Compositions and methods for use in depleting senescent cells and treating aging

Abstract

Methods of depleting senescent cells in a subject, thereby treating aging and senescence- related diseases and disorders, are presented. The methods comprise administering immunoresponsive cells comprising an NKG2D polypeptide, a fusion polypeptide comprising a DNAX-activating 10 (DAP10) polypeptide and a DNAX-activating protein 12 (DAP12) polypeptide, and, optionally, a CXCR2 polypeptide.

Description

COMPOSITIONS AND METHODS FOR USE IN DEPLETING SENESCENT CELLS AND TREATING AGING1. BACKGROUND

[0001] Aging is characterized by the accumulation of proinflammatory senescent cellswithin tissues (“inflammaging”). Senesence is a stress response that leads to permanent arrest of cell growth with maintained viability and metabolic activity. The Hayflick limit describes the number of cell division cycles that may be completed prior to onset of senescence.

[0002] The senescence process is triggered by several insults including DNA damage,telomere loss, oxidative stress and oncogenic signalling and is mediated by transcription factors that include p53 and cyclin-dependent inhibitors (e.g., p16INK4Aand p21CIP). Inflammatory cells that accumulate as a result of senescence-induced inflammaging are immunosuppressive and include myeloid-derived suppressor cells, regulatory T-cells and M2 polarised macrophages.

[0003] At one level, senescence may be viewed as an innate protective mechanism that limitsthe accumulation of damaged and potentially dangerous cells in the body. However, senescence is also intimately linked to aging. A number of underlying mechanisms are likely to be relevant to this association. First, senescence limits tissue regenerative potential. Second, senescent cells drive chronic and tissue damaging low-grade inflammation via their senescence-associated secretory phenotype (SASP). Third, the accumulation of senescent cells in aged tissues can crowd out their healthy counterparts, compromising end-organ function. For these reasons, senescence is believed to contribute to the development of numerous diseases.

[0004] One characteristic of the inflammatory SASP phenotype is the release of chemokinesand cytokines, including ligands of the CXCR2 receptor. Senescent cells themselves are characterized by the expression of a family of stress ligands that bind the NKG2D receptor.2. SUMMARY

[0005] In a first aspect, described herein is an immunoresponsive cell comprising an NKG2Dpolypeptide, a CXCR2 polypeptide, and a fusion polypeptide comprising a DNAX-activating 10 (DAP10) polypeptide or a functional variant thereof and a DNAX-activating 12 (DAP12) polypeptide or a functional variant thereof. In some embodiments, the NKG2D, CXCR2, DAP10, and DAP12 polypeptides are each mammalian polypeptides. In some embodiments,the NKG2D, CXCR2, DAP10, and DAP12 polypeptides are each human polypeptides. In some embodiments, the sequence of the NKG2D polypeptide has at least about 85% sequence identity to the sequence of SEQ ID NO: 14. In some embodiments, the NKG2D polypeptide is a functional variant of human NKG2D polypeptide and has one or more mutations that add, delete, or substitute at least one of the amino acids of SEQ ID NO: 14. In some embodiments, the functional variant of NKG2D polypeptide is a truncated version of the polypeptide having the sequence of SEQ ID NO: 14. In some embodiments, the functional variant is a chimeric NKG2D polypeptide. In some embodiments, the functional variant is a human-murine chimeric NKG2D polypeptide.

[0006] In some embodiments, the DAP10 polypeptide is a functional variant of DAP10comprising 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 is a functional variant of DAP10 that has one or more point mutations that add, delete, or substitute any of the amino acids of SEQ ID NO: 1. In some embodiments, the DAP10 polypeptide is a functional variant of DAP10 which is a truncated version of the polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the DAP10 polypeptide comprises or consists of the sequence of any one of SEQ ID NOs: 1-8.

[0007] In some embodiments, the DAP12 polypeptide is a functional variant of DAP12comprising 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 is a functional variant of DAP12 that has one or more point mutations that add, delete, or substitute any of the amino acids of SEQ ID NO: 9. In some embodiments, the DAP12 polypeptide is a functional variant of DAP12 which is a truncated version of the polypeptide having the amino acid sequence of SEQ ID NO: 9. In some embodiments, the DAP12 polypeptide comprises or consists of the sequence of any one of SEQ ID NOs: 9-13.

[0008] In some embodiments, the DAP10 polypeptide and the DAP12 polypeptide are joinedby a linker. In some embodiments, the linker comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 18-46.

[0009] In some embodiments, the fusion polypeptide comprises an N-terminal sequence. Insome embodiments, the fusion polypeptide comprises a C-terminal sequence. In some embodiments, the N-terminal or C-terminal sequence comprises one or more of a His-tag, FLAG-tag, Arg-tag, T7-tag, Strep-tag, S-tag, an AviTagTM, an aptamer-tag, a myc tag, CD8α leader sequence, a 4-1BB endodomain, a V5 tag, or a CD27 endodomain. In particular embodiments, the fusion polypeptide comprises or consists of the sequence of any one of SEQ ID NOs: 60-63.

[0010] In some embodiments, the CXCR2 polypeptide has a sequence that has at least about85% sequence identity to SEQ ID NO: 87. In particular embodiments, the CXCR2 polypeptide has the sequence of SEQ ID NO: 87.

[0011] In some embodiments, the immunoresponsive cell of the disclosure is a T cell,Natural Killer (NK) cell, or myeloid cell (e.g., monocyte, macrophage, dendritic cell). In some embodiments, the immunoresponsive cell is an αβ T cell, a γδ T cell, a CD4+T cell, a CD8+T cell, a Natural Killer T (NKT) cell, or any combination thereof.

[0012] In a second aspect, described herein is an immunoresponsive cell comprising achimeric NKG2D polypeptide and a CXCR2 polypeptide. In some embodiments, the chimeric NKG2D polypeptide comprises a human NKG2D extracellular domain or a variant thereof and a murine NKG2D transmembrane domain or a variant thereof. In particular embodiments, the human NKG2D extracellular domain has the sequence set forth in any one of SEQ ID NOs: 95-97. In some embodiments, the chimeric NKG2D polypeptide comprises a variant of the human NKG2D extracellular domain that has at least 80% sequence identity to the sequence of any one of SEQ ID NOs: 95-97. In some embodiments, the murine NKG2D transmembrane domain has the sequence set forth in any one of SEQ ID NOs: 98- 101. In some embodiments, the chimeric NKG2D polypeptide comprises a variant of the murine NKG2D transmembrane domain that has at least 80% sequence identity to the sequence of any one of SEQ ID NOs: 98-101. In some embodiments, the immunoresponsive cell further comprises at least one DAP12 polypeptide or a variant thereof. In some embodiments, the immunoresponsive cell further comprises at least one DAP10 polypeptide or a variant thereof. In a preferred embodiment, the cell comprises a polypeptide that has at least 90% sequence identity to SEQ ID NO: 102.

[0013] Also described herein is a nucleic acid molecule encoding a NKG2D polypeptide, aCXCR2 polypeptide, and, optionally, a DNAX-activating (DAP10) polypeptide, a DNAX- activating (DAP12) polypeptide, and / or a fusion polypeptide.

[0014] Also described herein is a method of making an immunoresponsive cell comprisingthe steps of (i) transducing a T-cell or a natural killer cell or a myeloid cell with a nucleic acid molecule or vector as described herein, and (ii) culturing the T-cell or natural killer cell such that the transduced cell expresses an NKG2D polypeptide, a CXCR2 polypeptide, and, optionally, a DAP10 / DAP12 fusion polypeptide. In preferred embodiments, the NKG2D polypeptide associates with the cell membrane.

[0015] Also described herein is a method of treating a subject who has cancer, comprisingadministering to the subject a therapeutically effective of an immunoresponsive cell described herein. In some embodiments, the immunoresponsive cells are manufactured from T or natural killer (NK) cells or myeloid cells autologous to the subject. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the solid tumor cancer is liver cancer, lung cancer, breast cancer, prostate cancer, lymphoid cancer, colon cancer, renal cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, thyroid cancer, cancer of the esophagus, cancer of the small intestine, or any combination thereof.3. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] These and other features, aspects, and advantages of the present invention willbecome better understood with regard to the following description, and accompanying drawings, where:

[0017] FIGS. 1A-1C are schematics of the CAR constructs N1012, N1012_CXCR2, andNKG2D (FIG.1A) and CYAD-01_10 (FIG.1B). In CYAD-01_10, a replica of CYAD-01 has been co-expressed with additional DAP10. FIG.1C shows the transduction percentage (left panel) and median fluorescence intensity (MFI) (right panel) of cell surface NKG2D expression in the CD4+subset of T-cells following transduction of activated unfractionated human T-cells with retroviral vectors that encode N1012, N1012_CXCR2, CYAD-01_10, CYAD-01 replica, or NKG2D. Results for untransduced (UT) T-cells are shown for comparison.

[0018] FIG. 2 shows viability of two ovarian cancer cell lines, Kuramochi_LT (left) andA2780_LT (right) following co-culture with N1012, N1012_CXCR2, CYAD-01_10, and untransduced T-cells at varying CAR T-cell:target ratios. Data are shown for two independent donors. Tumor cell viability was assessed by MTT assay after 72 hours and is expressed as a percentage viable cells cultured in the absence of T-cells.

[0019] FIG. 3 shows the improved ability of N1012 and N1012_CXCR2 T-cells to persistand proliferate following repeated rounds of stimulation on Ren tumor cell monolayers, compared to a replica of CYAD-01 or CYAD-01_10 T-cells.

[0020] FIGS. 4A-4B are flow cytometry plots showing the nearly complete shift in N1012and N1012_CXCR2 T-cells from a mixed population containing both CD4+and CD8+T- cells, to a population composed almost exclusively of CD8+T-cells following 16 re- stimulation cycles (FIG.4A) and 23 re-stimulation cycles (FIG.4B) on Ren tumor cell monolayers.

[0021] FIG. 5 shows flow cytometry results of N1012 T-cells assessed for CD45RO, CD62Land CD27 expression following 28 rounds of stimulation on Ren tumor cell monolayers.

[0022] FIG. 6 shows results of N1012 T-cells assessed for expression of CD45RO andCD62L following 31, 32, and 33 rounds of stimulation on Ren tumor cell monolayers.

[0023] FIG. 7 shows avidity plots of the indicated CAR T-cells for LO68-CD19 cells (leftpanel) and SKOV-3 cells (right panel) upon application of an acoustic force ramp.

[0024] FIGS. 8A-8B show the levels of cytokines, IFN-^ (FIG. 8A) and IL-2 (FIG. 8B)secreted by N1012, N1012_CXCR2, CYAD-01 replica and untransduced T-cells when co- cultured with four different tumor cell lines. Supernatants were removed from the co- cultured cells and assessed for cytokine presence by ELISA. UT – untransduced; n.d. – not detected.

[0025] FIG. 9 shows that fold expansion of unstimulated N1012 and N1012_CXCR2 T-cellsover 12 days exceeds expansion of NKG2D and untransduced T-cells whereas CYAD-01 replica and CYAD-01_10 T-cells demonstrate substantially poorer expansion compared to control T-cells.

[0026] FIG. 10 shows bioluminescence emission from intraperitoneal (i.p.) firefly luciferase / tdTomato red fluorescent protein (LT)-expressing A2780 tumor xenografts in mice treated with PBS or CAR-T cells (N1012, N1012_CXCR2, CYAD-01 replica, or untransduced). T-cells were administered i.p. on day 6 and intravenously (i.v.) on day 7 after tumor inoculation (indicated by adjacent vertical dotted lines). Mice that achieved a complete response were treated with a fresh i.p. bolus of 1 x 105A2780 cells 49 days after the final T-cell administration (separate vertical dotted line). Tumor development is measured using bioluminescence imaging as total flux (photons / sec).

[0027] FIG. 11 shows results of tumor bioluminescence imaging demonstrating therapeuticactivity in i.p. A2780_LT xenograft-containing mice treated intravenously (i.v.) on day 6 (vertical dotted line) with N1012 and N1012_CXCR2 T-cells (left and middle panels). Improved trafficking of N1012_CXCR2 T-cells to intraperitoneal tumor sites compared to N1012 T-cells is shown in the right panel. Tumor development is measured by firefly luciferase expression as total flux (photons / sec). T-cell trafficking is monitored by bioluminescence imaging of renilla luciferase (rLuc)-expressing CAR T-cells, quantified once again as total flux (photons / sec).

[0028] FIG. 12 shows development of i.p. firefly luciferase (ffLuc)-expressing SKOV-3tumor xenografts in mice treated i.v. with PBS or CAR-T cells (N1012, N1012_CXCR2, CYAD-01_10, or control NKG2D construct) 14 days after tumor inoculation. Tumor development is measured using bioluminescence imaging as total flux (photons / sec).

[0029] FIG. 13 shows results of ffLuc-expressing tumor growth (left panel) and rLuc-expressing T-cell trafficking (right panel) assessments in SKOV-3 xenograft-containing mice treated i.v. with CAR T-cells (N1012, N1012_CXCR2, or reporter control “Renilla Luciferase”) on day 14 (vertical dotted line).

[0030] FIG. 14 shows results of bioluminescence imaging of the indicated rLuc-expressingCAR T-cells following i.v. administration to NSG mice with an i.p. SKOV-3 tumor xenograft. Imaging of T-cells that express rLuc N1012, rLuc N1012_CXCR2, or rLuc alone at various time points is shown.

[0031] FIG. 15 shows development of subcutaneous (s.c.) CFPac-1 pancreatic tumorxenografts in mice following i.v. administration of PBS or CAR T-cells (N1012_CXCR2, CYAD-01 replica, N1012 or NKG2D) on day 28 (vertical dotted line).

[0032] FIGS. 16A-16B show CD3 immunohistochemistry images of s.c. CFPac-1 tumors(FIG.16A) and RNAScope of CA T-cells (FIG.16B) demonstrating improved infiltration of N1012_CXCR2 T-cells compared to N1012, CYAD-01 replica or NKG2D T-cells.

[0033] FIG. 17 shows development over time of s.c. CFPac-1 pancreatic tumor xenografts inmice treated 28 days after tumor inoculation (vertical dotted line) with an i.v. administration of PBS, high dose (10x106) CAR T-cells (N1012, N1012_CXCR2, CYAD-01 replica or untransduced) or low dose (4x106) CAR T-cells (N1012, or N1012_CXCR2).

[0034] FIG. 18 shows development of s.c. BxPC3 pancreatic tumor xenografts in micefollowing i.v. administration of PBS or 10 x 106CAR T-cells (CYAD-01 replica, N1012, N1012_CXCR2, or untransduced) 14 days following tumor inoculation (vertical dotted line).

[0035] FIG. 19 is a survival curve of s.c. BxPC3 tumor xenograft-containing mice treated i.v.as shown in FIG.18 with PBS or 10 x 106CAR T-cells (CYAD-01 replica, N1012, N1012_CXCR2, or untransduced).

[0036] FIG. 20 shows development of a s.c. mesothelioma patient derived xenograft (PDX)in mice (PDX PD_008) following i.v. administration of PBS or 10 x 106CAR T-cells (N1012_CXCR2, N1012, CYAD-01_10, or untransduced (UT)) 111 days following tumor engraftment (vertical dotted line).

[0037] FIG. 21 shows tumor volumes as assessed by caliper measurements in individualNSG mice with a s.c. mesothelioma PDX tumor following i.v. administration of PBS or 10 x 106CAR T-cells (N1012_CXCR2, N1012, CYAD-01_10, or untransduced T-cells) 111 days following tumor engraftment (vertical dotted line).

[0038] FIG. 22 is a Kaplan-Meier survival curve of NSG mice with a s.c. mesotheliomaPDX tumor that were treated i.v. with PBS or 10 x 106CAR T-cells (N1012_CXCR2, N1012, CYAD-01_10, or untransduced T-cells) 111 days following tumor engraftment.

[0039] FIG. 23 shows results of bioluminescence imaging of LT-expressing CAR T-cells. 10x 106CAR T-cells (N1012_LT, N1012_CXCR2_LT, or LT only control) were administered i.v. to mesothelioma PDX tumor-containing NSG mice. Imaging was performed at various time points following T-cell administration.

[0040] FIG. 24 shows a Kaplan-Meier survival curve of NSG mice bearing i.p. SKOV-3tumors that were treated i.p. with either PBS or 10 x 106CAR-T cells (N1012, N1012_CXCR2, CYAD-01_10, or control NKG2D) 14 days after tumor inoculation.

[0041] FIG. 25 shows development of i.p. ffLuc Kuramochi tumor xenografts in NSG micetreated with PBS or CAR-T cells (N1012, N1012_CXCR2, or untransduced).2 x 106T-cells were administered i.p. on day 17 post tumor inoculation and a further dose of 2 x 106T-cellswere administered i.v. on the following day (adjacent vertical dotted lines). Tumor-free mice were re-challenged with ffLuc Kuramochi cells 86 days after initial tumor inoculation (separate vertical dotted line).

[0042] FIG. 26 is a Kaplan-Meier survival curve of mice bearing i.p. ffLuc Kuramochitumors that were treated with PBS or CAR-T cells (N1012, N1012_CXCR2, or untransduced) 17 and 18 days after tumor inoculation, as described in Figure 26.

[0043] FIG. 27 shows development of i.p. ffLuc Kuramochi tumor xenografts in NSG micetreated with PBS or CAR-T cells (N1012, N1012_CXCR2, or untransduced).2 x 106T-cells were administered i.p. on day 17 post tumor inoculation and a further dose of 2 x 106T-cells were administered i.v. on the following day (adjacent vertical dotted lines). Tumor-free mice were re-challenged with ffLuc Kuramochi cells 86 days after initial tumor inoculation (separate vertical dotted line).

[0044] FIG. 28 shows results of CAR T-cell bioluminescence imaging in Kuramochi tumorxenograft-containing NSG mice treated i.v. with rLuc labeled CAR T-cells (N1012_rLuc, N1012_CXCR2_rLuc). T-cell infiltration into the peritoneal cavity was assessed over 4 days by bioluminescence imaging following coelenterazine administration.

[0045] FIG. 29 is a Kaplan-Meier survival curve of NSG mice bearing Kuramochi tumorsthat were treated with PBS or CAR-T cells (N1012, N1012_CXCR2, CYAD-01 replica or untransduced T-cells).2 x 106T-cells were administered i.p. on day 17 post tumor inoculation and a further dose of 2 x 106T-cells were administered i.v. on the following day.

[0046] FIGs. 30A-30C show expression of KLRG1 (median + interquartile range) (FIG.30A), CD57 (median + interquartile range) (FIG.30B), and CD27 (mean ± SEM) (FIG. 30C) on N1012 T-cells, untransduced T-cells (untrans), and NKG2D-transduced T-cells following expansion for 10 days in culture.

[0047] FIG. 31 is a lollipop plot highlighting significant findings of gene set enrichmentanalysis (GSEA) using the indicated gene set databases. Changes in N1012 CAR T-cells compared to three CD3ζ-containing NKG2D-based CAR T-cell populations (CYAD-01 analogue, CYAD-01_10 and NKG2D / Dap10-CD3ζ) are indicated.

[0048] FIGs. 32A-32B show results of mitochondrial stress testing in NKG2D-based CART-cells (N1012, CYAD-01, CYAD-01_10), NKG2D alone and untransduced T-cells using the Seahorse platform. FIG.32A shows oxygen consumption rate of each T-cell population. Specified inhibitors were added at the indicated timepoints (mean ± SEM, n = 3-7). *p<0.05;**p<0.01; ***p<0.001 by one-way ANOVA, comparing N1012 T-cells to the indicated comparator group (timepoints 7-9). FIG.32B shows spare respiratory capacity of each T- cell population (mean ± SEM, n = 3-7).4. DETAILED DESCRIPTION4.1. Definitions

[0049] Terms used in the claims and specification are defined as set forth below unlessotherwise specified.

[0050] The term “mammal” as used herein includes both humans and non-humans andinclude but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0051] The term “murine” refers to a rodent of the subfamily Murinae. The term “murine”comprises rat and mouse.

[0052] The term “chimeric NKG2D polypeptide” refers to an NKG2D receptor formed ofdomains from two or more different organisms. Chimeric NKG2D polypeptides are described in more detail in WO 2021 / 234163, the disclosure of which is herein incorporated by reference in its entirety.

[0053] The term percent "identity," in the context of two or more nucleic acid or polypeptidesequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[0054] For sequence comparison, typically one sequence acts as a reference sequence towhich test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0055] For purposes herein, percent identity and sequence similarity is performed using theBLAST algorithm, which is described in Altschul et al., J. Mol. Biol.215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0056] As used herein, the term “subject” broadly refers to any animal, including but notlimited to, human and non-human animals (e.g., dogs, cats, cows, horses, sheep, pigs, poultry, fish, crustaceans, etc.).

[0057] As used herein, the term “effective amount” refers to the amount of a composition(e.g., a synthetic peptide) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.

[0058] The term “therapeutically effective amount” is an amount that is effective toameliorate a symptom of a disease. A therapeutically effective amount can be a “prophylactically effective amount” as prophylaxis can be considered therapy.

[0059] As used herein, the terms “administration” and “administering” refer to the act ofgiving a drug, prodrug, or other agent, or therapeutic treatment (e.g., peptide) to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs. Exemplary routes of administration tothe human body can be through space under the arachnoid membrane of the brain or spinalcord (intrathecal), the eyes (ophthalmic), mouth (oral), skin (topical or transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal or lingual), ear, rectal, vaginal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.

[0060] As used herein, the term “treatment” means an approach to obtaining a beneficial orintended clinical result. The beneficial or intended clinical result can include alleviation of symptoms, a reduction in the severity of the disease, inhibiting an underlying cause of a disease or condition, steadying diseases in a non-advanced state, delaying the progress of a disease, and / or improvement or alleviation of disease conditions.

[0061] As used herein, the term “pharmaceutical composition” refers to the combination ofan active ingredient with a carrier, inert or active, making the composition especially suitable for therapeutic or diagnostic use in vitro, in vivo or ex vivo.

[0062] The terms “pharmaceutically acceptable” or “pharmacologically acceptable,” as usedherein, refer to compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject.

