WO2009083755A1

WO2009083755A1 - Use of t cells expressing cd16 receptors for enhancing the antibody-dependent cellular cytotoxiciy (adcc)

Info

Publication number: WO2009083755A1
Application number: PCT/IB2007/055408
Authority: WO
Inventors: Henri Vie; Béatrice CLEMENCEAU
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale)
Priority date: 2007-12-27
Filing date: 2007-12-27
Publication date: 2009-07-09

Abstract

A method is provided for enhancing ADCC in an individual in need thereof, comprising the administration of natural CD16+ T lymphocytes in said individual. Said method for enhancing ADCC may be used in particular for treating cancers.

Description

USE OF T CELLS EXPRESSING CDl 6 RECEPTORS FOR ENHANCING THE ANTIBODY-DEPENDENT CELLULAR CYTOTOXICIY (ADCC)

FIELD OF THE INVENTION

The present invention relates to a method for enhancing antibody-dependent cellular cytotoxicity (ADCC) and for treating cancer and to pharmaceutical compositions comprising T cells expressing a CD16 receptor.

BACKGROUND OF THE INVENTION

Treatments based on monoclonal antibodies (mAb) have been clinically successful. Adoptive immunotherapy with monoclonal antibodies targeting molecules such as CD20 or Her2/Neu recently have shown its capability to produce a clear clinical benefit. Such passively acquired antibodies can trigger apoptosis of tumor cells and activate complement-mediated (CDC) or antibody-dependent cellular cytotoxicity (ADCC) in treated patients. For rituximab, an anti-CD20 humanized mAb, several clinical observations suggested that ADCC mediated by FcγRIIIa (CD16) -bearing cells is a key mechanism of action. For the anti-Her2/Neu humanized mAb trastuzumab, which is widely used to treat Her2/neu+ breast cancer, mechanisms thought to be responsible for the antitumor activity include down- modulation of the receptor, an anti-angiogenic effect, complement-dependent cytotoxicity, a direct apoptotic effect and ADCC. In fact, in a recent pilot study to elucidate the mechanism by which trastuzumab mediates its antitumor effect, R. Gennari et al observed that patients with complete or partial remission had a higher in situ leukocyte infiltration and a higher capacity to mediate in vitro ADCC (Gennari R et al. Clin Cancer Res. 2004 ; 10 : 5650-5655) . The findings of these clinical studies thus suggest that cancer patients eligible for mAb treatment are likely to benefit from efforts to optimize ADCC in vivo.

Several effectors from both the innate and the adaptive immune system express CD16 receptors, including neutrophils, monocytes, a subset of natural killer (NK) cells, and rare T cells. In the early 1980, several mAb have been produced (anti-Leu-11, VEP 13, B73.1, and 3G8) that specifically react with the IgG FcR responsible for ADCC (FcgRIIIa, CD16) and the presence in most individuals of cells with the phenotype CD3+, CD16+ was confirmed. CD3+, CD16+ cells usually comprised less than 2% of total PBL with rare exceptions.

The existence of "normal" T cells harboring coexpression of CD16 and CcβTCR has been suggested in rare instances only, although this phenotype has been documented on cells from large granular lymphocyte leukemia. Lanier LL et al, described CD16+ CD3+ T lymphocytes most likely with a gamma-delta T cell receptor since they lacked detectable surface expression of either CD4 or CD8. Later on, it was confirmed that human lymphocytes expressing the gamma- delta TCR can express CD16 under stimulation with nonpeptidic Ags and that among circulating gamma-delta TCR-Vδ2 T lymphocytes two functionally diverse subsets of effector memory cells could be discriminated on the basis of CD16 expression. Moreover, it was suggested that due to their surface phenotype profile, this CD16+ Vδ2 T population corresponds to a late stage of Vδ2 T cell differentiation. The present invention aims to provide means for enhancing ADCC and therefore the efficiency of mAb treatment in vivo. The Applicant previously described the use of genetically modified T cells expressing a transgenic CD16/γ receptor for improving ADCC potential (Clemenceau et al . , Blood 2006, 107 (12) 4669-4677) .

While working on "natural" CD16+ T cells, which refer to non- modified T cells expressing naturally a CD16 receptor, the Applicant made the following observations: 1/ natural CD16+ T-cells are present in all individual at low frequency,

2/ natural CD16+ T-cells show a surface phenotype that belongs to terminally differentiated effector memory T cells: in particular they express CD45RA and CD57, the former considered as a marker of replicative senescence,

3/ natural CD16+ T-cells retain the capacity of proliferating and are capable to mediate ADCC immediately ex vivo. This third observation was surprising as, considering the surface phenotype of these CD16+ T cells indicating that these cells are in a state of replicative senescence, it was unexpected that these cells retain the capacity of expansion.

Therefore, the invention aims to use natural CD16+ T cells for improving ADCC in an individual in need thereof. In the meaning of the invention, "CD16+ T cells" refers to natural CD16+ T cells that express naturally an endogenous CD16 receptor and that are not genetically modified. SUMMARY OF THE INVENTION

An object of the invention is to provide natural CD16+ T cells for enhancing ADCC in an individual in need thereof, and in particular for treating cancer. Another object of the invention is to provide isolated natural CD16+ T cell clones.

An object of the invention is also to provide method for producing said natural CD16+ T cell clones.

Another object of the invention is a kit comprising natural CD16+ T cells and at least one immuno-therapeutic agent such as tumor antigens or monoclonal therapeutic antibodies.

DESCRIPTION OF THE FIGURES

Figure 1: Distribution of CDl6-expressing lymphocytes in the peripheral blood of healthy donors.

(A) Cytometric panels refer to a representative healthy donor. Peripheral blood mononuclear cells were stained with antibodies to αβ TCR, γδ TCR and CD16. Three subsets of CDl 6- expressing cells could be identified based on the analysis of gated lymphocytes: CD16+ NK cells, CD16+ αβ T-lymphocytes and CDl 6+ γδ T-lymphocytes .

(B) Analysis of the absolute number of CD16+ NK cells, CD16+ ocβ T-lymphocytes and CD16+ γδ T-lymphocytes in the peripheral blood of 26 healthy donors. Bars indicate means. (C) May-Grunwald-Giemsa stained cytospins of FACS-sorted CD16+ ocβ T-lymphocytes showing morphology typical of large granular lymphocytes (LGL) .

