD3ζ (Zhang et al, 2005, Blood 106:1544-1551). Nominally a first-generation CAR, it associates with endogenous DAP10 in T cells, meaning that both signal 1 and signal 2 are provided. This CAR is currently in clinical development as CYAD-01 by Celyad S.A., and is therefore provided in these examples only for comparison purposes.

[0129] Surface expression of CYAD-01 was confirmed in primary human T cells when evaluated by flow cytometry (Figure 13).

[0130] Briefly, N1012 or CYAD-01 T cells were co-cultured with fresh monolayers twice a week until no single-layer disruption was observed. To achieve this, 1×10 5 individual tumor cells were plated in triplicate wells of a 24-well plate and incubated at 37°C and 5% CO2 for 24 hours. After 24 hours, 1×10 5 individual CAR+ T cells were added at 1×10 5Added at a final concentration of [[number]] CAR+ / mL. After 72 hours, the T cells were gently removed and the wells were washed with 1 mL of PBS. After removal of the PBS, 1 mL of MTT (final concentration 500 μg / mL) was added to each well and the plate was incubated at 37 °C and 5% CO2 for approximately 1 hour. The plate was read and tumor cell viability was calculated as detailed above. Restimulation was considered successful if tumor cell viability was measured to be less than 50%.

[0131] To examine T cell proliferation in response to recognition of target cells, the T cells removed from the plate were spinoculated at 400 x g for 5 minutes and the supernatant was removed. The pellet was resuspended in 3.2 mL of R5 medium and 1 mL was added in triplicate to each well of a fresh tumor monolayer. The total number of T cells was evaluated by trypan blue dye exclusion of the remaining 200 μL aliquot.

[0132] When compared to CYAD-01, NKG2D CAR, and control T cells, N1012 + T cells received significantly more rounds of restimulation against BxPC3_LT cells (Figure 14A). Furthermore, N1012 T cells also showed substantially greater proliferation than CYAD-01 T cells or controls (Figure 14B).

[0133] When co-cultured with tumor spheroids, a significant decrease in spheroid viability was observed with N1012 + T cells but not with UT control T cells or T cells expressing a functional CYAD-01 CAR (Figure 15A). N1012 T cells also showed significant proliferation compared to UT or CYAD-01 T cells (Figure 15B).

[0134] Sequence Listing SEQ ID NO: 1 (Full human DAP10 sequence) MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRG

[0135] Accession number 2 (DAP10 aa19-93, lacking the leader sequence) QTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRG

[0136] Accession number 3 (DAP10 aa19-69, extracellular / membrane-spanning domain) QTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVF

[0137] Accession number 4 (DAP10 aa1-71) MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLC

[0138] Accession number 5 (DAP10 aa19-71) QTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLC

[0139] Accession number 6 (DAP10 aa70-93, intracellular domain) LCARPRRSPAQEDGKVYINMPGRG

[0140] Accession number 7 (DAP10 aa49-93, membrane-spanning and intracellular domains) LLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRG

[0141] Accession number 8 (DAP10 aa49-69, membrane-spanning domain) LLAGLVAADAVASLLIVGAVF

[0142] Accession number 9 (full-length human DAP12 sequence) MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK

[0143] Sequence number 10 (DAP12 aa22 - 113 with the leader sequence deleted) LRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK

[0144] Sequence number 11 (DAP12 aa62 - 113 - cytoplasmic / intracellular domain) YFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK

[0145] Sequence number 12 (DAP12 aa41 - 61 - transmembrane domain) GVLAGIVMGDLVLTVLIALAV

[0146] Sequence number 13 (DAP12 aa22 - 61 - extracellular and transmembrane domains) LRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAV

[0147] Sequence number 14 (full - length human NKG2D) MGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNW YESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV

[0148] Accession No. 15 (Human NKG2D aa73-216 - extracellular domain) IWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV

[0149] Accession No. 16 (Human NKG2D aa82-216 - extracellular domain) LFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV

[0150] Accession No. 17 (Human NKG2D aa52-216 - transmembrane and extracellular domains) PFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV

