WO2016048872A1

WO2016048872A1 - Compositions, methods and kits used to determine potency of dendritic cells in cancer immunitherpay

Info

Publication number: WO2016048872A1
Application number: PCT/US2015/051174
Authority: WO
Inventors: Andrew CORNFORTH; Monica SIEGENTHALER
Original assignee: NeoStem Oncology LLC
Current assignee: NeoStem Oncology LLC
Priority date: 2014-09-23
Filing date: 2015-09-21
Publication date: 2016-03-31
Anticipated expiration: 2017-03-23

Abstract

The described invention provides compositions, methods and kits for determining the potency of dendritic cells (DC) used in cancer immunotherapy.

Description

COMPOSITIONS, METHODS AND KITS USED TO DETERMINE POTENCY OF DENDRITIC

CELLS IN CANCER IMMUNOTHERAPY

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] This application claims priority from U.S. provisional patent application serial number

62/054,229, filed September 23, 2014. The entire disclosure of this application is incorporated herein by reference.

FIELD OF INVENTION

[21 The described invention generally relates to compositions, methods and kits for determining the potency of dendritic cells (DC) used in cancer immunotherapy.

BACKGROUND OF THE INVENTION

[3] Immune Response to Cancer

The immune system encompasses cellular immunity, humoral immunity, and complement response.

Animal models have demonstrated that, although humoral mechanisms may be relevant, it is cellular immunity that is of critical importance in transplanted tumors (Dermime S, et al., British Medical Bulletin (2002) 62: 149-162; Melief C J M, Adv. Cancer.Res. (1992) 58: 143-175). Cellular immunity includes a network of cells and events involving dendritic cells (DC), CD8⁺ T-cells (a.k.a., cytotoxic T-cells or cytotoxic lymphocytes), and CD4⁺T-cells (a.k.a., helper T-cells). T-cells recognize antigens on the cell surface of target T-cells as small peptides presented by class I or class II of the major histocompatibility complex (MHC) molecules. CD8⁺ cytotoxic T-cells recognize short peptides derived from intracellular cytoplasmic proteins and presented at the cell surface by class I MHC molecules that are expressed by virtually all nucleated cells (Dermime S, et al., British Medical Bulletin (2002) 62: 149-162; Grey H M, et al.. Cancer Ser. (1995) 22: 3749). Conversely, CD4⁺ helper T-cells recognize longer peptides derived from engulfed extracellular proteins and presented at the cell surface by class II MHC molecules by antigen presenting cells (APCs) (Dermime S, et al., British Medical Bulletin (2002) 62: 149-162; Pieters J, Adv. Immunol. (2000) 75: 159-208). APCs are central to the priming of T-cells by specific antigens, and dendritic cells (DCs) are the most potent stimulatory APCs (Dermime S, et al., British Medical Bulletin (2002) 62: 149-162). |4] Dendritic Cells

[5] Dendritic cells (DC) are bone marrow-derived cells that are seeded in all tissues (Palucka K and Banchereau J, Nature Reviews Cancer (April 2012) 12: 265-277; Banchereau J and Steinman R M, Nature (1998) 392: 245-252; Steinman R M and Banchereau J, Nature (2007) 449: 419-426). DCs exhibit several features necessary for the generation of T-cell- mediated antitumor immunity (Dermime S, et al., British Medical Bulletin (2002) 62: 149162; Cella M, et al., Curr. Opin. Immunol. (1997) 9: 10-16). They efficiently capture and take up antigens in peripheral tissues and transport these antigens to the primary and secondary lymphoid organs where they express high levels of MHC class I and II molecules that present the processed peptides to T-cells for the priming of antigen-specific responses. Specifically, a DC acquires polypeptide antigens, where these antigens can be acquired from outside of the DC, or biosynthesized inside of the DC by an infecting organism. The DC processes the polypeptide, resulting in peptides of about ten amino acids in length, transfers the peptides to either MHC class I or MHC class II to form a complex, and shuttles the complex to the surface of the DC. When a DC bearing a MHC class I/peptide complex contacts a CD8⁺ T-cell, the result is activation and proliferation of the CD8⁺ T-cell.

Regarding the role of MHC class II, when a DC bearing a MHC class Il/peptide complex contacts a CD 4⁺ T- cell, the outcome is activation and proliferation of the CD4⁺ T-cell (Munz, et al. (2010) Curr. Opin. Immunol. 22:89-93; Monaco (1995) J. Leukocyte Biol. 57:543-547; Robinson, et al (2002) Immunology 105:252-262). Although dendritic cells presenting antigen to a T-cell can "activate" that T-cell, the activated T-cell might not be capable of mounting an effective immune response. Effective immune response by the CD8⁺ T-cell, for example, often requires prior stimulation of the DC by one or more of a number of interactions. These interactions include direct contact of a CD4⁺ T-cell to the DC (by way of contact of the CD4⁺ T-cell's CD40 ligand to the DCs CD40 receptor), or direct contact of a toll-like receptor (TLR) agonist to one of the dendritic cell's toll-like receptors (TLRs).

[6] In addition, DCs express high levels of co-stimulatory molecules. The trafficking of immature DCs to sites of inflammation and or mature DCs to the T-cell area of secondary lymphoid organs is regulated by the expression of different chemokines and chemokine receptors (Rubio M T, et al., International Immunology (2005) 17(12): 1561 -1572; Sallusto F and Lanzavecchia A, Immunol. Rev. (2000) 177: 134-140; Dieu, M, et al, J. Ex. Med. (1998) 188: 373-386; Sallusto F, et al, Eur. J. Immunol. (1999) 29: 1617-1625). Immature DCs express inflammatory chemokines (monocyte chemoattractant protein- 1 (CCL2/MCP- 1), macrophage inflammatory protein-la (CCL3/MlP-la), macrophage inflammatory protein-ip (CCL4/MIP-lf3), regulated on activation, normal T- cell expressed and secreted (CCL5/RANTES) and macrophage inflammatory protein-3a (CCL20/MIP-3a)) and chemokine receptors that bind to inflammatory chemokines (chemokine (C-C motif) receptor 1 (CCRl), chemokine (C-C motif) receptor 2 (CCR2), chemokine (C-C motif) receptor 5 (CCR5), chemokine (C-C motif) receptor 6 (CCR6) and chemokine (C-X-C motif) receptor 1 (CXCRl )). Upon maturation, DCs down-regulate the inflammatory chemokines and their receptors and up-regulate constitutive chemokines such as interferon-y-inducible protein 10 (CXCLl O/IP-10), thymus and activation regulated chemokine (CCL 17/TARC), pulmonary and activation regulated chemokine (CCL 18/PARC), macrophage inflammatory protein-3p (CCL 19/MIP-3P), macrophage derived chemokine (CCL22/MDC), the chemokine receptor chemokine (C-C motif) receptor 7 (CCR7) and secondary lymphoid-tissue chemokine

(CCL21/SLC) (Rubio M T, et al.. International Immunology (2005) 17(12): 1561 - 1572).

[007] The co-stimulatory chemokines expressed by mature DCs function to attract T-cells and to interact with receptors on T-cells to provide a second signal required to optimally activate antigen-specific T-cells (Dermime S, et al, British Medical Bulletin (2002) 62: 149162; June C, et al, Immunol. Today (1994) 15: 321 -331). DCs have been shown to attract T helper 1 (Thl) and T helper 2 (Th2) cells, as well as naive T- cells and memory T-cells. T helper cells express CCR4, a G-coupled protein that is the receptor for the chemokines MCP- 1 , MIP-1 , RANTES, macrophage-derived chemokine and thymus and activation regulated chemokine (CCL 17/TARC). DCs are known to express high levels of CCL 17/TARC, which acts on the CCR4 receptor, and thus attracts and activates downstream immune cells (Vissers J L, et al, J. Leukoc. Biol. (2001) 69: 785-793). A particular advantage of attracting or "recruiting" CD4+ T-cells is that they provide "help" for cross-priming naive T-cells by expression of CD40L. The expression of CD40L permits DCs to activate CD8⁺ cytotoxic T- lymphocytes (Schoenberger S P, et al., Nature (1998) 393: 480-483). DCs also produce heterodimer IL- 12p70, which is the major determinant of their ability to promote Thl differentiation (Rubio M T, et al., International Immunology (2005) 17(12): 1561-1572; Hilkens C M, et al., Blood (1997) 90: 1920-1926).

|8] CCL17/TARC

|9] Thymus and activation regulated chemokine (CCL17/TARC) is a chemokine secreted from monocyte-derived DCs and endothelial cells, and is responsible for selective recruitment and migration of activated Th2 lymphocytes to affected tissue (Dallos T, et al.. Arthritis & Rheumatism (2010) 62(1 1): 3496-3503). The expression of both of its receptors (CCR4 and CCR8) is transiently up-regulated on activated T-cells and preferentially on cells of the Th2 subset, and conversely, production of CCL 17/TARC by DCs is stimulated by Th2 cytokines (Dallos T, et al., Arthritis & Rheumatism (2010) 62(1 1): 3496- 3503; DAmbrosio D, et al., J. Immunol. (1998) 161 : 51 1 1 -51 15; De Lavareille A, et al., Eur. J. Immunol. (2001) 31 : 1037-1046). Analogous to previously reported observations in a murine model of atopic dermatitis (Dallos T, et al., Arthritis & Rheumatism (2010) 62(1 1): 3496-3503; Xiao T, et al., Cytokine (2003) 23: 126- 132), CCL17/TARC production by DCs in affected tissue may be up-regulated by Th2 cytokines and thus provide additional chemoattraction for Th2 cells (Dallos T, et al.. Arthritis &

Rheumatism (2010) 62(1 1): 3496-3503; DAmbrosio D, et al, J. Immunol. (1998) 161 : 51 1 1 -51 15). Expression of CCL 17/TARC has been demonstrated in Langerhans' cells and keratinocytes in the skin of patients with atopic dermatitis, which, in its acute phase, is considered to be a Th2-dominated disease (Dallos T, et al.. Arthritis & Rheumatism (2010) 62(11): 3496-3503; Kakinuma T, et al., J. Allergy Clin. Immunol. (2001 ) 107: 535-541). CCL 17/TARC is expressed in CD 1 lc⁺ DCs in murine lung tissue and plays a crucial role in Th2-mediated experimental allergen-induced asthma in mice, and development of murine asthma could be inhibited by a monoclonal antibody against CCL 17/TARC (Dallos T, et al., Arthritis & Rheumatism (2010) 62(1 1): 3496-3503; Lieberman I and Forster I, J. Immunol. (1999) 29: 2684-2694; Kawasaki S, et al., J.

Immunol. (2001 ) 166: 2055-2062). CCL 17/TARC's role in Th2-mediated allergen-induced asthma is also supported by the finding of production of high levels of CCL 17/TARC by bronchial epithelial cells of asthma patients (Dallos T, et al., Arthritis & Rheumatism (2010) 62(1 1): 3496- 3503; Sekiya T, et al, J. Immunol. (2000) 165: 2205-2213).

[10] DC vaccine

[11 ] DCs that are generated ex vivo by culturing hematopoietic progenitor cells or monocytes with cytokine combinations have been tested as therapeutic vaccines in cancer patients for more than a decade (Ueno H, et al., Immunol. Rev. (2010) 234: 199-212). The therapeutic use of DC cancer vaccines has recently been revived owing to a series of clinical trials that have yielded encouraging clinical outcomes.

[12] For example, treatment of metastatic prostate cancer with sipuleucel-T (also known as APC 801 ), which is a cellular product based on enriched blood APCs that are briefly cultured with a fusion protein of prostatic acid phosphatase (PAP) and granulocyte macrophage colony-stimulating factor (GM-CSF), resulted in an approximately 4-month- prolonged median survival in Phase III trials (Higano C S, et al..

