ES2668538T3 - Innovative artificial antigen presenting cells and uses thereof - Google Patents

Abstract

In vitro method of activation or stimulation of T cells, the method comprising contacting said T cells with a transduced K562 cell using a lentiviral vector expressing CD64 and 4-1BBL, wherein said K562 cell is loaded with an anti-antibody CD3 and an anti-CD28 antibody, and wherein the binding of said anti-CD3 antibody and said anti-CD28 antibody with said T cells activates or stimulates said T cells.

Description

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DESCRIPTION

Innovative artificial antigen presenting cells and their uses Declaration on federally supported research or development

This research was supported in part by US state funds. (grants from the National Institutes of Health R21 AI060477, R01 CA105216 and R01 AI 057838 and, therefore, the US government may have certain rights in the invention.

Background of the invention

Adoptive transfer is a term coined by Medawar (1954, Proc. Royal Soc. 143: 58-80) to study allograft rejection. The term adoptive immunotherapy indicates the transfer of immunocompetent cells for the treatment of cancer or infectious diseases (June, CH, ed., 2001, In: Cancer Chemotherapy and Biotherapy: Principles and Practice, Lippincott Williams & Wilkins, Baltimore; Vonderheide et al. , 2003, Immun. Research 27: 1-15). Adoptive therapy can be considered a strategy aimed at replacing, repairing or enhancing the biological function of a damaged tissue or system by means of autologous or allogeneic cells. The first satisfactory infusion of polyclonal CD4 T cells, infected with HIV, expanded ex vivo, which allowed a high degree of grafting after infusion, was performed using magnetic beads coated with anti-CD3 and anti-CD28 beads (beads coated with CD3 / 28) to expand ex vivo T cells of HIV-infected individuals. Eight patients were given 51 infusions of costimulated CD4 cells according to this protocol with minimal adverse events (Levine et al., 2002, Nature Med. 8: 47-53).

HIV infection induces a pronounced expansion of HIV-specific CD8 T cells. These CD8 T cells release soluble factors (Walker et al., 1986, Science 234: 1563-1566; Zhang et al., 2002, Science 298: 995-1000; Cocchi et al., 1995, Science 270: 1811-1815) which limit HIV replication as well as directly lyse HIV-infected cells (Walker et al., 1987, Nature 328: 345-348; Koup et al., 1994, J. Virol. 68: 4650-4655). The reduction of CD8 T cells before exposure to VIS leads to uncontrolled viral replication and rapid death, indicating that the activity of CD8 T cells is necessary to make HIV a chronic disease (Schmitz et al. 1999 , Science 283: 857-860; Jin et al., 1999, J. Exp. Med. 189: 991-998). However, CD8 T cells cannot ultimately control HIV infection. HIV-specific T cells have been shown to have a highly reduced perforin expression (Zhang et al., 2003, Blood 101: 226-235; Appay, et al., 2000, J. Exp. Med. 192: 63- 75), regulation by decrease of two key signaling receptors, CD3 ^ and CD28 (Trimble et al., 2000, Blood 96: 1021-1029), a biased maturation pattern (Appay et al., 2002, Nature Med. 8 , 379-385) and a high sensitivity to Fas-induced apoptosis (Mueller et al., 2001, Immunity, 15: 871-882). Therefore, it is believed that optimal activation of HIV-specific CD8 T cells will restore effector functions.

The anti-CD3 and anti-CD28 coated (CD3 / 28) beads were the first generation of artificial APCs (aAPC) that allowed the expansion of HIV-infected CD4 T cells (Levine et al., 1996, Science 272: 1939-1943 ). In addition to providing the necessary signals for the activation and growth of T cells, stimulation with beads with CD3 / 28 makes T cells resistant to R5 infection by regulating CCR5 decrease and regulating the expression of their ligands, the p -RANTES chemokines, macrophage-1 alpha inflammatory protein (MIP-1a) and MIP-1P (Riley et al., 1997, J. Immunol. 158: 5545-5553 Carroll et al., 1997, Science 276: 273-276 ). Several phase I and II trials have shown that the infusion of expanded autologous CD4 T cells using beads coated with CD3 / 28 in individuals infected with R5 is both safe and feasible (Carroll et al., 1997, Science 276: 273-276; Levine et al., 2002, Nature Med. 8: 47-53; Walker et al., 2000, Blood 96: 467-474; Ranga et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1201- 1206). More importantly, sustained increases in total lymphocyte count, the ratio of CD4 T cells to CD8, the fraction of cytokine-secreting T cells and the ability to respond to recall antigens were observed, suggesting that adoptive cell immunotherapy T can restore at least one limited immune function again in HIV-infected individuals (Levine et al., 2002, Nature Med. 8: 47-53). Despite the success of these initial trials, several limitations were observed, including the difficulty of (1) expanding CD8 T cells, (2) adding additional costimulatory signals that may be required to expand certain subsets of T cells, (3) removing the beads before infusion and (4) generate antigen specific T cells with high graft potential.

Others have used CD3 / 28 coated T-cell expansion beads to introduce genetically modified T cells into HIV-infected patients. In these studies (Walker et al., 2000, Blood 96: 467-474; Mitsuyasu et al., 2000, Blood 96: 785-793; Deeks et al., 2002, Mol. Ther. 5: 788-797), a chimeric molecule consisting of the extracellular domain of CD4 and the intracellular domain of the zeta chain of CD3 was introduced (CD4 ^ was introduced into CD4 T cells by retroviral transduction). Modified CD4 ^ T cells were detected by DNA PCR in the peripheral blood of all patients after infusion, and mean levels of 1-3% of total peripheral blood mononuclear cells (PBMC) were maintained at all points of time after infusion. In an extended follow-up, CD4 ^ was detected in the blood of 17 of 18 patients one year after the infusion. These levels of sustained grafting are several orders of magnitude greater than what was previously observed for infusions of human T-cells, perhaps because previous cell culture techniques may have induced susceptibility to apoptosis or replicative senescence (Rosenberg et al., 1990 , N. Engl. J. Med. 323: 570-578; Yee et al., 2002, Proc.

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Natl Acad. Sci. U.S.A. 99: 16168-16173; Brodie et al., 1999, Nature Med. 5: 34-41; Riddell et al., 1996, Nature Med. 2: 216-223; Riddell et al., 2000, Cancer Journal 6: S250-S258). These clinical results indicate that costimulated T cells propagated with pearl-based aAPC are safe and have the ability for prolonged grafting. However, due to the limited number of study subjects and the length of time required to achieve a clinical assessment criterion in an HIV therapeutic trial, the statistical significance of the clinical benefit of autologous CD4 T cell transfer to individuals could not be measured. HIV infected.

Although they are potentially effective in limiting immunodeficiency, polyclonal CD4 T cells are likely to have only a moderate effect on the specific response to HIV. Immunotherapy of viral infection in humans has been studied using adoptive transfer of antigen-specific CD8 T cells in the environment of CMV, EBV and HIV infection. This approach has been evaluated using T-cell clones with HLA-restricted antigen specificity for CMV (Riddell et al., 1992, Science 257: 238-241; Walter et al., 1995, N. Engl. J. Med. 333: 1038-1044). CMV-specific CD8 + T cells isolated from identical MHC bone marrow donors were expanded ex vivo and administered to 14 allogeneic bone marrow transplant recipients. Recovery of CMV-specific CTL activity was observed in each case and CTL undergoing adoptive transfer persisted in vivo for up to 12 weeks. In a similar study, donor-specific CD8 + and CD4 + T cells derived from donors, genetically labeled with the neomycin resistance gene, were administered to 6 allogeneic bone marrow allograft recipients with T-cell reduction (Rooney et al., 1995 , Lancet 345: 9-13; Heslop et al., 1996, Nature Med. 2: 551-555). Gene-labeled CD4 + and CD8 + T cells that respond to exposure in vivo or ex vivo to EBV persisted at low frequencies in vivo for up to 18 months after infusion. Infusion of CD8 T cells with a single specificity for HIV (Nef) (Koenig et al., 1995, Nature Med. 1: 330-336) in a patient demonstrated CTL selection of viral variants indicating that the infusion of cells HIV-specific T versus multiple targets may be necessary to control HIV replication. In all these studies, the inability of the vast majority of these T cells to graft has limited the study of the long-term effects of immunotherapy of antigen-specific CD8 T cells. An important challenge in the field is to expand CD8 T cells that will be grafted and will have potent long-term effector functions to more effectively fight chronic infections. However, despite the long-term need for sufficient numbers of therapeutic T cells, there are no methods available to expand these cells.

HIV-specific T cells may contain but not eradicate HIV. Some studies that have removed CD8 T cells before, or during, HIV infection have shown uncontrolled viral replication and a much faster progression to AIDS, indicating that CD8 T cells are important in HIV control (Schmitz et al. 1999, Science 283: 857-860; Jin et al., 1999, J. Exp. Med. 189: 991-998). However, HIV-specific T cells generally lack perforin expression (Gandhi et al., 2002, Annu. Rev. Med. 53: 149-172) and other effector functions required to eliminate host HIV. Some studies of patients without long-term progression indicated that they are more likely to proliferate HIV-specific T cells from these individuals and contain perforin, demonstrating that CD8 T cells with enhanced effector functions may slow the progression to AIDS (Migueles et al., 2002, Nature Immunol. 3: 1061-1068). Other researchers have shown that two key signaling receptors, CD3 ^ and CD28 are regulated by decrease in HIV-specific T cells (Trimble et al., 2000, Blood 96: 1021-1029; Trimble et al., 1998, Blood 91: 585-594; Trimble et al., 2000, J. Virol. 74: 7320-7330), and that HIV-specific T cells are more sensitive to Fas-induced apoptosis (Mueller et al., 2001, Immunity, 15 : 871-882). Appay et al. (2002, Nature Med. 8: 379-385) examined the differences between HIV-specific CD8 T cells, VeB and CMV based on the expression of CD27 and CD28. T cells with early differentiation expressed both CD27 and CD28 and had few effector functions but excellent proliferative capabilities. Intermediate T cells were positive for CD27 but negative for CD28, and these cells had limited proliferative and effector functions. The most differentiated T cells lack both CD27 and CD28, and these cells had poor proliferative capacity but enhanced effector functions. It was observed that most of the HIV-specific T cells had the intermediate phenotype. Therefore, the fact that cells are "trapped" in this intermediate T cell phenotype that lacks both proliferative capacity and effector functions may be the contributing factor to the inefficiency of HIV-specific T cells (Appay et al., 2002 , Nature Med. 8, 379-385). In addition, the function of CD8 T cells depends greatly on the function of CD4 T cells (Zajac et al., 1998, J. Exp. Med. 188: 2205-2213; Shedlock et al., 2003, Science 300: 337 -339; Sun et al., 2003, Science 300: 339-342) and since HIV selects CD4 T cells as target, the defects of CD8 T cells observed in HIV infection could be the result of a lack of help by T cells.

US 2003/224520 A1 and WO 03/057171 A1 refer to methods for activating and expanding cells, preferably to a method of cell activation and stimulation using a multiple length signaling platform, obtained by genetic engineering. Compositions of activated and expanded cells by the methods are further described. Stimulated and / or expanded T cells can be used for therapeutic purposes. US 2003/147869 A1 refers to a system and methods to promote the expansion of antigen specific and polyclonal cytotoxic T lymphocytes in response to artificial antigen presenting cells. Thomas A. K. et al: "A cell-based artificial antigen-presenting cell coated with anti-CD3 and CD28 antibodies enable rapid expansion and long-term growth of CD4 T lymphocytes", Clinical Immunology, Academic Press, USA. vol. 105, No. 3, December 3, 2002, pages 259-272 discloses the ability of two genetically modified myeloid cells, K562 and U937 to serve as antigen presenting cells

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artificial. Maus MV et al .: "Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T cell receptor, CD28 and 4-1 BB", Nature Biotechnology, Nature Publishing Group, New York, NY, USA, vol. 20, No. 2, February 2002, pages 143-184 discloses the ex vivo priming and expansion of human cytotoxic T lymphocytes and their potential for use in immunotherapeutic applications for cancerous and infectious diseases.

Therefore, there has been a long-felt need to provide ways to stimulate T cells to combat various acute and chronic diseases and to promulgate sufficient numbers of therapeutic T cells for adoptive immunotherapy. The present invention satisfies this and other needs.

Brief summary of the invention

The present invention includes an in vitro method of activation or stimulation of T cells, the method comprising contacting said T cells with a transduced K562 cell using a lentiviral vector expressing CD64 and 4-1BBL, in which said K562 cell is loaded with an anti-CD3 antibody and an anti-CD28 antibody, and wherein the binding of said anti-CD3 antibody and said anti-CD-218 antibody with said T cells activates or stimulates said T cells. The present invention further relates to a K562 cell transduced using a lentiviral vector expressing CD64 and 4-1BBL, wherein said K562 cell is loaded with an anti-CD3 antibody and an anti-CD28 antibody, for use in the activation or stimulation of T cells.

Brief description of the drawings

In order to illustrate the invention, certain embodiments of the invention are shown in the drawings. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments represented in the drawings.

Figure 1 is a diagram illustrating a model for the construction of a T-cell culture system based on artificial antigen presenting cells (aAPC) using a parental K562 human erythromyloid cell line. Figure 1 depicts an engine-modified aAPC K32 / 4-1BBL that interacts with a CD8 + T cell.

Figure 2, comprising Figures 2A to 2C, depicts the expansion of central memory T cells specific for influenza (influenza) viruses classified by tetramers using aAPC K32 / 4-1BBL / CD3 / 28. Figure 2A depicts a series of images demonstrating the classification of CD8 T cells from an HLA-A2 donor who had been exposed to the influenza virus. Figure 2B is a graph that shows that these cells could be maintained in culture for 70 days. The total number of cells that would have accumulated is represented if cells had not been discarded as a semi-logarithmic graphical representation of the total number of cells versus days in culture. Figure 2C demonstrates a chromium release assay using TLA-positive TLA-deficient T2 cells loaded with either the flu peptide or leaving them unchecked in the effector ratios with respect to target indicated.

Figure 3, comprising Figures 3A to 3E, illustrates the creation of an aAPC that can be used to expand influenza-specific T cells. Figure 3 represents a series of five (5) images that demonstrate the analysis by fAcS for each of the markers CD32 (figure 3A), KA2 (figure 3B), 4-1BBL (figure 3D) and FluGFP (green fluorescent protein, Figure 3E) by the aAPC and also representing an isotype control.

Figure 4 is a diagram illustrating an experimental model demonstrating methods of expanding HIV-specific CD8 T cells with broad specificity.

Figure 5 is a graph showing that K562-based aAPCs (for example, K32 / CD3 / 28, K32 / 86 / CD3) mediate the long-term growth of CD4 T cells and do so more effectively than aAPC based on U937 (U32 / CD3 / 28) or aAPC based on pearls (pearls coated with CD3 / 28).

Figure 6, comprising Figures 6A through 6D, is a graph demonstrating the induction of costimulatory and cytokine gene expression in aAPC based on K562 but not in aAPC based on U937. Figure 6A is a graph depicting that the level of induction of interleukin 15 (IL-15) by K32 / CD3 / 28, K32 / CD3, K32, U32 / CD3 / 28, U32 / CD3, U32 and T CD4 cells in repose. Figure 6B depicts the induction of PD-L1 in cells, demonstrating that aAPC K32 / CD3 / 28 express substantially higher levels of PD-L1. Figure 6C is a graph depicting the induction of PD-L2 by various aAPCs as described above. Figure 6D is a graph depicting the induction of B7-H3 by various aAPC.

Figure 7 is a diagram representing the growth rate of aAPC transduced with lentivirus (LV) grown in Aim V media (Invitrogen, Carlsbad, CA) supplemented with 3% human Ac serum (Valley Biomedical, Winchester, VA). Various aAPCs based on K562 were produced by transducing parental K562 (k562cc; dark rhombus) using the following LV vector constructs: KT32 (1 gene; dark squares); KT32 / 4-1BBL / CD86 (3 genes; light triangles); KT32 / 4-1BBL / CD86 / A2 / Flu-GFP (5 genes; "X"). The total number of cells that would have accumulated is represented if cells had not been discarded as a semi-logarithmic graphical representation of the total number of cells versus days in culture.

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Figure 8, comprising Figures 8A through 8E, is a graph depicting the stable co-expression of at least eight (8) genes in a single aAPC K562 (8-THREAT). The following genes were transduced in a K562 cell and stably expressed, as detected using flow cytometry: Flu-GFP (Figure 8A); CD80 (Figure 8B); CD86 (Figure 8C); 4-1BBL (Figure 8D); and HLA ABC (figure 8E).

Figure 9 is a graph demonstrating the long-term expansion of polyclonal CD8 T cells using aAPC transduced with LV.

Figure 10, comprising Figures 10A-10E, is a series of graphs showing that aAPC K32 / 4-1BBL expanded hTERT-specific cytotoxic lymphocytes (CTLs). Figures 10A to 10C are graphs representing the increasing percentage of CTL CD8 tet + during expansion by aAPC K32 / 4-1BBL. The timing of the MoFlo classification corresponding to each figure 10A-10C is indicated in the graph showing population duplications (figure 10D). Figure 10D is a graph depicting the expansion of hTERT-specific CTLs by aAPC, while CTLs were obtained from a patient with hTERT-vaccinated breast cancer. Figure 10E is a graph demonstrating that hTERT-specific CTLs expanded using aAPC specifically lyse carcinoma cells that express HLA-A2 and telomerase + (OV-7) but not carcinoma cells that are telomerase + but that are HLA-A2- (SK-OV-3).

Figure 11, comprising Figure 11A to Figure 11D, depicts data comparing aAPC transfection using a plasmid with expression of transduced molecules in an otherwise identical aAPC using an LV. Figure 11A is a diagram representing an exemplary LV used herein, representing the particular modifications disclosed elsewhere in this document. Figures 11B and 11C represent the efficiency of transduction of K562 cells from a single virus inoculum expressing GFP (monocistronic) or mCD8 IRES GFP (bicistronic). The surface expression of mCD8 and GFP was measured 5 days after transduction.

Figure 12, comprising Figures 12A through 12F, represents a representative experiment, in which it was observed that 8,000 antigen-specific T cells produced 2X106 cells after one month of culture. In summary, purified T cells obtained from an HLA donor A * 0201 were stained, with anti-CD8 AcM and an MHC A * 0201 tetramer complexed to an A * 0201 restricted epitope of the influenza matrix protein (tetramer of Flu virus MP). The positive population for the tetramer (approximately 8000 cells) was classified and stimulated with aAPC KTA2 / CD32 / 4-1BBL / FLU GFP irradiated that were loaded with anti-CD28 antibody. The cells were re-stimulated with aAPC KTA2 / CD32 / 4-1BBL / fLu GFP (Figures 12A-D) approximately every 10-12 days. Interleukin 2 (IL-2) was added to the culture in each cell feed (every 2-3 days). Figures 12E and 12F are graphs demonstrating the purity of the reactive cells with the flu tetramer before and after 26 days of expansion. That is, an expansion of approximately 250 times of the population positive for the tetramer was observed in these culture conditions.

Figure 13 is a bar graph depicting the level of cytokine expression using an aAPC K562 transduced with CD32 IRES IL-7 (KT32-IL7) and K562 transduced with CD32 IRES IL-15 (KT32-IL15) in which They classified the cells for high CD32 expression.

Figure 14 is a graphical representation demonstrating the stable expression of CD64 on the surface of a K562 cell transduced with a lentiviral vector expressing CD64.

Figure 15 is a graph illustrating the increase in antibody binding capacity of K562 cells transfected with a lentiviral vector expressing CD64 (K64 cells).

Figure 16 is a series of graphs illustrating that K64 cells loaded with antibody and washed multiple times are superior in stimulation of T cells compared to K562 cells expressing CD32 (K32 cells), although both K64 cells and K32 cells can stimulate T cells.

Figure 17, comprising Figures 17A to 17D, is a series of images illustrating that much less antibody is required to optimally load K64 cells compared to K32 cells. Figures 17A and 17B represent CD4 cells, Figures 17C and 17D represent CD8 cells.

Figure 18 is a graph illustrating the increase in the expansion of Treg cells stimulated with K32 cells loaded with anti-CD3 and anti-CD28 antibodies compared to Treg cells stimulated with anti-CD3 and anti-CD28 coated beads.

Figure 19 is a graph depicting the ability of Treg cells to suppress an allogeneic mixed lymphocyte reaction (MLR).

Figure 20 is a graph showing that CD4 + CD25 + Treg cells stimulated using K32 cells expressing OX40L make non-suppressor Treg cells.

Figure 21, comprising Figures 21A through 21C, is a series of images illustrating the expansion of antigen-specific CD8 cells using K32 loaded with anti-CD3 antibody and expressing IL-15, 4-1BBL and CD80.

