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Definitions

• the present invention relates to novel methods for the isolation and the selective ex vivo expansion of V52 " TCRY5 + T cells and to their clinical application.
• the immune system of jawed vertebrates includes various lymphocyte populations capable of recognizing and eliminating tumor cells, which constitutes the basis of cancer immunotherapy.
• One population is characterized by the expression of a T-cell receptor (TCR) formed by the junction of a gamma ( ⁇ ) chain and a delta ( ⁇ ) chain.
• TCR T-cell receptor
• TCRY5 + T cells account for 1 -10% of human peripheral blood lymphocytes (PBLs) but are substantially enriched in epithelial tissues of healthy individuals, where they reach up to 50% of T cells.
• PBLs peripheral blood lymphocytes
• 1 TCRY5 + T cells have potent Major Histocompatibility Complex (MHC)-unrestricted cytotoxicity against malignant and infected cells, while leaving unharmed healthy cells and tissues. Therefore, they are usually considered a first-line surveillance mechanism against infection and tumors.
• MHC Major Histocompatibility Complex
• TCRY5 + T cells In humans, different subsets or subpopulations of TCRY5 + T cells are identified and classified based on the genes that encode their ⁇ chain. Around 60-95% of TCRY5 + T cells in the peripheral blood express the V52 chain in association with ⁇ 9 chain, while most of the remainder TCRY5 + T cells express the V51 chain in association with various ⁇ elements: ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5 or ⁇ 8. Other (rarer) human TCRY5 + T cell populations express V53, V55, V56, V57 and V58 chains. 2"4
• V52 + T cells Current adoptive immunotherapy approaches based on TCRY5 + T cells are limited to the V52 + TCRY5 + T cell subpopulation (here also designated as V52 + T cells). 5 ' 6 Most V52 + T cells specifically respond to nonpeptide alkylphosphates such as isopentenyl pyrophosphate (IPP), which is produced at abnormal levels in tumor cells and in individuals exposed to bone-strengthening aminobisphosphonates, such as zoledronate and pamidronate. These compounds, when combined with interleukin-2, stimulate the proliferation and anti-tumour cytotoxic function of V52 + T cells in vitro, and generate purified cell populations for clinical applications. 5"7 However, the clinical trials completed to date have shown a low percentage of objective responses in cancer patients. 8 Thus, current ⁇ T cell-based treatments, although feasible and safe, have obvious limitations. 6 V51 + TCRY5 + T cells
• V51 + ⁇ ⁇ ⁇ + T cells (here also designated as V61 + T cells), constitute 1 -40% of all ⁇ ⁇ ⁇ + PBLs but are the major ⁇ T cell population at epithelial sites, such as the intestine and the skin. While it has never been evaluated in clinical trials, this cell subset can also display strong antitumor activity. V51 + T cells infiltrating skin, colon, kidney and lung tumours were cytotoxic against both autologous and allogeneic cancer cells. 9-13 An interesting correlation was also found between the increase in number of donor-derived peripheral blood ⁇ 1 + T cells and improved 5-10 -year disease-free survival following bone marrow transplantation for acute lymphoblastic leukaemia (ALL).
• ALL acute lymphoblastic leukaemia
• V51 + T cells can exhibit immunosuppressive and regulatory properties, a function that can also be exploited for therapeutic purposes. 22
• a small number of methods to specifically expand V51 + T cells in vitro have been described, although none of them could be adapted for clinical applications (reviewed in Siegers, G. et al., 2014, ref. 22 ).
• Meeh et al. first showed that V51 + T cells from healthy donors could expand ex vivo in response to ALL leukemic blasts. 23 Knight, A. and colleagues and Merims, S.
• peripheral blood ⁇ 1 + T cells isolated peripheral blood ⁇ 1 + T cells and treated them in the presence of phytohemagglutinin (PHA) or anti-CD3 monoclonal antibody (mAb), IL-2 and irradiated allogeneic peripheral blood mononuclear cells (PBMCs), for 3 weeks. 24, 25 In a more recent study, PHA was used in combination with interleukin-7 (IL-7). 26 Siegers, G. et al, reported a two-step culture protocol, in which sorted ⁇ ⁇ ⁇ + T cells were first treated with concanavalin-A (ConA), IL-2 and IL-4 for 6-8 days, followed by stimulation with IL-2 and IL-4 for another 10 days.
• ConA concanavalin-A
• IL-2 and IL-4 irradiated allogeneic peripheral blood mononuclear cells
• V61 + T cells were expanded in 59% of cultures and were the dominant subset (average 70% of ⁇ 1 + T cells) in half of the cultures; however, a maximum 25 fold increase of ⁇ 1 + T cells could be achieved with this method.
• This 2-step culture method was also described in International Patent Application Number PCT/CA99/01024 (published as WO 00/26347). According to those inventors, for continued cell proliferation following the removal of the mitogen, both IL-2 and IL-4 in the second culture medium were essential.
• TCRv5 + V53 + T cells account for -0.2% of circulating T cells, 30 but are enriched in the peripheral blood of renal and stem cell transplant recipients with CMV activation, 4, 21 in patients with HIV infection 31 or with B-CLL, 17 and in healthy livers. 32 Activated TCRv5 + V53 + T cells were able to kill CD1 d + cells and epithelial tumors in vitro. 4 However, available cell culture methods cannot produce large numbers of TCRv5 + V53 + T cells for clinical applications.
• TCRv6 + T cell populations were treated with plate-bound anti-TCRv6 mAb and IL-2 for 3 weeks, resulting in about 90% of TCRv5 + T cells, most of which were V52 + T cells, and a small percentage of ⁇ 1 + T cells.
• 33 Lopez, et al generated apoptosis- resistant TCRv6 + T cells by treating cells with high dose interferon- ⁇ (IFN- ⁇ ), IL-12, anti-CD2 mAb and soluble anti-CD3 mAb, in the presence of IL-2.
• IFN- ⁇ interferon- ⁇
• IL-12 interferon- ⁇
• anti-CD2 mAb anti-CD2 mAb
• soluble anti-CD3 mAb soluble anti-CD3 mAb
• polyclonal TCRv5 + T cells were expanded in the presence of ⁇ -irradiated artificial antigen-presenting cells (aAPC) of tumor origin genetically modified to co-express CD19, CD64, CD86, CD137L, and membrane bound IL- 15. 3 ' 35 Cells were further cultured in the presence of soluble IL-2 and IL-21.
• aAPC ⁇ -irradiated artificial antigen-presenting cells
• V62 TCRv6 + T cells (here also designated as V62 ⁇ T cells) exhibited higher anti-tumor cytotoxicity and increased survival capacities than V52 + T cells, both in vitro and in vivo. 26 ' 29, 37 Consequently, a medicinal product highly enriched in V52 ⁇ T cells is expected to generate more potent anti-tumor effects and to provide improved benefits to treated patients.
• V52 + T cells Previous clinical data on infused V52 + T cells suggested that a clinically relevant dose of TCRy6 + T cells for treating a disease comprises at least about 1 x10 9 live cells. 39 Consequently, methods aiming to generate V52 ⁇ T cells for clinical applications should be able to expand these cells in vitro by at least 1 .000 fold.
• V52 ⁇ T cells do not respond to alkylphosphates and the very limited knowledge about the antigens they recognize has limited their expansion in vitro. Plant lectins such as PHA and Con-A have been used to expand and enrich (to very high purity levels) these cells in vitro, at a preclinical scale.
• V52 ⁇ T cells must be isolated by magnetic- activated cell sorting (MACS) or by fluorescence-activated cell sorting (FACS) prior to stimulation with anti-CD3 mAb.
• MCS magnetic- activated cell sorting
• FACS fluorescence-activated cell sorting
• lnterleukin-2, interleukin-7 and interleukin-15 are very pleiotropic molecules, with strong stimulatory effects on multiple immune cells, including TCRa + T and V52 + T cells. Consequently, these reagents are not appropriate for expanding V52 ⁇ T cells in vitro, as contaminating cells will also expand in culture, compromising cell purity. Furthermore, the typical combination of these pro-inflammatory cytokines with Y5TCR agonists often leads to activation-induced-cell-death (AICD) of stimulated cells and to smaller numbers of cells obtained.
• AICD activation-induced-cell-death
• IL-2 and IL-4 have been used with some success to expand V61 + T cells in vitro.
• IL-4 in the culture medium induces a strong downregulation of natural killer (NK) activating receptors (such as NKG2D and NCRs) on ⁇ 2 " ⁇ T cells, weakening their anti-tumor responses (Table 3).
• NK natural killer
• Mao, Y. and colleagues recently demonstrated that cultured ⁇ 1 + T cells treated with IL-4 and anti-TCRV61 mAb secreted significantly less IFN- ⁇ and more IL-10 relative to V52 + T cells.
• V52 ⁇ T cells ex vivo require the presence of natural or artificial feeder cells, usually in the form of virus-infected or transformed cells or cell lines, bacteria and parasites. These culture methods are more complex, more prone to microbial contamination and less suitable for clinical applications.
• the present invention provides novel methods for expanding and differentiating human V62 TCRY6 + T cells in vitro, without the need for the use of feeder cells or microbial or viral components.
• the inventors aimed at improving the expansion and purity levels of cultured V52 TCRY5 + T cells.
• a novel, more efficient and selective method to expand these cells in culture was developed, in the presence of a T cell mitogen and IL-4, and in the absence of IL-2, IL-7 and IL-15.
• the obtained V52 TCRY6 + T cells were differentiated towards a more cytotoxic phenotype through additional in vitro optimization steps.
• V62 TCRY5 + T cells After the removal of IL-4 and the addition of a T cell mitogen and IL-15, IL-2, or IL-7 to the culture medium, the previously obtained V62 TCRY5 + T cells produced pro-inflammatory cytokines and expressed high levels of activating Natural Killer receptors (NKR), which mediated tumor cell killing in vitro. Importantly, upon infusion in mice, the differentiated TCRY6 + T cells maintained their cytotoxic phenotype and inhibited tumor growth in vivo.
• NLR Natural Killer receptors
• the cell culture method described herein is very robust, highly reproducible and fully compatible with large-scale clinical applications. It generates sufficient numbers of differentiated V52 TCRY6 + T cells for use in adoptive immunotherapy of cancer, and in a variety of experimental, therapeutic and commercial applications.