Figure 2. CDl6-expressing lymphocytes are able to mediate ADCC ex-vivo . The cytotoxic activity of FACS-sorted CD16+ NK cells, CD16+ αβ T-cells and CDlβneg αβ T-cells from the same donor was evaluated against a ⁵¹Cr-labeled K562 cell line and ⁵¹Cr-labeled autologous BLCL in the absence or presence of either rituximab (anti-CD20, 2 μg/ml) or herceptin (anti-HER-2, 10 μg/ml) as a negative control. Results are expressed as percentage of specific lysis (effector-to-target ratio = 10:1, mean of triplicate wells) . Similar results were obtained with two other donors . (B) Intracellular expresssion of perforin by CD16+ αβ T lymphocytes ex-vivo. Similar results were obtained with six other donors .

Figure 3. Limiting dilution analysis and functional characterization of CD16+ αβ T-lymphocytes .

(A) PBMC were stained with PE-anti-αβ and PC5-anti-CDl 6 antibodies. Next CD16+ αβ T-lymphocytes were isolated using a FACSVantageTM and cloned by limiting dilution (see method section) . Cloning efficiency was approximatively 0.75 and 0.30 (according to the Poisson Distribution) .

(B) upper panel: maintenance of CD16 expression by a CD16+ αβ T-cell clone. T-cell clones were analysed by flow cytometry for CD16 expression over a 2.5 month period, a = Day 28 after cloning, b and c = Days 27 and 52 after the first restimulation and d = after freezing and thawing, 38 days after stimulation.

(B) lower panel: the same T-cell clone was tested for ADCC against 51Cr-labeled autologous BLCL, in the presence of either rituximab (anti-CD20, 0.02 μg/ml or 2 μg/ml) or herceptin (anti-HER-2, 10 μg/ml) as a negative control. Results are expressed as percentage of specific lysis (effector-to-target ratio = 30:1, mean of triplicate).

Figure 4. Functional characterization of CD16+ αβ T- lymphocytes .

CD16+ αβ T-cell clones can proliferate only when the CD16 molecule is crosslinked in the presence of mAbs and target cells. Two CD16+ αβ T-cell clones and one CDlβneg αβ T-cell clone were tested in a 72-h proliferation assay in the presence IL-2 (40 UI /ml) . Assays were performed in the presence of soluble anti-CD20 mAb alone or against the autologous BLCL in the absence or the presence of rituximab.

Figure 5. Functional characterization of CD16+ αβ T- lymphocytes.

CD16+ αβ T-cell clone produce cytokines only when the CD16 molecule is crosslinked in the presence of mAbs and target cells. (Dl) The CD16+/CD8+ T cell clone #14 and #21 (Dl and D2 respectively) (which do not recognize the autologous BLCL through their TCR) produced TNFα after PMA+ ionomycin stimulation (a) were activated (to produce TNFα) only after CDl 6-crosslinking in the presence of the BLCL and 0.02 or 2 μg/ml of anti-CD20 (b, c and d) but remained unstimulated by the soluble mAb at concentrations up to 1000 μg/ml (e,f,g) .

Figure 6. Increase in CDl6+ αβ T-lymphocytes during hyperlymphocytosis in vivo.

Frequency of CD16+ αβ T-lymphocytes in healthy control donors and patients with hyperlymphocytosis (superior to 4000 lymphocytes/μl) . Peripheral blood mononuclear cells were stained with antibodies to αβ TCR and CD16. The percentage of CD16+ αβ double-positive cells as a fraction of all αβ T- lymphocytes is shown for each sample. Bars indicate means. (A) The CD16+/CD8+ T cell clone #14 from donor 1 and (B) the CD16+/CD4+ T-cell clone #21 from donor 2 (which do recognize the autologous BLCL through their TCR) produced TNFCC after PMA+ ionomycin stimulation (a) was activated only after CD16- crosslinking in the presence of the autologous BLCL and 0.02 or 2 μg/ml of anti-CD20 (b, c and d) but remained unstimulated by the soluble mAb at concentrations up to 1000 μg/ml (e,f,g) .

Figure 7. EBV-specific cytotoxic T cell lines (CTL) contain CD16+ αβ T-cells that mediate ADCC.

EBV-specific CTLs were selected against the aulogous BLCL and stained with PE-anti-cφ antibody and PC5-anti-CD16 antibody. The ADCC activity of two EBV-specific CTLs was evaluated against ⁵¹Cr-labeled allogeneic BLCL (not recognized by the TCR of the CTL) in the presence of either rituximab (anti-CD20, 2 μg/ml) or herceptin (anti-HER-2, 10 μg/ml) as a negative control. Results are expressed as percentage of specific lysis (effector-to-target ratio = 30:1, mean of triplicate wells).

DETAILED DESCRIPTION OF THE INVENTION I . Definitions The term "T cell", equivalent to "T lymphocytes", refers to a class of lymphocytes, so called because they mature in the thymus and have the ability to recognize specific antigens through the receptors on their cell surface. T cells can be a monoclonal or polyclonal population. They can express TCROcβ or TCRγδ and CD4 or CD8 or both coreceptors, and their TCR specificity can be known or unknown. The term "endogenous" is known in the art, and, as used herein, generally means developing or originating from within the organism or arising from causes within the organism. A T cell expressing an endogenous receptor means a T cell expressing naturally this endogenous receptor.

By "natural CD16+ T cells" it is intended non-modified T cells expressing an endogenous CD16 receptor.

The term "transformed cell line" is known in the art, and, as used herein, generally refers to a permanently established cell culture, wherein cells are transformed and/or immortalized. For example, Jurkat cells refer to a transformed cell line derived from human T cell leukaemia.

The term "T cell clone" is known in the art, and, as used herein, generally includes T cells derived from a single T cell. T cells can be cloned using numerous methods known in the art including limiting dilution assays (LDA) and cell sorting using flow cytometry.