[0063] As used herein, the words “comprise” and “contain” and variations of the words, forexample, “comprising” and “comprises”, mean “including but not limited to”, and do not exclude other components, integers or steps. Moreover, the singular encompasses the plural unless the context otherwise requires; in particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0064] It must be noted that, as used in the specification and the appended claims, thesingular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. 4.2. Immunoresponsive cells comprising an NKG2D polypeptide, aDAP10 / DAP12 fusion polypeptide, and, optionally, a CXCR2 polypeptide

[0065] Immunoresponsive cells of the disclosure comprise an NKG2D polypeptide, aCXCR2 polypeptide, and, optionally, a DNAX-activating protein 10 (DAP10) polypeptide or functional variant thereof, and, optionally, a CXCR2 polypeptide. Immunoresponsive cells comprising an NKG2D polypeptide, a DAP10 / DAP12 fusion polypeptide, and, optionally, CXCR2, are described in more detail in WO 2021 / 0585663; WO 2021 / 234163; and WO 2022 / 200442, the disclosure of each of which is herein incorporated by reference in its entirety. 4.2.1. DAP10 polypeptides and functional variants thereof

[0066] DAP10 polypeptide may be endogenously expressed in certain organisms and certaincell types. In an embodiment, the DAP10 polypeptide or variant thereof is endogenous. Alternatively, the DAP10 polypeptide or variant thereof may be exogenous.

[0067] The DAP10 polypeptide of the disclosure may be mammalian, for example human.Wild-type human DAP10 is encoded by the amino acid sequence having UniProt accession no: Q9UBK5 (SEQ ID NO: 1). This is a 93 amino acid polypeptide. The first 18 amino acids are considered to be a signal / leader sequence, amino acids 19-48 the extracellular domain, amino acids 49-69 the transmembrane domain, and amino acids 70-93 the cytoplasmic / intracellular domain.

[0068] In one embodiment, a DAP10 polypeptide used in the fusion polypeptide of thedisclosure 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, aDAP10 polypeptide used in the fusion polypeptide of the disclosure comprises an amino acid sequence of SEQ ID NO: 1.

[0069] In another embodiment, a functional variant DAP10 polypeptide used in the fusionpolypeptide of the 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 of the amino acids of the amino acids of DAP10 (such as that of wild-type human DAP10 (SEQ ID NO: 1)).

[0070] Truncated versions of a DAP10 polypeptide may also be used in fusion polypeptidesof the disclosure. For example, a truncated version of DAP10 comprising only amino acids 19-93 of SEQ ID NO: 1 (i.e. lacking amino acids 1-18, the signal / leader sequence) may be used in the fusion polypeptide of the disclosure. Such a sequence is referred to as SEQ ID NO: 2 herein. Other truncated versions may comprise amino acids 19-69 of SEQ ID NO: 1, such a sequence comprising merely the extracellular and transmembrane domains of DAP10, and referred to herein as SEQ ID NO: 3. A further truncated version of DAP10 used in the invention may comprise amino acids 1-71 of SEQ ID NO: 1 (i.e. the signal / leader sequence, extracellular domain, transmembrane domain and 2 amino acids from the cytoplasmic / intracellular domain), referred to as SEQ ID NO: 4 herein. A further truncated version of DAP10 used in the invention may comprise amino acids 19-71 of SEQ ID NO: 1 (i.e. the extracellular domain, transmembrane domain and 2 amino acids from the cytoplasmic / intracellular domain), referred to as SEQ ID NO: 5 herein. A further truncated version of DAP10 used in the invention may comprise amino acids 70-93 of SEQ ID NO: 1 (i.e. the intracellular domain), referred to as SEQ ID NO: 6 herein. A yet further truncated version of DAP10 used in the invention may comprise amino acids 49-93 of SEQ ID NO: 1 (i.e. the transmembrane and cytoplasmic / intracellular domains), referred to as SEQ ID NO: 7 herein. A yet further truncated version of DAP10 used in the invention may comprise amino acids 49-69 of SEQ ID NO: 1 (i.e. the transmembrane domain), referred to as SEQ ID NO: 8 herein.

[0071] Other mutated versions or truncated versions of the DAP10 polypeptide are alsosuitable for use in the disclosure. In some embodiments, mutated versions or truncated versions that are used as functional variants of DAP10 polypeptides in the disclosure retain the activity of the wild type polypeptide shown in SEQ ID NO: 1.

[0072] In one embodiment, a functional variant of a DAP10 polypeptide of the disclosureretains at least 10% (for example 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 may be measured by assessment of tyrosine phosphorylation of DAP10 and / or recruitment and activation of the p85 subunit of phosphatidylinositol 3-kinase and the downstream anti-apoptotic kinase, AKT.

[0073] As the skilled person will appreciate, DAP10 typically exists as a homodimer. Thus,in an embodiment, the immunoresponsive cell comprises a DAP10 homodimer comprising two DAP10 polypeptides according to the disclosure. In an embodiment, the immunoresponsive cell comprises a DAP10 heterodimer, each peptide of the DAP10 heterodimer comprising a different DAP10 polypeptide of the disclosure. 4.2.2. DAP12 polypeptides and functional variants thereof

[0074] DAP12 polypeptide may be endogenously expressed in certain organisms and certaincell types. In an embodiment, the DAP12 polypeptide or variant thereof is endogenous. Alternatively, the DAP12 polypeptide or variant thereof may be exogenous.

[0075] The DAP12 polypeptide of the disclosure may be mammalian, for example human.Wild-type human DAP12 is encoded by the amino acid sequence having UniProt accession no: O43914 (SEQ ID NO: 9). The first 21 amino acids are considered to be a signal / leader sequence, amino acids 22-40 the extracellular domain, amino acids 41-61 the transmembrane domain, and amino acids 62-113 the cytoplasmic / intracellular domain.

[0076] In one embodiment, a DAP12 polypeptide used in the fusion polypeptide of thedisclosure 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, a DAP12 polypeptide used in the fusion polypeptide of the disclosure comprises an amino acid sequence of SEQ ID NO: 9.

[0077] In another embodiment, a functional variant DAP12 polypeptide used in the fusionpolypeptide of the 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 of the amino acids of the amino acids of DAP12 (such as that of wild-type human DAP12 (SEQ ID NO: 9)).

[0078] Truncated versions of a DAP12 polypeptide may also be used in fusion polypeptidesof the disclosure. For example, a truncated version of DAP12 comprising only amino acids 22-113 of SEQ ID NO: 9 (i.e. lacking amino acids 1-21, the signal / leader sequence) may be used in the fusion polypeptide of the disclosure. Such a sequence is referred to as SEQ IDNO: 10 herein. Other truncated versions may comprise amino acids 62-113 of SEQ ID NO: 9, such a sequence comprising merely the cytoplasmic / intracellular domain of DAP12 and referred to herein as SEQ ID NO: 11. Other truncated versions may comprise amino acids 41- 61 of SEQ ID NO: 9 (i.e. the transmembrane domain), referred to as SEQ ID NO: 12 herein. Another truncated version may comprise amino acids 22-61 of SEQ ID NO: 9 (i.e. the extracellular and transmembrane domains), referred to as SEQ ID NO: 13 herein.

[0079] Other mutated versions or truncated versions of the DAP12 polypeptide are alsosuitable for use in the disclosure. In some embodiments, mutated versions or truncated versions that are used as functional variants of DAP12 polypeptides in the disclosure retain the activity of the wild type polypeptide shown in SEQ ID NO: 9.

[0080] In one embodiment, a functional variant of a DAP12 polypeptide of the disclosureretains at least 10% (for example 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 may be measured using functional assays, such as MTT and measuring cytokine secretion by ELISA.

[0081] The skilled person will appreciate that DAP12 typically exists as a homodimer. Thus,in an embodiment, the immunoresponsive cell comprises a DAP12 homodimer comprising two DAP12 polypeptides according to the disclosure. In an embodiment, the immunoresponsive cell comprises a DAP12 heterodimer, each peptide of the DAP12 heterodimer comprising a different DAP12 polypeptide of the disclosure. 4.2.3. Polypeptides that associate with DAP10 / DAP12 fusionpolypeptides

[0082] The fusion polypeptide of the invention may associate with other polypeptides. Suchassociation may be due to electrostatic forces, such as provided by complementary charged amino acids. Such association may be due to the presence of one or more chemical or peptide linkers. Such association may be due to direct fusion of the DAP10 / DAP12 fusion polypeptide of the invention to a second polypeptide.

[0083] One example of such a polypeptide that may associate with a fusion polypeptide ofthe 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).

[0084] In some embodiments, a functional variant of NKG2D polypeptide may associatewith a DAP10 / DAP12 fusion polypeptide disclosed herein. In some embodiments, thefunctional variant of NKG2D polypeptide is a truncated NKG2D polypeptide. In some embodiments, the functional variant of NKG2D polypeptide is a mutated NKG2D polypeptide when compared to wild-type NKG2D.

[0085] In one embodiment, such polypeptides may be genetically encoded as part of acontiguous chimeric construct with the gene that encodes for the fusion polypeptide of the disclosure. The fusion polypeptide and other polypeptide may then be separated during translation (e.g. using a ribosomal skip peptide) or by post translation cleavage (e.g. using a furin cleavage site). The fusion polypeptide and other polypeptide may therefore be joined by an optional linker. Such a linker may comprise a cleavage site to facilitate cleavage. 4.2.3.1. NKG2D polypeptides and functional variants thereof

[0086] The NKG2D polypeptide of the disclosure may be mammalian, for example human.Wild-type human NKG2D is encoded by the amino acid sequence having UniProt accession no: P26718 (SEQ ID NO: 14). The polypeptide is considered to comprise a cytoplasmic domain (amino acids 1-51), a transmembrane domain (amino acids 52-72) and an extracellular domain (amino acids 73-216).

[0087] In one embodiment, a NKG2D polypeptide used in the disclosure comprises an aminoacid 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, a NKG2D polypeptide used in the disclosure comprises an amino acid sequence of SEQ ID NO: 14.

[0088] In another embodiment, a functional variant NKG2D polypeptide used in thedisclosure 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 of the amino acids of the amino acids of NKG2D (such as that of wild-type human NKG2D (SEQ ID NO: 14)).

[0089] Truncated versions of a NKG2D polypeptide may also be used in polypeptides of thedisclosure. For example, a truncated version of NKG2D comprising only amino acids 73-216 of SEQ ID NO: 14 (i.e., the extracellular domain) may be used in the disclosure. Such a sequence is referred to as SEQ ID NO: 15 herein. Other truncated versions may comprise amino acids 82-216 of SEQ ID NO: 14, such a sequence comprising part of the extracellular domain of NKG2D and referred to herein as SEQ ID NO: 16. A further truncated version comprises amino acids 52-216 of SEQ ID NO: 14 (i.e. the transmembrane and extracellular domains), referred to as SEQ ID NO: 17 herein.

[0090] Other mutated versions or truncated versions of the NKG2D polypeptide are alsosuitable for use in the disclosure. Of course, any such mutated versions or truncated versions that are used as functional variants of NKG2D polypeptides in the disclosure should preferably retain the activity of the wild type polypeptide shown in SEQ ID NO: 14.

[0091] In one embodiment, a functional variant of a NKG2D polypeptide of the disclosureretains at least 10% (for example 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 may be measured using flow cytometry to confirm continued binding to NKG2D ligands and through various cell culture assays (such as MTT and ELISA) aimed at confirming target cell lysis, cytokine secretion and co-stimulation.

[0092] In some embodiments, the NKG2D polypeptide is a chimeric polypeptide. Inparticular embodiments, the NKG2D polypeptide is a human-murine chimeric polypeptide. In an embodiment, the chimeric NKG2D polypeptide comprises from N terminus to C terminus the murine NKG2D transmembrane domain or a variant thereof and a human NKG2D extracellular domain or a variant thereof. Chimeric NKG2D polypeptides are described in more detail in WO 2021 / 234163, the disclosure of which is herein incorporated by reference in its entirety. 4.2.3.2. CXCR2 polypeptides and functional variants thereof

[0093] The CXCR2 polypeptide of the present disclosure may be mammalian, for examplehuman. Wild-type human CXCR2 is encoded by the amino acid sequence having UniProt accession no: P25025 (SEQ ID NO: 87).

[0094] In one embodiment, the CXCR2 polypeptide of the disclosure comprises an aminoacid 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 CXCR2 polypeptide of SEQ ID NO: 87. In one embodiment, the CXCR2 polypeptide of the disclosure has the amino acid sequence of SEQ ID NO: 87.

[0100] In one embodiment, the CXCR2 polypeptide of the disclosure is a functional variantof the CXCR2 polypeptide of SEQ ID NO: 87. In one embodiment, a functional variant CXCR2 polypeptide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) point mutations that add, delete, or substitute amino acids of CXCR2 (e.g., SEQ ID NO: 87).

[0101] In one embodiment, a functional variant CXCR2 polypeptide of the disclosure retainsat least 10% (for example, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%,99% or more) of the activity of wild-type human CXCR2 (SEQ ID NO: 87). In one embodiment, activity of the polypeptide is measured using flow cytometry to confirm binding of CXCR2 to ligand (e.g., IL-8). In one embodiment, activity of the polypeptide is measured through cell culture assays known in the art. 4.2.4. Linkers

[0102] The DAP10 and DAP12 moieties of the DAP10 / DAP12 fusion polypeptidesdescribed herein can be directly bonded to each other in a contiguous polypeptide chain or may be indirectly bonded to each other through 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, e.g., Chen X et al., 2013, Adv. Drug Deliv. Rev. 65(10):135701369 and Wriggers W et al., 2005, Biopolymers 80:736-746.). Linkers may also be used to join the fusion polypeptide of the disclosure to another polypeptide (such as a NKG2D polypeptide) in a chimeric construct as described above.

[0103] Peptide linkers generally are categorized as i) flexible linkers, ii) helix forminglinkers, 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 polypeptides described herein. Flexible linkers may contain a majority of amino acids that are sterically unhindered, such as glycine and alanine. The hydrophilic amino acid Ser is also conventionally used in flexible linkers. Examples of flexible linkers include, without limitation: polyglycines (e.g., (Gly)4 and (Gly)5), polyalanines poly(Gly-Ala), and poly(Gly-Ser) (e.g., (Glyn-Sern)n or (Sern-Glyn)n, wherein each n is independently an integer equal to or greater than 1).

[0104] Peptide linkers can be of a suitable length. The peptide linker sequence may be atleast 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 in length. For example, a peptide linker can be from about 5 to about 50 amino acids in length; from about 10 to about 40 amino acids in length; from about 15 to about 30 amino acids in length; or from about 15 to about 20 amino acids in length. Variation in peptide linker length may retain or enhance activity, giving rise to superior efficacy in activity studies. The peptide linker sequence may be comprised of naturally or non-naturally occurring amino acids, or a mixture of both naturally and non- naturally occurring amino acids.

[0105] In some aspects, the amino acids glycine and serine comprise the amino acids withinthe linker sequence. In certain aspects, the linker region comprises sets of glycine repeats(GSG3)n(SEQ ID NO: 18), where n is a positive integer equal to or greater than 1 (for example 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 aspects, the linker region orientation comprises sets of glycine repeats (SerGly3)n, where n is a positive integer equal to or greater than 1 (for example 1 to about 20) (SEQ ID NO: 21).

[0106] In other embodiments, a linker may contain glycine (G) and serine (S) in a random ora repeated pattern. For example, the linker can be (GGGGS)n(SEQ ID NO: 22), wherein n is an integer ranging from 1 to 20, for example 1 to 4. In a particular example, n is 4 and the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 23). In another particular example, n is 3 and the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 24).

[0107] In other embodiments, a linker may contain glycine (G), serine (S) and proline (P) ina random or repeated pattern. For example, the linker can be (GPPGS)n, wherein n is an integer ranging from 1 to 20, for example 1-4. In a particular example, n is 1 and the linker is GPPGS (SEQ ID NO: 25).

[0108] In general, the linker is not immunogenic when administered in a subject, such as ahuman. Thus, linkers may be chosen such that they have low immunogenicity or are thought to have low immunogenicity.

[0109] The linkers described herein are exemplary, and the linker can include other aminoacids, such as Glu and Lys, if desired. The peptide linkers may 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), if desired. In certain aspects, the peptide linkers may 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).

[0110] In other aspects, the peptide linkers may include combinations and multiples ofrepeating amino acid sequence units, such as (G3S)+(G4S)+(GlySer) (SEQ ID NO: 26 +SEQ ID NO: 27 +SEQ ID NO: 29). In other aspects, Ser can be replaced with Ala e.g., (G4A) (SEQ ID NO: 35) or (G3A) (SEQ ID NO: 36). In yet other aspects, the linker comprises the motif (EAAAK)n, where n is a positive integer equal to or greater than 1, for example 1 to about 20 (SEQ ID NO: 37). In certain aspects, peptide linkers may also include cleavable linkers.

[0111] The linkers may comprise further domains and / or features, such as a furin cleavagesite (RRKR) (SEQ ID NO: 38), a P2A ribosomal skip peptide(ATNFSLLKQAGDVEENPGP) (SEQ ID NO: 39) and / or a T2A ribosomal skip peptide (EGRGSLLTCGDVEENPGP) (SEQ ID NO: 40). Examples of linkers comprising these domains include SGSG + a P2A ribosomal skip peptide (SGSGATNFSLLKQAGDVEENPGP) (SEQ ID NO: 41), SGSG + a T2A ribosomal skip peptide (SGSGEGRGSLLTCGDVEENPGP) (SEQ ID NO: 42), and versions also including a furin cleavage site, i.e. furin cleavage site + SGSG + a P2A ribosomal skip peptide (RRKRSGSGATNFSLLKQAGDVEENPGP) (SEQ ID NO: 43) and furin cleavage site + SGSG + a T2A ribosomal skip peptide (RRKRSGSGEGRGSLLTCGDVEENPGP) (SEQ ID NO: 44). Alternative ribosomal skip peptides that may be used in the invention include F2A (VKQTLNFDLLKLAGDVESNPGP) (SEQ ID NO: 45) and E2A (QCTNYALLKLAGDVESNPGP) (SEQ ID NO: 46). 4.2.5. N-terminal sequences and C-terminal sequences

[0112] Various sequences may be attached to the N- or C-terminus of the fusion polypeptidesof the 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 a function, such as a T- cell stimulatory function. 4.2.5.1. Purification tags and markers

[0113] A variety of tags or markers may be attached to the N- or C-terminus of the fusionpolypeptides of the disclosure to assist with purification. Any affinity tag may be combined with the fusion polypeptides of the disclosure to assist with purification. Examples of such affinity tags are a His-tag, a FLAG-tag, Arg-tag, T7-tag, Strep-tag, S-tag, aptamer-tag, V5 tag, AviTagTM, myc epitope tag or any combination of these tags. In one embodiment the affinity tag is a His-tag (usually comprising 5-10 histidine residues), for example a 6 His 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 AviTagTM (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 disclosure are well known in the art.

[0114] Combinations of such affinity tags may also be used, either comprising one or moretags at the N-terminus, one or more tags at the C-terminus, or one or more tags at each of the N-terminus and the C-terminus. 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 N-AH-C or N-HA-C, while the Avi / His / FLAG tag may have the orientation N- AHF-C, N-FHA-C, etc.

[0115] In one embodiment, a fusion polypeptide according to the disclosure comprises an“AHF” tag having the sequence “GLNDIFEAQKIEWHEGGHHHHHHDYKDDDDK” (SEQ ID NO: 53). In another embodiment, a fusion polypeptide according to the disclosure comprises an “FHA” tag having the sequence “DYKDDDDKHHHHHHGGGLNDIFEAQKIEWHE” (SEQ ID NO: 54).

[0116] The CD8α leader sequence (amino acids 1-21 of UniProt: P01732 or a shortenedderivative comprising amino acids 1-18), is a commonly used leader sequence and is referred to as SEQ ID NO: 55 herein. 4.2.5.2. Co-stimulatory sequences

[0117] Various T-cell co-stimulatory activation sequences are known from previous work toenhance the function of CAR-T cells. These may also be added to fusion polypeptides of the disclosure.

[0118] The 4-1BB endodomain (amino acids 214-255 of UniProt: Q07011) may also be usedas an N- or C-terminal sequence. The 4-1BB endodomain is referred to as SEQ ID NO: 56 herein. The 4-1BB endodomain may act as a co-stimulatory domain.

[0119] The CD27 endodomain (amino acids 213-260 of UniProt: P26842) may also be usedas an N- or C-terminal sequence. The CD27 endodomain is referred to as SEQ ID NO: 57 herein. The CD27 endodomain may act as a co-stimulatory domain.

[0120] The human IgG1 hinge (amino acids 218-229 or 218-232 of UniProt: P0DOX5) mayalso be used as an N- or C-terminal sequence. The human IgG1 hinge is referred to as SEQ ID NO: 58 or SEQ ID NO: 104.