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Detailed description of the invention

The application refers to the surprising discovery that lentivirus vectors can be used to efficiently produce aAPC that stably express numerous T-cell stimulating and costimulatory ligands, and antibodies thereof, as well as antigens, cytokines, among other molecules. The application also refers to the novel aAPCs produced and methods for their use to expand a desired T cell, activate and / or expand subsets of specific T cells, identify stimulatory molecules, costimulatory molecules, and combinations thereof, which can promote expansion of subsets of specific T cells, as well as numerous therapeutic uses related to the expansion and stimulation of T cells using novel aAPCs.

As demonstrated by the data disclosed herein, upon activation of T cells, factors such as IFNy are secreted which in turn induce the expression of cytokines such as IL-15 and costimulatory ligands such as B7-H3 in K562 cells (Thomas et al., 2002, Clin. Immunol. 105: 259-272). The exchange or "interference" between aAPC and a T cell is one reason why cell-based aAPCs are more efficient T-cell expansion systems than pearl-based aAPCs. K562 cells are engineered so that these cells are a continuously commercially available "commercially available" dendritic cell (DC) replacement system. The use of aAPC would obviate the time and expense required to generate autologous DC as a source of APC for cell culture. Additional costimulatory signals may be necessary to rescue effector functions of HIV-specific CD8 T cells and as demonstrated by the data herein, aAPC cells may be modified to express such signals as desired. Again, this is an advantage over pearl-based systems that do not encompass adding additional costimulatory signals that may be required to expand certain subsets of T cells.

Previously, cell-based aAPCs were created by electroporation of K562 cells with 4-1BBL and CD32 expression plasmids. Using a combination of drug selection, cell classification and limiting dilution, high-expression clones were isolated (Maus et al., 2002, Nature Biotechnol. 20: 143-148). Although effective, this approach takes a lot of time and is limited by the need to use drug selection markers. The dependence on drug selection restricts the number of constructs that can be introduced into K562 cells and poses compliance problems with GMP when clinical use is contemplated.

Definitions

As used herein, each of the following terms has the meaning associated with it in this section.

The articles "a" and "a" are used herein to refer to one or more than one (ie, at least one) of the grammatical object of the article. As an example, "an element" means an element or more than one element.

As used herein, "relieving" a disease means reducing the severity of one or more symptoms of the disease.

As used herein, "amino acids" are represented by their full name, by the three letter code corresponding to them, or by the one letter code corresponding to them, as indicated in the next table:

Full name Aspartic acid Glutamic acid Lysine Arginine Histidine Tyrosine Cysteine Asparagine Glutamine Serine Threonine Glycine Alanine Valine Leucine Isoleucine Methionine Proline Phenylalanine

Three-letter code Asp Glu Lys Arg

His

Tyr

Cys

Asn

Gln

Be

Thr

Gly

To

Val

Leu

Ile

Met

Pro

Phe

One letter code D E K R

H

Y C N Q S T G A

V L I

M

P

F

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Tryptophan Trp W

"Antisense" refers particularly to the nucleic acid sequence of the non-coding chain of a double-stranded DNA molecule encoding a protein, or to a sequence that is substantially homologous to the non-coding chain. As defined herein, an antisense sequence is complementary to the sequence of a double stranded DNA molecule that encodes a protein. It is not necessary that the antisense sequence be complementary only to the coding part of the DNA molecule coding chain. The antisense sequence may be complementary to regulatory sequences specified in the coding chain of a DNA molecule encoding a protein, whose regulatory sequences control the expression of the coding sequences.

By the term "applicator," as the term is used herein, is meant any device that includes, but is not limited to, a hypodermic syringe, a pipette, and the like, to administer the compounds and compositions of the invention. .

A "disease" is a state of health of an animal in which the animal cannot maintain homeostasis, and in which if the disease is not improved, then the animal's health continues to deteriorate. Instead, a "disorder" in an animal is a state of health in which the animal can maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. If left untreated, a disorder does not necessarily cause an additional decrease in the animal's health status.

By the term "effective amount", as used herein, it is meant an amount that when administered to a mammal, causes a detectable level of T-cell response compared to the T-cell response detected in the absence of compound. The T cell response can be easily evaluated by a multitude of methods recognized in the art.

The person skilled in the art would understand that the amount of the compound or composition administered herein varies and can easily be determined based on several factors such as the disease or condition being treated, the age and the health and physical state of the mammal being treated. treats, the severity of the disease, the particular compound that is administered, and the like.

"Instructional material," as that term is used herein, includes a publication, a record, a diagram, or any other means of expression that may be used to communicate the usefulness of the composition and / or compound of the invention in the kit to alleviate or treat the various diseases or disorders listed herein. Optionally, or alternatively, the instructional material may describe one or more methods of relieving diseases or disorders in a cell or a tissue or a mammal, including as disclosed elsewhere herein.

The instructional material of the kit can, for example, be fixed to a container containing the compound and / or composition of the invention or sent together with a container containing the compound and / or composition. Alternatively, the instructional material can be sent separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively.

As used herein, the term "pharmaceutically acceptable carrier" means a chemical composition with which the active ingredient can be combined and which, after combination, can be used to administer the active ingredient to a subject.

As used herein, the term "physiologically acceptable" ester or salt means an ester or salt form of the active ingredient that is compatible with any other component of the pharmaceutical composition, which is not detrimental to the subject to which The composition will be administered.

By "complementary to a part or all of the nucleic acid encoding for" a protein of the invention, is meant a nucleic acid sequence that does not encode, for example, a costimulatory ligand protein. Rather, the sequence that is expressed in the cells is identical to the non-coding chain of the nucleic acid that codes for the protein and therefore does not code for the protein.

The terms "complementary" and "antisense" as used herein are not completely synonymous. "Antisense" refers particularly to the nucleic acid sequence of the non-coding chain of a double-stranded DNA molecule encoding a protein, or to a sequence that is substantially homologous to the non-coding chain.

"Complementary" as used herein refers to the broad concept of sequence complementarity of subunits between two nucleic acids, for example, two DNA molecules. When a nucleotide position in both molecules is occupied by nucleotides normally capable of performing base pairing with each other, then the nucleic acids are considered complementary to each other in this position. Therefore, two nucleic acids are complementary to each other when a substantial number (at least 50%) of the corresponding positions in each of the molecules is occupied by nucleotides that normally perform base pairing with each other (for example, pairs of nucleotides A: T and G: C). As defined herein

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document, an antisense sequence is complementary to the sequence of a double-stranded DNA molecule that encodes a protein. It is not necessary that the antisense sequence be complementary only to the coding part of the DNA molecule coding chain. The antisense sequence may be complementary to regulatory sequences specified in the coding chain of a DNA molecule encoding a protein, whose regulatory sequences control the expression of the coding sequences.

A "coding region" of a gene consists of the nucleotide residues of the gene coding chain and the nucleotides of the non-coding chain of the gene that are homologous with or complementary to, respectively, the coding region of an mRNA molecule that is produced by gene transcription.

A "coding region" of an mRNA molecule also consists of nucleotide residues of the mRNA molecule that match an anticodon region of a transfer RNA molecule during translation of the mRNA molecule or that encode a codon of termination. The coding region can, therefore, include nucleotide residues corresponding to amino acid residues that are not present in the mature protein encoded by the mRNA molecule (eg, amino acid residues in a protein export signal sequence).

"Coding for" refers to the inherent property of specific nucleotide sequences in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for the synthesis of other polymers and macromolecules in biological processes that have or either a defined nucleotide sequence (ie, rRNA, tRNA and mRNA) or a defined amino acid sequence and the resulting biological properties. Therefore, a gene encodes a protein if the transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding chain, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in sequence lists, as the non-coding chain, used as a template for the transcription of a gene or cDNA, can be referred to as encoding the protein. or another product of that gene or cDNA.

Unless otherwise specified, a "nucleotide sequence that codes for an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that code for the same amino acid sequence. Nucleotide sequences encoding proteins and RNA can include introns.

"Expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient elements that act in cis for expression; The host cell can provide other elements for expression or in an in vitro expression system. Expression vectors include all known in the art, such as cosmids, plasmids (for example, naked or contained in liposomes) and viruses (for example, retroviruses, lentiviruses, adenoviruses and adeno-associated viruses) that incorporate the recombinant polynucleotide.

A first region of an oligonucleotide "flanks" a second region of the oligonucleotide if the two regions are adjacent to each other or if the two regions are separated by no more than approximately 1000 nucleotide residues, and preferably no more than approximately 100 nucleotide residues. .

As used herein, the term "fragment" as applied to a nucleic acid, may normally be at least about 18 nucleotides in length, preferably, at least about 24 nucleotides, more usually, about 24 at about 50 nucleotides, preferably, at least about 50 to about 100 nucleotides, even more preferably, at least about 100 nucleotides at about 200 nucleotides, even more preferably, at least about 200 to about 300, even more preferably, from at least about 300 nucleotides to about 400 nucleotides, even more preferably, from at least about 400 to about 500, and most preferably, the nucleic acid fragment will not be greater than about 500 nucleotides in length.

As applied to a protein, a "fragment" of a stimulant or costimulatory ligand protein or an antigen is approximately 6 amino acids in length. More preferably, the fragment of a protein is about 8 amino acids, even more preferably, of at least about 10, even more preferably, of at least about 15, even more preferably, of at least about 20, even more preferably, of at less about 30, even more preferably, about 40, and more preferably, at least about 50, more preferably, at least about 60, even more preferably, at least about 70, even more preferably, at least about 80 , and more preferably, at least about 100 amino acids in length.

A "genomic DNA" is a DNA chain that has a nucleotide sequence homologous to a gene as it exists in the natural host. As an example, a fragment of a chromosome is a genomic DNA.

"Homologous" as used herein refers to the similarity of subunit sequences between two polymeric molecules, for example, between two nucleic acid molecules, for example, two DNA molecules.

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or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, for example, if a position is occupied in each of the two DNA molecules per adenine, then they are 100% homologous or completely in that position. The percentage of homology between two sequences is a direct function of the number of matching or homologous positions, for example, if half (for example, five positions in a polymer of ten subunits in length) of the positions in two compound sequences are homologous , then the two sequences are 50% identical, if 90% of the positions, for example, 9 of 10, coincide or are homologous, the two sequences share 90% homology. As an example, the 5'ATTGCC3 'and 5'TATGGC3' DNA sequences share 50% homology.

In addition, when the terms "homology" or "identity" are used herein to refer to nucleic acids and proteins, it should be construed to apply to homology or identity in both nucleic acid and amino acid sequence levels.

An "isolated nucleic acid" refers to a nucleic acid fragment or segment that has been separated from sequences flanking it in a naturally occurring state, for example, a DNA fragment that has been removed from the sequences that They are normally adjacent to the fragment, for example, sequences adjacent to the fragment in a genome in which it occurs naturally. The term also applies to nucleic acids that have been substantially purified from other components that naturally accompany the nucleic acid, for example, RNA or DNA or proteins, that naturally accompany them in the cell. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into a virus or plasmid that replicates autonomously, or into the genomic DNA of a prokaryotic or eukaryotic, or that exists as a separate molecule ( for example, as cDNA or genomic fragment or cDNA produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene that codes for an additional polypeptide sequence.

In the context of the present invention, the following abbreviations for commonly produced nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytidine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

In describing two polynucleotides as "operably linked" it is meant that a single-stranded or double-stranded nucleic acid moiety comprises the two polynucleotides disposed within the nucleic acid moiety such that at least one of the two polynucleotides can exert a physiological effect by which It is characterized on the other. By way of example, a promoter operatively linked to the coding region of a gene can promote transcription of the coding region.

Preferably, when the nucleic acid encoding the desired protein further comprises a promoter / regulatory sequence, the promoter / regulator is positioned at the 5 'end of the sequence encoding a desired protein so as to drive the expression of the desired protein. in a cell Together, the nucleic acid encoding the desired protein and its promoter / regulatory sequence comprise a "transgene."

As used herein, the term "promoter / regulatory sequence" means a nucleic acid sequence that is required for the expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, this sequence may be the central promoter sequence and in other cases, this sequence may also include an enhancer sequence and other regulatory elements that are required for the expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a living human cell in most or all physiological conditions of the cell.

An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a living human cell substantially only when an inducer corresponding to the promoter is present. in the cell

A "tissue specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a living human cell substantially only if the cell is a cell. of the type of tissue corresponding to the promoter.

A "polyadenylation sequence" is a polynucleotide sequence that directs the addition of a poly A tail to a transcribed messenger RNA sequence.

A "polynucleotide" means a single chain or parallel or antiparallel chains of a nucleic acid. Thus, a polynucleotide can be either a single stranded or double stranded nucleic acid.

The term "nucleic acid" normally refers to large polynucleotides.

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The term "oligonudeotide" normally refers to short polynucleotides, generally, not greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T."

Conventional notation is used herein to describe polynucleotide sequences: the left end of a single stranded polynucleotide sequence is the 5 'end; The left direction of a double stranded polynucleotide sequence is called the 5 'address.

The direction of the 5 'to 3' addition of nucleotides to nascent RNA transcripts is called the transcription address. The DNA chain that has the same sequence as an mRNA is called the "coding chain"; the sequences in the DNA chain that are located at 5 'with respect to a reference point in the DNA are called "sequences towards 5'"; the sequences in the DNA chain that are 3 'from a reference point in the DNA are called "sequences towards 3'."

A "part" of a polynucleotide means at least about twenty sequential nucleotide residues of the polynucleotide. It is understood that a part of a polynucleotide can include each nucleotide residue of the polynucleotide.

"Primer" refers to a polynucleotide that can specifically hybridize with a designated polynucleotide template and that provides a starting point for the synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions where synthesis is induced, that is, in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization such as DNA polymerase. A primer is normally single stranded, but it can be double stranded. The primers are normally deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers are useful for many applications. A primer is complementary to the template with which it is designed to hybridize to serve as a site for the initiation of the synthesis, but does not need to reflect the exact sequence of the template. In such a case, the specific hybridization of the primer with the template depends on the rigor of the hybridization conditions. The primers can be labeled with, for example, chromogenic, radioactive or fluorescent moieties and used as detectable moieties.

"Probe" refers to a polynucleotide that can specifically hybridize with a designated sequence of another polynucleotide. A probe specifically hybridizes with a complementary target polynucleotide, but does not need to reflect the exact complementary sequence of the template. In such a case, the specific hybridization of the probe with the target depends on the rigor of the hybridization conditions. Probes can be labeled with, for example, chromogenic, radioactive or fluorescent moieties and used as detectable moieties.

"Recombinant polynucleotide" refers to a polynucleotide that has sequences that are not linked together naturally. An amplified or assembled polynucleotide can be included in a suitable vector, and the vector can be used to transform a suitable host cell.

A recombinant polynucleotide can also serve a non-coding function (eg, promoter, origin of replication, ribosome binding site, etc.).

A "recombinant polynucleotide" is one that occurs after the expression of a recombinant polynucleotide.

"Polypeptide" refers to a polymer composed of amino acid residues, naturally occurring related structural variants, and synthetic, non-naturally occurring analogs thereof linked by peptide bonds, naturally occurring structural variants related, and analogs that are produced in a non-natural way. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer.

The term "protein" normally refers to large polypeptides.

The term "peptide" normally refers to short polypeptides.

Conventional notation is used herein to represent polypeptide sequences: the left end of a polypeptide sequence is the amino end; the right end of a polypeptide sequence is the carboxyl end.

As used herein, the term "reporter gene" means a gene, the expression of which can be detected using a known method. By way of example, the Escherichia coli lacZ gene can be used as an indicator gene in a medium because expression of the lacZ gene can be detected using known methods by adding a chromogenic substrate such as o-nitrophenyl-p-galactoside to the medium (Gerhardt et al. , eds., 1994, Methods for General and Molecular Bacteriology, American Society for Microbiology, Washington, DC, p. 574).

A "restriction site" is a part of a double stranded nucleic acid that is recognized by a restriction endonuclease.

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A first oligonucleotide is hybridized with a second oligonucleotide "with high stringency" if the two oligonucleotides hybridize under conditions where only oligonucleotides that are at least about 73%, more preferably, at least about 75%, even more preferably, at least about 80%, even more preferably, at least about 85%, even more preferably, at least about 90%, and most preferably, at least about 95%, complementary hybridize each. The stringency of the conditions used to hybridize two oligonucleotides is a function of, among other factors, the temperature, the ionic strength of the hybridization medium, the incubation period, the length of the oligonucleotides, the GC content of the oligonucleotides, and the expected degree of non-homology between the two oligonucleotides, if known. Methods for adjusting the stringency of hybridization conditions are known (see, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

As used herein, the term "transgene" means an exogenous nucleic acid sequence whose exogenous nucleic acid is encoded by a mammal or transgenic cell.

A "recombinant cell" is a cell that comprises a transgene. Such a cell can be a eukaryotic cell or a prokaryotic cell. In addition, the transgenic cell encompasses, but is not limited to, an aAPC, an embryonic stem cell comprising the transgene, a cell obtained from a chimeric mammal derived from a transgenic ES cell in which the cell comprises the transgene, a cell obtained of a transgenic mammal, or fetal or placental tissue thereof, and a prokaryotic cell comprising the transgene.

By the term "exogenous nucleic acid" is meant that the nucleic acid has been introduced into a cell or an animal using technology that has been developed in order to facilitate the introduction of a nucleic acid into a cell or an animal.

By "tag" polypeptide is meant any protein that, when linked by a peptide to a protein of interest, can be used to locate the protein, to purify it from a cell extract, to immobilize it for use in binding assays, or to otherwise study its biological properties and / or function.

As used herein, "treating" means reducing the frequency with which a patient experiences the symptoms of a disease (ie, viral infection, tumor growth and / or metastasis, or other effect mediated by diminished numbers and / or decreased activity of T cells, and the like).

By the term "vector" as used herein, is meant any plasmid or virus encoding an exogenous nucleic acid. The term should also be construed as including non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into virions or cells, such as, for example, polylysine compounds and the like. The vector may be a viral vector that is suitable as a delivery vehicle for the administration of a nucleic acid encoding a protein and / or antibody of the invention, to the patient, or to the aAPC, or the vector may be a non-viral vector. That is suitable for the same purpose.

Examples of viral and non-viral vectors for the administration of DNA to cells and tissues are well known in the art and are described, for example, in Ma et al. (1997, Proc. Natl. Acad. Sci. U.S.A. 94: 12744-12746). Examples of viral vectors include, but are not limited to, a lentiviral vector, a recombinant adenovirus, a recombinant retrovirus, a recombinant adeno-associated virus, a recombinant avian smallpox virus, and the like (Cranage et al., 1986, EMBO J. 5: 3057-3063; International Patent Application No. WO94 / 17810, published August 18, 1994; International Patent Application No. WO94 / 23744, published October 27, 1994). Examples of non-viral vectors include, but are not limited to, liposomes, DNA polyamine derivatives, and the like.

A "therapeutic" treatment is a treatment administered to a patient who shows signs of pathology in order to decrease or eliminate those signs and / or decrease the frequency, duration and intensity of the signs.

An "effective amount" of a compound is the amount of a cell (for example, an aAPC or T cell stimulated and / or expanded in this way) that is sufficient to provide a detectable effect to a population of T cells, or to a mammal , to which the aAPC is administered and / or contacted when compared to an otherwise identical population of T cells, or mammal, to which the aAPC, or thus expanded T cell, is not administered.

The person skilled in the art would understand that the effective amount varies and can easily be determined based on several factors such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound or cell that is administered, the level of activity or expression of the aAPC or T cell thereby expanded, and the like. Generally, the effective amount will be set between about 0.1 mg / kg and about 100 mg / kg, more preferably about 1 mg / kg and 25 mg / kg. The compound or cell (for example, a cytokine, a ligand or stimulatory molecule thereof, a ligand or costimulatory molecule thereof, an antibody that specifically binds with a ligand, a nucleic acid encoding these proteins, an aAPC , a T cell thus expanded, and the like) may be administered through intravenous injection, or administered to a tumor site, and includes, among other things, a bolus injection. However, the invention is not limited to this, or any other method of administration.

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A "therapeutic" treatment is a treatment administered to a patient who shows signs of pathology in order to decrease or eliminate those signs and / or decrease or decrease the frequency, duration and intensity of the signs.

By the term "stimulation," it is meant a primary response induced by the binding of a stimulatory molecule (eg, a TCR / CD3 complex) with its related ligand thereby mediating a signal transduction event, such as, but without limited to, signal transduction through the TCR / CD3 complex. Stimulation can mediate the altered expression of certain molecules, such as regulation by decrease of TGF-p, and / or reorganization of cytoskeleton structures, and the like.

"Activation," as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term "activated T cells" refers to, among other things, T cells that are undergoing cell division.

By the term "specifically binds," as used herein, is meant an antibody, or a ligand, that recognizes and binds with a protein (for example, a stimulatory and / or costimulatory molecule present in a cell T) of related binding partner present in a sample, but that the antibody or ligand does not recognize or binds substantially to other molecules in the sample.

A "stimulator ligand," as used herein, means a ligand that when present in a cell that has an antigen (for example, an aAPC, a dendritic cell, a B cell, and the like) can specifically bind with a related binding partner (referred to herein as "stimulatory molecule") in a T cell, thereby mediating a primary response by the T cell, which includes, but is not limited to, activation, initiation of an immune response , proliferation, and the like. Stimulant ligands are well known in the art and encompass, among others, a class I MHC molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.