Cancer (2009) 1 15: 36703679; Kantoff P W, et al., N. Engl. J. Med. (2010) 363: 41 1 -422). This study concluded that DC-based vaccines are safe and can induce the expansion of circulating CD4⁺ T-cells and

CD8⁺ T-cells specific for tumor antigens. As a result of this and similar studies, sipuleucel-T has been approved by the US Food and Drug Administration (FDA) for the treatment of metastatic prostate cancer, thereby paving the clinical development and regulatory path for the next generation of cellular immunotherapy products (Palucka K and Banchereau J,

Nature Reviews Cancer (April 2012) 12: 265-276).

[13] For example, advanced melanoma patients receiving autologous antigen-loaded dendritic cell vaccinations in a phase II clinical trial had better overall and progression-free survival rates when their CCL 17/TARC serum levels were increased (Cornforth A N, et al., J. Clin. Immunol. (2009) 29: 657-664), leading to the observation that dendritic cell potency can be associated with the ability of DCs to secrete CCL 17/TARC which recruits CD47CD40L expressing T-cells to sites of antigen presentation and permits DCs to activate CD8⁺ cytotoxic lymphocytes. [14] Considering the long history and renewed interest in the therapeutic use of DC cancer vaccines, a need exists for the development a method to determine the potency of DCs generated for use as a therapeutic vaccine in cancer patients.

[15] The described invention provides compositions, methods and kits useful for the reliable and reproducible detection of potent DCs generated for use as a cancer vaccine. The potency of DCs can be determined by measuring (i) the ability of these cells to respond to maturation signals such as cytokines and toll-like receptor (TLR) ligands; (ii) changes in the expression of CD80, CD83, MHC 11 and/or CD54 on the surface of these cells; (iii) secretion of immune stimulating factors such as IL-12 and/or CCL 17/TARC; or (iv) a combination thereof.

SUMMARY OF THE INVENTION

116] The described invention provides compositions, methods and kits for determining potency of dendritic cells used in cancer immunotherapy.

117| According to one aspect, the described invention provides a method for preparing a population of immunopotent dendritic cells activated in vitro with a tumor-specific antigen derived from a population of purified cultivated tumor cells derived from a patient comprising: (a) obtaining peripheral blood mononuclear cells (PBMCs) by leukapheresis from the patient from whom the tumor cells were derived; (b) optionally shipping the collected PBMCs from (a) to a manufacturing facility; (c) purifying the PBMCs from (a)

from other lymphocytes; (d) incubating the purified PBMCs from (c) with GM-CSF and IL-4 for 6 days to generate dendritic cells; and (e)contacting the dendritic cells from (d) with the purified cultivated rumor cells derived from the patient for 18-24 hours to form immunopotent dendritic cells, wherein

immunopotency of the dendritic cells formed in (e) is measured by an amount of a biomarker produced by the dendritic cells formed in (e), and wherein the dendritic cells formed in (e) are effective to generate an effective immune response against the tumor-specific antigen comprising activation and proliferation of CD4+ T-cells, CD8+ T-cells, B-cells or a combination thereof. [18] According to one embodiment, the amount of the biomarker is measured by an immunoassay. According to another embodiment, the immunoassay is selected from the group consisting of Western blot, ELISA and flow cytometry. According to another embodiment, the biomarker is CCL 17/TARC. According to another embodiment, the amount of the biomarker is at least 274.3 pg/mL/day.

[19] According to one aspect, the described invention provides a method for treating a subject suffering from a cancer comprising: (a) preparing for a cancer patient a patient- specific immunogenic composition comprising an immunopotent amount of an isolated population of dendritic cells contacted ex vivo with a cancer cell expressing a cancer-specific antigen by the method according to claim 1 ; (b) administering the immunogenic composition to the cancer patient; and (c) generating an effective immune response against the cancer- specific antigen comprising activation and proliferation of CD4+ T-cells, CD8+ T-cells, B- cells or a combination thereof, wherein the effective immune response is effective to improve a clinical parameter selected from the group consisting of progression-free survival, disease- free survival, time to progression, time to distant metastasis and overall survival of the subject when compared to a control.

BRIEF DESCRIPTION OF THE DRAWINGS

120] Figure 1 depicts a CCL17/TARC standard curve for Sample # 1955 and Sample # 1 893.

[21] Figure 2 depicts calculated concentrations of Sample # 1955 and Sample # 1893 from diluted concentrations within an acceptable percent linearity. Data is expressed as mean ± standard error.

[22] Figure 3 depicts calculated concentrations of Sample # 1955 and 1893 from all diluted concentrations tested. Data is expressed as mean ± standard error.

[23] Figure 4 depicts a CCL17/TARC standard curve for Sample #2353.

|24] Figure 5 depicts a bar graph representing CCL17/TARC concentrations for Sample # 2353 calculated from a 1 :8 dilution.

[25| Figure 6 depicts an IL-12 standard curve for Sample # 1955 and Sample # 1 893. |26] Figure 7 depicts a bar graph representing IL-12 concentrations for Sample # 1955 and 1893 calculated from 1 :2 and 1 :20 dilutions.

[27] Figure 8 depicts an IL-12 standard curve for Sample #2353.

|28] Figure 9 depicts a bar graph representing IL-12 concentrations for Sample #2353 calculated from undiluted, 1 :2 and 1 :4 dilutions.

[29] Figure 10 depicts cytometry data for various dendritic cell media conditions. Representative flow cytometry of DC-TC incubated under control conditions (DC media plus 5% FBS only) or under dendritic cell maturation conditions (DC media plus 5% FBS plus 0.5|j.g/mL CD40L, 1000 lU/mL gIFN and 1 |ag/mL LPS) with three different Goli-Stop treatment conditions (Brefeldin-A) none (0), 4 hours and 24 hours. The extracellular/intracellular flow cytometry optimization consisted of applying extracellular antigen antibodies (CD40 and CD83) prior to fixation and permeabilization (Pre-fix) or after fixation and permeabilization (Post-fix) followed by staining with intracellular antigen antibodies (CCL 17 and IL12).

[30] Figure 11 depicts AIM-V media conditions. Representative flow cytometry of DC- TC incubated under control conditions (AIMV media plus 5% FBS only) or under dendritic cell maturation conditions (AIMV media plus 5% FBS plus 0.5|j.g/mL CD40L, lOOOIU/mL gIFN and 1 ug/mL LPS) with three different Goli-Stop treatment conditions (Brefeldin-A) none (0), 4 hours and 24 hours. The extracellular/intracellular flow cytometry optimization consisted of applying the extracellular antigen antibodies (CD40 and CD83) prior to fixation and permeabilization (Pre-fix) or after fixation and permeabilization (Post-fix) followed by staining with intracellular antigen antibodies (CCL 17 and IL12). 131 1 Figure 12 depicts a bar graph representing flow cytometry results for Sample # 1986 for

CD40/CCL 17 double positive cells for each of the eight conditions tested. Each bar represents a single experiment. CTL = control, CK = cytokine matured. AIM = AIMV plus 5%FBS. DC = Dendritic cell media plus 5%FBS.

[321 Figure 13 depicts a bar graph representing flow cytometry results for Sample # 1986 for CD83/IL- 12 double positive cells for each of the eight conditions tested. Each bar represents a single experiment. CTL = control, CK = cytokine matured. AIM = AIMV plus 5%FBS. DC = Dendritic cell media plus 5%FBS. [33] Figure 14 depicts a bar graph representing flow cytometry results for Sample # 1955 for

[34] Figure 15 depicts a bar graph representing flow cytometry results for Sample # 1955 for CD83/IL- 12 double positive cells for each of the eight conditions tested. Each bar represents a single experiment. CTL = control, CK = cytokine matured. AIM = AIMV plus 5%FBS. DC = Dendritic cell media plus 5%FBS.

[35] Figure 16 depicts a bar graph representing flow cytometry results for Sample #1718 for

|36| Figure 17 depicts a bar graph representing flow cytometry results for Sample #1718 for CD83/IL-12 double positive cells for each of the eight conditions tested. Each bar represents a single experiment. CTL = control, CK = cytokine matured. AIM = AIMV plus 5%FBS. DC = Dendritic cell media plus 5%FBS. |37| Figure 18 depicts flow cytometry results for CD40, CD80, MHC-II and CD54. Incubation of normal donor dendritic cells generated in Cell Genix or AIMV media followed by standard maturation cytokine cocktail or toll-like receptor ligand with CD40L/IFNgamma overnight in 5%FBS/AIMV. CTL is equal to 5% FBS/AIMV only. CK1 is equal to 5% FBS/AIMV plus 0.5 Lj,g/mL CD40L, 1000 lU/mL IFNgamma, IOng/mL TNFalpha, lOng/mL ILl-beta, and 15ng/mL IL6 and CK2 is equal to 5% FBS/AIMV plus 0.5|a,g/mL CD40L, 1000 lU/mL IFNgamma and 30|ig/mL poly IC.

[381 Figure 19 depicts flow cytometry results for CD83 and IL- 12. Incubation of normal donor dendritic cells generated in Cell Genix or AIMV media followed by standard maturation cytokine cocktail or toll-like receptor ligand with CD40L/IFNganima overnight in 5% FBS/AIMV. CTL is equal to 5% FBS/AIMV only. C 1 is equal to 5% FBS/AIMV plus 0.5|ag/mL CD40L, 1000 lU/mL IFNgamma, IOng/mL TNFalpha, !Ong/mL ILl-beta, and

15ng/mL IL6 and CK2 is equal to 5%FBS/AIMV plus 0.5|ig/mL CD40L, 1000 lU/mL IFNgamma and 30|jg/mL poly IC. |39] Figure 20 depicts flow cytometry results for CD54, CD40, CD83 and MHC-class II. Incubation of normal donor dendritic cells generated in AIM-V dendritic cell media followed by sequential treatment with toll-like receptor ligand and maturation cytokines. Normal donor dendritic cells were exposed to 1 (ig/mL LPS, 30 MglmL poly IC, or control conditions for 24 hours followed by the addition of 0.5 [j.g/mL CD40L and 1000 lU/mL IFNgamma for additional 24 hours. Brefeldin-A was added for the last 4 hours of incubation. Control consisted of AIM-V plus 5% FES only. A single donor dendritic cell sample was used. |40] Figure 2 1 depicts flow cytometry results for CD54, CD40, CD83 and MHC-class II. Incubation of autologous antigen-loaded dendritic cells generated in AIMV dendritic cell media followed by combinatorial treatment with toll-like receptor ligand and maturation cytokines. Antigen -loaded dendritic cells were exposed to 1 [ig/mL LPS, 30 |ag/mL poly IC, 0.5 [j.g/mL CD40L and 1000 lU/mL IFNgamma or in combination for 24 hours. Brefeldin-A was added for the last 4 hours of incubation. Control consisted of AIMV plus 5% FBS only. Three separate antigen-loaded dendritic cells were assayed, only a single sample is shown.

141 j Figure 22 depicts flow cytometry results of for IL-12. Incubation of autologous antigen-loaded dendritic cells generated in AIM-V dendritic cell media followed by combinatorial treatment with toll-like receptor ligand and maturation cytokines. Antigen- loaded dendritic cells were exposed to 1 |a.g/mL LPS, 30 |j.g/mL poly IC, 0.5 [j,g/mL CD40L and 1000 lU/mL IFNgamma or in combination for 24 hours followed by 4 hours incubation with brefeldin-A. Control consisted of AIMV plus 5% FBS only. Three separate antigen- loaded dendritic cells were assayed, only a single sample is shown.

[42] Figure 23 depicts a bar graph representing expression of CD83 and CD40 in monocytes, unloaded dendritic cells (DC only) and antigen-loaded dendritic cells (DC-TC) after incubation for approximately 24 hours in either unstimulated (media only),

CD40L/gIFN or CD40L/gIFN/LPS maturation conditions. N = 5 for monocytes and DC only and N = 1 1 for DC-TC.

[43] Figure 24 depicts a bar graph representing CCL 17/TARC ELISA results for monocyte, unloaded dendritic cells (DC only) and antigen-loaded dendritic cells (DC-TC). N = 5 for monocytes and DC only and N = 1 1 for DC-TC. [44] Figure 25 depicts CCL 17/TARC ELISA results normalized on a per day basis.