[0121] A truncated CD8α hinge (amino acids 138-182 of Uniprot: P01732) may also be usedas an N- or C-terminal sequence. The truncated CD8a hinge is referred to as SEQ ID NO: 59.4.2.6. Exemplary constructs

[0122] The present disclosure provides the following exemplary fusion polypeptideconstructs in Table 1: Table 1: Exemplary DAP10 / DAP12 fusion polypeptide constructs Name Sequence DAP10 (full sequence MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGS – aa1-93)-DAP12 LSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYI endodomain (aa62- NMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQ 113) (SEQ ID NO: RSDVYSDLNTQRPYYK 60) CD8α leader-FLAG- MALPVTALLLPLALLLHAARPDYKDDDDKQTTPGERSSLPAF DAP10 (extracellular YPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFYFLGR and TM – aa19-69)- LVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQ DAP12 endodomain RPYYK (aa62-113) (SEQ ID NO: 61) CD8α leader–FLAG– MALPVTALLLPLALLLHAARPDYKDDDDKEPKSCDKTHTCPL human IgG1 hinge (aa LAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPG 218-229 Uniprot ref RGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDV P0DOX5)–DAP10 YSDLNTQRPYYK (TM and endodomain aa 49-93)–DAP12 endodomain (aa 62- 113) (SEQ ID NO: 62) CD8α leader– MALPVTALLLPLALLLHAARPDYKDDDDKTTTPAPRPPTPAP truncated CD8a hinge TIASQPLSLRPEACRPAAGGAVHTRGLDFACDLLAGLVAADA (aa 138-182 Uniprot VASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLV reference P01732)–DAP10 (TM and PRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRP endodomain-aa 49- YYK 93)–DAP12 endodomain (aa 62- 113) (SEQ ID NO: 63)

[0123] Furthermore, as mentioned above, fusion polypeptides of the disclosure may beexpressed as a single chimeric construct with a NKG2D polypeptide, for translation- associated or post-translational cleavage. In such constructs, following expression, the translated polypeptides are cleaved to create the separate polypeptides which then self- associate to form a CAR. In one embodiment, a fusion polypeptide of the disclosure is cleaved from a NKG2D polypeptide. Examples of such constructs are shown in Table 2: Table 2: Exemplary chimeric constructs Name Sequence DAP10 (aa1-93)- MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGS DAP12 endodomain LSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYI NMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQ (aa62-113)-Furin RSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPG cleavage-linker-P2A- PMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCP VVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFN NKG2D (aa1-216) QEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQAS (Construct 1) (SEQ ID CMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSW QWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYI NO: 64) (N1012) CMQRTV CD8α leader-FLAG- MALPVTALLLPLALLLHAARPDYKDDDDKQTTPGERSSLPAF DAP10 (aa19-69)- YPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFYFLGR LVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQ DAP12 (aa62-113) - RPYYKRRKRSGSGEGRGSLLTCGDVEENPGPMIHLGHILFLLL Furin cleavage-linker- LPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVA ADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGRRKR T2A-DAP10 (aa1- SGSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEF 93)-Furin cleavage- HNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIA VAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWI linker-P2A- NKG2D CYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDL(aa1-216) (Construct LKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKG 3) (SEQ ID NO: 65) DCALYASSFKGYIENCSTPNTYICMQRTV DAP10 (aa1-93)- MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGS DAP12 endodomain LSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYI NMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQ (aa62-113)-Furin RSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPG cleavage-linker-P2A- PMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCP VVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFN NKG2D (aa1-216)- QEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQAS Furin cleavage-linker- CMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSW QWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYI T2A-DAP10 (aa1- CMQRTVRRKRSGSGEGRGSLLTCGDVEENPGPMIHLGHILFLL 71)-4-1BB LLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLV AADAVASLLIVGAVFLCKRGRKKLLYIFKQPFMRPVQTTQEE endodomain (aa214- DGCSCRFPEEEEGGCEL 255) (Construct 8) (SEQ ID NO: 66) DAP10 (aa1-93)- MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGS DAP12 endodomain LSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYI NMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQ (aa62-113)-Furin RSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPG cleavage-linker-P2A- PMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCP VVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFN NKG2D (aa1-216)- QEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQAS Furin cleavage-linker- CMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSW QWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYI T2A-CD8α leader CMQRTVRRKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLL (aa1-21)-FLAG- PLALLLHAARPDYKDDDDKQTTPGERSSLPAFYPGTSGSCSGC GSLSLPLLAGLVAADAVASLLIVGAVFLCKRGRKKLLYIFKQP DAP10 (aa19-71)-4- FMRPVQTTQEEDGCSCRFPEEEEGGCEL 1BB endodomain (aa214-255) (Construct 9) (SEQ ID NO: 67) DAP10 (aa1-93)- MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGS DAP12 endodomain LSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYI NMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQ (aa62-113)-Furin RSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPG cleavage-linker-P2A- PMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCP VVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFN NKG2D (aa1-216)- QEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQAS CMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWFurin cleavage-linker- QWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYI T2A-DAP10 (aa1- CMQRTVRRKRSGSGEGRGSLLTCGDVEENPGPMIHLGHILFLL LLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLV 71)-CD27 AADAVASLLIVGAVFLCQRRKYRSNKGESPVEPAEPCHYSCP endodomain (aa213- REEEGSTIPIQEDYRKPEPACSP 260) (Construct 10) (SEQ ID NO: 68) DAP10 (aa1-93)- MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGS DAP12 endodomain LSLPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYI NMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQ (aa62-113)-Furin RSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPG cleavage-linker-P2A- PMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCP VVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFN NKG2D (aa1-216)- QEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQAS Furin cleavage-linker- CMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSW QWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYI T2A-CD8α leader CMQRTVRRKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLL (aa1-21)-FLAG- PLALLLHAARPDYKDDDDKQTTPGERSSLPAFYPGTSGSCSGC GSLSLPLLAGLVAADAVASLLIVGAVFLCQRRKYRSNKGESP DAP10 (aa19-71)- VEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP CD27 endodomain (aa213-260) (Construct 11) (SEQ ID NO: 69) 4.2.7. Nucleic acid molecules encoding NKG2D, CXCR2, DAP10,DAP12, and DAP10 / DAP12 fusion polypeptides of the disclosure

[0124] Another aspect of the disclosure pertains to nucleic acid molecules that encode one ormore polypeptides of the disclosure. This may be as DNA or RNA. Unless specifically limited herein, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphorates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, as detailed below, degenerate codon substitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted with mixed-base 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).

[0125] Thus, the disclosure also provides a nucleic acid comprising a nucleotide sequenceencoding the polypeptide sequence of any one or more of SEQ ID NOs: 60-69, 87, 90, and 102.

[0126] The disclosure further provides a nucleic acid comprising a nucleotide sequencehaving 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 of SEQ ID NOS: 60-69, 87, 90, and 102. Sequence identity is typically measured along the full length of the reference sequence.

[0127] The disclosure further provides a nucleic acid comprising a nucleotide sequencehaving 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 any one of SEQ ID NOs: 70-79, 88, 91, and 103.

[0128] The disclosure also provides a nucleic acid comprising the nucleotide sequence of anyone of SEQ ID NOs: 70-79, 88, 91, and 103. The disclosure also provides a nucleic acid consisting of the nucleotide sequence of any one of SEQ ID NOs: 70-79, 91, and 103.

[0129] The polynucleotide sequences can be produced by de novo solid-phase DNAsynthesis or by PCR mutagenesis of an existing sequence (e.g., sequences as described in the Examples below). Direct chemical synthesis of nucleic acids can be accomplished 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. Pat. No.4,458,066. Introducing mutations to a polynucleotide sequence by PCR can be performed as described in, e.g., 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 1:17.4.2.8. Vectors

[0130] The present disclosure also provides vectors comprising one or more nucleic acidmolecules of the disclosure.

[0131] For expression in host cells, the nucleic acid encoding one or more polypeptides ofthe disclosure can be present in a suitable vector and after introduction into a suitable host, the sequence can be expressed to produce the encoded polypeptide(s) according to standard cloning and expression techniques, which are 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 disclosure also relates to such vectors comprising a nucleic acid sequence according to the disclosure.

[0132] Various expression vectors can be employed to express the polynucleotides encodingthe polypeptide(s) of the disclosure. Both viral-based and non-viral expression vectors can be used to produce the polypeptide(s) in a host cell, such as a mammalian host cell. Non-viral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al., 1997, Nat Genet.15:345). For example, non-viral vectors useful for expression of the polynucleotides and polypeptides of the 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 vectors, and numerous other vectors known in the art for expressing other proteins. Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, vectors based on SV40, papilloma virus, 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 a lentiviral vector is used, these include self-inactivating lentiviral vectors (so-called SIN vectors).

[0133] The choice of expression vector depends on the intended host cells in which thevector is to be expressed. Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer(see, e.g., Queen, et al., 1986, Immunol. Rev.89:49-68), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type-specific, stage-specific, and / or modulatable or regulatable. 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 (such as the human immediate-early CMV promoter), the constitutive CMV promoter, the EF1 alpha promoter, the phosphoglycerate kinase (PGK) promoter and promoter-enhancer combinations known in the art.

[0134] Cultures of transformed organisms can be expanded under non-inducing conditionswithout biasing the population for coding sequences whose expression products are better tolerated by the host cells. In addition to promoters, other regulatory elements may also be required or desired for efficient expression of the polypeptide(s) of the disclosure. These elements typically include an ATG initiation codon and adjacent ribosome binding site or other sequences. In addition, the efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (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 CMV enhancer may be used to increase expression in mammalian host cells.

[0135] The disclosure provides a cloning or expression vector comprising a nucleic acidcomprising 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 of SEQ ID NOS: 60-69, 87, 90, and 102. Furthermore, the disclosure provides a cloning or expression vector comprising a nucleic acid encoding one or more of SEQ ID NOs: 60-69, 87, 90, and 102. The disclosure provides a cloning or expression vector comprising the nucleic acid sequence of any one of SEQ ID NOs: 70-79, 88, 91, and 103.4.2.9. Host cells

[0136] Host cells comprising a polypeptide of the disclosure, nucleic acid of the disclosure,vector of the disclosure, or combinations of either or both thereof are provided. Such cells are generally utilized for the expression of the polypeptide(s) according to the disclosure.

[0137] The nucleic acid or vector may be transfected into a host cell by standard techniques.

[0138] The various forms of the term "transfection" are intended to encompass a wide varietyof techniques commonly used for the introduction of exogenous DNA and RNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.

[0139] Alternatively, the nucleic acid or vector may be delivered into the host cell bytransduction. For example, a viral vector, as disclosed above, may be used for delivery of the nucleic acid or vector.

[0140] It is possible to express the polypeptide(s) of the disclosure in either prokaryotic oreukaryotic host cells. Representative host cells include many E. coli strains, 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 a NK cell (either a primary NK cell, or a NK cell line) or a T cell (either a primary T-cell, or a T-cell line). Other types of host cells include macrophages, induced pluripotent stem cells (iPSCs), neutrophils and invariant NKT (iNKT) cells. The T- cell may 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 which may be used include the NK cell line NK-92.

[0141] Mammalian host cells for expressing the polypeptide(s) of the disclosure includeChinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220 used with a DH FR selectable marker, e.g., as described in R.J. Kaufman and P.A. Sharp, 1982, Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. In one embodiment the host cells are CHO K1PD cells. In another embodiment the host cells are NSO1 cells. In particular, for use with NSO myeloma cells, another expression system is the GS gene expression system shown in WO 87 / 04462, WO 89 / 01036 and EP 338,841. When recombinant expression vectors encoding polypeptides are introduced into mammalian host cells, the polypeptides may be produced by culturing the host cells for a period of time sufficient to allow for expression ofthe polypeptides in the host cells or secretion of the polypeptides into the culture medium in which the host cells are grown. Polypeptides can be recovered from the culture medium using standard protein purification methods. 4.3. Methods of producing immunoresponsive cells of the disclosure

[0142] The present disclosure also provides methods of producing immunoresponsive cellscomprising polypeptides of the disclosure. Such a method may comprise transducing a cell with a nucleic acid or vector encoding polypeptide(s) of the disclosure. The method may further comprise culturing the cell, such that the polypeptides are expressed and associates with a NKG2D polypeptide to form a CAR.

[0143] In one embodiment the present disclosure provides a method for preparing animmunoresponsive cell comprising the steps of (i) transducing a nucleic acid molecule or vector of the disclosure into the immunoresponsive cell, and (ii) culturing the immunoresponsive cell such that the transduced cell expresses a NKG2D polypeptide, a DAP10 / DAP12 fusion polypeptide, and a CXCR2 polypeptide, wherein the NKG2D polypeptide and the DAP10 / DAP12 fusion polypeptide associate in the cell membrane.

[0144] In a further embodiment, the present disclosure provides a method comprising, (i)obtaining T-cells and / or NK cells from a subject, (ii) transducing a nucleic acid or vector of the disclosure into the T-cells and / or NK cells, and (iii) culturing the T-cells and / or NK cells such that the transduced cell expresses a NKG2D polypeptide, a DAP10 / DAP12 fusion polypeptide, and a CXCR2 polypeptide, wherein the NKG2D polypeptide and the DAP10 / DAP12 fusion polypeptide associate in the cell membrane.

[0145] Various methods for the culture of immunoresponsive 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. 4.4. Pharmaceutical compositions

[0146] The disclosure also provides pharmaceutical compositions comprising polypeptides,nucleic acid, vector, immunoresponsive cell or host cell as described herein. Such pharmaceutical compositions can comprise a pharmaceutically or physiologically acceptable diluent and / or carrier. The carrier is generally selected to be suitable for the intended mode of administration and can include agents for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odour, sterility, stability,rate of dissolution or release, adsorption, or penetration of the composition. Typically, these carriers include aqueous or alcoholic / aqueous solutions, emulsions or suspensions, including saline and / or buffered media.

[0147] Suitable agents for inclusion in the pharmaceutical compositions include, but are notlimited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen- sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as free serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as Polysorbate 20 or Polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides, such as sodium or potassium chloride, or mannitol sorbitol), delivery vehicles, diluents, excipients and / or pharmaceutical adjuvants.

[0148] Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose andsodium chloride and lactated Ringer's. Suitable physiologically-acceptable thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates may be included. Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. In some cases one might include agents to adjust tonicity of the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in a pharmaceutical composition. For example, in many cases it is desirable that the composition is substantially isotonic. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present. The precise formulation will depend on the route of administration. Additional relevant principle,methods and components for pharmaceutical formulations are well known (see, e.g., Allen, Loyd V. Ed, (2012) Remington's Pharmaceutical Sciences, 22nd Edition).

[0149] A pharmaceutical composition of the present disclosure can be administered by one ormore routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results. Routes of administration for pharmaceutical compositions of the disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intra-sternal injection and infusion. In one embodiment, the pharmaceutical composition is administered intratumorally. When parenteral administration is contemplated, the pharmaceutical compositions are usually in the form of a sterile, pyrogen-free, parenterally acceptable composition. A particularly suitable vehicle for parenteral injection is a sterile, isotonic solution, properly preserved. The pharmaceutical composition can be in the form of a lyophilizate, such as a lyophilized cake.

[0150] Alternatively, the pharmaceutical composition described herein can be administeredby a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

[0151] In certain embodiments, the pharmaceutical composition is for subcutaneousadministration. Suitable formulation components and methods for subcutaneous administration of polypeptide therapeutics (e.g., antibodies, fusion polypeptides and the like) are known in the art, see, for example, US2011 / 0044977, US8465739 and US8476239. Typically, the pharmaceutical compositions for subcutaneous administration contain suitable stabilizers (e.g, amino acids, such as methionine, and or saccharides such as sucrose), buffering agents and tonicifying agents.

[0152] Typically, in cell therapy, the composition comprising the host cell orimmunoresponsive cell is administered to the subject by intravenous infusion.

[0153] Administration of the pharmaceutically useful composition of the present disclosure ispreferably in a “therapeutically effective amount” or “prophylactically effective amount” (as the case can be, although prophylaxis can be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of protein aggregation disease being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0154] A composition can be administered alone or in combination with other treatments,either simultaneously or sequentially dependent upon the condition to be treated. 4.5. Methods of Treatment

[0155] In some embodiments, the polypeptides, nucleic acids, vectors, cells and / orpharmaceutical compositions of the disclosure are used for treating a disease or disorder, prophylaxis, and / or for delaying the onset of disease symptoms. The methods include administering a therapeutically effective amount of the polypeptides, nucleic acids, vectors, cells, and / or pharmaceutical compositions of the disclosure.

[0156] The disclosure provides a polypeptide, nucleic acid, vector, immunoresponsive cell,host cell or pharmaceutical composition of the disclosure for use in therapy or as a medicament. The disclosure further provides a polypeptide, nucleic acid, vector, immunoresponsive cell, host cell or pharmaceutical composition of the disclosure for use in a method of depleting senescent cells in a subject. The disclosure further provides a polypeptide, nucleic acid, vector, immunoresponsive cell, host cell or pharmaceutical composition of the disclosure for use in the treatment of a pathological disorder. The disclosure further provides the use of a polypeptide, nucleic acid, vector, immunoresponsive cell, host cell or pharmaceutical composition of the disclosure in the manufacture of a medicament for depleting senescent cells in a subject. The disclosure also provides the use of a polypeptide, nucleic acid, vector, immunoresponsive cell, host cell or pharmaceutical composition of the disclosure in the manufacture of a medicament for the treatment of a pathological disorder. The disclosure further provides an immunoresponsive cell whichexpresses a chimeric antigen receptor comprising (a) an NKG2D polypeptide, (b) a fusion polypeptide comprising (i) a DNAX-activating 10 (DAP10) polypeptide, or a functional variant thereof and (ii) a DNAX-activating protein 12 (DAP12) polypeptide, or a functional variant thereof; and, optionally, (c) a CXCR2 polypeptide or a population of such immunoresponsive cells for use in a method of depleting senescent cells in a subject. The method may be any of those set out in the appended claims. The disclosure further provides use of an immunoresponsive cell which expresses a chimeric antigen receptor comprising (a) an NKG2D polypeptide, (b) a fusion polypeptide comprising (i) a DNAX-activating 10 (DAP10) polypeptide, or a functional variant thereof and (ii) a DNAX-activating protein 12 (DAP12) polypeptide, or a functional variant thereof; and, optionally, (c) a CXCR2 polypeptide or a population of such immunoresponsive cells in the manufacture of a medicament for depleting senescent cells in a subject. In some embodiments, the method of depleting senescent cells in a subject or the medicament for depleting senescent cells in a subject is for treating a disease or pathological condition in the subject. The disclosure further provides a method of treating a subject suffering from a pathological disorder comprising administering a therapeutically effective amount of a polypeptide, nucleic acid, vector, immunoresponsive cell, host cell or pharmaceutical composition of the disclosure to said subject.

[0157] As used herein, the terms “disease” and “pathological disorder” include aging andchronic age-related pathology as well as diseases and disorders associated aging and accumulation of senescent cells. The diseases and disorders include, but are not limited to, arthritis or osteoarthritis, osteoporosis, atherosclerosis, dysplastic or preneoplastic lesions, benign prostatic hyperplasia, normal and / or tumor tissues following DNA-damaging therapy, Alzheimer's disease, Parkinson's disease, cataracts, macular degeneration, glaucoma, atherosclerosis, acute coronary syndrome, myocardial infarction, stroke, hypertension, pulmonary fibrosis, kidney fibrosis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, osteoarthritis, osteoporosis, type 2 diabetes, obesity, fat dysfunction, coronary artery disease, cerebrovascular disease, vascular cognitive impairment and dementia, periodontal disease, cancer treatment-related disability (e.g. atrophy and fibrosis in various tissues), brain and heart injury, and therapy-related myelodysplastic syndromes, Hutchinson-Gilford progeria syndrome, Werner syndrome, Cockayne syndrome, xeroderma pigmentosum, ataxia telangiectasia, Fanconi anemia, dyskeratosis congenita, aplastic anemia; cardiovascular diseases such as angina, aortic aneurysm, arrhythmia, brain aneurysm, cardiacdiastolic dysfunction, cardiac fibrosis, cardiac stress resistance, cardiomyopathy, carotid artery disease, coronary thrombosis, endocarditis, hypercholesterolemia, hyperlipidaemia, mitral valve prolapse, peripheral vascular disease, inflammatory or autoimmune diseases such as herniated intervertebral disc, inflammatory bowel disease, kyphosis, oral mucositis, lupus, interstitial cystitis, scleroderma, and alopecia, neurodegenerative diseases such as dementia, Huntington's disease, motor neuron dysfunction, age-related memory decline, and depression / mood disorders, metabolic diseases such as diabetic ulcer and metabolic syndrome, pulmonary diseases such as age-related loss of pulmonary function, asthma, bronchiectasis, cystic fibrosis, emphysema, and age-associated sleep apnoea, gastrointestinal diseases such as Barrett's oesophagus; age-related disorders such as liver fibrosis, Non- Alcoholic Fatty Liver Disease (NAFLD) & Non-Alcoholic Steatohepatitis (NASH), toxin (e.g. alcohol) or viral-related hepatic fibrosis, primary biliary cirrhosis, primary biliary cholangitis, primary sclerosing cholangitis, end-stage hepatic cirrhosis, muscle fatigue, oral submucosa fibrosis, pancreatic fibrosis, benign prostatic hyperplasia, and age-related sleep disorders, dermatological diseases such as atopic dermatitis, cutaneous lupus, cutaneous lymphomas, dysesthesia, eczema, eczematous eruptions, eosinophilic dermatosis, fibrohistocytic proliferations of skin, hyperpigmentation, immunobullous dermatosis, nevi, pemphigoid, pemphigus, pruritus, psoriasis, rashes, reactive neutrophilic dermatosis, urticaria and other diseases such as diabetic wound healing, post-transplant kidney fibrosis, and carotid thrombosis.

[0158] In some embodiments, the administration of host cells or immunoresponsive cells tothe subject results in a reduction in the number of senescent cells within one or more tissues in the subject, thereby reducing and / or preventing inflammation within the one or more tissues. In some embodiments, the administration of host cells or immunoresponsive cells to the subject results in a reduction in damage in one or more tissues when compared to the health of the tissue prior to administration.

[0159] In some embodiments, the administration of host cells or immunoresponsive cells tothe subject results in a reduction in the expression level of one or more cellular senescence markers (e.g., KLRG1, CD57, P16 (CDKN2A), P21 (CDKN1A), p53, SA-^-galactosidase and a range of inflammatory cytokines, including IL-6, IL-1^ and IL-8) in the subject compared to the expression level prior to administration of the host cells or immunoresponsive cells.

[0160] In some embodiments, the administration of host cells or immunoresponsive cells tothe subject results in a reduction in senescence-associated secretory phenotype (SASP)-associated symptoms in the subject compared to the subject’s SASP-associated symptoms prior to administration of the host cells or immunoresponsive cells.

[0161] In some embodiments, the administration of host cells or immunoresponsive cells tothe subject results in reduction in mRNA expression of SASP-related cytokines (e.g., IL1A, IL1B, IL6, IL8, CCL2, CXCL1) compared to the expression level prior to administration of the host cells or immunoresponsive cells.