[0151] Accession No. 18 (Linker) GSG

[0152] Accession No. 19 (Linker) GSGGG

[0153] Accession No. 20 (Linker) GSGG

[0154] Accession No. 21 (Linker) SGGG

[0155] Accession No. 22 (Linker) GGGGS

[0156] SEQ ID NO: 23 (Linker) GGGGSGGGGSGGGGSGGGGS

[0157] SEQ ID NO: 24 (Linker) GGGGSGGGGSGGGGS

[0158] SEQ ID NO: 25 (Linker) GPPGS

[0159] SEQ ID NO: 26 (Linker) GGGS

[0160] SEQ ID NO: 27 (Linker) GGGGS

[0161] SEQ ID NO: 28 (Linker) GYS

[0162] SEQ ID NO: 29 (Linker) GS

[0163] SEQ ID NO: 30 (Linker) SGGGG

[0164] SEQ ID NO: 31 (Linker) SGGG

[0165] SEQ ID NO: 32 (Linker) SGG

[0166] SEQ ID NO: 33 (Linker) SGSG

[0167] SEQ ID NO: 34 (Linker) SG

[0168] SEQ ID NO: 35 (Linker) GGGGA

[0169] SEQ ID NO: 36 (Linker) GGGA

[0170] Array number 37 (Linker) EAAAK

[0171] Array number 38 (Furin cleavage site) RRKR

[0172] Array number 39 (P2A skip peptide) ATNFSLLKQAGDVEENPGP

[0173] Array number 40 (T2A skip peptide) EGRGSLLTCGDVEENPGP

[0174] Array number 41 (SGSG + P2A) SGSGATNFSLLKQAGDVEENPGP

[0175] Array number 42 (SGSG + T2A) SGSGEGRGSLLTCGDVEENPGP

[0176] Array number 43 (Furin + SGSG + P2A) RRKRSGSGATNFSLLKQAGDVEENPGP

[0177] Array number 44 (Furin + SGSG + T2A) RRKRSGSGEGRGSLLTCGDVEENPGP

[0178] Array number 45 (F2A skip peptide) VKQTLNFDLLKLAGDVESNPGP

[0179] Array number 46 (E2A skip peptide) QCTNYALLKLAGDVESNPGP

[0180] Array number 47 (His tag) HHHHHH

[0181] Array number 48 (FLAG tag) DYKDDDDK

[0182] Sequence number 49 (Avi tag) GLNDIFEAQKIEWHE

[0183] Sequence number 50 (V5 tag) GKPIPNPLLGLDST

[0184] Sequence number 51 (V5 tag) IPNPLLGLD

[0185] Sequence number 52 (Myc tag) EQKLISEEDL

[0186] Sequence number 53 (AHF tag) GLNDIFEAQKIEWHEGGHHHHHHDYKDDDDK

[0187] Sequence number 54 (FHA tag) DYKDDDDKHHHHHHGGGLNDIFEAQKIEWHE

[0188] Sequence number 55 (CD8α leader sequence) MALPVTALLLPLALLLHAARP

[0189] Sequence number 56 (4-1BB end domain) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0190] Sequence number 57 (CD27 end domain) QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP

[0191] Sequence number 58 (human IgG1 hinge) EPKSCDKTHTCP

[0192] Sequence number 59 (truncated CD8α hinge) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0193] Sequence number 60 MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK

[0194] Sequence number 61 MALPVTALLLPLALLLHAARPDYKDDDDKQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK

[0195] Sequence number 62 MALPVTALLLPLALLLHAARPDYKDDDDKEPKSCDKTHTCPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK

[0196] Sequence number 63 MALPVTALLLPLALLLHAARPDYKDDDDKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK

[0197] Sequence number 64 (Construct 1 / N1012) MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV

[0198] Sequence number 65 (Construct 3) MALPVTALLLPLALLLHAARPDYKDDDDKQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRSGSGEGRGSLLTCGDVEENPGPMIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV

[0199] Sequence number 66 (Construct 8) MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVRRKRSGSGEGRGSLLTCGDVEENPGPMIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0200] Sequence number 67 (Construct 9) MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVRRKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAARPDYKDDDDKQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0201] Sequence number 68 (Construct 10) MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVRRKRSGSGEGRGSLLTCGDVEENPGPMIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP

[0202] SEQ ID NO: 69 (Construct 11) MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVRRKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAARPDYKDDDDKQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP

[0203] SEQ ID NO: 70 (encoding the polypeptide of SEQ ID NO: 60) ATGATCCACCTGGGCCACATCCTGTTCCTGCTGCTGCTGCCCGTGGCCGCTGCCCAGACCACCCCTGGCGAGCGGAGCAGCCTGCCTGCCTTCTACCCTGGCACCAGCGGCAGCTGCAGCGGCTGCGGCAGCCTGAGCCTGCCCCTGCTGGCCGGCCTGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATCGTGGGCGCCGTGTTCCTGTGCGCCAGGCCCAGGCGGAGCCCtGCCCAGGAGGACGGCAAGGTGTACATCAACATGCCCGGCCGGGGCTACTTCCTGGGCAGGCTGGTGCCCAGGGGCAGGGGCGCTGCCGAGGCTGCCACCCGGAAGCAGCGGATCACCGAGACCGAGAGCCCCTACCAGGAGCTGCAGGGCCAGCGGAGCGACGTGTACAGCGACCTGAACACCCAGAGGCCCTACTACAAG

[0204] SEQ ID NO: 71 (encoding the polypeptide of SEQ ID NO: 61) ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCTAGACCCGATTATAAGGACGACGACGACAAGCAGACCACCCCTGGCGAGCGGAGCAGCCTGCCTGCCTTCTACCCTGGCACCAGCGGCAGCTGCAGCGGCTGCGGCAGCCTGAGCCTGCCCCTGCTGGCtGGCCTGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATCGTGGGCGCCGTGTTCTACTTCCTGGGCAGGCTGGTGCCCAGGGGCAGGGGCGCTGCCGAGGCTGCCACCCGGAAGCAGCGGATCACCGAGACCGAGAGCCCCTACCAGGAGCTGCAGGGCCAGCGGAGCGACGTGTACAGCGACCTGAACACCCAGAGGCCCTACTACAAG

[0205] SEQ ID NO: 72 (encoding the polypeptide of SEQ ID NO: 62) ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCTAGACCCGATTATAAGGACGACGACGACAAGGAGCCCAAGAGCTGCGACAAGACACACACATGCCCTCTTctggccggCCTGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATCGTGGGCGCCGTGTTCCTGTGCGCCAGGCCCAGGCGGAGCCCtGCCCAGGAGGACGGCAAGGTGTACATCAACATGCCCGGCCGGGGCTACTTCCTGGGCAGGCTGGTGCCCAGGGGCAGGGGCGCTGCCGAGGCTGCCACCCGGAAGCAGCGGATCACCGAGACCGAGAGCCCCTACCAGGAGCTGCAGGGCCAGCGGAGCGACGTGTACAGCGACCTGAACACCCAGAGGCCCTACTACAAG

[0206] SEQ ID NO: 73 (encoding the polypeptide of SEQ ID NO: 63) ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCTAGACCCGATTATAAGGACGACGACGACAAGACCACAACACCTGCTCCTAGACCTCCCACCCCTGCTCCCACCATCGCCAGCCAGCCCCTGAGCCTGAGACCCGAGGCCTGCAGACCCGCTGCTGGCGGCGCTGTGCATACCAGAGGCCTGGATTTCGCCTGCGACCTTctggccggCCTGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATCGTGGGCGCCGTGTTCCTGTGCGCCAGGCCCAGGCGGAGCCCtGCCCAGGAGGACGGCAAGGTGTACATCAACATGCCCGGCCGGGGCTACTTCCTGGGCAGGCTGGTGCCCAGGGGCAGGGGCGCTGCCGAGGCTGCCACCCGGAAGCAGCGGATCACCGAGACCGAGAGCCCCTACCAGGAGCTGCAGGGCCAGCGGAGCGACGTGTACAGCGACCTGAACACCCAGAGGCCCTACTACAAG

[0207] SEQ ID NO: 74 (encoding the polypeptide of SEQ ID NO: 64 / Construct 1 / N1012)

[0208] Array number 75 (encoding the polypeptide of array number 65 / structure 3)

[0209] SEQ ID NO: 76 (encoding the polypeptide of SEQ ID NO: 66 / Construct 8)

