Gamma delta t cell expansion method

Abstract

A method for expanding a population of gamma delta T-cells is provided, wherein isolated activated peripheral blood mononuclear cells (PBMCs) are cultured in a medium comprising transforming growth factor beta (TGF-beta) under conditions favoring the production of effector gamma delta T-cells having therapeutic activity against a malignant disease. The use of TGF-beta in the production of effector cells, in particular Vgamma9Vdelta2 T-cells, is also described and claimed.

Description

[0001] This invention relates to methods for expanding γδ T cells (specifically, human Vγ9Vδ2 T cells with antitumor effector functions), reagents and compositions used in these methods, products of these methods, and their use in therapy. Additionally, in some cases, these methods are suitable for enhancing cell expansion efficiency and effector function. Background of the Invention

[0003] γδ T cells comprise up to 10% of circulating lymphocytes and act as a bridge between innate and adaptive immunity. Four properties of these pluripotent cells make them well-suited for therapeutic use and specifically for cancer immunotherapy. First, γδ T cells recognize genomic, metabolic, and signaling disturbances associated with the transformation state [1,2]. Second, they possess a diverse network of immune effector activities that overlaps with, yet differs from, those deployed by “conventional” αβ T cells. γδ T cells release perforin and granzymes, express both FAS and TRAIL, participate in Fc receptor-dependent effector functions, and produce a range of immunomodulatory cytokines, including tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and IL-17. Third, γδ T cells act as effective antigen-presenting cells, enabling immune attack perpetuity through adaptive mechanisms [3]. Finally, because these cells are not HLA-restricted, they do not cause graft-versus-host disease. This enhances their future prospects for use in allogeneic “existing” settings [4].

[0004] Most circulating γδ T cells in humans exhibit Vγ9Vδ2 receptors that recognize non-peptide phosphate antigens (PAg), with the best example being IPP and its stereoisomer DMAPP. Figure 1 [5] Because PAg is an intermediate in the metabolism of mevalonate, Vγ9Vδ2T-cells provide an innate mechanism for detecting excess activity of this key metabolic pathway. This monitoring is evolutionarily reasonable, as excess mevalonate pathway flux promotes cell transformation and thus works synergistically with p21Ras [6]. This reflects the fundamental role of this network in the biosynthesis of isoprene-like substances required for post-translational modifications of several GTPases, including p21Ras, Cdc42, Rho, Rab, and Rac.

[0005] Amino-bisphosphonate (NBP) drugs such as zoledronic acid (ZA), alendronate (AA), pamidronate (PA), and ibandronic acid (IA) exert their antitumor activity through a combination of direct cytotoxic and immunomodulatory mechanisms [7]. A key example of the latter is the ability of these drugs to activate Vγ9Vδ2T cells. This is achieved by inhibiting FPP synthase in the mevalonate pathway, leading to increased PAg accumulation ( Figure 1 [8]. Tumor cells treated with NBP pulses rapidly require a large PAg load and thus become more sensitive to the recognition of Vγ9Vδ2T cells [5,9]. This principle provides an opportunity to enhance the sensitivity of tumors to γδ T-cell immunotherapy.

[0006] The clinical development of γδ T-cell immunotherapy is built on two established findings. First, patients with various malignancies have been treated with ZA and low-dose IL-2 in pursuit of in vivo expansion of Vγ9Vδ2 T-cells. In many cases, these small studies have linked circulating Vγ9Vδ2 T-cell numbers with delayed disease progression

[10] . Second, ex vivo expanded Vγ9Vδ2 T-cells have been tested as autologous adoptive immunotherapy in several early clinical trials involving a variety of cancers, including epithelial ovarian cancer (EOC) [11-13]. Although these studies have demonstrated the safety of infused γδ T-cells, clinical efficacy has been limited (even when combined with ZA). This highlights the need for better systems to expand these cells efficiently, thereby producing cells that exhibit enhanced antitumor activity.

[0007] Transforming growth factor-β (TGF-β) is a secreted protein present in at least three isoforms, referred to as TGF-β1, TGF-β2, and TGF-β3. It is a cytokine that functions in a variety of processes, including proliferation and cell differentiation, as well as immunity and cancer. It is generally understood to have a regulatory immune function in this context, and this may partially explain why it is upregulated in certain cancers where overexpression of cytokines reduces the host immune response. Numerous papers have shown that adding TGF-β to T cells promotes a regulatory phenotype. For example, two independent groups have shown that culturing human peripheral blood mononuclear cells (PBMCs) in the presence of cytokines including TGF-β induced the generation of regulatory γδ T cells that expressed high levels of immunosuppressive Foxp3 and CD25 [14,15].

[0008] The applicant has conducted research on different protocols for expanding γδ T-cells and has identified a specific set of conditions for producing high levels of cells with enhanced effector activity. Invention Overview

[0010] Surprisingly, the applicant has discovered that the presence of TGF-β under certain culture conditions can generate effector T cells with enhanced yield and immunostimulatory activity specifically targeting cancer cells. Furthermore, the anticancer efficacy of cells produced using this method can be increased.

[0011] According to the present invention, a method for expanding a population of γδ T-cells is provided, the method comprising culturing isolated activated peripheral blood mononuclear cells (PBMCs) in a medium containing transforming growth factor β (TGF-β) under conditions conducive to the generation of effector γδ T-cells with therapeutic activity against malignant diseases.