Values shown are an average of three data points per sample. N = 1 1.

[45] Figure 26 depicts a Kaplan Meier survival curve correlating cancer patient outcome with

CCL 17/TARC concentration.

DETAILED DESCRIPTION OF THE INVENTION

[461 The invention can be better understood from the following description of exemplary embodiments, taken in conjunction with the accompanying figures and drawings. It should be apparent to those skilled in the art that the described embodiments of the present invention provided herein are merely exemplary and illustrative and not limiting.

Definitions;

[47] Various terms used throughout this specification shall have the definitions set out herein.

|48] The term "activation" or "lymphocyte activation" as used herein, refers to stimulation of lymphocytes by specific antigens, nonspecific mitogens, or allogeneic cells resulting in synthesis of RNA, protein and DNA and production of lymphokines; it is followed by proliferation and differentiation of various effector and memory cells. For example, a mature B-cell can be activated by an encounter with an antigen that expresses epitopes that are recognized by its cell surface immunoglobulin Ig). The activation process may be a direct one, dependent on cross-linkage of membrane Ig molecules by the antigen (cross-linkage- dependent B-cell activation) or an indirect one, occurring most efficiently in the context of an intimate interaction with a helper T-cell ("cognate help process"). T-cell activation is dependent on the interaction of the TCR/CDS complex with its cognate ligand, a peptide bound in the groove of a class I or class II MHC molecule. The molecular events set in motion by receptor engagement are complex. Among the earliest steps appears to be the activation of tyrosine kinases leading to the tyrosine phosphorylation of a set of substrates that control several signaling pathways. These include a set of adapter proteins that link the TCR to the ras pathway, phospholipase Cyl, the tyrosine phosphorylation of which increases its catalytic activity and engages the inositol phospholipid metabolic pathway, leading to elevation of intracellular free calcium concentration and activation of protein kinase C, and a series of other enzymes that control cellular growth and differentiation. Full responsiveness of a T-cell requires, in addition to receptor engagement, an accessory cell-delivered costimulatory activity, e.g., engagement of CD28 on the T-cell by CD80 and/or CD86 on the antigen presenting cell (APC). The soluble product of an activated B lymphocyte is immmunoglobulins (antibodies). The soluble product of an activated T lymphocyte is lymphokines.

[49] The term "CCL2/MCP-1 " as used herein, refers to monocyte chemoattractant protein- 1 , which is a member of the C-C chemokine family, and a potent chemotactic factor for monocytes. CCL2/MCP-1 is produced by many cell types, including dendritic, endothelial, fibroblasts, epithelial, smooth muscle, mesangial, astrocytic, monocytic and microglial cells. CCL2/MCP-1 regulates the migration and infiltration of monocytes, memory T-cells and natural killer (NK) cells.

|50[ The term "CCL3/MIP-la" as used herein, refers to macrophage inflammatory protein-la, which is a member of the C-C family of chemokines. CCL3/MIP-la expression can be induced in a variety of cell types, including dendritic cells, Langerhans cells, fibroblasts and T-cells. CCL3/MIP-la induces migration of monocytes and T-cells. It is primarily chemotactic for B-cells and activated CD8⁺ T-cells.

|51] The term "CCL4/MIP-1 P" as used herein, refers to macrophage inflammatory protein-ip, which is a member of the C-C family of chemokines. CCL4/MIP-1P is secreted by a variety of cell types, including dendritic cells and is the principal regulator of macrophage migration. CCL4/MIP- I P binds to and signals through the CCR5 receptor.

1521 The term "CCL5/RANTES" as used herein, refers to Regulated on Activation Normal T-cell Expressed and Secreted, which is a member of the C-C family of chemokines. CCL5/RANTES is secreted by many hematopoietic and non-hematopoietic cells, including dendritic cells. CCL5/RANTES plays an important role in homing and migration of effector and memory T- cells by binding to and signaling through CCR5 receptor.

[53] The term "CCL20/MIP-3a" as used herein, refers to macrophage inflammatory protein-3a, which is a member of the C-C family of chemokines. CCL20/MIP-3a is predominantly expressed in

extralymphoid tissue and is known to direct migration of dendritic cell precursors and memory

lymphocytes to sites of antigen invasion.

|54| The term "CCR1 " as used herein, refers to chemokine (C-C motif) receptor 1. CCR1 is a member of the beta chemokine receptor family, predicted to be a seven transmembrane protein. Ligands for CCR1 include CCL3/MIP-la, CCL5/RANTES, monocyte chemoattractant protein 3 (MCP-3) and myeloid progenitor inhibitory factor-1 (MPIF-1 ). CCR1 -mediated signal transduction is critical for recruitment of effector immune cells to the site of inflammation and plays a role in host protection from inflammatory response and susceptibility to virus and parasite.

[55] The term "CCR2" as used herein, refers to chemokine (C-C motif) receptor 2. CCR2 is a receptor for monocyte chemoattractant protein- 1 (CCL2/MCP-1), a chemokine involved in monocyte infiltration in inflammatory diseases. CCR2 mediates agonist-dependent calcium mobilization and inhibition of adenylyl cyclase.

|56| The term "CCR5" as used herein, refers to chemokine (C-C motif) receptor 5. CCR5 is a member of the beta chemokine receptor family, predicted to be a seven transmembrane protein. Ligands for CCR1 include CCL3/MlP-la, CCL5/RANTES, monocyte chemoattractant protein 3 (MCP-3) and myeloid progenitor inhibitory factor- 1 (MPIF-1 ). CCRl -mediated signal transduction is critical for recruitment of effector immune cells to the site of inflammation and plays a role in host protection from inflammatory response and susceptibility to virus and parasite.

|57| The term "CCR6" as used herein, refers to chemokine (C-C motif) receptor 6. CCR6 is a member of the beta chemokine receptor family, predicted to be a seven transmembrane protein. The ligand for CCR6 is macrophage inflammatory protein 3 alpha (MI -3a). CCR6 has been shown to be important for B-Iineage maturation and antigen-driven B-cell differentiation, and it may regulate the migration and recruitment of dendritic and T cells during inflammatory and immunological responses.

[58] The term "CD 54" (also known as ICAM-1 ) as used herein, refers to cluster of differentiation 54. CD54 is a type I transmembrane protein present on leukocytes and endothelial cells and inducible on lymphocytes, dendritic cells, keratinocytes, chondrocytes, fibroblasts and epithelial cells. CD54 acts as a ligand for CD 1 1 and CD 18 and aids in intercellular adhesion.

|59] The term "CD80" as used herein, refers to cluster of differentiation 80. CD80 is a membrane receptor activated by the binding of CD28 or CTLA-4. Activated CD80 induces T-cell proliferation and cytokine production.

[601 The term "CD83 " as used herein, refers to cluster of differentiation 83. CD83 is a single-pass type I membrane protein thought to be involved in the regulation of antigen presentation. A soluble form of this protein can bind to dendritic cells and inhibit their maturation.

[61 ] The term "CXCR1" as used herein, refers to chemokine (C-X-C motif) receptor 1. CXCR1 is a member of the G-protein-coupled receptor family. This protein is a receptor for interleukin 8 (IL-8). It binds to IL-8 with high affinity, and transduces the signal through a G- protein activated second messenger system. Knockout studies in mice suggested that this protein inhibits embryonic oligodendrocyte precursor migration in developing spinal cord.

|62| The term "cellular immunity" as used herein, refers to T lymphocyte-mediated immunity. T lymphocytes, or T-cells, are known to directly kill cells, to provide "help" for such killers, to activate other immune system cells (e.g., macrophages), to help B-cells make an antibody response, to down-modulate the activities of various immune system cells, and to secrete cytokines, chemokines, and other mediators. T- cells are divided into two (2) major classes: T helper cells (Th) and regulatory T-cells (T_reg). T helper cells are further subdivided into T helper 1 (Thl) cells and T helper 2 (Th2) cells. The type 1 and type 2 helper classes are defined by their cytokine secretion profiles. T-helper 1 (Thl) cells, which are implicated in the stimulation of inflammation, produce IFN-gamma, GM-CSF, TNF-beta, and TNF alpha. TNF and IFN- gamma signals synergize in inducing an activated state in the macrophage, and lead to increased expression of adhesion and homing molecules in the vascular endothelium, which recruit additional blood-bom leukocytes to the site of inflammation. (See, Paul, Fundamentals of Immunol. 4^th Ed., p. 397 (1999)). T helper 2 (Th- 2) cells produce IL-4, IL-5, IL-10, and iL-13, and provide help for B-cells in their activation and differentiation leading to the humoral immune response, (de Waal Malefyt, Immunity 31 : 700-702 (2009)). Regulatory T-cells, either natural, induced, or Trl cells, produce IL-10 and TGFp, suppress the activation of effector T-cells, and provide a counter-balance against uncontrolled and harmful T-cell responses. Id. Th9 cells may provide additional help for mast-cells through the production of IL-9. Id. Thl 7, an additional T-cell subset, produces IL-17A, 17- 17F, IL-22 and CCL20, which act on stromal and epithelial cells to induce a number of secondary effector molecules, such as G-CSF, which stimulates the production and mobilization of neutrophils, acute phase proteins, chemokines, and antimicrobial peptides. Id. Naive T- cells can differentiate into any of the distinct T-cell subsets when activated in the presence of appropriate signals and cytokines. The induction of a maturation process in dendritic cells is a crucial step for efficient priming of naive T-cells. There is an extensive cross-regulation between subsets to ensure that the appropriate T-cell subset is activated. Id.

|63| The term "chemokine" as used herein refers to a class of chemotactic cytokines that signal leukocytes to move in a specific direction. The terms "chemotaxis" or "chemotactic" refer to the directed motion of a motile cell or part along a chemical concentration gradient towards environmental conditions it deems attractive and/or away from surroundings it finds repellent.

[64| The term "cytokine" as used herein, refers to small soluble protein substances secreted by cells which have a variety of effects on other cells. Cytokines mediate many important physiological functions including growth, development, wound healing, and the immune response. They act by binding to their cell-specific receptors located in the cell membrane, which allows a distinct signal transduction cascade to start in the cell, which eventually will lead to biochemical and phenotypic changes in T-cells. Generally, cytokines act locally. They include type I cytokines, which encompass many of the interleukins, as well as several hematopoietic growth factors; type II cytokines, including the interferons and interleukin- 10; tumor necrosis factor ("TNF")-related molecules, including TNFa and lymphotoxin; immunoglobulin super-family members, including interleukin 1 ("IL- 1 "); and the chemokines, a family of molecules that play a critical role in a wide variety of immune and inflammatory functions. The same cytokine can have different effects on a cell depending on the state of the cell.

Cytokines often regulate the expression of, and trigger cascades of, other cytokines.

(65] The term "dendritic cell" or "DC" as used herein refers to bone marrow -derived cells that are seeded in all tissues. DCs exhibit several features which are necessary for the generation of T-cell-mediated antitumor immunity. That is, they efficiently capture and take up antigens in peripheral tissues and transport these antigens to the primary and secondary lymphoid organs where they express high levels of MHC class I and II molecules that present the processed peptides to T-cells for the priming of antigen-specific responses.

|66] The term "enzymatic activity" as used herein refers to the action of an enzyme (meaning a protein that catalyzes a specific chemical reaction) on its target. It is quantified as the amount of substrate consumed (or product formed) in a given time under given conditions. The term "turnover number" as used herein refers to the number of molecules of substrate that can be converted into product per catalytic site of a given enzyme.

|67] The terms "immune response" and "immune-mediated" are used interchangeably herein to refer to any functional expression of a subject's immune system, against either foreign or self antigens, whether the consequences of these reactions are beneficial or harmful to the subject. [68| The term "immunomodulatory cell(s)" as used herein refer(s) to cell(s) that are capable of augmenting or diminishing immune responses by expressing chemokines, cytokines and other

mediators of immune responses.

[69] The term "immunopotent" as used herein, refers to the ability to activate and guide a naive immune system to mount a response toward a foreign protein.