A "stimulatory molecule," as the term is used herein, means a molecule in a T cell that specifically binds to a related stimulator ligand present in a cell that has an antigen (eg, an aAPC of the invention , among others).

"Loaded" with a peptide, as used herein, refers to a presentation of an antigen in the context of an MHC molecule. "Loaded" as used herein also means the binding of an antibody to an Fc binding receptor in a cell, such as CD32 and / or CD64.

"Costimulatory ligand," as the term is used herein, includes a molecule in a cell that has antigen (eg, aAPC, dendritic cell, B cell, and the like) that specifically binds to a related costimulatory molecule in a T cell, thus providing a signal that, in addition to the primary signal provided by, for example, the binding of a TCR / CD3 complex with a peptide-loaded MHC molecule, mediates a T-cell response, which includes, but It is not limited to, proliferation, activation, differentiation, and the like. A costimulatory ligand may include, but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L) , intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, beta lymphotoxin receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds to Toll-like ligand receptor and a ligand that specifically binds with B7-H3. A costimulatory ligand also encompasses, among others, an antibody that specifically binds to a costimulatory molecule present in a T cell, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1 , ICOS, lymphocyte-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.

A "costimulatory molecule" refers to the related binding partner in a T cell that specifically binds with a costimulatory ligand, thereby mediating the T-cell a costimulatory response, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to a MHC class I molecule, BTLA and a Toll-like ligand receptor.

"Supersragon antibody," as used herein, means an antibody that specifically binds to a molecule in a T cell and can mediate a primary activation signal event in a T cell without interaction of the TCR / CD3 complex. or CD2 in the T cell. Such a superagonist antibody includes, but is not limited to, a superagonist anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.

Unless referred to as "superagonist," an anti-CD2 antibody, an anti-CD28 antibody, and the like, is a costimulatory ligand as defined elsewhere herein, and provides a costimulatory signal instead of a primary activation signal.

"Treating" a disease as the term is used herein means reducing the frequency of the disease or disorder by reducing the frequency with which a patient experiences a symptom of the one or more symptoms of the disease or disorder.

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By the term "vaccine" as used herein, is meant a composition, a protein or a nucleic acid encoding a protein, or an aAPC of the invention, which serves to protect an animal against disease and / or to treat an animal already affected by a disease by inducing an immune response, compared to an animal otherwise identical to the one that is not given the vaccine or compared to the animal before the administration of the vaccine.

"Immunovacuna," as used herein, means an aAPC that can elicit a detectable immune response when administered to an animal. More preferably, an immunovacuna is an aAPC that stimulates and activates T cells when administered to the animal, so that it generates an immune response of T cells to a pathogen, a tumor cell, and the like, when the response is compared with a T cell. immune, if it exists, in an animal otherwise identical to the one that is not given the immunovacuna.

Description

The invention relates to the surprising finding that a human erythromyloid cell line, K562, which does not express MHC class I or class II molecules, and which was previously believed to be resistant to genetic manipulation techniques, can be easily transduced using vectors of lentivirus to express numerous molecules, which includes, but is not limited to, stimulant ligands, costimulatory ligands, antigens (eg, tumor, viral, and the like), cytokines, etc.

In addition, the data disclosed herein demonstrate that several (at least nine) exogenous nucleic acids expressing several proteins can be easily introduced and expressed in these cells, but that the level of protein expression is greater than that achieved using Plasmid-based expression systems and expression is stable and continues for many months to decrease detectable. In addition, the K562-based artificial antigen presenting cell (aAPC), which does not express CHM class I or II molecules, can be transduced with and easily expressed. Notably, aAPC transduced with a nucleic acid encoding an antigen of interest processed the antigen and properly presented it to a T cell thereby producing antigen-specific T cells without identifying the epitope recognized by the T cell. Surprisingly, as demonstrated by the data disclosed herein, the aAPC cell was properly processed and presented the antigen described above.

I. Compositions

An isolated artificial antigen presenting cell (aAPC) is disclosed, in which the cell comprises a transduced K562 cell using a lentiviral vector (LV). In addition, the LV codes for at least one immune stimulatory and costimulatory ligand. Although the data disclosed herein demonstrates that approximately nine nucleic acids encoding approximately nine different transduced molecules in a K562 cell were highly and stably expressed in long-term culture, there is nothing to suggest that this is a limit of the number or classes of molecules that can be introduced into these cells. Instead, any molecule or ligand, either stimulator, costimulator, cytokine, antigen, Fcy receptor, and the like, can be introduced into these cells to produce an aAPC of the invention.

The person skilled in the art will appreciate, based on the disclosure provided herein, that numerous immunoregulatory molecules can be used to produce an almost unlimited variety of aAPC once endowed with the teachings provided herein. That is, there is extensive knowledge in the art regarding the events and molecules involved in T cell activation and induction, and treaties that comment on T-cell mediated immune responses, and the factors that mediate them, are well known in The technique. There is extensive disclosure provided in WO 03/057171 and US2003 / 0147869. More specifically, a primary signal, usually mediated through the T / CD3 cell receptor complex in a T cell, initiates the T cell activation process. Additionally, numerous costimulatory molecules present on the surface of a T cell are involved in the regulation of the transition from resting T cell to cell proliferation. Such costimulatory molecules, also called "costimulators," which specifically bind to their respective ligands, include, but are not limited to, CD28 (which binds with B7-1 [CD80], B7-2 [CD86]), PD- 1 (which binds to PD-L1 and PD-L2 ligands), B7-H3, 4-1BB (binds to 4-1BBL ligand), OX40 (binds to oX40L ligand), ICOS (binds to ICOS ligand -L) and LfA (binds to the ICAM ligand). Therefore, the primary primary stimulatory signal in T cell stimulation, but the costimulatory signal is then required for T cell activation, as demonstrated by proliferation.

Therefore, the aAPC disclosed is a cell comprising a stimulant ligand that specifically binds with a TCR / CD3 complex such that a primary signal is transduced. Additionally, as one skilled in the art will appreciate, based on the disclosure provided herein, the aAPC further comprises at least one costimulatory ligand that specifically binds with at least one costimulatory molecule present in a T cell, costimulatory molecule that includes , but not limited to, CD27, CD28, CD30, CD7, a ligand that specifically binds with CD83, 4-1bB, PD-1, OX40, ICOS, LFA-1, CD30L, NKG2C, B7-H3, MHC Class I, BTLA, Toll and LIGHT type ligand receptor. This is because, as previously discussed and as demonstrated by the data disclosed elsewhere herein, a costimulatory signal is required to induce T cell activation and associated proliferation.

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60

Other costimulatory ligands are encompassed in the invention, as will be understood by one skilled in the art endowed with the teachings provided herein. Such ligands include, but are not limited to, a mutant, a variant, a fragment and a homologue of the natural ligands described previously.

These and other ligands are well known in the art and have been well characterized as described, for example, in Schwartz et al., 2001, Nature 410: 604-608; Schwartz et al., 2002, Nature Immunol. 3: 427-434; and Zhang et al., 2004, Immunity. 20: 337-347. Using the extensive knowledge in the art concerning the ligand, the person skilled in the art, endowed with the teachings provided herein will appreciate that a mutant or a variant of the ligand is also described and can be transduced into a cell using an LV to produce the aAPC and such mutants and variants are discussed in more detail elsewhere in this document.

Thus, the aAPC disclosed comprises at least one stimulatory ligand and at least one costimulatory ligand, such that aAPC can stimulate and expand a T cell comprising a related binding partner stimulating molecule that specifically binds with the ligand stimulator in the aAPC and a related binding partner costimulatory molecule that specifically binds with the costimulatory ligand in the aAPC in such a way that the interaction between the ligands in the aAPC and the corresponding molecules in the T cell mediates, among other things, in T cell proliferation, expansion and immune response as desired. One skilled in the art will appreciate that when particular stimulatory and costimulatory molecules are known in a T cell of interest, an aAPC of the invention can easily be produced to expand that T cell. Conversely, when stimulatory and costimulatory molecules are not known In a T cell of interest, an aAPC panel can be used to determine which molecules, and combinations thereof, can expand that T cell. Therefore, tools for the expansion of desired T cells are provided, as well as tools to elucidate the molecules in particular T cells that mediate the activation and proliferation of T cells.

The person skilled in the art will understand that nucleic acids encompass an RNA or DNA sequence encoding a protein of the invention, and any modified form thereof, including chemical modifications of the DNA or RNA that make the nucleotide sequence more stable when free of cells or when associated with a cell. Chemical modifications of nucleotides can also be used to enhance the efficiency with which a nucleotide sequence is captured by a cell or the efficiency with which it is expressed in a cell. Each and every one of the combinations of modifications of the nucleotide sequences are contemplated herein.

In addition, any number of methods can be used for the generation of mutant, derivative or variant forms of a protein using a recombinant DNA methodology that is well known in the art such as, for example, that described in Sambrook and Russell (2001, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY), and Ausubel et al. (2002, Current Protocols in Molecular Biology, John Wiley & Sons, NY). Procedures for the introduction of amino acid changes in a protein or a polypeptide by altering the DNA sequence encoding the polypeptide are well known in the art and are also described in these treaties, and others.

In addition, a nucleic acid encoding a costimulatory ligand, or antigen, is described in which a nucleic acid encoding a tag polypeptide covalently binds to it. That is, a chimeric nucleic acid is described in which the nucleic acid sequences encoding a tag polypeptide covalently bind to the nucleic acid encoding at least one protein of the invention, or biologically active fragment thereof. Such tag polypeptides are well known in the art and include, for example, green fluorescent protein (GFP), an influenza virus hemagglutinin tag polypeptide, a herpes virus tag polypeptide, myc, myc-pyruvate kinase (myc -PK), His6, maltose binding protein (MBP), a FLAG tag polypeptide and a glutathione-S-transferase (GST) tag polypeptide. However, the invention should not be considered in any way limited to the nucleic acids encoding the tag polypeptides listed above. Rather, it should be considered that any nucleic acid sequence encoding a polypeptide that can function in a manner substantially similar to these tag polypeptides is disclosed herein.

The nucleic acid comprising a nucleic acid encoding a tag polypeptide can be used to locate a protein, or a biologically active fragment thereof, within a cell, a tissue and / or a whole organism (eg, a being human, and the like), and to study the role (s) of protein in a cell. In addition, the addition of a tag polypeptide facilitates the isolation and purification of the "tagged" protein such that proteins can be easily produced and purified. More importantly, as demonstrated elsewhere in this document, the expression of a costimulatory ligand comprising a tag allows the detection of ligand expression, and also allows the isolation of cells expressing the ligand using many methods , including, but not limited to, cell classification.

Also described are protein or peptide analogs comprising a costimulatory ligand as disclosed herein. The analogs may differ from proteins or peptides that occur naturally in conservative differences of the amino acid sequence or in modifications that do not affect the sequence, or both. For example, conservative amino acid changes can be made, which although they alter the primary sequence of the protein or peptide, normally do not alter its function. The substitutions of

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Conservative amino acids normally include substitutions within the following groups:

glycine, alanine;

valine, isoleucine, leucine;

aspartic acid, glutamic acid;

asparagine, glutamine;

serine, threonine;

lysine, arginine;

phenylalanine, tyrosine.

Modifications (which normally do not alter the primary sequence) include, in vivo or in vitro, chemical derivatization of polypeptides, for example, acetylation or carboxylation.

Also included are glycosylation modifications, for example, those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in additional processing steps; for example, by exposing the polypeptide to enzymes that affect glycosylation, for example, glycosylation or deglycosylation enzymes of mammals. Sequences that have phosphorylated amino acid residues, for example, phosphotyrosine, phosphoserine or phosphotreonin, are also encompassed.

Polypeptides that have been modified using standard molecular biology techniques are also included so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to make them more suitable as a therapeutic agent. Analogues of such polypeptides include those that contain residues other than naturally occurring L-amino acids, for example, D-amino acids or synthetic amino acids that do not occur naturally. The peptides of the invention are not limited to products of any of the specific exemplary processes listed herein.

The application also encompasses "mutants", "derivatives" and "variants" of the peptides (or of the DNA encoding them) mutants, derivatives and variants that are costimulatory ligands, cytokines, antigens (eg, tumor cell, viral, and other antigens), which are altered in one or more amino acids (or, when referring to the nucleotide sequence encoding them, are altered in one or more base pairs) such that the peptide (or DNA) ) resulting is not identical to the sequences cited herein, but has the same biological properties as the peptides disclosed herein, because the peptide has the biological / biochemical properties of a costimulatory ligand, cytokine, antigen, and similar, of the present invention (eg, expression by an aAPC when the aAPC expressing the protein is contacted with a T cell, mediates proliferation of, or af ecta otherwise a, cell T).

Among a "biological activity", as used herein, a costimulatory ligand is included which, when transduced into a K562 cell, is expressed and, when the cell contacts a T cell that expresses a related costimulatory molecule on its surface, it mediates the activation and stimulation of the T cell, with induced proliferation.

Indeed, a powerful novel test assay for the identification of mutants, variants, fragments and homologs of costimulatory ligands in which a possible novel form of a costimulatory ligand can be transduced and expressed in the aAPC of the invention is described. The ability of aAPC to stimulate and / or activate a T cell can be evaluated and compared with the ability of an aAPC comprising the wild or "natural" costimulatory ligand to stimulate and / or otherwise activate an identical T cell. In this way, functional variants can be easily identified, isolated and characterized that demonstrate the ability to activate / stimulate the T cell to a greater, lesser or equal extent than the wild-type control ligand. Such novel variants of costimulatory ligands are possible research tools for elucidation of T-cell processes, and also provide important therapeutic potential based on the inhibition or induction of T-cell activation / stimulation, such as, but not limited to, the administration of a variant with inhibitory activity that can compete with the natural ligand to inhibit unwanted T cell responses such as, but not limited to, transplant rejection. Conversely, a variant that demonstrates increased activity as a costimulatory ligand can be used to increase a desired T-cell response, such as, but not limited to, administration to an immunosuppressed patient. For example, an example variant ligand can be engineered to be more effective than the natural ligand or to favor the binding of a positive costimulatory molecule (CD28) at the expense of a negative regulator (CTLA-4). These and many other variations are encompassed in the invention.

One skilled in the art will appreciate, based on the disclosure provided herein, that a costimulatory ligand encompasses an antibody that specifically binds with the costimulatory molecule present in a T cell with which the ligand also binds. That is, the invention encompasses an aAPC comprising not

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only a costimulatory ligand (for example, CD80 and CD86, among others) that binds to a costimulatory molecule in a T cell (for example, CD28), but also encompasses at least one antibody that specifically binds with the costimulatory molecule ( for example, anti-CD28). Numerous antibodies against costimulatory molecules are currently available, or can be produced following procedures that are well known in the art.

The person skilled in the art will understand, based on the disclosure provided herein, that an aAPC comprising an antibody may be produced, as exemplified elsewhere herein, by introducing a nucleic acid encoding CD32, the receptor. of human Fcy, in the aAPC. In addition, as disclosed elsewhere herein, an aAPC that binds to an antibody, such as an antibody against CD3 or an antibody against CD28, can be produced by expressing a nucleic acid encoding CD64 in the aAPC . CD64 is the high affinity human FcyRI receptor. The CD32 and / or CD64 expressed on the surface of the aAPC can then be "loaded" with any desired antibody that binds with CD32 and / or CD64, including, but not limited to, antibody that specifically binds with CD3 and antibody that binds specifically binds with CD28. In addition, an aAPC is disclosed in which a nucleic acid encoding the antibody ligand of interest, perhaps bound to an IRES sequence, is transduced and expressed on the surface of the aAPC thereby eliminating the need for expression of CD32 and / or CD64 and the loading thereof. Thus, an aAPC transduced with a nucleic acid encoding at least one antibody that specifically binds to CD3, CD28, PD-1, B7-H3, 4-1BB, OX40, ICOS, CD30, HLA- is disclosed. DR, MHCII, Toll-type ligand receptor and LFA, among others, as well as an aAPC transduced with CD32 and / or CD64 and loaded with at least one antibody that specifically binds with the molecules mentioned above.

In addition, an aAPC is encompassed in which the costimulatory ligand is a related binding partner that specifically binds with a costimulatory molecule, as well as in which the ligand is an antibody that specifically binds with a costimulatory molecule, and any combination of the same, such that a single aAPC may comprise both nucleic acids encoding costimulatory ligands and / or as specific antibodies to costimulatory molecules present in the T cell, and any combination thereof.

An aAPC comprising a nucleic acid encoding an antigen of interest is also encompassed. A huge variety of antigens are included, such as, but not limited to, tumor antigens, for example, telomerase, T-cell recognized melanoma antigen (MART-1), genes encoding melanoma antigen, 1, 2 and 3 (MAGE-1, -2, -3), melanoma GP100, carcinoembryonic antigen (CEA), HER-2 / Neu breast cancer antigen, prostate specific serum antigen (PSA), Wilm 1 tumor (WT-1) , mucin antigens (MUC-1, 2, 3, 4) and B-cell lymphoma idiotypes. This is because, as demonstrated by the data disclosed elsewhere herein, the aAPC based in K562 comprising an antigen, they can process and present the antigen in the context of MHC (in which the cell is also transduced with a nucleic acid encoding a class I or class II MHC molecule) thereby producing cells T antigen specific and expanding a population thereof. The data disclosed demonstrate that hTERT-specific CTLs were produced by expanding hTERT + T cells using an aAPC transduced with CD32 and 4-1BBL (K32 / 4-1BBL). Therefore, aAPCs can be used to expand and produce sufficient antigen-specific T cells to administer the T cells to a patient in need, thus providing an immunovaccine treatment directed against tumor cells carrying the antigen. Thus, an antigen of interest can be introduced into an aAPC of the invention, in which the aAPC then presents the antigen in the context of the MCH class I or II complex, that is, the MHC molecule is "charged" with the antigen, and aAPC can be used to produce an antigen specific T cell.

Similarly, a viral antigen, or any other pathogen, can also be transduced and expressed by the aAPC. Data disclosed elsewhere herein demonstrate that matrix protein positive T cells (flu MP tetramer) classified and stimulated with irradiated aAPC cells (K2 / CD3 / 4-1BBL / FLU-GFP) loaded with anti-CD28 antibody expanded T cells providing large numbers of antigen-specific CTLs, specific for the viral antigen. These data demonstrate that the aAPCs of the invention can be used to expand and produce antigen-specific T cells that are to be used to treat viral infections, and other pathogens.

Additionally, the application encompasses an aAPC transduced with a nucleic acid encoding at least one cytokine, at least one chemokine, or both. This is because the data disclosed elsewhere in this document broadly demonstrates that an aAPC transduced with a nucleic acid encoding an interleukin (eg, IL-7, IL-15, and the like) expressed so stable interleukin. In addition, using an LV vector comprising an internal ribosome entry site (IRES), interleukin can be secreted from aAPCs (for example, a K562 cell transduced with an LV vector such as, but not limited to, pCLPS CD32 - IRES-IL-7, -12, -15, -18 and -21). Other cytokines that can be expressed by aAPC include, but are not limited to, interferon-y (IFNy), tumor necrosis factor-a (TNFa), SLC, IL-2, IL-4, IL-23, IL-27 and Similar. The invention further includes, but is not limited to, RANTES chemokine, MIP-1a, MIP-1b, SDF-1, eotaxin, and the like.

Therefore, the application encompasses a cytokine, including a full-length cytokine, fragment, homologue, variant or mutant of the cytokine. A cytokine includes a protein that can affect the biological function of another

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cell. A biological function affected by a cytokine may include, but is not limited to, cell growth, cell differentiation or cell death. Preferably, a disclosed cytokine can bind to a specific receptor on the surface of a cell, thereby affecting the biological function of a cell.

A preferred cytokine includes, among others, a hematopoietic growth factor, an interleukin, an interferon, a molecule of the immunoglobulin superfamily, a molecule of the family of tumor necrosis factor and / or a chemokine. A more preferred cytokine includes a granulocyte and macrophage colony stimulating factor (GM-CSF), tumor tumor necrosis factor alpha (TNFa), tumor tumor necrosis factor beta (TNFp), macrophage colony stimulating factor (M-CSF), interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-10 (IL -10), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21), interferon alpha (IFNa), beta interferon (IFNp), gamma interferon (IFNy) and IGIF , among many others.

A chemokine, including a homologue, variant, mutant or fragment thereof, encompasses an alpha-chemokine or a beta-chemokine, including, but not limited to, a C5a, interleukin-8 (IL-8), monocyte chemotactic protein 1 alpha (MIP1a), 1 beta monocyte chemotactic protein (MIP1P), monocyte chemoattractant protein I (MCP-1), monocyte chemoattractant protein 3 (MCP-3), platelet activation factor (PAFR), N-formyl -methionyl-leucyl- [3 H] phenylalanine (FMLPR), leukotriene B4 (LTB4R), gastrin release peptide (GRP), RANTES, eotaxin, lymphotactin, IP10, I-309, ENA78, GCP-2, NAP-2 and / or MGSA / gro. One skilled in the art will appreciate, once endowed with the teachings provided herein, that the invention encompasses a chemokine and a cytokine, as is well known in the art, as well as any discovered in the future.