[0012] Specifically, the T-cell population generated using the method of the present invention is rich in γδ cells with therapeutic activity against malignant diseases and is specifically rich in Vγ9Vδ2 cells. In this context, malignant diseases specifically include proliferative disorders such as cancer, including solid tumors, hematologic malignancies, or other circulatory system cancers. Examples of solid tumors include breast cancer, ovarian cancer, colon cancer and typically G (gastrointestinal) tract cancer, cervical cancer, lung cancer (specifically small cell lung cancer and non-small cell lung cancer), head and neck cancer, bladder cancer, prostate cancer, or Kaposi's sarcoma. Examples of circulatory system cancers include leukemias such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), myeloproliferative disorders (MPD), chronic myeloid leukemia (CML), T-cell acute lymphoblastic leukemia (T-ALL), B-cell acute lymphoblastic leukemia (B-ALL), non-Hodgkin lymphoma (NHL), and B-cell lymphoma.

[0013] As used herein, 'effective T-cells' refers to T-cells that, in addition to their regulatory or immunosuppressive effects on the immune response, also possess anticancer or antileukemic properties.

[0014] It is evident that the presence of TGF-β in the culture medium under certain conditions increases both the yield and efficacy of effector T cells. This contradicts the prevailing understanding that this cytokine induces the production of major regulatory T cells.

[0015] In the method of the present invention, PBMCs used as starting materials are suitably primate PBMCs, such as human PBMCs. They are suitably separated from blood samples from humans or other primates such as apes using conventional methods.

[0016] These cells can be obtained from a patient and then reintroduced into that patient (autologous therapy). However, in some cases, cell expansion from patients who have been pre-treated with solid tumors such as triple-negative breast cancer has been found to be minimal or nonexistent. In such cases, it may be necessary to obtain PBMCs, which are used as starting materials in the method of this invention, from a healthy donor and employ an allogeneic treatment approach. In this case, it is preferable to purify γδ T cells from the expanded product, specifically by removing potentially harmful B-cells (CD19). + ) and αβT- cells, in order to promote the safe allogeneic use of γδT cells.

[0017] TGF-β is suitably present in the culture medium at concentrations ranging from 0.1 to 100 ng / mL, for example, at a concentration of about 5 ng / mL. However, the precise amount of TGF-β added depends on the biological activity of the TGF-β used. This can be determined using a suitable bioassay that produces an ED50 value equivalent to the unit of activity. For example, the ED50 of TGF-β can be determined by the ability of TGF-β to inhibit the mouse IL-4-dependent proliferation of mouse HT-2 cells. Typically, a concentration of 5 ng / mL equals 2 × 10⁻⁶. 5 Specific activity per unit. Therefore, it is suitable to reduce from 4 × 10 3 Up to 4×10 6 One unit of TGF-β was added to the culture medium, wherein the unit was determined as described above.

[0018] The applicant has found that the properties of the culture medium can be important in this context. Specifically, the culture medium used by the applicant is produced under good manufacturing processes (GMP) and does not contain fetal calf serum or fetal bovine serum, which are often included in conventional T-cell culture media [14,15 (personal communication, Dr. Rita Casetti)]. These specific properties of the culture medium appear to influence T-cell development in the presence of TGF-β, thereby favoring the expansion of effector cells with antitumor activity over regulatory T-cells.

[0019] Specifically, the culture media include serum-free media, such as synthetic media like TexMACS (Miltenyi) or RPMI, and can be used in the presence of additional human AB serum. The culture media are preferably GMP-grade media.

[0020] Additionally, the culture medium used may further contain interleukin-2 (IL-2). Other cytokines may be present, as long as they do not alter their nature as products of T-cells, which are primarily effector cells with antitumor and antileukemic activities. However, in one specific embodiment, the culture medium does not contain any additional cytokines.

[0021] Interleukin-2 is suitably present in culture medium at amounts ranging from 1 to 1000 U / mL, for example, at approximately 100 U / mL, where U is a unit. In this context, one unit of IL-2 can be defined as the amount of IL-2 in 1 ml that will induce IL-2-dependent mouse T cells to incorporate at 50% of their maximum level after 24 hours of incubation. 3 H-TdR.

[0022] TGF-β and IL-2 (when present) are suitably added repeatedly at regular intervals during the culture process, specifically in response to cell expansion, which is suitably monitored throughout the process by cell counting.

[0023] The cells used as starting material are activated. In one specific embodiment, this can be achieved by adding an activator capable of activating specifically Vγ9Vδ2T cells. Suitable activators may include amino-bisphosphonate drugs such as zoledronic acid (ZA), alendronate (AA), pamidronate (PA), and ibandronic acid (IA). In one specific embodiment, the activator is zoledronic acid or a salt thereof. Alternatively, cells may be activated using phosphate antigens such as BRHPP or IPP.

[0024] The activator is suitably added in an effective amount. The addition may occur together with the initial addition of TGF-β and IL-2 (if present). The concentration of the added activator will depend on various factors such as the specific type of activator used, but typically will be in the range of 0.1–10 μg / ml, for example, at about 1 μg / ml.

