Vdelta1 + t cells for the treatment of myeloid malignancies

Abstract

To provide a composition for use in treating a patient having a myeloid malignancy.SOLUTION: An allogeneic composition comprising γ δ 1 + T cells is provided. Preferably, it is an allogeneic composition comprising at least about 60% γ δ T cells with respect to total viable cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to compositions comprising V51 T cells for use in the treatment of myeloid malignancies. The present invention also relates to methods of treatment using said compositions. [Background technology]

[0002] Acute myeloid leukemia (AML) remains a clinical challenge due to the high incidence of chemotherapy resistance and fatal relapse. The survival rate for AML in elderly patients (≥65 years) is low (10%), primarily due to resistance to standard treatment. Available treatment consists of combining cytarabine with anthracyclines. Although effective in inducing complete remission, this treatment ultimately selects for chemotherapy-resistant clones that cause refractory relapse. Promising alternatives to chemotherapy are targeted therapies and upcoming immunotherapies, which have been successful against B-cell malignancies.

[0003] Measurable residual disease (MRD) is an independent post-diagnosis prognostic indicator in AML and myelodysplastic syndromes (MDS), and this indicator is important for risk stratification and treatment planning because MRD+ patients have a high tendency to relapse and poor survival even when morphological complete remission occurs. Eliminating MRD in AML and MDS is an area of ​​great unmet need, but is difficult due to the lack of specific antigens expressed on leukemic blasts.

[0004] The presence of γδ T cells has been shown to positively correlate with the prognosis of several solid and hematological cancers (Deniger at al. Clin. Cancer Res. (2014) 20(22):5708-5719, Gentles et al. Nat. Med. (2015) 21(8):938-945). While the use of V52 T cells in such therapies is being explored, clinical manipulation of V51 T cells has been hampered by their relatively low abundance (<0.5%) in peripheral blood lymphocytes. However, recent improvements in the yield of V51 T cells suitable for clinical use, by methods such as those described in WO2016 / 198480, have fulfilled the need for the first treatment of myeloid malignancies described herein. Summary of the Invention

[0005] According to a first aspect of the present invention there is provided an allogeneic composition comprising V51+ T cells for use in treating patients with myeloid malignancies.

[0006] According to another aspect of the present invention, there is provided a dose comprising an allogeneic composition for use as described herein.

[0007] According to another aspect of the invention, there is provided a method of treating a myeloid malignancy, the method comprising administering to a patient having the myeloid malignancy a therapeutically effective amount of an allogeneic composition comprising V51 T cells. [Brief explanation of the drawings]