The person skilled in the art will appreciate, once endowed with the teachings provided herein, that aAPC is in no way limited to any antigen, cytokine, costimulatory ligand, antibody that specifically binds to a particular costimulatory molecule, and the like. Rather, the invention encompasses an aAPC comprising numerous molecules, either expressed all under the control of a single promoter / regulatory sequence or under the control of more than one such sequence. In addition, the application encompasses the administration of one or more aAPCs disclosed in which the various aAPCs encode for different molecules. That is, the various molecules (for example, costimulatory ligands, antigens, cytokines, and the like) can act in cis (i.e., in the same aAPC and / or encoded by the same contiguous nucleic acid or in independent nucleic acid molecules within of the same aAPC) or in trans (that is, the various molecules are expressed by different aAPC).

Thus, as one skilled in the art will understand, based on the disclosure provided herein, the dose and timing of administration of the medications comprising the aAPC can be tailored specifically for each application. More specifically, when it is desired to provide stimulation to a T cell using certain molecules expressed by an aAPC, or several aAPC, followed by stimulation using another aAPC, or several aAPC, which expresses a different set of molecules, although overlapping, then combination can be used. of approaches in cis and trans. In essence, the aAPC disclosed, and the methods disclosed herein, provide an almost unlimited number of variations. The person skilled in the art, endowed with the teachings provided herein and the knowledge available in the art, can easily determine the desired approach for each particular T cell. Alternatively, based on the disclosure provided herein, which provides methods for evaluating the effectiveness of the T cell stimulation and expansion methods disclosed herein, the person skilled in the art can determine which approach (s) can apply to particular T cells that are going to expand or stimulate.

The person skilled in the art will understand, based on the disclosure provided herein, that various combinations of molecules that are to be expressed in the disclosed aAPCs may be favored. Although several of these combinations of molecules are indicated throughout the specification, including, but not limited to, the combinations exemplified in Tables 1, 2, 3, and 4. Rather, one skilled in the art will appreciate, based on the teachings provided herein, that a wide variety of combinations of molecules can be transduced into a cell to produce the aAPC of the invention. The molecules encompass those known in the art, such as those discussed herein, as well as the molecules that will be discovered in the future.

The application encompasses the preparation and use of pharmaceutical compositions comprising an aAPC of the invention as an active ingredient. Such a pharmaceutical composition may consist of the active ingredient alone, as a combination of at least one active ingredient (e.g., an effective dose of an APC) in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active substance and one or more pharmaceutically acceptable carriers, one or more additional components (active and / or inactive), or some combination thereof.

As used herein, the term "pharmaceutically acceptable carrier" means a chemical composition with which the active ingredient can be combined and which, after combination, can be used to administer the active ingredient to a subject.

The formulations of the pharmaceutical compositions described herein can be prepared.

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by any method known or developed from now on in the pharmacology technique. In general, such preparatory methods include the step of putting the active ingredient in association with a carrier or one or more other auxiliary components, and then, if necessary or desirable, forming or packaging the product in a single or multiple dose unit. desired.

Although the descriptions of pharmaceutical compositions provided herein refer primarily to pharmaceutical compositions that are suitable for ethical administration to humans, the person skilled in the art will understand that such compositions are generally suitable for administration to animals of all kinds. The modification of pharmaceutical compositions suitable for administration to humans in order to return the compositions suitable for administration to various animals is well understood, and the usual expert veterinary pharmacologist can design and perform such modification with merely usual experimentation, if required. . Subjects for which administration of the disclosed pharmaceutical compositions is contemplated includes, but is not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats. and dogs, birds including commercially relevant birds such as chickens, ducks, geese and turkeys, fish including farmed fish and aquarium fish, and crustaceans such as shellfish shellfish.

Pharmaceutical compositions that are useful in the methods of the invention can be prepared, packaged or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, intralesional, buccal, ophthalmic, intravenous, intraorganic or other administration. route of administration Other formulations contemplated include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active substance and immunologically based formulations.

A pharmaceutical composition may be prepared, packaged or sold in bulk, as a single dose unit, or as a plurality of individual unit doses. As used herein, a "unit dose" is a differentiated amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such dosage such as, for example, half or a third of such dosage.

The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional component in a pharmaceutical composition of the invention will vary, depending on the identity, size and condition of the treated subject and also depending on the route by which it is to be administered. the composition. By way of example, the composition may comprise between 0.1% and 100% (w / w) of active ingredient.

In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents. Additional agents particularly contemplated include antiemetics and scavengers such as cyanide and cyanate and AZT scavengers, protease inhibitors, reverse transcriptase inhibitors, interleukin-2, interferons, cytokines, and the like.

Controlled or sustained release formulations of a pharmaceutical composition of the invention can be prepared using conventional technology.

As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by the formation of a physical gap of a tissue of a subject and the administration of the pharmaceutical composition through the gap in the tissue. Therefore, parenteral administration includes, but is not limited to, the administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a non-surgical wound. tissue penetrant, and the like. In particular, it is contemplated that parenteral administration includes, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and renal dialysis infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations can be prepared, packaged or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations can be prepared, packaged or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, implantable or biodegradable or sustained release formulations. Such formulations may further comprise one or more additional components including, but not limited to, suspending, stabilizing or dispersing agents.

The pharmaceutical compositions can be prepared, packaged or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known technique, and may comprise, in addition to the active ingredient, additional components such as dispersing agents, wetting agents or suspending agents described herein. Such sterile injectable formulations can be prepared using a parenteral, non-toxic, acceptable diluent or solvent, such

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as water or 1,3-butanediol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic solution of sodium chloride and fixed oils such as synthetic mono or diglycerides. Other formulations that can be administered parenterally that are useful include those comprising the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a moderately soluble polymer or a moderately soluble salt.

The aAPC of the invention and / or expanded T cells using the aAPC, can be administered to an animal, preferably a human being. When expanded T cells are administered using an aAPC of the invention, the amount of cells administered can range from about 1 million cells to about 300 billion. When aAPC itself is administered, either with or without expanded T cells, they can be administered in an amount ranging from about 100,000 to about 1 billion cells in which the cells are infused into the animal, preferably a Human patient who needs it. Although the precise dosage administered will vary depending on any number of factors, including but not limited to, the type of animal and the type of pathological condition being treated, the age of the animal and the route of administration.

The aAPC can be administered to an animal as frequently as several times a day, or it can be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be apparent to those skilled in the art and will depend on any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc. .

An aAPC (or cells expanded by it) can be co-administered with the various other compounds (cytokines, chemotherapeutic and / or antiviral drugs, among many others). Alternatively, the compound (s) can be administered one hour, one day, one week, one month, or even more, in advance of the aAPC (or cells expanded by it), or any permutation thereof. . In addition, the compound (s) may be administered one hour, one day, one week, or even more, after administration of aAPC (or cells expanded therein), or any permutation thereof. The frequency and regimen of administration will be readily apparent to those skilled in the art and will depend on any number of factors such as, but not limited to, the type and severity of the disease being treated, age and health status. of the animal, the identity of the compound or compounds being administered, the route of administration of the various compounds and the aAPC (or cells expanded therein), and the like.

In addition, one skilled in the art will appreciate, based on the disclosure provided herein, that when aAPC is to be administered to a mammal, the cells are treated so that they are in a "non-growing state"; that is, cells cannot divide when administered to a mammal. As disclosed elsewhere herein, cells can be irradiated to make them unable to grow or divide once they are administered to a mammal. Other methods, including haptenization (for example, using dinitrophenyl and other compounds), are known in the art to make the cells to be administered, especially to a human being, unable to grow and these methods are not discussed further herein. document. In addition, the safety of administration of aAPC has been established which has been made that they cannot be divided in vivo in phase I clinical trials using aAPC transfected with plasmid vectors encoding some of the molecules discussed herein.

II. Methods

The application encompasses a method to specifically induce the proliferation of a T cell that expresses a known costimulatory molecule. The method comprises contacting a T cell that is to be expanded with an aAPC comprising a lentivirus vector encoding a ligand that specifically binds with that costimulatory molecule. As demonstrated elsewhere in this document, contacting a T cell with an aAPC based on K562 comprising, inter alia, a costimulatory ligand that specifically binds to a related costimulatory molecule expressed on the cell surface T, stimulates the T cell and induces the proliferation of T cells in such a way that large numbers of specific T cells can easily be produced. The aAPC expands the T cell "specifically" because only the T cells that express the particular costimulatory molecule expand through the aAPC. Therefore, when the T cell to be expanded is present in a mixture of cells, some or most of which do not express the costimulatory molecule, only the T cell of interest will be induced to proliferate and expand in the number of cells. The T cell can be further purified using a wide variety of cell separation and purification techniques, such as those known in the art and / or described elsewhere herein.

As will be appreciated by the person skilled in the art, based on the disclosure provided herein, it is not necessary to identify or isolate the T cell of interest prior to expansion using the aAPC. This is because the aAPC is selective and will only expand the T cell (s) that express the related costimulatory molecule.

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Preferably, the expansion of certain T cells is achieved using several aAPC or a single aAPC, which express various molecules, including, but not limited to, an antigen, a cytokine, a costimulatory ligand, an antibody ligand that specifically binds to the costimulatory molecule present in the T cell. As disclosed elsewhere herein, the aAPC may comprise a nucleic acid encoding CD32 and / or CD64 such that CD32 and / or CD64 expressed in The surface of aAPC can be "loaded" with any desired antibody as long as they bind to CD32 and / or CD64, which are Fcy receptors. This makes the "commercially available" aAPC easily adapt to stimulate any desired T cell.

The application encompasses a method to specifically induce the proliferation of a T cell that expresses a known costimulatory molecule. The method comprises contacting a population of T cells comprising at least one T cell expressing the known costimulatory molecule with an aAPC comprising an LV encoding a ligand of the costimulatory molecule. As disclosed elsewhere herein, when an aAPC expresses at least one costimulatory ligand that specifically binds with a costimulatory molecule in a T cell, the binding of the costimulatory molecule with its related costimulatory ligand induces proliferation. of the T cell. Therefore, the T cell of interest is induced to proliferate without first having to purify the cell from the cell population. In addition, this method provides a rapid test to determine if any cell in the population is going to express a particular costimulatory molecule of interest, since contacting the cells with the aAPC will induce the proliferation and detection of the growing cells, identifying that so that a T cell expressing a costimulatory molecule of interest was present in the sample. Thus, any T cell of interest in which at least one costimulatory molecule is known on the cell surface can be expanded and isolated.

The invention includes a method for specifically expanding a subset of the T cell population, namely the method of claim 1. As previously demonstrated elsewhere herein, the binding of the costimulatory molecule with its partner costimulatory ligand of binding induces T cell proliferation, thereby specifically expanding a subset of the T cell population. One skilled in the art will understand, based on the disclosure provided herein, that T cell subsets include helper T cells. (TH1 and TH2) CD4 expressing, cytotoxic T lymphocyte (CTL) (Tc1 or Tc2), regulatory T cells (Treg), Tc / s, virgins, memory, central memory, effector memory and yST cells. Therefore, enriched cell populations can easily be produced for a particular subset of T cells using the method of the invention.

The application also includes a method for identifying a costimulatory ligand, or a combination thereof, that specifically induces the activation of a subset of T cells. In summary, the method comprises contacting a population of T cells with an aAPC comprising a LV coding for at least one costimulatory ligand, and comparing the proliferation level of the T-cell subset brought into contact with the aAPC with the proliferation level of an otherwise identical subset of T cells not brought into contact with the aAPC. A higher level of proliferation of the T-cell subset brought into contact with the aAPC compared to the otherwise identical proliferation level of the subset of T-cell that did not contact the aAPC is an indication that the costimulator ligand induces specifically the activation of the subset of T cells to which the T cell belongs.

The method allows the identification of a costimulatory ligand that specifically expands a subset of T cells when it was not previously known what factor (s) expands that subset of T cells. The person skilled in the art will appreciate that in order to minimize the number of tests, it is preferred to transduce so many nucleic acids that code for costimulatory ligands such that the number of assays can be reduced. In addition, the method allows, by combining the various proteins (e.g., stimulator ligand, costimulatory ligand, antigen, cytokine, and the like), to evaluate which combination / combinations of factors will produce the most effective aAPC, or a combination of aAPC, to expand the subset of T cells. Thus, the various requirements for growth and activation for each subset of T cells can be examined.

In one aspect, the method comprises contacting various aAPCs with the subset of T cells without first characterizing the costimulatory molecules on the surface of the subset of T cells. In addition, the invention encompasses a method in which the molecule (s) ) costimulator (s) present on the surface of the subset of T cells is examined before bringing the aAPC into contact with the cell. Therefore, the present invention provides a novel assay to determine the growth requirements for various subsets of T cells.

The application encompasses a method to induce a T-cell response against an antigen in a mammal. The method comprises administering an aAPC that specifically induces the proliferation of a T cell specific for the antigen. Once sufficient numbers of antigen specific T cells are obtained using the aAPC to expand the T cell, the antigen specific T cells thus obtained are administered to the mammal according to the methods disclosed elsewhere herein, inducing from that way a T cell response to the antigen in the mammal. This is because, as demonstrated by the data disclosed herein, antigen-specific T cells can be easily produced by stimulating resting T cells using the aAPC of the invention.

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The application includes a method for inducing a T-cell response against an antigen in a mammal that needs it, the method comprising obtaining a population of mammalian cells, in which the population comprises T cells, contacting T cells with an aAPC presenting the antigen in the context of an MHC complex, in which contacting the T cells with the aAPC induces the proliferation of T cells specific for the antigen. Antigen-specific T cells are administered to the mammal, thereby inducing a T-cell response to the antigen in the animal that needs it. As previously established elsewhere in this document, the data disclosed elsewhere demonstrates widely that antigen-specific CTLs can easily be produced by contacting a T cell with an aAPC in which the aAPC presents the antigen in the context of an MHC complex. As previously indicated elsewhere herein, a wide variety of aAPC may be used, comprising numerous combinations of various molecules (costimulatory ligands, antibodies, antigens, MHC, and the like), to determine the optimal method for expanding the Antigen-specific T cells for administration to a mammal that needs it.

III. Kits

The application includes various kits comprising an aAPC of the invention, a nucleic acid encoding various proteins, an antibody that specifically binds to a costimulatory molecule on the surface of a T cell and / or a nucleic acid encoding the antibody. of the invention, an antigen or a cytokine, an applicator and instructional materials that describe the use of the kit to perform the methods of the invention. Although exemplary kits are described below, the content of other useful kits will be apparent to those skilled in the art in light of the present disclosure. Each of these kits is included in the invention.

The application includes a kit to specifically induce the proliferation of a T cell expressing a known costimulatory molecule. This is because contacting the T cell with an aAPC, specifically induces proliferation of the T cell. The kit is used according to the methods disclosed in the invention. In summary, the kit can be used to administer an aAPC of the invention to a T cell that expresses at least one costimulatory molecule. This is because, as disclosed in more detail elsewhere in this document, the data disclosed herein demonstrates that contacting a T cell with an aAPC comprising a costimulatory ligand that It specifically binds to the related costimulatory molecule present in the T cell, mediating the stimulation and activation of the T cell. In addition, the T cells produced using this kit can be administered to an animal to achieve therapeutic results.

The kit further comprises an applicator useful for administering the aAPC to T cells. The particular applicator included in the kit will depend, for example, on the method used to administer the aAPC, as well as on the T cells expanded by the aAPC, and such applicators. They are well known in the art and may include, among other things, a pipette, a syringe, an eyedropper, and the like. In addition, the kit comprises an instructional material for the use of the kit. These instructions simply make the disclosure provided in this document.

The kit includes a pharmaceutically acceptable carrier. The composition is provided in an appropriate amount as set forth elsewhere herein. In addition, the route of administration and the frequency of administration are as previously discussed elsewhere in this document.

The kit encompasses an aAPC comprising a multitude of molecules, such as, but not limited to those set forth in Tables 1, 2, 3 and 4 elsewhere in this document. However, the expert endowed with the teachings provided herein will readily appreciate that the application is not limited in any way to this or any other combination of molecules. In contrast, the combinations set forth herein are for illustrative purposes and do not limit in any way the combinations encompassed by the present invention. In addition, the kit comprises a kit in which each molecule to be transduced to the aAPC is provided as an isolated nucleic acid encoding a molecule, a vector comprising a nucleic acid encoding a molecule, and any combination thereof. same, including when at least two molecules are encoded by a contiguous nucleic acid and / or encoded by the same vector. The usual expert will understand that the application encompasses a multitude of constructs that code for the molecules of interest to be introduced into an aAPC of the invention.

The application is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Therefore, it should not be considered in any way that the invention is limited to the following examples, but rather should be considered as encompassing each and every variation that will become apparent as a result of the teachings provided herein.

Examples

Example 1: Development of artificial antigen presenting cells (aAPC) based on cells for adoptive immunotherapy

The intrinsic growth requirements of CD4 and CD8 T cells have been shown to differ (Deets et al., 1997,

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Eur. J. Immunol. 27: 598-608; Laux et al., 2000, Clin. Immunol 96: 187-197; Foulds et al., 2002, J. Immunol. 168: 1528-1532). A cell-based aAPC was designed to allow genetic manipulation of the expression of different costimulatory molecules in addition to CD28 for long-term growth of CD8 cells. The culture system was based on the fact that costimulatory signals, in addition to those provided by CD28, are required for optimal growth of CD8 cells. The human erythromyloid LMC cell line k562 (Lozzio et al., 1975, Blood 45: 321-334) was used as the basis for cellular aAPCs, because this cell line does not express HLA proteins that will promote allogeneic responses. However, K562 cells do express ICAM (CD54) and LFA-3 (CD58), both of which foster interactions with T cells (Figure 1). Other advantages of using K562 cells include, but are not limited to, the fact that irradiated K562 cells can be introduced into the clinical environment since these cells are free of mycoplasma, spread in serum-free medium and are easily destroyed by cytolytic lymphocytes. natural (NK). Although it is desirable to use K562 cells to produce such aAPC, K562 cells have been notoriously difficult to transduce (Kahl et al., 2004, J. Virol. 78: 1421). Surprisingly, the data disclosed herein demonstrate, for the first time, that K562 cells can be transduced, in series and / or in parallel, with a large amount of exogenous nucleic acids to express several molecules thus obtaining an aAPC library with desired phenotypes Such LV-based transduction is performed in series and / or in parallel, and MoFlo is used to clone cells that demonstrate a desired phenotype. In addition, the data demonstrates that an optimal promoter can be selected and promoter competition can be evaluated and eliminated if necessary or desired. The aAPC library produced is then evaluated using the methods of the invention to determine biological function in vivo, using a model recognized in the art, such as, but not limited to, a mouse NOD / SCID model.

With respect to the use of K562 cells to produce aAPC, the disclosures of U.S. Patent Application No. 10 / 336,135 (now published as U.S. Patent Application Publication No. US2003 / 0147869A1) and international patent application are mentioned No. PCT / US03 / 00339 (now published as international publication No. WO 03/057171A2).

Production of lentivial vectors

To avoid the limitations of transfection-based approaches described above to introduce genes into K562 cells, a series of high-title lentiviral vectors, as disclosed elsewhere herein, was used to stably introduce a broad network of costimulant ligands and MHC molecules in K562 cells. This allows the rapid and systematic production of a variety of aAPC and allows the determination of the combination of costimulatory molecules that provides the optimal effector and expansion functions to HIV-specific T cells. The data disclosed in this document demonstrate this approach.

As a non-limiting example, an aAPC may comprise some or all of the ligands, among other things, described herein. These various constructs are used to transduce K562 cells using LV. These are merely by way of example and the invention is not limited to these constructs, or any other particular construct, for the transduction of the aAPCs of the invention with a known molecule of interest to be expressed in the cells.

CD32: An LAP-transduced aAPC was produced comprising CD32 using CD32 (SEQ ID NO: 8) amplified from cDNA prepared from neutrophil RNA. In summary, neutrophils were isolated by Ficoll gradient from an apheresis product obtained from a normal anonymous donor. This PCR product was cloned into pcDNA3.1 by restriction sites of Kpn I and Not I that were added to the ends of each amplification primer. This vector was digested with XbaI and Sal I and cloned into pCLPS (Parry et al., 2003, J. Immunol. 171: 166-174) to create pCLPS / CD32. Supernatant containing high titre lentiviral vector was obtained by collecting transfected 293T cells that had been transfected using a split genome transfection method as described in Dull et al. (1998, J. Virol. 72: 8463-8471) and Parry et al. (2003, J. Immunol. 171: 166-174).