[0025] Following the amplification process described above, γδ T-cells can then be obtained by purifying the amplification product. Specifically, CD19 and αβ T-cells can be removed from the product through negative selection or by using a suitable isolation technique or kit. The applicant has discovered that the amplification process may be ineffective if γδ T-cells are isolated from PBMCs prior to amplification.

[0026] Using the method described above, the yield of in vitro expanded effector T-cells can be increased, and thus the application of this method to increase the T-cell expansion yield forms another aspect of the present invention.

[0027] Similarly, as described below, the efficacy of T-cells obtained using this method, specifically their anti-cancer efficacy, is improved. Therefore, the present invention further provides a method for assessing the anti-cancer efficacy of T-cells expanded in vitro using the amplification method described above.

[0028] Another aspect of the invention provides the use of TGF-β for enhancing the expansion of effector T cells and, specifically, human Vγ9Vδ2 T cells suitable for treating malignant diseases as described above.

[0029] In another aspect, the present invention provides the use of TGFβ for enhancing the anticancer effector capabilities of T-cells.

[0030] The T-cells obtained by the method described above constitute another aspect of the present invention. These cells can be used in treatment, and specifically for the treatment of cancer.

[0031] These cells can be used to treat patients in a conventional manner. Specifically, the present invention also provides a method for treating patients in need by administering T-cells obtained as described above. Specifically, the T-cells are adoptively transferred into the patient according to standard clinical practice.

[0032] Specifically, these cells can be administered with activators such as those described herein and / or chemotherapeutic agents. Suitable activators include bisphosphonate drugs such as zoledronic acid, alendronate, and pamidronate. They can activate T cells and also sensitize the tumor to T cells.

[0033] Certain chemotherapeutic agents have also been found to sensitize tumors to γδ T cells

[18] , and therefore these chemotherapeutic agents can be administered pre- or co-administered with the γδ T cells of the present invention. Specific examples of such chemotherapeutic acids include cisplatin, etoposide, anthracyclines, and cytarabine, which is described below.

[0034] The applicant first administers cytarabine followed by γδ T-cells sequentially to produce an anti-tumor effect, and this novel treatment constitutes another aspect of the invention. In this treatment, an effective amount of γδ T-cells and cytarabine is administered to the patient in need. Specifically, the γδ T-cells are obtained according to the invention.

[0035] Another hope is to co-administer cytokines such as IL-2 in order to prolong the survival of T cells.

[0036] Detailed Description of the Invention

[0037] The invention will now be described in detail by way of example with reference to the accompanying drawings, wherein... Figure 1This is a schematic diagram illustrating the mevalonate pathway. Phosphoantigens (PAg) recognized by Vγ9Vδ2T cells include DMAPP, IPP, and Apppl. Inhibition sites of this pathway by amino-bisphosphonates and statins are indicated by circles, where IPP = isopentened diphosphate and DMAPP = dimethylallyl diphosphate.

[0038] Figure 2 The results of in vitro expansion of Vγ9 and Vδ2 T-cells using a comparative method (Method 1) are shown. After culture using the conditions described above, the percentage (A) and absolute number (B) of γδ T-cells per 20 ml blood sample were assessed at the start of the culture cycle and after 15 days. (C) Expected expression of Vγ9 and Vδ2 T-cell receptor subunits was determined by flow cytometry. Pooled immunophenotypic data (D) and representative immunophenotypic data (E) of γδ T-cells expanded in vitro for 15 days from healthy donors and women recently diagnosed with EOC (donor number indicated in parentheses).

[0039] Figure 3 The results of expanding Vγ9Vδ2T-cells using Comparative Method 1 in different culture media with and without human AB serum are shown. Each figure shows the total number of γδ T-cells (A), the percentage of γδ T-cells present (B), and the yield (C) after 14 days of culture.

[0040] Figure 4 The results of cytotoxicity assays using cells expanded using comparative method 1 are shown in assays against a series of ovarian cancer cell lines: (A) IGROW-1; (B) KOC7C; (C) PEO1; (D) PEA; (E) SKOV-3; (F) TOV-21G.

[0041] Figure 5 Results obtained using the method for in vitro expansion of Vγ9Vδ2T-cells according to the present invention are shown. Results show enrichment (A) and expansion (B) of Vγ9Vδ2T-cells (mean ± SEM, n = 13 independent replicates). The percentage of γδ T-cells present at the beginning and end of fabrication is also shown (mean ± SD, n = 10). *p = 0.03, obtained by Mann-Whitney test.

[0042] Figure 6Comparative antitumor activity of γδ T-cells expanded using Method 1 and Method 2 is shown. Cytotoxicity assays were performed three times in 96-well plates at an effector:target ratio of 5:1, two weeks after expansion of γδ T-cells using Method 1 or 2. Tumor cells were cultured with the indicated aminobisphosphonates for 24 hours before cytotoxicity assays. Residual tumor cell viability was measured by MTT or luciferase assays after overnight co-culture with Vγ9Vδ2 T-cells. Data show tumor cell killing from 2–5 independent replicates, mean ± SEM, using the indicated cell lines: ovarian cancer cell lines (A) IGROW-1, (B) SKOV-3, (C) Kuramochi, and (D) TOV-21G; myeloid leukemia cell lines (E) U937 and (F) KG-1; and breast cancer cell lines (G) MDA-MB-231, (H) MDA-MB-468, and (I) BT-20.