• the present invention provides a method for expanding and differentiating V62 TCRY5 + T cells in a sample comprising: (1 ) culturing cells in the sample in a first culture medium comprising a T cell mitogen and at least one growth factor having interleukin-4-like activity; in the absence of growth factors having interleukin-15-like activity; and
• step (1 ) (2) culturing the cells obtained in step (1 ) in a second culture medium comprising a T cell mitogen and at least one growth factor having interleukin-15-like activity, in the absence of growth factors having interleukin-4-like activity.
• the present invention provides a method for expanding and differentiating V52 ⁇ ⁇ ⁇ + T cells in a sample comprising:
• step (1 ) (2) culturing the cells obtained in step (1 ) in a second culture medium comprising a T cell mitogen and interleukin-15, in the absence of interleukin-4.
• the present invention relates to novel methods for the isolation and the selective in vitro lex vivo expansion and differentiation of V52 TCRY5 + T cells, and to their clinical application.
• the inventors tested multiple combinations of clinical-grade agonist antibodies and cytokines for their capacity to expand and differentiate (over 2-3 weeks) peripheral blood V52 TCRy5 + T cells in culture.
• TCRy6 + T cells were isolated and expanded in culture in the absence of feeder cells and molecules of microbial origin.
• V62 TCRy5 + T cells (here also designated as V52 ⁇ T cells), can be selectively expanded in vitro by culturing these cells in a first culture medium comprising a T cell mitogen and interleukin-4, in the absence of interleukin-2, interleukin-7 and interleukin-15, and sub-culturing these cells in a second culture medium containing a T cell mitogen and interleukin-15, interleukin-2, or interleukin-7, in the absence of interleukin-4.
• Other key growth factors such as interferon- ⁇ , interleukin-21 and interleukin-1 ⁇ were also added to one or both culture media to further increase the expansion and purity levels of cultured V52 ⁇ T cells.
• the first culture medium supported the selective survival and expansion of V62 ⁇ T cells (up to 8.000-fold increase of V61 + T cells in 14 days; Table 3).
• the absence of interleukin-2, interleukin-7 and interleukin-15 during the first days of culture contributed to the starvation and apoptosis of contaminant cells (including TCRa + T and V62 + T cells), which critically depend on these cytokines for survival.
• V52 ⁇ T cell population which expanded in vitro a total of several thousand fold and reached >90% of total cells after 21 days of culture (Tables 3 and 7).
• This second culture step is necessary to change the physiological properties of the cells towards a more appropriate phenotype for use as an anti-tumor or anti-viral treatment.
• Expanded and differentiated V52 ⁇ T PBLs obtained after the second culture step expressed high levels of activating Natural Killer receptors (NKR), including NKp30 and NKp44, which synergized with the T-cell receptor to mediate tumor cell targeting in vitro, while not targeting healthy cells.
• NLR Natural Killer receptors
• cells were cultured with minimum manipulation in a serum-free cell culture medium. Closed, large-scale, gas-permeable plastic cell culture bags were used as recipients for cell culture, instead of open culture plates or flasks. All used reagents and materials (or equivalent reagents and materials) are currently available and manufactured in clinical-grade quality, or in Good Manufacturing Practice (GMP) quality, free of animal-derived components. Therefore, cells produced by this method can be used in a variety of experimental, therapeutic and commercial applications.
• GMP Good Manufacturing Practice
• the present invention provides a method for expanding and differentiating V62 " TCRY6 + T cells from a sample containing TCRY5 + T cells or precursors thereof, comprising:
• step (1 ) (2) culturing the cells obtained in step (1 ) in a second culture medium comprising a T cell mitogen and at least one growth factor having interleukin-15-like activity, in the absence of growth factors having interleukin-4-like activity.
• the method of the first aspect of the invention results in expanded cell populations of V62 TCRY6 + T cells.
• expanded it is meant that the number of the desired or target cell type in the final preparation is higher than the number in the initial or starting cell population.
• a T cell mitogen means any agent that can stimulate T cells through 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 OKT-3 and UCHT-1 clones, anti-TCRY6 antibodies such as B1 and IMMU510, or anti-TCRV51 antibodies such as 5TCS1.
• 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.
• the antibody is an anti-CD3 monoclonal antibody (mAb).
• Other mitogens include phorbol 12-myristate-13-acetate (TPA) and its related compounds, such as mezerein, or bacterial compounds (e.g., Staphylococcal enterotoxin A (SEA) and Streptococcal protein A).
• T cell mitogen may be soluble or immobilized and more than one T cell mitogen may be used in the method of the invention.
• a growth factor having interleukin-4-like activity means any compound that has the same activity as IL-4 with respect to its ability to promote similar physiological effects on V52 ⁇ T cells in culture and includes, but is not limited to, IL-4 and IL-4 mimetics, or any functional equivalent of IL-4.
• the physiological effects promoted by IL-4 on V62 ⁇ T cells include the decrease of NKG2D and NCR expression levels, the inhibition of cytotoxic function and improved selective survival.
• Some of the referred activities of IL-4 on V52 ⁇ T cells were also reported independently by another group. In that study, IL-4 significantly inhibited the secretion of pro-inflammatory cytokines, including IFN- ⁇ , TNF-a, from activated TCRY5 + T cells. 45
• a growth factor having interleukin-15-like activity means any compound that has the same activity as IL-15 with respect to its ability to promote similar physiological effects on ⁇ 2 " ⁇ T cells in culture and includes, but is not limited to, IL-15 and IL-15 mimetics, or any functional equivalent of IL-15, including IL-2 and IL-7.
• the physiological effects promoted by IL-15, IL-2 and IL-7 on cultured V62 ⁇ T cells were essentially equivalent, namely, the induction of cell differentiation towards a more cytotoxic phenotype, including the upregulation of NKG2D and NCR (NKp30 and NKp44) expression levels, increased anti-tumor cytotoxic function and increased production of pro-inflammatory cytokines, such as IFN- ⁇ .
• interleukin-15, interleukin-2 and interleukin-7 refer not only to the complete absence of these cytokines in the culture medium, but also include the use of such cytokines at concentration levels so low that they cannot produce a measurable response or physiological effect in target cells and thus can be considered absent for practical purposes.
• a measurable physiological effect in target cells refers to any measurable change in the cells' physiological state, within the scope of the present invention and according to standard definitions.
• changes in the cell's physiological state can be detected by changes in their activation state (recognized by the up-regulation or downregulation of the expression levels of the early-activation cell marker CD69); or detected by changes in their differentiation state (recognized by the up-regulation or downregulation of NKG2D or NCRs), a few hours or a few days after contact with such cytokines.
• a measurable physiological effect may also be a change in the cell's proliferation rate, as measured by CFSE staining or by other techniques known in the art. It should be apparent for any one skilled in the art that cells cultured in the first culture medium must not receive a functionally relevant stimulus by IL-2, IL-7 and IL-15 or functionally similar growth factors.
• cells in the second culture medium must not receive a functionally relevant stimulus by IL-4 or functionally similar growth factors.
• these cytokines must not be present in the cell culture medium at a final concentration higher than 2ng/ml; more preferably, not higher than 1 ng/ml, more preferably not higher than 0,1 ng/ml, more preferably, they should be absent.
• the present invention provides a method for expanding and differentiating V52 ⁇ T cells in a sample comprising:
• step (1 ) (2) culturing the cells obtained in step (1 ) in a second culture medium comprising a T cell mitogen and interleukin-15, in the absence of interleukin-4.
• the present invention provides a method for expanding and differentiating V62 ⁇ T cells in a sample comprising:
• step (1 ) (2) culturing the cells obtained in step (1 ) in a second culture medium comprising a T cell mitogen and interleukin-2, in the absence of interleukin-4.
• the present invention provides a method for expanding and differentiating V62 ⁇ T cells in a sample comprising:
• the first or second culture medium, or both culture media may additionally include other ingredients that can assist in the growth and expansion of the V62 ⁇ T cells.
• other ingredients that may be added include, but are not limited to, plasma or serum, purified proteins such as albumin, a lipid source such as low density lipoprotein (LDL), vitamins, amino acids, steroids and any other supplements supporting or promoting cell growth and/or survival.
• the first or second culture medium, or both culture media may also contain other growth factors, including cytokines that can further enhance the expansion of V62 ⁇ T cells.
• cytokines include, but are not limited to: (i) lnterferon- ⁇ and any growth factor having interferon-Y-like activity, (ii) interleukin-21 and any growth factor having interleukin-21 -like activity and (Mi) IL-1 ⁇ and any growth factor having interleukin-1 ⁇ -like activity.
• growth factors that can be added include co-stimulatory molecules such as a human anti-SLAM antibody, any soluble ligand of CD27, or any soluble ligand of CD7. Any combination of these growth factors can be included in the first or second culture medium, or in both media.
• a growth factor having interferon-Y-like activity means any compound that has the same activity as IFN- ⁇ with respect to its ability to promote survival or proliferation of V62 ⁇ T cells in culture and includes, but is not limited to, IFN- ⁇ and IFN- ⁇ mimetics, or any functional equivalent of IFN- ⁇ .
• a growth factor having interleukin-21 -like activity means any compound that has the same activity as IL-21 with respect to its ability to promote survival or proliferation of V62 ⁇ T cells in culture and includes, but is not limited to, IL-21 and IL-21 mimetics, or any functional equivalent of IL-21.
• a growth factor having interleukin-1 ⁇ -like activity means any compound that has the same activity as IL-1 ⁇ with respect to its ability to promote survival or proliferation of V62 ⁇ T cells in culture and includes, but is not limited to, IL-1 ⁇ and IL-1 ⁇ mimetics, or any functional equivalent of IL-1 ⁇ .
• the inventor has found that the addition of a second growth factor having interferon-Y-like activity to the first or second culture medium, or to both culture media, resulted in enhanced expansion of V62 ⁇ T cells as compared to the expansion obtained using one growth factor.
• the method of the first aspect of the invention comprises:
• culturing cells in the sample in a first culture medium comprising a T cell mitogen, interleukin-4 and interferon- ⁇ , in the absence of interleukin-2, interleukin-7 and interleukin-15;
• the growth factor having IFN-Y-like activity is present in an amount from about 1 to about 1000 ng/ml. More preferably, this growth factor is present in an amount from about 2 to about 500 ng/ml. More preferably, this growth factor is present in an amount from about 20 to about 200 ng/ml.
• the second culture medium comprises about 70 ng/mL of a growth factor having IFN-Y-like activity, such as IFN- ⁇ .
• the inventor has also found that the addition of a second growth factor having IFN-Y-like- activity and a third growth factor having IL-21 -like-activity to the first or second culture medium, or to both culture media, resulted in enhanced expansion of V52 " ⁇ T cells as compared to the expansion obtained using one or two growth factors.