An "isolated" biological component (such as a nucleic acid molecule, protein, vascular tissue or haematological material, such as blood components) is known in the art, and, as used herein, generally refers to a biological component which has been substantially separated or purified away from other biological components of the cell in the organism in which the component naturally occurs. An isolated cell is one which has been substantially separated or purified away from other biological components of the organism in which the cell naturally occurs.

The term "enhance" as used herein means to improve the quality, amount, or strength of a phenomenon, especially a biological response.

The term "ADCC" or "antibody-dependent cell mediated cytotoxicity" is known in the art, and, as used herein, generally refers to a form of lymphocyte mediated cytotoxicity that functions only if antibodies are bound to the target cell. Antibody-coated target cells are killed by cells bearing Fc receptors specific for the Fc regions of the antibodies, especially NK cells.

The term "transfection" is known in the art, and, as used herein, is generally used to refer to the uptake of foreign DNA by a cell. The term "transduction" is known in the art, and, as used herein, generally denotes the delivery of a DNA molecule to a recipient cell either in vivo or in vitro, via a replication-defective viral vectors, such as retroviral gene transfer vector.

A recipient cell which has been "modified" has been generally transfected or transduced, either in vivo or in vitro, with a gene transfer vector comprising a DNA molecule of interest or with a RNA molecule of interest or with a protein of interest. By "vector" or "gene transfer vector" is generally meant any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells. Thus, the term "vector" generally includes cloning and expression vehicles, as well as viral vectors. By "individual", it is meant mammal, in particular a human being. By "effective amount", it is meant an amount sufficient to effect a beneficial or desired clinical result (e.g. improvement in clinical condition) .

As used herein, "treatment" or "treating" generally refers to a clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and may be performed either for prophylaxis or during the course of clinical pathology. Desirable effects include, but are not limited to, preventing occurrence or recurrence of disease, alleviating symptoms, suppressing, diminishing or inhibiting any direct or indirect pathological consequences of the disease, preventing metastasis, lowering the rate of disease progression, ameliorating or palliating the disease state, and causing remission or improved prognosis.

The term "chemotherapy" as used herein generally refers in cancer treatment to the administration of one or a combination of compounds to kill or slow the reproduction of rapidly multiplying cells. Chemotherapeutic agents include those known by those skilled in the art, including, but not limited to: 5- fluorouracil (5-FU) , azathioprine, cyclophosphamide, antimetabolites (such as fludarabine) , antineoplastics (such as etoposide, doxorubicin, methotrexate, and vincristine) , carboplatin, cis-platinum and the taxanes, such as taxol. The term "immuno-depleting agent" generally refers to a compound which results in a decrease in the number of cells of the immune system (such as lymphocyte) when administrated to an individual. Examples include, but are not limited to, chemotherapeutic agents. The term "immuno-therapeutic agent" generally refers to a compound which results in the activation of an immune response when administrated to an individual. Examples include, but are not limited to, tumor antigens or monoclonal therapeutic antibodies .

II. The present invention

The present invention relates to a method for enhancing ADCC in an individual in need thereof, said method comprising the administration of an effective amount of natural CD16+ T cells . Natural CD16+ T cells are able to bind the constant region of antibodies via their CD16 receptor, activating by this way their mechanism of antibody-dependent cellular toxicity. Without wanting to be bound to any theory, the administration of an effective amount of natural CD16+ T cells should increase the number of effector cells capable of activating ADCC and therefore enhance patient ADCC potential.

An object of the invention is natural CD16+ T cells for enhancing ADCC.

In a preferred embodiment of the invention, said effective amount of natural CD16+ T cells is administrated in an individual in need thereof via a parenteral route. A parenteral administration mode includes subcutaneous, intramuscular, intravenous, intraperitoneal, intranasal and intradermal administration. Administration can be systemic or local .

In a more preferred embodiment of the invention, said natural CD16+ T cells are intravenously administrated in an individual in need thereof.

In another embodiment of the invention, said natural CD16+ T cells are administrated at a dose of about 1 to 5xlO⁶ cells per kilogram to about 10⁹ cells per kilogram. Preferably, said natural CD16+ T cells are administrated at a dose of about 10⁷ cells per kilogram to 10⁹ cells per kilogram, more preferably to about 10⁸ cells per kilogram to 10⁹ cells per kilogram.

According to the invention, said method for enhancing

ADCC permits the treatment of cancers, and infectious diseases. Indeed ADCC plays a major role in such diseases or conditions for the elimination of infected cells, tumor cells...

An object of the invention is to provide natural CD16+ T cells for treating cancer or for preventing and/or treating infectious diseases.

Certain embodiments of this invention relate to combination therapies. According to the invention, said method for enhancing ADCC further comprises the administration of at least one immuno-therapeutic agent such as tumor antigens for antitumoral vaccination or monoclonal therapeutic antibodies for monoclonal antibody therapy. The administration of natural CD16+ T cells should indeed enhance the effect of said immuno- therapeutic agents via the enhancement of ADCC.

In one embodiment of the invention, said immuno- therapeutic agent comprises tumor antigens. Tumor antigens include but are not limited to peptides derived from the MAGE, BAGE, GAGE and LAGEl/NY-ESO-1 gene families. These tumor antigens can be administrated alone or can be presented by an antigen presenting cells such as dendritic cells or can be contain in a delivery system such as exosomes, apoptotic bodies, or tumor cells. Such agents can be administrated before administration of the natural CD16+ T cells in a time delay required to obtain immunisation, for example 1 month to 3 months .

However, it is understood that the regimen of administration of said natural CD16+ T cells is within the judgment of the managing physician, and depends on the clinical condition of the individual, the objectives of treatment, and concurrent therapies also being administrated. In another embodiment of the invention, said immuno- therapeutic agent comprises monoclonal therapeutic antibodies. Examples of monoclonal antibodies include, but are not limited to, Infliximab (anti-TNFCC) , Basiliximab, Daclizumab (anti- CD25), Trastuzumab (anti-Her2/neu) , Rituximab, Ibritumomab tiutexan (anti-CD20), Tositumomab (anti-CD122) , Gemtuzumab ozogamicin (anti-CD33) , Alemtuzumab (anti-CD52) . Such agents can be administrated before, during or after administration of the natural CD16+ T cells.