IL-7: An aAPC comprising IL7 was produced using IL-7 nucleic acid (SEQ ID NO: 9) amplified from cDNA and cloned into pcDNA3.1 / hygromycin. SOE was performed by PCR (consisting of three independent reactions) using primers designed with 5'-CD32-XbaI and 3'-IL-7-SalI. Additional templates used during the reaction included CD32-pcDNA3.1 and pCLPS / m8h28-IRES-YFP followed by digestion with restriction enzymes of the PCR products to produce CD32-IRES-IL-7 / pCLPS. The lentivirus was prepared as previously described elsewhere in this document.

IL-15: Plasmid pYAX / Hum-IL-15 (comprising SEQ ID NO: 10) was used, which comprises an IgE leader peptide bound to a mature IL-15 sequence. PCR primers were designed to add restriction sites of MluI and SalI to the plasmid sequence. The PCR product was digested with the respective enzymes and cloned into CD32-IRES-IL-7 / pCLPS (which was also cut with MluI and SalI to remove the IL-7 gene). The lentivirus was prepared as described herein.

IL-21: IL-21 (SEQ ID NO: 11) was amplified from activated human PBMC and cloned into the TOPO 3 vector (Invitrogen, Carlsbad, CA). Using BamHI and xhoI, the human IL-21 gene was cleaved from the vector and then cloned

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the insert in the first position of the NKG2D-IRES-DAP12 / pCLPS construct (which was produced using BamHI and Xhol). Human CD32 was amplified from CD32-pCLPS (as described above) using PCR primers flanking the ends comprising sites of MluI and SalI. The PCR product was digested with the respective enzymes and cloned into the second position to create IL21-IRES-CD32 / pCLPS. The lentivirus was produced as previously described elsewhere in this document.

OX40L: OX40L (SEQ ID NO: 12) was amplified from cDNA obtained from mature dendritic cells (Schlienger et al., 2000, Blood) and cloned into pcDNA3.1 / hygromycin. MluI and SalI restriction enzyme sites were added to the ends of PCR primers and the PCR product was digested with the respective enzymes and cloned into CD32-IRES-IL-7 / pCLPS, which was also cut with MluI and SalI to remove the IL-7 insert. The CD32-IRES-OX40L / pCLPS lentiviral vector was prepared as described elsewhere elsewhere herein.

4-1BBL: 4-1BBL (SEQ ID NO: 13) was amplified from cDNA obtained from activated B cells that were purified by negative selection as described above, and the cells were activated with PMA and ionomycin. The PCR product in which Kpn I and NotI sites were introduced at the ends was cloned into pcDNA3.1 / hygromycin digested with KpnI / Not I. The vector was digested with XbaI and Salt I and cloned into pCLPS lentiviral vector digested with XbaI / Salt I. The lentiviral vector pCLPS / 4-1BBL was prepared as described elsewhere elsewhere herein.

CD80: CD80 (SEQ ID NO: 14) was amplified from cDNA obtained from an immortalized B cell line (Vonderheide et al., 1999, Immunity 10: 673-679) using PCR primers that introduced BamHI sites and SalI at the ends of the PCR product. After digestion with these enzymes, CD80 was cloned into pCLPS lentiviral vector digested with BamHI / SalI. CD80-pCLPS lentiviral vector was produced as previously described elsewhere herein.

CD83: CD83 (SEQ ID NO: 15) was amplified from cDNA prepared from the Ramesh cell line, using primers to introduce XbaI and XhoI restriction sites at the ends of the PCR product. After digestion with these enzymes, CD83 PCR product was ligated into pCLPS (digested with XbaI / SalI). The CD83-pCLPS lentiviral vector was produced as previously described elsewhere herein.

CD86: CD86 (SEQ ID NO: 16) was amplified from cDNA obtained from mature dendritic cells (which were prepared as described in Schlienger et al., 2000, Blood) using PCR primers that had restriction sites of BamHI and Not I at the ends. The PCR product was digested with BamHI and Not I and ligated into pcDNA3.1 / digested hygromycin in a similar manner. This vector was digested with BamHI and Salt I and cloned into pCLPS. The pCLPS / CD86 lentiviral vector was produced as previously described elsewhere herein.

ICOS-L: ICOS-L (SEQ ID NO: 17) was amplified using PCR primers comprising restriction sites of Kpn I and Not I at the ends, using cDNA obtained from dendritic cells. The PCR product was cloned into pcDNA3.1 / hygromycin digested with KpnI and Not I. The plasmid was digested with BamHI and Xho I to cleave the ICOS-L insert that was cloned into pCLPS (digested with BamHI / XhoI) to generate ICOS-L-pCLPS. The lentiviral vector was produced as previously described elsewhere herein.

HLA-A * 0201: HLA-A * 0201 cDNA clone was obtained from The International Cell and Gene Bank, which is available to the public from the website of the International Histocompatibility Working Group (ihwg.org) in the shared resources of cell banks and genes (cbankover). HLA-A * 0201 was amplified by PCR using primers comprising Bam HI and Sal restriction sites at the ends and the amplification product was cloned into pCLPS. The pCLPS / HLA-A2 lentiviral vector was produced as previously described elsewhere herein.

Flu-GFP: A Flu-GFP fusion vector was used that comprised the entire region encoding the enhanced green fluorescent protein (BD Biosciences, Palo Alto, CA) fused with nucleotides 113-290 of the flu matrix protein 1 (SEQ ID NO: 18). This construct was digested with BamHI and Xho I and cloned into pCLPS digested with Bam HI and Salt I. The lentiviral vector pCLPS / GFP-Flu was produced as described elsewhere elsewhere herein.

DRa: DRa (SEQ ID NO: 19) and DRB4 (SEQ ID NO: 20) were cloned using cDNA obtained from CD4 T cells activated with CD3 / 28 using conventional techniques and each nucleic acid was cloned into pCLPS. To produce K562 cells expressing DR4, both vectors were simultaneously transduced into K562 and HLA-DR cells and cells expressing DR4 were isolated using flow cytometry as described elsewhere herein.

In addition, ILT3 (SEQ ID NO: 21) and ILT4 (SEQ ID NO: 22) were expressed in the aAPC disclosed essentially by methods disclosed elsewhere herein and known in the art.

Third generation, high efficiency and high titer lentiviral vectors (LV) were used to effectively produce aAPC. These vectors have several built-in safety features that make them ideally suited for therapeutic products for humans. Specifically, approximately 90% of

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HIV-1 sequences have been removed from the transfer vector leaving only the packaging and integration sequences physically linked to the payload gene. Incompetent packaged LVs are generated for replication using a split genome approach. Specifically, 293T cells are transfected with four independent plasmids encoding HIV gag / pol, VEV G protein (env), HIV rev and transfer vector. Lentiviral vectors were produced after transfection of HEK 293T cells cultured in RPMI 1640 (BioWhittaker, Inc. Rockville MD), 10% FCS, 2 mM glutamine and 100 IU / ml penicillin, 100 pg / ml streptomycin. Cells were seeded at 5x106 by T150 tissue culture flask 24 hours before transfection. All plasmid DNA was purified twice using a CsCl gradient. Cells were transfected with 7 pg of pMDG.1 (enveloped G of VEV), 18 pg of pRSV.rev (plasmid encoding HIV-1 Rev), 18 pg of pMDLg / p.RRE (packaging plasmid) and 15 pg of pCLS transfer plasmid using Fugene 6 (Roche Molecular Biochemicals, Indianapolis, IN). The media was changed 6 hours after transfection and the viral supernatant was collected at 24 hours and at 48 hours after transfection. Viral particles were concentrated 10 times by ultracentrifugation for 3 hours at 28,000 RPM with a Beckmann SW28 rotor as described in Reiser (2000, Gene Ther. 7: 910-913).

As a result of this strategy, three independent and highly unlikely recombination events would have to occur to create a competent vector for replication. As an additional safety precaution, this vector was made to become self-activating by removing the 3'LTR promoter (Zufferey et al., 1998, J. Virol. 72: 9873-9880). Therefore, after integration, the only functional promoter is the internal promoter supplied (in this case, CMV) juxtaposed with the payload gene and therefore no HIV sequence is transcribed.

Using this lentiviral vector transduction approach, several high-title lentiviral vectors have been created for aAPC CD83 and ICOS-L and KA2 / 32/86 / 4-1BBL and the parental KA2 / 32/86 cell has been created (Figure 3 ), as well as other aAPCs described elsewhere in this document. In summary, K562 cells were transduced with lentiviral expression vectors encoding CD32, HLA-A2, 4-1BBL and an influenza MP1GFP fusion protein, classified to detect individual clones expressing the four markers and expanded for four weeks. (figure 3). In addition, a wide variety of lentiviral vectors have been produced (P) or designed (D), comprising numerous combinations of molecules useful for transduction of aAPC based on k562 cells, as set forth in Table 1.

TABLE 1

 pCLPS / CD32 (P)

 pCLPS / siRNA-PD-L1 (D)

 pCLPS / CD32 / IRES / GM-GCSF (D)

 pCLPS / siRNA-B7-H3 (D)

 pCLPS / CD32 / IRES / IL-7 (P)

 pCLPS / siRNA-TGFbeta (D)

 pCLPS / CD32 / IRES / IL-12 (D)

 pCLPS / IDO (D)

 pCLPS / CD32 / IRES / IL-15 (P)

 pCLPS / GFP-flu matrix (P)

 pCLPS / CD32 / IRES / IL-18 (D)

 pCLPS / GFP / IRES / pol (P)

 pCLPS / CD32 / IRES / IL-21 (P)

 pCLPS / HLA DR0101 (D)

 pCLPS / CD32 / IRES / Interferon alpha (D)

 pCLPS / HLA A201 (P)

 pCLPS / CD32 / SLC (D)

 pCLPS / ICOSL (P)

 pCLPS / CD30L (D)

 pCLPS / CD86 (P)

 pCLPS / OX40L (P)

 pCLPS / CD83 (P)

 pCLPS / 4-1BBL (P)

 pCLPS / CD80 (P)

 pCLPS / GITRL (D)

 pCLPS / CD70 (D)

 pCLPS / CD40 (D)

K562 cells

K562 cells were isolated from a patient with chronic myelogenous leukemia in terminal hemoblastic crisis (Lozzio et al., 1975, Blood 45: 321-334). K562 cells may represent a DC precursor that does not express MHC molecules or costimulatory T-cell ligands, but retains many other attributes that make DCs effective APCs, such as, but not limited to, cytokine production, expression of Adhesion molecules and macropinocytosis. These attributes may be unique to K562 cells, since the U-937 monocytic cell line could not function as an effective aAPC. Thus, K562 cells represent ideal bases on which the desired MHC molecules and costimulatory ligands can be introduced to establish a DC type aAPC. An aAPC of this type has the advantages of DC, including high levels of MHC expression, a wide range of costimulatory ligands, and the ability to engage cytokine interference with a T cell. AAPCs based on K562 cells also lack the Disadvantages of CDs, such as their limited shelf life, lack of replicative capacity and poorly defined maturation requirements (Lee et al., 2002, Vaccine 20: A8-A22).

K562 cell transduction to produce aAPC

The data disclosed herein demonstrate the creation of a K562 aAPC by lentivirus-mediated introduction of costimulant ligands that allow K562 cells to better mimic the potent T-cell stimulating capacity of DC.

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K562 cells were transfected with the human CD32 Fc receptor ("K32 cell") to allow loading with anti-CD3 and anti-CD28 antibodies, and the cell was also transfected with human 4-1BBL ("K32 / 4-1BBL cells ”) To obtain added costimulation (figure 1). AAPC KA2 / 32/86 / 4-1BBL / CD83 were produced with a CD83 lentiviral vector by spinoculation (O'Doherty et al., 2000, J. Virol. 74: 10074-10080) to transduce the KA2 / 32/86 cell / 4-1BBL parental. Approximately 5 million KA2 / 32/86 / 4-1BBL cells were mixed with 500 µl of concentrated virus (5 x 107-5 x 108 UFI / ml) and centrifuged at 1200g for 2 hours. Five days after transduction, the cells were stained with a specific Ac for CD83 and a Moflo classifier was used to isolate high-expression clones. 15-20 days after classification, there were colonies of individual clones visible and these colonies of individual clones were examined by CD83 expression. High-expression clones were further expanded and expression levels of the other introduced markers (HLA-A2, 4- 1BBL, CD86 and cD32) were measured to ensure that the descendants were similar to the parental cell line in all but the Cd83 expression. In this way the aAPC K32 / 86 / 4-1BBL / ICOS-L and K32 / 86 / 4-1BBL / ICOS-L / CD83 were created using the appropriate viruses.

Using the methods described herein, stable expression of at least nine (9) genes in an aAPC K562 has been achieved. The following genes were transduced in a K562 cell and stably expressed, as detected using flow cytometry: Flu-GFP (Figure 8A); CD80 (Figure 8B); CD86 (Figure 8C); 4- 1BBL (Figure 8D); and HLA ABC (figure 8E). The KT32 / A2 / 4-1BBL / 40L / CD80 / CD83 / CD86 cell also stably expressed detectable levels of CD32, CD83, CD40L and ICOS-L. These expression levels remained constant for more than 3 months of continuous culture without any selection. In addition, the production of several aAPCs is illustrated in Figure 3. These aAPCs comprise the expression of all transgenes driven by the CMV promoter. Although reductions in transgene expression levels may occur due to the sequestration of CMV-specific transcription factors, (Cahill et al., 1994, FEBS Lett. 344: 105-108; Kang et al., 1992, Science 256: 1452- 1456) to date, no evidence of any problem with the serial transduction of K562 cells with five different lentiviral vectors has been detected (Figure 3).

Using the methods described above to transduce a K562 cell, a parental K562cc cell was compared with K562 cells transduced with LV (eg, transduced with and expressing five and eight genes). As illustrated in Figures 7 and 8, the LV transduced cells show favorable growth kinetics compared to otherwise identical but not transduced parental cells.

In addition to the expression of costimulatory ligands, the disclosed aAPCs can be used to produce various cytokines, as shown by way of example by producing IL-7 and IL-15 by K562 cells transduced with CD32 / IRES / IL-7 vectors and CD32 / IRES / IL-15. Cells were classified to detect high expression of CD32 and the production of the respective interleukin was evaluated (Figure 13), demonstrating that the appropriate cytokine was produced.

In addition, using the methods to transduce a K562 cell described above, aAPCs express CD32 from an LV at a level greater than the expression of CD32 in an otherwise identical K562 cell transfected using a plasmid vector (Figure 11). These data demonstrate that, surprisingly, K562 cells were easily transduced using lentiviral vectors. Figure 11D is a graph depicting that the expression of CD32 using an LV to transduce K562 cells is greater than the level of CD32 expression in an otherwise identical K562 cell transfected using a plasmid vector. In addition, the data disclosed herein demonstrate that the level of CD32 expression was maintained for more than nine months. In addition, this level of CD32 expression was maintained for more than nine months. The characterization of aAPC cells expressing CD64 is described below.

In addition, the data disclosed herein demonstrate that aAPCs are grown in culture in fetal calf serum-free medium (FCS), an important consideration for the production of aAPC for use in the treatment of human patients (see figure 7). These data demonstrate that the novel aAPCs of the invention grow in defined medium (Aim V) comprising 3% AB serum. That is, various aAPCs based on K562 cells were produced by transduction of parental K562 cells (k562cc) using lentivirus vectors ("LV"): KT32 (1 gene); KT32 / 4-1BBL / CD86 (3 genes); KT32 / 4-1BBL / CD86 / A2 / Flu-GFP (5 genes). In addition, as illustrated in Figure 7, the introduction of a lentiviral vector does not significantly alter the growth rates of K562 cells. These data demonstrate that master cell banks can be produced using aAPC K562 transduced with LV and that aAPC grow just as well as parental cells.

The present data have demonstrated the methods for transducing K562 cells and the expression and growth properties of these aAPC. In addition, the long-term stability and sufficient expression of a cytokine / costimulant molecule transduced in an aAPC based on K562 cells has been evaluated. CD32 has been stably expressed in a transduced K562 cell for more than nine months. In addition, the detectable and stable expression of at least eight exogenous molecules introduced into an aAPC based on K562 cells (KT32-A2-41BBL-40L-80-83-86) has also been achieved, and there is no data to suggest that molecules Additional will not be expressed in a similar manner. In fact, an aAPC has been produced that expressed nine genes (including ICOS-L) for more than 60 days at this time. Therefore, at present, the ability of aAPC to express a variety of molecules in a single aAPC single is not limited. In addition, the aAPCs of the invention are negative for mycoplasma and replication competent lentivirus (CSF), and their

safety and lack of any contaminating pathogen.

The present application comprises numerous aAPCs based on K562 cells produced, according to the methods set forth herein, including, but not limited to, those set forth in Table 2. These aAPCs, which comprise combinations of various immunostimulatory molecules, can be used for methods. both ex vivo 5 and in vivo that comprise the expansion of certain subsets of T cells, identification of combinations of factors that expand subsets of T cells, as well as gene and cell-based therapy in which aAPCs are administered, and / or cells T expanded through them, to a patient who needs it. Obviously, the list set out in Table 2 is simply illustrative of the teachings provided in this document.

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TABLE 2

 aAPC of polyclonal T cell expansion (called “KT32” series)

 aAPC for expansion of antigen-specific T cells (called the "KTA2" series)

 KT32

 KTA2

 KT32 / 4-1BBL

 KTA2 / 86

 KT86

 ktA2-86-ICOSL

 KT83

 ktA2-41 BBL

 KT80

 ktA2-41 BBL-FLU-GFP

 KT86-80

 ktA2-41 BBL-86-FLU-GFP

 KT83-80

 ktA2-32-41 BBL-FLU-GFP

 KT32 / 86/83

 ktA2-86-FLU-GFP-CD40L

 kt32-ICOSL

 ktA2-41 BBL-86-FLU-GFP-83

 kt86-ICOSL

 ktA2-41 BBL-86-FLU-GFP-CD40L

 kt32-41 BBL-80

 ktA2-86-FLU-GFP-CD40L

 kt32-41 BBL-86 (hi, lo)

 * ktA2-41BBL-86-83-40L-80-Flu-GFP

 kt32-41 BBL-83

 kt32-IL7

 kt32-IL15

 ktIL-21

 kt32-41 BBL-86-83

 kt32-41 BBL-86-83-IL15

 kt32-CD30L

 kt32-OX40L

 kt32-HLA-DR (MHC class II)

Example 2: Therapeutic in vivo use of aAPC

The application includes aAPC K562 engineered by LV for therapeutic vaccination in vivo and for ex vivo expansion of T cells for therapeutic uses. Antigens, cytokines and / or costimulatory molecules can be transduced into a K562 cell under the control of identical or independent promoters / regulatory sequences. In addition, a nucleic acid encoding the tumor cell antigen in the cell can be transduced or the antigen in the cell can be loaded differently so that the cell processes and presents the appropriate epitope in the context of an MHC protein. This is because it has been demonstrated elsewhere in this document that K562 cells have the ability to process and present antigens without the need to first identify or isolate the specific antigen or epitope required. Thus, a cell extract (comprising at least one membrane component of a tumor cell) can be loaded into the aAPC based on K562 cells and the natural ability of the cell to process and present the relevant antigen is exploited. Although customized APCs are set forth herein, these are for illustrative purposes only, and the invention is not limited to these embodiments set forth in Table 3. This is because, as the expert endowed with the teachings provided in another will appreciate In this document, a multitude of molecules can be transduced and expressed by the aAPC in a virtually unlimited combination.

TABLE 3

 Indication

 Epithelial (breast / colon / lung / ovaries / prostate) Hematic neoplasia (CML / AML / lymphoma / ALL) Skin (melanoma / Merkel) Autoimmune / transplant (SR / SLE / EICH / organ transplant)

 aAPC

 KT-BCLOP KT-CALA KT-MM KT-Treg

 coestim.

 CD80 / 83 / 41BBL / OX40L CD80 / 83 / 41BBL / OX40L CD80 / 83 / 41BBL / OX40L CD86 dim / B7H-3 / MHCII

 antigens

 gp100 / MAGE To be determined To be selected

 cytokines

 GM-CSF / IL-15 / SLC GM-CSF / IL-15 / SLC GM-CSF / IL-15 / SLC TGFbeta / IL-10

Example 3: Therapeutic ex vivo uses of aAPC

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Other aAPCs can be prepared for ex vivo use, such as, but not limited to, adoptive immunotherapy and gene therapy. Among the customized versions of aAPC for such ex vivo uses are, among others, the constructs disclosed in Table 4 below. That is, T cells isolated from a subject can be stimulated and expanded in vitro using these, or a large number of others, aAPC and then T cells can be introduced into the subject thus providing adoptive immunotherapy to it. Additionally, expanded T cells can be engineered to express an exogenous protein that was not expressed, or expressed at a lower level, compared to the expression of the protein in the T cell before, or in the absence, of the modification by genetic engineering. Therefore, the present application provides both adoptive immunotherapy and gene therapy based on ex vivo cells using the aAPC disclosed to expand T cells used for autologous transplantation of a subject in need. Table 4 simply sets forth several illustrative examples of aAPC that can be used for such cell / gene therapy, but the invention as defined in the claims is not limited to those commercially available aAPCs by way of example.