[0210] Sequence number 77 (encoding the polypeptide of sequence number 67 / construct 9)

[0211] SEQ ID NO: 78 (encoding the polypeptide of SEQ ID NO: 68 / Construct 10)

[0212] SEQ ID NO: 79 (encoding the polypeptide of SEQ ID NO: 69 / Construct 11)

[0213] SEQ ID NO: 80 (A20FMDV2 peptide) NAVPNLRGDLQVLAQKVART

[0214] SEQ ID NO: 81 (CD124 signal peptide) MGWLCSGLLFPVSCLVLLQVASSGN

[0215] SEQ ID NO: 82 (CD28 aa114~220) IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0216] SEQ ID NO: 83 (CD247 aa52~164) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0217] SEQ ID NO: 84 (SEQ ID NO: 1 of WO2019 / 182425) MGWSCIILFLVATATGVHSQIQLVQSGPELKKPGETVKISCKTSGYTFTDYSMHWVNQAPGKGLKWMGWINTETGEPTYTDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARTAVYWGQGTTLTVSSGSTSGSGKPGSGEGSDIQMTQSPSSLSASLGERVSLTCRASQEISGSLSWLQQKPDGTIKRLIYAASTLNSGVPKRFSGRRSGSDYSLTISSLESEDFVDYYCLQYSSYPWSFGGGTKLEIKEPKSPDKTHTCPPCPSHTQPLGVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGKFWVLVVVGGVLACYSLLVTVAFIIFWVARPRRSPAQEDGKVYINMPGRGGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0218] Sequence number 85 (Sequence number 9 of WO2019 / 182425) ARPRRSPAQEDGKVYINMPGRG

[0219] Sequence number 86 (Sequence number 11 of WO2019 / 182425) GRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK

Claims

Claim 1 A fusion polypeptide comprising (i) a DNAX-activating protein 10 (DAP10) polypeptide, or a functional variant thereof, and (ii) a DNAX-activating protein 12 (DAP12) polypeptide, or a functional variant thereof. Claim 2 The fusion polypeptide according to claim 1, having, from the N-terminus to the C-terminus, the formula: A - B - C - D - E wherein here A = an optional N-terminal sequence B = a DAP10 polypeptide or a functional variant thereof C = an optional linker sequence D = a DAP12 polypeptide or a functional variant thereof E = an optional C-terminal sequence and being a fusion polypeptide. Claim 3 The fusion polypeptide according to claim 1 or 2, wherein the DAP10 polypeptide and / or the DAP12 polypeptide is a mammalian sequence. Claim 4 The fusion polypeptide according to any one of the preceding claims, wherein the DAP10 polypeptide and / or the DAP12 polypeptide is a human sequence. Claim 5 The fusion polypeptide according to any one of the preceding claims, wherein the DAP10 polypeptide is a functional variant of DAP10 comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% sequence identity to the DAP10 polypeptide of SEQ ID NO:

1. Claim 6 The fusion polypeptide according to any one of the preceding claims, wherein the DAP10 polypeptide is a functional variant of SEQ ID NO: 1 having one or more (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) point mutations that add, delete or substitute any of the amino acids of the DAP10 polypeptide of SEQ ID NO:

1. Claim 7 The fusion polypeptide according to any one of the preceding claims, wherein the DAP10 polypeptide is a functional variant of a truncated form of the DAP10 polypeptide of SEQ ID NO:

1. Claim 8 The fusion polypeptide according to claim 7, wherein the truncated form of DAP10 comprises or consists of amino acids 19 - 93, 19 - 69, 1 - 71, 19 - 71, 19 - 48, 49 - 69, 49 - 93, or 70 - 93 of SEQ ID NO:

1. Claim 9 The fusion polypeptide according to any one of claims 1 to 4, wherein the DAP10 polypeptide comprises, or consists of, any one of SEQ ID NOs: 1 to 8.

10. The fusion polypeptide according to any one of the preceding claims, wherein the DAP12 polypeptide is a functional variant of DAP12 comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% sequence identity to the DAP12 polypeptide of SEQ ID NO:

9.