According to the invention, said method for enhancing ADCC further comprises the administration of at least one immuno-depleting agent. As shown for example in Dudley et al . Science. 2002 Oct 25;298 (5594) :850-4 and in Nat Med. 2005 Nov; 11 (11) : 1230-7, lymphodepletion can have a marked effect on the efficacy of T cell transfer therapy. Preferably, such chemotherapeutic agents are administrated before the administration of natural CD16+ T cells.

In one embodiment of the invention, said immuno- depleting agents comprise at least one chemotherapeutic agent. Examples of chemotherapeutic agents include, but are not limited to, 5-fluorouracil, aziathioprine, cyclophosphamide, anti-metabolites (such as fludarabine) , anti-neoplasties (such as etoposide, doxorubicin, methotrexate, vincristine) , prednisone, carboplatin, cis-platinum and the taxanes such as taxol. Immuno-depleting agent such as chemotherapeutic agents defined hereabove can be administrated 2 days, preferably 1 day, before the administration of natural CD16+ T cells In a preferred embodiment, the method for enhancing ADCC according to the invention permits the treatment of cancer, optionally in combination with antitumoral vaccination. Said method comprises the administration in an individual in needs thereof of natural CD16+ T cells in combination with at least one tumor antigen. Tumor antigens such as peptides derived from the MAGE, BAGE, GAGE and LAGEl/NY-ESO-1 gene families are used for treating many melanomas, transitional bladder cancers, head and neck squamous cells carcinomas, non small cell lung cancers, oesophageal cancers, multiple myelomas.

In a preferred embodiment, the method for enhancing ADCC according to the invention permits the treatment of cancer, especially solid tumors, optionally in combination with monoclonal antibody therapy. Said method comprises the administration in an individual in need thereof of natural CD16+ T cells in combination with at least one monoclonal antibody used for treating solid tumors. Solid tumors, such as sarcomas and carcinomas, comprise fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, renal cell carcinoma, melanoma, CNS tumors... Examples of monoclonal antibody used for treating solid tumors include but are not limited to Trastuzumab used for treating breast cancer or Rituximab, Ibritumomab tiutexan or Tositumomab for treating lymphoma.

In a preferred embodiment, the method for enhancing

ADCC according to the invention permits the treatment of cancer, especially haematological tumors, optionally in combination with monoclonal antibody therapy. Said method comprises the administration in an individual in needs thereof of natural CD16+ T cells in combination with at least one monoclonal antibody used for treating hematologic or lymphoid malignancies.

Hematological tumors comprise acute lymphocytic leukaemia, acute myelogenous leukaemia, chronic lymphocytic leukaemia, chronic myelogenous leukaemia, indolent non Hodgkin' s lymphoma, high-grade Hodgkin' s lymphoma, Hodgkin' s lymphoma, multiple myeloma or myelodysplastic syndrome. Examples of monoclonal antibody used for treating hematologic or lymphoid malignancies include, but are not limited to, Gemtuzumab ozogamicin used for treating acute myelogenous leukaemia, or Alemtuzumab used for treating chronic lymphocytic leukaemia.

In another embodiment, the method for enhancing ADCC according to the invention permits also the treatment of infectious diseases, especially bacterial and viral infections .

It is another object of the present invention to provide an isolated natural CD16+ T cell clone.

In a preferred embodiment of the invention, said natural CD16+ T cell clone expresses an antigen specific receptor (TCR) of known specificity.

Knowing the specificity of the TCR will permit to anticipate that the T cells will be unable to recognize non infected or non transformed host tissues. Indeed, from an immunological point of view, the use of specific T cells whose TCR specificity is known should be safer than the use of a bulk population and will avoid the risk of a graft versus host reaction when allogeneic T cells are used. The specificity of the antigen specific receptor of the T cells can be defined by any methods known in the art, for example by flow cytometry, cytotoxicity assay or proliferation assay.

In one embodiment of the invention, the specificity of said natural CD16+ T cell clone is directed against a virus selected from the group consisting in Epstein Barr viruses (EBV) , cytomegaloviruses (CMV) , human papilloma viruses (HPV) , and herpes simplex virus (HSVl, HSV2) . Preferably, the specificity of said T cell clone expressing endogenous CD16 receptor is directed against EBV.

In another embodiment of the invention, the specificity of said T cell clone expressing an endogenous CD16 receptor is directed against the human leukocyte antigen system (HLA) . HLA is the general name of a group of genes in the human major histocompatibility complex (MHC) region on human chromosome 6

(mouse chromosome 17) that encodes the cell-surface antigen presenting proteins. HLA molecules comprise HLA-A, HLA-B, HLA- C, HLA-DPAl, HLA-DPBl, HLA-DQAl, HLA-DQBl, HLA-DRA, and HLA- DRBl.

In a preferred embodiment of the invention, a natural CD16+ T cell clone expresses an antigen specific receptor (TCR) of known specificity, and a specific HLA combination, that is widespread in the recipient individuals. For example, the natural CD16+ T cell clone e is derived from an individual being heterozygous, preferably homozygous, for the haplotype HLA A1B8DR3-DQ2 and can preferably be administrated in a Caucasian individual, for which this haplotype is widespread. Preferably, the natural CD16+ T clone is derived from an individual being homozygous for the haplotype HLA A1B8DR3-DQ2. Indeed, from an immunological point of view, the use of an allogeneic T cell clone further expressing a specific HLA combination, being widespread in the recipient individuals should allow an increased lifetime of this clone as the T cell clone expressing said HLA combination would be less recognized as non-self by the immune system of said individual.

It is an object of the invention to provide a method for isolating said natural CD16+ T cell clone, wherein said method comprises: isolating natural CD16+ T cells from PBL, purifying said natural CD16+ T cells, cloning said natural CD16+ T cells, - optionally further expanding the at least one

T cell clone thus obtained.

In a preferred embodiment of the invention, natural CD16+ T cells are isolated from PBL by using monoclonal antibodies and flow cytometry. T cells can be autologous or allogenic .