[0008] [Figure 1A]γδ T cell compositions exhibit higher clonal diversity than ex vivo Vδ1 T cells. Graphical representation of the TRGV and TRDV repertoires and CDR3 length (number of nucleotides) distribution of FACS-sorted Vδ1+ T cells derived from peripheral blood / PB or DOT cell products obtained from four individual healthy donors (HD#A-D). Each box represents a different clonotype (with a different nucleotide sequence), and its area is proportional to its relative abundance in the sample. Color represents the classification of clonotypes by chain. [Figure 1B] γδ T cell compositions exhibit higher clonal diversity than ex vivo Vδ1 T cells. Graphical representation of the TRGV and TRDV repertoires and CDR3 length (number of nucleotides) distribution of FACS-sorted Vδ1+ T cells derived from peripheral blood / PB or DOT cell products obtained from four individual healthy donors (HD#A-D). Each box represents a different clonotype (with a different nucleotide sequence), and its area is proportional to its relative abundance in the sample. Color represents the classification of clonotypes by chain. [Figure 1C] γδ T cell compositions exhibit higher clonal diversity than ex vivo Vδ1 T cells. Graphical representation of the TRGV and TRDV repertoires and CDR3 length (number of nucleotides) distribution of FACS-sorted Vδ1+ T cells derived from peripheral blood / PB or DOT cell products obtained from four individual healthy donors (HD#A-D). Each box represents a different clonotype (with a different nucleotide sequence), and its area is proportional to its relative abundance in the sample. Color represents the classification of clonotypes by chain. [Figure 1D] γδ T cell compositions exhibit higher clonal diversity than ex vivo Vδ1 T cells. Graphical representation of the TRGV and TRDV repertoires and CDR3 length (number of nucleotides) distribution of FACS-sorted Vδ1+ T cells derived from peripheral blood / PB or DOT cell products obtained from four individual healthy donors (HD#A-D). Each box represents a different clonotype (with a different nucleotide sequence), and its area is proportional to its relative abundance in the sample. Color represents the classification of clonotypes by chain. [Figure 2]Influence of CD27 expression phenotype on TCR repertoire diversity and AML reactivity of γδ T cell composition. (A) In vitro killing of AML KG-1 cells by DOT cells derived from pre-sorted CD27+V51+ or CD27-V51+ T cells (cultured for 21 days). Cells were co-incubated at a ratio of 10:1 (E:T) for 3 hours and then analyzed by Annexin V staining (percent positive events among pre-labeled KG-1 cells). Data represent the mean of two technical replicates for each donor. (B) Expression of NKp30 and NKp44 in CD27+ cells (black) and CD27- cells (white) after expansion of DOT cells. Mean values ​​of technical replicates are shown. [Figure 3] Reactivity of clonal γδ T cell compositions against AML cells. (A) and (B) show in vitro killing of AML KG-1 cells by DOT cell clones generated from a single Vδ1 T cell sorted from a healthy donor. Cells were co-incubated at a 10:1 (E:T) ratio for 3 hours and then analyzed by Annexin V staining (percent positive events among pre-labeled KG-1 cells). Each bar represents KG-1 cell killing upon co-incubation with an individual clone. The horizontal dashed line represents the mean basal tumor cell death (without DOT cells). In B, either an anti-Vδ1 TCR-specific mAb or an isotype control was added to the culture. Clones with more clearly reduced KG-1 targeting upon blockade are indicated. Data represent the mean of two technical replicates and are derived from four individual healthy donors (HD). [Figure 4A]The γδ T cell composition targets multiple AML cell types but not healthy leukocytes. In in vitro killing assays, DOT cells generated from three to four healthy donors were co-incubated with the indicated AML cell lines (A), primary AML samples (B), or normal leukocyte populations FACS-sorted from peripheral blood (C) at a 10:1 (E:T) ratio for 3 hours. In A, the horizontal dashed line represents the mean basal tumor cell death; in B, the CTR also represents tumor cells alone (no DOT cells). Experiments were performed in technical triplicate. (D) In ​​in vitro killing assays, non-expanded fresh ("ex vivo") Vδ1 T cells collected from three healthy donors were co-incubated with the indicated HEL or KG-1 cell lines at a 10:1 (E:T) ratio for 3 hours. DOT cells generated from HD#1 are shown as a positive control. (E) Granzyme B and perforin expression in DOT cells was assessed by intracellular flow cytometry. (F) Percentage of CD107a+DOT cells after co-incubation with AML tumor targets or upon stimulation with PMA / ionomycin (positive control) or without (negative control). Results are from two healthy donors and were tested in duplicate. [Figure 4B]The γδ T cell composition targets multiple AML cell types but not healthy leukocytes. In in vitro killing assays, DOT cells generated from three to four healthy donors were co-incubated with the indicated AML cell lines (A), primary AML samples (B), or normal leukocyte populations FACS-sorted from peripheral blood (C) at a 10:1 (E:T) ratio for 3 hours. In A, the horizontal dashed line represents the mean basal tumor cell death; in B, the CTR also represents tumor cells alone (no DOT cells). Experiments were performed in technical triplicate. (D) In ​​in vitro killing assays, non-expanded fresh ("ex vivo") Vδ1 T cells collected from three healthy donors were co-incubated with the indicated HEL or KG-1 cell lines at a 10:1 (E:T) ratio for 3 hours. DOT cells generated from HD#1 are shown as a positive control. (E) Granzyme B and perforin expression in DOT cells was assessed by intracellular flow cytometry. (F) Percentage of CD107a+DOT cells after co-incubation with AML tumor targets or upon stimulation with PMA / ionomycin (positive control) or without (negative control). Results are from two healthy donors and were tested in duplicate. [Figure 4C]The γδ T cell composition targets multiple AML cell types but not healthy leukocytes. In in vitro killing assays, DOT cells generated from three to four healthy donors were co-incubated with the indicated AML cell lines (A), primary AML samples (B), or normal leukocyte populations FACS-sorted from peripheral blood (C) at a 10:1 (E:T) ratio for 3 hours. In A, the horizontal dashed line represents the mean basal tumor cell death; in B, the CTR also represents tumor cells alone (no DOT cells). Experiments were performed in technical triplicate. (D) In ​​in vitro killing assays, non-expanded fresh ("ex vivo") Vδ1 T cells collected from three healthy donors were co-incubated with the indicated HEL or KG-1 cell lines at a 10:1 (E:T) ratio for 3 hours. DOT cells generated from HD#1 are shown as a positive control. (E) Granzyme B and perforin expression in DOT cells was assessed by intracellular flow cytometry. (F) Percentage of CD107a+DOT cells after co-incubation with AML tumor targets or upon stimulation with PMA / ionomycin (positive control) or without (negative control). Results are from two healthy donors and were tested in duplicate. [Figure 4D]The γδ T cell composition targets multiple AML cell types but not healthy leukocytes. In in vitro killing assays, DOT cells generated from three to four healthy donors were co-incubated with the indicated AML cell lines (A), primary AML samples (B), or normal leukocyte populations FACS-sorted from peripheral blood (C) at a 10:1 (E:T) ratio for 3 hours. In A, the horizontal dashed line represents the mean basal tumor cell death; in B, the CTR also represents tumor cells alone (no DOT cells). Experiments were performed in technical triplicate. (D) In ​​in vitro killing assays, non-expanded fresh ("ex vivo") Vδ1 T cells collected from three healthy donors were co-incubated with the indicated HEL or KG-1 cell lines at a 10:1 (E:T) ratio for 3 hours. DOT cells generated from HD#1 are shown as a positive control. (E) Granzyme B and perforin expression in DOT cells was assessed by intracellular flow cytometry. (F) Percentage of CD107a+DOT cells after co-incubation with AML tumor targets or upon stimulation with PMA / ionomycin (positive control) or without (negative control). Results are from two healthy donors and were tested in duplicate. [Figure 4E]The γδ T cell composition targets multiple AML cell types but not healthy leukocytes. In in vitro killing assays, DOT cells generated from three to four healthy donors were co-incubated with the indicated AML cell lines (A), primary AML samples (B), or normal leukocyte populations FACS-sorted from peripheral blood (C) at a 10:1 (E:T) ratio for 3 hours. In A, the horizontal dashed line represents the mean basal tumor cell death; in B, the CTR also represents tumor cells alone (no DOT cells). Experiments were performed in technical triplicate. (D) In ​​in vitro killing assays, non-expanded fresh ("ex vivo") Vδ1 T cells collected from three healthy donors were co-incubated with the indicated HEL or KG-1 cell lines at a 10:1 (E:T) ratio for 3 hours. DOT cells generated from HD#1 are shown as a positive control. (E) Granzyme B and perforin expression in DOT cells was assessed by intracellular flow cytometry. (F) Percentage of CD107a+DOT cells after co-incubation with AML tumor targets or upon stimulation with PMA / ionomycin (positive control) or without (negative control). Results are from two healthy donors and were tested in duplicate. [Figure 4F]The γδ T cell composition targets multiple AML cell types but not healthy leukocytes. In in vitro killing assays, DOT cells generated from three to four healthy donors were co-incubated with the indicated AML cell lines (A), primary AML samples (B), or normal leukocyte populations FACS-sorted from peripheral blood (C) at a 10:1 (E:T) ratio for 3 hours. In A, the horizontal dashed line represents the mean basal tumor cell death; in B, the CTR also represents tumor cells alone (no DOT cells). Experiments were performed in technical triplicate. (D) In ​​in vitro killing assays, non-expanded fresh ("ex vivo") Vδ1 T cells collected from three healthy donors were co-incubated with the indicated HEL or KG-1 cell lines at a 10:1 (E:T) ratio for 3 hours. DOT cells generated from HD#1 are shown as a positive control. (E) Granzyme B and perforin expression in DOT cells was assessed by intracellular flow cytometry. (F) Percentage of CD107a+DOT cells after co-incubation with AML tumor targets or upon stimulation with PMA / ionomycin (positive control) or without (negative control). Results are from two healthy donors and were tested in duplicate. [Figure 5] Cytotoxic activity of V51+ T cells against hematological tumor lines and sparing of healthy PBMCs. In vitro cytotoxicity assays of V51+ T cells against various AML (MV4-11, Kasumi-1, HL-60), NHL (Raji), and ALL (NALM-6) tumor targets, as well as healthy allogeneic PBMCs, were performed at various effector:target ratios for 20 hours. Percent target cell lysis is shown. N=2. [Figure 6A]Targeting AML in vivo with γδ T cell compositions. (A) Irradiated (200–225 rad) 8–12-week-old NOD-SCIDγc− / −−SGM3 (NSGS) mice were anesthetized and implanted with 1 × 10 primary human AML cells into the right tibia (intramedullary—ibm). (B) Irradiated (200–225 rad) 8–12-week-old NSG NOD-SCIDγc− / − (NSG) mice were injected intravenously (iv) with 2 × 10 human KG-1 cells. (C) Irradiated (225–250 rad) 8–12-week-old NOD.Rag1-γc− / −−SGM3 (NRGS) mice were anesthetized and implanted with 1 × 10 human HEL cells into the right tibia (ibm). In (A) and (C), tumor engraftment was assessed by detecting at least 100 tumor cells in the blood (tumor trigger) one week after tumor cell injection. Treatment began immediately once 100 tumor cells were detected in the mouse's blood (tumor trigger). In (B), treatment with either PBS or DOT cells began 10 days after intravenous tumor cell injection. Animals were intravenously injected with PBS or 2 x 107 DOT cells. This intravenous injection was performed three times, 5 days apart. Survival curves for NRGS hosts with HEL (CTR (n = 5), DOT-treated mice (n = 4); p < 0.05). DOT cells (three injections of 2 × 10 cells) were transferred into NSG mice (CTR (n = 6), DOT-treated mice (n = 7)) previously injected with KG-1 AML cells (D–E) or into NSGS mice bearing primary AML cells (CTR (n = 5), DOT-treated mice (n = 5)) (F–G; patient-derived xenografts (PDX)). Tumor burden was assessed in the blood and liver one week after the final DOT cell transfer (D) or by weekly blood sampling (F). Survival curves are shown in panel E (P < 0.05) and panel G (P < 0.01). (H–I) A second primary AML model was developed. (H) Tumor burden in hematologic progression. (I) Survival curves of NSGS hosts bearing primary AML (CTR (n = 5), DOT-treated mice (n = 5)). Animals were sacrificed when progression of disease symptoms (such as paralysis of hind legs) was observed.Mean values ​​± SEM are shown; *, P<0.05; ***, P<0.001; ****, P<0.0001. [Figure 6B]Targeting AML in vivo with γδ T cell compositions. (A) Irradiated (200–225 rad) 8–12-week-old NOD-SCIDγc− / −−SGM3 (NSGS) mice were anesthetized and implanted with 1 × 10 primary human AML cells into the right tibia (intramedullary—ibm). (B) Irradiated (200–225 rad) 8–12-week-old NSG NOD-SCIDγc− / − (NSG) mice were injected intravenously (iv) with 2 × 10 human KG-1 cells. (C) Irradiated (225–250 rad) 8–12-week-old NOD.Rag1-γc− / −−SGM3 (NRGS) mice were anesthetized and implanted with 1 × 10 human HEL cells into the right tibia (ibm). In (A) and (C), tumor engraftment was assessed by detecting at least 100 tumor cells in the blood (tumor trigger) one week after tumor cell injection. Treatment began immediately once 100 tumor cells were detected in the mouse's blood (tumor trigger). In (B), treatment with either PBS or DOT cells began 10 days after intravenous tumor cell injection. Animals were intravenously injected with PBS or 2 x 107 DOT cells. This intravenous injection was performed three times, 5 days apart. Survival curves for NRGS hosts with HEL (CTR (n = 5), DOT-treated mice (n = 4); p < 0.05). DOT cells (three injections of 2 × 10 cells) were transferred into NSG mice (CTR (n = 6), DOT-treated mice (n = 7)) previously injected with KG-1 AML cells (D–E) or into NSGS mice bearing primary AML cells (CTR (n = 5), DOT-treated mice (n = 5)) (F–G; patient-derived xenografts (PDX)). Tumor burden was assessed in the blood and liver one week after the final DOT cell transfer (D) or by weekly blood sampling (F). Survival curves are shown in panel E (P < 0.05) and panel G (P < 0.01). (H–I) A second primary AML model was developed. (H) Tumor burden in hematologic progression. (I) Survival curves of NSGS hosts bearing primary AML (CTR (n = 5), DOT-treated mice (n = 5)). Animals were sacrificed when progression of disease symptoms (such as paralysis of hind legs) was observed.Mean values ​​± SEM are shown; *, P<0.05; ***, P<0.001; ****, P<0.0001. [Figure 6C]Targeting AML in vivo with γδ T cell compositions. (A) Irradiated (200–225 rad) 8–12-week-old NOD-SCIDγc− / −−SGM3 (NSGS) mice were anesthetized and implanted with 1 × 10 primary human AML cells into the right tibia (intramedullary—ibm). (B) Irradiated (200–225 rad) 8–12-week-old NSG NOD-SCIDγc− / − (NSG) mice were injected intravenously (iv) with 2 × 10 human KG-1 cells. (C) Irradiated (225–250 rad) 8–12-week-old NOD.Rag1-γc− / −−SGM3 (NRGS) mice were anesthetized and implanted with 1 × 10 human HEL cells into the right tibia (ibm). In (A) and (C), tumor engraftment was assessed by detecting at least 100 tumor cells in the blood (tumor trigger) one week after tumor cell injection. Treatment began immediately once 100 tumor cells were detected in the mouse's blood (tumor trigger). In (B), treatment with either PBS or DOT cells began 10 days after intravenous tumor cell injection. Animals were intravenously injected with PBS or 2 x 107 DOT cells. This intravenous injection was performed three times, 5 days apart. Survival curves for NRGS hosts with HEL (CTR (n = 5), DOT-treated mice (n = 4); p < 0.05). DOT cells (three injections of 2 × 10 cells) were transferred into NSG mice (CTR (n = 6), DOT-treated mice (n = 7)) previously injected with KG-1 AML cells (D–E) or into NSGS mice bearing primary AML cells (CTR (n = 5), DOT-treated mice (n = 5)) (F–G; patient-derived xenografts (PDX)). Tumor burden was assessed in the blood and liver one week after the final DOT cell transfer (D) or by weekly blood sampling (F). Survival curves are shown in panel E (P < 0.05) and panel G (P < 0.01). (H–I) A second primary AML model was developed. (H) Tumor burden in hematologic progression. (I) Survival curves of NSGS hosts bearing primary AML (CTR (n = 5), DOT-treated mice (n = 5)). Animals were sacrificed when progression of disease symptoms (such as paralysis of hind legs) was observed.Mean values ​​± SEM are shown; *, P<0.05; ***, P<0.001; ****, P<0.0001. [Figure 6D]Targeting AML in vivo with γδ T cell compositions. (A) Irradiated (200–225 rad) 8–12-week-old NOD-SCIDγc− / −−SGM3 (NSGS) mice were anesthetized and implanted with 1 × 10 primary human AML cells into the right tibia (intramedullary—ibm). (B) Irradiated (200–225 rad) 8–12-week-old NSG NOD-SCIDγc− / − (NSG) mice were injected intravenously (iv) with 2 × 10 human KG-1 cells. (C) Irradiated (225–250 rad) 8–12-week-old NOD.Rag1-γc− / −−SGM3 (NRGS) mice were anesthetized and implanted with 1 × 10 human HEL cells into the right tibia (ibm). In (A) and (C), tumor engraftment was assessed by detecting at least 100 tumor cells in the blood (tumor trigger) one week after tumor cell injection. Treatment began immediately once 100 tumor cells were detected in the mouse's blood (tumor trigger). In (B), treatment with either PBS or DOT cells began 10 days after intravenous tumor cell injection. Animals were intravenously injected with PBS or 2 x 107 DOT cells. This intravenous injection was performed three times, 5 days apart. Survival curves for NRGS hosts with HEL (CTR (n = 5), DOT-treated mice (n = 4); p < 0.05). DOT cells (three injections of 2 × 10 cells) were transferred into NSG mice (CTR (n = 6), DOT-treated mice (n = 7)) previously injected with KG-1 AML cells (D–E) or into NSGS mice bearing primary AML cells (CTR (n = 5), DOT-treated mice (n = 5)) (F–G; patient-derived xenografts (PDX)). Tumor burden was assessed in the blood and liver one week after the final DOT cell transfer (D) or by weekly blood sampling (F). Survival curves are shown in panel E (P < 0.05) and panel G (P < 0.01). (H–I) A second primary AML model was developed. (H) Tumor burden in hematologic progression. (I) Survival curves of NSGS hosts bearing primary AML (CTR (n = 5), DOT-treated mice (n = 5)). Animals were sacrificed when progression of disease symptoms (such as paralysis of hind legs) was observed.Mean values ​​± SEM are shown; *, P<0.05; ***, P<0.001; ****, P<0.0001. [Figure 6E]Targeting AML in vivo with γδ T cell compositions. (A) Irradiated (200–225 rad) 8–12-week-old NOD-SCIDγc− / −−SGM3 (NSGS) mice were anesthetized and implanted with 1 × 10 primary human AML cells into the right tibia (intramedullary—ibm). (B) Irradiated (200–225 rad) 8–12-week-old NSG NOD-SCIDγc− / − (NSG) mice were injected intravenously (iv) with 2 × 10 human KG-1 cells. (C) Irradiated (225–250 rad) 8–12-week-old NOD.Rag1-γc− / −−SGM3 (NRGS) mice were anesthetized and implanted with 1 × 10 human HEL cells into the right tibia (ibm). In (A) and (C), tumor engraftment was assessed by detecting at least 100 tumor cells in the blood (tumor trigger) one week after tumor cell injection. Treatment began immediately once 100 tumor cells were detected in the mouse's blood (tumor trigger). In (B), treatment with either PBS or DOT cells began 10 days after intravenous tumor cell injection. Animals were intravenously injected with PBS or 2 x 107 DOT cells. This intravenous injection was performed three times, 5 days apart. Survival curves for NRGS hosts with HEL (CTR (n = 5), DOT-treated mice (n = 4); p < 0.05). DOT cells (three injections of 2 × 10 cells) were transferred into NSG mice (CTR (n = 6), DOT-treated mice (n = 7)) previously injected with KG-1 AML cells (D–E) or into NSGS mice bearing primary AML cells (CTR (n = 5), DOT-treated mice (n = 5)) (F–G; patient-derived xenografts (PDX)). Tumor burden was assessed in the blood and liver one week after the final DOT cell transfer (D) or by weekly blood sampling (F). Survival curves are shown in panel E (P < 0.05) and panel G (P < 0.01). (H–I) A second primary AML model was developed. (H) Tumor burden in hematologic progression. (I) Survival curves of NSGS hosts bearing primary AML (CTR (n = 5), DOT-treated mice (n = 5)). Animals were sacrificed when progression of disease symptoms (such as paralysis of hind legs) was observed.Mean values ​​± SEM are shown; *, P<0.05; ***, P<0.001; ****, P<0.0001. [Figure 6F]Targeting AML in vivo with γδ T cell compositions. (A) Irradiated (200–225 rad) 8–12-week-old NOD-SCIDγc− / −−SGM3 (NSGS) mice were anesthetized and implanted with 1 × 10 primary human AML cells into the right tibia (intramedullary—ibm). (B) Irradiated (200–225 rad) 8–12-week-old NSG NOD-SCIDγc− / − (NSG) mice were injected intravenously (iv) with 2 × 10 human KG-1 cells. (C) Irradiated (225–250 rad) 8–12-week-old NOD.Rag1-γc− / −−SGM3 (NRGS) mice were anesthetized and implanted with 1 × 10 human HEL cells into the right tibia (ibm). In (A) and (C), tumor engraftment was assessed by detecting at least 100 tumor cells in the blood (tumor trigger) one week after tumor cell injection. Treatment began immediately once 100 tumor cells were detected in the mouse's blood (tumor trigger). In (B), treatment with either PBS or DOT cells began 10 days after intravenous tumor cell injection. Animals were intravenously injected with PBS or 2 x 107 DOT cells. This intravenous injection was performed three times, 5 days apart. Survival curves for NRGS hosts with HEL (CTR (n = 5), DOT-treated mice (n = 4); p < 0.05). DOT cells (three injections of 2 × 10 cells) were transferred into NSG mice (CTR (n = 6), DOT-treated mice (n = 7)) previously injected with KG-1 AML cells (D–E) or into NSGS mice bearing primary AML cells (CTR (n = 5), DOT-treated mice (n = 5)) (F–G; patient-derived xenografts (PDX)). Tumor burden was assessed in the blood and liver one week after the final DOT cell transfer (D) or by weekly blood sampling (F). Survival curves are shown in panel E (P < 0.05) and panel G (P < 0.01). (H–I) A second primary AML model was developed. (H) Tumor burden in hematologic progression. (I) Survival curves of NSGS hosts bearing primary AML (CTR (n = 5), DOT-treated mice (n = 5)). Animals were sacrificed when progression of disease symptoms (such as paralysis of hind legs) was observed.Mean values ​​± SEM are shown; *, P<0.05; ***, P<0.001; ****, P<0.0001. [Figure 6G]Targeting AML in vivo with γδ T cell compositions. (A) Irradiated (200–225 rad) 8–12-week-old NOD-SCIDγc− / −−SGM3 (NSGS) mice were anesthetized and implanted with 1 × 10 primary human AML cells into the right tibia (intramedullary—ibm). (B) Irradiated (200–225 rad) 8–12-week-old NSG NOD-SCIDγc− / − (NSG) mice were injected intravenously (iv) with 2 × 10 human KG-1 cells. (C) Irradiated (225–250 rad) 8–12-week-old NOD.Rag1-γc− / −−SGM3 (NRGS) mice were anesthetized and implanted with 1 × 10 human HEL cells into the right tibia (ibm). In (A) and (C), tumor engraftment was assessed by detecting at least 100 tumor cells in the blood (tumor trigger) one week after tumor cell injection. Treatment began immediately once 100 tumor cells were detected in the mouse's blood (tumor trigger). In (B), treatment with either PBS or DOT cells began 10 days after intravenous tumor cell injection. Animals were intravenously injected with PBS or 2 x 107 DOT cells. This intravenous injection was performed three times, 5 days apart. Survival curves for NRGS hosts with HEL (CTR (n = 5), DOT-treated mice (n = 4); p < 0.05). DOT cells (three injections of 2 × 10 cells) were transferred into NSG mice (CTR (n = 6), DOT-treated mice (n = 7)) previously injected with KG-1 AML cells (D–E) or into NSGS mice bearing primary AML cells (CTR (n = 5), DOT-treated mice (n = 5)) (F–G; patient-derived xenografts (PDX)). Tumor burden was assessed in the blood and liver one week after the final DOT cell transfer (D) or by weekly blood sampling (F). Survival curves are shown in panel E (P < 0.05) and panel G (P < 0.01). (H–I) A second primary AML model was developed. (H) Tumor burden in hematologic progression. (I) Survival curves of NSGS hosts bearing primary AML (CTR (n = 5), DOT-treated mice (n = 5)). Animals were sacrificed when progression of disease symptoms (such as paralysis of hind legs) was observed.Mean values ​​± SEM are shown; *, P<0.05; ***, P<0.001; ****, P<0.0001. [Figure 6H]Targeting AML in vivo with γδ T cell compositions. (A) Irradiated (200–225 rad) 8–12-week-old NOD-SCIDγc− / −−SGM3 (NSGS) mice were anesthetized and implanted with 1 × 10 primary human AML cells into the right tibia (intramedullary—ibm). (B) Irradiated (200–225 rad) 8–12-week-old NSG NOD-SCIDγc− / − (NSG) mice were injected intravenously (iv) with 2 × 10 human KG-1 cells. (C) Irradiated (225–250 rad) 8–12-week-old NOD.Rag1-γc− / −−SGM3 (NRGS) mice were anesthetized and implanted with 1 × 10 human HEL cells into the right tibia (ibm). In (A) and (C), tumor engraftment was assessed by detecting at least 100 tumor cells in the blood (tumor trigger) one week after tumor cell injection. Treatment began immediately once 100 tumor cells were detected in the mouse's blood (tumor trigger). In (B), treatment with either PBS or DOT cells began 10 days after intravenous tumor cell injection. Animals were intravenously injected with PBS or 2 x 107 DOT cells. This intravenous injection was performed three times, 5 days apart. Survival curves for NRGS hosts with HEL (CTR (n = 5), DOT-treated mice (n = 4); p < 0.05). DOT cells (three injections of 2 × 10 cells) were transferred into NSG mice (CTR (n = 6), DOT-treated mice (n = 7)) previously injected with KG-1 AML cells (D–E) or into NSGS mice bearing primary AML cells (CTR (n = 5), DOT-treated mice (n = 5)) (F–G; patient-derived xenografts (PDX)). Tumor burden was assessed in the blood and liver one week after the final DOT cell transfer (D) or by weekly blood sampling (F). Survival curves are shown in panel E (P < 0.05) and panel G (P < 0.01). (H–I) A second primary AML model was developed. (H) Tumor burden in hematologic progression. (I) Survival curves of NSGS hosts bearing primary AML (CTR (n = 5), DOT-treated mice (n = 5)). Animals were sacrificed when progression of disease symptoms (such as paralysis of hind legs) was observed.Mean values ​​± SEM are shown; *, P<0.05; ***, P<0.001; ****, P<0.0001. [Figure 6I]Targeting AML in vivo with γδ T cell compositions. (A) Irradiated (200–225 rad) 8–12-week-old NOD-SCIDγc− / −−SGM3 (NSGS) mice were anesthetized and implanted with 1 × 10 primary human AML cells into the right tibia (intramedullary—ibm). (B) Irradiated (200–225 rad) 8–12-week-old NSG NOD-SCIDγc− / − (NSG) mice were injected intravenously (iv) with 2 × 10 human KG-1 cells. (C) Irradiated (225–250 rad) 8–12-week-old NOD.Rag1-γc− / −−SGM3 (NRGS) mice were anesthetized and implanted with 1 × 10 human HEL cells into the right tibia (ibm). In (A) and (C), tumor engraftment was assessed by detecting at least 100 tumor cells in the blood (tumor trigger) one week after tumor cell injection. Treatment began immediately once 100 tumor cells were detected in the mouse's blood (tumor trigger). In (B), treatment with either PBS or DOT cells began 10 days after intravenous tumor cell injection. Animals were intravenously injected with PBS or 2 x 107 DOT cells. This intravenous injection was performed three times, 5 days apart. Survival curves for NRGS hosts with HEL (CTR (n = 5), DOT-treated mice (n = 4); p < 0.05). DOT cells (three injections of 2 × 10 cells) were transferred into NSG mice (CTR (n = 6), DOT-treated mice (n = 7)) previously injected with KG-1 AML cells (D–E) or into NSGS mice bearing primary AML cells (CTR (n = 5), DOT-treated mice (n = 5)) (F–G; patient-derived xenografts (PDX)). Tumor burden was assessed in the blood and liver one week after the final DOT cell transfer (D) or by weekly blood sampling (F). Survival curves are shown in panel E (P < 0.05) and panel G (P < 0.01). (H–I) A second primary AML model was developed. (H) Tumor burden in hematologic progression. (I) Survival curves of NSGS hosts bearing primary AML (CTR (n = 5), DOT-treated mice (n = 5)). Animals were sacrificed when progression of disease symptoms (such as paralysis of hind legs) was observed.Mean values ​​± SEM are shown; *, P<0.05; ***, P<0.001; ****, P<0.0001. [Figure 7A] γδ T cell compositions (re)target chemotherapy-resistant AML. Comparison of the in vitro anti-AML activity of DOT cells and standard chemotherapy. (A) DOT cells and standard AML chemotherapy (doxorubicin + cytarabine) protocols were tested on chemotherapy-naive (wild-type (wt)) AML cells or chemotherapy-relapsed (CR, repopulation after >99% HEL cell elimination) AML cells. The percentage of Annexin V+ HEL cells 3 hours after treatment is shown. (B) Number of AML HEL cells before or 72 hours after treatment with DOT cells (performed at an E:T ratio of 5:1). Viable cells (<1%) were re-sorted and repopulated to obtain DOT-treated (DT) samples (C-E). (C) DOT cells were co-incubated with non-treated (NT) or previously DOT-treated (DT) AML HEL cells at a ratio of 5:1 or 10:1 (E:T) for 3 hours. The percentage of Annexin V+ HEL cells is shown. (D) Number of barcoded AML single-cell lineages in untreated (NT), chemotherapy-treated (CT), or DOT-treated (DT) AML HEL cells. (E) Pearson correlation for the distribution of barcoded AML single-cell lineages among different treatments. Dashed lines represent low correlation (0.2), moderate correlation (0.4), and high correlation (0.8), respectively. Mean ± SEM (**, P<0.01; ***, P<0.001; ****, P<0.0001) is shown. [Figure 7B]γδ T cell compositions (re)target chemotherapy-resistant AML. Comparison of the in vitro anti-AML activity of DOT cells and standard chemotherapy. (A) DOT cells and standard AML chemotherapy (doxorubicin + cytarabine) protocols were tested on chemotherapy-naive (wild-type (wt)) AML cells or chemotherapy-relapsed (CR, repopulation after >99% HEL cell elimination) AML cells. The percentage of Annexin V+ HEL cells 3 hours after treatment is shown. (B) Number of AML HEL cells before or 72 hours after treatment with DOT cells (performed at an E:T ratio of 5:1). Viable cells (<1%) were re-sorted and repopulated to obtain DOT-treated (DT) samples (C-E). (C) DOT cells were co-incubated with non-treated (NT) or previously DOT-treated (DT) AML HEL cells at a ratio of 5:1 or 10:1 (E:T) for 3 hours. The percentage of Annexin V+ HEL cells is shown. (D) Number of barcoded AML single-cell lineages in untreated (NT), chemotherapy-treated (CT), or DOT-treated (DT) AML HEL cells. (E) Pearson correlation for the distribution of barcoded AML single-cell lineages among different treatments. Dashed lines represent low correlation (0.2), moderate correlation (0.4), and high correlation (0.8), respectively. Mean ± SEM (**, P<0.01; ***, P<0.001; ****, P<0.0001) is shown. [Figure 7C]γδ T cell compositions (re)target chemotherapy-resistant AML. Comparison of the in vitro anti-AML activity of DOT cells and standard chemotherapy. (A) DOT cells and standard AML chemotherapy (doxorubicin + cytarabine) protocols were tested on chemotherapy-naive (wild-type (wt)) AML cells or chemotherapy-relapsed (CR, repopulation after >99% HEL cell elimination) AML cells. The percentage of Annexin V+ HEL cells 3 hours after treatment is shown. (B) Number of AML HEL cells before or 72 hours after treatment with DOT cells (performed at an E:T ratio of 5:1). Viable cells (<1%) were re-sorted and repopulated to obtain DOT-treated (DT) samples (C-E). (C) DOT cells were co-incubated with non-treated (NT) or previously DOT-treated (DT) AML HEL cells at a ratio of 5:1 or 10:1 (E:T) for 3 hours. The percentage of Annexin V+ HEL cells is shown. (D) Number of barcoded AML single-cell lineages in untreated (NT), chemotherapy-treated (CT), or DOT-treated (DT) AML HEL cells. (E) Pearson correlation for the distribution of barcoded AML single-cell lineages among different treatments. Dashed lines represent low correlation (0.2), moderate correlation (0.4), and high correlation (0.8), respectively. Mean ± SEM (**, P<0.01; ***, P<0.001; ****, P<0.0001) is shown. [Figure 7D]γδ T cell compositions (re)target chemotherapy-resistant AML. Comparison of the in vitro anti-AML activity of DOT cells and standard chemotherapy. (A) DOT cells and standard AML chemotherapy (doxorubicin + cytarabine) protocols were tested on chemotherapy-naive (wild-type (wt)) AML cells or chemotherapy-relapsed (CR, repopulation after >99% HEL cell elimination) AML cells. The percentage of Annexin V+ HEL cells 3 hours after treatment is shown. (B) Number of AML HEL cells before or 72 hours after treatment with DOT cells (performed at an E:T ratio of 5:1). Viable cells (<1%) were re-sorted and repopulated to obtain DOT-treated (DT) samples (C-E). (C) DOT cells were co-incubated with non-treated (NT) or previously DOT-treated (DT) AML HEL cells at a ratio of 5:1 or 10:1 (E:T) for 3 hours. The percentage of Annexin V+ HEL cells is shown. (D) Number of barcoded AML single-cell lineages in untreated (NT), chemotherapy-treated (CT), or DOT-treated (DT) AML HEL cells. (E) Pearson correlation for the distribution of barcoded AML single-cell lineages among different treatments. Dashed lines represent low correlation (0.2), moderate correlation (0.4), and high correlation (0.8), respectively. Mean ± SEM (**, P<0.01; ***, P<0.001; ****, P<0.0001) is shown. [Figure 7E]γδ T cell compositions (re)target chemotherapy-resistant AML. Comparison of the in vitro anti-AML activity of DOT cells and standard chemotherapy. (A) DOT cells and standard AML chemotherapy (doxorubicin + cytarabine) protocols were tested on chemotherapy-naive (wild-type (wt)) AML cells or chemotherapy-relapsed (CR, repopulation after >99% HEL cell elimination) AML cells. The percentage of Annexin V+ HEL cells 3 hours after treatment is shown. (B) Number of AML HEL cells before or 72 hours after treatment with DOT cells (performed at an E:T ratio of 5:1). Viable cells (<1%) were re-sorted and repopulated to obtain DOT-treated (DT) samples (C-E). (C) DOT cells were co-incubated with non-treated (NT) or previously DOT-treated (DT) AML HEL cells at a ratio of 5:1 or 10:1 (E:T) for 3 hours. The percentage of Annexin V+ HEL cells is shown. (D) Number of barcoded AML single-cell lineages in untreated (NT), chemotherapy-treated (CT), or DOT-treated (DT) AML HEL cells. (E) Pearson correlation for the distribution of barcoded AML single-cell lineages among different treatments. Dashed lines represent low correlation (0.2), moderate correlation (0.4), and high correlation (0.8), respectively. Mean ± SEM (**, P<0.01; ***, P<0.001; ****, P<0.0001) is shown. [Figure 8] Repeated cytotoxicity of expanded V51+ T cell populations against hematological tumor lines. The left side of the graph shows the percent of CTV+ve events (HL-60 tumor targets) that were Sytox+ve during challenge 1, and the right side of the graph shows the percent of CTV+ve events that were Sytox+ve during challenge 2. Mean values ​​with standard deviation for two donors. [Figure 9A]Cytokine production by stimulated V51 expanded cells. Cytokine production (pg / million cells / hour) of V51 expanded cells upon TCR stimulation (A). Pie chart representation of the top cytokines produced by V51 expanded cells stimulated with physiological levels of OKT3 and IL-15 (B) or by supraphysiological stimulation with IL-15 (C). IL-6 and TNFα production upon co-culture of blood samples (PBMCs or buffy coats) with V51 expanded cells (D). [Figure 9B] Cytokine production by stimulated V51 expanded cells. Cytokine production (pg / million cells / hour) of V51 expanded cells upon TCR stimulation (A). Pie chart representation of the top cytokines produced by V51 expanded cells stimulated with physiological levels of OKT3 and IL-15 (B) or by supraphysiological stimulation with IL-15 (C). IL-6 and TNFα production upon co-culture of blood samples (PBMCs or buffy coats) with V51 expanded cells (D). [Figure 9C] Cytokine production by stimulated V51 expanded cells. Cytokine production (pg / million cells / hour) of V51 expanded cells upon TCR stimulation (A). Pie chart representation of the top cytokines produced by V51 expanded cells stimulated with physiological levels of OKT3 and IL-15 (B) or by supraphysiological stimulation with IL-15 (C). IL-6 and TNFα production upon co-culture of blood samples (PBMCs or buffy coats) with V51 expanded cells (D). [Figure 9D] Cytokine production by stimulated V51 expanded cells. Cytokine production (pg / million cells / hour) of V51 expanded cells upon TCR stimulation (A). Pie chart representation of the top cytokines produced by V51 expanded cells stimulated with physiological levels of OKT3 and IL-15 (B) or by supraphysiological stimulation with IL-15 (C). IL-6 and TNFα production upon co-culture of blood samples (PBMCs or buffy coats) with V51 expanded cells (D). [Figure 10] Selective cytotoxic activity of expanded V51 T cell populations against NALM-6 cells and healthy B cells. Graph shows both the percentage of CTV events (healthy B cells) that were Sytox+ve and the percentage of CFSE events (NALM-6 tumor cells) that were Sytox+ve at various E:T ratios. Mean and SD (2 technical replicates). One experiment representative of 3 biological donors. [Figure 11] (A) PBLs isolated from buffy coat blood preparations and irradiated to inhibit their cell division potential were cocultured with CTV-stained allogeneic or autologous blood T cell populations at a 1:1 ratio for 5 days without cytokine support. Cell division in response to coculture with irradiated PBLs was then assessed by flow cytometric analysis of CTV dye dilution. The total percentage of dividing αβ T cells is shown. N=3. (B) PBLs isolated from buffy coat blood preparations and irradiated to inhibit their cell division potential were cocultured at a 1:1 ratio with either CTV-stained blood T cells or GDX012 cells prepared from two different donors (LK008, LK009). The blood T cells and GDX012 cells were from the same donor. The cocultures were incubated for 5 days without cytokine support. Cell division in response to co-culture with irradiated PBLs was then assessed by flow cytometric analysis of CTV dye dilutions. The total % of αβ T cells (for blood T cells) or the total % of viable GDX023 cells that divided is shown. Data are shown from three technical replicates. ND = not detected. [Figure 12]Tumor control in an in vivo model after a single intravenous administration of GDX012. NSG mice were inoculated with 0.5x106 NALM-6-FLuc / GFP cells via intravenous injection, followed by whole-body BLI to track tumor growth in mice treated with or without a single intravenous injection of 20x106 GDX012 cells the following day. All control and treated mice were injected intraperitoneally with recombinant human IL-15 (1µg / mouse every 3 days for the duration of the study). Mean values ​​± SEM (n=8) from days 14 to 28 are shown, with individual data points and ventral whole-body BLI images also shown for days 17 and 28. [Figure 13] Bone marrow homing in an in vivo model after a single intravenous administration of GDX012. NSG mice inoculated with 0.5 × 10 or 1 × 10 NALM-6-FLuc / GFP cells were either intravenously injected or not with a single intravenous injection of 20 × 10 GDX012 cells either 24 hours or 6 days later. All control and treated mice received intraperitoneal injections of recombinant human IL-15 (1 μg / mouse every 2–3 days for the duration of the study). The study was terminated 4 weeks later to assess the biodistribution of GDX012 in the bone marrow and tumor burden. Flow cytometry was performed on bone marrow obtained from hind leg long bones to assess the percentage of TCRγδ+ cells (GDX012) and CD19+ cells (NALM-6 cells) within the viable single-cell population. Representative flow cytometry plots and individual data points are shown. DETAILED DESCRIPTION OF THE INVENTION