• the method of the first aspect of the invention comprises:
• culturing cells in the sample in a first culture medium comprising a T cell mitogen, interleukin-4, interferon- ⁇ and interleukin-21 , in the absence of interleukin-2, interleukin-7 and interleukin-15;
• step (1 ) (2) culturing the cells obtained in step (1 ) in a second culture medium comprising a T cell mitogen, interleukin-15 and interferon- ⁇ , in the absence of interleukin-4, to expand and differentiate V62 ⁇ T cells.
• a second culture medium comprising a T cell mitogen, interleukin-15 and interferon- ⁇ , in the absence of interleukin-4, to expand and differentiate V62 ⁇ T cells.
• the growth factor having IL-21 -like-activity is present in an amount from about 1 to about 500 ng/ml. More preferably, this growth factor is present in an amount from about 2 to about 200 ng/ml. More preferably, this growth factor is present in an amount from about 5 to about 100 ng/ml. Most preferably, the second culture medium comprises about 15 ng/mL of a growth factor having IL-21 -like-activity, such as IL-21.
• the inventor has also found that the addition of a second growth factor having IFN-Y-like- activity, and a third growth factor having IL-21 -like-activity, and a fourth growth factor having IL-1 ⁇ -like activity, to the first or second culture medium, or to both culture media, resulted in enhanced expansion of the ⁇ / ⁇ 2 " ⁇ T cells as compared to the expansion obtained using one, two or three growth factors.
• the method of the first aspect of the invention comprises:
• culturing cells in the sample in a first culture medium comprising a T cell mitogen, interleukin-4, interferon- ⁇ , interleukin-21 and interleukin-1 ⁇ , in the absence of interleukin-2, interleukin-7 and interleukin-15; and
• step (1 ) (2) culturing the cells obtained in step (1 ) in a second culture medium comprising a T cell mitogen, interleukin-15, interferon- ⁇ and interleukin-21 , in the absence of IL-4, to expand V62 ⁇ T cells.
• a second culture medium comprising a T cell mitogen, interleukin-15, interferon- ⁇ and interleukin-21 , in the absence of IL-4, to expand V62 ⁇ T cells.
• the growth factor having IL-1 ⁇ -like-activity is present in an amount from about 1 to about 500 ng/ml. More preferably, this growth factor is present in an amount from about 2 to about 200 ng/ml. More preferably, this growth factor is present in an amount from about 5 to about 100 ng/ml.
• the second culture medium comprises about 15 ng/mL of a growth factor having IL-1 ⁇ -like-activity, such as IL-1 ⁇ .
• the inventor has also found that the addition of a co-stimulatory molecule to the first or second culture medium, or to both culture media, resulted in enhanced expansion of the V52 " ⁇ T cells as compared to the expansion obtained without using such molecule.
• the method of the first aspect of the invention comprises:
• culturing cells in the sample in a first culture medium comprising a T cell mitogen, interleukin-4, and any molecular ligand of CD27, or any molecular ligand of SLAM or any molecular ligand of CD7 receptors, in the absence of interleukin-2, interleukin-7 and interleukin-15; and
• step (1 ) (2) culturing the cells obtained in step (1 ) in a second culture medium comprising a T cell mitogen and interleukin-15, in the absence of IL-4, to expand V62 ⁇ T cells.
• a molecular ligand means any molecule or compound that binds to a specific target receptor.
• a soluble ligand of CD27 or a soluble ligand of CD7 or a soluble ligand of SLAM resulted in enhanced expansion of V62 ⁇ T cells.
• More than one subculture step can be performed during the total culture period.
• each of the previously described subculture steps can be further divided into two subculture steps (1 a) and (1 b) and (2a) and (2b), and different combinations of ingredients can be used according to the originally described method.
• the method of the first aspect of the invention comprises:
• culturing cells in the sample in a first culture medium comprising a T cell mitogen, interleukin-4, interferon- ⁇ , interleukin-1 ⁇ and interleukin-21 , in the absence of interleukin-2, interleukin-7 and interleukin-15;
• step (2a) culturing the cells obtained in step (1 ) in a second culture medium comprising a T cell mitogen, interleukin-15 and interleukin-21 , in the absence of interleukin-4; and (2b) culturing the cells obtained in step (2a) in a third culture medium comprising a T cell mitogen, interleukin-15 and interferon- ⁇ , in the absence of interleukin-4, to expand V62 ⁇ T cells.
• the method of the first aspect of the invention comprises:
• step (1 b) culturing the cells obtained in step (1 a) in a second culture medium comprising a T cell mitogen, interleukin-4, interferon- ⁇ , and interleukin-1 ⁇ , in the absence of interleukin-2, interleukin-7 and interleukin-15; and (2) culturing the cells obtained in step (1 b) in a third culture medium comprising a T cell mitogen and interleukin-15, in the absence of interleukin-4; to expand V62 ⁇ T cells.
• the TCRy5 + T cells obtained by the method of the invention can be used in a variety of experimental, therapeutic and commercial applications. This includes, but is not limited to, subsequent genetic modification or genetic editing of such cells, for example with the objective of improving their therapeutic potential. For example, with the objective of redirecting the specificity of the TCRy5 + T cells through the expression of a chimeric antigen receptor (CAR) or TCR on these cells. CAR expression can be induced through electroporation of TCRv5 + cells for the insertion of genetic material, or by infecting these cells with viral vectors, such as lentiviruses or retroviruses containing the desired genetic material. Such genetic editing may improve the potency of the TCRv5 + T cells by improving homing, cytokine production, recycle killing, and/or improved engraftment.
• CAR chimeric antigen receptor
• the present invention provides novel methods for selectively expanding V52 TCRv5 + T cells in culture.
• the methods of the first aspect of the invention are carried out on a sample, which is also referred to herein as a "starting sample".
• the methods can use either unfractionated samples or samples which have been enriched for TCRv5 + T cells.
• the sample can be any sample that contains TCRv6 + T cells or precursors thereof including, but not limited to, blood, bone marrow, lymphoid tissue, epithelia, thymus, liver, spleen, cancerous tissues, lymph node tissue, infected tissue, fetal tissue and fractions or enriched portions thereof.
• 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).
• PBMCs peripheral blood mononuclear cells
• LDMCs low density mononuclear cells
• the sample is human blood or a fraction thereof.
• the cells may be obtained from a sample of blood using techniques known in the art such as density gradient centrifugation.
• whole blood may be layered onto an equal volume of Ficoll- HypaqueTM followed by centrifugation at 400xg for 15-30 minutes at room temperature.
• the interface material will contain low density mononuclear cells which can be collected and washed in culture medium and centrifuged at 200xg for 10 minutes at room temperature.
• Isolated or unpurified TCRv5 + T cells can be cultured or maintained in any suitable mammalian cell culture medium such as AIM-VTM, RPMI 1640, OPTMIZER CTSTM (Gibco, Life Technologies), EXVIVO-10, EXVIVO-15 or EXVIVO-20 (Lonza), in the presence of serum or plasma.
• Cells can be transferred, for example, to VueLife ® clinical-grade gas- permeable cell culture static bags (Saint Gobain) or to Miltenyi Biotec's clinical-grade cell culture bags. Cell bags containing cells and culture medium can be placed in an incubator at 37 Q C and 5% CO2, in the dark.
• the sample or fraction thereof Prior to culturing the sample or fraction thereof (such as PBMCs) in the first culture medium, the sample or fraction thereof may be enriched for certain cell types and/or depleted for other cell types.
• the sample or fraction thereof may be enriched for T cells, or enriched for TCRY6 + T cells, or depleted of TCRa + T cells or depleted of non-TCRv5 + T cells.
• the sample is first depleted of TCRa + T cells and then enriched for CD3 + cells.
• the sample may be enriched or depleted of certain cell types using techniques known in the art.
• the cells of a particular phenotype may be depleted by culturing the sample or fraction thereof with an antibody cocktail containing antibodies that bind to specific molecules on the cells to be depleted.
• the antibodies in the cocktail are coupled to magnetic microbeads that can be used to magnetically deplete or enrich target cells when these cells are forced to pass through a magnetic column.
• the cells in the sample have been fractionated and enriched, if desired, the cells are cultured in a first culture medium comprising a T cell mitogen and at least one growth factor having interleukin-4-like activity, such as interleukin-4, in the absence of growth factors having interleukin-15-like activity, such as interleukin-15, interleukin-2 and interleukin-7.
• a first culture medium comprising a T cell mitogen and at least one growth factor having interleukin-4-like activity, such as interleukin-4, in the absence of growth factors having interleukin-15-like activity, such as interleukin-15, interleukin-2 and interleukin-7.
• the T cell mitogen in the first culture medium is present in an amount from about 10 to about 5000 ng/ml. More preferably, the T cell mitogen is present in an amount from about 20 to about 2000 ng/ml. More preferably, the T cell mitogen is present in an amount from about 50 to about 1000 ng/ml. Most preferably, the medium comprises 70 ng/mL of a T cell mitogen.
• the growth factor having interleukin-4-like activity is present in an amount from about 1 to about 1000 ng/ml. More preferably, this growth factor is present in an amount from about 5 to about 500 ng/ml. More preferably, this growth factor is present in an amount from about 20 to about 200 ng/ml. Most preferably, the medium comprises 100 ng/mL of a growth factor having interleukin-4-like activity, such as IL-4.
• the cells are preferably cultured in the first culture medium for a period of time ranging from about 2 days to about 21 days. More preferably, from about 3 days to about 14 days. More preferably, from about 4 days to 8 days.
• cells are sub-cultured in a second culture medium comprising a T cell mitogen and at least one growth factor having interleukin-15-like activity, such as IL-15, IL-2 or IL-7, in the absence of growth factors having interleukin-4-like activity, such as IL-4.
• a second culture medium comprising a T cell mitogen and at least one growth factor having interleukin-15-like activity, such as IL-15, IL-2 or IL-7, in the absence of growth factors having interleukin-4-like activity, such as IL-4.
• the subculture step consists in culturing the cells obtained in step 1 in a new culture medium. This can be achieved through the addition of fresh culture medium to the first culture medium, preferably after the removal of a fraction of the first culture medium. This can be done by centrifuging and/or decanting the cells, removing a fraction of the first culture medium and resuspending the cells in the second culture medium. Preferably, the subculture step involves the removal of at least 3/4 of the first culture medium.