The isolated natural CD16+ T cells can further be substantially purified by any well known method in the art. A substantially purified population of cells refers to a population of cells that are at least 80%, 90%, 95%, 96%, 97%, 98% or 99% pure. Preferably, isolated natural CD16+ T cells are sorted by flow cytometry using anti-αβ antibody and anti- CD16 antibody. However, for clinical use of these natural CD16+ T cell, these isolated cells are purified by using immunomagnetic methods.

Purified natural CD16+ T cells are further cloned by any method well known in the art, for example by a non-specific amplification procedure described in Gaschet et al . [Gaschet et al. Blood 1996, 87:2345-2353]. Finally, natural CD16+ T cell clones are further expanded by cell culture. The expansion of the T cell clones can be realized by in vitro non specific stimulation such as those provided by exposure to CD3 and CD28 mAb or lectins such as PHA, or by specific stimulation such as those provided by coculture of T cells with allogeneic or virally infected cells or with a soluble antigen. The soluble antigen may be a peptide corresponding to a viral epitope that stimulates αβ T cells or a non-peptidic molecule capable of stimulating γδ T cells.

The specificity of the TCR of the natural CD16+ T cell clones thus obtained can be further assessed by any well-known method in the art, for example by cytotoxicity assay, cytotoxicity assay or proliferation assay.

It is also an object of the invention to provide a method for producing a natural CD16+ T cell clone expressing a

TCR of known specificity, and optionally expressing a specific HLA combination that is widespread in the recipient individuals, comprising: isolating and expanding at least one (known- antigen) -specific natural CD16+ T cell optionally expressing a specific HLA combination that is widespread in the recipient individuals, cloning said (known-antigen) -specific T cell, isolating at least one (known-antigen) - specific natural CD16+ T cell clone, - and optionally expanding said (known- antigen) -specific natural CD16+ T cell clone. In one embodiment, the isolation and expansion of at least one

(known-antigen) -specific T cell is realized according to standard methods by stimulating PBL with said known-antigen or with a cell line expressing said known antigen. For example, the isolation and expansion of an EBV specific cytotoxic T cell is realized by stimulating PBL with an EBV B lymphoblastoid cell line (BLCL) according to standard methods.

Another example of CMV specific cytotoxic T cells is described in Gallot et al . [Gallot et al . , JI 2001, 167, 4196:4206]. In a preferred embodiment, the known-antigen is selected from the group consisting of EBV, CMV, HPV, HSVl and HSV2, or is directed against HLA molecules. Said (known-antigen) -specific T cell optionally expresses a specific HLA combination , that is widespread in the recipient individuals and can be obtained by using PBL derived from an individual being heterozygous, preferably homozygous, for this specific HLA combination. The (known-antigen) -specific T cell are further cloned by any method well known in the art, for example by a non-specific amplification procedure described in Gaschet et al . [Gaschet et al. Blood 1996, 87:2345-2353].

Among (known-antigen) -specific T cell clones thus obtained, is isolated a (known-antigen) -specific natural CD16+ T cell clone. Such isolation can be realized by immunostaining using flow cytometry. Finally, natural CD16+ T cell clones can optionally be expanded by cell culture. The expansion of the T cell clones can be realized by in vitro non specific stimulation such as those provided by exposure to CD3 and CD28 mAb or lectins such as PHA, or by specific stimulation such as those provided by coculture of T cells with allogeneic or virally infected cells or with a soluble antigen. The soluble antigen may be a peptide corresponding to a viral epitope that stimulates αβ T cells or a non-peptidic molecule capable of stimulating γδ T cell.

Another object of the invention is to provide a pharmaceutical composition comprising natural CD16+ T cells.

Another object of the invention is to provide a pharmaceutical composition comprising at least one natural CD16+ T cell clone as described above.

Another object of the invention is the use of natural CD16+ T cells or at least one natural CD16+ T cell clone as described here above for the preparation of a pharmaceutical composition for the treatment of cancer or infectious diseases .

In a preferred embodiment, said pharmaceutical composition includes an effective amount of natural CD16+ T cells with a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carriers useful herein are conventional. Remington's Pharmaceutical Sciences 16^th edition, Osol, A. Ed. (1980) describes composition and formulations suitable for pharmaceutical delivery of the natural CD16+ T cell clone herein disclosed. In general, the nature of the carrier will depend on the mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, sesame oil, glycerol, ethanol, combinations thereof, or the like, as vehicle. The carrier and composition can be sterile, and the formulation suits the mode of administration.

In addition to biological neutral carriers, pharmaceutical compositions to be administrated can contain minor amounts of non toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. The composition can be a liquid solution, suspension, emulsion.

The amount of natural CD16+ T cell clone effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each individual's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

In a preferred embodiment, said pharmaceutical composition includes an effective amount of natural CD16+ T cells with human albumin.

In a preferred embodiment, said pharmaceutical composition is administrated in an individual in need thereof by intravenous injections.

In a preferred embodiment, said pharmaceutical composition is used for treating diseases or conditions requiring an ADCC enhancement such as cancers.

Another object of the present invention is to provide a pharmaceutical kit comprising: at least one pharmaceutical composition comprising: natural CD16+ T cells or at least one isolated natural CD16+ T cell clone, and at least one immuno-therapeutic agent such as: a tumor antigen selected from the group consisting of peptides derived from the

MAGE, BAGE, GAGE and LAGEl/NY-ESO-1 gene families, and/or a monoclonal antibody selected from the group consisting in Infliximab, Basiliximab, Daclizumab, Trastuzumab,

Rituximab, Ibritumomab tiutexan, Tositumomab, Gemtuzumab ozogamicin, Alemtuzumab .

Optionally associated with the kit can be included a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration Instructions for use of the composition.

In a preferred embodiment, said kit further comprises at least one chemotherapeutic agent selected from the group consisting of etoposide, doxorubicin, vincristine, cyclophosphamide, prednisone, fludarabine.