[0009] According to a first aspect, an allogeneic composition comprising V51 T cells for use in treating patients with myeloid malignancies is provided. Data presented herein demonstrate that V51 T cells expanded from allogeneic donors are highly polyclonal and lack dominant clones, making them suitable as a therapy for use across a broad range of donors. Additional experiments have shown that such compositions have limited potential to induce cytokine release syndrome and do not mediate mixed lymphocyte reactions, important safety aspects when considering adoptive cell therapy. Furthermore, the V51 T cells of the present invention are highly selective and cytotoxic against myeloid cell lines and primary cells, while sparing non-malignant "healthy" cells of the same type.

[0010] Myeloid malignancies Myeloid malignancies are clonal disorders arising in hematopoietic stem or progenitor cells. They can be characterized by uncontrolled proliferation and / or differentiation blockage of abnormal myeloid progenitor cells. Several mutations associated with these malignancies have been identified, and these mutations belong to five main classes: signaling pathway proteins (e.g., CBL, FLT3, JAK2, RAS), transcription factors (e.g., CEBPA, ETV6, RUNX1), epigenetic regulators (e.g., ASXL1, DNMT3A, EZH2, IDH1, IDH2, SUZ12, TET2, UTX), tumor suppressors (e.g., TP53), and spliceosome components (e.g., SF3B1, SRSF2) (Murati et al. (2012) BMC Cancer 12:304).