• the subculture step should be carried out because it is important for the expansion and differentiation of V62 ⁇ T cells that the growth factor having interleukin-4-like activity is removed during the subculture step.
• the T cell mitogen is present in an amount from about 0,1 to about 50 ⁇ 9/ ⁇ . More preferably, the T cell mitogen is present in an amount from about 0,3 to about 10 ⁇ 9 ⁇ . More preferably, the T cell mitogen is present in an amount from about 0,5 to about 5 ⁇ 9 ⁇ . Most preferably, the medium comprises 1 ⁇ g/mL of a T cell mitogen.
• the growth factor having interleukin-15-like activity is present in an amount from about 1 to about 1000 ng/ml. More preferably, this growth factor is present in an amount from about 2 to about 500 ng/ml. More preferably, this growth factor is present in an amount from about 20 to about 200 ng/ml. Most preferably, the second culture medium comprises about 70 ng/mL of a growth factor having interleukin-15-like activity, such as IL-15, IL-2 or IL-7.
• the cells are preferably cultured in the second culture medium for a period of time ranging from about 2 days to about 30 days. More preferably, from about 5 days to about 21 days. More preferably, from about 10 days to 15 days.
• both the first and second culture media are supplemented with serum or plasma.
• the amount of plasma in the first and second culture media is preferably from about 0,5% to about 25% by volume, for example from about 2% to about 20% by volume or from about 2,5% to about 10% by volume, for example is about 5% by volume.
• the serum or plasma can be obtained from any source including, but not limited to, human peripheral blood, umbilical cord blood, or blood derived from another mammalian species.
• the plasma may be from a single donor or may be pooled from several donors. If autologous ⁇ + T cells are to be used clinically, i.e.
• autologous plasma i.e. from the same patient
• hazardous products e.g. viruses
• the plasma should be human-derived to avoid the administration of animal products to the patient.
• the ⁇ + T cells obtained according to the method of the first aspect of the invention 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 cellular panning.
• TCRy6 + T cells obtained by the method of the first aspect of the invention are also of use. Accordingly, the inventor describes a cell preparation of ⁇ + T cells.
• the present invention provides a cell preparation enriched in TCRy5 + T cells prepared according to the method of the first aspect of the invention.
• the present invention provides a cell preparation enriched in TCRv6 + T cells wherein greater than 80% of the total cells are TCRv6 + T cells.
• the TCRv5 + T cells comprise greater than 80%, more preferably greater than 90% and most preferably greater than 95%, of the total cells in the enriched population.
• TCRv5 + T cells obtained by the method of the first and second aspect of the invention may be used in any and all applications.
• TCRv5 + T cells are thought to be a first line of defense against infectious pathogens.
• TCRv5 + T cells possess intrinsic cytolytic activity against transformed cells of various origins including B-cell lymphomas, sarcomas and carcinomas.
• the TCRv5 + T cells obtained and cultured ex vivo according to the methods of the invention can be transfused into a patient for the treatment or prevention of infections, cancer or diseases resulting from immunosuppression.
• the present invention provides a method of modulating an immune response comprising administering an effective amount of TCRv6 + T cells prepared according to a method of the first or second aspect of the invention or obtained from a cell preparation according to the second or third aspect of the invention to an animal in need thereof.
• effective amount means an amount effective, at dosages and for periods of time necessary to achieve the desired results.
• animal as used herein includes all members of the animal kingdom. Preferably, the animal is a mammal, more preferably a human.
• the present invention provides a method of treating an infection comprising administering an effective amount of TCRv5 + T cells prepared according to the method of the first or second aspect of the invention or obtained from a cell preparation according to the second or third aspect of the invention to an animal in need thereof.
• infections examples include, but are not limited to, bacterial infections such as those caused by Mycobacteria (e.g. tuberculosis), viral infections such as those caused by herpes simplex virus (HSV), human immunodeficiency virus (HIV) or the hepatitis viruses, and parasitic infections such as those caused by Plasmodium (e.g. malaria).
• bacterial infections such as those caused by Mycobacteria (e.g. tuberculosis)
• viral infections such as those caused by herpes simplex virus (HSV), human immunodeficiency virus (HIV) or the hepatitis viruses
• parasitic infections such as those caused by Plasmodium (e.g. malaria).
• the present invention provides a method for treating cancer comprising administering an effective amount of TCRv5 + T cells prepared according to the method of the first or second aspect of the invention or obtained from a cell preparation according to the second or third aspect of the invention to an animal in need thereof.
• cancers include, but are not limited to, leukemias including chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia, and T cell and B cell leukemias, lymphomas (Hodgkin's and non-Hodgkins), lymphoproliferative disorders, plasmacytomas, histiocytomas, melanomas, adenomas, sarcomas, carcinomas of solid tissues, hypoxic tumors, squamous cell carcinomas, genitourinary cancers such as cervical and bladder cancer, hematopoietic cancers, head and neck cancers, and nervous system cancers.
• leukemias including chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia, and T cell and B cell leukemias
• lymphomas Hodgkin's and non-Hodgkins
• the cancer to be treated is chronic lymphocytic leukemia.
• PBMCs can be obtained from a patient with chronic lymphocytic leukemia (CLL). After culturing and expanding for TCRY5 + T cells, the expanded cells will not significantly contain cancerous CLL cells making them well suited for re-infusion back to the patient.
• CLL chronic lymphocytic leukemia
• aspects of the invention also extend to the TCRY5 + T cells obtained by a method of the first or second aspect of the invention or obtained from a cell preparation according to the second or third aspect of the invention for use in a method of modulating an immune response, treating an infection or treating cancer as described herein above.
• the invention further includes the use of the TCRY6 + T cells obtained according to methods of the first or second aspect of the invention in the manufacture of a medicament or pharmaceutical composition to modulate an immune response, to treat an infection or to treat cancer as described hereinabove.
• the TCRY5 + T cells obtained according to the present invention can also be used in experimental models, for example, to further study and elucidate the function of the cells. Additionally, these cells may be used for studies directed towards the identification of the antigens/epitopes recognized by TCRY6 + T cells and for the design and development of vaccines.
• the present invention provides a method for vaccinating an animal comprising administering an effective amount of TCRY5 + T cells obtained by a method of the first aspect of the invention or obtained from a cell preparation according to the second or third aspect of the invention to an animal in need thereof.
• Such vaccine can be given to immunocompromised patients or individuals with elevated risk of developing an infectious disease or cancer.
• This aspect of the invention also extends to the use of TCRY5 + T cells prepared according to the first aspect of the invention or obtained from a cell preparation according to the second or third aspect of the invention for the manufacture of a vaccine, and to TCRy5 + T cells prepared according to the method of the first aspect of the invention or obtained from a cell preparation according to the second or third aspect of the invention for use in a method of vaccinating an animal.
• the obtained ⁇ + T cells, according to the invention may be immediately used in the above therapeutic, experimental or commercial applications or the cells may be cryopreserved for use at a later date.
• Patent Application n Q PCT/CA1999/001024 (WO/2000/026347; Filing Date of 04.1 1.1999), describes a method for the production of ⁇ + T cells.
• the method involves two steps, wherein ⁇ + T cells in the starting sample are cultured in a first culture medium comprising a T cell mitogen and at least two cytokines, preferably interleukin-2 and interleukin-4. Cells obtained in the first step are then cultured in a second culture medium comprising at least two cytokines, which are preferably interleukin-2 and interleukin-4.
• cytokines used in each step can be the same or different.
• the present invention discloses a 2-step method for selectively expanding and differentiating V62 ⁇ T cells in culture, wherein the first and second culture steps are necessarily different from each other.
• the first step comprises culturing TCRy5 + T cells in the starting sample in a culture medium comprising a T cell mitogen and a growth factor having IL-4-like activity, preferably interleukin-4, in the absence of interleukin-2, interleukin-7 and interleukin-15.
• IL-4 and IL-2 or IL-7 or IL-15
• the first culture medium contains a growth factor having IL-4-like activity, which cannot be mixed with (IL-2 /IL-7 /IL-15-like) growth factors, otherwise cell viability and proliferation will decrease and the expected cell product will not be produced.
• the first cell culture step is used to expand ⁇ + T cells many fold in an exceptional short period of time, generating a cell product highly enriched in V62 ⁇ T cells.
• the absence of IL- 2/IL-7-IL15 -like growth factors in the culture medium allows the elimination of many impurities (other cell types) that would not be eliminated without this innovation.
• the expanded ⁇ 2 " ⁇ T cells are in a less differentiated state since they have not contacted interleukin-2 /-15 /-7-like growth factors.
• V62 ⁇ T cells obtained in the first step must then be cultured in a second culture medium comprising 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. Again, it is critical that these different growth factors are not mixed, since the presence of IL-4 in the second culture medium decreased cell viability and cytotoxic function.
• the second culture step further expands V62 ⁇ T cells in culture and makes them better effector cells, generating a distinctive cell product that can be used for many therapeutic purposes.
• Patent n°- US2003/0157060 A1 (Application n°- US10239854); and Filing Date of 03.04.2000)
• ⁇ ⁇ ⁇ + T cells in the starting sample are cultured in a first culture medium comprising (1 ) a T cell mitogen, (2) a growth factor having interleukin-2-like activity and (3) a growth factor having interleukin-7-like activity.
• Cells obtained in the first step are then cultured in a second culture medium comprising (1 ) a growth factor having interleukin-2-like activity and a (2) growth factor having interleukin-7-like activity to expand ⁇ ⁇ + T cells.
• the present invention is different since the two culture steps are necessarily different, as they serve distinct purposes.
• the invention clearly excludes the use of growth factors having interleukin-2-like activity or interleukin-7-like activity in the first culture step, due to reasons already explained (see above).
• the present invention describes the presence of other growth factors in the first and/or second culture medium, including cytokines such as lnterferon- ⁇ , IL-1 ⁇ and IL-21 , these cytokines cannot be considered to have interleukin-2-like or interleukin-7-like activity since the physiological effects that they exert on V62 ⁇ T cells are very different.
• lnterferon- ⁇ , IL-1 ⁇ and IL-21 in the absence of IL-2, IL-7 and IL-15
• IL-2, IL-7 and IL-15 could not acquire their full cytotoxic function and differentiated state
• lnterferon- ⁇ , IL-1 ⁇ and IL-21 could not mimic the high pro-inflammatory effects induced by IL-2, IL-7 and IL-15 on cultured V62 ⁇ T cells (Table 2).