TABLE 4

 Indication

 CTL (melanoma, HIV, CRC) Genetically engineered T cells (HIV / cancer)

 AAPC

 KT-A2 KT32

 Coestim.

 CD80 / 83/83 / 4-1BBL Anti-CD3 antibody, 28 / 4-1BBL / 83

 Antigens

 Of choice None

 cytokines

 IL-7 / IL-15 / SLC IL-7, IL-15

Example 4: Stimulation of human CD 4 T cells with aAPC

The data disclosed herein demonstrate that long-term growth of CD4 T cells was obtained using aAPC K32 / CD3 / 28 and K32 / 86CD3 in the absence of exogenous cytokines, but aAPCs based on U937 cells were not effective ( figure 5). In addition, the data demonstrate that aAPCs based on K562 cells (e.g., K32 / CD3 / 28, K32 / 86 / CD3) mediate the long-term growth of CD4 T cells more efficiently than pearl-based aAPCs. coated with CD3 / 28) in which both the beads and the cells were loaded with CD3 and CD28. These results demonstrate that detectable "interference" occurs between aAPCs based on K562 cells and T cells, which is not possible using pearl-based systems. As illustrated in Figure 5, not all tumor cell lines have the ability to serve as artificial APCs and the data further demonstrates the ability of K562 cells to serve as potent APCs, which was not expected. These surprising results support the significant improvement over prior art methods that are possible using aAPC based on K562 cells.

The data disclosed herein further demonstrate the usefulness of aAPC based on K562 cells to induce the expression of cytokines and / or costimulatory molecules by CD4 T cells (Figures 6A-6D). All these data demonstrate that aAPCs based on K562 cells are far superior to aAPCs based on U937 cells in terms of induction of cytokine gene expression and costimulatory components (costimulation) by T cells and that some aAPC constructs are better. than others, depending to some extent on the cytokine and / or costimulant molecule that is being expressed. More specifically, although K32 / CD3 / 28 was generally superior compared to the other aAPCs, B7-H3 expression was actually higher by K32 / CD3 in the presence of CD4 compared to K32 / CD3 / 28 under similar conditions. These data demonstrate that aAPC K562, by interacting with T cells, produces a series of additional cytokines and costimulatory molecules (interference from APC and T cells) that can further enhance the activation and expansion of T cells. More specifically, they were tested. various aAPCs based on K562 and U937 cells transduced with various vectors encoding certain molecules (e.g., K32 / CD3 / 28, K32 / CD3, K32, U32 / CD3 / 28, U32 / CD3, U32) to determine their ability to induce the expression of molecules of interest (for example, IL-15, PD-L-1, PD-L2 and B7-H3). The data disclosed herein demonstrate that aAPCs based on K562 cells induced detectable expression of these molecules and did so to a much greater extent than cells based on U937 cells. These data further demonstrate the utility of novel aAPCs, and the significant improvement over prior art methods since all these data demonstrate that aAPCs based on K562 cells are far superior to aAPCs based on U937 cells in induction of Gene expression of cytokines and costimulatory components (costimulation) by T cells. These results are particularly notable given the previous teachings that demonstrated that the K562 parental cell line demonstrates poor T cell stimulating activity (Britten et al., 2002, J Immunol. Methods 259: 95-110).

Given the superior aAPC results of the present application compared to parental K562 cells, aAPC based on U-937 cells and beads, the relative capabilities of the various aAPCs disclosed herein are evaluated. Some aAPC constructs are better than others in mediating an effect on certain T cells, and the efficacy varies to some extent depending on the cytokine and / or costimulatory molecule, or combinations thereof, that are being expressed. More specifically, although K32 / CD3 / 28 was generally superior compared to the other aAPCs, the expression of B7-H3 was actually higher by K32 / CD3 in the presence of CD4 compared to K32 / CD3 / 28 under similar conditions. These

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data further demonstrate that certain combinations of molecules expressed in aAPCs based on novel K562 cells have a greater effect on certain populations of T cells. Therefore, these data provide a novel system for evaluating the effectiveness of various combinations of molecules to achieve effect ( s) desired and / or to stimulate and expand subsets of T cells of interest, none of which was possible before the present invention.

Example 5: Stimulation of human CD8 T cells with aAPC

In summary, 50,000 KT32 / 4-1BBL / CD86 cells irradiated with anti-CD3 Ac were coated and mixed with 100,000 freshly isolated CD8 T cells from a healthy donor. Every 10-12 days, newly irradiated CD8 T cells were re-stimulated with aAPC KT32 / 4-1BBL / CD86. The total number of cells that would have accumulated if no cells had been discarded is represented as a semi-logarithmic plot of the total cell number versus days in culture (Figure 9). As illustrated in Figure 9, polyclonal CD8 T cells expanded by aAPC transduced with CD32 and 4-1BBL (K32 / 4-1BBL) were expanded 18,600 times after 43 days.

The data disclosed herein further demonstrate the expansion of antigen-specific CD8 Tcm cells using an aAPC (eg, aAPC K32 / K-41BBL). In summary, both T-cells positive for flu tetramer and negative for flu tetramer (Figures 10A-10C) were expanded as a function of days in culture in which interleukin 2 (IL-2) was added to the culture medium on day 20. The data demonstrates a shift on day 16 of stained cells to determine CD8 expression using flow cytometry in which the cells were classified as negative for influenza tetramer or positive for influenza tetramer, demonstrating a specific antigen proliferation of CD8 Tcm cells cultured with aAPC K32 / 4-1BBL. On day 26, the cells were tested for their ability to specifically lysis positive cells for influenza tetramer or negative for influenza tetramer that were zero T2 or expressed T2-influenza. The data demonstrate that cell destruction was specific for tetramer-positive T2-influenza target cells as demonstrated by a chromium release assay (Figure 10E). The specific lysis in percentage was a function of the ratio of effector cells: target (E: T), detecting a maximum specific cell lysis at a ratio of 10: 1, and decreasing thereafter as the E: T ratio decreased up to 1: 1. At all E: T reasons examined, specific cell lysis was observed for tetramer positive T2-influenza cells using expanded cells using aAPC.

To determine whether the aAPCs of the present application can act in a specific antigen / MHC manner, K562 cells were transduced with HLA-A2, CD86, 4-1BBL, CD32, CD64 and influenza MP1 minigene encoding the restricted epitope A2 GILGFVFTL (SEQ ID NO: 1) attached to GFP. FACS analysis of this aAPC KA2 / 32/86 / 4-1BBL / Flu-GFP showed that the five markers are expressed at high levels (Figures 3 and 12), and stable expression of all transgenes was observed for up to nine months of continuous cultivation.

To demonstrate that these aAPCs were sufficient to expand antigen-specific cells, 1500 flu-tetramer positive cells were isolated from an HLA-A2 donor and mixed with 3000 aAPC KA2 / 32/86 / 4-1 BBL / Irradiated Flu-GFP. Freshly irradiated aAPC KA2 / 32/86 / 4-1BBL / Flu-GFP was added every 10 days to the culture so that there would be approximately 1 aAPC for every two T cells. After 22 days, there were approximately 10 million CD8 T cells. These cells were stained with a flu-specific A2 tetramer and more than 90% of the cells were influenza-specific (figure 12F), which represents an enrichment of approximately 250 times compared to the cells before classification (figure 12E). These data demonstrate that K562 cells have the ability to process and present antigen and expand antigen-specific T cells without the use of an antibody. It is important that the aAPC KA2 / 32/86 / 4-1BBL / Flu-GFP could not expand T cells from the tetramer negative cell fraction.

A similar experimental protocol was used to expand influenza-specific T cells, but anti-CD3 antibody was used to deliver a "one" signal instead of a peptide bound to MHC class I (Figure 2). In summary, cells were stained with amino acid sequence of influenza matrix protein peptide loaded with tetrameric MHC A * 0201 (GILGFVTVL; SEQ ID NO: 1), and classified into positive and negative fractions. After 17 days of expansion using aAPC K32 / 4-1BBL / CD3 / 28, each cell population was stained with the same tetramer used for initial classification. Only about 60% of the cells were positive for flu tetramer and the overall level of staining was lower. This suggests that aAPC KA2 / 32/86 / 4-1BBL / Flu-GFP selectively expand a T-cell receptor (TCR) that has the highest affinity for the GILGFVFTL peptide (SEQ ID NO: 1) presented by HLA-A2.

To determine whether aAPC K32 / 4-1BBL coated with anti-CD3 Ac and CD28 could be used to expand antigen-specific CD8 T cells, a population of primary CD8 + T cells classified by MHC tetramer with aAPC K32 / 4-1BBL was cultured / CD3 / 28 for 10 weeks (Figure 2A). T cells from individuals A * 0201 with influenza immunity were stained with anti-CD8 AcM and an MHC A * 0201 tetramer complexed with a restricted peptide epitope for A * 0201 of the influenza matrix protein (MP tetramer of the flu). The positive population for low frequency tetramer (less than about 0.1%) was classified and stimulated with irradiated aAPC K32 / 4-1BBL coated with anti-CD3 Ac and CD28. All cells were again stimulated with aAPC K32 / 4-1BBL at intervals of approximately 10 days. No specific flu stimulation was provided during the culture. Exponential growth curves were obtained during several months of culture. In a

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Representative experiment, approximately 8,000 antigen-specific T cells provided 1.5 x 109 cells after one month of culture (Figure 2B), which is a sufficient number for effective immunotherapy (Riddell et al., 1995, Annu. Rev. Immunol. 13: 545-586). A phenotypic analysis of cultures showed that irradiated aAPCs mixed with resting human T cells provided a population of pure T cells within one week. In addition, influenza MP positive tetramer cells exhibited potent cytotoxicity for T2 targets pulsed with influenza MP peptide (Figure 2C). This strategy can be adapted to expand HIV-specific CD8 T cells and use these aAPC to expand CD8 T cells with broad specificity.

AAPC K32 / 4-1BBL was also used to expand hTERT-specific cytotoxic lymphocytes (CTL). HTERT-specific CTLs expanded using a K32 / 4-1BBL aAPC specifically lysed carcinoma cells expressing HLA-A2 and telomerase positive (OV-7) but not carcinoma cells that were telomerase positive and HLA-A2 negative (SK-OV-3) (figure 10E). Thus, aAPC induced the expansion of antigen-specific CTLs that require recognition of the antigen in the context of HLA-A2. In addition, during expansion, CTLs, which were obtained from a patient with hTERT-vaccinated breast cancer, demonstrated a detectable increase, as assessed using MoFlo classification, in the percentage of CTL CD8 tet + during expansion by aAPC K32 / 4-1BBL. The moment of classification by MoFlo corresponding to each of figures 10A-10C is indicated in the graph showing duplications of the population as indicated by an arrow (figure 10D).

Surprisingly, the data disclosed herein demonstrate, for the first time, that aAPCs based on K562 cells have the ability to process an antigen that is then presented to T cells thereby expanding antigen-specific T cells in which the epitope Particularly responsible for the expansion is not previously known. More specifically, purified T cells were obtained from an HLA A * 0201 donor and the cells were stained with anti-CD8 AcM and an MHC A * 0201 tetramer complexed with a restricted epitope for A * 0201 of the protein of the influenza matrix (MP tetramer of influenza). The tetramer positive population of approximately 1,500 cells was classified and stimulated using irradiated aAPC KTA2 / CD32 / 4-1BBL / FLU-GFP loaded with anti-CD28 antibody. The cells were re-stimulated with aAPC KTA2 / CD32 / 4- 1BBL / FLU-GFP approximately every 10-12 days. Interleukin 2 was added to the culture at each feed, approximately every 2-3 days.

After twenty-six days of culture, most T cells were positive for MP tetramer of influenza compared to the initial population before classification, demonstrating that aAPC processed and presented the influenza specific antigen and effectively expanded specific CTLs of the flu (Figures 12A-12F). These results demonstrate that the aAPCs of the application can be used to expand and produce antigen-specific T cells even when the precise epitope of the antigen required to produce the cells is unknown. This is important for the development of transfer therapy in which the precise antigen that stimulates a T cell is not known. This is the case, among other things, for tumor specific antigens, in which very few are known. Therefore, the present application relates to providing a pathogen (for example, a virus) or another molecule against which a specific T cell response is desired, to a K562 cell and allowing the cell to process and present the antigen by generating thus the specific antigen response desired.

In an additional experiment, specific ligands were shown to promote an unexpected expansion of antigen-specific CD8 T cells. Figure 21 illustrates CMV-specific T cells isolated by tetramer classification and stained with CFSE and mixed with aAPC at a ratio of 2: 1. Figure 21A illustrates CMV specific CD8 cells, Figure 21B illustrates CMV specific CD8 cells contacted with K32 cells loaded with anti-CD3 antibody. Figure 21C depicts CMV-specific CD8 cells contacted with aAPC expressing CD32, IL-15, 4-1BBL, CD80 and anti-CD3 antibody. These data demonstrate that the addition of costimulatory ligands (in this case CD80, IL-15 and 4-1BBL) unexpectedly encourages the expansion of antigen-specific CD8 T cells.

Example 6: AAPC expansion of HIV-1 specific CD8 T cells with effector functions restored

Attempts to increase the response of HIV-specific T cells by autologous transfer of antigen-specific CD8 T cells have not resulted in long-term containment of HIV infection (Tan et al., 1999, Blood 93: 1506; Koenig et al., 1995, Nature Med. 1: 330-336; Brodie et al., 1999, Nature Med. 5: 34-41; Riddell et al., 1996, Nature Med. 2: 216-223; Lieberman et al. ., 1997, Blood 90: 2196-2206). The inability of these cells to survive in vivo ruled out any attempt to measure an anti-HIV response in a clinically meaningful way. In some cases the early disappearance of these cells was easily explained as immune recognition of a selectable marker (Riddell et al., 1996, Nature Med. 2: 216-223). In other cases, the reasons for T cell death after infusion were less clear. These initial trials used slightly different variations of peripheral mononuclear blood cell (PMBC) or lymphoblast cell (LCL) cell lines with HIV-specific peptides, limiting dilution to isolate clones, and expansion of ex vivo T cells for several months using high activation levels with exogenous IL-2 and TCR in the absence of costimulation to produce up to 1 x 109 HIV-specific T cells. Without wishing to limit itself to any particular theory, prolonged ex vivo culture, coupled with a dependence on high levels of IL-2, may lead to the onset of apoptosis in these cells once infused back into their host.

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Although pearl-based aAPCs (pearls coated with CD3 / 28) are effective vehicles for expanding CD4 T cells from HIV-infected individuals (Levine et al., 1996, Science 272: 1939-1943), there are several possible advantages of use cell-based aAPC (genetically modified K562 cells) for use in clinical trials of adoptive HIV transfer. First, cell-based aAPCs expand T cells much more robustly than pearl-based systems (Parry et al., 2003, J. Immunol. 171: 166-174). This reduces the time required to obtain therapeutic amounts of T cells, reducing the cost of these therapies and perhaps improving the function of the cells once they are introduced by infusion back into the patient. Subsequently, additional costimulant molecules can be easily introduced into the aAPC by lentiviral transduction. It is important that the beads coated with CD3 / 28 are only effective for expanding CD4 T cells (Laux et al., 2000, Clin. Immunol. 96: 187-197; Deets et al., 1999, J. Immunol. 163: 102 -110). Therefore, in order to test the potential for immune reconstitution of the infusion expanded T cells ex vivo both CD4 and CD8 back in HIV-infected individuals, new expansion systems must be developed and such systems are disclosed in the present document

It is useful to create APCs that optimally expand CD8 T cells from HIV-infected individuals. Previously, K562 cells were transfected with CD32 (to bind Ac stimulant) and 4-1BBL as minimal aAPC that induces long-term expansion of CD8 T cells. In addition, it was demonstrated that CD86 triggered T cells endowed with CD28 with the same proliferative capacity as the triggering of T cells endowed with CD28 with an anti-CD28 Ac (Thomas et al., 2002, Clin. Immunol. 105: 259-272) . Since it was desired to develop independent culture systems of Ac, CD86 was used instead of the anti-CD28 antibody to trigger both costimulatory effects of CD28 on T-cell expansion and HIV replication. Thus, the following five aAPCs can be used to expand and functionally test CD8 T cells of HIV-infected patients: KA2 / 32/86, KA2 / 32/86 / 4-1BBL, KA2 / 32/86 / 4-1BBL / CD83, KA2 / 32/86 / 4-1BBL / ICOS-L and KA2 / 32/86 / 4-

1BBL / CD83 / ICOS-L. KA2 / 32/86 can stimulate CD8 T cells but does not give them long-term growth potential (Maus et al., 2002, Nature Biotechnol. 20: 143-148). This aAPC serves as a negative (or reference) control with which other costimulatory ligands can be compared. All aAPCs created express both HLA-A2 and CD32. This allows the use of the same aAPC to expand polyclonal CD8 T cells causing the first signal to be supplied by anti-CD3 Ac bound to CD32 (Figure 1) or antigen-specific T cells causing the first signal to be initiated by peptide bound in HLA-A2

KA2 / 32/86 / 4-1BBL is the minimum aAPC for expanding CD8 T cells from healthy donors (Maus et al., 2002, Nature Biotechnol. 20: 143-148). Additional costimulatory signals may be required to expand HIV-specific T cells with enhanced effector functions. Comparison of expanded cells with this aAPC with the expanded cells with the aAPC indicated below allows the identification of any additional desirable costimulation signal.

KA2 / 32/86 / 4-1BBL / CD83 is used because CD83 is a mature DC marker whose role in T cell activation has been investigated recently. Stimulation of T cells with magnetic beads coated with anti-CD3 antibody and CD83Ig fusion protein enhanced the ratio of CD8 T cells to CD4, suggesting that CD83 ligation preferably activates CD8 T cells. In addition, tumor cells expressing CD83 were more efficiently destroyed by CD8 T cells and sensitized the immune system to also reject tumors deficient in CD83 (Scholler et al., 2001, J. Immunol. 166: 3865-3872; Scholler et al. , 2002, J. Immunol. 168: 2599-2602).

KA2 / 32/86 / 4-1BBL / ICOS-L is used because ICOS-L binds to the CD28-related molecule, inducible costimulant protein (ICOS), providing a potent costimulant signal to T cells that enhances the production of effector cytokines (IFN-y, IL-4 and IL-13) but interestingly it cannot produce high levels of IL-2 (Hutloff et al., 1999, Nature 397: 263-266) or induce the survival factor Bcl-xL (Parry et al., 2003, J. Immunol. 171: 166-174). The precise roles that ICOS and CD28 play in the immune system are still unclear, but comparing the outcome of blocking ICOS and CD28 in several disease models has revealed clues. The blocking of either ICOS or CD28 interferes with both the production and the generation of IFN-and protective immunity in models of infection by lymphocytic choriomeningitis virus (VCML) (Kopf et al., 2000, J. Exp. Med. 192: 53-61) and Toxoplasma gondii (Villegas et al., 2002, J. Immunol. 169: 937-943), suggesting a non-redundant relationship between costimulation of ICOS and CD28. In addition, the examination of when the costimulatory block is administered has revealed that CD28 is crucial for sensitization, while ICOS is more important for maintaining a T-cell response (Gonzalo et al., 2001, Nature Immunol. 2: 597-604 ; Coile et al.,

2000, Immunity 13: 95-105). The stimulation of ICOS-L promotes effector functions in both CD4 and CD8 T cells (Villegas et al., 2002, J. Immunol. 169: 937-943; Mittrucker et al., 2002, J. Immunol. 169: 5813-5817 ; Wallin et al.,

2001, J. Immunol. 167: 132-139).

KA2 / 32/86 / 4-1BBL / CD83 / ICOS-L allows, among other things, the examination of whether there is synergy between the signaling of CD83 and ICOS-L in the generation of an immune response.

These aAPCs allow experimentation of whether effector functions can be restored to T cells from HIV-1 infected donors by optimal ex vivo expansion. After the creation of aAPC was completed, its ability to expand polyclonal CD8 T cells isolated from HIV-infected patients was evaluated and it was determined whether and how ex vivo expansion had impaired the ability of HIV-specific T cells to

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respond to antigen stimulation. A similar analysis was then performed using specific Pol cells isolated from HIV-infected and uninfected individuals. These studies demonstrate the possible use of aAPC for clinical trials and allows the study of how HIV infection influences the development of HIV-specific CD8 T cells.

The methods described in this example refer to the purification of polyclonal and Pol-specific CD8 T cells from HIV-infected and uninfected individuals. These cells serve as source material used in methods disclosed herein.

Source and purification of HIV-1 infected T cells

HLA-A2 donors are used due to the high prevalence of this allele in the population. Initially, the viral phenotype is not used as a patient selection criterion for HLA-A2 donors. Virgin CD8 T cells have been shown to express low amounts of CD4 on their cell surface after activation, making them prone to HIV infection (Yang et al., 1998, J. Exp. Med. 187: 1139-1144; Kitchen et al., 1998, J. Virol. 72: 9054-9060; Flamand et al., 1998, Proc. Natl. Acad. Sci. USA 95: 3111-3116; Imlach et al., 2001, J. Virol. 75: 11555-116464). However, only virgin CD8 T cells appear to have this plasticity and the Pol-specific T cells used are by definition memory T cells. Therefore, without wishing to limit themselves to any particular theory, these cells will not become infected and will not be infected after ex vivo expansion.