[0011] Myeloid malignancies can include chronic stages (including myelodysplastic syndromes, myeloproliferative neoplasms, and chronic myelomonocytic leukemia) as well as acute stages (acute myeloid leukemia).

[0012] Based on the morphology, cytochemistry, immunophenotype, genetics, and clinical characteristics of myeloid disorders, the World Health Organization (WHO) classifies myeloid malignancies into five major types: (1) acute myeloid leukemia, (2) myelodysplastic syndrome, (3) myeloproliferative neoplasm, (4) myelodysplastic and myeloproliferative neoplasm, and (5) myeloid neoplasm with eosinophilia and platelet- or fibroblast-derived growth factor receptor abnormalities. This classification is further described in Tefferi and Vardiman (2008) Leukemia 22:14-22.

[0013] Thus, in one embodiment, the myeloid malignancy is selected from acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), myelodysplastic and myeloproliferative (MDS / MPN) neoplasms, and myeloid neoplasms with eosinophilia and platelet- or fibroblast-derived growth factor receptor abnormalities. In another embodiment, the myeloid malignancy is AML, MDS, or MPN, particularly AML or MDS.

[0014] In one embodiment, the myeloid malignancy is AML. AML occurs due to the clonal proliferation of myeloid blasts in peripheral blood, bone marrow, or other tissues. AML occurs when abnormal myeloid blasts arise from myeloid stem cells that do not become healthy white blood cells, or when too many myeloid stem cells develop into abnormal red blood cells or platelets. As a result, leukemic blasts or immature cell forms accumulate in the bone marrow, peripheral blood, and sometimes other tissues, resulting in a decrease in the production of normal red blood cells, platelets, and mature granulocytes.

[0015] In an alternative embodiment, the myeloid malignancy is MDS. MDS and MPN are often considered precursors to myeloid malignancies (such as AML). Low blood cell counts (also called "cytopenias") are a hallmark of MDS and are responsible for many of the symptoms associated with MDS (such as infections, anemia, spontaneous bleeding, or easy bruising).

[0016] MDS types include refractory cytopenia with single-lineage dysplasia (RCUD), refractory anemia with ringed sideroblasts (RARS), refractory cytopenia with multilineage dysplasia (RCMD), refractory anemia with excess blasts (RAEB-1 and RAEB-2), myelodysplastic syndrome with isolated deletion (5q), and unclassifiable myelodysplastic syndrome (MDS-U). RCUD affects a single blood cell type and can be divided into three subtypes: refractory anemia (low red blood cell count), refractory neutropenia (low white blood cell count), and refractory thrombocytopenia (low platelet count). RARS is similar to refractory anemia, but the number of early red blood cells with iron rings inside (ringed sideroblasts) increases in the bone marrow. RCMD affects multiple blood cell types and is characterized by very few or no immature cells (blasts) in the blood and low numbers of blasts in the bone marrow. With RAEB, levels of one or more blood cells are low, and many of these cells appear abnormal in the bone marrow. In RAEB-2, there are more blasts in the blood and bone marrow than in RAEB-1.

[0017] In one embodiment, the patient is minimal residual disease positive (MRD+).

[0018] Minimal residual disease (MRD) refers to the presence of small numbers of cancer cells in the body after cancer treatment. MRD is an independent post-diagnosis prognostic indicator for AML and MDS, and this indicator is important for risk stratification and treatment planning.

[0019] The low cell levels of MRD necessitate testing using highly sensitive tests. The most widely used tests are flow cytometry, polymerase chain reaction (PCR), and next-generation sequencing (NGS) on samples of bone marrow and / or peripheral blood cells. MRD patients can be diagnosed using methods known in the art. In one embodiment, an MRD+ patient is in complete remission and has no detectable leukemic blasts in the peripheral blood and / or fewer than 5% leukemic blasts in the bone marrow.

[0020] The patient or subject to be treated is preferably a human cancer patient (eg, a human cancer patient undergoing treatment for a hematological cancer).

[0021] In one embodiment, the patient has been previously treated with chemotherapy, for example, the patient may have been treated with chemotherapy at least 3 days prior to administration of the allogeneic composition.

[0022] In one embodiment, the chemotherapy is selected from fludarabine and cyclophosphamide.

[0023] Allogeneic composition In one embodiment, the allogeneic composition comprises at least about 90% CD45+ cells relative to total viable cells. In another embodiment, the allogeneic composition comprises at least about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 101%, about 102%, about 103%, about 104%, about 105%, about 106%, about 107%, about 108%, about 109%, about 1109%, about 1111%, about 112%, about 113%, about 114%, about 115%, about 116%, about 117%, about 118%, about 119%, about 120%, about 121%, about 122%, about 123%, about 124%, about 125%, about 126%, about 1 9%, containing approximately 99.5% CD45+ cells.

[0024] In one embodiment, the allogeneic composition comprises at least about 60% γδ T cells relative to total viable cells, hi another embodiment, the allogeneic composition comprises at least about 70%, 75%, 80%, 85%, 90%, or 95% γδ T cells relative to total viable cells.

[0025] In one embodiment, the allogeneic composition comprises an ex vivo expanded cell population enriched in V51 T cells relative to the non-expanded starting cell population. In one embodiment, the allogeneic composition comprises at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% V51 T cells relative to total viable cells. In another embodiment, the allogeneic composition comprises more than 30% (e.g., at least 33%) V51 T cells relative to total viable cells. In another embodiment, V51 T cells constitute at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of all γδ T cells of the allogeneic composition. In another embodiment, V51 T cells constitute at least 40%, at least 50%, or at least 60% of all γδ T cells of the allogeneic composition.

[0026] In one embodiment, the allogeneic composition comprises less than 0.1% αβ T cells relative to total viable cells. Preferably, the allogeneic composition comprises less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% αβ T cells.

[0027] The allogeneic composition may comprise a dose suitable for administration to a patient. According to another aspect, there is provided a dose of an allogeneic composition comprising V51 T cells for use in treating a patient with a myeloid malignancy.

[0028] In one embodiment, the dose of the allogeneic composition is about 1×10 10 Less than 10 total viable cells (approximately 9 × 10 9 Less than 8 x 10 9 Less than 7 x 10 9 Less than 6 x 10 9 Less than 5 x 10 9 Less than 4 x 10 9 Less than 3 x 10 9 Less than 2 x 10 9 Less than 1 x 10 9Less than 5 x 10 8 Less than 3 x 10 8 Less than 1 x 10 8 Less than 5 x 10 7 Less than 3 x 10 7 Less than 1 x 10 7 Less than 5 x 10 6 Less than 3 x 10 6 Less than or about 1 x 10 6 In one embodiment, the dose of the allogeneic composition comprises about 1 x 10 total viable cells. 8 In one embodiment, the dose of the allogeneic composition comprises less than about 1 x 10 total viable cells. 4 More than 10 living cells (approximately 3 × 10 4 More than 5 x 10 pieces 4 More than 1 x 10 pieces 5 More than 3 x 10 pieces 5 More than 5 x 10 pieces 5 More than 1 x 10 pieces 6 More than 3 x 10 pieces 6 More than 5 x 10 pieces 6 More than 1 x 10 pieces 7 More than 3 x 10 pieces 7 More than or about 5 x 10 7 In one embodiment, the dose of the allogeneic composition comprises about 1 x 10 whole viable cells. 6 In one embodiment, the dose of the allogeneic composition comprises more than about 1 x 10 whole viable cells. 4 cells ~ approx. 1 x 10 10 total viable cells (approximately 1 × 10 5 Total viable cells ~ approx. 1 x 10 9 cells, specifically, approximately 1 x 10 6 cells ~ approx. 1 x 10 8 In one embodiment, the dose of the allogeneic composition comprises about 4 x 10 total viable cells. 7 pieces~8×10 9 pieces (e.g., 4 x 10 7 pieces, 8×10 7 pieces, 4×10 8 pieces, 8×10 8 pieces, 1.2×10 9 pieces, 2.4×10 9 pieces, 4×10 9 pieces, or 8 x 10 9Contains all living cells (800).

[0029] The allogeneic composition may comprise a dose (such as a therapeutically effective dose) for administration to a patient. In one embodiment, a dose of V51 T cells calculated per kg of patient body weight is administered to the patient. In some embodiments, the dose of V51 T cells described herein is about 1 x 10 5 Individual cells / kg, approximately 5×10 5 Individual cells / kg, approximately 1×10 6 Individual cells / kg, approximately 1.5×10 6 Individual cells / kg, approximately 2×10 6 Individual cells / kg, approximately 3×10 6 Individual cells / kg, approximately 5× 10 6 Individual cells / kg, approximately 1×10 7 Individual cells / kg, approximately 1.5×10 7 Individual cells / kg, approximately 2×10 7 Individual cells / kg, approximately 3×10 7 Individual cells / kg, approximately 5×10 7 Individual cells / kg, approximately 1×10 8 Individual cells / kg, approximately 2×10 8 cells / kg, or approximately 5 x 10 8 In some embodiments, the dose of V51 T cells comprises at least about 1 x 10 cells / kg. 5 Individual cells / kg, approximately 5×10 5 Individual cells / kg, approximately 1×10 6 Individual cells / kg, approximately 1.5×10 6 Individual cells / kg, approximately 2×10 6 Individual cells / kg, approximately 3×10 6 Individual cells / kg, approximately 5×10 6 Individual cells / kg, approximately 1×10 7 Individual cells / kg, approximately 1.5×10 7 Individual cells / kg, approximately 2×10 7 Individual cells / kg, approximately 3×10 7 Individual cells / kg, approximately 5×10 7 Individual cells / kg, approximately 1×10 8 Individual cells / kg, approximately 2×10 8 cells / kg, or approximately 5 x 10 8In some embodiments, the dose of V51 T cells comprises up to about 1 x 10 cells / kg. 6 Individual cells / kg, approximately 1.5×10 6 Individual cells / kg, approximately 2×10 6 Individual cells / kg, approximately 3×10 6 Individual cells / kg, approximately 5×10 6 Individual cells / kg, approximately 1×10 7 Individual cells / kg, approximately 1.5×10 7 Individual cells / kg, approximately 2×10 7 Individual cells / kg, approximately 3×10 7 Individual cells / kg, approximately 5×10 7 Individual cells / kg, approximately 1×10 8 Individual cells / kg, approximately 2×10 8 cells / kg, or approximately 5 x 10 8 In some embodiments, the dose of V51 T cells comprises about 1 x 10 cells / kg. 6 ~1×10 8 Includes cells / kg.

[0030] The dose of the allogeneic composition is 5 x 10 4 αβT cells / kg (approximately 10 4 αβT cells / kg, approximately 10 3 ≤ 10 αβ T cells / kg, or approximately 10 2 Thus, in one embodiment, the dose may comprise about 5×10 4 In another embodiment, the dose comprises less than about 1 x 10 αβ T cells / kg. 4 Contains less than αβ T cells / kg.

[0031] In one embodiment, the allogeneic composition is frozen and then thawed prior to administration. In another embodiment, the dose of the allogeneic composition is calculated prior to freezing. In another embodiment, the dose is calculated after freezing. In another embodiment, the allogeneic composition is not frozen.

[0032] As used herein, the term "about" includes an uprange of up to and including 10% of the stated value and a downrange of up to and including 10% of the stated value, suitably including an uprange of up to and including 5% of the stated value and a downrange of up to and including 5% of the stated value, among others. The term "to" includes the stated boundary value.

[0033] Pharmaceutical compositions can include the expanded V51 T cell compositions described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers (such as neutral buffered saline, phosphate buffered saline, and the like), carbohydrates (such as glucose, mannose, sucrose, or dextran, mannitol), proteins, polypeptides, or amino acids (such as glycine), antioxidants, chelating agents (such as EDTA or glutathione), adjuvants (e.g., aluminum hydroxide), and preservatives. Cryopreservation solutions that can be used in pharmaceutical compositions of the invention include, for example, DMSO. Compositions can be formulated for any suitable administration (e.g., intravenous administration).

[0034] In one embodiment, the pharmaceutical composition is substantially free of (eg, free of detectable levels of) contaminants (eg, endotoxins or mycoplasma).

[0035] Gamma delta T cells In a preferred embodiment, the γδ T cells comprise a population of Vδ1+ T cells.

[0036] In some embodiments, V51 T cells express CD27. For example, V51 T The cells may have a frequency of CD27+ cells of more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90%. Alternatively, V51+ T cells may have a frequency of CD27+ cells of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In certain embodiments, V51+ T cells have a frequency of CD27+ cells of more than 10%. Thus, in one embodiment, V51+ T cells have a frequency of CD27+ cells of about 20%. In another embodiment, V51+ T cells have a frequency of CD27+ cells of more than 20%. In one embodiment, V51+ T cells have a frequency of CD27+ cells of about 20%.

[0037] In some embodiments, V51 T cells have a low proportion of TIGIT-expressing cells. For example, V51 T cells may have a frequency of TIGIT cells of less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%. Alternatively, V51 T cells may have a frequency of TIGIT cells of about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10%. In certain embodiments, V51 T cells have a frequency of TIGIT cells of less than 80%. Thus, in one embodiment, V51 T cells have a frequency of TIGIT cells of about 70%. In another embodiment, V51 T cells have a frequency of TIGIT cells of less than 60%. In yet another embodiment, the V51 T cells have a frequency of TIGIT cells of about 30%. Thus, in one embodiment, the V51 T cells do not substantially express TIGIT.

[0038] In another embodiment, V51+ T cells express CD27 and / or do not substantially express TIGIT.

[0039] Method of obtaining Vδ1+ T cell enriched composition V51 T cells can be obtained using methods known in the art. For example, V51 T cells can be obtained using the methods described in WO2016 / 198480, WO2017 / 072367, or WO2018 / 202808, which are incorporated herein by reference. These methods can selectively expand V51 T cells (specifically, V52 TCRγδ T cells) in culture. These methods are performed on a sample, which may also be referred to as a "starting sample." These methods can use either an unfractionated sample or a sample enriched in TCRγδ T cells.

[0040] The data provided in the Examples herein demonstrate that V51 T cell compositions expanded using exogenous growth factors have improved polyclonality compared to FACS-sorted, non-expanded V51 T cells simply obtained from peripheral blood (i.e., ex vivo V51 T cells). Thus, in one embodiment, the allogeneic composition comprises V51 T cells obtained using an expansion method, specifically the expansion method comprising culturing V51 T cells in the presence of exogenous growth factors.

[0041] The sample can be any sample containing γδ T cells or their progenitors, including, but not limited to, blood, bone marrow, lymphoid tissue, epithelium, thymus, liver, spleen, cancer tissue, lymph node tissue, infected tissue, fetal tissue, and fractions or enriched portions thereof. The compositions and methods of the present invention are particularly useful with V51 T cells obtained from hematological samples. Thus, in one embodiment, V51 T cells are obtained from a blood sample.