• some of these cytokines such as lnterferon- ⁇ and IL-1 ⁇ , belong to a structurally different family of cytokines.
• Figure 1 shows percentages of TCF?Y6 + T PBLS in a peripheral blood sample collected from a healthy donor, before and after magnetic activated cell sorting (MACS).
• 50-150ml of fresh peripheral blood was obtained from a healthy volunteer and diluted in a 1 :1 ratio (volume-to-volume) with PBS (Invitrogen Gibco) and centrifuged in Ficoll-Paque (Histopaque-1077; Sigma-Aldrich) in a volume ratio of 1 :3 (1 part ficoll to 3 parts of diluted blood) for 35 minutes at 1.500 rpm and 25°C.
• the interphase containing mononuclear cells (PBMCs) was collected and washed (in PBS).
• Total TCRy6 + T cells were labeled with an anti-TCRv6 mAb conjugated with magnetic microbeads and TCRy6 + T cells were isolated (to above 75% purity) with a magnetic column (Miltenyi Biotec, German).
• Cells were labeled with the following fluorescent monoclonal antibodies: anti-CD3-PerCP-Cy5.5 (Biolegend; clone SK7); anti- TCR-V51 -APC (Miltenyi Biotec, clone REA173); anti- Mouse IgGl K-APC Isotype Ctrl (Miltenyi Biotec; clone IS5-21 F5).
• Cells were analyzed on a Fortessa II flow cytometer (BD Biosciences). Shows representative results of 6 independent experiments.
• FIG. 2 shows percentages of TCRy6 + T PBLs in a peripheral blood sample collected from the previously selected healthy donor, before and after the 2-steps MACS sorting procedure.
• 50-150ml of fresh peripheral blood was obtained from healthy volunteers and diluted in a 1 :1 ratio (volume-to-volume) with PBS (Invitrogen Gibco) and centrifuged in Ficoll-Paque (Histopaque-1077; Sigma-Aldrich) in a volume ratio of 1 :3 (1 part ficoll to 3 parts diluted blood) for 35 minutes at 1.500 rpm and 25°C.
• the interphase containing mononuclear cells (PBMCs) was collected and washed (in PBS).
• Unwanted ⁇ ⁇ + T lymphocytes were then labelled by incubation in the presence of a murine anti-human TCRc ⁇ monoclonal antibody (mAb) conjugated to Biotin (Miltenyi Biotec Ref#701 -48, clone BW242/412). Cells were then labelled again with a murine anti- Biotin mAb coupled to magnetic microbeads (Miltenyi Biotec Ref#173-01 ). Finally, the cell suspension was loaded onto a magnetic column (Miltenyi Biotec, Germany) and TCRa + T lymphocytes were magnetically depleted (and discarded).
• mAb murine anti-human TCRc ⁇ monoclonal antibody conjugated to Biotin
• CD3 + cells (of which most of them were TCRY6 + T cells) present in the remaining cell population were labelled with a murine anti-human CD3 mAb (Miltenyi Biotec Ref#273-01 , clone OKT-3, targeting an epitope located on the CD3epsilon chain), conjugated to superparamagnetic iron dextran particles.
• a murine anti-human CD3 mAb Miltenyi Biotec Ref#273-01 , clone OKT-3, targeting an epitope located on the CD3epsilon chain
• Cells were loaded onto a magnetic MACS separation column and CD3 + cells were positively selected (purified). Cells were stained for TCRy5, CD3, TCRV51 and TCRV52 markers, and analysed on a Fortessa II flow cytometer (BD Biosciences). Shows representative results of 6 independent experiments.
• FIG. 3 shows a summary of tested culture conditions.
• A-C TCRv6 + PBLS from a healthy donor were isolated by MACS as previously described and cultured in 96-well plates at 1 million cells/ml in complete medium (Optmizer CTS, GIBCO) supplemented with 5% human serum, 1 mM L-glutamine, at 37 S C and 5% CO2. Cells were equally distributed by multiple wells and cultured in the presence of three fixed combinations of anti-CD3 mAb and IL-4, further supplemented with various concentrations of IL-2, IL-7, IL-15 or IFN- ⁇ . Fresh medium supplemented with the same growth factors was added every 5-6 days.
• FIG. 4 shows the percentages of V51 + T PBLs before and after in vitro culture for 15 days.
• TCRv5 + PBLs from a healthy donor were isolated by MACS as previously described and cultured in the presence of 200ng/ml IFN- ⁇ , 1 ug/ml a-CD3 and 100ng/ml IL-4.
• a fraction of fresh medium containing the same combination of growth factors was added every 5-6 days. Shows the FACS-plot analysis at day 15. Representative results of 3 independent experiments.
• FIG. 5 shows a panel of tested cell culture media.
• TCRy6 + T PBLS from a healthy donor were isolated by MACS as previously described and cultured in different commercially available serum-free, clinical grade cell culture media.
• Cells were cultured in a first culture medium in the presence of 70ng/ml IFN- ⁇ , ⁇ g/ml a-CD3 and 100ng/ml IL-4, followed by culture in a second culture medium in the presence of 100ng/ml IL-15 and 2 ⁇ g/ml anti-CD3 mAb, (in the absence of IL-4). Shows final number of cells and percentages of cells after FACS analysis at day 15. Shows average ⁇ SD of 3 technical replicates.
• FIG. 6 shows that V61 + T cells expand in vitro to become the dominant cell subset in culture.
• 70ml of concentrated peripheral blood (corresponding to 450ml of peripheral blood) was obtained from 8 Buffy Coat units collected from 8 healthy donors. Blood was centrifuged undiluted in Ficoll-Paque (Histopaque-1077; Sigma-Aldrich) in a volume ratio of 1 :3 (1 part ficoll to 3 parts of blood) for 35 minutes at 1.600 rpm and 25 . The interphase containing mononuclear cells (PBMCs) was collected and washed (in saline buffer).
• PBMCs mononuclear cells
• TCRv5 + PBLs were isolated by the previously described 2-step MACS and resuspended in serum-free culture medium (OPTMIZER, GIBCO) supplemented with 5% autologous plasma and 1 mM L-glutamine. Cells were seeded at a concentration of 0,5x10 6 cells/ml and expanded in closed gas-permeable, 1 L cell culture plastic bags in the incubator at 37 a C and 5% CO2.
• Growth factors were added to the cell culture media according to the previously described 2-step protocol: 70ng/ml anti-CD3 mAb, 100ng/ml IL-4, 70ng/ml IFN- ⁇ , 7ng/ml IL-21 and 15ng/ml IL- 1 ⁇ were added to the first culture medium and 70ng/ml IL-15, 100ng/ml IFN- ⁇ , 15ng/ml IL-21 and ⁇ g/ml anti- CD3 mAb were added to the second culture medium. A fraction of old medium was removed and fresh medium was added every 5-6 days, supplemented with growth factors. Accessory "feeder" cells were not required in this system. Left panel shows percentages of CD3 + V51 + cells among total live cells as analyzed by flow cytometry. Right panel shows fold increase in the absolute number of CD3 + V51 + T cells relative to the initial cell number. Live cells were counted using Trypan Blue-positive exclusion in a haemocyto meter. Shows an average of 3 measurements of number of cells of
• Figure 7 shows the expression of several cell surface markers in in vitro expanded V51 + T cells.
• Figure 8 shows that in vitro expanded TCRY5 + T cells are cytotoxic against CLL cells but not against autologous PBMCs. After 21 days of expansion and differentiation (as described in Figure 6), the resulting TCRv5 + T cells were co-incubated with target cells (pre-labelled with DDAO-SE) for 3h at 37 a C, and target cell death was assessed by Annexin V staining.
• A Flow cytometry analysis of susceptible MEC-1 CLL cells (upper panel) and non-susceptible autologous PBMCs (lower panel). Representative plots of 3 technical replicates.
• B Impact of different target:effector cell ratios and of blocking antibodies against NKG2D or NKp30 on MEC-1 leukemia cell killing.
• the MEC-1 CLL cell line 46 was obtained from the German Resource Center for Biologic Material (DSMZ). MEC-1 tumor cells were cultured in T25 flasks in complete 10% RPMI 1640 with 10% Fetal Bovine Serum, 2mM L-Glutamine and maintained at 10 5 up to 10 6 cells/mL by dilution and splitting in a 1 :3 ratio every 3-4 days. For cytotoxicity assays, in vitro expanded TCRy6 + T cells were plated in 96-well round-bottom plates.
• Tumor cell lines or leukemia primary samples were stained with CellTrace Far Red DDAO-SE (1 ⁇ ; Molecular Probes, Invitrogen) and incubated at the indicated target:effector ratio with TCRv5 + T cells in RPMI 1640 medium for 3 hours at 37°C and 5% CO2, in the presence of 70ng/ml IL-15. All cells were then stained with Annexin V-FITC (BD Biosciences) and analyzed by flow cytometry.
• FIG 9 shows that activated TCRv6 + T cells produce high levels of IFN- ⁇ .
• TCRv5 + T cells were produced from two healthy donors in cell culture bags for 21 days, following the 2-step culture protocol, as previously described. Cells were washed, plated in 96-well plate and re-stimulated with fresh medium supplemented with 100ng/ml IL-15 and 2 ⁇ g/ml anti-CD3 mAb. After 48h, cell culture supernatants were analyzed by flow cytometry by Cytometric bead array (BD Biosciences).
• FIG. 10 shows that TCRv6 + T cells from CLL/SLL patients expand robustly in vitro and are highly cytotoxic against CLL/SLL cells and CMV-infected cells.
• MACS-sorted TCRv5 + PBLs from three CLL/SLL patients were cultured for 21 days with cytokines and mAb as previously described.
• Left panel Cells were analysed by flow cytometry for TCRV61/CD3 co-expression and fold increase of CD3 + V 1 + T cells was calculated. Live cells were identified by flow cytometry with a viability dye (Zombie Violet; Biolegend) and counted with Trypan Blue in a haemocytometer.
• a viability dye Zombie Violet; Biolegend
• FIG 11 shows that in vitro expanded TCRy6 + T cells are cytotoxic against cancer cells of diverse tissue origins.
• Peripheral blood TCRv5 + T lymphocytes from one healthy donor were MACS-sorted and stimulated ex vivo in the presence of cytokines and anti-CD3 mAb for 21 days, according to the described two- step culture protocol.