In addition to using HLA-2 donors, PBMC can be used. PBMC are stained with HLA-A2 specific Ac labeled with FITC, BB7.2, (BD Pharmingen). Desired cells can be obtained from apheresis of HLA-A2 donors. A random and representative sample of CD4 T counts and viral loads is obtained from the apheresis product. A sample of the apheresis product is used to make a Ficoll / Hypaque gradient, and PBMC are frozen in 50 million aliquots / vial. With the remaining apheresis product, monocytes are removed by elutriation to create PBL and approximately 50 million CD4 T cells are isolated by negative selection (Maus et al., 2002, Nature Biotechnol. 20: 143-148). The rest of the PBLs are used to produce purified CD8 T cells (by negative selection) and are used in the methods and experiments described herein.

It has been measured that the percentage of HIV-specific Pol T cells by tetramer staining is approximately 0.7% ± 1.1 (Sun et al., 2003, Journal of immunological methods 272: 23-34 ; Kostense et al., 2002, Blood 99: 2505-2511; Rinaldo et al., 2000, J. Virol. 74: 4127-4138). Thus, from approximately 100 million CD8 T cells, approximately 700,000 HIV Pol-specific CD8 T cells are recovered. Short-lived HIV-infected T cells present several technical and safety problems. Vantage SE / DiVa is a classifier with all the features that can measure 12 colors plus direct and lateral dispersion. Vantage SE / DiVa has been equipped with improved safety features (Perfetto et al., 2003, Cytometry 52A: 122-130) that allow you to safely classify infectious materials into 4 populations at once.

Source and purification of Pol-specific T cells from a non-HIV-infected host

Seven patients with HLA-A2 breast cancer were vaccinated with the Pol peptide, ILKEPVHGV (SEQ ID NO: 3), as a control branch for a vaccine with hTERT peptide (Vonderheide, 2004, Clin. Cancer Res. 10: 828 -839). Vaccinated HLA-A2 breast cancer patient cells were used to expand Pol-specific cells from an HIV-negative host. Frozen PMBC from these patients are used to purify Pol-specific T cells. The frequency of these Pol-specific T cells is less than the expected frequency of HIV-infected donors (approximately 0.1%) so that fewer are obtained ( but still a sufficient number) of these cells to start the experiments. Patients with Pol-specific T cells can be obtained from any patient who has been vaccinated with a Pol peptide.

During ex vivo expansion, cultures are monitored for evidence of HIV infection using ELISA p24, since HIV infection skews the results. In the case where an infection is observed, patients presenting with R5 virus infection are used. R5 viruses cannot replicate in T-cells co-stimulated with CD3 / 28 due to the high levels of secretion of the natural ligands of CCR5 RANTES, MIP-1 and MIP-1 p as well as the regulation by decrease of the steady state levels of the CCR5 transcript (Riley et al., 1997, J. Immunol. 158: 5545-5553; Carroll et al., 1997, Science 276: 273-276). Therefore, costimulation with CD28 allows long-term growth of R5-infected T cells without the addition of antiviral components that can alter the expansion properties of T cells. Viral tropism is determined using the GHOST cell assay developed by Littman. and collaborators

In addition, the experiments disclosed herein do not require pure Pol specific T cells. HIV-1 Pol-specific CD8 T cells isolated by magnetic beads coated with tetramer can be used (Maus et al., 2003, Clin. Immunol. 106: 16-22). This method can provide sufficient enrichment of Pol specific T cells and can be used to isolate Pol specific cells.

Example 7: Characterization of expanded polyclonal CD8 T cells ex vivo

The ability to expand mass T cells of HIV-infected patients is examined before characterizing the

AAPC's ability to expand HIV-specific T cells. This allows the measurement of the expansion rate to determine whether a particular subset of T cells is preferably expanded by one aAPC with respect to another. In addition, the tetramer staining analysis coupled with IFN-y secretion and perforin expression is used to determine if a particular aAPC expands and / or confers an enhanced effector function 5 to influenza-specific CD8 T cells, CMV, VEB or HIV

A comparison of these studies with those that expand HIV-specific CD8 T cells in isolation to determine if HIV-specific T cells have single costimulatory ligand needs for the expansion and induction of effector functions. The following attributes are measured:

T cell expansion

10 To generate therapeutic levels of HIV-specific CD8 T cells, T cells are expanded to be between about 10,000 and about 1,000,000 times (about 13-20 population duplications) (Riddell et al., 1995, Annu. Rev Immunol. 13: 545-586). There is an inverse relationship between the amount of time that HIV-specific CD8 T cells spend in an ex vivo culture and the potential clinical benefit that these cells provide to HIV-infected patients. Therefore, aAPC that rapidly expands the CD8 T cells of HIV-infected patients is determined and used in the methods disclosed herein.

T cell survival and replication potential

The suitability of T cells after ex vivo expansion is an excellent predictive element of their ability to function in vivo. The ability of these aAPCs to induce the key cell survival gene 20 Bcl-xL is also measured. The percentage of apoptotic cells in a culture is used during the expansion process to determine if any of the cell-based aAPCs confers a particular survival advantage to the expanded T cells. Additionally, the telomere length of the cells is measured after ex vivo expansion to determine if a particular aAPC is more effective in preserving the replication potential of the cells it expands. At the end of each chromosome there is a large number of TTAGGG nucleotide repeats that are called telomeres. Each time a cell divides, it loses a part of its telomeres. Since most cells do not express the enzyme telomerase, which can restore copies of DNA repeat at the ends of chromosomes, it is believed that once a cell has lost a critical mass of its telomeric length, it loses its ability to divide Telomere lengths have been used in the art as a way of calibrating how many times a cell has been replicated and, by inference, to evaluate its future replication potential (Palmer et al., 1997, J. Exp. Med. 185: 1381-1386; Weng et al., 1997, J. Immunol. 158: 3215-3220). However, T lymphocytes are one of the few cell types that can induce telomerase activity (Weng et al., 1996, J. Exp. Med. 183: 2471-2479) and therefore the relative differences between expanded T cells that use Different methods reflect both the number of mitotic events of T cells as well as the degree that was induced by telomerase. Infusing cells that have the greatest potential for replication is essential to ensure that the adopted HIV-specific T cells transferred control HIV infection in the long term.

Cytokine production

Cytokines are important effector molecules and provide a perception of the differentiation of T cells. The ability of each aAPC to induce the following cytokines from CD8 T cells derived from HIV-infected individuals is quantified: IL-2 ( a key T cell growth factor for ex vivo expansion and the ability of a cell to induce IL-2 correlates well with its long-term growth potential); IL-4 (a marker for the differentiation of TH2); and IL-10 (an immunosuppressive cytokine that may be a substitute for the outcome of regulatory T cells). For HIV-infected patients, it is preferred that aAPCs that induce T cells produce low levels of IL-10. Other cytokines include, but are not limited to, TGF-p (for the same reason as IL-10); IFN-y (a marker for the differentiation of TH1 and an important effector cytokine); and TNFa (an important effector cytokine).

Tetramer and effector function analysis

The tetramer / IFN-and intracellular and perforin staining assay developed by Immunomics (according to the manufacturer's instructions) allows the detection of antigen-specific T cells coupled with phenotypic analysis as well as functional assays. This flow-based method is the most rigorous assay of the function of the 50 antigen-specific T cells currently available. This assay is used to determine how ex vivo expansion affects the total number and function of HIV-specific T cells.

T cell expansion

To assess how these aAPCs expand to VHI-1 infected T cells in bulk, each aAPC was irradiated, coated with anti-CD3 antibody and mixed with purified CD8 T cells from an HIV-1 infected patient at a rate of aAPC with respect to 1: 2 T cells. To compare the initial rate of cell expansion, the cells were subjected to CFSE staining instead of 3H-thymidine uptake to determine how well each aAPC induced the proliferation of all T cells, because staining with CFSE provides a criterion from

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much more quantitative assessment and allows simultaneous phenotyping of expanded cells. Approximately 20 million purified CD8 T cells from an HIV-infected individual are mixed with 3 pM CFSE for 8 minutes, washed thoroughly to remove unbound CFSE and stimulated with aAPC. Each day after stimulation, an aliquot of cells is removed from each culture and analyzed by flow cytometry. Staining with CFSE makes the cells highly fluorescent. After cell division, the fluorescence is reduced by half and therefore the more times a cell is divided the less fluorescent it will be. The ability of each aAPC to induce T cell proliferation is measured by measuring the number of cells that divide once, twice, three times, etc. The aAPC that induces the highest number of cell divisions at a particular time point is considered the most potent expansion factor of CD8 T cells of HIV-infected individuals (Wells et al, 1997, J. Clin. Invest. 100: 3173-3183).

However, staining with CFSE can only detect a limited number of T cell divisions (approximately 7) and to generate therapeutic amounts of T cells for immunotherapy, 13-20 population duplications may be necessary. Therefore, to determine how well these aAPC promote long-term growth of T cells, cell growth curves are generated. These experiments are configured exactly like the CFSE experiments as described elsewhere herein, but CFSE is not used. Every 2-3 days of culture, T cells are removed from the respective cultures and counted using a Coulter counter that measures how many cells are present and the average cell volume. The average cell volume is the best predictor of when to re-stimulate cells. In general, when T cells are stimulated properly, they triple their cell volume. When this volume is reduced to more than about half of the initial blast, it may be necessary to re-stimulate T cells to maintain a logarithmic linear expansion (Levine et al., 1996, Science 272: 1939-1943; Levine et al., 1997, J. Immunol .. 159: 5921-5930). The time it takes for each aAPC to induce 20 population duplications is calculated. The relative differences of each aAPC to induce this level of T-cell expansion is an important criterion on which particular aAPC to use to advance clinical trials.

The phenotypes of the expanded cells are characterized by each aAPC to determine if a particular subset preferably expands. Before each new stimulation, a phenotype analysis of the expanding T cell populations is performed to define the state of differentiation of the expanded T cells using the definitions of CD27 and CD28 proposed by Appay et al. (2002, Nature Med. 8, 379-385) and the definitions of CCR7 proposed by Sallusto et al. (1999, Nature 401: 708-712). Intracellular staining of perforin and granzyme B are used to perform a macroscopic measurement to estimate the cytolytic potential.

Apoptosis rate and telomere length

Annexin V / To-Pro staining (Molecular Probes, Eugene, OR) is performed before each new stimulation to determine whether differences in growth rate reflect differences in the number of cells undergoing apoptosis. The experimental details of this trial are described in detail in Maus et al. (2002, Nature Biotechnol. 20: 143-148). Cultivation conditions that lead to the least amount of apoptosis are desirable.

Telomere lengths are measured using various established techniques known in the art, but a preferred method is to use the FISH flow method since it can be performed relatively quickly using many fewer cells and is easier to quantify. In this method, approximately 1 million T cells (although much less required) are denatured using 75% heat and formamide, and then hybridized with a FITC-conjugated DNA probe for the TTAGGg sequence. The unbound probe is washed away and the DNA is counterstained with LDS 751. A mixture of 4 populations of FITC-labeled beads, each having known amounts of equivalent soluble fluorochrome molecule (MESF), is analyzed in each experiment to allow the creation of calibration and the determination of the relative telomere length of each crop over time (Baerlocher et al., 2002, Cytometry 47: 89-99). The relative telomere length is measured before each new stimulation and it is recorded if any of the aAPC expands T cells that have significantly longer telomeres.

Cytokine and expression of Bcl-xL

To investigate the production of cytokine and Bcl-xL expression levels, RNA is isolated from approximately 1 million cells 24 hours after each stimulation and subjected to quantitative RT-PCR to examine the relative expression of, but not limited to, IL-2, IL-4, IL-10, IFN-y, TNF-a and Bcl-xL. The experimental details of these established trials can be found in Maus et al. (2002, Nature Biotechnol. 20: 143-148), Thomas et al. (2002, Clin. Immunol. 105: 259-272) and Parry et al. (2003, J. Immunol. 171: 166-174). Many discrepancies have been observed between the levels of TGF-a mRNA and the secreted cytokine (Assoian et al., 1987, Proc. Natl. Acad. Sci. USA 84: 6020-6024) whereby TGF production is measured -a through ELISA.

Tetramer analysis and ELISPOT

The ability of expanded CD8 T cells to recognize common memory antigens and HIV is compared. Prior to expansion, purified CD8 T cells are stained using the following, among others, tetramers of HLA-A2 GLCTLVAML (SEQ ID NO: 4) (VEB BMLF), NLVPMVATV (SEQ ID NO: 5) (CMV p65), SLYNTVATL (SEQ ID

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NO: 6) (HIV gag p17), ILKEPVHGV (SEQ ID NO: 3) (HIV RT pol), GILGFVFTL (SEQ ID NO: 1) (flu matrix) and LLFGYPVYV (SEQ ID NO: 7) (HTLV Tax) and the frequency of these tetramers is determined before ex vivo expansion. Next, mass CD8 T cells are stimulated from an HIV-infected donor using the aAPC disclosed herein and the cells are expanded using methods described herein. Before each new stimulation, the expanded T cells are subjected to tetramer staining to determine if the relative frequency of EBV, CMV and HIV-specific T cells has been altered by stimulation by a particular aAPC.

It is important to determine the frequency of cells that secrete IFN-y and the frequency of cells that express high levels of perforin after antigen recognition. The tetramer / IFN- and intracellular staining assay developed by Immunomics, which combines an intracellular cytokine assay with tetramer staining can be used to perform flow-based functional assays using antigen specific cells. The assay may include the incorporation of an intracellular stain for perforin expression. Before freezing the PBMC isolated from each patient, a tetramer / IFN-and intracellular and perforin staining assay is used using each of the peptides listed elsewhere herein to determine the initial positive population for tetramers / secreting IFN-y / that produces perforin. For each peptide with a corresponding tetramer, approximately 1 million PBMC is placed in three tubes for the following controls and experimental conditions: 1) stimulated non-peptide control, 2) control tetramer staining without peptide stimulation, and 3) more tetramer tube peptide Next, the appropriate tetramer is added to each tube and incubated for 30 minutes at room temperature. Two micrograms of peptide are added to the third tube and the sample is incubated for 1 hour at 37 ° C. Brefeldin A is added to the three tubes and the tubes are incubated for another 4 hours. Since all tetramers are labeled with P5-Cy5.5 and APC-Cy7, anti-CD27 and CD28 antibodies labeled with PerCP and APC can be used to determine the state of differentiation of virus-specific T cells. Cells are lysed, fixed and permeabilized and IFN-and-FITC labeling protocols are performed using methods known in the art and methods disclosed herein. These have been satisfactory in measuring perforin expression by co-incubating an anti-perforin antibody labeled with PE with the anti-IFN- y antibody, which allows simultaneous measurement of IFN-y expression and perforin expression. Given the reported discrepancy between HIV-specific T cells that secrete IFN-y and those that have elimination potential (Zhang et al., 2003, Blood 101: 226-235), this analysis is used to determine how ex vivo expansion With the various cell-based aAPCs, it alters any of these functional attributes. The cells are fixed in PFA and analyzed by flow cytometry. This analysis establishes an initial level phenotype of cells expressing IFN-and perforin. To determine whether the percentage of tetramer positive cells or the phenotype of IFN-y secreting cells, or expressing high levels of perforin, is altered, after ex vivo expansion, the tetramer / IFN-y staining test is performed intracellular using differentially expanded CD8 T cells. To do this, autologous PBMCs are used and the percentage of CD8 T cells is determined and CD8 T cells are removed by reduction of magnetic beads. Reduced CD8 T cells are reconstituted with those expanded by the K562-based aAPCs described elsewhere herein and are subjected to the tetramer / IFN- and intracellular staining assay as described elsewhere herein. These experiments provide an indication of what aAPC could alter the functional phenotype of HIV-specific T cells during ex vivo expansion.

The data disclosed herein demonstrate the phenotype of CD8 T cells isolated from HIV-infected individuals expanded by cell-based aAPCs. Although not wanting to restrict itself to any particular theory, it is anticipated that a subset of aAPC can expand HIV-specific T cells with enhanced effector functions. This result would confirm that these effector functions can be restored by ex vivo expansion. Through the elimination process, it is determined which signals are necessary for this transformation according to the methods described herein. This finding is confirmed by growing HIV-specific T cells in isolation and provides a rationale for making aAPC a GMP reagent and for conducting the phase I clinical trial to check if enhanced ex vivo enhanced CD8 T effector cells can help control HIV infection in patients. Additionally, this result establishes an experimental system to study the mechanisms that mediate the defect (s) of HIV-specific T cells and how a costimulatory ligand can overcome or reverse this defect.

Example 8: Characterization of Ex-Expanded Pol-specific T cells

The methods disclosed herein provide important insights into which aAPC is best for expanding HIV-specific CD8 T cells by examining the expansion and function of these cells within the polyclonal T cell expansion environment. However, the translation value of ex vivo expanded polyclonal T cells of HIV-infected individuals is low since the number of total CD8 T cells is increased in most HIV-infected patients since homeostatic mechanisms adjust the loss. of CD4 T cells and there appears to be no macroscopic abnormalities in non-HIV-specific CD8 T cells (Gandhi et al., 2002, Annu. Rev. Med. 53: 149-172). Therefore, to improve the cellular response of HIV-specific CD8 T cells by autologous adoptive transfer, systems that expand only HIV-specific T cells and confer effector functions that can eliminate long-term HIV infection are desired.

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It is desirable to expand Pol-specific T cells isolated with tetramers using each of the cell-based aAPCs and determine which one generates Pol-specific T cells that best eliminate HIV-infected cells with the greatest potential for replication and survival. These studies are conducted using Pol-specific cells isolated from an HIV-infected individual with Pol-specific T cells isolated from cancer patients vaccinated with Pol-specific peptide. This comparison provides a unique insight into the effects that HIV has on generation of antigen-specific cells, since the same reagents and assays can be used to study these Pol-specific T cells isolated from these two types of disease. These studies can provide insight into the nature of the defect of HIV-specific T cells and lead to new hypotheses on how to overcome these defects.

T cell expansion and phenotype

On average, 700,000 Pol-specific T cells from an HIV-positive individual infected with HLA-A2 and approximately 100,000 Pol-specific T cells from a vaccinated cancer patient are isolated. Specific antigen expansion can be carried out using only 1,500 T cells (Figure 3). To obtain equivalent culture conditions, cultures are started by mixing 7,500 Pol-specific T cells and 15,000 of the aAPCs described elsewhere herein (it has been observed that reversing the ratio of T cells with respect to APC is important when expand so few cells) with 0.5 ng / ml of anti-CD3 antibody in a total of 100 pl of media in a 96-well plate. To compare the ability of the processed anti-CD3 and K562 antibodies and the antigen presented to stimulate these cells, an identical set of cultures whose aAPC have also been transduced with a Pol-GFP expression vector is also expanded. Therefore, 10 cultures are available for each of the donor cells. Recently irradiated aAPC are added to the growing polyclonal T cells every 10-12 days at an estimated rate of 1 aAPC for every 2 T cells. Once the population expands to a point where a precise quantification of the number can be calculated of cells present using a Coulter counter, the expansion rate of each population is monitored. The CD28 / CD27 phenotype of Pol-specific T cells isolated from patients with HIV and cancer before and after T-cell expansion is compared.

Cytokine production, Bcl-xL expression, apoptosis rate and telomere length evaluation

These studies are performed using methods disclosed elsewhere herein, except that the analysis comprises using several million cells present (approximately 30 days). However, these studies should confirm the results disclosed elsewhere herein using polyclonal T cells.

Elimination of HIV-infected targets

An advantage of expanding antigen-specific T cells in isolation is that multiple elimination and other functional assays can be performed very quantitatively. The first test is the tetramer / IFN- and intracellular assay and perforin expression assay. As disclosed elsewhere herein, expanded CD8 T cells ex vivo are mixed with autologous PBMCs with reduced CD8. Since most CD8 T cells are positive for tetramers, quantitative data is obtained regarding which aAPC produces T cells that produce the highest levels of IFN-y and perforin after contact with the Pol peptide. Additionally, since the number of cells with tetrameters is not limiting, a complete phenotype analysis of these cells is performed using CCR7, CD27, CD28, CD62L, CD45 RO, CD45 Ra and CD57 (Brenchley et al., 2003, Blood 101: 2711-2720), thereby correlating the effector function (s) with the phenotype of the T cells.

The ability of differentially expanded Pol-specific T cells to eliminate T2 cells loaded with the Pol peptide is evaluated through the 51 Cr release assay. In this assay, T2 cells are loaded with the Pol iLkEPVHGV peptide before 51Cr uptake. After thorough washing, labeled T2 cells are incubated with antigen-specific T cells at ratios of 1:30, 1:10, 1: 3 and 1: 1 and 1: 3 for 4 hours. If the antigen-specific T cells recognize the peptide presented in the T2 cells and have the ability to remove these cells, then 51 Cr is released and can be detected. No peptide control or detergent lysis control allows the determination of specific lysis. The lower target ratio with respect to the effector in which a high degree of specific lysis is observed indicates which aAPC expands T cells with the highest elimination capacity.