[0042] The sample is preferably blood, including peripheral blood or umbilical cord blood or fractions thereof, including buffy coat cells, leukapheresis products, peripheral blood mononuclear cells (PBMCs), and low density mononuclear cells (LDMCs). The sample is human blood or a fraction thereof. In some embodiments, the sample is human blood or a fraction thereof. Cells can be obtained from a blood sample using techniques known in the art, such as density gradient centrifugation. For example, whole blood can be layered onto an equal volume of FICOLL-HYPAQUE, followed by centrifugation at 400 x g at room temperature for 15-30 minutes. The interface material will contain low-density mononuclear cells, which can be collected in culture medium, washed with culture medium, and centrifuged at 200 x g at room temperature for 10 minutes. The sample can be fresh or frozen.

[0043] In one embodiment, the V51 T cells are obtained from a human sample.

[0044] As described herein, the compositions and methods of the invention may be used with allogeneic V51 T cells, i.e., cells derived from a sample obtained from another donor. In one embodiment, the V51 T cells are obtained from a healthy donor.

[0045] Prior to culturing a sample or a fraction thereof (such as PBMCs), such sample or fraction may be enriched for certain cell types and / or depleted of other cell types. In one embodiment, the sample is enriched for T cells. The sample may be enriched for TCRγδ T cells. For example, the sample may be depleted of TCRαβ T cells, non-TCRγδ T cells, and / or enriched for CD3 cells. In one embodiment, the sample is first depleted of TCRαβ T cells, followed by enrichment of CD3 cells.

[0046] A sample can be enriched or depleted of certain cell types using techniques known in the art. In one embodiment, cells of a particular phenotype can be depleted by incubating the sample, or a fraction thereof, with an antibody cocktail containing antibodies that bind to specific molecules on the cells to be depleted. Preferably, the antibodies in the cocktail are coupled to magnetic microbeads that can be used to magnetically deplete or enrich such cells when the target cells are forced through a magnetic column. In one embodiment, the sample is depleted of αβ T cells.

[0047] Harvesting V51 T cells may involve physical collection of V51 T cells from culture, isolation of V51 T cells from other lymphocytes (e.g., αβ T cells, γδ T cells, and / or NK cells), or isolation and / or separation of V51 T cells from stromal cells (e.g., fibroblasts). In one embodiment, V51 T cells are collected by mechanical means (e.g., pipetting). In another embodiment, V51 T cells are collected by magnetic separation and / or labeling. In yet another embodiment, V51 T cells are collected by flow cytometry techniques (such as FACS). Thus, in certain embodiments, V51 T cells are collected by specifically labeling V51 T cells. It will be understood that such collection of V51 T cells may involve physical removal from culture, transfer to another culture vessel, or transition to alternative or different culture conditions.

[0048] Upon isolation from a sample, V51 T cells are generally part of a larger population of lymphocytes, which includes, for example, αβ T cells, B cells, and natural killer (NK) cells. In some embodiments, 0.1%-10% of the isolated lymphocyte population are V51 T cells, e.g., 1-10% of the isolated lymphocyte population are V51 T cells. In some embodiments, the percent of V51 T cells is measured as the proportion of CD45 cells (leukocyte common antigen). In some embodiments, the isolated population is depleted of other cell types (e.g., αβ T cells are depleted). In some embodiments, an isolated CD45 cell population that has been depleted of αβ T cells comprises at least 0.1% V51 T cells (e.g., at least 0.5% V51 T cells). Often, a γδ T cell population (e.g., a blood-derived γδ T cell population) will comprise a large population of V51 T cells. In some embodiments, In some cases, fewer than 10% of γδ T cells are Vδ2+ T cells (e.g., fewer than 10% of γδ T cells are Vδ2+ T cells).

[0049] Once the cells in the sample have been fractionated and concentrated, the cells can be cultured, if desired.

[0050] In certain embodiments, the invention features methods for expanding V51 T cells. Such methods can be performed in vitro. In some embodiments, V51 T cells are expanded from a population of γδ T cells isolated from a sample as described herein.

[0051] As used herein, reference to "expansion" or an "expanded population of V51 T cells" includes a larger population of cells or containing a greater number of cells compared to a non-expanded population. Such a population may be enriched, diminished, or a mixed population with an increased proportion of a particular cell type within the population. It will be understood that the term "expansion step" refers to the process by which an expansion or expanded population occurs. Thus, an expansion or expanded population may be enriched or contain a greater number of cells compared to a population that has not undergone an expansion step or the population prior to any expansion step. It will be further understood that any numbers (e.g., fold increase or fold expansion) provided herein to indicate expansion are illustrative of the increase in number or size of a cell population or number of cells, as well as the amount of increase.

[0052] In one embodiment, V51 T cells are obtained from a sample by a method comprising culturing the sample in a medium comprising a T cell mitogen and a growth factor with interleukin-4-like activity in the absence of a growth factor with interleukin-15-like activity.

[0053] In one embodiment, V51 T cells are obtained from a sample by a method comprising culturing the sample in a medium comprising a T cell mitogen and a growth factor with interleukin-15-like activity in the absence of a growth factor with interleukin-4-like activity.

[0054] In one embodiment, (1) culturing cells in the sample in a first culture medium comprising a T cell mitogen and a growth factor having interleukin-4-like activity in the absence of a growth factor having interleukin-15-like activity; and (2) culturing the cells obtained in step (1) in a second culture medium containing a T cell mitogen and a growth factor having interleukin-15-like activity in the absence of a growth factor having interleukin-4-like activity; V51 T cells are obtained from the sample by a method comprising:

[0055] The terms "in the absence of interleukin-15, interleukin-2, and interleukin-7" and "in the absence of interleukin-4" refer not only to the complete absence of these cytokines in the culture medium, but also to the use of such cytokines at concentration levels so low that they cannot produce a measurable response or physiological effect in the target cells and therefore can be considered absent for practical purposes. Furthermore, a "measurable physiological effect in the target cells" refers to any measurable change in the physiological state of the cells according to standard definitions. For example, a change in the physiological state of the cells can be detected by a change in their activation state (recognized by upregulation or downregulation of the expression level of the early activated cell marker CD69) or a change in their differentiation state (recognized by upregulation or downregulation of NKG2D or NCR) several hours or days after contact with such cytokines. A measurable physiological effect can also be a change in the proliferation rate of the cells measured by CFSE staining or other techniques known in the art. Cells cultured in a first culture medium are exposed to IL-2, IL-7, and IL-15. It will be apparent to those skilled in the art that the cells in the second culture medium should not be subjected to functionally relevant stimulation with IL-15 or functionally similar growth factors. Furthermore, the cells in the second culture medium should not be subjected to functionally relevant stimulation with IL-4 or functionally similar growth factors. Preferably, these cytokines should not be present in the cell culture medium at a final concentration greater than 2 ng / ml, more preferably not greater than 1 ng / ml, more preferably not greater than 0.1 ng / ml, and more preferably not present.

[0056] The term "growth factor with interleukin-15-like activity" refers to any compound that has the same activity as IL-15 with respect to its ability to promote similar physiological effects on γδ T cells in culture, and includes, but is not limited to, IL-15 and IL-15 mimetics, or any functional equivalents of IL-15, including IL-2 and IL-7. Physiological effects on cultured γδ T cells promoted by IL-15, IL-2, and IL-7 include the induction of cell differentiation toward a more cytotoxic phenotype (e.g., upregulation of the expression levels of NKG2D and NCRs (NKp30 and NKp44)), increased anti-tumor cytotoxic function, and increased production of pro-inflammatory cytokines (e.g., IFN-γ).

[0057] In one embodiment, the growth factor having interleukin-15-like activity is either interleukin-15 (IL-15), interleukin-2 (IL-2), or interleukin-7 (IL-7), preferably IL-15.

[0058] As used herein, "IL-15" refers to native or recombinant IL-15 or variants thereof (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof) that act as agonists for one or more IL-15 receptor (IL-15R) subunits. IL-15 is a known T cell growth factor that, like IL-2, can support the proliferation of the IL-2-dependent cell line CTLL-2.

[0059] IL-15 can also refer to IL-15 derived from various mammalian species, including, for example, humans, monkeys, cows, pigs, horses, and mice. An IL-15 "mutein" or IL-15 "variant" referred to herein is a polypeptide that is substantially homologous to the sequence of a native mammalian IL-15 but has an amino acid sequence that differs from the native mammalian IL-15 polypeptide due to amino acid deletions, insertions, or substitutions. Variants can include conservatively substituted sequences, meaning that a given amino acid residue has been replaced by a residue with similar physiochemical characteristics. Examples of conservative substitutions include the substitution of one aliphatic residue for another (e.g., the substitution of Ile, Val, Leu, or Ala for another residue) or the substitution of one polar residue for another (e.g., the substitution of Lys for Arg, Glu for Asp, or Gln for Asn). Other such conservative substitutions are well known, such as the substitution of entire regions with similar hydrophobic characteristics. Naturally occurring IL-15 variants are also encompassed by the present invention. Examples of such variants are proteins resulting from alternative mRNA splicing events or proteins resulting from proteolytic cleavage of the IL-15 protein, which retain the binding properties of IL-15. Alternative splicing of the mRNA can result in a truncated but biologically active IL-15 protein. Variations that may result from proteolysis include, for example, differences at the N- or C-terminus resulting from the proteolytic removal of one or more terminal amino acids (generally 1 to 10 amino acids) from the IL-15 protein upon expression in different types of host cells.

[0060] As used herein, "IL-2" refers to natural or recombinant IL-2, or any IL-2 receptor that acts as an agonist for one or more IL-2 receptor (IL-2R) subunits. It refers to variants (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof) that can support the growth of the IL-2-dependent cell line CTLL-2 (33; American Type Culture Collection (ATCC®) TIB 214).

[0061] IL-2 can also refer to IL-2 derived from various mammalian species, including, for example, humans, monkeys, cows, pigs, horses, and mice. Variants can include conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue with similar physiochemical characteristics. Examples of conservative substitutions include the substitution of one aliphatic residue for another (e.g., Ile, Val, Leu, or Ala for another) or the substitution of one polar residue for another (e.g., Lys for Arg, Glu for Asp, or Gln for Asn). Other such conservative substitutions are well known, such as the substitution of entire regions with similar hydrophobic characteristics. Naturally occurring IL-2 variants are also encompassed by the present invention. Examples of such variants are proteins resulting from alternative mRNA splicing events or proteins resulting from proteolytic cleavage of the IL-2 protein, which retain the binding properties of IL-2. Alternative splicing of mRNA can yield a truncated but biologically active IL-2 protein. Variations that can result from proteolysis include, for example, differences at the N- or C-terminus due to the proteolytic removal of one or more terminal amino acids (generally 1 to 10 amino acids) from the IL-2 protein upon expression in different types of host cells.

[0062] As used herein, "IL-7" refers to native or recombinant IL-7 or variants thereof (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof) that act as agonists for one or more IL-7 receptor (IL-7R) subunits. Mature human IL-7 occurs as a 152 amino acid sequence (excluding a signal peptide consisting of an additional 25 N-terminal amino acids).

[0063] IL-7 can also refer to IL-7 derived from various mammalian species, including, for example, humans, monkeys, cows, pigs, horses, and mice. Variants can include conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue with similar physiochemical characteristics. Examples of conservative substitutions include the substitution of one aliphatic residue for another (e.g., the substitution of Ile, Val, Leu, or Ala for another) or the substitution of one polar residue for another (e.g., the substitution of Lys for Arg, Glu for Asp, or Gln for Asn). Other such conservative substitutions are well known, such as the substitution of entire regions with similar hydrophobic characteristics. Naturally occurring IL-7 variants are also encompassed by the present invention. Examples of such variants are proteins resulting from alternative mRNA splicing events or proteins resulting from proteolytic cleavage of the IL-7 protein, which retain the binding properties of IL-7. Alternative splicing of mRNA can yield a truncated but biologically active IL-7 protein. Variations that can result from proteolysis include, for example, differences at the N- or C-terminus due to the proteolytic removal of one or more terminal amino acids (generally 1 to 10 amino acids) from the IL-7 protein upon expression in different types of host cells.

[0064] The term "growth factor with interleukin-4-like activity" refers to any growth factor that has the same activity as IL-4 with respect to its ability to promote similar physiological effects on γδ T cells in culture. "The term IL-4 refers to any compound, including, but not limited to, IL-4 and IL-4 mimetics, or any functional equivalent of IL-4. Physiological effects on γδ T cells promoted by IL-4 have been shown to include reduced expression levels of NKG2D and NCR, suppression of cytotoxic function, and selective improvement of survival. IL-4 has also been shown to significantly suppress the secretion of pro-inflammatory cytokines (including IFN-γ and TNF-α) from activated TCRγδ T cells.

[0065] In one embodiment, the growth factor having interleukin-4-like activity is interleukin-4 (IL-4).

[0066] As used herein, "IL-4" refers to natural or recombinant IL-4 or variants thereof (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof) that act as agonists for one or more IL-4 receptor (IL-4R) subunits. Such substances can support the differentiation of naive helper T cells (Th0 cells) to Th2 cells. Mature human IL-4 occurs as a 129 amino acid sequence (excluding a signal peptide consisting of an additional 24 N-terminal amino acids).

[0067] IL-4 can also refer to IL-4 derived from various mammalian species, including, for example, humans, monkeys, cows, pigs, horses, and mice. Variants can include conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue with similar physiochemical characteristics. Examples of conservative substitutions include the substitution of one aliphatic residue for another (e.g., the substitution of Ile, Val, Leu, or Ala for another) or the substitution of one polar residue for another (e.g., the substitution of Lys for Arg, Glu for Asp, or Gln for Asn). Other such conservative substitutions are well known, such as the substitution of entire regions with similar hydrophobic characteristics. Naturally occurring IL-4 variants are also encompassed by the present invention. Examples of such variants are proteins resulting from alternative mRNA splicing events or proteins resulting from proteolytic cleavage of the IL-4 protein, which retain the binding properties of IL-4. Alternative splicing of mRNA can yield a truncated but biologically active IL-4 protein. Variations that can result from proteolysis include, for example, differences at the N- or C-terminus due to the proteolytic removal of one or more terminal amino acids (generally 1 to 10 amino acids) from the IL-4 protein upon expression in different types of host cells.

[0068] In one embodiment, (1) culturing cells in the sample in a first culture medium containing a T cell mitogen and interleukin-4 in the absence of interleukin-15, interleukin-2, and interleukin-7; and (2) culturing the cells obtained in step (1) in a second culture medium containing a T cell mitogen and interleukin-15 in the absence of interleukin-4, thereby obtaining Vδ1+ T cells from the sample.

[0069] The method for obtaining V51 T cells from a sample may include additional growth factors. Thus, in one embodiment, the first culture medium or the second culture medium, or both culture media, further comprises one or more additional growth factors. The additional growth factors may be selected from interferon-γ (IFN-γ), interleukin-21 (IL-21), interleukin-1β (IL-1β), and combinations thereof. Preferably, the additional growth factor is IFN-γ. These growth factors may be added to one or both culture media to increase the number of V51 T cells in the culture. Further increasing the proliferation and purity levels of V51+ T cells. Additional growth factors may include IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, human platelet lysate (HPL), and stromal cell-derived factor-1 (SDF-1). In one embodiment, such factors are used in the expansion to selectively promote the proliferation of V51+ T cells.