[0043] Figure 7 This illustrates cytokine production in γδ T-cells expanded via Method 1 and Method 2. γδ T-cells were expanded using Method 1 or Method 2 and then... Figure 6 Tumor cells, either pulsed-treated or un-pulsed-treated with bisphosphonates, were co-cultured. The supernatant was then harvested after 24 hours of co-culture, and interferon-γ (AI) and interleukin-2 (JO) were analyzed by ELISA. Interferon (IFN)-γ production is shown in the following cell lines: ovarian cancer cell lines (A) Kuramochi, (B) IGROW-1, (C) SKOV-3, (D) TOV-21G; breast cancer cell lines (E) MDA-MB-468, (F) MDA-MB-231, (G) BT-20; and myeloid leukemia cell lines (H) U937, (I) KG-1. Interleukin-2 production is shown in co-culture experiments using the following cells: (J) Kuramochi, (K) U937, (L) KG-1, (M) MDA-MB-231, (N) MDA-MB-468, and (O) BT-20 tumor cells. The data are the mean ± SEM from 3-5 independent replicate experiments.

[0044] Figure 8 The results of immunophenotypic analysis of Vγ9Vδ2T cells expanded by Method 1 and Method 2 are shown. (A) Cells expanded by Method 2 express a distinct immunophenotype with higher levels of memory receptors (CD45RO, CD27) and homing receptors (CCR7, CXCR4), skin leukocyte antigen (CLA), and E-selectin binding receptor (using E-selectin-IgG fusion protein detection-B). (C) Compared to Method 1, primary cells (CD45RA... +CCR7 + ) and central memory cells (CD45-CD27) + The proportion of NS- was higher in cells expanded by method 2. NS- was not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0045] Figure 9 The results show the evaluation of the number (A) and percentage (B) of γδ T-cells present in cultures obtained using Method 1 and the method of the present invention in different basal media (RPMI + 10% human AB serum).

[0046] Figure 10 This study demonstrates the in vivo therapeutic activity of expanded Vγ9Vδ2T-cells obtained by intravenous administration in SCID brown mice using method 1 and the method of the present invention against a defined malignant disease (U937 leukemia) load.

[0047] Figure 11 This paper illustrates the in vivo therapeutic activity of expanded Vγ9Vδ2T-cells obtained using the method of the present invention, administered intravenously in SCID brown mice, against a defined malignant disease (U937 leukemia) load, wherein (A) shows the tumor load indicated by bioluminescence; and (B) shows the mouse weight, thereby providing an indication of therapeutic toxicity.

[0048] Figure 12 This illustrates the in vivo therapeutic activity of expanded Vγ9Vδ2T cells obtained via intravenous administration using method 2 against a defined malignant disease load (MDA-MB-231 triple-negative breast cancer implanted in the mammary fat pads of SCID brown mice). (A) Tumor load indicated by bioluminescence. (B) Mouse survival. (C) Mouse body weight providing an indication of therapeutic toxicity.

[0049] Figure 13The results are shown using different purification methods, where (A) shows the extent to which Vγ9Vδ2T- cells were purified from freshly isolated PBMCs by negative selection using a CD19 and αβT- cell microbead separation kit; (B) shows the results of attempts to expand these cells; and (C) shows the percentage of cell types obtained in these experiments, with γδ2T- cells being the most abundant. T-cells were expanded from PBMCs using the method of the present invention, followed by negative selection to consume CD19 and αβ T-cells; (D) shows the results of flow cytometry analysis of these cells after consumption of contaminated CD19 and αβ T-cells; (E) shows the results of a 24-hour cytotoxicity test against MDA-MB-231 (231), MDA-MB-468 (468), or BT20 triple-negative tumor cells, or (F) against U937 or KG-1 myeloid leukemia cells (effector:target ratio of 5:1); (G) shows the cytokine concentrations in the supernatant harvested from the treated breast cancer coculture and (H) shows the cytokine concentrations in the supernatant harvested from the treated leukocyte coculture (n=2).

[0050] Figure 14 The flow cytometry results of genetically engineered γδ T-cells obtained using the method of the present invention, the technique of preloading viral vectors onto a solid phase coated with recombinant fibronectin (B), or by adding viral supernatant to the cells (C), are shown compared to an untransduced control (A).

[0051] Figure 15 The results of in vitro cytotoxicity assays against tumor cells ((A) U937 cells and (B) KG1 cells) are shown after 24 hours of treatment with different concentrations of the chemotherapeutic agent cytarabine before the addition of γδ T-cells (including some cells obtained using the method (M2) of the present invention).

[0052] Figure 16 This is a set of graphs showing the in vivo test results of the combination administration of the present invention to γδ T-cells with cytarabine and IL-2 compared to the use of cytarabine and IL-2 alone, wherein (A) shows the tumor burden indicated by bioluminescence from malignant cells on days 4, 11, 19, and 26 after administration and (B) shows the mouse body weight during the test period. In each case, cytarabine was injected as a single dose 24 hours prior to the infusion of γδ T-cells.

[0053] Comparison Example A

[0054] In previous studies, the applicant has demonstrated that healthy donors have 19,916 ± 29,887 (mean ± SD, n = 21) circulating γδ T cells. By comparison, patients recently diagnosed with EOC have 14,240 ± 15,215 γδ cells / ml of blood (mean ± SD, n = 13; not statistically significant (NS))

[16] .