[70| The term "inflammatory cytokines" or "inflammatory mediators" as used herein refers to the molecular mediators of the inflammatory process, which may modulate being either pro- or antiinflammatory in their effect. These soluble, diffusible molecules act both locally at the site of tissue damage and infection and at more distant sites. Some inflammatory mediators are activated by the inflammatory process, while others are synthesized and/or released from cellular sources in response to acute inflammation or by other soluble inflammatory mediators. Examples of inflammatory mediators of the inflammatory response include, but are not limited to, plasma proteases, complement, kinins, clotting and fibrinolytic proteins, lipid mediators, prostaglandins, leukotrienes, platelet- activating factor (PAF), peptides and amines, including, but not limited to, histamine, serotonin, and neuropeptides, pro-inflammatory cytokines, including, but not limited to, interleukin-l-beta (IL-ip), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-a), interferon-gamma (IF-y), and interleukin- 12 (IL- 12).

[71] The term "interleukin (IL)" as used herein refers to a cytokine secreted by, and acting on, leukocytes. Interleukins regulate cell growth, differentiation, and motility, and stimulates immune responses, such as inflammation. Examples of interleukins include interleukin- 1 (IL- 1 ), interleukin 2 (IL- 2), interleukin- 1 (3 (IL- 1 (3), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin- 12 (IL- 12).

[72] The term "interleukin-12" or "IL-12" as used herein refers to a cytokine that regulates the differentiation of naive T-cells into Thl cells. It stimulates the growth and function of T- cells and alters the normal cycle of apoptotic cell death. IL-12 is one of a large group of cytokines that folds into a bundle of four alpha-helices. It is a heterodimer of 70kDa that is composed of two disulfide-linked subunits, of mass 35kDa and 40kDa.

|73] The term "Kaplan Meier plot" or "Kaplan Meier survival curve" as used herein refers to the plot of probability of clinical study subjects surviving in a given length of time while considering time in many small intervals. The Kaplan Meier plot assumes that: (i) at any time subjects who are censored (i.e., lost) have the same survival prospects as subjects who continue to be followed; (ii) the survival probabilities are the same for subjects recruited early and late in the study; and (iii) the event (e.g., death) happens at the time specified. Probabilities of occurrence of event are computed at a certain point of time with successive probabilities multiplied by any earlier computed probabilities to get a final estimate. The survival probability at any particular time is calculated as the number of subjects surviving divided by the number of subjects at risk. Subjects who have died, dropped out, or have been censored from the study are not counted as at risk.

|74| The term "lymphocyte" refers to a small white blood cell formed in lymphatic tissue throughout the body and in normal adults making up about 22-28% of the total number of leukocytes in the circulating blood that plays a large role in defending the body against disease. Individual lymphocytes are specialized in that they are committed to respond to a limited set of structurally related antigens. This commitment, which exists before the first contact of the immune system with a given antigen, is expressed by the presence on the lymphocyte's surface membrane of receptors specific for determinants (epitopes) on the antigen. Each lymphocyte possesses a population of receptors, all of which have identical combining sites. One set, or clone, of lymphocytes differs from another clone in the structure of the combining region of its receptors and thus differs in the epitopes that it can recognize. Lymphocytes differ from each other not only in the specificity of their receptors, but also in their functions.

|75] Two broad classes of lymphocytes are recognized: the B-lymphocytes (B-cells), which are precursors of antibody-secreting cells, and T-lymphocytes (T-cells), B-lymphocytes

[76| B-lymphocytes are derived from hematopoietic cells of the bone marrow. A mature B-cell can be activated with an antigen that expresses epitopes that are recognized by its cell surface. The activation process may be direct, dependent on cross-linkage of membrane Ig molecules by the antigen (cross-linkage-dependent B-cell activation), or indirect, via interaction with a helper T-cell, in a process referred to as cognate help. In many physiological situations, receptor cross-linkage stimuli and cognate help synergize to yield more vigorous B-cell responses. (Paul, W. E., "Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4^lh Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)).

[77] Cross-linkage dependent B-cell activation requires that the antigen express multiple copies of the epitope complementary to the binding site of the cell surface receptors because each B-cell expresses Ig molecules with identical variable regions. Such a requirement is fulfilled by other antigens with repetitive epitopes, such as capsular polysaccharides of microorganisms or viral envelope proteins. Cross-linkage-dependent B-cell activation is a major protective immune response mounted against these microbes. (Paul, W. E., "Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4^lh Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)).

[78] Cognate help allows B-cells to mount responses against antigens that cannot crosslink receptors and, at the same time, provides co-stimulatory signals that rescue B-cells from inactivation when they are stimulated by weak cross-linkage events. Cognate help is dependent on the binding of antigen by the B-cell's membrane immunoglobulin (Ig), the endocytosis of the antigen, and its fragmentation into peptides within the endosomal/lysosomal compartment of the cell. Some of the resultant peptides are loaded into a groove in a specialized set of cell surface proteins known as class II major

histocompatibility complex (MHC) molecules. The resultant class II/peptide complexes are expressed on the cell surface and act as ligands for the antigen-specific receptors of a set of T-cells designated as CD4⁺ T-cells. The CD4⁺ T-cells bear receptors on their surface specific for the B-cell's class Il/peptide complex. B-cell activation depends not only on the binding of the T-cell through its T-cell receptor (TCR), but this interaction also allows an activation ligand on the T-cell (CD40 ligand) to bind to its receptor on the B-cell (CD40) signaling B- cell activation. In addition, T helper cells secrete several cytokines that regulate the growth and differentiation of the stimulated B-cell by binding to cytokine receptors on the B-cell. (Paul, W. E., "Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4^th Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)).

|79| During cognate help for antibody production, the CD40 ligand is transiently expressed on activated CD4⁺ T helper cells, and it binds to CD40 on the antigen-specific B- cells, thereby tranducing a second co- stimulatory signal. The latter signal is essential for B- cell growth and differentiation and for the generation of memory B-cells by preventing apoptosis of germinal center B-cells that have encountered antigen. Hyperexpression of the CD40 ligand in both B and T-cells is implicated in the pathogenic autoantibody production in human SLE patients. (Desai-Mehta, A. et al., "Hyperexpression of CD40 ligand by B and T- cells in human lupus and its role in pathogenic autoantibody production," J. Clin. Invest., 97(9): 2063-2073 (1996)).

T-lymphocytes

(80 J T-lymphocytes derive from precursors in hematopoietic tissue, undergo differentiation in the thymus, and are then seeded to peripheral lymphoid tissue and to the recirculating pool of lymphocytes. T- lymphocytes or T-cells mediate a wide range of immunologic functions. These include the capacity to help B-cells develop into antibody- producing cells, the capacity to increase the microbicidal action of monocytes/macrophages, the inhibition of certain types of immune responses, direct killing of target cells, and mobilization of the inflammatory response. These effects depend on their expression of specific cell surface molecules and the secretion of cytokines. (Paul, W. E., "Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4^th Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)). [81 ] T-cells differ from B-cells in their mechanism of antigen recognition. Immunoglobulin, the B-cell's receptor, binds to individual epitopes on soluble molecules or on particulate surfaces. B-cell receptors see epitopes expressed on the surface of native molecules. Antibody and B-cell receptors evolved to bind to and to protect against microorganisms in extracellular fluids. In contrast, T-cells recognize antigens on the surface of other cells and mediate their functions by interacting with, and altering, the behavior of these antigen-presenting cells (APCs). There are three main types of antigen-presenting cells in peripheral lymphoid organs that can activate T-cells: dendritic cells, macrophages and B- cells. The most potent of these are the dendritic cells, whose only function is to present foreign antigens to T-cells. Immature dendritic cells are located in tissues throughout the body, including the skin, gut, and respiratory tract. When they encounter invading microbes at these sites, they endocytose the pathogens and their products, and carry them via the lymph to local lymph nodes or gut associated lymphoid organs. The encounter with a pathogen induces the dendritic cell to mature from an antigen-capturing cell to an antigen- presenting cell (APC) that can activate T-cells. APCs display three types of protein molecules on their surface that have a role in activating a T-cell to become an effector cell: (1) MHC proteins, which present foreign antigen to the T-cell receptor; (2) co-stimulatory proteins which bind to complementary receptors on the T-cell surface; and (3) cell-cell adhesion molecules, which enable a T-cell to bind to the antigen-presenting cell (APC) for long enough to become activated. ("Chapter 24: The adaptive immune system," Molecular Biology of the Cell, Alberts, B. et al., Garland Science, NY, 2002).

1821 T-cells are subdivided into two distinct classes based on the cell surface receptors they express. The majority of T-cells express T-cell receptors (TCR) consisting of a and p chains. A small group of T- cells express receptors made of y and 5 chains. Among the a/p T-cells are two important sublineages: those that express the coreceptor molecule CD4 (CD4⁺T-cells); and those that express CDS (CD8⁺ T-cells). These cells differ in how they recognize antigen and in their effector and regulatory functions.

[83] CD4⁺ T-cells are the major regulatory cells of the immune system. Their regulatory function depends both on the expression of their cell-surface molecules, such as CD40 ligand whose expression is induced when the T-cells are activated, and the wide array of cytokines they secrete when activated.

[84| T-cells also mediate important effector functions, some of which are determined by the patterns of cytokines they secrete. The cytokines can be directly toxic to target cells and can mobilize potent inflammatory mechanisms.

185] In addition, T-cells, particularly CD8⁺ T-cells, can develop into cytotoxic T- lymphocytes (CTLs) capable of efficiently lysing target cells that express antigens recognized by the CTLs. (Paul, W. E., "Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4^lh Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)).

[86] T-cell receptors (TCRs) recognize a complex consisting of a peptide derived by proteolysis of the antigen bound to a specialized groove of a class II or class IMHC protein. The CD4+ T-cells recognize only peptide/class II complexes while the CD8⁺ T-cells recognize peptide/class I complexes. (Paul, W. E., "Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4^lh Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)).

|87| The TCR's ligand (i.e., the peptide/MHC protein complex) is created within antigen- presenting cells (APCs). In general, class II MHC molecules bind peptides derived from proteins that have been taken up by the APC through an endocytic process. These peptide- loaded class II molecules are then expressed on the surface of the cell, where they are available to be bound by CD4⁺ T-cells with TCRs capable of recognizing the expressed cell surface complex. Thus, CD4⁺ T-cells are specialized to react with antigens derived from extracellular sources. (Paul, W. E., "Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4¹ Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)).

|88) In contrast, class I MHC molecules are mainly loaded with peptides derived from internally synthesized proteins, such as viral proteins. These peptides are produced from cytosolic proteins by proteolysis by the proteosome and are translocated into the rough endoplasmic reticulum. Such peptides, generally nine amino acids in length, are bound into the class I MHC molecules and are brought to the cell surface, where they can be recognized by CD8+ T-cells expressing appropriate receptors. This gives the T- cell system, particularly CD8+ T-cells, the ability to detect cells expressing proteins that are different from, or produced in much larger amounts than, those of cells of the remainder of the organism (e.g., vial antigens) or mutant antigens (such as active oncogene products), even if these proteins in their intact form are neither expressed on the cell surface nor secreted. (Paul, W. E.,

"Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4^th Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia ( 1999)).

|89| T-cells can also be classified based on their function as helper T-cells; T-cells involved in inducing cellular immunity; suppressor T-cells; and cytotoxic T-cells.

Helper T-cells

[90] Helper T-cells are T-cells that stimulate B-cells to make antibody responses to proteins and other T-cell-dependent antigens. T-cell-dependent antigens are immunogens in which individual epitopes appear only once or a limited number of times such that they are unable to cross-link the membrane

immunoglobulin (Ig) of B-cells or do so inefficiently. B- cells bind the antigen through their membrane Ig, and the complex undergoes endocytosis. Within the endosomal and lysosomal compartments, the antigen is fragmented into peptides by proteolytic enzymes and one or more of the generated peptides are loaded into class II MHC molecules, which traffic through this vesicular compartment. The resulting peptide/class II MHC complex is then exported to the B-cell surface membrane. T-cells with receptors specific for the peptide/class II molecular complex recognize this complex on the B-cell surface. (Paul, W. E., "Chapter 1 : The immune system: an introduction,"

Fundamental Immunology, 4^th Edition, Ed. Paul, W. E., Lippincott-Raven Publishers,

Philadelphia (1999)).