11. The fusion polypeptide according to any one of the preceding claims, wherein the DAP12 polypeptide is a functional variant of SEQ ID NO: 8 having one or more (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) point mutations that add, delete or substitute any of the amino acids of the DAP12 polypeptide of SEQ ID NO:

9.

12. The fusion polypeptide according to any one of the preceding claims, wherein the DAP12 polypeptide is a functional variant of a truncated form of the DAP12 polypeptide of SEQ ID NO:

9.

13. The fusion polypeptide according to claim 12, wherein the truncated form of DAP12 comprises, or consists of, amino acids 22 - 113, 62 - 113, 22 - 61 or 41 - 61 of SEQ ID NO:

9.

14. The fusion polypeptide according to any one of claims 1 to 9, wherein the DAP12 polypeptide comprises, or consists of, any one of SEQ ID NOs: 9 to 13.

15. The fusion polypeptide according to any one of the preceding claims, wherein the DAP10 polypeptide and the DAP12 polypeptide are linked by a linker.

16. The fusion polypeptide according to claim 15, wherein the linker comprises, or consists of, an amino acid sequence set forth in any of SEQ ID NOs: 18 to 46.

17. The fusion polypeptide according to claim 15 or 16, wherein the linker comprises, or consists of, an amino acid sequence set forth in SEQ ID NO: 33 or any of SEQ ID NOs: 38 to 44.

18. The fusion polypeptide according to any one of the preceding claims, wherein the fusion polypeptide comprises an N-terminal sequence.

19. The fusion polypeptide according to any one of the preceding claims, wherein the fusion polypeptide comprises a C-terminal sequence.

20. The fusion polypeptide according to claim 18 or 19, wherein the N-terminal or C-terminal sequence comprises one or more of a His tag, a FLAG tag, an Arg tag, a T7 tag, a Strep tag, an S tag, an AviTag (trademark), an aptamer tag, a myc tag, a CD8α leader sequence, a 4-1BB end domain, a V5 tag, or a CD27 end domain.

21. The fusion polypeptide according to claim 20, wherein the N-terminal or C-terminal sequence comprises one or more of a CD8α leader sequence, a 4-1BB end domain or a CD27 end domain.

22. The fusion polypeptide according to any one of the preceding claims, comprising, or consisting of, the sequence set forth in any one of SEQ ID NOs: 60-63.

23. A fusion polypeptide according to any one of claims 1-4, wherein (a) does not include SEQ ID NO: 84; and / or (b) does not include an anti-EpCAM peptide; and / or (c) does not include SEQ ID NO: 85; and / or (d) does not include SEQ ID NO: 86; and / or (e) does not include both SEQ ID NO: 85 and SEQ ID NO:

86. Fusion polypeptide.

24. The fusion polypeptide according to any one of the preceding claims, which is part of a continuous chimeric polypeptide further comprising an NKG2D polypeptide.

25. The fusion polypeptide according to any one of claims 1-23, wherein the fusion polypeptide is electrostatically associated with an NKG2D polypeptide.

26. The fusion polypeptide according to claim 24 or 25, wherein the NKG2D polypeptide comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% sequence identity to the human NKG2D polypeptide of SEQ ID NO:

14.

27. The fusion polypeptide according to any one of claims 24-26, wherein the NKG2D polypeptide is a functional variant of SEQ ID NO: 14 having one or more (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) point mutations that add, delete or substitute any of the amino acids of the NKG2D polypeptide of SEQ ID NO:

14.

28. The fusion polypeptide according to any one of claims 24 to 27, wherein the NKG2D polypeptide is a functional variant of the truncated NKG2D polypeptide of SEQ ID NO:

14.

29. The fusion polypeptide according to any one of claims 24 to 28, wherein the truncated form of NKG2D comprises or consists of amino acids 52 to 216, 73 to 216 or 82 to 216 of SEQ ID NO:

14.

30. The fusion polypeptide according to any one of claims 24 or 26 to 29, wherein the continuous chimeric polypeptide comprises or consists of any one of the sequences of SEQ ID NOs: 64 to 69.

31. An isolated nucleic acid sequence encoding the fusion polypeptide according to any one of the preceding claims.

32. The isolated nucleic acid according to claim 31, comprising a nucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% sequence identity with a nucleic acid encoding any one of SEQ ID NOs: 60 to 69.