[0055] To enrich these cells, peripheral blood mononuclear cells (PBMCs) were activated with ZA and cultured in RPMI 1640 medium supplemented with IL-2 / IL-15 and containing AB serum. Specifically, PBMCs isolated from normal (healthy) donors (n=21 individual donors) and patients with EOC (n=13 individual donors) were cultured with ZA (1 μg / ml, day 1 only), IL-2 (100 U / ml), and IL-15 (10 ng / ml). Cytokines and culture medium were added daily.

[0056] The percentage and absolute number of γδ T cells per 20 ml blood sample were assessed at the start of the culture period and after 15 days. Results are shown in... Figure 2 (A) and Figure 2 (B) In this study, this "research-grade" method resulted in an average 97-fold increase in γδ T-cells (EOC patients) or 172-fold increase (healthy donors; NS). Figure 2 B).

[0057] The expected expression of Vγ9 and Vδ2 T-cell receptor subunits was determined by flow cytometry and the results were shown in Figure 2 In (C), it is clear that expanded γδ T-cells from patients and healthy donors express the Vγ9Vδ2 T-cell receptor.

[0058] Pooled and representative immunophenotypic data of 15-day-old expanded γδ T-cells from healthy donors and newly diagnosed patients with EOC (donor numbers are indicated in parentheses) were also obtained, and the results are shown separately in Figure 2 (D) and Figure 2 (E) A small number of contaminated γδ T cells and natural killer cells (CD16+56) were observed. + CD3 - γδ T-cells showed an advantage. The expanded cells primarily exhibited effector and effector memory phenotypes, similar to those in patients and healthy volunteers. We subsequently found that the addition of IL-15 did not significantly differ the yield of the obtained cells, and this was omitted from subsequent expansion runs (data not shown).

[0059] To prepare for the manufacture of γδ T-cell products for clinical use, we tested the ability of commercially available GMP-compliant media to support the expansion of these cells using ZA+IL-2. The procedure described above was repeated using clinical-grade serum-free media. PBMCs were cultured in RPMI+10% human AB serum or two commercially available GMP-compliant media with or without 10% human AB serum. In each case, ZA (1 μg / ml) was added to activate γδ T-cells, followed by expansion of these cells by the addition of IL-2 (100 U / ml). Results are shown in... Figure 3 These results show that the TexMACS medium specifically enables these cells to expand under the serum-free conditions of "Method 1".

[0060] Cytotoxicity assays were performed three times in 96-well plates at an effector:target ratio of 5:1, and the results are shown below. Figure 4 In this study, tumor cells were pulsed with the indicated concentration of zoledronic acid (ZA) or pamidronate (PA) for 24 h, followed by the addition of γδ T cells. Residual tumor cell viability was measured by MTT assay after overnight co-culture with Vγ9Vδ2T cells in the following cell lines: (A) IGROWV-1; (B) KOC7C; (C) PEO1; (D) PEA; (E) SKOV-3; (F) TOV-21G. Results showed that Vγ9Vδ2T cells expanded using Method 1 exhibited broad-spectrum and NBP-enhanced antitumor activity against a range of ovarian tumors and other tumor cell lines.

[0061] Example 1

[0062] The present invention expands T-cells.

[0063] Next, we improved Method 1 so that transforming growth factor (TGF)-β was always added together with IL-2. This method is referred to as Method 2 below.

[0064] In a variation of the method in Example A above, blood from a healthy donor or patient is collected in a test tube containing a citrate anticoagulant. PBMCs are isolated using a Ficoll-Paque (GE) according to a previously disclosed method

[17] .

[0065] Then, in GMP TexMACS medium (Mittenia), at 3 × 10⁻⁶... 6100 U / mL of reconstituted PBMC cells were added to the reconstituted cells. 1 μg / mL zoledronic acid (Zometa, Novartis) as an activator, along with 100 U / mL IL-2 and 5 ng / mL TGF-β. The cells were incubated at 37°C in air containing 5% carbon dioxide.

[0066] On day 3, cells were supplied with 100 U / mL IL-2 and 5 ng / mL TGF-β. Subsequently, on days 4, 7, 9, 11, 13, and 15, cells were counted using a hematology counter via trypan blue exclusion assay. If the T-cell count was less than 1 × 10⁻⁶, the cells were considered T-cells. 6 Add 100 U / mL IL-2 and 5 ng / mL TGF-β. If the T-cell count is 1×10⁻⁶ cells / mL, then add 100 U / mL IL-2 and 5 ng / mL TGF-β. 6 With 2×l0 6 Between cells / mL, add an equal volume of TexMACS medium, along with 100 U / mL IL-2 and 5 ng / mL TGF-β. If the T-cell count is greater than 2 × 10⁻⁶ cells / mL... 6 Cells / mL, with double the volume of TexMACS medium and 100 U / mL IL-2 and 5 ng / mL TGF-β added.

[0067] Fifteen days later, the cells were analyzed by flow cytometry using the panγδ antibody to confirm the enrichment of γδ T cells in these cultures. Results were shown in... Figure 5 These results show that the improved method achieved enrichment (A) and improved expansion (B) of Vγ9Vδ2T- cells (mean ± SEM, n = 13 independent replicates).