[91] B-cell activation depends both on the binding of the T-cell through its TCR and on the interaction of the T-cell CD40 ligand (CD40L) with CD40 on the B-cell. T-cells do not constitutively express CD40L. Rather, CD40L expression is induced as a result of an interaction with an APC that expresses both a cognate antigen recognized by the TCR of the T-cell and CD80 or CD86. CD80/CD86 is generally expressed by activated, but not resting, B-cells so that the helper interaction involving an activated B-cell and a T-cell can lead to efficient antibody production. In many cases, however, the initial induction of CD40L on T- cells is dependent on their recognition of antigen on the surface of APCs that constitutively express CD80/86, such as dendritic cells. Such activated helper T-cells can then efficiently interact with and help B-cells. Cross-linkage of membrane Ig on the B-cell, even if inefficient, may synergize with the CD40L/CD40 interaction to yield vigorous B-cell activation. The subsequent events in the B-cell response, including proliferation, Ig secretion, and class switching (of the Ig class being expressed) either depend or are enhanced by the actions of T-cell-derived cytokines. (Paul, W. E., "Chapter 1 : The immune system; an introduction," Fundamental Immunology, 4^th Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)).

[92] CD4⁺ T-cells tend to differentiate into cells that principally secrete the cytokines IL- 4, IL-5, IL- 6, and I L- l 0 (Th2 cells) or into cells that mainly produce IL-2, IFN-y, and lymphotoxin (Thl cells). The Th2 cells are very effective in helping B-cells develop into antibody-producing cells, whereas the THl cells are effective inducers of cellular immune responses, involving enhancement of microbicidal activity of monocytes and macrophages, and consequent increased efficiency in lysing microorganisms in intracellular vesicular compartments. Although the CD4⁺ T-cells with the phenotype of TH 2 cells (i.e., IL-4, IL-5, IL-6 and IL-l 0) are efficient helper cells, THl cells also have the capacity to be helpers. (Paul, W. E., "Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4^th Edition, Ed. Paul, W. E., Lippincott-Raven Publishers, Philadelphia (1999)).-

T-cells involved in Induction of Cellular Immunity

[93] T-cells also may act to enhance the capacity of monocytes and macrophages to destroy intracellular microorganisms. In particular, interferon-gamma (IFN-y) produced by helper T-cells enhances several mechanisms through which mononuclear phagocytes destroy intracellular bacteria and parasitism including the generation of nitric oxide and induction of tumor necrosis factor (TNF) production. The Thl cells are effective in enhancing the microbicidal action because they produce IFN-y. By contrast, two of the major cytokines produced by Th2 cells, IL-4 and IL- 10, block these activities. (Paul, W. E., "Chapter 1 : The immune system: an introduction," Fundamental Immunology, 4^th Edition, Ed. Paul, W. E., Lippincott-Raven

Publishers, Philadelphia (1999)).

Suppressor or Regulatory T (Treg) cells

|94| A controlled balance between initiation and downregulation of the immune response is important to maintain immune homeostasis. Both apoptosis and T-cell anergy (a tolerance mechanism in which the T-cells are intrinsically functionally inactivated following an antigen encounter (Scwartz, R. H., "T-cell anergy," Annu. Rev. Immunol., 21 : 305-334 (2003)) are important mechanisms that contribute to the downregulation of the immune response. A third mechanism is provided by active suppression of activated T-cells by suppressor or regulatory CD4⁺ T (T,_¾) cells. (Reviewed in ronenberg, M. et al.,

"Regulation of immunity by self-reactive T-cells," Nature 435: 598-604 (2005)). CD4⁺

constitutively express the IL-2 receptor alpha (IL-2Ra) chain (CD4⁺ CD25⁺) are a naturally occurring T-cell subset that are anergic and suppressive. (Taams, L. S. et 1.,

"Human anergic/suppressive CD4⁺CD25⁺ T-cells: a highly differentiated and apoptosis- prone population," Eur. J. Immunol, 31 : 1122-1 131 (2001)). Depletion of CD4⁺CD25⁺

Tregs results in systemic autoimmune disease in mice. Furthermore, transfer of these

development of autoimmune disease. Human CD4⁺CD25⁺ Tc_gs similar to their murine counterpart, are generated in the thymus and are characterized by the ability to suppress proliferation of responder T-cells through a cell-cell contact-dependent mechanism, the inability to produce 1L-2, and the anergic phenotype in vitro. Human CD4⁺CD25⁺ T- cells can be split into suppressive (CD25^hlsh) and nonsuppressive (CD25^low) cells, according to the level of CD25 expression.

Cytotoxic TLymphocytes (CTL)

[95] The CD8⁺ T-cells that recognize peptides from proteins produced within the target cell have cytotoxic properties in that they lead to lysis of the target cells. The mechanism of CTL-induced lysis involves the production by the CTL of perforin, a molecule that can insert into the membrane of target cells and promote the lysis of that target cell. Perforin-mediated lysis is enhanced by a series of enzymes produced by activated CTLs, referred to as granzymes. Many active CTLs also express large amounts of fas ligand on their surface. The interaction of fas ligand on the surface of CTL with fas on the surface of the target cell initiates apoptosis in the target cell, leading to the death of these cells. CTL-mediated lysis appears to be a major mechanism for the destruction of virally infected cells.

|96) The term "major histocompatibility complex" or "MHC" as used herein refers to a complex of vertebrate genes coding for a large family of cell-surface proteins that bind peptide fragments of foreign proteins and present them to T-Iymphocytes to induce an immune response. The MHC also plays a role in resistance to infection and in susceptibility to a number of autoimmune diseases. The MHC complex is divided into three subgroups: MHC class I (MHC I); MHC class II (MHC II); and MHC class III (MHC III). MHC I molecules are present on nearly every nucleated cell of the body. MHC I presents peptides derived from cytosolic proteins and/or peptides from infectious agents. MHC II molecules are found only on specialized, antigen-presenting cell types such as macrophages, dendritic cells, activated T-cells and B- cells. MHC II presents peptides derived from extracellular proteins that are internalized by the cell from its environment, digested by lysosomes and bound by MHC II before its migration to the plasma membrane. MHC II interacts with helper (CD4⁺) T-cells to trigger an appropriate immune response. MHC III molecules include several secreted proteins comprising components of the complement system (e.g., C2, C and B factor), cytokines (e.g., TNF-a, LTA and LTB) and heat shock proteins (hsp).

Priming

[97] The term "unprimed cells" (also referred to as virgin, naive, or inexperienced cells) as used herein refers to T-cells and B-cells that have generated an antigen receptor (TCR for T- cells, BCR for B-cells) of a particular specificity, but have never encountered the antigen. The term "priming" as used herein refers to the process whereby T-cells and B-cell precursors encounter the antigen for which they are specific.

[98] For example, before helper T-cells and B-cells can interact to produce specific antibody, the antigen-specific T-cell precursors must be primed. Priming involves several steps: antigen uptake, processing, and cell surface expression bound to class II MHC molecules by an antigen presenting cell, recirculation and antigen-specific trapping of helper T-cell precursors in lymphoid tissue, and T-cell proliferation and differentiation. Janeway, CA, Jr., "The priming of helper T-cells, Semin. Immunol. 1(1): 13-20 (1989). Helper T- cells express CD4, but not all CD4⁺ T-cells are helper cells. Id. The signals required for clonal expansion of helper T-cells differ from those required by other CD4⁺ T-cells. The critical antigen-presenting cell for helper T-cell priming appears to be a macrophage; and the critical second signal for helper T-cell growth is the macrophage product interleukin 1 (IL- 1 ). Id. If the primed T-cells and/or B- cells receive a second, co-stimulatory signal, they become activated T-cells or B-cells.

[99] The term "modulate" as used herein, refers to the regulation, alteration, adaptation or adjustment to a certain measure or proportion.

[ 100] The terms "peripheral blood mononuclear cells" or "PBMCs" are used interchangeably herein to refer to blood cells having a single round nucleus such as, for example, a lymphocyte or a monocyte. [1011 The term "potency" as used herein refers to the relationship between a therapeutic effect of a therapeutic substance and the dose necessary to achieve that effect; the amount of a substance required to produce a given percentage of its maximal effect, irrespective of the size of maximal effect; or the relative pharmacologic activity of a substance.

11021 The terms "TARC", "CCL17", "TARC/CCL 17" and "CCL 17/TARC" are used interchangeably herein to refer to chemokine (C-C motif) 17 (also known as thymus and activation regulated chemokine). CCL17/TARC is a chemokine that is secreted from monocyte-derived dendritic cells and endothelial cells and is responsible for selective recruitment and migration of activated Th2 lymphocytes to affected tissue.

[103| The described invention provides methods, compositions and kits useful in determining the potency of dendritic cells used in cancer immunotherapy.

1104] According to one embodiment, the described invention provides a dendritic cell (DC) that presents a processed peptide within the dendritic cell. According to another embodiment, the described invention provides a dendritic cell that presents a processed peptide on the dendritic cell surface. According to another embodiment, the processed peptide is a tumor antigen. According to another embodiment, the tumor antigen is from a tumor that includes, but is not limited to, a melanoma, a tissue of endodermal, mesodermal, or ectodermal origin (e.g., melanoma of neural crest origin, colon cancer of endoderm origin, renal cancer of mesoderm origin, glioblastoma of ectoderm origin, ovarian cancer of mixed mesoderm plus extra-embryonic origin) a hepatocellular carcinoma, colon carcinoma,

ovarian carcinoma, glioblastoma multiforme and the like.

1105 J According to one embodiment, the described invention provides methods for determining the potency of dendritic cells used in cancer immunotherapy. These methods include, but are not limited to, immunoassays. Exemplary immunoassays include Western blot, enzyme-linked immunosorbent assay (ELISA), flow cytometry and the like. Flow cytometry includes, but is not limited to, fluorescence-activated cell sorting (FACS®), magnetic-activated cell sorting (MACS®), high-dimensional flow cytometry and cytometric bead array.

[1061 The enzyme-linked immunosorbent assay (ELISA) employs highly-purified capture antibodies that are non-covalently adsorbed ("coated") onto plastic microwell plates. After washings, the immobilized antibodies capture specifically soluble proteins (e.g., chemokine) present in samples applied to the plate. After washing away unbound material, the captured proteins are detected by biotin-conjugated detection antibodies followed by an enzyme-labeled avidin or streptavidin reporter. Following addition of a chromogenic (color- developing) substrate-containing solution, the level of colored product generated by the bound, enzyme-linked, detection reagents can be measured spectrophotometrically using an ELISA- plate reader at an appropriate optical density.

1107J Flow cytometry, a technique that may be used for counting and examining cells, allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of each individual cell. Briefly, a beam of light (usually laser light) of a single wavelength is directed onto a

hydrodynamically-focused stream of fluid. A number of detectors are aimed at the point where the stream passes through the light beam: one in line with the light beam (Forward Scatter (FSC)), several perpendicular to it (Side Scatter (SSC)), and one or more fluorescence detectors. Each suspended cell (from 0.2 y.m to 150 |_im) passing through the light beam scatters the light in some way, and fluorescent molecules (naturally occurring or as part of an attached label or dye) may be excited into emitting light at a longer wavelength than the light source. This combination of scattered and fluorescent light is recorded by the detectors. The FSC correlates with the cell volume; SSC depends upon the inner complexity of the cell (i.e., shape of the nucleus, type of cytoplasmic granules, etc.). The data generated by flow cytometers may be plotted as a histogram. The regions on these plots can be separated sequentially based on fluorescence intensity by creating a series of subset extractions ("gates"). Specific gating protocols have been developed for diagnostic and clinical purposes. [108] Flow cytometers may use either light scattering in combination with fluorescence or light scattering only for analysis. Flow cytometers are available from a variety of commercial sources, including BD Biosciences (San Jose, CA), EMD Millipore (Billerica, MA), Life Technologies (Carlsbad, CA), Agilent (Santa Clara, CA), Miltenyi Biotec (Cambridge, MA) and the like.