[0162] The amount of host cells or immunoresponsive cells administered to the subjectshould take into account the route of administration, the disease and / or disorder being treated, the weight of the subject and / or the age of the subject. In general, about 1 x 106to about 1 x 1011cells are administered to the subject. In one embodiment, about 1 x 107to about 1 x 1010cells, or about 1 x 108to about 1 x 109cells are administered to the subject. 4.6. Kits

[0163] In another aspect, components or embodiments described herein for the system areprovided in a kit. For example, any of the plasmids, as well as the mammalian cells, related buffers, media, triggering agents, or other components related to cell culture and virion production can be provided, with optional components frozen and packaged as a kit, alone or along with separate containers of any of the other agents and optional instructions for use. In some embodiments, the kit may comprise culture vessels, vials, tubes, or the like. 4.7. Examples

[0164] Below are examples of specific embodiments for carrying out the present invention.The examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0165] The practice of the present invention will employ, unless otherwise indicated,conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. 4.7.1. MethodsT cell Isolation and Retroviral Transduction

[0166] Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples fromhealthy volunteers through density-mediated centrifugation. T-cells were activated withphytohemagglutinin, TransAct or CD3 + CD28-coated paramagnetic beads for 48-72 hours, with 100IU / mL IL-2 added for the final 24 hours.1x106activated PBMC were plated onto a RetroNectin-coated plate that had been pre-treated with 3mL retroviral supernatant. Each well was subsequently treated with 3mL fresh viral supernatant and 100IU / mL IL-2. Retroviral transduction was performed with viral particles produced by stable gibbon ape leukemia virus (GALV)-pseudotyped 293TVec stable packaging cells or RD114-pseudotyped retrovirus generated from transient transfection of HEK293T cells. Thereafter, T-cells were fed with 100IU / mL in RPMI1640 medium + 5% normal human AB serum, with fresh medium and IL-2 (100IU / mL) provided thrice weekly. Flow Cytometry

[0167] T-cell transduction and transfection of 293T cells was assessed by flow cytometry,making comparison, where indicated, with an appropriate isotype control or untransduced / untransfected control. To assess the expression of the NKG2D-based constructs, cells were stained with mouse anti-human CD4-FITC, mouse anti-human NKG2D-PE and mouse anti-human CD8-APC and compensated appropriately. Due to high levels of endogenous NKG2D expression in CD8+T-cells, transduction efficiency was compared against NKG2D expression in untransduced CD4+T-cells. Transduction efficiency was calculated by comparison with N1012+T-cells stained using the same reagents. Prior to use, the transduction efficiency was normalized between constructs by spiking in the requisite proportion of untransduced T-cells. This ensured that all conditions were identical for the total number of CAR+T-cells and overall T-cell concentration.

[0168] To assess T-cell differentiation and subset distribution after multiple rounds ofstimulation, T-cells were removed from tumor cell co-cultures and stained with FITC- conjugated anti-human CD4 and allophycocyanin (APC)-conjugated anti-human CD62L, PE- conjugated anti-human CCR7, APCCy7-conjugated anti-human CD8α and phycoerythrin cyanine7 (PECy7)-conjugated anti-human CD27 antibodies. After washing in 2 mL ice-cold PBS the cells were re-suspended in 0.5 mL ice-cold PBS and assessed by flow cytometry. Staining efficiency was assessed using T-cells stained with the appropriate isotype and fluorescence minus one (FMO) controls. Dose response assays

[0169] 1x104 tumor cells were plated per well (100µL) of a 96-well plate and incubated at37°C and 5% CO2 overnight. Twenty-four hours later T-cells were added at log2 CAR T- cell:tumor cell ratios ranging from 1:1 to 1:64. After 72 hours, the T-cells were removed and 100µL MTT solution (500µg / mL) added, before the plates were incubated at 37°C and 5% CO2 for approximately 1 hour. Following removal of the MTT solution, the resulting formazan crystals were solubilized in DMSO (100µL / well) and the absorbance measured at 560nm. Tumor cell viability was calculated as follows: (Absorbance of monolayer with T- cells / absorbance of monolayer without T-cells)*100. Re-stimulation assays

[0170] 1x105 tumor cells were plated in triplicate wells of a 24-well plate and incubated at37°C and 5% CO2. Twenty-four hours later, 1mL medium containing 1x105CAR+T-cells were added per well. After 72 hours, the T-cells were gently removed and the well was washed with 1mL PBS. Following removal of the PBS, 1mL of MTT (at a final concentration of 500µg / mL) was added to each well and the plate incubated at 37°C and 5% CO2 for approximately 1 hour. Absorbance was measured in the appropriate wells at 560nm and tumor cell viability calculated as detailed in the ‘dose response’ section. A re-stimulation was considered successful if the tumor cell viability was measured as less than 60%.

[0171] The T-cells that had been removed from the plate were centrifuged at 400xg for 5minutes and the supernatant removed. The pellet was re-suspended in 3.2mL R5 medium and 1mL added to each well of fresh tumor monolayer (1x105tumor cells per well of a 24-well plate) in triplicate. Total T-cell number was assessed by trypan blue exclusion of a small aliquot of the remaining 200µL. Binding avidity

[0172] Binding avidity of CAR T-cells for target cells was assessed by measuring thepercentage of T-cells bound to target cells at increasing levels of acoustic force. CD19- engineered LO68 or SKOV-3 tumor cells were seeded in a z-Movi microfluidic chip (Lumicks, Amsterdam, Netherlands) coated with poly-L-lysine and cultured for 16 hours. The next day, flow sorted CAR-T cells were serially flowed in the chips and incubated with the target cells for 5 minutes prior to initializing a 3-minute linear force ramp. During the force ramp, the z-Movi device (Lumicks) captured a time series of images using a bright field microscope integrated into the platform. Detached cells were levitated towards the acousticnodes, allowing the tracking of cells based on their XY positions. Changes in the Z-position resulted in a change in the diffraction pattern, which permitted distinction between cells adhered to the substrate and cells suspended to the acoustic nodes. This information was used to correlate cell detachment events with a specific rupture force. Cell detachment was acquired using (z-Movi Tracking_v1.6.0) and post experiment image analysis performed using Cell Tracking offline analysis_v2.1. ELISA

[0173] Secretion of IFN-γ and IL-2 by T-cells were assessed in supernatant aliquots removed24 hours after the initiation of co-culture using Duo-set and Ready-Steady-Go ELISA kits respectively. In vivo studies Bioluminescence imaging

[0174] NSG mice were inoculated i.p. with 1x105 ffLuc-expressing A2780 or 5x105 ffLuc-expressing SKOV-3 cells. Mice inoculated with A2780 cells were treated with either PBS or 5x106CAR T-cells (N1012, N1012_CXCR2, CYAD-01 replica, or untransduced) via intraperitoneal injection (6 days after tumor inoculation) and / or intravenous injection (7 days after inoculation). Mice inoculated with SKOV-3 cells were treated intravenously with either PBS or 1x107CAR T-cells (N1012, N1012_CXCR2, or CYAD-01_10) 14 days after tumor inoculation.

[0175] In order to evaluate T-cell trafficking, T-cells were double transduced to express aCAR construct (N1012 or N1012_CXCR2) and a reporter construct, either renilla luciferase (rLuc) alone or a dual reporter which encodes both rLuc and GFP. Mice inoculated with A2780 cells were treated with 5x106CAR T-cells (N1012_rLuc or N1012_CXCR2_rLuc) by intravenous injection 7 days after tumor inoculation. Mice inoculated with SKOV-3 cells were treated with 1x107CAR T-cells (N1012_CXCR2_rLuc or N1012_rLuc) by intravenous injection 14 days after tumor inoculation. Mice inoculated with Kuramochi cells were treated with 2x106CAR T-cells cells (N1012_CXCR2_rLuc or N1012_rLuc) by intravenous injection 18 days after tumor inoculation. Coelenterazine, a substrate for renilla luciferase, was administered i.p. to the mice in order to monitor T-cell tracking into the peritoneal cavity.

[0176] Tumor growth was monitored by BLI, with all data presented as total flux(photons / second) or average total flux (photons / second) per treatment. Mice were monitored closely and weighed three times per week for signs of ill health. Tumor volume

[0177] 1x105 CFPac-1 or BxPC3 cells were injected subcutaneously in 50 μl of Matrigel (1:1PBS) into the left flank of NSG mice. Twenty-nine days after inoculation with CFPac-1 cells or fourteen days after inoculation with BxPC3 cells, mice were treated intravenously with either PBS or 1x107or 4x106CAR T-cells (N1012_CXCR2, N1012, NKG2D, CYAD-01 replica) or untransduced (UT) T-cells as control.

[0178] Tumor growth was measured weekly by caliper measurements and presented as tumorvolume (mm3). RNA Analysis

[0179] Non-tissue culture-treated 24-well plates were coated with 0.1 ^g / mL of humanMICA-Fc fusion protein in PBS. The next day, excess PBS was removed and 2 x 105of the indicated CAR T-cell populations (n=3 independent donors) were added per well in 1 mL of R5 medium. After 24h, T-cells were harvested into 15mL Falcon tubes and washed with 5 mL of PBS. Cells (1:2 dilution with Trypan Blue) were then counted using the LUNA-FL^ Dual Fluorescence Cell Counter (Logos Biosystems). A total of 1x106T cells per condition were aliquoted and centrifuged at 800 g for 10 minutes. Pellets were stored at -20^C until shipment for RNA sequencing.

[0180] Nucleic acid extraction, sample QC, library preparation and sequencing wereperformed by Genewiz (Genomics from Azenta Life Sciences). Paired-end sequencing was performed on the Illumina NovaSeq, with a 2x150bp configuration and an estimated 20 million paired-end reads per sample.

[0181] FASTQ files were generated by Genewiz and initial sample QC was run, providing areport detailing RNA concentration as well as RNA quality numbers. Quality of the sequencing data was studied by the package fastqc (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / , accessed August 1st, 2023). Transcript quantification was performed using Salmon (v.1.9.0) against a decoy-awaretranscriptome generated from the GENCODE reference assembly (v.41) of the human genome. Transcript-level data were imported into R (v.4.2.1; https: / / www.r-project.org, accessed 01.08.2022) and gene-level count data were generated with the tximeta package.

[0182] Data for Ensembl genes with no associated ENTREZ gene identifier were discarded;counts for Ensembl genes mapped to the same ENTREZ gene identifier were summed up in each sample. Differential expression analysis was performed in R using DESeq2 and adjusting for donor identity. Only genes with at least 0.5 counts per million in at least 2 samples and not coding for immunoglobulin light and heavy chains and known B-cell markers were tested. The Benjamini and Hochberg procedure was applied for multiple testing correction.

[0183] Enrichment of signatures in MSigDB was assessed using gene set enrichment analysis(GSEA). Genes were ranked by decreasing scores calculated by taking the geometric mean between the absolute value of the log fold change and the p-value from DEseq2 following log10 transformation and change of sign. The sign of the log fold change was finally multiplied to the ranking measure. These calculations were executed in R using the packages msigdbr and fsgsea with default parameters.

[0184] Heatmaps of z-score transformed counts per millions were generated using thepheatmap package (https: / / cran.r-project.org / web / packages / pheatmap / index.html, accessed March 1st, 2024). All plots were generated using the ggplot2 package. Genes were labeled as differentially upregulated if the log2 fold change in their expression was > 0.6, with an adjusted p-value < 0.05. Conversely, genes were labelled as differentially downregulated if the log2 fold change in their expression was < -0.6, with an adjusted p-value < 0.05. Seahorse Metabolic Analysis

[0185] Real-time analysis of oxygen consumption rates (OCR) and extracellular-acidificationrates (ECAR) of untransduced T-cells and CAR T-cells post-transduction and expansion for 10 days were assessed using a Seahorse XFe-96 analyser (Agilent Technologies). Cells were resuspended in Agilent Seahorse XF RPMI medium pH 7.4 supplemented with glucose (10mM), glutamine (2mM), and sodium pyruvate (1mM) and 1 x 105cells / well were seeded in a Seahorse XFe96 Cell Culture PDL-coated Microplate (all Agilent Technologies). Measurements of ECAR and OCR were performed prior to and following the sequential addition of Oligomycin A (1.5μM), BAM15 (2.5μM) and Rotenone (0.5μM) plus AntimycinA (0.5μM). Data was analyzed using Seahorse Analytics (Agilent Technologies) to calculate basal OCR, basal ECAR and spare respiratory capacity (SRC). Immunohistochemistry

[0186] 1x105 CFPac-1 cells were injected subcutaneously in 50 μl of Matrigel (1:1 PBS) intothe left flank of NSG mice. Twenty-nine days after inoculation, mice were treated intravenously with either PBS or 1x107CAR T-cells (N1012_CXCR2, N1012, NKG2D, CYAD-01 replica or untransduced (UT) T-cells). Three days following T-cell injections, three mice from each of the N1012, N1012_CXCR2, and NKG2D T-cell treatment groups were sacrificed and tumors were harvested for immunohistochemistry (IHC) analysis. Briefly, tissues were fixed in formalin, embedded in paraffin, sectioned, mounted on slides, and stained with anti-human CD3 antibody (Abcam) using standard methods. 4.7.2. Example 1: Generating N1012 T-cells and N1012_CXCR2 T-cells

[0187] T-cells expressing a CAR construct (e.g., N1012, N1012_CXCR2, CYAD-01 replica,CYAD-01_10) were generated by isolating peripheral blood mononuclear cells (PBMCs), activating the T-cells, and transducing the T-cells with virus containing nucleic acid encoding the CAR (FIGS.1A-1B).

[0188] Briefly, to generate virus, 1.65x106 HEK293T cells were seeded in a 10cm2 tissueculture dish in 10mL of IMDM medium containing 10% FBS and 2mM L-glutamine (I10 medium) and incubated for 24 hours at 37^C at 5% CO2. The following day, transfection mix was generated for each CAR construct according to the protocol in Table 3. The HEK293T cells were separately transfected with the following plasmids: N1012 (encoding DAP10 / 12 fusion protein and human NKG2D receptor (SEQ ID NO: 74)), N1012_CXCR2 (encoding DAP10 / 12 fusion protein, human NKG2D receptor, and CXCR2 (SEQ ID NO: 91)), CYAD- 01 replica (encoding NKG2D receptor fused to CD3ζ (SEQ ID NO: 93)), and CYAD-01_10 (encoding CYAD-01 replica and DAP10 (SEQ ID NO: 94). Table 3: Transfection protocol for CAR constructs (volumes per 10cm2dish) Reagent Volume / Amount Incubation Time Serum-free medium 470 µL Incubate for 5 minutes atGenejuice 30 µL room temperature. This is Mix A PeqPam-3 4.6875 µgRDF 3.125 µg Add all three plasmids toSFG vector containing CAR 4.6875 µg Mix A, gently mix and then (e.g. N1012_CXCR2) incubate for 15 minutes at room temperature. This is now Mix B

[0189] Upon completion of the 15 minute incubation, Mix B was added dropwise to theHEK293T cells and gently swirled. The cells were then placed back in the incubator. Supernatant was harvested 48 hours after transfection, collected into pre-chilled 50 mL Falcon tubes, and stored at 4˚C.

[0190] HEK293T cells were fed with 10 mL of fresh I10 medium and returned to theincubator. After an additional 24 hours, the supernatant was harvested a second time from the HEK293T cells and combined with the supernatant harvested 48 hours after transfection. The combined supernatant was aliquoted into pre-labeled tubes, snap frozen, and stored at - 80˚C.

[0191] PBMCs were isolated using standard Ficoll Paque-mediated density centrifugation.Once re-suspended at a concentration of 3x106cells / mL in RPMI + 5% normal human AB serum and 2mM L-glutamine (‘R5’ medium), the T-cells were activated using paramagnetic beads coated with anti-human CD3 and anti-human CD28 antibodies (1:2 cell:bead ratio), or phytohemagglutinin (PHA) at a concentration of 5µg / mL. Forty-eight hours after activation, 1x106T-cells were plated onto RetroNectin-coated non-tissue culture treated plates and mixed with 3mL viral supernatant harvested from transiently transfected HEK 293T cells. T- cells were fed with 100IU / mL IL-2 in RPMI1640 medium + 5% normal human AB serum, with fresh medium and IL-2 (100IU / mL) provided thrice weekly.

[0192] Surface expression of the N1012, N1012_CXCR2, CYAD-01 replica and CYAD-01_10 CARs on T-cells was assessed by flow cytometry. Briefly, T-cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-human CD4, phycoerythrin (PE)- conjugated anti-human NKG2D, Alexafluor_647 (AF_647)-conjugated anti-human CXCR2 and allophycocyanin cyanine7 (APC / Cy7)-conjugated anti-human CD8^ antibodies on ice for 30 minutes. As a control for background CXCR2 expression, one tube was stained with FITC-conjugated CD4, PE-conjugated NKG2D, APC / Cy7-conjugated CD8^ and AF_647- conjugated isotype antibodies. After washing in 2 mL ice-cold PBS, the cells were re- suspended in 0.5 mL ice-cold PBS and assessed by flow cytometry. Due to endogenous expression of NKG2D in CD8+T-cells, gene transfer efficiency was calculated within theCD4+T-cells and compared to that seen in untransduced (UT) T-cells. Results shown in FIG.1C demonstrate that high levels of gene transfer are achieved with all constructs (FIG. 1C, left panel) but that N1012 has significantly higher surface expression compared to other CARs, including N1012_CXCR2 (FIG.1C, right panel). 4.7.3. Example 2: Evaluating efficacy of N1012_CXCR2 T-cells againsthuman ovarian cancer cells

[0193] Cytotoxicity (MTT) assays were performed to assess anti-tumor efficacy of CAR T-cells in vitro against ovarian cancer cells. Briefly, two epithelial ovarian cancer cell lines – Kuramochi and A2780 – were separately co-cultured with CAR T-cells (N1012, N1012_CXCR2, CYAD-01 replica, or CYAD-01_10) at log2 effector:target ratios ranging from 1:1 to 1:64. Tumor cell viability was assessed 48 hours after T-cell addition by performing MTT assays as described above. Tumor cell viability was expressed as a percentage of tumor cells grown in the absence of T-cells (FIG.2). Results show that N1012, N1012_CXCR2, CYAD-01 replica, and CYAD-01_10 T-cells demonstrated potent cytotoxicity in both tumor cell lines (FIG.2). By contrast, untransduced T-cells mediated negligible tumor cell death. 4.7.4. Example 3: N1012 and N1012_CXCR2 T-cells demonstratesuperior proliferation

[0194] Re-stimulation assays were performed to assess the ability of CAR T-cells to undergomultiple rounds of target cell lysis. Briefly, CAR-T cells were co-cultured with Ren mesothelioma tumor cells at a 1:1 effector ratio. After 72 hours, the T-cells were gently removed and each well gently washed with 1mL PBS. Following removal of the PBS, 0.5mL MTT solution was added per well and the plates incubated at 37°C and 5% CO2 for approximately 1 hour. Following removal of the MTT solution, the resulting formazan crystals were solubilized in DMSO (0.5mL / well) and the absorbance measured at 560nm. Tumor cell viability was calculated as follows: (Absorbance of monolayer with T- cells / absorbance of monolayer without T-cells)*100. The T-cells that had been removed from the monolayer were centrifuged at 400xg for 5 minutes and the supernatant removed. The pellet was re-suspended in 3.2 mL R5 media and 1 mL was added to each well of fresh tumor monolayer in triplicate. The assay was repeated twice weekly until the T-cells failed to mediate greater than 40% target cell lysis. To investigate T-cell proliferation in response to target cell recognition, T-cell number was assessed by trypan blue exclusion of a smallaliquot of the remaining 200µL. Fold-expansion was calculated as follows (highest total T- cell number achieved during re-stimulation / initial T-cell number seeded).

[0195] Results shown in FIG. 3 demonstrate significantly improved proliferation andpersistence of N1012 and N1012_CXCR2 T-cells during re-stimulation assays compared to CYAD-01 replica and CYAD-01_10 T-cells. Whereas no expansion of untransduced T-cells or T-cells expressing NKG2D was observed, all CAR+T-cells proliferated from the number initially seeded. The level of expansion of N1012 and N1012_CXCR2 T-cells was substantially greater than that observed with either CYAD-01 replica or CYAD-01_10 T- cells. Furthermore, while the number of CYAD-01 replica and CYAD-01_10 T-cells rapidly diminished upon additional rounds of stimulation, both N1012 and N1012_CXCR2 T-cells demonstrated robust proliferation. Indeed, N1012 and N1012_CXCR2 T-cells demonstrated a 59.7-fold and 58.1-fold maximum expansion, respectively, in T-cell number, when compared to the number of cells initially seeded. It was also apparent that both N1012 and N1012_CXCR2 T-cells demonstrated bursts of proliferation, followed by a contraction in T- cell number, which were then followed by additional rounds of proliferation (FIG.3). This pattern of proliferation and contraction mirrors that of an endogenous immune response. 4.7.5. Example 4: Assessing T-cell differentiation, CD4:CD8 ratio, andexpression of co-stimulatory molecules following T- cell stimulation

[0196] A small aliquot of T-cells was removed from ongoing re-stimulation assays followingstimulation 16 and stimulation 23 and the T-cells were stained with FITC-conjugated anti- human CD4 and APC / Cy7-conjugated CD8^ antibodies. T-cells were stained separately for CD27 using a PE / Cy7-conjugated antibody. T-cells were assessed by flow cytometry. At both timepoints, N1012 and N1012_CXCR2 T-cells demonstrated an almost complete shift towards the CD8+subset, with very few CD4+cells detected (FIGS.4A-4B). The shift to CD8+was more pronounced following stimulation 23 than stimulation 16 (comparing FIG. 4B to FIG.4A). Critically, the N1012 T-cells retained expression of a key co-stimulatory molecule, CD27, even after 28 rounds of re-stimulation (FIG.5, right panel). Results also demonstrate that N1012 T-cells maintain some memory T-cells (CD45RO+CD62L+) following 28 rounds of stimulation (FIG.5, left panel).

[0197] N1012 T-cells further appeared to cycle between central memory T-cells (CD45RO+CD62L+) and effector memory T-cells (CD45RO+CD62L-). Minimal evidence of terminalexhaustion (CD45RO- CD62L-) was observed, even at advanced rounds of re-stimulation (FIG.6). 4.7.6. Example 5: Assessing avidity of CAR T-cells

[0198] In order to assess binding avidity of NKG2D-targeted CAR T-cells for target cells, thepercentage of T-cells bound to target cells at increasing levels of acoustic force was measured. Briefly, CD19-engineered LO68 or SKOV-3 tumor cells (both of which express NKG2D ligands) were seeded in a z-Movi microfluidic chip (Lumicks, Amsterdam, Netherlands) coated with poly-L-lysine and cultured for 16 hours. The next day, flow sorted CAR-T cells were serially flowed in the chips and incubated with the target cells for 5 minutes prior to initializing a 3-minute linear force ramp. During the force ramp, the z-Movi device (Lumicks) captured a time series of images using a bright field microscope integrated into the platform. Detached cells were levitated towards the acoustic nodes, allowing the tracking of cells based on their XY positions. Changes in the Z-position resulted in a change in the diffraction pattern, which permitted distinction between cells adhered to the substrate and cells suspended to the acoustic nodes. This information was used to correlate cell detachment events with a specific rupture force. Cell detachment was acquired using z-Movi Tracking_v1.6.0 and post experiment image analysis performed using Cell Tracking offline analysis_v2.1.