[0070] The term "T cell mitogen" refers to any substance capable of stimulating T cells via TCR signaling, including, but not limited to, plant lectins (such as phytohemagglutinin (PHA) and concanavalin A (ConA)) and lectins of non-plant origin, antibodies that activate T cells, and other non-lectin / non-antibody mitogens. Preferred antibody clones include anti-CD3 antibodies (such as the OKT-3 and UCHT-1 clones), anti-γδ antibodies (such as B1 and IMMU510), or anti-Vδ1 antibodies. In the context of the present invention, antibodies are understood to include monoclonal antibodies (mAbs), polyclonal antibodies, antibody fragments (e.g., Fab and F(ab')2), single-chain antibodies, single-chain variable fragments (scFv), and recombinantly produced binding partners. In one embodiment, the antibody is an anti-CD3 monoclonal antibody (mAb). In another embodiment, the antibody is an anti-Vδ1 antibody. Other mitogens include phorbol 12-myristate-13-acetate (TPA) and its related compounds, such as mezerein, or bacterial compounds, such as Staphylococcal enterotoxin A (SEA) and Streptococcal protein A. The T cell mitogen can be soluble or immobilized, and multiple T cell mitogens can be used in the method.

[0071] In one embodiment, the T cell mitogen is an antibody or a fragment thereof. The antibody or fragment thereof may be an anti-CD3 antibody (e.g., OKT-3). Alternatively or additionally, the antibody or fragment thereof may be an anti-TCRγδ antibody (such as a pan-γδTCR antibody) or an anti-TCRVδ1 antibody.

[0072] References herein to "culturing" include adding cells to culture medium containing growth factors and / or essential nutrients required and / or preferred for those cells and / or the non-hematopoietic tissue sample. Culturing can be by selective expansion, such as by selecting culture conditions that preferentially expand V51 T cells over other cell types present in the sample. Alternatively, the expansion conditions can be non-selective, and culturing can be followed by depletion of non-target cells (e.g., cells other than V51 T cells, such as αβ T cells). Alternatively, the expansion conditions can be non-selective, and culturing is preceded by depletion of non-target cells (e.g., cells other than V51 T cells, such as αβ T cells).

[0073] In one embodiment, the culturing is carried out in the absence of feeder cells.

[0074] In one embodiment, the culturing is carried out in the substantial absence of contact with stromal cells, hi another embodiment, the culturing is carried out in the substantial absence of contact with fibroblasts.

[0075] In one embodiment, the V51 T cells are harvested after culturing for at least 11 days (such as culturing for at least 14 days). In certain embodiments, the period of culturing according to the methods defined herein is at least 14 days. In certain embodiments, the period of culturing according to the methods defined herein is less than 45 days, such as less than 30 days, such as less than 25 days. In another embodiment, the period of culturing according to the methods defined herein is between 14 and 35 days, such as between 14 and 21 days. In yet another embodiment, the period of culturing according to the methods defined herein is about 21 days.

[0076] In another embodiment, the culturing is performed in an amount effective to generate an expanded population of V51+ T cells for a period of time (e.g., at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days, at least 28 days, or a longer period, e.g., between 5 and 40 days, between 7 and 35 days, between 14 and 28 days, or about 21 days). In some embodiments, culturing is carried out for a few hours (e.g., about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 15 hours, about 18 hours, or about 21 hours) to about 35 days (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, or 35 days). In one embodiment, culturing is carried out for 14 to 21 days.

[0077] It will be understood that when two culture media are used, the length of time for culturing in each medium may vary. For example, cells may be cultured in the first culture medium for about 2 to about 21 days, more preferably about 3 to about 14 days, and more preferably about 4 to about 8 days. Cells may be cultured in the second culture medium for about 2 to about 30 days, more preferably about 5 to about 21 days, and more preferably about 10 to 15 days.

[0078] In one embodiment, culturing is performed in a vessel comprising a gas-permeable material. Such materials allow gases (such as oxygen, carbon dioxide, and / or nitrogen) to pass through, thereby permitting gas exchange between the contents of the vessel and the surrounding atmosphere. References herein to "vessel" will be understood to include culture dishes, culture plates, single-well dishes, multi-well dishes, multi-well plates, flasks, multi-layer flasks, bottles (such as roller bottles), bioreactors, bags, tubes, and the like. Such vessels for use in methods involving the expansion of non-adherent cells and other lymphocytes are known in the art. Containers comprising gas-permeable materials have been shown to increase the yield of isolated V51 T cells. Such vessels have also been shown to preferentially support V51 T cells and other lymphocytes over fibroblasts and other stromal cells (e.g., epithelial cells), including adherent cell types. In another embodiment, fibroblasts and / or other stromal cells (e.g., epithelial cells) are absent from cultures performed in vessels comprising gas-permeable materials.

[0079] Such containers that include a gas permeable material may further include a non-porous gas permeable material. Thus, in one embodiment, the gas permeable material is non-porous. In some embodiments, the gas permeable material is a membrane film (such as silicone, fluoroethylene polypropylene, polyolefin, or ethylene vinyl acetate copolymer). Furthermore, such containers may include only a portion of the gas permeable material, gas permeable membrane film, or non-porous gas permeable material. Thus, according to yet another embodiment, a container includes an upper portion, a lower portion, and at least one sidewall, wherein at least a portion of the lower portion of the container includes a gas permeable material that forms a substantially horizontal surface when the upper portion is placed on top of the lower portion. In one embodiment, a container includes an upper portion, a lower portion, and at least one sidewall, wherein at least a portion of the lower portion includes a gas permeable material that forms a horizontal surface when the upper portion is placed on top of the lower portion. In another embodiment, a container includes a top, a bottom, and at least one sidewall, the at least one sidewall comprising a gas permeable material that can be a vertical surface when the top overlies the bottom, or a horizontal surface when the top does not overlie the bottom. In such an embodiment, only a portion of the bottom or the sidewall can comprise a gas permeable material. It will be understood that, alternatively, the entire lower portion or the entire sidewall may comprise a gas permeable material. In yet another embodiment, the upper portion of the container comprising a gas permeable material may be sealed, for example, by utilizing an O-ring. It will be understood that such an embodiment prevents leakage or reduces evaporation of the container contents. Thus, in certain embodiments, the container comprises a liquid-tight container that comprises a gas permeable material to allow gas exchange. In an alternative embodiment, the upper portion of the container comprising a gas permeable material is a horizontal surface, is located above the lower portion, and is not sealed. Thus, in certain embodiments, the upper portion is configured to allow gas exchange from the upper portion of the container. In another embodiment, the lower portion of the gas permeable container is configured to allow gas exchange from the lower portion of the container. In yet another embodiment, the container comprising a gas permeable material may be a liquid-tight container and may further include inlet and outlet ports or inlet and outlet tubes. Thus, in certain embodiments, a container comprising a gas permeable material comprises a top, a bottom, and optionally at least one sidewall, wherein at least a portion of the top and bottom comprise a gas permeable material, and at least a portion of at least one sidewall, if present, comprises a gas permeable material. Examples of containers are described in WO 2005 / 035728 and US 9,255,243, which are incorporated herein by reference. Such containers are also commercially available, such as the G-REX® cell culture devices (such as the G-REX 6-well plate, G-REX 24-well plate, and G-REX 10 container) supplied by Wilson Wolf Manufacturing.

[0080] In certain embodiments, the sample is cultured in a medium that is substantially free of serum (e.g., serum-free medium or medium containing serum replacement (SR)). Thus, in one embodiment, the sample is cultured in a serum-free medium. Such serum-free medium can also include serum replacement medium, where serum replacement is based on chemically defined components to avoid the use of serum derived from humans or animals. In an alternative embodiment, the sample is cultured in a medium containing serum (e.g., human AB serum or fetal bovine serum (FBS)). In one embodiment, the sample is cultured in a medium containing serum replacement. In one embodiment, the sample is cultured in a medium that does not contain animal-derived products.

[0081] It will be appreciated that embodiments in which samples are cultured in serum-free media have the advantage of avoiding the problems associated with serum filtration, precipitation, contamination, and delivery. Furthermore, animal-derived products are not preferred for use in clinical-grade manufacturing of human therapeutics.

[0082] Many basal culture media suitable for use in expanding γδ T cells are available, including AIM-V, Iscove's medium, and RPMI-1640 (Life Technologies). The medium may also be supplemented with other media factors, such as serum, serum proteins, and selection agents (e.g., antibiotics), as defined herein. For example, in some embodiments, RPMI-1640 medium contains 2 mM glutamine, 10% FBS, 10 mM HEPES (pH 7.2), 1% penicillin-streptomycin, sodium pyruvate (1 mM; Life Technologies), non-essential amino acids (e.g., 100 μM Gly, Ala, Asn, Asp, Glu, Pro, and Ser; 1×MEM Non-Essential Amino Acids (Life Technologies)), and 10 μl / L β-mercaptoethanol. In an alternative embodiment, AIM-V medium may be supplemented with CTS immune serum replacement and amphotericin B. Conveniently, cells are cultured during isolation and / or expansion in an appropriate culture medium at 37° C. in a humidified atmosphere containing 5% CO 2 .

[0083] Examples of other components that may be added to the culture medium include, but are not limited to, plasma or serum, purified proteins (such as albumin), lipid sources (such as low-density lipoprotein (LDL)), vitamins, amino acids, steroids, and other components that support or promote cell growth and / or survival. Any other additives that may be used.

[0084] V51 T cells obtained according to the described methods can be separated from other cells that may be present in the final culture using techniques known in the art, including fluorescence-activated cell sorting, immunomagnetic separation, affinity column chromatography, density gradient centrifugation, and cell panning.

[0085] The resulting V51 T cells can be used immediately for therapeutic, experimental, or commercial applications as described herein, or the cells can be cryopreserved for later use.

[0086] Treatment method According to another aspect of the invention, there is provided a method of treating a myeloid malignancy, the method comprising administering to a patient having the myeloid malignancy a therapeutically effective amount of an allogeneic composition comprising V51 T cells.

[0087] As used herein, the term "therapeutically effective amount" means an amount effective to achieve a desired result, at dosages and for periods of time necessary to achieve that result.

[0088] As previously described herein, the myeloid malignancy may be selected from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). The present invention is particularly useful for patients who are minimal residual disease positive (MRD+).

[0089] In one embodiment, the method further comprises administering chemotherapy.

[0090] In one embodiment, the patient is treated with chemotherapy at least 3 days prior to administration of the allogeneic composition. The chemotherapy may be selected from, for example, fludarabine and cyclophosphamide.

[0091] In one embodiment, a dose of V51 T cells calculated per kg of patient body weight is administered to the patient. In some embodiments, a therapeutically effective amount is about 1 x 10 5 Individual cells / kg, approximately 5×10 5 Individual cells / kg, approximately 1×10 6 Individual cells / kg, approximately 1.5×10 6 Individual cells / kg, approximately 2×10 6 Individual cells / kg, approximately 3×10 6 Individual cells / kg, approximately 5×10 6 Individual cells / kg, approximately 1×10 7 Individual cells / kg, approximately 1.5×10 7 Individual cells / kg, approximately 2×10 7 Individual cells / kg, approximately 3×10 7 Individual cells / kg, approximately 5×10 7 Individual cells / kg, approximately 1×10 8 Individual cells / kg, approximately 2×10 8 cells / kg, or approximately 5 x 10 8 In some embodiments, the therapeutically effective amount comprises about 1 x 10 cells / kg. 6 Individual cells / kg, approximately 1.5×10 6 Individual cells / kg, approximately 2×10 6 Individual cells / kg, approximately 3×10 6 Individual cells / kg, approximately 5×10 6 Individual cells / kg, approximately 1×10 7 Individual cells / kg, approximately 1.5×10 7 Individual cells / kg, approximately 2×10 7 Individual cells / kg, approximately 3×10 7 Individual cells / kg, approximately 5×10 7 Individual cells / kg, approximately 1×10 8 Individual cells / kg, approximately 2×10 8 cells / kg, or approximately 5 x 10 8 In some embodiments, the therapeutically effective amount comprises up to about 1 x 10 cells / kg. 6 Individual cells / kg, approximately 1.5×10 6Individual cells / kg, approximately 2×10 6 Individual cells / kg, approximately 3×10 6 Individual cells / kg, approximately 5×10 6 Individual cells / kg, approximately 1×10 7 Individual cells / kg, approximately 1.5×10 7 Individual cells / kg, approximately 2×10 7 Individual cells / kg, approximately 3×10 7 Individual cells / kg, approximately 5×10 7 Individual cells / kg, approximately 1×10 8 Individual cells / kg, approximately 2×10 8 cells / kg, or approximately 5 x 10 8 In some embodiments, the therapeutically effective amount comprises about 1 x 10 cells / kg. 6 ~1×10 8 In one embodiment, the therapeutically effective amount comprises about 1 x 10 cells / kg. 8 Contains less than 10 cells / kg.

[0092] In some embodiments, a therapeutically effective amount is about 1×10 10 Less than 10 total viable cells (approximately 1 × 10 9 Less than 10 total viable cells or approximately 1 x 10 8 In some embodiments, a therapeutically effective amount comprises about 8×10 total viable cells. 9 pieces, about 4×10 9 pieces, approx. 2.4× 10 9 pieces, approximately 1.2×10 9 pieces, about 8×10 8 pieces, about 4×10 8 pieces, about 8×10 7 pieces, or approximately 4 x 10 7 Contains all living cells.

[0093] In some embodiments, a therapeutically effective amount is about 5×10 4 In another embodiment, the therapeutically effective amount comprises less than about 1 x 10 αβ T cells / kg. 4 Contains less than αβ T cells / kg.

[0094] In one embodiment, V51 T cells (e.g., 10 per kg of the subject's body weight) 6 ~108 Vδ1+ T cells, e.g., 10 per kg of subject body weight 6 ~10 7 V51 T cells) is initially administered to the subject, followed by one or more (e.g., two, three, four, or five) subsequent administrations of V51 T cells. In one embodiment, the one or more subsequent administrations are administered less than 15 days (e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days) since the previous administration (e.g., less than 4, 3, or 2 days since the previous administration).

[0095] In some embodiments, one or more additional therapeutic agents may be administered to the subject. The additional therapeutic agent may be selected from the group consisting of an immunotherapeutic agent, a cytotoxic agent, a growth inhibitory agent, a radiotherapeutic agent, an anti-angiogenic agent, or a combination of two or more of these agents. The additional therapeutic agent may be administered simultaneously with, before, or after administration of the expanded V51 T cells. The additional therapeutic agent may be an immunotherapeutic agent, which may act on a target within the subject (e.g., the subject's own immune system) and / or on the transferred V51 T cells.