1109] Fluorescence activated cell sorting (FACS) provides a method of sorting a heterogeneous mixture of cells into two or more containers, a single cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. Briefly, the cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid and the flow is arranged such that there is a large separation between cells relative to their diameter. The stream of individual cells passes through a fluorescence detector, and an electrical charge is assigned to each cell (based on the cell's fluorescence) just as the stream is broken into individual drops (usually via vibration) such that there is a low probability of more than one cell per droplet. Each charged droplet (containing an individual cell) may be sorted, via electrostatic deflection, into separate containers.

[110] MACS provides a cell separation technique in which cells that express a specific surface antigen may be isolated from a heterogeneous mixture of cells using magnetic particles coated with a binding agent (e.g., antibody) that recognizes the specific surface antigen. For example, in a positive cell selection MACS technique, cells expressing the specific surface antigen bind to the magnetic particles. After incubation with the magnetic particles, the heterogeneous mixture of cells is transferred to a column placed in a magnetic field. The magnetic field captures the magnetic particles (including magnetic particles bound to cells expressing the specific surface antigen) while cells not expressing the specific surface antigen (i.e., not bound to magnetic particles) may be eluted as flow through.

11 11 It is understood by those in the art that MACS also provides negative selection of cells.

Negative selection, for example, involves the isolation and removal of undesired cells expressing a specific surface antigen from a heterogeneous mixture of cells by binding the cells expressing the specific surface antigen to magnetic particles coated with a binding agent (e.g., antibody) that recognizes the specific surface antigen. A magnetic field captures the magnetic particles (including magnetic particles bound to undesired cells expressing the specific surface antigen) while cells not expressing the specific surface antigen (i.e., not bound to magnetic particles) may be eluted and collected.

1112] One skilled in the art recognizes that various MACS products are commercially available. These products include, but are not limited to, MACS microbeads (Miltenyi Biotec, Cambridge, MA), autoMACS® columns (Miltenyi Biotec, Cambridge, MA), autoMACS Pro Separator Instrument (Miltenyi Biotec, Cambridge, MA), and the like.

1113] High-dimensional flow cytometry provides a method of sorting a heterogeneous mixture of cells into two or more containers, a single cell at a time, using 6- 12 fluorescent colors (i.e., fluorophores). For example, the following protocol may be used to perform FACS to detect antigen-specific B lymphocytes. Cryopreserved peripheral blood mononuclear cell (PBMC) samples may be thawed and washed in deficient RPMI media supplemented with 4% FCS. Biotin-coupled antigen (DBY-2 or DBX-2) may be added to the cells and 20 minutes later, a "cocktail" of fluorochrome conjugated monoclonal antibodies detecting CD19, CD2r, CD43, CD5, CD23, IgM and IgG, CD27 and dead cells may be added. Following 20 minute incubation, cells may be spun and washed and incubated for 20 min with fluorochrome-conjugated streptavidin. Data may be collected for 1 -5 x 10⁶ cells on a LSRII flow cytometer (BDBiosciences.com). The data may be analyzed using FlowJo (TreeStar.com) and further analyzed with Excel and Prism (GraphPad software, Inc).

[1 14] Cytometric bead array (BD Biosciences, San Jose, CA) is a flow cytometry application that allows users to quantify multiple proteins simultaneously. The system employs the broad dynamic range of fluorescence detection offered by flow cytometry and antibody-coated beads to efficiently capture analytes. Each bead in the array has a unique fluorescence intensity so that beads can be mixed and run

simultaneously in a single tube. This method significantly reduces sample requirements and time to results in comparison with traditional ELISA and Western blot techniques.

[ 115] The data generated by flow cytometers may be plotted in a single dimension to produce a histogram or in two-dimensional or three-dimensional plots. The regions on these plots may be sequentially separated, for example, based on fluorescence intensity, by creating a series of subset extractions termed "gates." One skilled in the art recognizes that specific gating protocols exist for diagnostic and clinical purposes, including, but not limited to, classification of immune system cells. By way of example, and without limitation, one skilled in the art would recognize that it is possible to define a light scattering gate to include only B lymphocytes by placing upper and lower limits on the forward and side scatter distributions.

11 16| Most parameters measurable by flow cytometry can also be measured by other techniques well- known in the art. These techniques include, but are not limited to, analytical cytology (e.g., microfluorimetry), standard microscopic-based cytometric analysis, physical sorting (e.g., panning), standard

immunohistochemical techniques and the like.

[117] The surfaces of all cells in the body are coated with specialized protein receptors that selectively can bind or adhere to other signaling molecules. These receptors and the molecules that bind to them are used for communicating with other cells and for carrying out proper cell functions in the body. Each cell type has a certain combination of receptors (or surface markers) on its surface that makes it distinguishable from other kinds of cells. Cells may be fluorescently labeled, i.e., a reactive derivative of a fluorophore may be covalently attached to a cell. The most commonly used labeled molecules are antibodies; their specificity towards certain surface markers on a cell surface allows for more precise detection and monitoring of particular cells. The fluorescence labels that can be used will depend upon the lamp or laser used to excite the fluorochromes and on the detectors available. For example, when a blue argon laser (448 nm) is used, fluorescent labels used may include, but are not limited to, fluorescein isothiocyanate (FITC), Alexa Fluor® 488, green fluorescent protein (GFP), carboxyfluorescein (CFSE), carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), DyLight® 488 (Dyomics), phycoerythrin (PE), propidium iodide (PI), peridinin chlorophyll protein (PerCP), PerCP-Cy™5.5, PE-AlexaFluor 700, PE- Cy™5; PE-Cy™5.5, PE- AlexaFluor® 750 and PE-Cy™7; when a red diode laser (635 run) is used, fluorescent labels used may include, but are not limited to, allophycocyanin (APC), APC-Cy™7, APC-eFluor® 780, AlexFluor® 700, Cy™5, and Draq-5; when a violet laser is used (405 nm), fluorescent labels may include, but are not limited to. Pacific Orange™,

amine aqua. Pacific Blue™, 4'-6-diamidino-2-phenylindole (DAPI), AlexFluor® 405, and eFluor® 450.

[118] Conjugation of a label to a binding agent may be accomplished by covalent or non-covalent (including hydrophobic) bonding, or by adsorption. Techniques for conjugation are well-known in the art and may be readily adapted for the particular reagents employed.

111 | Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise,

between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

1120] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and described the methods and/or materials in connection with which the publications are cited. [121 ] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. All technical and scientific terms used herein have the same meaning.

[ 1221 The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application and each is incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

EXAMPLES

[123] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

METHODS

Dendritic Cell Preparation

[124] Peripheral blood mononuclear cells (PBMCs) are obtained by leukapheresis from a patient from whom a tumor sample (e.g., a needle biopsy, a lavage of a tumor-containing tissue, or a bulk tumor) was derived. Optionally the collected PBMCs are shipped to a manufacturing facility. The collected PBMCs are purified from other lymphocytes. According to one embodiment, DCs are purified from PBMCs by counter flow density centrifugation (elutriation), meaning a process by which monocytes are purified from other lymphocytes in order to enrich for cells that can be turned into APCs or DCs). According to another embodiment, low density DCs can be prepared by gradient separation over a density gradient to obtain PBMCs, T-lymphocyte depletion (optional), overnight incubation for 16 hours in autologous serum/medium (± cytokines), gradient separation, and positive selection (e.g., flow cytometry, immunomagnetic beads).

According to one embodiment, positive selection is for activation antigens, e.g., CD80, CD83, CD86, and CMRF-44 (Feamley, DB et al. Blood 89; 3708 (1997); Caux, C. et al.., J. Exp. Med. 180 (5); 1841 -47 (1994)),CD80. Hart, DNJ, Blood 90(9); 3245-87 (1997); Hock, B.D. et al, Immunol. 83; 573-81 (1994). According to another embodiment, low density DCs can be prepared by gradient separation over a density gradient to obtain PBMCs, T-lymphocyte depletion (optional), overnight incubation for 16 hours in autologous serum/medium (± cytokines), metrizamide gradient separation, and positive selection (e.g., flow cytometry, immunomagnetic beads) to obtain a lineage-negative, CD83+ cell population. Zhou, L and Tedder, TF, J. Immunol. 154; 382135 (1995).

[125] To generate DCs, purified DCs are incubated with, e.g., GM-CSF and IL-4 for 6 days. ELISA

CCL17/TARC

| 126| Detection and quantitation of human CCL17/TARC was accomplished by using a human CCL 17/TARC Quantikine ELISA Kit (R&D Systems, catalog # DDN00,

Minneapolis, MN) according to manufacturer's protocol. Briefly, reagents, standard dilutions and samples were prepared as directed in the product insert. Excess microplate strips were removed from the plate frame and returned to the foil pouch containing the desiccant pack and the pouch was resealed. Next, 100 |.iL of Assay Diluent was added to each well followed by 50 |iL of Standard (human CCL 17/TARC, R&D Systems, part # 890078), control or sample. The plate was covered with a plate sealer and incubated at room temperature for 2 hours. Following incubation, each well was aspirated and washed for a total of 3 washes. Next, 200 (aL of HRP-conjugated anti-CCLl 7/TARC polyclonal antibody ("Conjugate") was added to each well, the plate was covered with a new plate sealer and the plate was incubated at room temperature for 1 hour. After incubation, the plate was aspirated and washed 3 times. Following aspiration and washing, 200 |iL of Substrate Solution was added to each well and the plate was incubated in the dark at room temperature for 30 minutes. Next, 50 |j,L of Stop Solution was added to each well and the plate was read on a microtiter plate reader (Molecular Devices, Sunnyvale, CA) at a wavelength of 450 nm within 30 minutes.

IL-12 P70

[127] Detection and quantitation of human Interleukin 12 (IL-12) p70 was accomplished by using a human IL-12 p70 Quantikine ELISA Kit (R&D Systems, catalog # D1200, Minneapolis, MN) according to manufacturer's protocol. Briefly, reagents, standard dilutions and samples were prepared as directed in the product insert. Excess microplate strips were removed from the plate frame and returned to the foil pouch containing the desiccant pack and the pouch was resealed. Next, 50 [iL of Assay Diluent was added to each well followed by 200 |iL of Standard (human IL- 12 p70, R&D Systems, part # 890214), control or sample. The plate was covered with a plate sealer and incubated at room temperature for 2 hours. Following incubation, each well was aspirated and washed for a total of 3 washes. Next, 200 |LlL of HRP-conjugated anti-IL-12 p70 polyclonal antibody was added to each well, the plate was covered with a new plate sealer and the plate was incubated at room temperature for 2 hours. After incubation, the plate was aspirated and washed 3 times. Following aspiration and washing, 200 ]aL of Substrate Solution was added to each well and the plate was incubated in the dark at room temperature for 20 minutes. Next, 50 |iL of Stop Solution was added to each well and the plate was read on a microtiter plate reader (Molecular Devices, Sunnyvale, CA) at a wavelength of 450 nm within 30 minutes.

Flow Cytometry/FACS

1. FACS Compensation - Instrument Settings with CaliBRITE™ Beads

1128 J Prepare all bead suspensions immediately prior to use. 1129] Label two tubes, Tube A and Tube B. Tube A for PMT adjustment and Tube B for fluorescence compensation and sensitivity testing. Note: Do not dilute PerCP-Cy5.5 beads in sheath fluid.

[130] Dispense approximately 0.5 mL sheath fluid into Tube A (fill to just above the bulb of the FACS tube bottom).