[0199] Results shown in FIG. 7 demonstrate that avidity of the CAR T-cells appeared tomirror the level of cell surface expression of the CAR (FIG.1C). N1012 CAR T-cells showed higher avidity than N1012_CXCR2, CYAD-01 replica, and CYAD-01_10 CAR T- cells for both LO68-19 and SKOV3 cells (FIG.7). All NKG2D CAR T-cells demonstrated lower binding avidity than the CD19-specific T-cells (FMC63) for LO68-19 cells, reflecting the lower affinity of NKG2D compared to an FMC63 scFv for its ligand(s). 4.7.7. Example 6: Assessing cytokine secretion from N1012 andN1012_CXCR2 T-cells

[0200] Secretion of Interferon-gamma (IFN-γ) and Interleukin-2 (IL-2) by CAR T-cells wasassessed by ELISA following co-culture with various tumor cell lines. CAR T-cells were separately co-cultured with cells derived from ovarian cancer (Kuramochi, A2780), mesothelioma (Ren) and pancreatic cancer (CFPac-1) for 24 hours. Supernatant was subsequently removed and assessed by ELISA as previously described.

[0201] Results demonstrate that N1012_CXCR2 T-cells secrete substantially less IFN-γ(FIG.8A) and IL-2 (FIG.8B) than N1012 T-cells across all cell lines tested. 4.7.8. Example 7: Unstimulated N1012 and N1012_CXCR2 T-cellsdemonstrate improved expansion ex vivo compared to CYAD-01 replica or CYAD-01_10 T-cells

[0202] Experiments were performed to evaluate fold expansion and, consequentially, scale-up potential of N1012, N1012_CXCR2, CYAD-01 replica, and CYAD-01_10 T-cells. Briefly, T-cells were transduced as previously described, and fed every 48-72 hours with 100% volume R5 medium supplemented with 100 IU / mL recombinant human IL-2. Cell expansion was calculated as total number of T-cells at the end of the 12 day culture period / initial number of T-cells transduced.

[0203] Results show both N1012 and N1012_CXCR2 T-cells demonstrate levels ofexpansion that slightly exceed those of untransduced T-cells (FIG.9). In contrast, CYAD-01 replica and CYAD-01_10 T-cells demonstrate substantially poorer expansion compared to control T-cells (FIG.9). 4.7.9. Example 8: Evaluating anti-tumor efficacy of N1012_CXCR2 T-cells in vivo in mouse models of ovarian cancer

[0204] In order to evaluate efficacy of CAR T-cells on ovarian tumors in vivo, CAR T-cellswere injected into mice bearing firefly luciferase (ffLuc)-expressing A2780_LT ovarian tumor xenografts, ffLuc-expressing SKOV-3 ovarian tumor xenografts, or ffLuc-expressing Kuramochi tumor xenografts. A2780 tumor cells express low levels of IL-8; SKOV-3 tumor cells express moderate levels of IL-8. Tumor growth was subsequently monitored by bioluminescence imaging.

[0205] Briefly, 1x105 ffLuc-expressing A2780 or Kuramochi or 5x105 ffLuc-expressingSKOV-3 cells were injected into the intraperitoneal cavity of NSG mice. Mice inoculated with A2780 cells were treated with either PBS [n=5] or 5x106CAR T-cells (N1012 [n=7], N1012_CXCR2 [n=7], CYAD-01 replica [n=4], or untransduced T-cells) administered via intraperitoneal injection (6 days after tumor inoculation) and / or intravenous injection (7 days after inoculation). Mice inoculated with SKOV-3 cells were treated intravenously with either PBS [n=5] or 1x107CAR T-cells (N1012 [n=11], N1012_CXCR2 [n=11], or CYAD-01_10 [n=8]) 14 days after tumor inoculation. On day 17 following inoculation, mice inoculated with Kuramochi cells were treated via intraperitoneal injection with PBS or 2x106CAR T- cells (N1012 or N1012_CXCR2) or untransduced T-cells. On day 18, mice inoculated withKuramochi cells were treated via intravenous injection with PBS or 2x106CAR T-cells (N1012 or N1012_CXCR2) or untransduced T-cells.

[0206] Results show an initial anti-tumor effect on A2780 cell tumors in mice treated withN1012, N1012_CXCR2, and CYAD-01 replica T-cells (FIG.10). Mice that achieved tumor- free status (e.g. tumor-derived bioluminescence emission at or below baseline levels) were re- challenged on Day 56 following initial tumor inoculation by intraperitoneal injection of 1x105ffLuc-expressing A2780 cells. Following tumor re-challenge, sustained tumor growth was not observed in mice that had been treated with N1012 or N1012_CXCR2 T-cells (FIG.10). By comparison, mice that had been treated with CYAD-01 replica T-cells showed a marked increase in tumor growth following tumor re-challenge (FIG.10). The results demonstrate the superior and more durable anti-ovarian tumor activity of N1012_CXCR2 and N1012 T- cells compared to CYAD-01 replica T-cells.

[0207] SKOV-3 xenograft mice treated with N1012_CXCR2 T-cells showed superior anti-tumor activity (FIG.12) and survival (FIG.24) compared to mice treated with N1012 or CYAD-01_10 T-cells.

[0208] Treatment with N1012 or N1012_CXCR2 T-cells also demonstrated anti-tumoractivity in Kuramochi xenograft mice (FIG.25 and FIG.27). N1012 and N1012_CXCR2- treated mice that had rejected tumors were re-challenged with tumor on day 86 by intraperitoneal injection of 1x105ffLuc-expressing Kuramochi cells. Tumor growth was inhibited in some of the re-challenged mice, demonstrating retained anti-tumor activity in the mice (FIG.25 and FIG.27). FIGS.26 and 29 are Kaplan-Meier survival curves of the Kuramochi xenograft-bearing mice treated in the experiments detailed in FIG.25 and FIG. 27, respectively. The T-cells used in a single experiment were from the same donor. T-cells used in the experiment detailed in FIG.27 were from a different donor than the T-cells used in the experiment detailed in FIG.25.

[0209] In order to evaluate T-cell trafficking, T-cells were double transduced to express aCAR construct (N1012 or N1012_CXCR2) and a reporter construct (rLuc).

[0210] Mice inoculated with intraperitoneal A2780 cells as previously described were treatedwith 5x106CAR T-cells (N1012_rLuc [n=3] or N1012_CXCR2_rLuc [n=3]) by intravenous injection 7 days after tumor inoculation. Results of bioluminescence imaging (FIG.11, right panel) show that between days 1-3 following T-cell injection, N1012_CXCR2-rLuc T-cells trafficked to the peritoneum more efficiently than N1012_rLuc T-cells while equivalent anti-tumor activity was observed in the two treatment groups (FIG.11, left and middle panels). Given that A2780 cells express IL-8 at moderate to low levels, the increased trafficking of N1012_CXCR2_ rLuc T-cells may be due to CXCR2 ligands other than IL-8.

[0211] Mice inoculated with intraperitoneal SKOV-3 cells as previously described weretreated with 1x107CAR T-cells (N1012_CXCR2_rLuc or N1012_rLuc) by intravenous injection 14 days after tumor inoculation. Results shown in FIG.13 and FIG.14 demonstrate that N1012_CXCR2_rLuc T-cells trafficked more efficiently than N1012-rLuc T-cells in the first four days following T-cell administration (FIG.13, right panel and FIG. 14). Results shown in FIG.13 (left panel) further demonstrate that mice treated with N1012_rLuc and N1012_CXCR2_rLuc T-cells maintain anti-tumor activity more than 60 days following T-cell administration.

[0212] Mice inoculated with intraperitoneal Kuramochi cells as previously described weretreated intravenously on Day 18 post tumor inoculation with 2x106CAR T-cells co- transduced with either N1012 or N1012_CXCR2 and rLuc. Results shown in FIG.28 demonstrate that N1012_CXCR2-rLuc T-cells trafficked more efficiently into the peritoneal cavity than N1012-rLuc T-cells in the first four days following T-cell administration. 4.7.10. Example 9: Evaluating anti-tumor efficacy of N1012_CXCR2 Tcells in vivo in mouse models of pancreatic cancer

[0213] In order to evaluate efficacy of CAR T-cells on pancreatic tumors in vivo, CAR T-cells were injected into mice bearing either CFPac-1 or BxPC3 tumor xenografts.

[0214] Briefly, 1x105 CFPac-1 or BxPC3 cells were injected subcutaneously in 50 μl ofMatrigel (1:1 PBS) into the left flank of NSG mice. Twenty-nine days after inoculation with CFPac-1 cells or fourteen days after inoculation with BxPC3 cells, mice were treated intravenously with either PBS or 1x107CAR T-cells (N1012_CXCR2, N1012, NKG2D, CYAD-01 replica or untransduced (UT) T-cells). Three days following T-cell injections, three mice from each of the N1012, N1012_CXCR2, and UT T-cell treatment groups in the CFPac1 model were sacrificed and tumors were harvested for immunohistochemistry (IHC) analysis. Tumor growth was measured weekly by caliper measurements and presented as tumor volume (mm3).

[0215] Results show that tumors in CFPac-1 xenograft mice treated with N1012_CXCR2 T-cells were completely eradicated following T-cell administration and no tumor relapse was observed (FIG.15). In contrast, tumor growth in CFPac-1 xenograft mice treated with N1012and CYAD-01 replica T-cells was initially inhibited following T-cell administration prior to loss of anti-tumor activity and tumor relapse (FIG.15). IHC analysis and RNAScope showed improved infiltration of N1012_CXCR2 T-cells into CFPac-1 tumors compared to N1012 or NKG2D T-cells (FIG.16).

[0216] BxPC3 xenograft mice treated with N1012_CXCR2 T-cells showed delayed tumorprogression (FIG.18) and enhanced survival (FIG.19) compared to mice treated with N1012 or CYAD-01 replica T-cells, suggesting that the presence of CXCR2 improves homing of the CAR T-cells to IL-8-producing BxPC3 tumors.

[0217] Additional studies were performed to compare treatment with high and lower doses ofCAR T-cells. Briefly, 1x105CFPac-1 cells were injected subcutaneously in 50 μl of Matrigel (1:1 PBS) into the left flank of NSG mice. Twenty eight days after tumor inoculation, mice were treated with either PBS [n=5], a high dose of 10x106CAR T-cells (N1012 [n=7], N1012_CXCR2 [n=7], CYAD-01 replica [n=5]) or a low dose of 4x106CAR T-cells (N1012 [n=6], N1012_CXCR2 [n=7]). Results shown in FIG.17 demonstrate that efficacy of treatment with a low dose of N1012_CXCR2 T-cells tracks treatment with a higher dose of N1012 T-cells. 4.7.11. Example 10: Evaluating anti-tumor efficacy of N1012_CXCR2 T-cells in a mesothelioma patient derived xenograft (PDX) tumor model

[0218] A mesothelioma patient derived xenograft (PDX) mouse tumor model was generatedto evaluate efficacy of N1012 and N1012_CXCR2 CAR T-cell treatment of mesothelioma in vivo. In order to establish the mesothelioma PDX model, primary patient mesothelioma tumor fragments (2 x 2 mm) were engrafted subcutaneously into the left flank of NSG mice using 15G trocar implant needles. The tumors were grown over a period of six months and were passaged three times into new cohorts. At passage 4, an experimental cohort of male NSG mice were implanted with PDX material into the left flank. On Day 111 following tumor engraftment, mice were treated intravenously with either PBS [n=8] or 1x107CAR T- cells (untransduced (UT), N1012_CXCR2 [n=8], N1012 [n=9] or CYAD-01_10 [n=5]). A subgroup of mice was treated with T-cells transduced with luciferase and tdTomato (LT) with or without a CAR in order to track T-cells by bioluminescence imaging (N1012_CXCR2_LT [n=3], N1012_LT or LT alone [n=3]). Tumor growth was measured weekly by caliper measurements and presented as tumor volume (mm3).

[0219] Results in FIG. 20 and FIG. 21 show that anti-tumor activity was observed in micetreated with N1012 and N1012_CXCR2 T-cells. In contrast, mice treated with CYAD-01_10 T-cells did not respond well to treatment. FIG.23 shows bioluminescence imaging of T- cells, demonstrating that N1012 and N1012_CXCR2 T-cells localized to the tumor site in the mesothelioma PDX mice whereas T-cells transduced with only the LT reporter (no CAR) did not localize to tumor. A Kaplan-Meier survival curve of the mice in the study is shown in FIG.22. 4.7.12. Example 11: Assessing senescence-associated gene expression inN1012 T-cells

[0220] The expression levels of CD27 as well as the senescence markers CD57 and KLRG1were evaluated in unstimulated N1012 T-cells, NKG2D T-cells, and untransduced T-cells all of which had been cultured for 10 days post gene transfer. In addition, RNA-sequencing and gene set enrichment analysis (GSEA) were performed following activation of the cells for 24 hours on immobilized MICA (an NKG2D ligand) to evaluate and compare gene expression in activated N1012 T-cells with that of other NKG2D-based CAR T-cells, including T-cells expressing the CYAD-01 replica CAR.

[0221] Results show that compared to untransduced T-cells or T-cells engineered to expressNKG2D alone, N1012 T-cells expressed significantly reduced levels of the senescence markers KLRG1 (FIG.30A) and CD57 (FIG.30B). The results further show that the level of CD27, a marker lost in senescent T-cells, was elevated compared to control untransduced or NKG2D engineered T-cells (FIG.30C).

[0222] Results of GSEA using KEGG, REACTOME, and HALLMARK gene sets show thatgenes associated with cellular senescence pathways were downregulated in activated N1012 T-cells compared to three poorly functional NKG2D-based CAR T-cells that conferred little to no therapeutic benefit in in vivo studies (CYAD-01 replica, CYAD-01_10 and NKG2D / Dap10-CD3^) (FIG.31). Moreover, genes involved in p53 signaling (also implicated in senescence) were downregulated in N1012 T-cells (FIG.31).4.7.13. Example 12: Assessing mitochondrial fitness of NKG2D-basedCAR T-cells

[0223] One component of cell health that impacts efficacy of CAR T cells is mitochondrialfitness. In order to assess mitochondrial fitness, mitochondrial stress testing was performed on NKG2D-based CAR T-cells using the Seahorse platform.

[0224] Real-time analysis of oxygen consumption rates (OCR) and extracellular acidificationrates (ECAR) of CAR T-cells and untransduced T-cells were assessed using a Seahorse XFe- 96 analyzer (Agilent Technologies).

[0225] Results in FIG. 32A show that N1012 T-cells had significantly greater maximalrespiratory capacity compared to T-cells that express the CYAD-01 replica CAR. By comparison, CYAD-01_10 CAR T-cells had an intermediate capacity (FIG.32A). An increase in spare respiratory capacity in N1012 T-cell cultures was also observed (FIG.32B). 4.7.14. Example 13: N1012 and N1012_CXCR2 T-cells for use in thetreatment of senescence-related disease

[0226] A clinical study is performed to investigate the safety and efficacy of N1012 CAR T-cells (LEU002) and N1012_CXCR2 CAR T-cells (LEU011) when administered to adult human subjects (patients) to treat senescence-related disease.

[0227] LEU002 CAR T-cells or LEU011 CAR T-cells are administered IV at fixed doses of1 x 108, 3 x 108, 1 x 109, 2 x 109or 3 x 109CAR T-cells, according to study protocol, optionally following the administration of lymphodepleting chemotherapy comprising fludarabine and cyclophosphamide.

[0228] Evaluation

[0229] Patients are monitored immediately prior to administration of CAR T-cells and for atleast 7 days following initial administration of CAR T-cells according to study protocol. Time (T) = 0 is defined as the time at which CAR T-cells are administered. On each day in which CAR T-cells are administered, the patient’s oxygen saturation and heart rate are monitored continuously from T = -30 min to T = 4 hours. Blood samples are collected for cytokine and / or RNA analysis at T = -30 min, 30 min, 1 hour, 4 hours on Day 0 (day that the first dose of CAR T-cells is administered), Day 7, and every seven days thereafter for six months. Blood pressure, temperature, and respiratory rate are monitored as follows: every half hour for T = -30 min to T = 4 hours; every hour for T = 4 hours to T = 10 hours; everytwo hours for T = 10 hours to T = 16 hours; every four hours for T = 16 hours to T = 24 hours.

[0230] Patients body weight is measured and recorded immediately prior to administration ofthe initial dose of CAR T-cells and weekly thereafter for at least six months.

[0231] Results

[0232] Depletion of senescent cells in patients treated with LEU002 CAR T-cells or LEU011CAR T-cells is demonstrated by a decrease in mRNA and protein expression of cellular senescence markers following treatment. Decreased mRNA and protein expression of senescence associated secretory phenotype (SASP)-related cytokines is also measured in samples collected from patients following CAR T-cell treatment compared with levels prior to administration of CAR T-cells.

[0233] Improvements in patients’ physical endurance and end-organ function is observedfollowing treatment with LEU002 CAR T-cells or LEU011 CAR T-cells. Biochemical analysis of blood samples and other functional studies (e.g. echocardiography, pulmonary function tests etc.) further demonstrates improvements in liver, kidney, heart, and / or metabolism-related biomarkers in some patients following treatment with LEU002 CAR T- cells or LEU011 CAR T-cells.5. EQUIVALENTS AND INCORPORATION BY REFERENCE

[0234] All references cited herein are incorporated by reference to the same extent as if eachindividual publication, database entry (e.g. Genbank sequences or GeneID entries), patent application, or patent, was specifically and individually indicated to be incorporated by reference in its entirety, for all purposes. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. §1.57(b)(1), to relate to each and every individual publication, database entry (e.g. Genbank sequences or GeneID entries), patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. §1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