• MOLT-4 acute lymphoblastic leukemia
• HL-60 acute myeloid leukemia; AML
• K562 Choronic myeloid leukemia; CML
• MEC-1 Choronic lymphocytic leukemia; CLL
• HELA Cervical carcinoma
• Daudi Baudi (Burkitt's lymphoma)
• THP-1 Acute monocytic leukemia; AMoL
• MDA-231 Breast carcinoma
• PA-1 Ovarian carcinoma
• PC3 Prostate carcinoma
• HCT116 Cold carcinoma
• Figure 12 shows that infused TCRY5 + T cells home and survive in xenograft tumor models.
• TCRY5 + T cells were transferred into tumor-bearing (MEC-1 CLL ⁇ SLL cell-line) NSG immunodeficient hosts. 30 days after transfer of TCRY6 + T cells, animals were sacrificed and TCRy6 + T cell -progeny was evaluated by FACS in the indicated tissues. Note that TCRv6 + T cells were present in all tissues analyzed and also that CD3 + V61 + T cells were highly enriched, suggesting preferential fitness and/or activation in presence of CLL tumor. Dot-Plots are from a representative animal of 6 animals analyzed.
• B. Infused TCRy5 + T cells express NCRs in vivo. TCRv5 + T cells recovered from the liver at day 30 after transfer were analyzed by FACS for NCR expression.
• NKp30 and NKG2D Top dot plots are isotype controls. A representative animal of 3 animals analyzed is shown.
• C Infused TCRv5 + T cells are able to home into BRG immunodeficient hosts. 10 7 TCRv5 + T cells from a different donor were transferred into CLL tumor-bearing BRG hosts and quantified by FACS 72 hours after transfer. TCRv5 + T cells could be recovered from both the lung and liver and TCRV51 + -expressing cells were found at proportions similar to the initial transfer. One animal out of two is shown.
• FIG. 13 shows that TCF?Y6 + T cells can limit tumour growth in vivo.
• FIG. 14 shows that TCRve + T cells limit CLL tumour spread.
• Plots shown in B depict FACS analysis of organs recovered at the end of the experiment from NSG hosts receiving TCRy5 + T cells transfer or control PBS. We observed a generalized reduction in CLL/SLL tumour cells recovered in different organs but this effect was more pronounced in the liver, a major organ for tumour spreading in this model.
• C histological (H&E) analysis of a representative animal from each group, showing CLL/SLL tumour metastasis in the indicated anatomical sites of PBS treated animals and absence of tumour infiltrates in treated animals. ( * p ⁇ 0.05; ** p ⁇ 0.01 ).D.
• FIG. 15 shows that TCRv6 + T cells can infiltrate primary tumour and are activated in vivo.
• Figure 16 shows biochemical analysis of blood samples.
• Balb/c Rag _ ⁇ _ 47 animals were obtained from Taconic (USA); NOD-SCIDTC' - 48 mice were obtained from the Jackson Laboratories (USA).
• BRG or NSG mice were injected subcutaneously with MEC-1 cells and treated after 6 and 11 days with two intravenous transfers of 10 7 or 2x10 7 DOT cells, and then analyzed (tumor size, histology, flow cytometry of tumor or organ infiltrates, and blood biochemistry) as detailed. All animal procedures were performed in accordance to national guidelines from the Direcao Geral de Veterinaria and approved by the relevant Ethics Committee.
• mice were euthanized using Eutasil in order for blood collection via cardiac puncture; and quickly perfused with PBS + Heparin. Organs were homogenized and washed in 70 ⁇ cell strainers. Femurs were flushed and then filtered. Cells were then stained with the following antibodies from ebioscience, Biolegend, Myltenyi Biotec or Beckton Dickinson: anti-mouse CD45 (30-F11 ), and anti-Human: CD45 (HI30). Other antibodies used are common with the in vitro studies.
• Antibodies were coupled to FITC, PE, PerCP, PerCP-Cy5, PE-Cy7, APC, APC-Cy7, Pacific Blue, Brilliant Violet 421 and Brilliant Violet 510 fluorochromes.
• Statistical analysis was performed using Graphpad-Prism software. Sample means were compared using the unpaired Student's f-test. In case variances of the two samples were found different using F-test, the data was log transformed and if variances were then found not to be different, the unpaired t-test was applied to the log-transformed data. For Survival data, log-rank (Mantel-Cox) test was used.
• tumour load as a function of luminescence, before ascribing treatment (or PBS control) to the animals.
• Animals were distributed randomly in cages and assigned to each treatment (PBS or DOT-Cells) according to luminescence measured at day 7, in such a way that animal with highest luminescence received treatment, second highest received PBS, third highest received treatment, etc. This resulted in a non-randomized distribution into groups but randomized distribution in the different cages.
• 2 additional animals received DOT-Cells in the indicated experiments for initial homing analysis.
• Luminescence Analysis After transduction of MEC-1 Cell line with GFP-firefly luciferase, growing cells were screened and sorted according to GFP expression using a FACS-Aria (Becton Dickinson, USA), up to >95% GFP positive cells. These cells were then kept in culture until transferred subcutaneous into host animals (in 50 ⁇ PBS). At the indicated time points after transfer, animals were anesthetized (Ketamin/Medetomidine) and Luciferin was injected (i.p.). 4 min later luciferase activity was detected and acquired using I VIS Lumina (Calliper LifeSciences) at the IMM bioimaging facilities. Anesthesia was then reverted and animals returned to previous housing.
• FACS-Aria Becton Dickinson, USA
• Lymphocyte counts Cell counts were performed with a hemocytometer or using Accuri Flow cytometer (Becton Dickinson, USA). Counts per organ were estimated when parts of the organ were sampled for histological analysis by weighting organs before and after the samples were split. Numbers presented are then corrected for the whole organ. In Bone Marrow data, absolute numbers were calculated and are displayed for one femur.
• mice were sacrificed with anesthetic overdose, necropsies were performed and selected organs (lung, heart, intestine, spleen, liver, kidney, reproductive tract, brain, cerebellum, spinal cord, and femur) were harvested, fixed in 10% neutral-buffered formalin, embedded in paraffin and 3 ⁇ sections were stained with hematoxylin and eosin (H&E). Bones were further decalcified in Calci-ClearTM (Fisher Scientific) prior to embedding. Tissue sections were examined by a pathologist, blinded to experimental groups, in a Leica DM2500 microscope coupled to a Leica MC170 HD microscope camera.
• Immunohistochemical staining for CD3 was performed by the Histology and Comparative Pathology Laboratory at the IMM, using standard protocols, with a Dako Autostainer Link 48. Antigen heat-retrieval was performed in DAKO PT link with low pH solution (pH 6), and incubation with ENVISION kit (Peroxidase/DAB detection system, DAKO, Santa Barbara, CA) was followed by Harri's hematoxylin counterstaining (Bio Otica, Milan, IT). Negative control included the absence of primary antibodies; and CD3 staining was not observed in the negative controls. Images were acquired in a Leica DM2500 microscope, coupled with a Leica MC170 HD microscope camera.
• Mouse Blood Biochemistry Mice were deeply anaesthetised and blood was collected from the heart to heparin-coated tubes, sent for analysis of biochemical parameters shown at an independent laboratory. Biochemical parameters were measured in serum, in a RX monaco clinical chemistry analyser (RANDOX).
• RANDOX RX monaco clinical chemistry analyser
• Figure 17 shows TCRv6 + PBL enrichment after two-step MACS-sorting.
• TCRY + T cells were isolated by MACS: first, TCRa depletion was performed and then CD3 + cells were positively selected. Cells were stained for TCRY5, CD3 and TCRV51 and analyzed by flow cytometry. Dot plots show fractions of TCRY5 + PBLS before and after each MACS-sorting step (left panels); and initial and final percentages of V51 + T cells (right panels).
• Figure 18 shows the characterization of the activation and maturation phenotype of the obtained V51 + T cells.
• (A) Flow cytometry comparison of the cell surface phenotype of V61 + T cells at day 21 of culture (using the previously described 2-step culture protocol); (full lines) with freshly-isolated V51 + T cells (dotted lines), as analyzed using the LEGENDScreen kit (Biolegend). Shown are histogram overlays for several markers related to lymphocyte activation and differentiation, and markers implicated in adhesion and migration. Cells from one healthy donor are shown.
• Figure 19 shows TCR/ NCR-dependent cytotoxicity of TCRY6 + T cells against leukemic (but not healthy) cells.
• Expanded and differentiated TCRY6 + T cells produced from two healthy donors were tested in different experiments against MEC-1 (CLL) target cells at increasing effector/target ratios (left plot, gray bars) and also in presence of blocking antibodies for (a, anti-) the indicated molecules, either individually (Expt 1 ) or in combinations (Expt 2).
• the highest Effector/Target ratio (10:1 ) was used in blocking experiments and gray bar at this ratio (with IgG isotype antibody) serves as control.
• * and # indicate significant differences relative to IgG isotype control or a-TCRV51 , respectively (Mean+SD; * # p ⁇ 0.05; ** ## p ⁇ 0.01 ; Student's t-test).
• MEC-1 tumor cells were cultured in T25 flasks in complete 10% RPMI 1640 with 10% Fetal Bovine Serum, 2mM L-Glutamine and maintained at 10 5 up to 10 6 cells/mL by dilution and splitting in a 1 :3 ratio every 3-4 days.
• In vitro expanded TCRY6 + T cells were plated in 96-well round-bottom plates. Tumor cells were stained with CellTrace Far Red DDAO-SE (1 ⁇ ; Molecular Probes, Invitrogen) and incubated at the indicated target:effector ratio with TCRY6 + T cells in RPMI 1640 medium for 3 hours at 37°C and 5% CO2, in the presence of 70ng/ml IL-15.
• ⁇ PBLs were pre-incubated for 1 hour with blocking antibodies: human anti-TCRY6 (clone B1 ); human anti-NKG2D (clone 1 D11 ); human anti-CD2 (clone RPA- 2.10); human anti-CD3 (clone OKT-3); human anti-NKp30 (clone P30-15); human anti-NKp44 (clone P44-8), mouse lgG1 ,k (clone MOPC-21 ), mouse lgG2b (clone MPC-11 ), mouse lgG3k (clone MG3-35), all from Biolegend.
• Human anti-CD226 (clone DX11 ) was from BD Biosciences.
• Human anti-V51 TCR (clones TCS-1 or TS8.2) were from Fisher Scientific, and human anti-TCRv5 (clone IMMU510) was from BD Biosciences. The blocking antibodies were maintained in the culture medium during the killing assays.
• Figure 20 shows the expression of activating cell surface NK receptors in in vitro expanded V51 + and V51 ' V62 ' T cells.