EXAMPLES

In the following description, all molecular biology experiments for which no detailed protocol is given are performed according to standard protocol. The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Materials and Methods

Samples and Cell lines. Peripheral blood mononuclear cells

(PBMCs) were prepared by ficoll (PAA laboratories, Les

Mureaux, France) gradient centrifugation of blood obtained from adult volunteers or patients with hyperlymphocytosis that were recruited non-selectively from the Department of Infectious Diseases. All individuals gave informed consent. Epstein-Barr Virus B lymphoblastoid cell lines (BLCL) were derived from PBMCs by in vitro infection using EBV-containing culture supernatant from the Marmoset B95.8 cell line purchased from the American Type Culture Collection (ATCC; Rockville, MD) in the presence of lμg/ml cyclosporine-A. The K562 cell line was cultured in complete medium consisting of RPMI 1640 (Sigma Aldrich, St Quentin Fallavier, France), 10% heat inactivated fetal calf serum, 2mM glutamine (Sigma Aldrich) , lOOU/ml penicillin and lOμg/ml streptomycin (Sigma Aldrich) . Monoclonal antibodies (rnAb) and flow cytometric analysis. The following mAbs and their isotype controls were used : anti-αβ - FITC (BMA031, Serotec, Cergy Saint-Christophe, France), anti- CCR7-PE (150503, RD systems, Lille, France), anti-CD28-PE (L293), and anti-perforin-PE (δ69) (BD Biosciences, Le ponts de Claix, France). Anti-CD16-PC5 (3G8,), anti-αβ-PE (BMA031), anti-γδ-FITC (IMMU510), anti-CD4-PE (13B8.2), anti-CD8-PE (B9.ll), anti-CD27-PE (1A4), anti-CD45-RA-PE (ALBIl), anti- CD45-RO-PE (UCHLl), anti-CD57-PE (NCl), anti-CD62L-PE

(DREG56), anti-CD32-PE (2El) and anti-CD64-PE (22), all from

Beckman Coulter, Roissy, France. Five hundred thousand (0.5 x

10⁶) PBMC or 50 μl of whole blood were incubated for 12 min at

RT in 1 ml tubes in the presence of optimal concentrations of antibodies diluted with PBS supplemented with 5% human serum

(HS) . After staining, tubes were centrifuged, the supernatant was discarded and cells were washed twice with 900 μl PBS.

Data were acquired by a FACSCalibur™ instrument (BD

Biosciences, Mountain View, CA) and analyzed using CELLquest™ software (BD Immunocytometry Systems) . The absolute number of each population was then calculated from the total lymphocyte count within the blood sample determined using an automated cell counter (Sysmex HSTXE 2100, Roche) .

Cell sorting. PBMC (15 x 10⁶) were stained with PE-anti- αβ. antibody (BMA031, Beckman Coulter) and PC5-anti-CDl 6 antibody (3G8, Beckman Coulter). Cell sorting was performed on a FACSVantage™ or FACSAria flow cytometer (BD Biosciences) .

Cell Cultures. Sorted CD16-expressing αβ T lymphocytes were cloned using a non-specific amplification procedure: T-cells were seeded at 3.0, 1.0 and 0.3 cells/well in 96-well U-bottom plates together with irradiated (35 Gy) pooled allogeneic feeder cells (1 X 10⁵ PBMC and 1 X 10⁴ B cells from a B lymphoblastoid cell line (BLCL) ) , lμg/ml leucoagglutinin PHA-L (Sigma-Aldrich) and 300 UI/ml of recombinant IL-2 (Roussel- Uclaff, Romainville, France) in a final volume of 200 μl . After cloning, T cells were further expanded: first, in 24- well culture plates, then in culture flasks using the same culture conditions (feeder cell concentration, recombinant IL2 and PHA concentration) that allow for maximal proliferation of the T cell clones for several weeks.

Generation and expansion of EBV-specific cytotoxic cell lines.

Donor PBMCs were plated in 24-well culture plates at 2 X 10⁶ cells/well in RPMI1640 with glutamax (Invitrogen, Cergy Pointoise, France) culture medium supplemented with 8% pooled human serum (HS), and stimulated with 5x 10⁴ 40 Gray-irradiated autologous BLCL (PBMC :BLCL ratio of 40 :1). After 10 days, T cells were collected and restimulated at a T :B ratio of 4 : 1 (5 x 10⁵ T cells and 1.25 X 10⁵ BLCL/well) . IL-2 was added 4 days after the second stimulation (4 OUI /ml) . A third and a fourth stimulation were performed each 7 days in the presence of IL2 and at the same T:B cell ratio (4:1).

Cytotoxicity assay. Cytotoxic activity was assessed using a standard ⁵¹Cr release assay. Target cells were labeled with 100 Ci ⁵¹Cr for Ih at 37°C, washed four times with culture medium, and then plated at the indicated effector-to-target cell ratio in a 96-well flat or U-bottom plate. An autologous BLCL was used as a model of an autologous tumor and the humanized anti-CD20 mAb Rituximab (Roche, UK) was used (at 2 μg/ml) to induce ADCC. In some experiments, the anti-Her2/neu mAb Trastuzumab (Roche, UK) was used (at 10 μg/ml) as a control. For ADCC assays, the indicated monoclonal antibody was incubated with target cells for 20 min before addition of effector cells. After a 4h incubation at 37°C, 25 μl of supernatant were removed from each well, mixed with 100 μl scintillation fluid, and ⁵¹Cr activity was counted in a scintillation counter. Each test was performed in triplicate. The results are expressed as the percentage of lysis, which is calculated according to the following equation: (experimental release-spontaneous release) / (maximal release-spontaneous release) X 100, where experimental release represents the mean counts per minute (cpm) for the target cells in the presence of effector cells, spontaneous release represents the mean cpm for target cells incubated without effector cells, and maximal release represents the mean cpm for target cells incubated with 1% Triton X 100.

Statistical analysis

Differences between subjects with hyperlymphocytosis and healthy controls were analyzed using the t-test. A p value of <0.05 was considered significant.

Results

Frequency and phenotype of CD16+ αβ T-lymphocytes in the blood of healthy donors .