The ability of ex vivo expanded Pol-specific T cells to eliminate T2 cells is compared with the ability of these cells to remove CD4 T cells expressing Pol. This scenario more closely mimics the in vivo targets of ex vivo expanded CD8 T cells . The ability of these cells to eliminate HIV-infected CD4 T cells is the definitive proof of principle. In addition, cells that are infected at this level are likely to generate a very high background in the 51 Cr release assay making it difficult to determine the efficacy of expanded CD8 T cells. Alternatively, autologous CD4 T cells are transduced with the lentiviral Pol expression vector IRES GFP that allows the monitoring of Pol-expressing cells by GFP expression. It was observed that approximately 50% of the T cells transduced by these vectors (see Parry et al., 2003, J. Immunol. 171: 166-174, for details of how T cells are transduced with lentiviral vectors) produce

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Sufficient targets for 51Cr release assays. CD4 T cells transduced with Pol are labeled as well as not transduced with 51 Cr. CD4 T cells do not capture 51 Cr as well as T2 cells, so the target is changed to the effector ratio of 1: 100, 1:30, 1:10 and 1: 3 to improve the sensitivity of the assay. The differences between expanded and unexpanded CD8 T cells in their ability to eliminate CD4 T cells expressing Pol provide a strong basis for further developing one aAPC with respect to another.

CD28 expression is required for long-term ex vivo expansion of CD8 T cells. Recently it has been observed that the restoration of CD28 expression by retrovirus transduction (Topp et al., 2003, J. Exp. Med. 198: 947-955) restores the ability of CD28 negative cells to produce IL-2 and to expand without the presence of CD4 T cells. Recently, IL-12 has been shown to restore cD28 expression for CD28 T negative cells (Warrington et al., 2003, Blood 101: 3543-3549). To determine if IL-12 can enhance CD28 expression and improve the long-term growth potential of Pol-specific T cells, 10 ng / ml of IL-12 are added to the various aAPC / Pol specific cultures are evaluated. the cells to determine how well the expression of CD28 in Pol-specific cells is improved and if any of the aAPC can expand Pol-specific cells in a long-term culture. Other cytokines, such as IL-21 and IL-15, can work in conjunction with IL-12 to induce HIV-specific T-cell expansion. IL-21 is a multifunctional cytokine that induces T and B cell proliferation and differentiation of natural cytolytic lymphocytes (NK) (Parrish-Novak et al., 2002, J. Leukoc. Biol. 72: 856-863). IL-21 is produced exclusively by activated CD4 T cells and performs a synergistic action with IL-2 and IL-15 to promote the growth of CD8 T cells. Likewise, IL-15 is a survival factor for key CD8 T cells and is produced by CDs and active macrophages (Waldmann et al., 2001, Immunity 14: 105-110) whose addition can also enhance the expansion of CD8 T cells.

In the event that cytokines do not allow long-term expansion of HIV-specific T cells, Pol-specific T cells are transduced with a lentivirus that expresses CD28. As explained briefly in Parry et al., 2003, J. Immunol. 171: 166-174, more than 90% of T cells can be transduced with a single transgenic vector (as disclosed herein, vectors containing IRES are less effective). Therefore, to test whether CD28 transduction restores long-term growth for Pol-specific T cells, Pol-specific T-cell spinoculation is performed (O'Doherty et al., 2000, J. Virol. 74: 10074-10080) with lentiviral vector expressing CD28 immediately before mixing these cells with aAPC. The activation of the T cells induced by these aAPCs facilitates the integration of this vector and the expression of the transgene can be observed 12 hours after transduction. These cells are cultured as disclosed elsewhere herein, and Pol-specific T cells are recovered. This is an indication that CD28 costimulation is required for long-term expansion of these cells. If lentiviral transduction is used as a necessary step to expand HIV-specific T cells, the translation impact of these results can be slowed down. However, it has recently been shown that lentiviral T-cell transduction from an HIV-infected individual may be a viable therapeutic option.

Example 9: Expansion of HIV-specific CD8 T cells with broad specificity

Infusion of a single CD8 clone that recognized a Nef epitope led to the selection of viruses that did not express this epitope (Koenig et al., 1995, Nature Med. 1: 330-336) indicating that T cells are required with multiple specificities to prevent HIV escape mutants. A potentially powerful way to generate T cells that recognize multiple epitopes of a specific virus from a patient is to have aAPC uptake based on cells and present antigens similar to natural APCs. Loading chemically inactivated viruses from a patient in aAPC based on K562, expressing MHC and mixing them in autologous T cells, patient-specific anti-HIV T cells can be expanded. By providing this optimal situation whereby HIV-specific T cells find HIV-specific antigens, T cells that recognize immunodominant and cryptic antigens can be expanded, thus resulting in a greater potential to control HIV infection. In addition, the T cells of a healthy person not infected with shared class I HLA alleles can be vaccinated ex vivo with the chemically inactivated virus of the recipient, spread and infused in the HIV-infected patient. This represents a possible powerful treatment option for an individual with advanced disease and with a limited repertoire of T cells.

Recently, Lu et al. (2003, Nature Med. 9: 27-32), demonstrated that macaques that had been infused with DC loaded with a chemically inactivated form of VIS had significantly less levels of RNA and viral DNA, suggesting that the T-cell response initiated using this immunotherapy approach can control VIS infection. These data suggest that properly sensitized T cells can control VIS infection and reinforce the prospects of a cell-based HIV vaccine in humans (Bhardwaj et al., 2003, Nature Med. 9: 13-14). The inactivated virus is used to load aAPC based on K562 that express MHC in a manner similar to the study with chemically inactivated VIS described elsewhere herein. The complexes are used to expand HIV-specific T cells ex vivo to create a therapeutic patient-specific T-cell vaccine. The complexes can also be administered to a patient who uses an in vivo approach.

CD107a and 107b are associated lysosomal proteins that are not normally found on the surface of the T cell. When TCR is activated, degranulation of CD8 and CD107 T cells occurs rapidly and other lysosomal proteins are transported to the cell membrane to facilitate release. of perforin and granzyme. Betts et al.

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(2003, J. Immunol. Methods 281: 65-78), demonstrated that CD107 expression can be detected in specific T cells of CD8 antigen already at 30 minutes after stimulation with maximum expression 4 hours after stimulation. Thus, antigen specific effectors can be identified without eliminating the desired T cells, thereby identifying which antigen is activating the cells. Similar studies are conducted to identify and expand HIV-specific T cells as disclosed herein.

Figure 4 illustrates, without wanting to restrict itself to any particular theory, an experimental approach to expand HIV-specific CD8 T cells with broad specificity. Autologous, high-grade viral T-cell banks and isolates are available from a recently completed adoptive transfer clinical trial (Levine et al., 2002, Nature Med. 8: 47-53) and are used in the methods disclosed in the present document. A patient viral isolate is inactivated using 250 pM of aldritiol-2 (AT-2) in order to preserve the fusogenic potential of the virus (Lu et al., 2001, J. Virol. 75: 8949-8956). The ability of AT-2 to inactivate HIV by infecting PHA blasts with this treated virus and measuring p24 production over a period of two weeks is validated. In order to allow HIV fusion, the cell-based aAPC that best allows the expansion of Pol-specific T cells is transduced with lentiviral expression vectors with CD4 and CCR5 that are already available (Simmons et al., 2003, Virology 305: 115-123). K562 are cells that express CXCR4 naturally (Gupta et al., 1999, J. Leukoc. Biol. 66: 135-143). These aAPCs are pulsed with the inactivated viruses (50 ng of p24 / million cell-based aAPCs) for 2 hours at 37 ° C (Lu et al., 2001, J. Virol. 75: 8949-8956). Fifty million patient CD8 T cells are mixed with duplicated antigen-loaded aAPCs. With a culture, the utility of using CD107 mobilization as a substitute for antigen-specific T cells to which enhanced effector functions have been conferred is evaluated. Four hours after stimulation, the cells are stained for CADS and CD107, the CD8 + CD107 + cells are classified using the BLS 3 classification device and expanded in isolation using "infected" aAPCs. With the other set of cells, the ability of these aAPCs to selectively expand HIV-specific T cells from the polyclonal population is tested. Chemically inactivated HIV-infected aAPCs can be used to stimulate mass CD8 T cells of the HIV-infected individual every 10 days. As a control, both aAPC cells that have not been pulsed with viruses are used, which should only expand cells that recognize K562 antigens, such as an aAPC coated with aCD3 that expands all cells. After two weeks of expansion, the cultures are monitored and the cells are evaluated to determine whether HIV-specific T cells that recognize the patient's own virus have been enriched better than a reference HIV strain. PHA is used to stimulate and superinfect the pBmC with reduced patient CD8 T cells or with high-grade patient virus (approximately 5 x 10 TCID50 / ml) or similarly with high-tit Bal virus (for patients with R5 ) or NL4-3 (for patients with X4) for 3 days. The majority of patients who received autologous, expanded ex vivo CD4 T cells had undetectable viral loads (Levine et al., 2002, Nature Med. 8: 47-53) and therefore after only 3 days, the vast majority of viruses in replication they will represent the virus that was superinfected. Approximately 400,000 PBMc obtained from a patient infected with reduced CD8 were mixed with 100,000 expanded CD8 T cells from an aAPC infected with the patient's own virus. After 24 hours, the percentage of CD8 T cells expressing INF-X and perforin is measured by intracellular flow cytometry. If more CD8 T cells expressing IFN-X and perforin are observed when mixed with the PBMC superinfected with the patient's own virus compared to those superinfected with the reference strains Bal or NL4-3, the aAPC presentation of the virus patient probably allowed the expansion of T cells that selectively recognize the patient's viral epitopes. This approach has tremendous potential to increase the amplitude of a patient's HIV response against their own virus or can potentially cause responses against cryptic epitopes not well presented during a natural HIV infection (Sewell et al., 1999, J. Immunol. 162: 7075-7079). In addition, these experiments provide a determination of whether CD107 staining provides a more robust way to identify and expand HIV-specific T cells.

It was previously shown that high affinity CTLs were generated from APC expressing lower antigen levels (Alexander-Miller et al., 1996, Proc Natl. Acad. Sci USA 93: 4102-4107; Oh et al., 2003, J. Immunol. 170: 2523-2530). It is evaluated whether aAPCs that express lower levels of MHC class I are more effective in generating high avid T cells that have a greater potential to eliminate their targets. Based on the disclosure herein, the amount of virus used to load KA2 cells is titrated, or an aAPC is used that expresses smaller amounts of HLA-A2, to ensure that high affinity T-cell responses are generated that are useful for adoptive transfer immunotherapy (Alexander-Miller et al., 1996, Proc Natl. Acad. Sci. USA 93: 4102-4107). Yes

If aAPC loaded with chemically inactivated viruses is not an effective method for expanding patient-specific T cells, this method of generating HIV-specific T cells is compared with cross-priming methods described by Larsson et al. (2002, AIDS 16: 1319-1329). For this method to work, the K562-based aAPC has the ability to capture dying or dead T cell antigens. The T cells are transduced either with GFP or with LV construct of influenza virus matrix fusion - GFP. These cells are subjected to UVB radiation as described in Schlienger et al. (2003, Clin. Cancer Res. 9: 1517-1527), and mixed with the optimal aAPC that expresses HLA-A2. If aAPC properly processes dead influenza-infected T cells, KA2 cells incubated with apoptotic T cells that express the influenza virus matrix fusion protein - GFP, but not those incubated with T cells expressing only GFP, may expand specific influenza virus cells as described elsewhere herein. If KA2 processes and exhibits dead cell antigens, a patient's T cells can be superinfected with their virus. These cells undergo

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UVB radiation and incubate with aAPC. The ability of aAPCs loaded with apoptotic HIV-infected cells to promote the expansion of HIV-specific T cells is then evaluated as described elsewhere herein.

Alternatively, HIV-specific T cells with multiple specificities are expanded using a panel of HIV-specific tetramers. In this approach, a series of HIV-specific tetramers labeled with the same fluorochrome are mixed with T cells from an HIV-infected donor and the T cells that bind to these ordered tetramers are classified to give a unique population. This population of antigen-specific T cells is expanded using optimal aAPC, as described elsewhere herein, and the ability of expanded T cells in this way to recognize and respond to autologous virus against strains is evaluated. of reference as described elsewhere in this document. Currently, there are defined tetrameters of HLA-A2 that recognize epitopes conserved in gag, pol and nef and new tetramers can be created that present HIV-specific peptides. Although this approach expands a limited number of antigen-specific T cells, it must be sufficient to prevent viral escape. In addition, the methods disclosed herein can be quickly transferred to phase I clinical trials.

Example 10: Production and evaluation of K562 cells comprising CD32 or CD64

K562 cells were stably transfected with (i) the human Fcy CD32 receptor to allow exogenous loading of anti-CD3 and anti-CD28 antibodies, and an independent population of cells was transduced with the human Fcy CD64 receptor, which allows high affinity loading of antibodies to anti-CD3, anti-CD28, and other receptors, and (ii) human 4-1BB ligand. 4-1BB, also known as CD137, is a member of the TNF receptor family that promotes the survival of CD8 T cells (Hurtado et al., 1997, J. Immunol. 158: 2600- 2609; Takahashi et al., 1999, J. Immunol. 162: 5037-5040; Tran et al., 1995, J. Immunol. 155: 1000-1009). Stimulation of 4-1BB preferably activates CD8 T cells in vitro, amplifies CTL responses in vivo and improves the survival of activated CTL (Shuford et al., 1997, J. Exp. Med. 186: 47-55). 4-1BB is a candidate molecule that can promote the long-term ex vivo growth of CD8 T cells. The initial growth rate of cD8 T cells stimulated with either CD3 / 28 beads or aAPC K32 / 4-1BBL coated with anti-CD3 and CD28 antibodies (CD3 / 28 K32 / 4-1BBL) was equivalent. However, after stimulation again, only CD8 T cells activated with aAPC CD3 / 38 K32 / 4-1BBL continued to expand. This ability to expand correlates with the increased regulation of the Bcl-xL cell survival gene and the cytokine IL-2. In the absence of induction of these genes, a large percentage of cultures become positive for annexin V, an early sign of apoptosis (Maus et al., 2002, Nature Biotechnol. 20: 143-148). "Interference" was observed between cell-based aAPCs and T cells (Thomas et al., 2002, Clin. Immunol. 105: 259-272).

The transduction of K562 cells with CD64 was carried out as follows: K562 cells were transduced with a lentiviral vector expressing CD64 (SEQ ID NO: 2) according to the methods described herein. High expression cells were classified and individual clones were examined to determine CD64 expression. A clone was selected for further characterization (Figure 14).

The binding capacity of K562 cells expressing CD64 (K64 cells) was evaluated as follows. One million K64 cells were loaded with 0.5-50 pl of anti-IgG2a antibodies labeled with FITC for 1 hour at 4 ° C, then washed once and fixed. Using the known antibody concentration (50 pg / ml), a known ratio of FITC / protein (3.5) and Immuno-Brite beads (Beckman Coulter), it was calculated that the amount of antibody bound to K64 cells was between approximately 2000 and 6000 antibodies bound to each cell (figure 15).

To assess the ability of K64 cells to load antibodies and stimulate T cells, K64 and K32 cells were irradiated at 100 Gy, and loaded with 1 pg / ml of anti-CD3 / anti-CD28 antibody mixture per 106 cells in the medium Protein-free PFHMII (Gibco / Invitrogen, Carlsbad, CA) and were rotated at 4 ° C for 1 hour. The cells were then washed three times with the same protein-free medium, resuspended in the same medium and added to CD4 T cells at a ratio of 2: 1. T cells were resuspended in RPMI + 10% HSAB at a concentration of 106 cells per milliliter. As a control, K64 and K32 cells were also loaded using the conventional method (10 minutes at room temperature without washes), and then mixed with CD4 T cells.

As illustrated in Figure 16, when K64 cells are compared with K32 cells, K64 cells loaded with anti-CD3 / CD28 antibodies and washed three times to remove excess unbound antibodies can still effectively stimulate cells. T. Specifically, as depicted in Figure 16, resting CD4 T cells have an average cell volume of ~ 140 fl. The disappearance of this population of cells indicates that CD4 T cells have been activated. Therefore, the aAPC cells of the present application can be used in in vivo applications, as described elsewhere herein because they can initiate stimulation and proliferation of T cells without the presence of excess antibody that could give as A HAMA (human anti-mouse antibody) response resulted if monoclonal antibodies are used in an in vivo application of the present invention.

In addition, as shown in Figure 17, much less antibody is required to optimally load K64 cells, but washing K64 cells to prevent a HAMA reaction when administered to a mammal has a minimal effect, in case of have any, about the ability of an aAPC to stimulate a T cell.

Claims (3)

1. In vitro method of activation or stimulation of T cells, the method comprising contacting said T cells with a transduced K562 cell using a lentiviral vector expressing CD64 and 4-1BBL, wherein said K562 cell is loaded with an antibody anti-CD3 and an anti-CD28 antibody, and in the 5 that the binding of said anti-CD3 antibody and said anti-CD28 antibody with said active T cells or stimulates said T cells. 2. K562 cell transduced using a lentiviral vector expressing CD64 and 4-1BBL, wherein said K562 cell is loaded with an anti-CD3 antibody and an anti-CD28 antibody, for use in the activation or stimulation of T cells. 10 image 1 MBBl Anti-CD28 Anti-CD3 CD3 / 28 TCD8 cell K32 / 4-1BBL FIG. one % of specific lysis image2 3 I FIG. 2B Days Total cell number image3 image4 Ll e! Q image5 -1BBL image6 í .. 'j / .//VCD86 i_-Í. ^ CD4 CXCR4 • '’* -v 4-1 BBL image7 4 ^ .. '<CD4 CXCR4 -1BBL image8 / P§ - • S ‘^ CD4 image9 0KT3 and T cells of patients Expand for 1 month Patient T cells " Expand for 1 month ^ ■ Patient T cells * Expand for 1 month / Chemically inactivated virus FIG. 4 Superinfect PBMC with reduced CD8 of the patient with strain either R5 or autologous X4; of high title. Mix in expanded T cells with infected PBMC. Detect reactivity using IFN-y and perforin by intracellular staining. Population Duplications image10 image11 £ = -OR 'or JO _Q 0 Q_ 01 "O Wl 0) C O o co o Q. D Q 18 16 14 12 10 8 6 4 2 0 image12 0 2 4 7 12 14 18 21 23 25 28 Cultivation day FIG. 7 —K562cc -i- kt32 kt32 4-1BBL CDB6 -X- kt32 4-1 BBL CD86 A2 Flu GFP FLU-GFP image13 image14 8-THREAT K562CC PARENTAL FIG * 8A 4-IBBL image15 FIG. 8D image16 FIG. 8B FIG. 8C HLA ABC image17 ‘ALSO EXPRESS CD32, CD83, CD40L FIG. 8E OR) co Total number of cells Long-term expansion of CD8 T cells using aAPC transduced with LV image18 FIG. 9 Population duplications FIG. J OA Before the classification image19 coa FIG. 10B you will go classification n01

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CDfl FIG. 10C image20 32.7 * s CD8 image21 FIG. 10D image22 image23 image24 Flu tetramer PE Fia) 2E image25 CD8 FITC FIG. 12F image26 h- Before the classification After classification and culture with aAPC ktA2-32-41BBL Flu-GFP image27 Atokine production UOOO-i 12000 10000 8000 □ IL-7 (pg / ml) pg / ml 6000 O L-15 (pg / ml) 4000 2000 ■ íh ', J. I Media alone KT32-IL7 KT32-IL15 KJ32 K562CC Samples FIG. 13 % of max. image28 FIG. 14 -saw -saw FIG. fifteen Medium fluorescence I heard or I heard o o o o image29 T = image30 CQ OR ho image31 ^ iáidí & rór «>? r 'ÍTOi ^ 2 V-V3 rfrHvffj PmmííIPw Differential number 1 Differential number Bwll Differential number 150-f ---- * ----------! - LC * 1 W rL UC ^ S31S1 IL (1 <71) ^ 9RMS Differential number Differential number 10 ioo 1000 1 * 10 Particle volume (fl) LO 1.317 a. Ufe 53151 M3118) CD32 Control Particle Volume (fl) Particle Volume (fl) Particle Volume (fl) 150 CD64 j'i • fifty 1 "10 Particle Volume (fl) LO 48917 Í Ut * 531514. Washed 3 times No washing FIG. 16 image32 image33 image34 FIG. 21A FIG. 21B

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to ° 102 102a 104 FFTC-H: CFSE FTTtH coa + 2e » 2 & -TSÓLQCMV1 Count (Je events 6691 image35 FITOH; CPSE FITC-H CBD + 28 + 28.KT32A3CMV3 Event Count: 7360 FIG. 21C image36 FITC-H: CFSE; FITC-li CD6 + 26 + 26- KT1541BBL-60 A3 CMV 4 Event count 49456