J 311 Dispense approximately 1.5 mL sheath fluid into Tube B (fill to about one inch from the bottom).

| 132| Gently mix the CaliBRITE™ bead (BD Biosciences, catalog # 349502, San Jose, CA) vials, and then add beads to each tube as indicated below. Invert bead vial completely when adding a drop to the tube. Make sure to obtain a full drop of beads. The drop should be cloudy, indicating the beads are properly mixed. For the four-color setup:

1133 J Tube A: add 1 drop unlabeled beads + 1 drop allophycocyanin (APC)-labeled bead

[ 134] Tube B: add 1 drop unlabeled beads + 1 drop allophycocyanin (APC)-labeled bead + 1 drop fluorescein isothiocyanate (FITC) labeled bead + 1 drop phycoerythrin (PE)- labeled bead + 1 drop peridinin chlorophyll protein (PerCP)-labeled bead.

1135J Keep prepared bead suspensions on ice or at 2° to 8°C and protect from direct light until ready to use.

J 1361 CaliBRITE® bead suspensions prepared in FACSFlow® sheath fluid are stable for 8 hours at 4°C, or if PerCP-labeled beads are included, then for 1 hour at 4°C.

1137] System Setup using FACSComp® software with CaliBRITE® beads:

Open FACSComp® software and click Accept

Enter CaliBRITE® Bead Lot IDs and click Run.

1138J Note: If already populated then simply verify the lot numbers for each bead.

[139] Set FACS machine to MED

11401 Place Tube A in machine and press RUN; click Start in FACSComp®

[ 141] Place Tube B in machine and press RUN; click Start in FACSComp®

[ 142 J Select Cytometer in FACSComp and from the drop down menu select Instrument Settings. ( 143 J The fluorescence sensitivity is determined by the amount of channel separation between the unlabeled and labeled bead populations. The light scatter sensitivity is determined by the amount of channel separation between the mixed bead population and instrument background signal.

2. Dendritic Cell (DC) Staining

[144] Obtain a fresh cell sample with cell concentration. Centrifuge at 1500 RPM for 5 minutes. Note: Prior to any centrifuging step the sample(s) must be pan balanced.

| 145| Depending on the vial, decant or aspirate media supernatant gently.

| 146I Resuspend cells to a concentration of at least 2.5xl0⁵ cells/100)jL in 10% HSA in PBS. A DC sample requires testing for CD45/14-FITC/PE, 7AAD-PerCP and CDl Ic-APC. 7AAD is a nucleic acid dye for non-viable cells and requires a fresh/live cell sample.

[147] Block cells in the 10% HSA in PBS for 15 minutes at room temperature. While blocking prepare tubes and antibodies for staining.

[148] Label round bottom BD FACS tubes. Each sample requires two tubes. Label one tube for the isotype control (ISO) and the other for the conjugated antibody of interest.

[149] Note for remaining steps minimize light exposure.

1150J Prepare isotype controls and conjugated antibodies. Refer to Table 1 - Dilutions of Antibodies for required antibodies and dilutions. Record antibody and isotype volume, lot number, part number, and expiration date in Attachment 1 - FACS Staining and Counting for an Autologous (e.g. Melanoma) Sample Worksheet, Section 5 Preparation of Antibodies. Place antibodies on chilled beads (4°C) during preparation.

[ 1511 If several samples are provided, then prepare separate mastermixes of the isotype controls and the conjugated antibodies. For example, if three different samples are to be stained with CD45/CD14- FITC/PE, 7AAD-PerCP and CDl lc-APC (therefore, 7.5x10^s cells), then add 30 [oL of CD45/14-FITC/PE, 15 pL of 7AAD-PerCP and 15 pL of CD l l c together in a clean BD tube, this is the conjugated antibody mastermix.

[152] Account for pipette error by adding antibody/isotype for an extra sample.

[153] Add isotype or conjugated antibody to its labeled BD FACS tube. If mastermix was prepared, then add volume to account for the number of isotypes or antibodies mixed. For example, staining for CD45/14, 7AAD and CD l lc requires lOpL of CD45/14, 5 )aL of 7AAD and 5 fiL of CD l l c therefore 20 |j,L of mastermix will be added to the antibody tube.

[154] Apply 100 )j,L of cell sample to its designated isotype and conjugated antibody BD FACS tubes.

1155] Stain with cells in blocking solution.

[156] Vortex cells to mix with antibody.

[157] Stain for 30 minutes in the dark at room temperature.

1158] Add 1 mL PBS to each sample tube and centrifuge at 1500 RPM for 5 minutes to wash excess antibody and block.

[159] Decant the BD FACS tubes gently.

| 160| Apply 500 [j,L PBS for analysis.

3. Dendritic Cell-loaded Tumor Cell (DC-TQ Staining

11611 Obtain a fresh cell sample with cell concentration.

[ 162] Centrifuge at 1500 RPM for 5 minutes. Note: Prior to any centrifuging step the sample(s) must be pan balanced.

1163] Depending on the vial, decant or aspirate media supernatant gently.

[ 164] Resuspend cells to a concentration of at least 2.5x10^s cells/lOOpL in 10% HSA in PBS. A DC- TC sample requires testing for CD45/14-FITC/PE, 7AAD-PerCP and CDl 1 c- APC. For information purposes only it is beneficial to test for CD40-FITC, CD80-PE, MHClI-PerCP, and CD54-APC. If testing for 7AAD, note that it is a nucleic acid dye for non-viable cells and requires a fresh/live cell sample.

[165] Block cells in the 10% HSA in PBS for 15 minutes at room temperature. While blocking prepare tubes and antibodies for staining.

| 166| Label round bottom BD FACS tubes. Each sample requires two tubes. Label one tube for the isotype control (ISO) and the other for the conjugated antibody of interest.

[167| Note for remaining steps minimize light exposure.

[ 1681 Prepare isotype controls and conjugated antibodies. Refer to Table 1 - Dilutions of Antibodies for required antibodies and dilutions. Place antibodies on chilled beads (4°C) during preparation. If several samples are provided, then prepare separate mastermixes of the isotype controls and the conjugated antibodies. For example, if three different samples are to be stained with CD45/CD 14-FITC/PE, 7AAD-PerCP and CD1 Ic-APC (therefore, 7.5x l 0⁵ cells), then add 30 \a.L of CD45/14-FITC/PE, 15 pL of 7AAD-PerCP and 15 |uL of CDl lc together in a clean BD tube, this is the conjugated antibody mastermix.

[169] Account for pipette error by adding antibody/isotype for an extra sample.

1170] Add isotype or conjugated antibody to its labeled BD FACS tube. If mastermix was prepared, then add volume to account for the number of isotypes or antibodies mixed. For example, staining for CD45/14, 7AAD and CDl l c requires 10|aL of CD45/14, 5 pL of 7AAD and 5 pL of CD 1 l c therefore 20 pL of mastermix will be added to the antibody tube.

[171] Apply 100 pL of cell sample to its designated isotype and conjugated antibody BD FACS tubes.

[172] Stain with cells in blocking solution.

[ 173] Vortex cells to mix with antibody.

[ 174| Stain for 30 minutes in the dark at room temperature.

[175] Add 1 mL PBS to each sample tube and centrifuge at 1500 RPM for 5 minutes to wash excess antibody and block.

[176] Decant the BD FACS tubes gently.

[177] Apply 500 pL PBS for analysis.

4. Data Acquisition

[ 178] Vortex sample to ensure homogenous mixture.

[179] Analyze sample using the CellQuest™ Pro Software. First, run the isotype control to assess the level of background staining, then run the conjugated antibody. Collect data for 120 seconds or until 10^s events have been gathered. Repeat analysis for all samples. Minimize light exposure of stained sample(s) between readings.

Example 1 : Development and Optimization of Enzyme-immunosorbent Assays

(ELISA) to Detect and Quantitate Secreted CCL17/TARC and 1L-12 in Supernatant from Activated Dendritic Cell (DC) Culture

[00180] Dendritic cells receive maturation signals in the form of cytokines and toll-like receptor ligands such as CD40 ligand, interferon-gamma and lipopolysaccride (LPS), respectively. Upon ligation, dendritic cells undergo maturation which induces the expression of maturation markers such as CD83 and the induction of T- cell activating cytokines such as interleukin-12 (IL-12) and CCL 17/TARC (Vissers J L, et al., J. Leukoc. Biol. (2001) 69: 785-793; Ping J, et al., J. Trans. Med. (2010) 8: 1 -15; Butterfield L H, et al, J. Immunother. (2008) 3 1 : 89-100).

[1811 In this study, optimal dilution ranges of activated dendritic cell culture supernatants were determined that yield quantifiable values of secreted CCL17/TARC and IL-12 in a linear fashion in an enzyme-linked immunosorbent assay (ELISA).

1182| Supernatant samples consisting of patient-derived irradiated tumor cells (DC-TC) (Sample # 1955 and 1893) that were exposed to known maturation cytokines combinations (CD40L/interferon- gamma(glFN) and CD40L/gIFN/lipopoly saccharide (LPS)) were thawed and used in a CCL17/TARC (R&D Systems, catalog # DDN00, Minneapolis, MN) and an IL-12 p70 (R&D Systems, catalog # D1200, Minneapolis, MN) ELISA. All ELISA reagents were brought to room temperature and prepared according to manufacturer's protocol. DC- TC supernatants (Sample # 1955 and 1893) were serially diluted according to the manufacturer's protocol to produce the following sample dilutions: 1 :2, 1 :4, 1 :8, 1 : 16, 1 :32, 1 :64 and 1 : 128. All standards and samples were assayed in triplicate. Monocytes, unloaded dendritic cells and loaded dendritic cells (DC-TC) from a patient derived sample (Sample # 2353) were cultured overnight in AIM-V media (Invitrogen, Grand Island, NY), AIM-V media containing CD40L/interferon-gamma(gIFN) and AIM-V media containing CD40L/gIFN/lipopolysaccharide (LPS). Supernatant was collected, diluted 1 :2, 1 :4, 1 :8, 1 : 16, 1 :32 and 1 :64 and assessed for CCL17/TARC (R&D Systems, catalog # DDN00,

Minneapolis, MN) and IL-12 (R&D Systems, catalog # D1200, Minneapolis, MN) chemokine levels by ELISA. Standard curves were created by 1) calculating the mean from the triplicates and correcting for the absorbance from zero standard, 2) plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis in Excel, and 3) drawing a best fit curve through the points on the graph with the equation of the line (y=mx+b) displayed on the graph. The corrected optical density for each sample was then calculated for a diluted concentration by using the equation of the line. The diluted concentrations were then used to calculate the linearity between dilutions. The linearity between each dilution was determined using the following equation: %Linearity = (Observed Concentration / (Previous observed value in the dilution series / Dilution Factor))* 100. The acceptable criteria for mean percent linearity for each interval was 80-120%. The concentrations of the samples were calculated using diluted concentration values that were found to be within acceptable linearity, and multiplying by the dilution factor (e.g., 2, 4, 8, 16, 32, 64, or 128). Mean and standard deviation were calculated using EXCEL functions. Standard error was calculated accordingly: standard error = standard deviation / square root (n-1). A student t-test was used to determine significant difference between culture conditions.

[183] The standard curves for the CCL17/TARC ELISA Immunoassays for determining the TARC concentration of Sample # 1955 and 1893 are shown in Figure 1. The equation for the standard line for the plate for Sample # 1955 was: y= 0.0014x + 0.1359 with an correlation coefficient (r²) value of 0.99464, demonstrating a good linear fit (Figure 1 a). The equation for the standard line for the plate for Sample #1893 was: y= 0.0012x + 0.1641 with an r value of 0.95722, demonstrating a good linear fit (Figure lb).

[ 184| The diluted concentrations of each sample are demonstrated in Table 2.

Table 2. Diluted Concentrations (pg/mL) of

CCL 17/TARC for Sample # 1955 and 1893

[00185] The percent linearity between each dilution for each sample is demonstrated in Table 3. The percent linearity values that were within range (80%-120%) are highlighted in gray.