Direct staining of freshly isolated PBMCs with antibodies to CD16, αβ TCR and γδ TCR led to the identification of three subsets of CDl 6-expressing lymphocytes in all healthy adult donors tested (Fig.l A): CD16+ NK cells, CD16+ αβ and CD16+ γδ. T-lymphocytes . Analysis of absolute cell numbers for each subset within the peripheral blood lymphocytes revealed that there were 30 CD16+ αβ T-lymphocytes per μl of blood (range 1- 198); 40 CD16+ γδ T-lymphocytes per μl of blood (range 0-218) and 194 CD16+ NK cells per μl of blood (range 50-577) (Fig. 1 B, n=26) . Thus, altogether αβ and γδ T-cells represented on average 25% of all CD16-expressing lymphocytes. Significant differences were found in terms of CD16 expression between these three populations: the mean fluorescence intensity for CD16 was 20 ± 7 for CD16+ αβ T-lymphocytes, 56 ± 35 for CD16+ γδ T-lymphocytes and 503 ± 120 for CDl 6+ NK cells. MayGrunwald Giemsa coloration of FACS-sorted CD16+ αβ T-lymphocytes showed that these cells are characterized by an abundant basophilic cytoplasm with azurophilic granules typical of large-granular lymphocytes (LGL) (Fig. 1C) .

The CD16+ αβ T-lymphocyte subset was further characterized for CD4 or CD8 expression and with a panel of mAb that discriminates between different memory T-lymphocyte subsets (CD27, CD28, CD45RO, CD45RA, CD57, CD62L and CCR7) . Staining for CD32a/CD32b (anti-FcγRIIa and -FcγRIIb) , CD64 (anti-FcγRI) , and three KIRs- (CD158a, h, CD158b and KIRp70) was also performed. Data from fifteen donors are reported in Table 1. The vast majority of CD16+ αβ T-lymphocytes (that were all negative for CD32 and CD64, data not shown) were CD8+ (89%) and mainly CD45RA+CCR7-, with only 42% of cells expressing CD62L, suggesting that they belonged to the effector memory T lymphocyte population. In addition, 74% of these cells expressed CD57 and only 12.9% expressed CD28. Since a lack of CD28 and presence of CD57 on CD8+ T cells are generally associated with a status of replicative senescence, altogether these results suggest that CD8+CD16+ αβ T-lymphocytes belong to a small population that has been previously described as TEMRA lymphocytes for terminally differentiated CD45RA+ effector memory T-cells.

The CD16+ αβ T-lymphocyte subset is capable of mediating ADCC ex-vivo .

The ADCC activity of CD16+ αβ T-lymphocytes from three healthy donors was tested and compared with that of autologous NK cells. CD16+ αβ T-lymphocytes, CD16⁺ NK cells and CDlβneg αβ T- lymphocytes from the same donor were sorted by FACS and their cytotoxic activity assessed using a 4h ⁵¹Cr release assay against the NK-sensitive K562 cell line and the autologous BLCL in the presence of absence of anti-CD20 or anti-Her2/neu humanized mAb (BLCL were all positive for CD20 and negative for Her2/neu antigens) . Figure 2 shows representative data obtained from one healthy donor. Only NK cells were able to kill the K562 cell line. In the absence of mAb, NK cells, CD16+ αβ and CDlβneg αβ T-lymphocytes did not recognize the autologous BLCL. In contrast, both CD16+ NK cells and CD16+ αβ T lymphocytes killed the BLCL incubated with anti-CD20 mAb. This cytotoxicity was not observed in the presence of anti- Her2/neu mAb. Together, these results demonstrate that despite expressing low levels of CD16, CD16+ αβ T lymphocytes all express perforin (data not shown) are able to mediate ADCC ex- vivo .

Limiting dilution analysis of the CD16+ αβ T-lymphocytes

CD16+ αβ T-lymphocytes from two healthy donors were sorted and cloned by limiting dilution using non-specific stimulation (lectin+feeder+IL2) . Cloning efficiency was 0.75 and 0.30 according to the Poisson distribution (Fig 3A) . Nineteen days after cloning, 47/52 clones from donor 1 and 18/19 clones from donor 2 were CD16-positive . Next, four clones from donor 1 and 6 from donor 2 were selected according to their CD16 expression levels and tested for changes in CD16 expression and ADCC activity over a 3-month culture period. One CDlβneg αβ T-cell clone from each donor was included in these experiments as a negative control for ADCC assays as well as a control for assessment of CD16 expression during culture. After stimulation (Lectin +feeder+IL2) CD16 on CD16-positive clones was down-regulated for 10-15 days but was eventually reexpressed and maintained for several weeks. Figure 3B shows one example of CD16 expression and ADCC activity monitoring for the clone #2 from donor 1. CD16 expression is shown at day 28 after cloning (a) , at days 27 (b) and 52 (c) after the first restimulation and then after freezing and thawing, 38 days after stimulation. Under the same conditions, the control CDlβneg αβ T-cell clone never expressed CD16. These results indicate that T-cells programmed to express CD16 maintained CD16 after TCR stimulation at a level depending on the state of activation. ADCC activity of CD16+ αβ T-cell clones was also assessed using a 4 h ⁵¹Cr release assay performed against the autologous target BLCL. Figure 3B shows the results obtained at different times points with the CD8+CD16+ αβ T-cell clone #2 that was derived by limiting dilution from the CD16+ αβ T-cell fraction of a healthy donor and whose TCR specificity was unknown. In the absence of mAb, clone #2 did not recognize the autologous BLCL. In contrast, addition of the humanized anti-CD20, but not the anti-Her2/neu mAb, induced lysis of the autologous BLCL, thus demonstrating that clone #2 was able to mediate ADCC (the same results were observed with the four clones tested) . One can note that despite the suggestion that anti-CD20 might be directly cytotoxic against target cells, this was never observed in our experiments using BLCL, even at high anti-CD20 concentrations, in the presence or absence of CDlβneg CTL (not shown) .

CD16+ αβ T-lymphocytes appear in vivo during hyperlymphocytosis Since CD16 was found to be expressed by memory αβ T-lymphocytes in normal, healthy donors, we anticipated that this particular subset would appear and be amplified during the T-cell response. To test whether this is indeed the case, we analyzed CD16 expression by αβ T-cells in blood samples from patients with hyperlymphocytosis that were retrieved non-selectively from the Department of Infectious Diseases (above 4000 lymphocytes/μl) . Twenty six healthy controls and fifteen subjects with hyperlymphocytosis were tested. The results shown in figure 6 demonstrate a significant increase (p=0.0002) in the proportion of CD16+ cells among the αβ T- lymphocyte subset in the patients (range 2.13-26.38%) compared to the healthy volunteer control group (range 0.04-15.0%) . These results demonstrate a systematic increase in the proportion of CD16+ cells among αβ T-lymphocytes during these T cell responses.