[0235] While the invention has been particularly shown and described with reference to apreferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.6. SEQUENCESSEQ ID NO: 1 (human DAP10 full sequence) MIHLGHILFL LLLPVAAAQT TPGERSSLPA FYPGTSGSCS GCGSLSLPLL AGLVAADAVA SLLIVGAVFL CARPRRSPAQ EDGKVYINMP GRG SEQ ID NO: 2 (DAP10 aa19-93 - lacking leader sequence) QTTPGERSSL PAFYPGTSGS CSGCGSLSLP LLAGLVAADA VASLLIVGAV FLCARPRRSP AQEDGKVYIN MPGRG SEQ ID NO: 3 (DAP10 aa19-69 - extracellular / transmembrane domain) QTTPGERSSL PAFYPGTSGS CSGCGSLSLP LLAGLVAADA VASLLIVGAV F SEQ ID NO: 4 (DAP10 aa1-71) MIHLGHILFL LLLPVAAAQT TPGERSSLPA FYPGTSGSCS GCGSLSLPLL AGLVAADAVA SLLIVGAVFL C SEQ ID NO: 5 (DAP10 aa19-71) QTTPGERSSL PAFYPGTSGS CSGCGSLSLP LLAGLVAADA VASLLIVGAV FLC SEQ ID NO: 6 (DAP10 aa70-93 – intracellular domain) LCARPRRSPA QEDGKVYINM PGRG SEQ ID NO: 7 (DAP10 aa49-93 – transmembrane and intracellular domain) LLAGLVAADA VASLLIVGAV FLCARPRRSP AQEDGKVYIN MPGRG SEQ ID NO: 8 (DAP10 aa49-69 – transmembrane domain) LLAGLVAADA VASLLIVGAV F SEQ ID NO: 9 (human DAP12 full sequence) MGGLEPCSRL LLLPLLLAVS GLRPVQAQAQ SDCSCSTVSP GVLAGIVMGD LVLTVLIALA VYFLGRLVPR GRGAAEAATR KQRITETESP YQELQGQRSD VYSDLNTQRP YYK SEQ ID NO: 10 (DAP12 aa22-113 - lacking leader sequence) LRPVQAQAQS DCSCSTVSPG VLAGIVMGDL VLTVLIALAV YFLGRLVPRG RGAAEAATRK QRITETESPY QELQGQRSDV YSDLNTQRPY YKSEQ ID NO: 11 (DAP12 aa62-113 - cytoplasmic / intracellular domain) YFLGRLVPRG RGAAEAATRK QRITETESPY QELQGQRSDV YSDLNTQRPY YK SEQ ID NO: 12 (DAP12 aa41-61 – transmembrane domain) GVLAGIVMGD LVLTVLIALA V SEQ ID NO: 13 (DAP12 aa22-61 – extracellular and transmembrane domains) LRPVQAQAQS DCSCSTVSPG VLAGIVMGDL VLTVLIALAV SEQ ID NO: 14 (human NKG2D full sequence) MGWIRGRRSR HSWEMSEFHN YNLDLKKSDF STRWQKQRCP VVKSKCRENA SPFFFCCFIA VAMGIRFIIM VAIWSAVFLN SLFNQEVQIP LTESYCGPCP KNWICYKNNC YQFFDESKNW YESQASCMSQ NASLLKVYSK EDQDLLKLVK SYHWMGLVHI PTNGSWQWED GSILSPNLLT IIEMQKGDCA LYASSFKGYI ENCSTPNTYI CMQRTV SEQ ID NO: 15 (human NKG2D aa73-216 - extracellular domain) IWSAVFLNSL FNQEVQIPLT ESYCGPCPKN WICYKNNCYQ FFDESKNWYE SQASCMSQNA SLLKVYSKED QDLLKLVKSY HWMGLVHIPT NGSWQWEDGS ILSPNLLTII EMQKGDCALY ASSFKGYIEN CSTPNTYICM QRTV SEQ ID NO: 16 (human NKG2D aa82-216 - extracellular domain) LFNQEVQIPL TESYCGPCPK NWICYKNNCY QFFDESKNWY ESQASCMSQN ASLLKVYSKE DQDLLKLVKS YHWMGLVHIP TNGSWQWEDG SILSPNLLTI IEMQKGDCAL YASSFKGYIE NCSTPNTYIC MQRTV SEQ ID NO: 17 (human NKG2D aa52-216 – transmembrane and extracellular domain) PFFFCCFIAV AMGIRFIIMV AIWSAVFLNS LFNQEVQIPL TESYCGPCPK NWICYKNNCY QFFDESKNWY ESQASCMSQN ASLLKVYSKE DQDLLKLVKS YHWMGLVHIP TNGSWQWEDG SILSPNLLTI IEMQKGDCAL YASSFKGYIE NCSTPNTYIC MQRTV SEQ ID NO: 18 (linker) GSG SEQ ID NO: 19 (linker) GSGGG SEQ ID NO: 20 (linker)GSGG SEQ ID NO: 21 (linker) SGGG SEQ ID NO: 22 (linker) GGGGS SEQ ID NO: 23 (linker) GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 24 (linker) GGGGSGGGGSGGGGS SEQ ID NO: 25 (linker) GPPGS SEQ ID NO: 26 (linker) GGGS SEQ ID NO: 27 (linker) GGGGS SEQ ID NO: 28 (linker) GYS SEQ ID NO: 29 (linker) GS SEQ ID NO: 30 (linker) SGGGG SEQ ID NO: 31 (linker) SGGG SEQ ID NO: 32 (linker)SGG SEQ ID NO: 33 (linker) SGSG SEQ ID NO: 34 (linker) SG SEQ ID NO: 35 (linker) GGGGA SEQ ID NO: 36 (linker) GGGA SEQ ID NO: 37 (linker) EAAAK SEQ ID NO: 38 (furin cleavage site) RRKR SEQ ID NO:39 (P2A skip peptide) ATNFSLLKQAGDVEENPGP SEQ ID NO: 40 (T2A skip peptide) EGRGSLLTCGDVEENPGP SEQ ID NO: 41 (SGSG + P2A) SGSGATNFSLLKQAGDVEENPGP SEQ ID NO: 42 (SGSG + T2A) SGSGEGRGSLLTCGDVEENPGP SEQ ID NO: 43 (furin + SGSG + P2A) RRKRSGSGATNFSLLKQAGDVEENPGP SEQ ID NO: 44 (furin + SGSG + T2A)RRKRSGSGEGRGSLLTCGDVEENPGP SEQ ID NO: 45 (F2A skip peptide) VKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 46 (E2A skip peptide) QCTNYALLKLAGDVESNPGP SEQ ID NO: 47 (His tag) HHHHHH SEQ ID NO: 48 (FLAG tag) DYKDDDDK SEQ ID NO: 49 (Avi tag) GLNDIFEAQKIEWHE SEQ ID NO: 50 (V5 tag) GKPIPNPLLGLDST SEQ ID NO: 51 (V5 tag) IPNPLLGLD SEQ ID NO: 52 (Myc tag) EQKLISEEDL SEQ ID NO: 53 (AHF tag) GLNDIFEAQKIEWHEGGHHHHHHDYKDDDDK SEQ ID NO: 54 (FHA tag) DYKDDDDKHHHHHHGGGLNDIFEAQKIEWHE SEQ ID NO: 55 (CD8^ leader sequence) MALPVTALLL PLALLLHAAR P SEQ ID NO: 56 (4-1BB endodomain)KRGRKKLLYI FKQPFMRPVQ TTQEEDGCSC RFPEEEEGGC EL SEQ ID NO: 57 (CD27 endodomain) QRRKYRSNKG ESPVEPAEPC HYSCPREEEG STIPIQEDYR KPEPACSP SEQ ID NO: 58 (human IgG1 hinge – aa 218-229 of UniProt: P0DOX5) EPKSCDKTHT CP SEQ ID NO: 59 (truncated CD8^ hinge) TTTPAPRPPT PAPTIASQPL SLRPEACRPA AGGAVHTRGL DFACD SEQ ID NO: 60 (full length DAP10, including leader sequence, fused to the DAP12 intracellular domain) MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPR RSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK SEQ ID NO: 61 (CD8α leader sequence, FLAG tag, DAP10 extracellular and transmembrane domain, DAP12 intracellular domain) MALPVTALLLPLALLLHAARPDYKDDDDKQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLI VGAVFYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK SEQ ID NO: 62 (CD8α leader sequence, FLAG tag, human IgG1 hinge, DAP10 transmembrane domain, DAP10 intracellular domain, DAP12 intracellular domain) MALPVTALLLPLALLLHAARPDYKDDDDKEPKSCDKTHTCPLLAGLVAADAVASLLIVGAVFLCARPRRSPAQED GKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK SEQ ID NO: 63 (CD8α leader sequence, truncated CD8α hinge, DAP10 transmembrane domain, DAP10 intracellular domain, DAP12 intracellular domain) MALPVTALLLPLALLLHAARPDYKDDDDKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDL LAGLVAADAVASLLIVGAVFLCARPRRSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQ ELQGQRSDVYSDLNTQRPYYK SEQ ID NO: 64 (Construct 1 / N1012) MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPR RSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPF FFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCM SQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENC STPNTYICMQRTV SEQ ID NO: 65 (Construct 3) MALPVTALLLPLALLLHAARPDYKDDDDKQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLI VGAVFYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRSGSGEGRGSLLTCG DVEENPGPMIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGA VFLCARPRRSPAQEDGKVYINMPGRGRRKRSGSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYN LDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCG PCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGS ILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV SEQ ID NO: 66 (Construct 8) MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPR RSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRS GSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPF FFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCM SQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENC STPNTYICMQRTVRRKRSGSGEGRGSLLTCGDVEENPGPMIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTS GSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC EL SEQ ID NO: 67 (Construct 9) MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPR RSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRS GSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPF FFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCM SQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENC STPNTYICMQRTVRRKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAARPDYKDDDDKQTTPGER SSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEEGGCEL SEQ ID NO: 68 (Construct 10) MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPR RSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRS GSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPF FFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENC STPNTYICMQRTVRRKRSGSGEGRGSLLTCGDVEENPGPMIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTS GSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYR KPEPACSP SEQ ID NO: 69 (Construct 11) MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPR RSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRS GSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPF FFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCM SQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENC STPNTYICMQRTVRRKRSGSGEGRGSLLTCGDVEENPGPMALPVTALLLPLALLLHAARPDYKDDDDKQTTPGER SSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCQRRKYRSNKGESPVEPAEPCHYSCPREEE GSTIPIQEDYRKPEPACSP SEQ ID NO: 70 (encoding polypeptide of SEQ ID NO: 60) ATGATCCACCTGGGCCACATCCTGTTCCTGCTGCTGCTGCCCGTGGCCGCTGCCCAGACCACCCCTGGCGAGCGG AGCAGCCTGCCTGCCTTCTACCCTGGCACCAGCGGCAGCTGCAGCGGCTGCGGCAGCCTGAGCCTGCCCCTGCTG GCCGGCCTGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATCGTGGGCGCCGTGTTCCTGTGCGCCAGGCCCAGG CGGAGCCCtGCCCAGGAGGACGGCAAGGTGTACATCAACATGCCCGGCCGGGGCTACTTCCTGGGCAGGCTGGTG CCCAGGGGCAGGGGCGCTGCCGAGGCTGCCACCCGGAAGCAGCGGATCACCGAGACCGAGAGCCCCTACCAGGAG CTGCAGGGCCAGCGGAGCGACGTGTACAGCGACCTGAACACCCAGAGGCCCTACTACAAG SEQ ID NO: 71 (encoding polypeptide of SEQ ID NO: 61) ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCTAGACCCGATTATAAGGAC GACGACGACAAGCAGACCACCCCTGGCGAGCGGAGCAGCCTGCCTGCCTTCTACCCTGGCACCAGCGGCAGCTGC AGCGGCTGCGGCAGCCTGAGCCTGCCCCTGCTGGCtGGCCTGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATC GTGGGCGCCGTGTTCTACTTCCTGGGCAGGCTGGTGCCCAGGGGCAGGGGCGCTGCCGAGGCTGCCACCCGGAAG CAGCGGATCACCGAGACCGAGAGCCCCTACCAGGAGCTGCAGGGCCAGCGGAGCGACGTGTACAGCGACCTGAAC ACCCAGAGGCCCTACTACAAG SEQ ID NO: 72 (encoding polypeptide of SEQ ID NO: 62) ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCTAGACCCGATTATAAGGAC GACGACGACAAGGAGCCCAAGAGCTGCGACAAGACACACACATGCCCTCTTctggccggCCTGGTGGCCGCCGAC GCCGTGGCCAGCCTGCTGATCGTGGGCGCCGTGTTCCTGTGCGCCAGGCCCAGGCGGAGCCCtGCCCAGGAGGAC GGCAAGGTGTACATCAACATGCCCGGCCGGGGCTACTTCCTGGGCAGGCTGGTGCCCAGGGGCAGGGGCGCTGCC GAGGCTGCCACCCGGAAGCAGCGGATCACCGAGACCGAGAGCCCCTACCAGGAGCTGCAGGGCCAGCGGAGCGAC GTGTACAGCGACCTGAACACCCAGAGGCCCTACTACAAGSEQ ID NO: 73 (encoding polypeptide of SEQ ID NO: 63) ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCTAGACCCGATTATAAGGAC GACGACGACAAGACCACAACACCTGCTCCTAGACCTCCCACCCCTGCTCCCACCATCGCCAGCCAGCCCCTGAGC CTGAGACCCGAGGCCTGCAGACCCGCTGCTGGCGGCGCTGTGCATACCAGAGGCCTGGATTTCGCCTGCGACCTT ctggccggCCTGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATCGTGGGCGCCGTGTTCCTGTGCGCCAGGCCC AGGCGGAGCCCtGCCCAGGAGGACGGCAAGGTGTACATCAACATGCCCGGCCGGGGCTACTTCCTGGGCAGGCTG GTGCCCAGGGGCAGGGGCGCTGCCGAGGCTGCCACCCGGAAGCAGCGGATCACCGAGACCGAGAGCCCCTACCAG GAGCTGCAGGGCCAGCGGAGCGACGTGTACAGCGACCTGAACACCCAGAGGCCCTACTACAAG SEQ ID NO: 74 (encoding polypeptide of SEQ ID NO: 64 / construct 1 / N1012) ATGATCCACCTGGGCCACATCCTGTTCCTGCTGCTGCTGCCCGTGGCCGCTGCCCAGACCACCCCTGGCGAGCGG AGCAGCCTGCCTGCCTTCTACCCTGGCACCAGCGGCAGCTGCAGCGGCTGCGGCAGCCTGAGCCTGCCCCTGCTG GCCGGCCTGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATCGTGGGCGCCGTGTTCCTGTGCGCCAGGCCCAGG CGGAGCCCtGCCCAGGAGGACGGCAAGGTGTACATCAACATGCCCGGCCGGGGCTACTTCCTGGGCAGGCTGGTG CCCAGGGGCAGGGGCGCTGCCGAGGCTGCCACCCGGAAGCAGCGGATCACCGAGACCGAGAGCCCCTACCAGGAG CTGCAGGGCCAGCGGAGCGACGTGTACAGCGACCTGAACACCCAGAGGCCCTACTACAAGAGGCGGAAAAGGTCT GGGAGTGGGGCTACCAATTTCTCTCTCCTCAAGCAAGCCGGAGACGTTGAGGAAAACCCTGGaCCcATGGGCTGG ATCCGGGGACGGAGGAGCCGGCACAGCTGGGAGATGAGCGAGTTCCACAACTACAACCTGGACCTGAAGAAGAGC GACTTCAGCACCCGGTGGCAGAAGCAGCGGTGCCCCGTGGTGAAGAGCAAGTGCCGGGAGAACGCCAGCCCCTTC TTCTTCTGCTGCTTCATCGCCGTGGCtATGGGCATCCGGTTCATCATCATGGTGGCCATCTGGAGCGCCGTGTTC CTGAACAGCCTGTTCAACCAGGAGGTGCAGATCCCCCTGACCGAGAGCTACTGCGGCCCCTGCCCCAAGAACTGG ATCTGCTACAAGAACAACTGCTACCAGTTCTTCGACGAGAGCAAGAACTGGTACGAGAGCCAGGCCAGCTGCATG AGCCAGAACGCCAGCCTGCTGAAGGTGTACAGCAAGGAGGACCAGGACCTGCTGAAGCTGGTGAAGAGCTACCAC TGGATGGGCCTGGTGCACATCCCCACCAACGGCAGCTGGCAGTGGGAGGACGGCAGCATCCTGAGCCCCAACCTG CTGACCATCATCGAGATGCAGAAGGGCGACTGCGCCCTGTACGCCAGCAGCTTCAAGGGCTACATCGAGAACTGC AGCACCCCCAACACCTACATCTGCATGCAGCGGACCGTG SEQ ID NO: 75 (encoding polypeptide of SEQ ID NO: 65 / construct 3) ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCTAGACCCGATTATAAGGAC GACGACGACAAGCAGACCACCCCTGGCGAGCGGAGCAGCCTGCCTGCCTTCTACCCTGGCACCAGCGGCAGCTGC AGCGGCTGCGGCAGCCTGAGCCTGCCCCTGCTGGCtGGCCTGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATC GTGGGCGCCGTGTTCTACTTCCTGGGCAGGCTGGTGCCCAGGGGCAGGGGCGCTGCCGAGGCTGCCACCCGGAAG CAGCGGATCACCGAGACCGAGAGCCCCTACCAGGAGCTGCAGGGCCAGCGGAGCGACGTGTACAGCGACCTGAAC ACCCAGAGGCCCTACTACAAGCGGAGAAAGCGCtccGGCTCCGGCGAGGGCcgcGGCAGCCTGCTGACCTGCGGC GACGTGGAAGAGAACCCCGGACCCATGATCCACCTGGGCCACATCCTGTTCCTGCTGCTGCTGCCCGTGGCCGCT GCCCAAACAACACCCGGCGAGAGATCCTCCTTGCCCGCTTTCTATCCCGGAACATCCGGAAGCTGTtccggaTGT GGATCCCTTTCTTTGcctttgCTTGCTGGATTGGTCGCAGCTGACGCTGTCGCTTCCCTCCTTATTGTCGGAGCT GTCTTCCTGTGCGCCAGGCCCAGGCGGAGCCCtGCCCAGGAGGACGGCAAGGTGTACATCAACATGCCCGGCCGG GGCAGGCGGaagcgctccGGGAGTGGGGCTACCAATTTCTCTCTCCTCAAGCAAGCCGGAGACGTTGAGGAAAACCCTGGaCCcATGGGCTGGATCCGGGGACGGAGGAGCCGGCACAGCTGGGAGATGAGCGAGTTCCACAACTACAAC CTGGACCTGAAGAAGAGCGACTTCAGCACCCGGTGGCAGAAGCAGCGGTGCCCCGTGGTGAAGAGCAAGTGCCGG GAGAACGCCAGCCCCTTCTTCTTCTGCTGCTTCATCGCCGTGGCtATGGGCATCCGGTTCATCATCATGGTGGCC ATCTGGAGCGCCGTGTTCCTGAACAGCCTGTTCAACCAGGAGGTGCAGATCCCCCTGACCGAGAGCTACTGCGGC CCCTGCCCCAAGAACTGGATCTGCTACAAGAACAACTGCTACCAGTTCTTCGACGAGAGCAAGAACTGGTACGAG AGCCAGGCCAGCTGCATGAGCCAGAACGCCAGCCTGCTGAAGGTGTACAGCAAGGAGGACCAGGACCTGCTGAAG CTGGTGAAGAGCTACCACTGGATGGGCCTGGTGCACATCCCCACCAACGGCAGCTGGCAGTGGGAGGACGGCAGC ATCCTGAGCCCCAACCTGCTGACCATCATCGAGATGCAGAAGGGCGACTGCGCCCTGTACGCCAGCAGCTTCAAG GGCTACATCGAGAACTGCAGCACCCCCAACACCTACATCTGCATGCAGCGGACCGTG SEQ ID NO:76 (encoding polypeptide of SEQ ID NO: 66 / Construct 8) ATGATCCACCTGGGCCACATCCTGTTCCTGCTGCTGCTGCCCGTGGCCGCTGCCCAGACCACCCCTGGCGAGCGG AGCAGCCTGCCTGCCTTCTACCCTGGCACCAGCGGCAGCTGCAGCGGCTGCGGCAGCCTGAGCCTGCCCCTGCTG GCCGGCCTGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATCGTGGGCGCCGTGTTCCTGTGCGCCAGGCCCAGG CGGAGCCCtGCCCAGGAGGACGGCAAGGTGTACATCAACATGCCCGGCCGGGGCTACTTCCTGGGCAGGCTGGTG CCCAGGGGCAGGGGCGCTGCCGAGGCTGCCACCCGGAAGCAGCGGATCACCGAGACCGAGAGCCCCTACCAGGAG CTGCAGGGCCAGCGGAGCGACGTGTACAGCGACCTGAACACCCAGAGGCCCTACTACAAGAGGCGGAAAAGGTCT GGGAGTGGGGCTACCAATTTCTCTCTCCTCAAGCAAGCCGGAGACGTTGAGGAAAACCCTGGaCCcATGGGCTGG ATCCGGGGACGGAGGAGCCGGCACAGCTGGGAGATGAGCGAGTTCCACAACTACAACCTGGACCTGAAGAAGAGC GACTTCAGCACCCGGTGGCAGAAGCAGCGGTGCCCCGTGGTGAAGAGCAAGTGCCGGGAGAACGCCAGCCCCTTC TTCTTCTGCTGCTTCATCGCCGTGGCtATGGGCATCCGGTTCATCATCATGGTGGCCATCTGGAGCGCCGTGTTC CTGAACAGCCTGTTCAACCAGGAGGTGCAGATCCCCCTGACCGAGAGCTACTGCGGCCCCTGCCCCAAGAACTGG ATCTGCTACAAGAACAACTGCTACCAGTTCTTCGACGAGAGCAAGAACTGGTACGAGAGCCAGGCCAGCTGCATG AGCCAGAACGCCAGCCTGCTGAAGGTGTACAGCAAGGAGGACCAGGACCTGCTGAAGCTGGTGAAGAGCTACCAC TGGATGGGCCTGGTGCACATCCCCACCAACGGCAGCTGGCAGTGGGAGGACGGCAGCATCCTGAGCCCCAACCTG CTGACCATCATCGAGATGCAGAAGGGCGACTGCGCCCTGTACGCCAGCAGCTTCAAGGGCTACATCGAGAACTGC AGCACCCCCAACACCTACATCTGCATGCAGCGGACCGTGAGAAGAAAGAGAAGCGGCAGCGGCGAGGGCAGAGGC AGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGACccATGATTCATCTCGGACATATTCTCTTTCTCTTG CTCTTGCCTGTCGCTGCCGCTCAAACAACTCCCGGAGAAAGATCTTCTCTCCCCGCTTTTTATCCCGGAACATCT GGATCTTGTTCTGGATGTGGATCTTTGTCTCTCCCTCTCCTCGCTGGACTCGTCGCAGCTGATGCTGTCGCTTCT CTCTTGATTGTCGGAGCTGTCTTTTTGTGTAAGAGAGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTC ATGAGACCCGTGCAGACCACCCAGGAGGAGGACGGCTGCAGCTGCAGATTCCCCGAGGAGGAGGAGGGCGGCTGC GAGCTG SEQ ID NO:77 (encoding polypeptide of SEQ ID NO: 67 / construct 9) ATGATCCACCTGGGCCACATCCTGTTCCTGCTGCTGCTGCCCGTGGCCGCTGCCCAGACCACCCCTGGCGAGCGG AGCAGCCTGCCTGCCTTCTACCCTGGCACCAGCGGCAGCTGCAGCGGCTGCGGCAGCCTGAGCCTGCCCCTGCTG GCCGGCCTGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATCGTGGGCGCCGTGTTCCTGTGCGCCAGGCCCAGG CGGAGCCCtGCCCAGGAGGACGGCAAGGTGTACATCAACATGCCCGGCCGGGGCTACTTCCTGGGCAGGCTGGTG CCCAGGGGCAGGGGCGCTGCCGAGGCTGCCACCCGGAAGCAGCGGATCACCGAGACCGAGAGCCCCTACCAGGAG CTGCAGGGCCAGCGGAGCGACGTGTACAGCGACCTGAACACCCAGAGGCCCTACTACAAGAGGCGGAAAAGGTCTGGGAGTGGGGCTACCAATTTCTCTCTCCTCAAGCAAGCCGGAGACGTTGAGGAAAACCCTGGaCCcATGGGCTGG ATCCGGGGACGGAGGAGCCGGCACAGCTGGGAGATGAGCGAGTTCCACAACTACAACCTGGACCTGAAGAAGAGC GACTTCAGCACCCGGTGGCAGAAGCAGCGGTGCCCCGTGGTGAAGAGCAAGTGCCGGGAGAACGCCAGCCCCTTC TTCTTCTGCTGCTTCATCGCCGTGGCtATGGGCATCCGGTTCATCATCATGGTGGCCATCTGGAGCGCCGTGTTC CTGAACAGCCTGTTCAACCAGGAGGTGCAGATCCCCCTGACCGAGAGCTACTGCGGCCCCTGCCCCAAGAACTGG ATCTGCTACAAGAACAACTGCTACCAGTTCTTCGACGAGAGCAAGAACTGGTACGAGAGCCAGGCCAGCTGCATG AGCCAGAACGCCAGCCTGCTGAAGGTGTACAGCAAGGAGGACCAGGACCTGCTGAAGCTGGTGAAGAGCTACCAC TGGATGGGCCTGGTGCACATCCCCACCAACGGCAGCTGGCAGTGGGAGGACGGCAGCATCCTGAGCCCCAACCTG CTGACCATCATCGAGATGCAGAAGGGCGACTGCGCCCTGTACGCCAGCAGCTTCAAGGGCTACATCGAGAACTGC AGCACCCCCAACACCTACATCTGCATGCAGCGGACCGTGAGAAGAAAGAGAAGCGGCAGCGGCGAGGGCAGAGGC AGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGACcTatggctctgcctgtgacagctctgctgctgcct ctggctctgctgctgcacgccgctagacccgattataaggacgacgacgacaagCAAACAACTCCCGGAGAAAGA TCTTCTCTCCCCGCTTTTTATCCCGGAACATCTGGATCTTGTTCTGGATGTGGATCTTTGTCTCTCCCTCTCCTC GCTGGACTCGTCGCAGCTGATGCTGTCGCTTCTCTCTTGATTGTCGGAGCTGTCTTTTTGTGTAAGAGAGGCAGA AAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCCGTGCAGACCACCCAGGAGGAGGACGGCTGCAGC TGCAGATTCCCCGAGGAGGAGGAGGGCGGCTGCGAGCTG SEQ ID NO:78 (encoding polypeptide of SEQ ID NO: 68 / construct 10) ATGATCCACCTGGGCCACATCCTGTTCCTGCTGCTGCTGCCCGTGGCCGCTGCCCAGACCACCCCTGGCGAGCGG AGCAGCCTGCCTGCCTTCTACCCTGGCACCAGCGGCAGCTGCAGCGGCTGCGGCAGCCTGAGCCTGCCCCTGCTG GCCGGCCTGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATCGTGGGCGCCGTGTTCCTGTGCGCCAGGCCCAGG CGGAGCCCtGCCCAGGAGGACGGCAAGGTGTACATCAACATGCCCGGCCGGGGCTACTTCCTGGGCAGGCTGGTG CCCAGGGGCAGGGGCGCTGCCGAGGCTGCCACCCGGAAGCAGCGGATCACCGAGACCGAGAGCCCCTACCAGGAG CTGCAGGGCCAGCGGAGCGACGTGTACAGCGACCTGAACACCCAGAGGCCCTACTACAAGAGGCGGAAAAGGTCT GGGAGTGGGGCTACCAATTTCTCTCTCCTCAAGCAAGCCGGAGACGTTGAGGAAAACCCTGGaCCcATGGGCTGG ATCCGGGGACGGAGGAGCCGGCACAGCTGGGAGATGAGCGAGTTCCACAACTACAACCTGGACCTGAAGAAGAGC GACTTCAGCACCCGGTGGCAGAAGCAGCGGTGCCCCGTGGTGAAGAGCAAGTGCCGGGAGAACGCCAGCCCCTTC TTCTTCTGCTGCTTCATCGCCGTGGCtATGGGCATCCGGTTCATCATCATGGTGGCCATCTGGAGCGCCGTGTTC CTGAACAGCCTGTTCAACCAGGAGGTGCAGATCCCCCTGACCGAGAGCTACTGCGGCCCCTGCCCCAAGAACTGG ATCTGCTACAAGAACAACTGCTACCAGTTCTTCGACGAGAGCAAGAACTGGTACGAGAGCCAGGCCAGCTGCATG AGCCAGAACGCCAGCCTGCTGAAGGTGTACAGCAAGGAGGACCAGGACCTGCTGAAGCTGGTGAAGAGCTACCAC TGGATGGGCCTGGTGCACATCCCCACCAACGGCAGCTGGCAGTGGGAGGACGGCAGCATCCTGAGCCCCAACCTG CTGACCATCATCGAGATGCAGAAGGGCGACTGCGCCCTGTACGCCAGCAGCTTCAAGGGCTACATCGAGAACTGC AGCACCCCCAACACCTACATCTGCATGCAGCGGACCGTGAGAAGAAAGAGAAGCGGCAGCGGCGAGGGCAGAGGC AGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGACccATGATTCATCTCGGACATATTCTCTTTCTCTTG CTCTTGCCTGTCGCTGCCGCTCAAACAACTCCCGGAGAAAGATCTTCTCTCCCCGCTTTTTATCCCGGAACATCT GGATCTTGTTCTGGATGTGGATCTTTGTCTCTCCCTCTCCTCGCTGGACTCGTCGCAGCTGATGCTGTCGCTTCT CTCTTGATTGTCGGAGCTGTCTTTTTGTGTCAGAGGCGGAAGTACCGGAGCAACAAGGGCGAGAGCCCCGTGGAG CCTGCCGAGCCCTGCCACTACAGCTGTCCCCGGGAGGAGGAGGGCAGCACCATCCCCATCCAGGAGGACTACCGG AAGCCCGAGCCTGCCTGCAGCCCCSEQ ID NO:79 (encoding polypeptide of SEQ ID NO: 69 / Construct 11) ATGATCCACCTGGGCCACATCCTGTTCCTGCTGCTGCTGCCCGTGGCCGCTGCCCAGACCACCCCTGGCGAGCGG AGCAGCCTGCCTGCCTTCTACCCTGGCACCAGCGGCAGCTGCAGCGGCTGCGGCAGCCTGAGCCTGCCCCTGCTG GCCGGCCTGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATCGTGGGCGCCGTGTTCCTGTGCGCCAGGCCCAGG CGGAGCCCtGCCCAGGAGGACGGCAAGGTGTACATCAACATGCCCGGCCGGGGCTACTTCCTGGGCAGGCTGGTG CCCAGGGGCAGGGGCGCTGCCGAGGCTGCCACCCGGAAGCAGCGGATCACCGAGACCGAGAGCCCCTACCAGGAG CTGCAGGGCCAGCGGAGCGACGTGTACAGCGACCTGAACACCCAGAGGCCCTACTACAAGAGGCGGAAAAGGTCT GGGAGTGGGGCTACCAATTTCTCTCTCCTCAAGCAAGCCGGAGACGTTGAGGAAAACCCTGGaCCcATGGGCTGG ATCCGGGGACGGAGGAGCCGGCACAGCTGGGAGATGAGCGAGTTCCACAACTACAACCTGGACCTGAAGAAGAGC GACTTCAGCACCCGGTGGCAGAAGCAGCGGTGCCCCGTGGTGAAGAGCAAGTGCCGGGAGAACGCCAGCCCCTTC TTCTTCTGCTGCTTCATCGCCGTGGCtATGGGCATCCGGTTCATCATCATGGTGGCCATCTGGAGCGCCGTGTTC CTGAACAGCCTGTTCAACCAGGAGGTGCAGATCCCCCTGACCGAGAGCTACTGCGGCCCCTGCCCCAAGAACTGG ATCTGCTACAAGAACAACTGCTACCAGTTCTTCGACGAGAGCAAGAACTGGTACGAGAGCCAGGCCAGCTGCATG AGCCAGAACGCCAGCCTGCTGAAGGTGTACAGCAAGGAGGACCAGGACCTGCTGAAGCTGGTGAAGAGCTACCAC TGGATGGGCCTGGTGCACATCCCCACCAACGGCAGCTGGCAGTGGGAGGACGGCAGCATCCTGAGCCCCAACCTG CTGACCATCATCGAGATGCAGAAGGGCGACTGCGCCCTGTACGCCAGCAGCTTCAAGGGCTACATCGAGAACTGC AGCACCCCCAACACCTACATCTGCATGCAGCGGACCGTGAGAAGAAAGAGAAGCGGCAGCGGCGAGGGCAGAGGC AGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGACcTatggctctgcctgtgacagctctgctgctgcct ctggctctgctgctgcacgccgctagacccgattataaggacgacgacgacaagCAAACAACTCCCGGAGAAAGA TCTTCTCTCCCCGCTTTTTATCCCGGAACATCTGGATCTTGTTCTGGATGTGGATCTTTGTCTCTCCCTCTCCTC GCTGGACTCGTCGCAGCTGATGCTGTCGCTTCTCTCTTGATTGTCGGAGCTGTCTTTTTGTGTCAGAGGCGGAAG TACCGGAGCAACAAGGGCGAGAGCCCCGTGGAGCCTGCCGAGCCCTGCCACTACAGCTGTCCCCGGGAGGAGGAG GGCAGCACCATCCCCATCCAGGAGGACTACCGGAAGCCCGAGCCTGCCTGCAGCCCC SEQ ID NO: 80 (A20FMDV2 peptide) NAVPNLRGDLQVLAQKVART SEQ ID NO: 81 (CD124 signal peptide) MGWLCSGLLFPVSCLVLLQVASSGN SEQ ID NO: 82 (CD28 aa114-220) IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLH SDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 83 (CD247 aa52-164) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSEQ ID NO: 84 (SEQ ID NO: 1 of WO 2019 / 182425) MGWSCIILFLVATATGVHSQIQLVQSGPELKKPGETVKISCKTSGYTFTDYSMHWVNQAP GKGLKWMGWINTETGEPTYTDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARTAV YWGQGTTLTVSSGSTSGSGKPGSGEGSDIQMTQSPSSLSASLGERVSLTCRASQEISGSL SWLQQKPDGTIKRLIYAASTLNSGVPKRFSGRRSGSDYSLTISSLESEDFVDYYCLQYSS YPWSFGGGTKLEIKEPKSPDKTHTCPPCPSHTQPLGVFLFPPKPKDQLMISRTPEVTCVV VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSL SLGKFWVLVVVGGVLACYSLLVTVAFIIFWVARPRRSPAQEDGKVYINMPGRGGRLVPRG RGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRVKFSRSADAPAYQQGQN QLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 85 (SEQ ID NO: 9 of WO 2019 / 182425) ARPRRSPAQEDGKVYINMPGRG SEQ ID NO: 86 (SEQ ID NO: 11 of WO 2019 / 182425) GRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK SEQ ID NO: 87 (sequence of human CXCR2 polypeptide expressed in N1012_CXCR2) MEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDAAPCEPESLEINKYFVVIIYALVFLLSLLGNSLVMLVILY SRVGRSVTDVYLLNLALADLLFALTLPIWAASKVNGWIFGTFLCKVVSLLKEVNFYSGILLLACISVDRYLAIVH ATRTLTQKRYLVKFICLSIWGLSLLLALPVLLFRRTVYSSNVSPACYEDMGNNTANWRMLLRILPQSFGFIVPLL IMLFCYGFTLRTLFKAHMGQKHRAMRVIFAVVLIFLLCWLPYNLVLLADTLMRTQVIQETCERRNHIDRALDATE ILGILHSCLNPLIYAFIGQKFRHGLLKILAIHGLISKDSLPKDSRPSFVGSSSGHTSTTL SEQ ID NO: 88 (nucleic acid sequence encoding polypeptide of SEQ ID NO: 87) atggaggatttcaatatggagagcgactccttcgaggatttttggaagggcgaggacctgtctaactacagctat agctccacactgcccccttttctgctggatgccgccccttgtgagccagagtccctggagatcaacaagtacttc gtggtcatcatctatgccctggtgtttctgctgtctctgctgggcaatagcctggtcatgctggtcatcctgtac tccagggtgggccgctctgtgaccgacgtgtatctgctgaatctggccctggccgatctgctgttcgcactgaca ctgccaatctgggcagcaagcaaggtgaacggctggatcttcggcacctttctgtgcaaggtggtgtctctgctg aaggaggtgaacttctacagcggcatcctgctgctggcctgtatctccgtggaccggtatctggccatcgtgcac gccaccaggacactgacccagaagcggtacctggtgaagttcatctgcctgagcatctggggactgtccctgctg ctggccctgcctgtgctgctgtttaggcgcacagtgtactctagcaacgtgtctccagcctgttatgaggatatg ggcaacaataccgccaattggaggatgctgctgcgcatcctgccacagagcttcggctttatcgtgcccctgctg atcatgctgttctgctacggctttacactgcggaccctgttcaaggcccacatgggccagaagcaccgggccatg agagtgatcttcgccgtggtgctgatctttctgctgtgctggctgccctataacctggtgctgctggccgacaca ctgatgcggacccaggtcatccaggagacatgcgagcggagaaaccacatcgacagagccctggatgccaccgag atcctgggcatcctgcactcctgtctgaatcctctgatctatgccttcatcggccagaagtttaggcacggcctg ctgaagatcctggccatccacggcctgatctccaaggactctctgcccaaggatagccgcccttccttcgtgggc tcctctagcggccacacctctaccacactg SEQ ID NO: 89 (nucleic acid sequence of SFG N1012_CXCR2)gatccggattagtccaatttgttaaagacaggatatcagtggtccaggctctagttttgactcaacaatatcacc agctgaagcctatagagtacgagccatagataaaataaaagattttatttagtctccagaaaaaggggggaatga aagaccccacctgtaggtttggcaagctagcttaagtaacgccattttgcaaggcatggaaaaatacataactga gaatagagaagttcagatcaaggtcaggaacagatggaacagctgaatatgggccaaacaggatatctgtggtaa gcagttcctgccccggctcagggccaagaacagatggaacagctgaatatgggccaaacaggatatctgtggtaa gcagttcctgccccggctcagggccaagaacagatggtccccagatgcggtccagccctcagcagtttctagaga accatcagatgtttccagggtgccccaaggacctgaaatgaccctgtgccttatttgaactaaccaatcagttcg cttctcgcttctgttcgcgcgcttctgctccccgagctcaataaaagagcccacaacccctcactcggggcgcca gtcctccgattgactgagtcgcccgggtacccgtgtatccaataaaccctcttgcagttgcatccgacttgtggt ctcgctgttccttgggagggtctcctctgagtgattgactacccgtcagcgggggtctttcacatgcagcatgta tcaaaattaatttggttttttttcttaagtatttacattaaatggccatagtacttaaagttacattggcttcct tgaaataaacatggagtattcagaatgtgtcataaatatttctaattttaagatagtatctccattggctttcta ctttttcttttatttttttttgtcctctgtcttccatttgttgttgttgttgtttgtttgtttgtttgttggttg gttggttaatttttttttaaagatcctacactatagttcaagctagactattagctactctgtaacccagggtga ccttgaagtcatgggtagcctgctgttttagccttcccacatctaagattacaggtatgagctatcatttttggt atattgattgattgattgattgatgtgtgtgtgtgtgattgtgtttgtgtgtgtgattgtgtatatgtgtgtatg gttgtgtgtgattgtgtgtatgtatgtttgtgtgtgattgtgtgtgtgtgattgtgcatgtgtgtgtgtgtgatt gtgtttatgtgtatgattgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgttgtgtatatat atttatggtagtgagaggcaacgctccggctcaggtgtcaggttggtttttgagacagagtctttcacttagctt ggaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcag cacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcc tgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgca ctctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccct gacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggt tttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatga taataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttct aaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaaga gtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacc cagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctca acagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctat gtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgact tggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgcca taaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttt tgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacg agcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctag cttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccgg ctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccag atggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacaga tcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattg atttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatccctt aacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttc tgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctac caactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagt taggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctg ccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggct gaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagc tatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggag agcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttg agcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggt tcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtatta ccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcgg aagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttc ccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttac actttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgacc atgattacgccaagctttgctcttaggagtttcctaatacatcccaaactcaaatatataaagcatttgacttgt tctatgccctagggggcggggggaagctaagccagctttttttaacatttaaaatgttaattccattttaaatgc acagatgtttttatttcataagggtttcaatgtgcatgaatgctgcaatattcctgttaccaaagctagtataaa taaaaatagataaacgtggaaattacttagagtttctgtcattaacgtttccttcctcagttgacaacataaatg cgctgctgagaagccagtttgcatctgtcaggatcaatttcccattatgccagtcatattaattactagtcaatt agttgatttttatttttgacatatacatgtgaaagaccccacctgtaggtttggcaagctagcttaagtaacgcc Ķķattttgcaaggcatggaaaaatacataactgagaatagaaaagttcagatcaaggtcaggaacagatggaacagc tgaatatgggccaaacaggatatctgtggtaagcagttcctgccccggctcagggccaagaacagatggaacagc tgaatatgggccaaacaggatatctgtggtaagcagttcctgccccggctcagggccaagaacagatggtcccca gatgcggtccagccctcagcagtttctagagaaccatcagatgtttccagggtgccccaaggacctgaaatgacc ctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgcgcttctgctccccgagctcaata aaagagcccacaacccctcactcggcgcgccagtcctccgattgactgagtcgcccgggtacccgtgtatccaat aaaccctcttgcagttgcatccgacttgtggtctcgctgttccttgggagggtctcctctgagtgattgactacc cgtcagcgggggtctttcatttgggggctcgtccgggatcgggagacccctgcccagggaccaccgacccaccac cgggaggtaagctggccagcaacttatctgtgtctgtccgattgtctagtgtctatgactgattttatgcgcctg cgtcggtactagttagctaactagctctgtatctggcggacccgtggtggaactgacgagttcggaacacccggc cgcaaccctgggagacgtcccagggacttcgggggccgtttttgtggcccgacctgagtcctaaaatcccgatcg tttaggactctttggtgcaccccccttagaggagggatatgtggttctggtaggagacgagaacctaaaacagtt cccgcctccgtctgaatttttgctttcggtttgggaccgaagccgcgccgcgcgtcttgtctgctgcagcatcgt tctgtgttgtctctgtctgactgtgtttctgtatttgtctgaaaatatgggcccgggctagactgttaccactcc cttaagtttgaccttaggtcactggaaagatgtcgagcggatcgctcacaaccagtcggtagatgtcaagaagag acgttgggttaccttctgctctgcagaatggccaacctttaacgtcggatggccgcgagacggcacctttaaccg agacctcatcacccaggttaagatcaaggtcttttcacctggcccgcatggacacccagaccaggtcccctacat cgtgacctgggaagccttggcttttgacccccctccctgggtcaagccctttgtacaccctaagcctccgcctcc tcttcctccatccgccccgtctctcccccttgaacctcctcgttcgaccccgcctcgatcctccctttatccagc cctcactccttctctaggcgcccccatatggccatatgagatcttatatggggcacccccgccccttgtaaactt ccctgaccctgacatgacaagagttactaacagcccctctctccaagctcacttacaggctctctacttagtcca gcacgaagtctggagacctctggcggcagcctaccaagaacaactggaccgaccggtggtacctcacccttaccg agtcggcgacacagtgtgggtccgccgacaccagactaagaacctagaacctcgctggaaaggaccttacacagt cctgctgaccacccccaccgccctcaaagtagacggcatcgcagcttggatacacgccgcccacgtgaaggctgc cgaccccgggggtggaccatcctctagactgccatgatccacctgggccacatcctgttcctgctgctgctgccc gtggccgctgcccagaccacccctggcgagcggagcagcctgcctgccttctaccctggcaccagcggcagctgc agcggctgcggcagcctgagcctgcccctgctggccggcctggtggccgccgacgccgtggccagcctgctgatc gtgggcgccgtgttcctgtgcgccaggcccaggcggagccctgcccaggaggacggcaaggtgtacatcaacatg cccggccggggctacttcctgggcaggctggtgcccaggggcaggggcgctgccgaggctgccacccggaagcag cggatcaccgagaccgagagcccctaccaggagctgcagggccagcggagcgacgtgtacagcgacctgaacacc cagaggccctactacaagaggcggaaaaggtctgggagtggggctaccaatttctctctcctcaagcaagccgga gacgttgaggaaaaccctggacccatgggctggatccggggacggaggagccggcacagctgggagatgagcgag ttccacaactacaacctggacctgaagaagagcgacttcagcacccggtggcagaagcagcggtgccccgtggtg aagagcaagtgccgggagaacgccagccccttcttcttctgctgcttcatcgccgtggctatgggcatccggttc atcatcatggtggccatctggagcgccgtgttcctgaacagcctgttcaaccaggaggtgcagatccccctgacc gagagctactgcggcccctgccccaagaactggatctgctacaagaacaactgctaccagttcttcgacgagagc aagaactggtacgagagccaggccagctgcatgagccagaacgccagcctgctgaaggtgtacagcaaggaggac caggacctgctgaagctggtgaagagctaccactggatgggcctggtgcacatccccaccaacggcagctggcag tgggaggacggcagcatcctgagccccaacctgctgaccatcatcgagatgcagaagggcgactgcgccctgtac gccagcagcttcaagggctacatcgagaactgcagcacccccaacacctacatctgcatgcagcggacagtgcgg agaaagagatccggatctggagagggaagaggaagcctgctgacctgcggcgacgtggaggagaacccaggaccc atggaggatttcaatatggagagcgactccttcgaggatttttggaagggcgaggacctgtctaactacagctat agctccacactgcccccttttctgctggatgccgccccttgtgagccagagtccctggagatcaacaagtacttc gtggtcatcatctatgccctggtgtttctgctgtctctgctgggcaatagcctggtcatgctggtcatcctgtac tccagggtgggccgctctgtgaccgacgtgtatctgctgaatctggccctggccgatctgctgttcgcactgaca ctgccaatctgggcagcaagcaaggtgaacggctggatcttcggcacctttctgtgcaaggtggtgtctctgctg aaggaggtgaacttctacagcggcatcctgctgctggcctgtatctccgtggaccggtatctggccatcgtgcac gccaccaggacactgacccagaagcggtacctggtgaagttcatctgcctgagcatctggggactgtccctgctg ctggccctgcctgtgctgctgtttaggcgcacagtgtactctagcaacgtgtctccagcctgttatgaggatatg ggcaacaataccgccaattggaggatgctgctgcgcatcctgccacagagcttcggctttatcgtgcccctgctg atcatgctgttctgctacggctttacactgcggaccctgttcaaggcccacatgggccagaagcaccgggccatg agagtgatcttcgccgtggtgctgatctttctgctgtgctggctgccctataacctggtgctgctggccgacaca ctgatgcggacccaggtcatccaggagacatgcgagcggagaaaccacatcgacagagccctggatgccaccgag atcctgggcatcctgcactcctgtctgaatcctctgatctatgccttcatcggccagaagtttaggcacggcctg ctgaagatcctggccatccacggcctgatctccaaggactctctgcccaaggatagccgcccttccttcgtgggc tcctctagcggccacacctctaccacactgtgacagccactcgag SEQ ID NO: 90 (protein encoded by SEQ ID NO: 89 (includes: (i) fusion of full length DAP10 / DAP12 intracellular domain; (ii) furin cleavage site (RRKR); (iii) SGSG linker; (iv) P2A skip sequence; (v) NKG2D; (vi) furin cleavage site (RRKR); (vii) SGSG linker; (viii) T2A skip sequence; (ix) CXCR2)MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPR RSPAQEDGKVYINMPGRGYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRS GSGATNFSLLKQAGDVEENPGPMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPF FFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCM SQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENC STPNTYICMQRTVRRKRSGSGEGRGSLLTCGDVEENPGPMEDFNMESDSFEDFWKGEDLSNYSYSSTLPPFLLDA APCEPESLEINKYFVVIIYALVFLLSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIWAASKVNG WIFGTFLCKVVSLLKEVNFYSGILLLACISVDRYLAIVHATRTLTQKRYLVKFICLSIWGLSLLLALPVLLFRRT VYSSNVSPACYEDMGNNTANWRMLLRILPQSFGFIVPLLIMLFCYGFTLRTLFKAHMGQKHRAMRVIFAVVLIFL LCWLPYNLVLLADTLMRTQVIQETCERRNHIDRALDATEILGILHSCLNPLIYAFIGQKFRHGLLKILAIHGLIS KDSLPKDSRPSFVGSSSGHTSTTL SEQ ID NO: 91 (encoding the polypeptide of SEQ ID NO: 90) ATGATCCACCTGGGCCACATCCTGTTCCTGCTGCTGCTGCCCGTGGCCGCTGCCCAG ACCACCCCTGGCGAGCGGAGCAGCCTGCCTGCCTTCTACCCTGGCACCAGCGGCAGCTGCAGCGGCTGCGGCAGCCTGAG CCTGCCCCTGCTGGCCGGCCTGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATCGTGGGCGCCGTGTTCCTGTGCGCCA GGCCCAGGCGGAGCCCtGCCCAGGAGGACGGCAAGGTGTACATCAACATGCCCGGCCGGGGCTACTTCCTGGGCAGGCTG GTGCCCAGGGGCAGGGGCGCTGCCGAGGCTGCCACCCGGAAGCAGCGGATCACCGAGACCGAGAGCCCCTACCAGGAGCT GCAGGGCCAGCGGAGCGACGTGTACAGCGACCTGAACACCCAGAGGCCCTACTACAAGAGGCGGAAAAGGTCTGGGAGTG GGGCTACCAATTTCTCTCTCCTCAAGCAAGCCGGAGACGTTGAGGAAAACCCTGGaCCcATGGGCTGGATCCGGGGACGG AGGAGCCGGCACAGCTGGGAGATGAGCGAGTTCCACAACTACAACCTGGACCTGAAGAAGAGCGACTTCAGCACCCGGTG GCAGAAGCAGCGGTGCCCCGTGGTGAAGAGCAAGTGCCGGGAGAACGCCAGCCCCTTCTTCTTCTGCTGCTTCATCGCCG TGGCtATGGGCATCCGGTTCATCATCATGGTGGCCATCTGGAGCGCCGTGTTCCTGAACAGCCTGTTCAACCAGGAGGTG CAGATCCCCCTGACCGAGAGCTACTGCGGCCCCTGCCCCAAGAACTGGATCTGCTACAAGAACAACTGCTACCAGTTCTT CGACGAGAGCAAGAACTGGTACGAGAGCCAGGCCAGCTGCATGAGCCAGAACGCCAGCCTGCTGAAGGTGTACAGCAAGG AGGACCAGGACCTGCTGAAGCTGGTGAAGAGCTACCACTGGATGGGCCTGGTGCACATCCCCACCAACGGCAGCTGGCAG TGGGAGGACGGCAGCATCCTGAGCCCCAACCTGCTGACCATCATCGAGATGCAGAAGGGCGACTGCGCCCTGTACGCCAG CAGCTTCAAGGGCTACATCGAGAACTGCAGCACCCCCAACACCTACATCTGCATGCAGCGGACAGTGCGGAGAAAGAGAT CCGGATCTGGAGAGGGAAGAGGAAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCAGGACCCATGGAGGATTTCAAT ATGGAGAGCGACTCCTTCGAGGATTTTTGGAAGGGCGAGGACCTGTCTAACTACAGCTATAGCTCCACACTGCCCCCTTT TCTGCTGGATGCCGCCCCTTGTGAGCCAGAGTCCCTGGAGATCAACAAGTACTTCGTGGTCATCATCTATGCCCTGGTGT TTCTGCTGTCTCTGCTGGGCAATAGCCTGGTCATGCTGGTCATCCTGTACTCCAGGGTGGGCCGCTCTGTGACCGACGTG TATCTGCTGAATCTGGCCCTGGCCGATCTGCTGTTCGCACTGACACTGCCAATCTGGGCAGCAAGCAAGGTGAACGGCTG GATCTTCGGCACCTTTCTGTGCAAGGTGGTGTCTCTGCTGAAGGAGGTGAACTTCTACAGCGGCATCCTGCTGCTGGCCT GTATCTCCGTGGACCGGTATCTGGCCATCGTGCACGCCACCAGGACACTGACCCAGAAGCGGTACCTGGTGAAGTTCATC TGCCTGAGCATCTGGGGACTGTCCCTGCTGCTGGCCCTGCCTGTGCTGCTGTTTAGGCGCACAGTGTACTCTAGCAACGT GTCTCCAGCCTGTTATGAGGATATGGGCAACAATACCGCCAATTGGAGGATGCTGCTGCGCATCCTGCCACAGAGCTTCG GCTTTATCGTGCCCCTGCTGATCATGCTGTTCTGCTACGGCTTTACACTGCGGACCCTGTTCAAGGCCCACATGGGCCAG AAGCACCGGGCCATGAGAGTGATCTTCGCCGTGGTGCTGATCTTTCTGCTGTGCTGGCTGCCCTATAACCTGGTGCTGCT GGCCGACACACTGATGCGGACCCAGGTCATCCAGGAGACATGCGAGCGGAGAAACCACATCGACAGAGCCCTGGATGCCA CCGAGATCCTGGGCATCCTGCACTCCTGTCTGAATCCTCTGATCTATGCCTTCATCGGCCAGAAGTTTAGGCACGGCCTG CTGAAGATCCTGGCCATCCACGGCCTGATCTCCAAGGACTCTCTGCCCAAGGATAGCCGCCCTTCCTTCGTGGGCTCCTC TAGCGGCCACACCTCTACCACACTGTGACAGCCACTCGAG SEQ ID NO: 92 (amino acid sequence of a replica of the Cyad-01 CAR (NKG2D fused to the intracellular domain of CD3ζ) MRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQ RCPVVKSKCRENASPFFFCCFIAVAMGIRFIIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQ FFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKG DCALYASSFKGYIENCSTPNTYICMQRTV SEQ ID NO: 93 (nucleic acid sequence encoding polypeptide of SEQ ID NO: 92) ATGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCcGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTC AATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCG AGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATT GGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGAC ACCTACGATGCATTGCACATGCAGGCCCTGCCCCCTCGCATGGGCTGGATCCGCGGCCGCAGGAGCCGGCACAGC TGGGAGATGAGCGAGTTCCACAACTACAACCTGGACCTGAAGAAGAGCGACTTCAGCACCCGGTGGCAGAAGCAGCGGTGCCCCGTGGTGAAGAGCAAGTGCCGGGAGAACGCCAGCCCCTTCTTCTTCTGCTGCTTCATCGCCGTGGCt ATGGGCATCCGGTTTATAATCATGGTGGCCATCTGGAGCGCCGTGTTCCTGAACAGCCTGTTCAACCAGGAGGTG CAGATCCCCCTGACCGAGAGCTACTGCGGCCCCTGCCCCAAGAACTGGATCTGCTACAAGAACAACTGCTACCAG TTCTTCGACGAGAGCAAGAACTGGTACGAGAGCCAGGCCAGCTGCATGAGCCAGAACGCCAGCCTGCTGAAGGTG TACAGCAAGGAGGACCAGGACCTGCTGAAGCTGGTGAAGAGCTACCACTGGATGGGCCTGGTGCACATCCCCACC AACGGCAGCTGGCAGTGGGAGGACGGCAGCATCCTGAGCCCCAACCTGCTGACCATCATCGAGATGCAGAAGGGC GACTGCGCCCTGTACGCCAGCAGCTTCAAGGGCTACATCGAGAACTGCAGCACCCCCAACACCTACATCTGCATG CAGCGGACCGTGtaa SEQ ID NO: 94 (nucleic acid sequence encoding CYAD-01_10 – polypeptides of SEQ ID NO: 92 and SEQ ID NO: 1) ATGATCCACCTGGGCCACATCCTGTTCCTGCTGCTGCTGCCCGTGGCCGCTGCCCAGACCACCCCTGGCGAGCGG AGCAGCCTGCCTGCCTTCTACCCTGGCACCAGCGGCAGCTGCAGCGGCTGCGGCAGCCTGAGCCTGCCCCTGCTG GCCGGCCTGGTGGCCGCCGACGCCGTGGCCAGCCTGCTGATCGTGGGCGCCGTGTTCCTGTGCGCCAGGCCCAGG CGGAGCCCtGCCCAGGAGGACGGCAAGGTGTACATCAACATGCCCGGCCGGGGCAGGCGGAAAAGGTCTGGGAGT GGGGCTACCAATTTCTCTCTCCTCAAGCAAGCCGGAGACGTTGAGGAAAACCCTGGaCCcATGAGAGTGAAGTTC AGCAGGAGCGCAGACGCCCCcGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGA GAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCT CAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAG CGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGgacacctacgatgcaTTG CACATGCAGGCCCTGCCCCCTCGCATGGGCTGGATCCGCGGCCGCAGGAGCCGGCACAGCTGGGAGATGAGCGAG TTCCACAACTACAACCTGGACCTGAAGAAGAGCGACTTCAGCACCCGGTGGCAGAAGCAGCGGTGCCCCGTGGTG AAGAGCAAGTGCCGGGAGAACGCCAGCCCCTTCTTCTTCTGCTGCTTCATCGCCGTGGCtATGGGCATCCGGTTT ATAATCATGGTGGCCATCTGGAGCGCCGTGTTCCTGAACAGCCTGTTCAACCAGGAGGTGCAGATCCCCCTGACC GAGAGCTACTGCGGCCCCTGCCCCAAGAACTGGATCTGCTACAAGAACAACTGCTACCAGTTCTTCGACGAGAGC AAGAACTGGTACGAGAGCCAGGCCAGCTGCATGAGCCAGAACGCCAGCCTGCTGAAGGTGTACAGCAAGGAGGAC CAGGACCTGCTGAAGCTGGTGAAGAGCTACCACTGGATGGGCCTGGTGCACATCCCCACCAACGGCAGCTGGCAG TGGGAGGACGGCAGCATCCTGAGCCCCAACCTGCTGACCATCATCGAGATGCAGAAGGGCGACTGCGCCCTGTAC GCCAGCAGCTTCAAGGGCTACATCGAGAACTGCAGCACCCCCAACACCTACATCTGCATGCAGCGGACCGTGtaa SEQ ID NO: 95 (human extracellular NKG2D domain) LFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMG LVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV SEQ ID NO: 96 (human extracellular NKG2D domain) IWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLK LVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV SEQ ID NO: 97 (SEQ ID NO: 95 minus 8 most N-terminal amino acids) PLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNG SWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV SEQ ID NO: 98 (mouse NKG2D TM domain; UniProt accession no: 054709) VVRVLAIALAIRFTLNTLMWLAI SEQ ID NO: 99 (mouse NKG2D TM domain) KISPMFVVRVLAIALAIRFTLNTLMWLAIFKETFQPV SEQ ID NO: 100 (rat NKG2D) MSKCHNYDLKPAKWDTSQEHQKQRSALPTSRPGENGIIRRRSSIEELKISPLFVVRVLVAAMTIRFTVITLTWLAVFITLLCNKEVSVSSREGYCGPCPNDWICHRNNCYQFFNENKAWN QSQASCLSQNSSLLKIYSKEEQDFLKLVKSYHWMGLVQSPANGSWQWEDGSSLSPNELTL VKTPSGTCAVYGSSFKAYTEDCSNPNTYICMKRAV SEQ ID NO: 101 (rat NKG2D TM domain; UniProt accession no: 070215 aa 52-74) LFVVRVLVAAMTIRFTVITLTWL SEQ ID NO: 102 (N5 polypeptide) MALPVTALLLPLALLLHAARPDYKDDDDKLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRL VPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKRRKRSGSGEGRGSLLTCGDVEENPGPMIH LGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPRRSP AQEDGKVYINMPGRGRRKRSGSGATNFSLLKQAGDVEENPGPMKISPMFVVRVLAIALAIRFTLNTLMWLAIFKE TFQPVLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKS YHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV SEQ ID NO: 103 (nucleic acid encoding polypeptide of SEQ ID NO: 102) ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCTAGACCCGATTATAAGGAC GACGACGACAAGCTGAGACCCGTGCAGGCCCAGGCCCAGAGCGACTGCAGCTGCAGCACCGTGAGCCCCGGCGTG CTGGCCGGCATCGTGATGGGCGACCTGGTGCTGACCGTGCTCATCGCCCTTGCCGTGTACTTCCTGGGCAGACTG GTCCCCAGGGGCAGAGGAGCTGCCGAGGCCGCTACCAGAAAGCAGAGGATCACCGAGACAGAGAGCCCCTACCAG GAGCTGCAGGGCCAGAGATCCGACGTGTACAGCGACCTCAACACCCAGAGACCCTATTACAAGAGGCGGAAGCGC TCCGGCTCCGGCGAGGGCCGCGGCAGCCTGCTGACCTGCGGCGACGTGGAAGAGAACCCCGGACCCATGATCCAC CTGGGCCACATCCTGTTCCTGCTGCTGCTGCCCGTGGCCGCTGCCCAAACAACACCCGGCGAGAGATCCTCCTTG CCCGCTTTCTATCCCGGAACATCCGGAAGCTGTTCCGGATGTGGATCCCTTTCTTTGCCTTTGCTTGCTGGATTG GTCGCAGCTGACGCTGTCGCTTCCCTCCTTATTGTCGGAGCTGTCTTCCTGTGCGCCAGGCCCAGGCGGAGCCCT GCCCAGGAGGACGGCAAGGTGTACATCAACATGCCCGGCCGGGGCAGGCGGAAGCGCTCCGGGAGTGGGGCTACC AATTTCTCTCTCCTCAAGCAAGCCGGAGACGTTGAGGAAAACCCTGGACCCATGAAAATATCTCCAATGTTCGTT GTTCGAGTCCTTGCTATAGCCTTGGCAATTCGATTCACCCTTAACACATTGATGTGGCTTGCCATTTTCAAAGAG ACGTTTCAGCCAGTACTGTTCAACCAGGAGGTGCAGATCCCCCTGACCGAGAGCTACTGCGGCCCCTGCCCAAAA AATTGGATCTGCTACAAGAACAACTGCTACCAGTTCTTCGACGAGAGCAAGAACTGGTACGAGAGCCAGGCCAGC TGCATGAGCCAGAACGCCAGCCTGCTGAAGGTGTACAGCAAGGAGGACCAGGACCTGCTGAAGCTGGTGAAGAGC TACCACTGGATGGGCCTGGTGCACATCCCCACCAACGGCAGCTGGCAGTGGGAGGACGGCAGCATCCTGAGCCCC AACCTGCTGACCATCATCGAGATGCAGAAGGGCGACTGCGCCCTGTACGCCAGCAGCTTCAAGGGCTACATCGAG AACTGCAGCACCCCCAACACCTACATCTGCATGCAGCGGACCGTG SEQ ID NO: 104 (human IgG1 hinge – aa 218-232 of UniProt: P0DOX5) EPK SCDKTHTCPP CP SEQ ID NO: 105 (amino acid sequence of CYAD-01_10 (NKG2D-CD3ζ + ribosomal skip peptide + DAP10)) MIHLGHILFLLLLPVAAAQTTPGERSSLPAFYPGTSGSCSGCGSLSLPLLAGLVAADAVASLLIVGAVFLCARPR RSPAQEDGKVYINMPGRGRRKRSGSGATNFSLLKQAGDVEENPGPMRVKFSRSADAPAYQQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPRMGWIRGRRSRHSWEMSEFHNYNLDLKKSDFSTRWQKQRCPVVKSKCRENASPFFFCCFIAVAMGIRF IIMVAIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKED QDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV

Claims

WHAT IS CLAIMED IS:

1. A method of depleting senescent cells in a subject, comprising: administering to the subject a therapeutically effective amount of an immunoresponsive cell which expresses a chimeric antigen receptor comprising: (a) an NKG2D polypeptide; (b) a fusion polypeptide comprising (i) a DNAX-activating 10 (DAP10) polypeptide, or a functional variant thereof and (ii) a DNAX-activating protein 12 (DAP12) polypeptide, or a functional variant thereof; and, optionally, (c) a CXCR2 polypeptide.

2. The method of claim 1, wherein the depletion of senescent cells is measured by a reduction in the expression level of one or more cellular senescence markers (e.g., KLRG1, CD57, P16 (CDKN2A), P21 (CDKN1A), p53, SA-^-galactosidase and a range of inflammatory cytokines, including IL-6, IL-1^ and IL-8) in a sample collected from the subject following administration of the immunoresponsive cell compared to the expression level in a sample collected from the subject prior to administration of the immunoresponsive cell.

3. The method of claim 1 or claim 2, wherein the depleting senescent cells reduces and / or prevents inflammation within one or more tissues, thereby reducing tissue damage.

4. The method of any one of the preceding claims, wherein the depleting senescent cells results in a reduction in the number of myeloid-derived suppressor cells, regulatory T-cells, and / or M2 polarized macrophages in the subject compared to the number of myeloid-derived suppressor cells, regulatory T-cells, and / or M2 polarized macrophages in the subject prior to administration of the therapeutically effective amount of the immunoresponsive cell.

5. The method of any one of the preceding claims, wherein an age-related cellular atrophy is attenuated in the subject following administration of the immunoresponsive cell.

6. The method of any one of the preceding claims, wherein an age-related bone loss is attenuated in the subject following administration of the immunoresponsive cell.

7. The method of any one of the preceding claims, wherein an age-related cognitive decline is attenuated in the subject following administration of the immunoresponsive cell.

8. The method of any one of the preceding claims, wherein an age-related parenchymal loss is attenuated in the subject following administration of the immunoresponsive cell.

9. The method of any one of the preceding claims, wherein an age-related cardiovascular disease is attenuated in the subject following administration of the immunoresponsive cell.

10. The method of any one of the preceding claims, wherein an age-related pulmonary disease is attenuated in the subject following administration of the immunoresponsive cell.

11. The method of any one of the preceding claims, wherein an age-related sensory loss is attenuated in the subject following administration of the immunoresponsive cell.

12. The method of any one of the preceding claims, wherein an age-related diabetes is attenuated in the subject following administration of the immunoresponsive cell.

13. The method of any one of the preceding claims, wherein an age-related arthritis is attenuated in the subject following administration of the immunoresponsive cell.

14. The method of any one of the preceding claims, wherein a fibrosis-related disease is attenuated in the subject following administration of the immunoresponsive cell.

15. The method of any one of the preceding claims, wherein the subject is human.

16. The method of any one of the preceding claims, wherein each of the NKG2D polypeptide, the DAP10 polypeptide, the DAP12 polypeptide, and the CXCR2 polypeptide is a mammalian polypeptide.

17. The method of claim 15, wherein each of the NKG2D polypeptide, the DAP10 polypeptide, the DAP12 polypeptide, and the CXCR2 polypeptide is a human polypeptide.

18. The method of any one of the preceding claims, wherein the amino acid sequence of the NKG2D polypeptide has at least about 85% sequence identity to the sequence of SEQ ID NO:

14.

19. The method of any one of the preceding claims, wherein the NKG2D polypeptide is a functional variant of the polypeptide having the amino acid sequence of SEQ ID NO:

14.

20. The method of claim 19, wherein the functional variant has one or more point mutations that add, delete, or substitute at least one of the amino acids of SEQ ID NO:

14.

21. The method of claim 19, wherein the functional variant is a truncated version of the polypeptide having the amino acid sequence of SEQ ID NO:

14.

22. The method of claim 19, wherein the functional variant is a chimeric NKG2D polypeptide.

23. The method of any one of the preceding claims, wherein the fusion polypeptide has the formula, from N-terminus to C-terminus: A-B-C-D-E, wherein A is an optional N-terminal sequence; B is a DAP10 polypeptide or functional variant thereof; C is an optional linker sequence; D is a DAP12 polypeptide or functional variant thereof; and E is an optional C-terminal sequence.

24. The method of 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.

25. The method of 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.

26. The method of any one of the preceding claims, wherein the DAP10 polypeptide is a functional variant of a DAP10 polypeptide which is a truncated version of the polypeptide having the amino acid sequence of SEQ ID NO:

1.

27. The method of any one of the preceding claims, wherein the truncated version 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.

28. The method of any one of claims 1-22, wherein the DAP10 polypeptide comprises or consists of any one of SEQ ID NOs: 1-8.

29. The method of 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.

30. The method of any one of the preceding claims, wherein the DAP12 polypeptide is a functional variant of SEQ ID NO: 9 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.

31. The method of any one of the preceding claims, wherein the DAP12 polypeptide is a functional variant of a DAP12 polypeptide which is a truncated version of the polypeptide having the amino acid sequence of SEQ ID NO:

9.

32. The method of claim 31, wherein the truncated version of DAP12 comprises or consists of amino acids 22-113, 62-113, 22-61, or 41-61 of SEQ ID NO: 9.

33. The method of any one of claims 1-28, wherein the DAP12 polypeptide comprises or consists of any one of SEQ ID NOs: 9-13.

34. The method of any one of the preceding claims, wherein the DAP10 polypeptide and the DAP12 polypeptide are joined by a linker.

35. The method of claim 33, wherein the linker comprises or consists of the amino acid sequence recited in any of SEQ ID NOs: 18-46.

36. The method of claim 34 or claim 35, wherein the linker comprises or consists of the amino acid sequence recited in any of SEQ ID NOs: 33 or 38-44.

37. The method of any one of the preceding claims, wherein the fusion polypeptide comprises an N-terminal sequence.

38. The method of any one of the preceding claims, wherein the fusion polypeptide comprises a C-terminal sequence.

39. The method of claim 37 or 38, wherein the N-terminal or C-terminal sequence comprises one or more of a His-tag, FLAG-tag, Arg-tag, T7-tag, Strep-tag, S-tag, an AviTagTM, an aptamer-tag, a myc tag, CD8α leader sequence, a 4-1BB endodomain, a V5 tag, or a CD27 endodomain.

40. The method of any claim 39, wherein the N-terminal or C-terminal sequence comprises one or more of a CD8α leader sequence, a 4-1BB endodomain, or a CD27 endodomain.

41. The method of any one of the preceding claims, wherein the fusion polypeptide comprises or consists of the sequence of any one of SEQ ID NOs: 60 to 63.

42. The method of any one of claims 1-22, wherein the fusion polypeptide: (a) does not comprise SEQ ID NO: 84; and / or (b) does not comprise an anti-EpCAM peptide; and / or (c) does not comprise SEQ ID NO: 85; and / or(d) does not comprise SEQ ID NO: 86; and / or (e) does not comprise both SEQ ID NO: 85 and SEQ ID NO:

86.

43. The method of any one of the preceding claims, wherein the fusion polypeptide comprises or consists of full length human DAP10 fused at its C-terminus to the endodomain of human DAP12 polypeptide, wherein the endodomain is encoded by amino acids 62-113 of human DAP12.

44. The method of any one of the preceding claims, wherein the fusion polypeptide has the sequence of SEQ ID NO:

60.

45. The method of any one of the preceding claims, wherein the amino acid sequence of the CXCR2 polypeptide has at least about 85% sequence identity to the sequence of SEQ ID NO:

87.

46. The method of claim 45, wherein the CXCR2 polypeptide has the amino acid sequence of SEQ ID NO:

87.

47. The method of any one of the preceding claims, wherein the immunoresponsive cell is generated from a population of T or NK cells that are autologous to the subject.

48. The method of any one of claims 1-47, wherein the immunoresponsive cell is generated from a population of T or NK cells that are allogeneic to the subject.

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