[0096] The administration of the composition can be carried out in any convenient manner. The administration of the composition described herein to a patient can be intraarterially, subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, by intravenous injection, or intraperitoneally, for example, by intradermal or subcutaneous injection. Specifically, the composition is administered as an intravenous infusion.

[0097] It will be understood that all of the embodiments described herein may be applied to all aspects of the present invention.

[0098] With reference to the above-mentioned figures, certain aspects and embodiments of the present invention will now be illustrated by way of example. [Example]

[0099] Materials and Methods Ethics Statement Primary acute myeloid leukemia (AML) cells were obtained from peripheral blood of patients with primary disease after informed consent and institutional review board approval. The study was conducted in accordance with the Declaration of Helsinki.

[0100] mouse NOD SCIDγ c - / - (NSG) mice, NOD SCIDγ c - / - SGM3(NSGS) mice and NOD Rag1 - / - gamma c - / - SGM3 (NRGS) mice were obtained from Jackson Laboratories. Age- and sex-matched mice were randomly assigned to different groups. Disease development was followed by weekly blood sampling (in the intramedullary model). The first evidence of decreased hind limb mobility was the disease endpoint. All animal procedures were performed in accordance with the guidelines of the Direcao Geral de Veterinaria and approved by the Animal Ethics Committee of the Instituto de Medicina Molecular João Lobo Antunes (Lisboa, Portugal). is.

[0101] γδ T cell composition and TCR repertoire analysis γδ T cell compositions were generated using the methods described in WO2016 / 198480. Specifically, the "Delta One T" (DOT) cell protocol refers to a research-scale version of the expansion protocol described in Almeida et al. (2016) Clin. Cancer Res. 22:5795-804, and "GDX012" refers to a scaled-up version of the expansion protocol using larger vessels (e.g., G-Rex vessels). Briefly, MACS-sorted γδ T cells were resuspended in serum-free culture medium (OpTmizer-CTS) supplemented with 5% autologous plasma and 2 mmol / L L-glutamine (Thermo Fisher Scientific). Animal-product-free human cytokines (recombinant IL-4 [rIL4] (100 ng / mL), recombinant interferon-γ [rIFNγ] (70 ng / mL), recombinant IL-21 [rIL21] (7 ng / mL), and recombinant IL-1β [rIL1β] (15 ng / mL)) and soluble anti-CD3 mAb (clone OKT-3, 70 ng / mL) were added to the medium. Cells were incubated at 37°C in a 5% CO atmosphere and periodically fed with fresh medium containing recombinant IL-15 [rIL15] (70 ng / mL), IFNγ (30 ng / mL), and anti-CD3 (1 mg / mL). Cells were optionally frozen after expansion and thawed before use.

[0102] For TRGV and TRDV repertoire analysis, Vδ1 T cells were sorted by FACS from either the initial blood sample (ex vivo) or the final DOT cell product generated as described above. Next-generation sequencing was performed as previously described (Verstichel et al. (2017) Sci. Immunol. 2:eaah4232; Ravens et al. (2017) Nat Immunol. 18:393-401; Di Lorenzo et al. (2019) Sci Data 6:115). For the generation of DOT cell clones, FACS-sorted CD3 TCR Vδ1 TCR Vδ2 single cells were transferred to 96-well plates and cultured at 1000 x g for 10 min. 4The cells were cultured for 21 days using the DOT cell protocol described above in the presence of irradiated autologous peripheral blood mononuclear cells (feeders) (which were replaced once a week).

[0103] Targeting AML cells in vitro and in vivo AML cell lines (THP-1, HEL, AML-193, MV4-11, HL-60, U-937, OCI-AML3, Kasumi-1, and KG-1) authenticated by the German Resource Center for Biologic Materials (DSMZ) were obtained from DSMZ and used at passages p3–p8. Lentiviral barcoding of AML cells was performed and analyzed as previously described (Naik et al. (2013) Nature 496:229–232). For in vitro targeting, AML cell lines or primary samples were co-incubated with DOT cells for 3 hours and stained with Annexin V as previously described (Nobrega-Pereira et al. (2018) Cancer Res. 78:731–741). For in vivo targeting, three xenograft hAML models were established, as shown in Figure 6A–C. Patient-derived xenografts (intratibial injection) were previously described (12). Tumor burden was assessed by staining with anti-human CD45 (HI30) and anti-human CD33 (P67.6). Flow cytometry data acquisition was performed on an LSR Fortessa (BD Biosciences), and data analysis was performed using FlowJo X software (Tree Star).

[0104] statistical analysis The data were analyzed using GraphPad Prism software. All data are averages. Values ​​were expressed as ± SEM. Comparisons of two groups were performed by Student's t-test, and comparisons of three or more groups were performed by ANOVA combined with Dunnett's post-hoc test. Comparisons of animal survival were performed using the log-rank (Cox-Mantel) test.

[0105] Example 1: γδ T cell compositions exhibit higher clonal diversity than ex vivo Vδ1 T cells. We first characterized the γδ T cell product upon expansion of αβ-depleted peripheral blood mononuclear cells using the DOT cell protocol described in the Materials and Methods section. Given reports that clonal expansion and reduced diversity of the adult peripheral blood Vδ1+ T cell repertoire can be induced by common pathogens, such as cytomegalovirus (CMV), we analyzed the effect of expansion on the TCR repertoire. Next-generation sequencing of the CDR3 regions of the TRGV and TRDV genes was performed before and after three weeks of cell culture. In contrast to fresh, non-expanded ex vivo Vδ1+ T cells from all analyzed donors, the expanded Vδ1+ cells were highly polyclonal and lacked dominant clones (Figure 1A-D).

[0106] This was demonstrated by the contribution of the top 20 expanded clones to the total V51 TCR repertoire. These 20 clones accounted for >60% of the total V51 TCR repertoire in peripheral blood, but <10% of the total V51 T cell product. Furthermore, very few clonotypes were shared between those identified in non-expanded ex vivo cells and those in expanded V51 cells, particularly for TRDV (Table 1).

[0107] [Table 1]

[0108] We investigated the basis for diversification of the expanding Vδ1+ T cell repertoire. Given that CD27 downregulation is a prerequisite for pre-expansion / differentiated Vδ1+ T cells, we compared the TCR clonality of expanded cells generated from pre-sorted CD27- and CD27+ subsets, demonstrating distinct proliferative capacities exhibited by our Vδ1+ T cell expansion protocol. The generation of diverse Vδ1+ T cells after expansion was revealed to be restricted to CD27+ progenitor cells. Furthermore, we demonstrated that the Vδ1+ T cell population (generated from bulk Vδ1 T cells) was predominantly composed of CD27+ cells. Vδ1+ T cell products derived from pre-sorted CD27+ cells expressed NKp30 and were highly cytotoxic against KG-1 AML cells (Figure 2A-B).

[0109] Example 2: Reactivity of γδ T cell compositions against AML cells To assess the functional relevance of V51 T cell polyclonality, clones were generated from single-cell sorted V51 T cells and expanded / differentiated using an adapted DOT cell expansion protocol, including the addition of feeder cells. The cytotoxicity of these cells against the AML cell line KG-1 was tested (Figure 3A). Most clones (derived from different donors) apoptotically inhibited KG-1 cells upon short-term (3 h) co-incubation in vitro. We found that Vδ1 T cells efficiently induced AML cell targeting (Fig. 3A). These results indicate that the expanded Vδ1 T cell population is composed of multiple clones with unique ability to target AML cells. To functionally test whether TCRs are involved in this reactivity, we performed killing assays in the presence of mAbs specifically blocking the Vδ1 TCR (or isotype control), and observed only a mild reduction in targeting of KG-1 cells across several clones derived from different donors (Fig. 3B).

[0110] To further evaluate anti-AML activity, bulk DOT cell products derived from multiple donors were tested against various other AML cell lines as well as primary samples obtained from patients at diagnosis. In all cases, expanded Vδ1 T cell populations rapidly (within 3 hours) killed AML cells in vitro (Figure 4A-B). This pattern was similar to that reported for CAR-T cells (Mardiros et al. (2013) Blood 122:3138-3148; Gill et al. (2014) Blood 123:2343-2354; Petrov et al. (2018) Leukemia 32:1317-1326) and distinct from unexpanded fresh ex vivo Vδ1 T cells (Figure 4D). Cytotoxicity was associated with degranulation and increased expression of perforin and granzyme B upon tumor cell recognition (Figure 4E-F). Expanded Vδ1 T cells did not target any normal leukocyte populations (myeloid or lymphoid) derived from the peripheral blood of healthy volunteers, including CD33 and CD123 myeloid progenitor cells (Figure 4C), whose on-target depletion by the respective CAR-T cells is known to cause unwanted myeloablation.

[0111] The allogeneic γδ T cell composition was also tested against other hematological tumor cell lines.

[0112] Generation of PBMCs from buffy coats The buffy coat was diluted with 1 part blood and 3 parts PBS and layered onto Leucosep tubes (20 ml of buffy coat-PBS mixture per tube). The Leucosep tubes were spun at 2000 rpm (approximately 800 g) for 20 minutes at room temperature with the centrifuge brake set to 1. The interface was collected, combined into one tube, and washed once more with PBS before use in downstream assays.

[0113] GDX012 cells ("effectors") derived from two donors were analyzed in flow cytometry cytotoxicity assays against the following tumor and healthy lines ("targets"): -NALM-6 -Raji -MV4-11 -Kasumi -HL-60 Healthy allogeneic peripheral blood mononuclear cells

[0114] Target cells were washed with PBS and stained with CellTrace Violet (CTV) for 20 minutes at room temperature. After 20 minutes, cells were washed with medium containing at least 10% serum and resuspended in cytokine-free target cell medium (RPMI). Target cells were then co-cultured with effector cells at 10:1, 5:1, 2:1, and 1:1 (effector:target) ratios in duplicate or triplicate at 37°C for 20 hours. This assay was performed in the absence of cytokines.

[0115] After 20 hours, dead cells were stained by adding SytoxAADvanced to the culture medium for 10 minutes at room temperature and analyzed immediately on a MACSQuant10. The percent dissolution was calculated.

[0116]

number

[0117] Overall, all tumor lines were sensitive to V51+ T cell-mediated targeting. Increasing the E:T ratio increased the level of cytotoxicity. Conversely, healthy PBMCs were completely spared, regardless of the E:T ratio. Thus, expanded V51+ T cells have the capacity to target a broad range of hematologic tumor targets, while sparing healthy allogeneic cells. The results are shown in Figure 5.

[0118] Example 3: Xenograft model for targeting AML in vivo with γδ T cell compositions To test DOT-expanded Vδ1 T cells against AML in vivo, we established various independent xenograft models of AML (Figures 6A-C). In both AML cell line models (Figures 6C-E) and two patient-derived xenografts (Figures 6F-I), administration of DOT-expanded Vδ1 T cells reduced tumor burden and increased host survival without significant toxicity. While CAR-T cells have been reported to confer greater survival benefits in AML xenografts (Mardiros et al. (2013), Gill et al. (2014), Petrov et al. (2018)), these models have been biased toward AML cell lines that uniformly express target antigens. However, it is not possible to assess the toxicity of strategies that are predicted to induce myeloablation in patients using xenografts. These data, combined with the safety and efficacy profiles of the VD1-enriched γδ T cell composition, support its potential as a candidate for adoptive cell therapy for AML.

[0119] Example 4: γδ T cell compositions target chemotherapy-resistant AML. Because chemotherapy resistance leads to fatal relapse in the setting of AML treatment, DOT-expanded V51 T cells were evaluated for targeting chemotherapy-resistant AML cells. To this end, AML cells were treated with cytarabine plus doxorubicin for 72 hours, which eliminated >99% of tumor cells. After allowing surviving cells to repopulate, the cultures were treated with chemotherapy or V51 T cells. While the cytotoxic efficacy of chemotherapy was reduced, the targeting efficacy of V51 T cells was unaffected (Figure 7A). This demonstrates the superior ability of V51 T cells to target chemotherapy-resistant AML cells.

[0120] Considering this and the polyclonal and polyreactive nature of the V51 T cell repertoire (as shown in Figure 1), we investigated the ability of V51 T cells to retarget AML cells after initial V51 T cell treatment, which eliminated >99% of tumor cells by 72 h (Figure 7B). The remaining approximately 0.1% of AML cells present at 72 h were sorted by FACS, repopulated, and then re-treated with V51 T cells. V51 T cells killed pre-treated AML cells with the same efficiency as untreated controls (Figure 7C). This suggests that treatment with V51 T cells did not select for a specific subset of V51 T-resistant AML cells. To track AML clonal dynamics during treatment (V51 T cell or chemotherapy), we tagged single AML cells with cell barcodes (non-coding DNA sequences trackable by NGS). Chemotherapy selectively targeted approximately half of all barcoded AML single-cell lineages, while Vδ1 T cells preserved the clonal architecture of the AML population (Figure 7D-E).

[0121] Collectively, these data suggest that the breadth of AML targeted by expanded Vδ1 T cells avoids the selection of resistant lineages and allows for efficient retreatment. Refractory relapse after chemotherapy should be prevented. Thus, this study provides evidence for the clinical application of γδ T cell compositions for the treatment of AML.

[0122] Example 5: Repeated cytotoxicity of V51+ T cell populations Repeated challenge cytotoxicity assay To determine the capacity of V51 T cells for repeated cytotoxicity against appropriate tumor cell lines, GDX012-expanded V51 T cells from two donors were analyzed in a flow cytometry cytotoxicity assay against AML HL-60 target cells. Briefly, HL-60 target cells were washed with PBS and stained with CellTrace Violet (CTV) for 20 minutes at room temperature. After 20 minutes, cells were washed with medium containing at least 10% serum and resuspended in cytokine-free target cell medium (RPMI). Target cells were then co-cultured with effector cells at a 10:1 (effector:target) ratio at 37°C in duplicate or triplicate for 48 hours. Assays were performed in the presence of 2 ng / ml IL-15. After 48 hours, dead cells were stained for 10 minutes at room temperature by adding SytoxAADvanced to the culture medium and immediately analyzed on a MACSQuant10. The percentage of Sytox+ve was calculated by quantifying the percentage of CTV+ve cells that were positive for SytoxAADvanced dye.

[0123] For the second, repeat killing assay, cells were collected from unused wells from the first killing assay by vigorous pipetting, sedimented (300 g, 5 min), the supernatant removed, and resuspended in fresh target cell culture medium. Cells were counted and added to new wells. Fresh HL-60 cells stained with CTV as above were resuspended and added to the newly seeded effector cells at a 10:1 effector:target ratio again. Fresh 2 ng / ml IL-15 was added back to the wells, and the wells were left for an additional 72 hours. Target cell killing was quantified using SytoxAADvanced as described above. Results are shown in Figure 8.

[0124] Overall, the HL-60 tumor line was sensitive to two rounds of Vd1 cell-mediated targeting over a 5-day period in the presence of IL-15 cytokine, suggesting that proliferating Vd1 cells have the potential to provide long-term targeting of tumor cells.