CD16+ αβ T-lymphocytes appear in cultures of EBV-specific cytotoxic T cells (EBV-CTL) .

The data presented above demonstrate that reactive T- lymphocytes can express CD16. Nevertheless, neither the specificity of these T-cells nor the time delay between their encounter with antigen and CD16 expression was known. To directly test CD16 levels and its kinetics of expression by memory T-cells of known specificity, we analyzed CD16 expression in EBV-specific T cell lines. In vitro stimulation of PBMC from an EBV seropositive donor with the autologous EBV B lymphoblastoid cell line allowed for the amplification of the EBV-specific memory T cell repertoire. In this situation it becomes possible to analyze antigen expression directly on virus-specific T-cells and at a documented time point after antigen exposure. We analyzed EBV-specific cytotoxic T cell lines from 6 healthy donors. The results shown in Figure 5 demonstrate that CD16+ αβ T-lymphocytes were detected in all EBV-specific cytotoxic T cell lines. CD16 expression became detectable from days 15 to 20 after restimulation with the autologous BLCL. The frequency of CD16+ EBV-CTL ranged from 1 to 27% (Fig7A) . Finally, the ADCC activity of these EBV-CTL containing significant numbers of CD16+ T-cells was revealed by their cytotoxic activity against an allogeneic BLCL in the presence of an anti-CD20 mAb (Fig7B) .

Claims

1. Natural CD16+ T cells for enhancing ADCC.

2. Natural CD16+ T cells according to claim 1, for treating cancer.

3. Natural CD16+ T cells according to anyone of claim 1 or 2, for treating cancer wherein said natural CD16+ T cells are administrated via a parenteral route.

4. Natural CD16+ T cells according to claim 3, for treating cancer, wherein said natural CD16+ T cells are administrated intravenously.

5. Natural CD16+ T cells according to anyone of claim 1 to 4, for treating cancer, wherein said natural CD16+ T cells are administrated in combination with at least one immuno- therapeutic agent such as tumor antigens or monoclonal therapeutic antibodies.

6. Natural CD16+ T cells according to claim 5, for treating cancer, wherein said immuno-therapeutic agent comprises at least one tumor antigen selected from the group consisting of peptides derived from the MAGE, BAGE, GAGE and LAGEl/NY-ESO-1 gene families.

7. Natural CD16+ T cells according to claim 5, for treating cancer, wherein said immuno-therapeutic agent comprises at least one monoclonal therapeutic antibody selected from the group consisting of Infliximab, Basiliximab, Daclizumab, Trastuzumab, Rituximab, Ibritumomab tiutexan, Tositumomab, Gemtuzumab ozogamicin, Alemtuzumab.

8. Natural CD16+ T cells according to anyone of claim 1 to 7, for treating cancer, wherein said natural CD16+ T cells are administrated in combination with at least one chemotherapeutic agent selected from the group consisting of 5-fluorouracil ; aziathioprine; cyclophosphamide; anti^¬ metabolites such as fludarabine; anti-neoplasties such as etoposide, doxorubicin, methotrexate, vincristine; prednisone; carboplatin; cis-platinum and the taxanes such as taxol.

9. Natural CD16+ T cells according to anyone of claim 1 to 4, for treating cancer, wherein said natural CD16+ T cells are administrated in combination with antitumoral vaccination.

10. Natural CD16+ T cells for treating and/or preventing infectious diseases.

11. Isolated natural CD16+ T cell clone.

12. Isolated natural CD16+ T cell clone according to claim 11, further expressing a TCR of known specificity.

13. Isolated natural CD16+ T cell clone according to anyone of claim 11 to 12, expressing a TCR directed against a virus selected from the group consisting of Epstein Barr viruses (EBV) , cytomegaloviruses (CMV) , human papilloma viruses (HPV) and herpes simplex virus (HSVl, HSV2) .

14. Isolated natural CD16+ T cell clone according to anyone of claim 11 to 13, expressing a TCR directed against HLA molecules .

15. Isolated natural CD16+ T cell clone according to anyone of claim 11 to 14, further expressing a specific HLA combination that is widespread in the recipient individuals.

16. Method for isolating a natural CD16+ T cell clone, comprising: isolating natural CD16+ T cells from PBL, purifying said natural CD16+ T cells, cloning said natural CD16+ T cells, optionally further expanding the at least one T cell clone thus obtained.

17. Method for producing a natural CD16+ T cell clone expressing a TCR of known specificity, and optionally expressing a specific HLA combination that is widespread in the recipient individuals, comprising: isolating and expanding at least one (known- antigen) -specific natural CD16+ T cell optionally expressing a specific HLA combination that is widespread in the recipient individuals , cloning said (known-antigen) -specific T cell, isolating at least one (known-antigen) - specific T cell clone, optionally purifying said (known-antigen) - specific T cell clone, and optionally expanding said (known- antigen) -specific T cell clone.

18. Pharmaceutical composition comprising natural CD16+ T cells.

19. Pharmaceutical composition comprising at least one isolated natural CD16+ T cell clone according to anyone of claims 11 to 15.

20. Pharmaceutical composition according to claim 18 or 19 for treating diseases or conditions requiring an ADCC enhancement such as cancers .

21. Kit comprising: at least one pharmaceutical composition comprising: o natural CD16+ T cells, or o at least one isolated natural CD16+ T cell clone according to anyone of claims 11 to 15, and at least one therapeutic agent such as: o a tumor antigen selected from the group consisting of peptides derived from the MAGE,

BAGE, GAGE and LAGEl/NY-ESO-1 gene families, and/or o a monoclonal antibody selected from the group consisting of Infliximab, Basiliximab, Daclizumab, Trastuzumab, Rituximab, Ibritumomab tiutexan, Tositumomab, Gemtuzumab ozogamicin, Alemtuzumab .

22. Kit according to claim 21, further comprising at least one chemotherapeutic agent selected from the group consisting of etoposide, doxorubicin, vincristine, cyclophosphamide, prednisone, fludarabine.