• Expanded and differentiated TCRy5 + T cells were produced from healthy donors, using the previously described 2-step culture protocol. Shows expression of the activating receptors NKp30 and NKp44 in CD3 + V51 + cells and in CD3 + V51 ⁇ V52 " T cells after 21 days of culture.
• CD3 + V52 + cells were identified (with anti-CD3 and anti-V52 mAbs conjugated to fluorochromes) and were excluded from the analysis. The remaining CD3+ cells were analyzed for the expression of V51 and NKp30, NKp44 and Isotype Control. Shows representative results of 4 independent experiments with 4 different donors.
• Figure 21 shows cytotoxicity of the expanded V51 V52 " T cell subset of TCRv5 + T cells against leukemic cells.
• TCRv5 + T cells produced from one healthy donor were stained for CD3, V51 and V52 T cell markers with monoclonal antibodies conjugated to fluorochromes, and the CD3+V51 V52- T cell population was isolated by flow cytometry. Isolated cells were then tested in a killing assay in vitro against acute myeloid leukemia (AML) target cell lines (KG-1 , THP-1 , HL-60 and NB-4) at an effector/target ratio of 10:1. The killing assay was performed as previously described.
• AML acute myeloid leukemia
• Table 1 describes the molecules used to stimulate the proliferation of V51 + T cells during the optimization stage. Reagents were used at a concentration range from 0,1 ng/ml to 8C ⁇ g/ml. The column on the right shows reagent distributor or manufacturer.
• Table 2 shows a summary of tested culture conditions.
• TCRy5 + PBLs were isolated by MACS from a healthy donor and cultured at 1 million cells/ml in 96 well plates, at 37 a C and 5% CO2. Cells were expanded in complete medium (Optmizer CTS, GIBCO) supplemented with 5% autologous plasma, 1 mM L-glutamine and with the described growth factors. At the end of the culture period, cells were counted and cell phenotype was analyzed by flow cytometry. Shows selected results of 4 consecutive experiments. The best culture conditions in each experiment are ranked by fold increase. Fold expansion rate of V51 + T cells was calculated as: (absolute number of V51 + T cells at the end of the culture) / (absolute number of V51 + T cells at day 0 of culture). Shows representative results of 2 independent experiments.
• Table 3 shows a summary of tested culture conditions.
• TCRy5 + PBLs were isolated by MACS from a healthy donor and cultured for 14 days in the presence of the described growth factors. At day 14 of culture, cells were split: one fraction of cells was cultured as before, while the other fraction of cells was cultured in the absence of IL-4 and in the presence of the indicated growth factors. At day 21 , cells were counted and cell phenotype was analyzed by FACS. Shows representative results from 2 independent experiments. A cytotoxicity assay was also performed at day 21 using the generated TCRy5 + cells against MOLT-4 leukemia targets (method is described in Figure 8).
• Apoptotic (dying) target cells were detected by positive staining with Annexin-V reagent in a Fortessa II flow cytometry machine (BD Biosciences). Basal tumor cell death (i.e., the percentage of apoptotic tumor cells in tumor samples that were not co-cultured with TCRv6 + cells) was 10 ⁇ 3%. Shows average of two technical replicates. Table 4 shows a summary of tested culture conditions. TCRy6 + PBLs were isolated by MACS from a healthy donor and cultured for 15 days in a two-step and three-step culture protocols, in the presence of the described growth factors. Cells were counted and cell phenotype was analyzed by FACS at the end of the culture period. Shows representative results of 2 independent experiments.
• Table 5 shows a summary of tested culture conditions.
• TCRy5 + PBLs were isolated by MACS from a healthy donor in a two-step protocol and cultured for 21 days in the presence of the described growth factors. Cells were counted and cell phenotype was analyzed by FACS at the end of the culture period. Shows representative results of 2 independent experiments
• Table 6 shows the purity and phenotype of TCRy6 + PBLs isolated via a two-step MACS protocol.
• PBMCs were obtained by density gradient centrifugation in ficoll from Buffy Coat products collected from 8 healthy donors.
• TCRy5 + T cells were further isolated by a two-step MACS protocol as described in Figure 2.
• Cell phenotype was characterized by flow cytometry analysis of cell surface antigens. Data correspond to percentages of total live cells.
• Table 7 shows the purity and phenotype of in vitro expanded TCRv5 + T cells.
• MACS-sorted TCRy6 + T cells from healthy donors (same as in Table 6) were cultured for 21 days in cell culture bags, according to the previously described two-step culture protocol. Cell populations were characterized by flow cytometry. Indicates percentages of TCRy5 + T cells and contaminant cells, relative to total live cells present in the cultures.
• Table 8 shows the expression of NCRs and NKG2D in freshly-isolated versus cultured V51 + T cells.
• Table 9 shows the purity and phenotype of pre- and post- MACS-sorted TCRv6 + PBLS from CLL/SLL patients.
• B-cell chronic lymphocytic leukemia (CLL) cells were obtained from the peripheral blood of patients at first presentation, after informed consent and institutional review board approval.
• TCRy5 + T cells were MACS-sorted from the peripheral blood of 3 CLL patients (CLL-1 -3) and cell population phenotype was characterized by flow cytometry analysis of cell surface antigens. Shows percentages of TCRv6 + T cells and contaminant cells, obtained immediately before and after the 2-step magnetic isolation procedure. Each cell subset was gated on total live cells.
• Table 10 shows that contaminant autologous B-CLL cells become a residual population in culture.
• TCRv5 + T cells were MACS-sorted from the peripheral blood of 3 CLL/SLL patients (CLL-1 -3; as in Table 9) and cultured in vitro for 16 days as previously described.
• Cell population phenotype was characterized by flow cytometry analysis of cell surface antigens. Shows percentages of TCRv6 + T cells and contaminant cells. Each cell subset is gated on total live cells, except NKp30 and NKG2D expression that were gated on V51 + T cells.
• Table 11 shows in more detail the tested culture conditions presented in Table 2 of a previous application.
• TCRv6 + PBLS were isolated by MACS from a healthy donor and cultured at 1 million cells/ml in 96 well plates, at 37 a C and 5% CO2.
• Cells were expanded in complete medium (Optmizer CTS, GIBCO) supplemented with 5% autologous plasma, 1 mM L-glutamine and with the described growth factors.
• Optmizer CTS GIBCO
• cells were counted and cell phenotype was analyzed by flow cytometry. Shows selected results of 4 consecutive experiments (the same experiments described in Table 2 of a previous application, but further discloses results of parallel control culture conditions, marked with an asterisk, for a more complete understanding of the results).
• TCRY5 + PBLS were isolated and expanded from a healthy donor, as described previously, in the presence of the indicated growth factors. Shows selected results of one experiment with multiple culture conditions.
• ⁇ + cells were cultured in culture medium and three different concentrations of IL-4 and anti-CD3 mAb, in the presence or absence of IL15 /IL-2 /IL-7 and IFN- ⁇ . Shows the detrimental effect of IL-15, IL-2 and IL-7 on ⁇ + T cell expansion, when these cells were cultured in the presence of IL-4 and IFN- ⁇ . Shows representative results of 2 independent experiments.
• Table 13 shows the total absolute number of TCF?Y6 + cells obtained before and after the large-scale 2- step cell culture protocol.
• MACS-sorted peripheral blood ⁇ + cells obtained from healthy donors (represented in Figure 6) were counted with a haemocyto meter before and after cell culture expansion / differentiation.
• Each Buffy Coat was concentrated from 450ml of peripheral blood and contained around 450- 550 million PBMCs. On average, 17 million TCRY5 + T cells could be collected by MACS from each Buffy Coat.
• starting samples will be Leukapheresis products that contain larger numbers of cells and are collected by an Apheresis machine.
• Table 14 shows reagents and materials used to produce pharmaceutical grade TCRy5 + T cells.
• the inventors performed a series of experiments aiming to improve the expansion and purity levels of in vitro cultured V52 ⁇ T cells. Since there was no commercially available antibody against the V53 + chain of the TCR, an anti-TCRV51 mAb was used to identify ⁇ 1 + T cells in cell samples, during the culture optimization stage.
• TCRY5 + T PBLS from a panel of healthy donors were isolated by MACS and tested for their reactivity to in vitro stimulation with IL-2 and PHA (i.e., detectable changes in cell activation and proliferation).
• One donor with reactive V51 + PBLs was selected to provide blood samples for the rest of the optimization study. The preference for a fixed healthy donor was important, since a more reliable comparison could be performed between results obtained in different experiments.
• the selected donor had a normal (but high) percentage of TCRY6 + T cells in the peripheral blood (10%-12% of total PBLs), although a very low percentage of V51 + PBLs (0,3% of total PBLs, or 3,0% of total TCRY6 + T PBLS; Figure 1 ). He was considered a suitable donor to use in these experiments since every minor improvement in the final number and purity levels of cultured V51 + T-cells could be readily detected by flow cytometry.
• the single-step MACS protocol used to isolate TCRY6 + T cells from PBMCs was very efficient, generating highly pure cell populations ( Figure 1 ).
• the inventors then tested multiple combinations of clinical-grade agonist antibodies and cytokines for their capacity to expand and differentiate (over 2-3 weeks) V51 + T cells from the peripheral blood.
• MACS-sorted TCRv5 + PBLS collected from the previously selected healthy donor were incubated in culture medium for 2-3 weeks in 96-well plates, at 37 Q C and 5% CO2, in the presence of 58 different T/NK cell activating molecules (Table 1 ). These included 13 different TCR agonists, 23 different co-receptor agonists, and 22 different cytokines, which were tested in 2.488 different combinations and concentrations.
• Antibodies were used in both soluble and plastic-bound presentations.
• Cytokines were tested at a concentration range from 0,1 ng/ml to 1000ng/ml; TCR agonists were used at 0,1 ng/ml to 40 g/ml, and co-receptor agonists were used at final concentration 0,5 g/ml to 80 g/ml.
• Lectin from Phaseolus vulgaris (red bean ; PHA-P), pur.
• TCR Concanavalin A (from Canavalia ensiformis; Con-A), pur.
• Lectin from Lens culinaris (lentil) ; purified Sigma-Aldrich, Co. (soluble) Lectin from Glycine max (soybean) ; purified
• anti-human NKG2D mAb (Clone 1 D11 ); purified Exbio Praha, a.s.