[0125] Example 6: γδ T cell compositions have limited potential to cause cytokine release syndrome Cytokine release syndrome (CRS) is a significant safety issue in other immunotherapies, such as αβ T cell therapy. To assess the potential risk of our product causing a cytokine burst, we thawed cryopreserved GDX012 cells and measured cytokine levels in the supernatants of 21-hour cultures under several different conditions. Indeed, physiological stimulation of GDX012 cells with either TCR (Figure 9A) or IL-15 (known to induce potent Vδ1 T cell responses) (Figure 9B) primarily induces the release of Th1-associated cytokines and barely induces detectable levels of key cytokines responsible for CRS. Even under supraphysiological stimulation with IL-15 (Figure 9C), IL-6 levels are undetectable over the course of our assay, while TNFα begins to be observed at some level. This behavior indicates a favorable safety profile for the claimed composition. Furthermore, additional studies demonstrated that there was very limited risk of a cytokine release burst when GDX012 cells were co-cultured with allogeneic blood-derived samples (PBMCs and buffy coats) (Figure 9D), with IL-6 and TNFα levels being virtually undetectable after 21 hours of co-culture.

[0126] Example 7: γδ T cell compositions preserve allogeneic B cells To determine cell selectivity, GDX012-expanded V51 T cells from three donors were analyzed in a flow cytometry cytotoxicity assay against a mixture of CFSE-labeled NALM-6 cells (tumorigenic B cells) and CTV-labeled B cells (non-tumorigenic primary B cells).

[0127] Isolation of primary B cells 100E6 PBMCs were obtained from freshly obtained buffy coats and centrifuged at 300g for 7 minutes. The supernatant was removed and 10 7The cells were resuspended in 40 μl of MACS buffer / 10 μl of Pan B cell biotin-antibody cocktail per cell. The cell suspension was left to stand in a refrigerator for 5 minutes. 7 30 μl of MACS buffer / 20 μl of anti-biotin microbeads per cell was added to the cell suspension and allowed to stand in a refrigerator for 10 minutes. Meanwhile, an LS column inserted into a quadroMACS on a magnetic stand was equilibrated by passing 3 ml of MACS buffer through the column. The cell suspension was applied to the column, and the flow-through was collected. A wash solution (3 ml of MACS buffer) was applied to the column and collected. This represented the negatively enriched B cell fraction.

[0128] Cytotoxicity assay B cells were washed with PBS and stained with CellTrace Violet (CTV) for 20 minutes at room temperature. NALM-6 cells were washed with PBS and stained with CFSE for 20 minutes at room temperature. After 20 minutes, cells were washed with medium containing at least 10% serum and resuspended in cytokine-free target cell medium (RPMI). Target cells were then co-cultured with effector cells at ratios of 10:1:1, 5:1:1, 2:1:1, and 1:1:1 (effector:NALM-6:B cells) in duplicate or triplicate at 37°C for 20 hours. This assay was performed in the absence of cytokines. After 20 hours, dead cells were stained by adding SytoxAADvanced to the culture medium for 10 minutes at room temperature and immediately analyzed using a MACSQuant10. The percentage of Sytox+ve cells was calculated by quantifying the percentage of CTV+ve or CFSE+ve cells that were positive for the SytoxAADvanced dye. The results are shown in Figure 10.

[0129] Overall, NALM-6 cells were specifically targeted, while healthy B cells were completely spared. Targeting of NALM-6 cells was dependent on the E:T ratio; increasing the E:T ratio increased the level of cytotoxicity. Conversely, healthy B cells were completely spared, regardless of the E:T ratio. Thus, expanded Vd1+ T cells specifically target B cell tumors without any collateral damage to healthy B cells cultured in the same plate.

[0130] Example 8: γδ T cell compositions do not mediate mixed lymphocyte reactions (MLR) To demonstrate the suitability of the culture system for detecting allogeneic responses, donor blood T cells were isolated, stained for CTV, and cultured with irradiated peripheral blood lymphocytes (PBLs) derived from either autologous or allogeneic sources. After 5 days of culture, αβ T cell division was assessed by analyzing the dilution of the CTV dye by flow cytometry. The method is presented here.

[0131] The results from this experiment (shown in Figure 11A) clearly demonstrate that irradiated PBLs can induce robust allogeneic responses from blood T cells in a mixed lymphocyte response culture system, while autologous matched cultures showed significantly lower levels of T cell proliferation, demonstrating the suitability of this culture system for determining the alloreactive potential of a given T cell population.

[0132] To determine whether expanded Vδ1+ T cells could drive an alloreactive response, GDX012 cells were cultured with irradiated PBLs from an allogeneic donor. As a control, matched blood T cells from the same individual from whom the GDX012 product was derived were CTV stained and cultured against irradiated PBLs from the same allogeneic donor. After 5 days of culture, cell division was assessed by flow cytometry.

[0133] The results shown in Figure 11B indicate that while blood αβ T cells clearly divided in the presence of allogeneic PBLs, GDX012 cells (expanded Vδ1 T cells) failed to persist in any meaningful numbers in culture. The αβ response elicited against allogeneic PBLs exhibited T cell proliferation typical of a mixed lymphocyte reaction. Despite this mismatch, GDX012 cells did not proliferate in the presence of the same irradiated PBLs, indicating that GDX012 cells are unable to initiate an allogeneic response in the same manner as αβ T cells. Together, these results indicate that GDX012 is unable to mediate GvHD in the same manner as αβ T cells.

[0134] Buffy coat preparation, CD14 event depletion, and irradiation treatment of resulting peripheral blood lymphocytes Buffy coat blood was subjected to density gradient separation to isolate the PBMC fraction. A small portion of the resulting PBMCs was frozen in 10% Cryostor10 cryopreservation medium and frozen at -80°C. The remaining PBMCs were washed, labeled for human CD14, and depleted using a Miltenyi MACS LS column. The resulting PBL fraction was then cultured overnight at 37°C in a 5% CO2 atmosphere in complete RPMI medium (RPMI medium containing 10% fetal bovine serum, 1% penicillin / streptomycin, 1% HEPES, 1% non-essential amino acids, and 1% sodium pyruvate).

[0135] The next day, PLBs were collected by pipetting and exposed to 40 Gy of x-ray irradiation to abrogate their cell proliferation potential. These cells represent the "stimulated" cell fraction in the MLR assay.

[0136] Isolation of blood T cells and preparation of MLR plates PBMCs derived from either buffy coat sources or refrozen leukopak material were removed from frozen storage and thawed. The PBMCs were then washed, labeled with pan T cell isolation beads, and blood T cells were isolated using a MACS LS column. The resulting blood T cell fraction was then washed and stained with Cell Tracker Violet (CTV). CTV+ blood T cells were then co-cultured with irradiated stimulator PBLs. In parallel, frozen vials of GDX12 cells were thawed, washed, and immediately stained with CTV. CTV+ GDX012 cells were then co-cultured with irradiated stimulator PBLs. In all cases, the effector-stimulator cell ratio per well was 1:1. Co-cultures were performed in complete RPMI medium. Cultures were then incubated at 37°C in a 5% CO2 atmosphere for 5 days. After this initial setup, the cultures were not fed with additional medium.

[0137] Example 8: Expanded V51+ T cell compositions inhibit tumor growth in vivo The biodistribution and efficacy of GDX012 in vivo was evaluated using a cell line-derived xenograft model. 0.5 × 10 cells of a human B-cell acute lymphoblastic leukemia (ALL) cell line (NALM-6), stably transduced to express firefly luciferase (FLuc) and green fluorescent protein (GFP) genes, were injected intravenously via the tail vein into immunodeficient NOD SCID gamma (NSG) mice. 6 pcs or 1 x 10 6 Then, 24 hours or 6 days after tumor inoculation, 20 × 10 6 Mice were either given a single intravenous injection of GDX012 cells via the tail vein or not. All control and treated mice received intraperitoneal injections of recombinant human IL-15 (1 μg / mouse every 2-3 days for the duration of the study) to support the survival of GDX012 cells. Tumor burden was assessed twice weekly by whole-body bioluminescence imaging (BLI) during and after in vivo administration. After 4 weeks, mice were sacrificed and the hind leg long bones were removed. Bone marrow was extruded from the hind leg long bones using RPMI-1640 and collected for flow cytometry analysis. Briefly, cells were stained with eFluor 780 fixable live / dead dye, followed by staining with FITC-conjugated anti-human CD45, PE-conjugated anti-human CD19, and APC-conjugated anti-human TCRγδ antibodies. Finally, cells were fixed in 4% paraformaldehyde and analyzed using a MACSQUANT 10 flow cytometer.

[0138] A single administration of GDX012 in an in vivo systemic ALL model reduced disseminated tumor growth compared to controls (Figure 12). Furthermore, GDX012 cells homed to the bone marrow and preferentially suppressed tumor burden within the bone marrow (Figure 13).

Claims

1. 1. An allogeneic composition comprising V51 T cells for use in treating patients with myeloid malignancies.

2. 2. The allogeneic composition for use according to claim 1, wherein the myeloid malignancy is selected from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS).

3. 3. The allogeneic composition for use according to claim 1 or claim 2, wherein the patient is minimal residual disease positive (MRD+).

4. 4. The allogeneic composition for use according to claim 3, wherein the MRD+ patient is in complete remission with no detectable leukemic blasts in the peripheral blood and less than 5% leukemic blasts in the bone marrow.

5. The allogeneic composition for use according to any one of claims 1 to 4, wherein the patient has been previously treated with chemotherapy.

6. 6. The allogeneic composition for use according to claim 5, wherein the patient has been treated with chemotherapy at least 3 days prior to administration of the allogeneic composition.

7. 7. The allogeneic composition for use according to claim 5 or claim 6, wherein the chemotherapy is selected from fludarabine and cyclophosphamide.

8. The allogeneic composition for use according to any one of claims 1 to 7, comprising at least about 90% CD45+ cells relative to total viable cells.

9. The allogeneic composition for use according to any one of claims 1 to 8, comprising at least about 60% γδ T cells relative to total live cells.

10. 10. The allogeneic composition for use according to any one of claims 1 to 9, comprising at least about 50% Vδ1+ T cells relative to total live cells.

11. Approximately 1×10 １０ 11. The allogeneic composition for use according to any one of claims 1 to 10, comprising less than 10 whole viable cells.

12. Approximately 1×10 ９ 12. The allogeneic composition for use according to claim 11, comprising less than 10 whole viable cells.

13. Approximately 1×10 ８ 13. The allogeneic composition for use according to claim 12, comprising less than 10 whole viable cells.

14. The allogeneic composition comprises about 8×10 ９ pieces, approximately 4 x 10 ９ pieces, approximately 2.4 x 10 ９ pieces, approximately 1.2 x 10 ９ pieces, approximately 8 x 10 ８ pieces, approximately 4 x 10 ８ pieces, approximately 8 x 10 ７ pieces, or about 4 x 10 ７ 12. An allogeneic composition for use according to any one of claims 1 to 11, comprising whole living cells.

15. A dosage comprising an allogeneic composition for use according to any one of claims 1 to 14.

16. Approximately 1×10 ５ 16. The dose of claim 15, comprising less than 10 cells / kg.

17. Approximately 1×10 ６ 16. The dose of claim 15, comprising less than 10 cells / kg.

18. Approximately 1×10 ７ 16. The dose of claim 15, comprising less than 10 cells / kg.

19. Approximately 3×10 ７ 16. The dose of claim 15, comprising less than 10 cells / kg.

20. Approximately 1×10 ８ 16. The dose of claim 15, comprising less than 10 cells / kg.

21. Approximately 5×10 ４ 16. The dose of claim 15, comprising less than αβ T cells / kg.

22. Approximately 1×10 ４ 22. The dose of claim 21 , comprising less than αβ T cells / kg.

23. 16. The allogeneic composition for use according to any one of claims 1 to 15, wherein said Vδ1 T cells are obtained from said sample by a method comprising culturing said sample in a medium comprising a T cell mitogen and a growth factor with interleukin-4-like activity in the absence of a growth factor with interleukin-15-like activity.

24. 16. The allogeneic composition for use according to any one of claims 1 to 15, wherein said Vδ1 T cells are obtained from said sample by a method comprising culturing said sample in a medium comprising a T cell mitogen and a growth factor with interleukin-15-like activity in the absence of a growth factor with interleukin-4-like activity.

25. 25. The allogeneic composition for use according to claim 23 or claim 24, wherein the V51 T cells are harvested after culturing for at least 11 days.

26. 26. The allogeneic composition for use according to any one of claims 23 to 25, wherein said culturing is carried out in a vessel comprising a gas permeable material.

27. 27. The allogeneic composition for use according to claim 26, wherein the container comprises a liquid-tight container comprising a gas permeable material to allow gas exchange.

28. 28. The allogeneic composition for use according to claim 26 or claim 27, wherein the lower part of the container is configured to allow gas exchange from the lower part of the container.

29. The allogeneic composition for use according to any one of claims 23 to 28, wherein the sample is cultured in the absence of serum.

30. The allogeneic composition for use according to any one of claims 23 to 28, wherein the sample is cultured in a medium comprising serum or a serum replacement.

31. 1. A method of treating a myeloid malignancy, the method comprising administering to a patient having said myeloid malignancy a therapeutically effective amount of an allogeneic composition comprising V51 T cells.

32. 32. The method of claim 31, wherein the myeloid malignancy is selected from AML and MDS.

33. 33. The method of claim 31 or claim 32, wherein the patient is minimal residual disease positive (MRD+).

34. The MRD+ patient is in complete remission with no detectable leukemic blasts in the peripheral blood; 34. The method of claim 33, wherein the bone marrow contains less than % leukemic blasts.

35. The method of any one of claims 31 to 34, further comprising administering chemotherapy.

36. 36. The method of claim 35, wherein the patient is treated with chemotherapy at least 3 days prior to administration of the allogeneic composition.

37. 37. The method of claim 35 or claim 36, wherein the chemotherapy is selected from fludarabine and cyclophosphamide. 【Request Item 38】 The therapeutically effective amount is about 8×10 ９ pieces, approximately 4 x 10 ９ pieces, approximately 2.4 x 10 ９ pieces, approximately 1.2 x 10 ９ pieces, approximately 8 x 10 ８ pieces, approximately 4 x 10 ８ pieces, approximately 8 x 10 ７ pieces, or about 4 x 10 ７ 32. The method of claim 31 , comprising whole living cells. 【Request Item 39】 The therapeutically effective amount is about 1×10 １０ 32. The method of claim 31 , comprising fewer than 100 total viable cells. 【Request Item 40】 The therapeutically effective amount is about 1×10 ９ 32. The method of claim 31 , comprising fewer than 100 total viable cells. 【Request Item 41】 The therapeutically effective amount is about 1×10 ８ 32. The method of claim 31 , comprising fewer than 100 total viable cells. 【Request Item 42】 The therapeutically effective amount is about 5×10 ４ 32. The method of claim 31 , comprising less than αβ T cells / kg. 【Request Item 43】 The therapeutically effective amount is about 1×10 ４ 43. The dose of claim 42, comprising less than αβ T cells / kg.

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