[0068] In addition, T-cells were immunophenotypically characterized and subjected to functional tests. The relative ability of T-cells obtained using Method 1 above or the method of the present invention to mediate cytotoxic destruction of tumor cells was evaluated. Two weeks after γδ T-cell expansion using Method 1 or 2, cytotoxicity assays were performed three times in 96-well plates at an effector:target ratio of 5:1. When indicated, tumor cells were pulsed with the indicated concentration of zoledronic acid (ZA), alendronate (AA), or pamidronate (PA) for 24 h, followed by the addition of γδ T-cells. Residual tumor cell viability was measured by MTT or luciferase assay after overnight co-culture with Vγ9Vδ2T- cells in the following cell lines: (A) IGROW-1, (B) SKOV-3, (C) Kuramochi, and (D) TOV-21G; myeloid leukemia cell lines (E) U937 and (F) KG-1; and breast cancer cell lines (G) MDA-MB-231, (H) MDA-MB-468, and (I) BT-20. Results were shown in... Figure 6 middle.

[0069] Activation of γδ T cells when co-cultured with tumor cells was assessed by measuring the release of IL-2 and IFN-γ. The ability of these expanded γδ T cells to control a defined malignant disease burden was also assessed in SCID light brown mice with a defined U937 myeloid leukemia burden.

[0070] The original basic principle of including TGF-β in the culture process was to attempt to improve the expression of homing receptors such as CXCR4 on these cells. However, completely unexpectedly, the addition of TGF-β caused a substantially increased yield of Vγ9Vδ2 T-cells, such as Figure 5 As shown.

[0071] The cell products amplified by Method 2 also showed equivalent or enhanced antitumor activity against EOC (IGROV-1, SKOV-3, Kuramochi, TOV-21G), breast cancer (MDA-MB-231), and myeloid leukemia cells (U937), even in the absence of NBP exposure. Figure 6 ; Figure 10 However, the antitumor activity was consistently enhanced by prior NBP sensitization. Figure 6 ).

[0072] Two weeks after γδ T-cell expansion using Method 1 or Method 2, co-culture was performed three times in 96-well plates at an effector:target ratio of 5:1. When indicated, tumor cells were pulsed with the indicated concentration (μg / ml) of zoledronic acid (ZA) or pamidronate (PA) for 24 h, followed by the addition of γδ T-cells. After another 24 h, the supernatant was harvested and analyzed by ELISA for interferon-γ or interleukin-2. Results are shown in... Figure 7 The following tumor cell monolayers are shown to produce interferon (IFN)-γ: ovarian cancer cell lines (A) Kuramochi, (B) IGROW-1, (C) SKOV-3, (D) TOV-21G; breast cancer cell lines (E) MDA-MB-468, (F) MDA-MB-231, (G) BT-20; and myeloid leukemia cells (H) U937 and (I) KG-1. Additionally, co-culture experiments using the following cells are shown to produce interleukin-2: (J) Kuramochi, (K) U937, (L) KG-1, (M) MDA-MB-231, (N) MDA-MB-468, and (O) BT-20 tumor cells.

[0073] Compared to cells expanded using Method 1, cells expanded using Method 2 produced significantly higher levels of IFN-γ when conjugated to tumor cell targets. This effect was most pronounced when the transformed cells had been pulsed with very low concentrations of NBP reagent. Figure 7 AG). Cells expanded using method 2 also produced IL-2 under these conditions, a finding that was not observed in cells expanded using method 1. Figure 7 HK).

[0074] Finally, the phenotypes of cells from Method 1 and Method 2 were studied using conventional methods, and the results are shown in... Figure 8 In this study, cells expanded using Method 2 were found to express a unique phenotype with high levels of homing receptors (CXCR4, CLA, E-selectin binding activity) and memory markers (CD27, CD45RO). Furthermore, compared to cells expanded using Method 1, primordial cells (CD45RA) showed increased expression of these markers. + and CCR7 + ) and central memory cells (CD45) - and CD27 + The proportion of cells amplified using method 2 was higher. Therefore, these cells were distinguishable from those produced using other amplification protocols.

[0075] Example 2

[0076] Alternative cell expansion methods

[0077] Using different basal media, the method described in Example 1 above was precisely repeated using RPMI + human AB serum. Specifically, PBMCs (3 × 10⁶ cells / mL) were cultured in RPMI + 10% human AB serum containing either zoledronic acid (1 μg / mL) + IL-2 (100 U / mL; Method 1) or zoledronic acid (1 μg / mL) + IL-2 (100 U / mL) + TGF-β (5 ng / mL; Method 2). 6 Cell counts were assessed on day 15 and results are shown in [data missing]. Figure 9 In A, the percentage of γδ T cells present in each culture was assessed on the day the culture began (Day 1) and after another 14 days (Day 15), and the results are shown in Figure 9 B in.

[0078] As before, it is clear that adding TGF-β can enhance cell proliferation.