• Human IFN- ⁇ (lnterferon- ⁇ ) ; PeproTech, inc. Human TGF- ⁇ (Transforming growth factor beta) PeproTech, inc. Human IL-1- ⁇ (interleukin-1 ⁇ ) ; PeproTech, inc. Human IL-2 (interleukin-2) ; PeproTech, inc. Human IL-3 (interleukin-3) ; PeproTech, inc. Human IL-4 (interleukin-4) ; PeproTech, inc. Human IL-6 (interleukin-6) ; Biolegend Human IL-7 (interleukin-7) ; PeproTech, inc. Human IL-9 (interleukin-9) ; PeproTech, inc. Human IL-10 (interleukin-10) PeproTech, inc.
• Human IL-18 (interleukin-18) Southern Biotech Human IL-21 (interleukin-21 ) PeproTech, inc. Human IL-22 (interleukin-22) PeproTech, inc. Human IL-23 (interleukin-23) PeproTech, inc. Human IL-27 (interleukin-27) Biolegend Human IL-31 (interleukin-31 ) PeproTech, inc. Human IL-33 (interleukin-33) PeproTech, inc. Human GM-CSF (Granul.-macroph. col. stimul. factor); PeproTech, inc. Human FLT3-L (FMS-like tyrosine kinase 3 ligand) ; PeproTech, inc. Table 2
• results of experiment n Q .2 demonstrated that IL-2 (when in the presence of IL-4 and PHA) must be used at low concentrations, for increased proliferation and enrichment of V51 + T cells in culture (conditions 3-7, Table 2). This effect was not observed in the previous experiment (exp. n Q 1 ), in which fold expansion was higher in the presence of PHA and high levels of IL-2, in the absence of IL-4. Furthermore, the anti-CD3 mAb was shown to be the most effective mitogen promoting survival and proliferation of V51 + T cells in culture (see condition 1 versus condition 2, Table 2).
• Results of experiment n Q 4 further indicated that the presence of high levels of IL-15 in the culture medium was detrimental for the proliferation of V51 + T cells. Indeed, even lower (than previously used) concentration levels of IL-15 promoted even higher expansion levels of V51 + T cells, when in the presence of IL-4 and a-CD3 (Conditions 3-5, Table 2).
• IFN- ⁇ was consistently found to be a much more effective reagent for promoting the selective expansion of ⁇ 1 + T cells in culture (when used in the presence of IL-4 and a T cell mitogen, such as anti-CD3 mAb; Figure 3).
• the higher performance of IFN- ⁇ in these cultures was independent of the concentrations of IL-4 and anti-CD3 mAb used ( Figure 3A- C and Figure 4).
• the use of IFN- ⁇ (but not of IL-2, IL-15 or IL-7), induced a drastic increase in the enrichment and expansion levels of V51 + T cells in bulk cultures of CD3 + T cells ( Figure 3D).
• IFN- ⁇ is a dimerized soluble cytokine and is the only member of the type II class of interferons. 50 It is structurally and functionally different from common ⁇ -chain cytokines such as IL-2, IL-4, IL-7 or IL-15 and is serologically distinct from Type I interferons: it is acid- labile, while the type I variants are acid-stable.
• NCRs Natural Cytotoxicity Receptors
• NK Natural Killer
• NKp30, NKp44 and NKG2D Activating Natural Killer receptors
• NKp44 and NKG2D Activating Natural Killer receptors
• Receptor-ligand binding triggers the production and release of granzymes and perforines by V51 + T cells, leading to the death of target cells.
• IL-4 in the culture medium induced a decrease in the levels of activating NK receptors located at the surface of cultured TCRv5 + T cells, thereby decreasing their cytotoxic function against MOLT-4 leukemia cells (Table 3).
• the inhibition induced by IL-4 seemed to affect all TCRY6 + T cell subsets present in the final cellular product, including both V51 ⁇ and ⁇ 1 + TCRY6 + T cells (Table 3).
• V51 + T cells secreted significantly less IFN- ⁇ and more IL-10 relative to V52 + T cells.
• V61 + T cells showed relatively low levels of NKG2D expression in the presence of IL-4, suggesting that V51 + T cells weaken the TCRv5 + T cell-mediated anti-tumor immune response. 42
• TCRY6 + T cells were cultured in a two-step protocol.
• the first step consisted of treating cells in culture medium in the presence of a T cell mitogen (such as anti-CD3 mAb or PHA) and IL-4, and in the absence of IL-2, IL-15 and IL-7, to promote the selective expansion of V51 + T-cells.
• the second culture step consisted of treating cells in a culture medium in the absence of IL-4, and in the presence of a T cell mitogen and IL-2, or IL-7 or IL-15, to promote cell differentiation, and NKR expression (Table 3 and Table 4).
• Conditions 1 -5 confirmed previous results, showing that V51 + T cells cultured in the presence of IL-4 could expand in culture several thousand fold, but could not differentiate, becoming inefficient killers of tumor cells.
• conditions 6- 13 when cells were subcultured in a second culture medium in the absence of IL-4 and in the presence of a T cell mitogen and IL-2, or IL-7, or IL-15, the ability to eliminate tumor cells increased radically.
• the presence of each one of these three cytokines IL-2, IL-7 and IL- 15
• CD27 receptor is typically required for the generation and long-term maintenance of T cell immunity. It binds to its ligand CD70, and plays a key role in regulating B-cell activation and immunoglobulin synthesis.
• CD7 receptor is a member of the immunoglobulin superfamily. This protein is found on thymocytes and mature T cells. It plays an essential role in T-cell interactions and also in T-cell /B-cell interaction during early lymphoid development.
• SLAM receptor is a member of the signaling lymphocytic activation molecule family of immunomodulatory receptors.
• N a (cultured 5x10 million cells/ml for 15 davs in 96-well plates): of V51 + T cells
• Davs 11-15 remove medium. 100na/ml IL-15 + 2ua ml a-CD3
• Davs 6-10 remove medium, 100na/ml IL-15 + 2ua/ml a-CD3 64,0 3 683 41 ,0 Days 11-15: 100ng/ml IL-15 + 2 ⁇ g/ml a-CD3 35
• V51 + TCRY5 + T cells comprised only about 1 % to 44% of the total viable cells initially present after MACS.
• V51 + T cells became the dominant cell subset in culture, varying between 60-80% of total cells between donors ( Figure 6).
• a very reproducible expansion was achieved and the composition of the final cellular product was remarkably similar across multiple donors ( Figure 6, Table 7).
• V51 + TCRv5 + T cells in the final products were found to be mostly V51 ⁇ V62 TCRv5 + T cells (which comprised around 17%-37% of total cells). These cells probably consisted of V63 + TCRv5 + T cells, since this is the third most abundant TCRY5 + T cell subset in the peripheral blood. It was possible to calculate the percentage of V51 " V52 TCRv5 + T cells in the final cellular products by subtracting the percentage of V51 + T cells and V52 + T cells from the percentage of total TCRv6 + T cells (Table 7). Fold expansions in these plastic bags were, as expected, of lower magnitude than those from plates, but still generated relevant numbers for clinical translation.
• V62 TCRv5 + T cells could be efficiently isolated and expanded from the PBLs of elderly CLL patients with very high tumour burden (Table 9 and Table 10 and Figure 10A), and displayed potent anti-tumour and anti-viral activities ( Figure 10B).
• contaminating autologous leukemic B cells were eliminated during the in vitro culture of TCRY6 + T cells (Table 10).
• V52 ⁇ T cells namely from cancer patients
• the method is robust enough to enrich (>60%) and expand (up to 2,000-fold) V51 + T cells from highly unpurified samples obtained from CLL patients, differentiating them into NKR-expressing and highly cytotoxic TCRY5 + T cells.
• CD20 + cells cells
• TCRaP + TCRY5 +
• CD3 + cells CD3 + cells
• CD3 + cells CD3 + cells
• CD3 + cells CD3 + cells
• CD3 + cells CD3 + cells
• DOT-cells functional TCRY6 + T cells
• DOT-cells functional TCRY6 + T cells
• the model relies on the adoptive transfer of CLL/SLL- derived MEC-1 cells into Balb/c Rag 7- yc 1' (BRG) animals, which lack all lymphocytes and thus do not immediately reject the human cells.
• BRG Balb/c Rag 7- yc 1'
• DOT-cells does not involve any genetic manipulation; and specifically targets leukemic but not healthy cells in vitro; and prevents wide-scale tumor dissemination to peripheral organs in vivo, without any signs of healthy tissue damage.
• Our results provide new means and the proof-of-principle for clinical application of DOT-cells in adoptive immunotherapy of cancer.
• interleukin-2 interleukin-2
• IL-4 interleukin-4
• NK natural killer
• IFN- ⁇ was consistently found to be a much more effective reagent for promoting the selective expansion of V51 + T cells in culture (when used in the presence of IL-4 and a T cell mitogen, such as anti-CD3 mAb).
• the cell culture protocol obtained with the previously described method is appropriate for use in clinical applications.
• several materials and reagents have been approved by at least one regulatory agency (such as the European Medicines Agency or the Food and Drug Administration) for use in clinical applications.
• the full list is detailed in Table 14.
• the 2-step method of cell isolation proposed by the described invention generates cell samples enriched in TCRY6 + T cells that are viable and can be further cultured.
• Figure 17 shows in more detail FACS-plots of TCRY5 + PBL enrichment after two-step MACS-sorting.
• expanded V61 + T cells increased the expression of NK cell-associated activating/ cytotoxicity receptors, namely NKp30, NKp44, NKG2D, DNAM-1 and 2B4, all previously shown to be important players in tumor cell targeting.
• NKp30, NKp44, NKG2D, DNAM-1 and 2B4 all previously shown to be important players in tumor cell targeting.
• key inhibitory and exhaustion-associated molecules such as PD-1 , CTLA-4 or CD94, were expressed either at very low levels or not expressed at all, demonstrating a striking "fitness" of expanded and differentiated TCRY5 + T cells even after 21 days of culture under stimulatory conditions.
• both V51 + and V61 " V62 TCRv5 + T cell subsets expanded in culture, but could not differentiate towards a cytotoxic phenotype.
• both cell subsets differentiated expressing high levels of activating NK receptors that, in turn, mediated the killing of tumor cells.
• MEC1 and MEC2 two new cell lines derived from B-chronic lymphocytic leukaemia in prolymphocytoid transformation. Leukemia research 1999; 23(2): 127-36. Traggiai E, Chicha L, Mazzucchelli L, et al. Development of a human adaptive immune system in cord blood cell-transplanted mice. Science 2004; 304(5667): 104-7.

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