33. The isolated nucleic acid according to claim 31, comprising or consisting of any one of the nucleotide sequences of SEQ ID NOs: 70 to 79.

34. A vector comprising the nucleic acid according to any one of claims 31 to 33.

35. The vector according to claim 34, which is a lentiviral vector or a retroviral vector.

36. A host cell comprising the fusion polypeptide according to any one of claims 1 to 30.

37. A host cell comprising the nucleic acid according to any one of claims 31 to 33 or the vector according to claim 34 or 35.

38. The host cell according to claim 36 or 37, which is a T cell or an NK cell.

39. A method for producing the fusion polypeptide according to any one of claims 1 to 30, comprising the step of maintaining the host cell according to claim 37 or 38 under conditions suitable for the expression of the nucleic acid, whereby the nucleic acid is expressed and the fusion polypeptide is produced. Method.

40. A method for producing immunoreactive cells, comprising the step of (i) transducing the immunoreactive cells with the nucleic acid according to any one of claims 31 to 33 or the vector according to claim 34 or 35, and (ii) culturing the immune-responsive cells such that the fusion polypeptide is expressed and associates with the NKG2D polypeptide to form a CAR comprising a method.

41. (i) obtaining T cells and / or NK cells from a patient, (ii) transducing the T cells and / or NK cells with the nucleic acid according to any one of claims 31 to 33 or the vector according to claim 34 or 35, and (iii) culturing the T cells and / or NK cells such that the fusion polypeptide is expressed and associates with the NKG2D polypeptide to form a CAR, a method.

42. A pharmaceutical composition comprising the fusion polypeptide according to any one of claims 1 to 30, the nucleic acid according to any one of claims 31 to 33, the vector according to claim 34 or 35, or the host cell according to any one of claims 36 to 38.

43. The pharmaceutical composition according to claim 42, further comprising a pharmaceutically or physiologically acceptable diluent and / or carrier.

44. For use in therapy or as a medicament, the fusion polypeptide according to any one of claims 1 to 30, the nucleic acid according to any one of claims 31 to 33, the vector according to claim 34 or 35, the host cell according to any one of claims 36 to 38, or the pharmaceutical composition according to claim 42 or 43.

45. For use in the treatment of a pathological disorder, the fusion polypeptide according to any one of claims 1 to 30, the nucleic acid according to any one of claims 31 to 33, the vector according to claim 34 or 35, the host cell according to any one of claims 36 to 38, or the pharmaceutical composition according to claim 42 or 43.

46. Use of the fusion polypeptide according to any one of claims 1 to 30, the nucleic acid according to any one of claims 31 to 33, the vector according to claim 34 or 35, the host cell according to any one of claims 36 to 38, or the pharmaceutical composition according to claim 42 or 43, in the manufacture of a medicament for the treatment of a pathological disorder.

47. A method of treating a patient suffering from a pathological disorder, comprising administering to the patient a therapeutically effective amount of the fusion polypeptide according to any one of claims 1 to 30, the nucleic acid according to any one of claims 31 to 33, the vector according to claim 34 or 35, the host cell according to any one of claims 36 to 38, or the pharmaceutical composition according to claim 42 or 43, Method.

48. wherein the pathological disorder is solid tumor cancer, soft tissue tumor, metastatic lesion and hematological cancer, such as liver cancer, lung cancer, breast cancer, prostate cancer, lymphatic cancer, colon cancer, kidney cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia including the following: acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, pediatric solid tumor, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis carcinoma, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell carcinoma, T cell lymphoma, myelodysplastic syndrome (MDS), chronic myeloid leukemia - chronic phase (CMLCP), diffuse large B cell lymphoma (DLBCL), cutaneous T cell lymphoma (CTCL), peripheral T cell lymphoma (PTCL), hepatocellular carcinoma (HCC), gastrointestinal stromal tumor (GIST), non-small cell lung carcinoma (NSCLC), squamous cell carcinoma of the head and neck (SCCHN), environmentally induced cancer including those induced by asbestos, and cancer selected from combinations of the foregoing cancers, the fusion polypeptide, nucleic acid, vector, host cell or pharmaceutical composition, use, or method according to any one of claims 44 to 47.

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