[0079] Example 3

[0080] In vivo therapeutic activity

[0081] In addition, the in vivo therapeutic activity of expanded Vγ9Vδ2T cells against a defined malignant disease burden was compared. 1×10⁻⁶ cells were administered via tail vein injection. 6 Twenty SCID light brown mice were inoculated with U937 leukemia cells expressing firefly luciferase and then divided into four groups of five mice each. Four days later, the mice were treated as follows: Group 1 was a control group receiving PBS alone; Group 2 received pamidronate alone (200 μg IV); and Group 3 received pamidronate (200 μg IV on day 4), followed by 20 × 10⁻⁶ styrosine phosphate. 6 One (day 5) and 10×10 6 Group 4 (day 6) received pamidronate (200 μg IV, day 4), followed by 20 × 10⁶ cells. 6 One (day 5) and 10×10 6 Vγ9Vδ2T-cells (IV-administered) expanded using Method 2 were used (day 6). Leukemia burden was then monitored using a series of bioluminescence imaging techniques.

[0082] The results are shown in Figure 10 It is clear that the efficacy of the cells obtained by method 2 of the present invention is significantly greater in this assay.

[0083] Example 4

[0084] In vivo activity of cells binding to IL-2 in this invention

[0085] In a separate experiment, the in vivo therapeutic activity of expanded Vγ9Vδ2T-cells obtained using the method (M2) of this invention, administered intravenously in SCID brown mice, against a defined malignant disease burden (U937 leukemia). Mice were divided into four groups of five mice, and each group received 1 million U937 cells IV on day 1. Subsequently, one group received treatment that can be summarized as follows:

[0086] Group deal with 1 PBS (control) 2 Zoledronic acid + IL-2 3 M2+IL-2 4 M2+IL-2+zoledronic acid

[0087] At administration, 20 μg of zoledronic acid was administered intravenously 24 hours after treatment with U937 cells. One day later, mice receiving M2 cells underwent a two-dose treatment with 15 million γδ T cells intravenously. Mice receiving IL-2 were administered 10,000 U IL-2 via the intraperitoneal (IP) route concurrently with M2 administration. Two days later, mice received 10,000 UIL-2 IP. The control group received phosphate-buffered saline (PBS) alone.

[0088] Bioluminescence from malignant cells, serving as an indicator of tumor burden, was measured on days 7, 15, 21, and 28. Results were shown in... Figure 11 In A, the results showed that Vγ9Vδ2T- cells obtained using the method of the present invention, specifically when administered with an activator, significantly reduced tumor burden.

[0089] Mice were weighed during treatment to provide an indication of treatment toxicity. Figure 11 The results shown in B indicate that there is no significant toxicity associated with the treatment.

[0090] Example 5

[0091] In vivo therapeutic effects against breast cancer

[0092] In this experiment, 20 SCID light brown mice with a defined malignant disease burden of MDA-MB-231 triple-negative breast cancer implanted in the mammary fat pads were used. Again, the mice were divided into four treatment groups. The mice were treated as follows: Group 1 was a control group receiving PBS alone. Group 2 received 20 μg of zoledronic acid intravenously. Group 3 received 20 × 10⁻⁶ ozoledronate intravenously. 6 One (day 2) and 10×10 6 Group 4 (day 3) had Vγ9Vδ2T- cells expanded using method 2. Group 4 received 20 μg pamidronate intravenously on day 1, followed by 20 × 10⁻⁶ cells. 6 One (day 2) and 10×10 6Vγ9Vδ2T-cells (day 2) were expanded using method 2. Tumor burden was measured over a 28-day period, as determined by bioluminescence. Results are shown in... Figure 12 In this case, the tumor burden generated by cells obtained using the method of the present invention is significantly reduced ( Figure 12 A), accompanied by prolonged survival ( Figure 12 B).

[0093] Mice were weighed during treatment to provide an indication of treatment toxicity. Figure 12 The result shown in C indicates that there is no significant toxicity associated with the treatment.

[0094] Example 6

[0095] Purification of expanded γδ T cells

[0096] In the first experiment, Vγ9Vδ2T cells were purified from freshly isolated PBMCs using a CD19 and / or αβT-cell microbead isolation kit via negative selection. When using both kits, residual contamination with CD19 and αβT-cells was <0.1%, as shown in the image. Figure 13 As shown in (A).

[0097] The purified cells were subjected to amplification using method 2 as described in Example 1. However, these cells could not be amplified, such as... Figure 13 As shown in (B). Therefore, it appears that the starting material must contain PBMC.

[0098] In other experiments, γδ T-cells were expanded from PBMCs for 15 days using method 2. At this time, flow cytometry analysis showed that a significant number of αβ T-cells were retained, accompanied by a small number of CD19 cells. + Cells (n=4) Figure 13 (C)).

[0099] The resulting product was then used to negatively select and consume CD19 and αβ T cells, as shown above relative to... Figure 13 As stated in (A). Figure 13 (D) shows two representative flow cytometry analyses to indicate the efficiency of the consumption process.

[0100] After purification using MACS beads (Medrin) via negative selection, γδ T-cells expanded using Method 2 were tested in a 24-hour cytotoxicity assay (5:1 effector:target ratio) against MDA-MB-231, MDA-MB-468, or BT20 triple-negative tumor cells or U937 or KG-1 myeloid leukemia cells, similar to the method described in Example 1. Cells were tested alone or in combination with zoledronic acid. Negative controls and controls using the active agent alone were present. Tumor cell viability was measured by luciferase assay and / or MTT assay (n=2). Results are shown separately in... Figure 13 (E) and Figure 13 (F) It is clear that the combination of T cells and activators leads to a significant reduction in tumor cell viability.

[0101] After 24 hours, supernatants were harvested from these co-cultures of breast cancer and leukemia, and the presence of IFN-γ and / or IL-2 was analyzed. Results are shown separately in... Figure 13 (G) and Figure 13 In (H), the combination of T cells expanded according to the present invention with an activator results in a substantial increase in cytokine levels.

[0102] These experiments demonstrate that the γδ T-cells expanded using Method 2 are fully functional after expansion, rather than after purification via negative selection. This purification facilitates the safe allogeneic use of these cells, as potentially harmful B-cells (CD19) are also present. + ) and αβT- cells have been removed.

[0103] Example 7

[0104] Genetic engineering of expanded cells

[0105] To further confirm the functionality of the γδ T-cells amplified according to the present invention, they were genetically engineered via retroviral transduction. Transduction was performed by preloading a viral vector onto a solid phase coated with recombinant fibronectin or by adding viral supernatant to the amplified cells.

[0106] It is clear that, in order to maintain the effective enrichment of these cells during the amplification process, it is preferable to preload the viral vector onto a solid phase coated with recombinant fibronectin. Figure 14 (B) instead of adding viral supernatant ( Figure 14 (C)). When a pre-loaded method is used to achieve gene transfer, this is indicated by a larger percentage of transduced cells and a larger percentage of γδ T cells present.

[0107] Example 8

[0108] The effect of combining γδ T cells with chemotherapy agents

[0109] Cytotoxicity assays were performed three times in 96-well plates containing U937 or KG-1 tumor cells at an effector:target ratio of 1:1. When indicated, tumor cells were pulsed with the indicated concentration of cytarabine for 24 h, followed by the addition of γδ T-cells generated using the method of the present invention (M2) or the method of the comparative examples above (M1). Three donors were available for M2 cells and two donors were available for M1 cells. The control group did not receive cytarabine.

[0110] Residual tumor cell viability was measured by luciferase assay after overnight co-culture with Vγ9Vδ2T- cells. Figure 15 The results showed that sublethal doses of cytarabine enhanced the antitumor activity of Vγ9Vδ2T-cells expanded using Method 2 against two cell models of AML (three donors for M2 cells and two donors for M1 cells).

[0111] In a separate experiment, 1×10 was administered via tail vein injection. 6 Fifteen SCID light brown mice were inoculated with U937 leukemia cells expressing firefly luciferase and then divided into three groups of five mice each. Four days later, the mice were treated as follows: Group 1 was a control group receiving PBS alone. Group 2 received cytarabine (480 mg / kg IV, day 4) and IL-2 (10000 IP, days 5, 6, 7, and 8). Group 3 received cytarabine (480 mg / kg IV, day 4), followed by 20 × 10⁻⁶ IL-2. 6 Vγ9Vδ2(IV) and IL-2 (10000 IP, on days 5, 6, 7 and 8) were amplified using method 2 (days 5 and 6).

[0112] Leukemia burden was then monitored using a series of bioluminescence imaging techniques. Bioluminescence from malignant cells, serving as an indicator of tumor burden, was measured on days 4, 11, 19, and 26. Results are shown in... Figure 16 (A) indicates that Vγ9Vδ2T-cells obtained using the method of the present invention most effectively reduce tumor burden when administered in combination with cytarabine. Mice are weighed during treatment to provide an indication of treatment toxicity. Figure 16 The results shown in (B) indicate that there is no significant toxicity associated with the treatment.

[0113] References

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Claims

1. A method for expanding a population of γδT cells, the γδT cells having antitumor or antileukemia effector activity, the method comprising culturing and isolating activated peripheral blood mononuclear cells (PBMCs) in a culture medium containing transforming growth factor β (TGF-β), interleukin-2 (IL-2) and free of fetal bovine serum or calf serum, thereby disadvantaging the production of immunosuppressive γδT cells and favoring the production of γδT cells with antitumor or antileukemia effector activity.

2. The method of claim 1, wherein the culture medium does not contain any additional cytokines.

3. The method according to claim 1 or claim 2, wherein the culture medium is a serum-free culture medium.

4. The method according to claim 1 or claim 2, wherein the culture medium contains human AB serum.

5. The method according to any one of the preceding claims, wherein the activator for Vγ9Vδ2T- cells is added in the initial step of the method.

6. The method according to claim 5, wherein the activator is an aminobisphosphonate.

7. The method of claim 6, wherein the bisphosphonate is zoledronic acid, alendronic acid, pamidronic acid, ibandronic acid, or a salt thereof.

8. The method according to any one of the preceding claims, wherein the PBMC is a human PBMC.

9. The method of claim 8, wherein the PBMC is derived from a healthy human.

10. The method of claim 8, wherein the PBMCs are derived from a human patient.

11. The method according to any one of the preceding claims, wherein CD19+B- cells and / or αβT- cells are removed from the amplification product.

12. A method for increasing the yield of in vitro expanded γδT-cells, the method comprising performing the method according to any one of claims 1 to 11.

13. A method for enhancing the anticancer efficacy of in vitro expanded γδT-cells, the method comprising performing the method according to any one of claims 1 to 11.

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