Table 3. Percent Linearity between Diluted Concentrations for Sample # 1955 and 1893

(00186) For Sample # 1955 DC-TCs cultured in control media, the acceptable linearity was between 1 :8 and 1 : 16. For Sample # 1955 DC-TCs cultured in media containing CD40L/gIFN, acceptable linearity was between 1 :8 and 1 : 16, 1 : 16 and 1 :32, and 1 :32 and 1 :64. For Sample # 1955 DC-TCs cultured in media containing CD40L/gIFN/LPS, acceptable linearity was between 1 :4 and 1 :8, 1 :8 and 1 : 16, and 1 : 16 and 1 :32.

[187] For Sample # 1893 DC-TCs cultured in control media, the acceptable linearity was between 1 :32 and 1 :64. For Sample # 1893 DC-TCs cultured in media containing CD40L/gIFN, there was no acceptable linearity between the tested dilutions, with the linearity between 1 .64 and 1 : 128 being the closest to the acceptable criteria (over by 2.16%). For Sample # 1893 DC-TCs cultured in media containing CD40L/gIFN/LPS, acceptable linearity was between 1 :32 and 1 :64.

1188] The TARC concentrations for Sample # 1955 and 1893 cultured in the various conditions were calculated from the diluted concentrations that were within acceptable percent linearity. The calculated TARC concentrations are shown in Figure 2.

1189] Sample # 1955 DC-TCs in control, CD40L/IFN -gamma, and CD40L/IFN- gamma/LPS conditions secreted an average of 8498.95 ± 664.41 pg/mL, 8568.33 ± 391.50 pg/mL, and 1619.17 ± 180.19 pg/mL, respectively (mean± standard error). The concentration of TARC in the CD40L/IF -gamma/LPS conditions was significantly less than that of the control condition (p=0.0026) and that of the CD40L/IFN- gamma condition (p=0.0048).

11901 Sample # 1893 DC-TCs in control and CD40L/IFN -gamma/LPS conditions secreted an average of 53733.78 ± 2945.96 pg/mL, and 42920.44 ± 4 14.80 pg/mL, respectively (mean ±

standard error).

[ 191] Figure 3 shows the calculated TARC concentrations for each sample from all diluted concentrations tested.

11921 The standard curve for the TARC ELISA Immunoassays for determining the TARC concentration of Sample # 2353 is shown in Figure 4. The equation for the standard line for the plate for Sample # 2353 was: y= 0.0014x + 0.21 84 with an r² value of 0.98386, demonstrating a good linear fit. [1931 Sample # 2353 was diluted 1 :8, 1 : 16, 1 :32, and 1 :64 for the TARC ELISA plate based on the percent linearity range from Samples 1955 and 1893. None of the Sample #2353 dilution ranges tested resulted in acceptable percentage of linearity (data not shown). Sample # 2353 was too dilute based on calculated concentrations and percent linearity. Only DC-TC samples diluted 1 :8 yielded TARC concentrations >0 pg/mL (Figure 5).

[194] The standard curve for the IL-12 ELISA Immunoassay for determining the IL-12 concentration of Sample #1955 and 1 893 is shown in Figure 6. The equation for the standard line for the plate was: y= 0.0056x + 0.0123 with an r² value of 0.99726, demonstrating a good linear fit. The diluted concentrations of each sample are listed in Table 4.

Table 4. Diluted Concentrations (pg/mL) of IL-12 for Sample # 1955 and 1893

1 1951 The percent linearity between the dilutions for samples 1955 and 1893 was not within the acceptable range (data not shown). Limited availability of the supernatants did not allow for a proper serial dilution, thus 1 :2 and 1 :20 dilutions were performed. Figure 7 shows the calculated concentrations of IL-12 for Sample # 1955 and 1893 at dilutions ranging from 1 :2 to 1 :20.

[196] Sample # 2353 was first tested in the IL-12 ELISA at dilutions 1 :2, 1 :4, 1 :8, and 1 : 16.

Sample#2353 was re-tested in the IL-12 ELISA undiluted, 1 :2, 1 :4. The standard curves from the IL-12 ELISA Immunoassays for determining the IL-12 concentration of sample 2353 are shown in Figure 8. The equation for the standard line for the plate for 1 :2 - 1 : 1 6 diluted sample 2353 was: y= 0.006 l x + 0.0295 with an r² value of 0.99656, demonstrating a good linear fit (Figure 8a). The equation for the standard line for the plate for undiluted and 1 :2 diluted sample 2353 was: y= 0.0007x + 0.0079 with an r² value of

0.99446, demonstrating a good linear fit (Figure 8b).

[197] Table 5 lists the calculated concentrations of IL-12 for Sample # 2353 undiluted, diluted 1 :2 and diluted 1 :4; despite the absence of an acceptable percent linearity. Figure 9 shows the calculated

concentrations from Sample # 2353 despite the absence of an acceptable percent linearity.

Table 5. Calculated Concentrations of IL-12 for Sam le # 2353

Dilution control CD40/ CD40/ control CD40/ CD40/ control CD40/ CD40/

Factor IFNG IFNG/ IFNG IFNG/ IFNG IFNG/

LPS LPS LPS

1 0 0 0 0 0 72.05 7.76 0 76.33

2 0 0 0 0 32.67 0 81.24 223.14 6.00

4 0 3.83 16.72 0 0 0 7.10 5.57 16.94

11 81 The results of this study indicate that a dilution range between 1 :8 tol :64 was acceptable for Sample # 1955 and 1893 tested in the CCL 17/TARC ELISA. However, a 1 :8 dilution was too dilute for Sample # 2353. Without being bound by theory, a difference in the number of dendritic cells in the test conditions could factor into the amount of secreted CCL 17/TARC. Therefore, the amount of secreted CCL 17/TARC calculated by ELISA can be normalized to the number of CD 1 lc+ cells in culture.

[199] All samples tested in the IL-12 ELISA failed to produce an acceptable dilution range for detection which may be indicative of the absence or low concentration of the IL-12 in the analyzed supernatants.

Example 2: Determination of Optimal Cytokine Maturation, Cell Culture and Flow

Cytometry Assay Conditions

[200] In this study, the optimal combination, concentration and type of maturation protocol necessary to induce maturation of dendritic cells was determined. In addition, optimal flow cytometry conditions necessary to detect CCL17/TARC and IL-12 within mature dendritic cells was obtained.

|2011 Three autologous tumor cell antigen-loaded dendritic cells (DC-TC) were thawed in AIM-V, counted and re-suspended at 1 x 10⁶ cells per mL in media consisting of AIM-V or dendritic cell media (AIM-V plus 1000 lU/mL GM-CSF or 400 lU/mL IL-4) with 5% FBS. The DC-TCs were incubated in the presence or absence of maturation conditions consisting 0.5lj,g/mL CD40L/1000 IU gammalFN/1 pg/mL LPS in their respective basal media conditions in mesh top FACS tube for 24 hours. Golgi-Stop (brefeldin-A; BD Biosciences, catalog # 554724, Franklin Lakes, NJ) was not added or was added either continuously or during the final 4 hours of incubation. At the end of the incubation period, the cells were centrifuged, supernatant collected for future assaying and the cell pellets transferred to 96-well plates for processing for flow cytometry. Two types of antibody

staining conditions were employed, pre-fixation staining of extracellular antigens (CD40 and CD83) and post-fixation staining of extracellular antigens. Sufficient cell numbers were achieved in all conditions tested (data not shown).

Table 6. Summary of Results Across All Three Samples Tested in AIM-V Media + 5% FBS

AIMY media plus 5%FBS Pre-fixation Pre-fixation Post-fixation Post-fixation

Control Cytokine Matured Control Cytokine Matured

% positive Golgi-Stop Ave sd Ave sd Ave sd Ave sd

CD83/IL12 None 6.10 1.27 9.40 3.74 3.68 2.68 3.23 2.36

4hr 3.80 0.63 2.74 0.96 8.42 7.62 9.29 8.12

24hr 1.93 0.49 2.02 0.78 5.39 3.75 14.61 11.96

CD40/CCL None 13.62 9.23 11.62 7.38 2.13 1.08 2.98 1.48

17 4hr 17.02 12.91 10.61 8.79 2.92 0.96 4.45 1.65

24hr 7.05 4.50 9.64 10.09 1.73 1.05 2.38 1.12 Table 7. Summary of Results Across All Three Samples Tested in Dendritic Cell Media +

5% FBS

[202] The results of this study indicate that DC media plus 5% FBS and AIM-V plus 5% FBS were comparable in supporting the response to maturation cytokines as measured by the induction of

CD83/IL12 positive cells. CD83/IL12 was detectable optimally only in the post-fixation, 24-hour Golgi- Stop conditions in only two out of three of the samples tested. CD40/CCL17 was not induced by cytokine maturation and was only marginally detectable in the pre-fixation conditions, which was comparable in either media formulation. There was slightly diminished detection rate in the cells treated for 24-hours with Golgi-Stop versus either 4-hours or without Golgi-Stop. Detecting double positive CD40/CCL17 and CD83/IL- 12 dendritic cells required completely separate incubation and fixation conditions for detection. Without being bound by theory, it may be preferential to measure cytokine maturation response for IL-12 and CCL 17/TARC by ELISA while measuring CD83 and CD40 by flow cytometry in the

absence of intracellular fixation protocols.

Example 3: Determination of Optimal Conditions for Maturation of Dendritic Cells

[203| In this study, conditions for maturation of dendritic cells were further optimized. Donor and patient derived dendritic cells with or without irradiated tumor cells were incubated with a panel of maturation cytokines to determine the optimal conditions necessary for producing IL-12 and CCL 17/TARC that are detectable by flow cytometry or ELISA. Incubation of dendritic cells in common maturation conditions such as ILl-beta, TNF-alpha, IL-6, or toll-like receptor ligands such as poly I:C and lipopolysaccharide as well as CD40 ligand and interferon- gamma were tested. The sequence of maturation steps (e.g., adding toll-like receptor ligands first, followed by the addition of cytokines) also was tested. Cell culture supernatants were collected and the expression of IL-12 and CCL17/TARC by ELISA was determined. In addition, a panel of IL-12 antibodies was used to determine which antibody is best suited for detecting IL-12 by flow cytometry. [204] First, dendritic cells were incubated in CellGenix DC Media (Portsmouth, NH) or AIM-V dendritic cell media containing 1000 lU/mL GM-CSF and 400 lU/mL IL-4 followed by combinatorial maturation conditions using either a standard cytokine cocktail of TNF alpha, IL-l-beta, IL-6 with CD40L/gIFN (CKl) or Poly IC with CD40L/gIFN (CK2) or AIM-V plus 5%FBS (CT) followed by 4 hours incubation with Golgi-stop (brefeldin-A).

|205| Similar levels of maturation were induced in dendritic cells generated in either CellGenix or AIM-V dendritic cells media (Figure 18), Maturation was induced comparably in either the standard cytokine (CKl) or toll-like receptor stimulated (CK2) cells plus CD40L/IFNgamma based on the increase in the expression of CD80, CD54 and MHC class II (HLA-DR). Intracellular flow cytometry using a eBioscience fixation and permeabilization kit failed to detect IL-12 in experiment 1 (Figure 19). Despite noticeable increases in other maturation markers such as CD80, CD54 and MHC-II (Figure 18), CD83 was not increased. Without being bound by theory, it is possible that the antibody for CD83 was quenched during the fixation step or lost affinity to the epitope after fixation.

[206] Levels of IL-12 as measured by ELISA were increased in response to either maturation conditions in dendritic cells generated with CellGenix or AIM-V dendritic cell media (Table 8). Levels of IL-12 as measured by ELISA were noticeably increased in those cells stimulated by the toll-like receptor ligand (CK2) generated AIMV media prior to maturation. Maximum detectable levels were reached for this sample in each replicate hence no standard deviation was calculated (Table 8).

Table i 5. ELISA Results for IL- 12

Ps/mL IL-12

DC Media Average s.d.

Control 2.25 0.56

AIMV CKl 51.88 8.55

CK2 529.85

Control 2.16 0.59

Cell Genix CKl 78.42 4.84

CK2 54.08 2.91 [207] Levels of CCL 17/TARC as measured by ELISA were noticeably high in all conditions tested. The levels represented in Table 9 had to be extrapolated because the absorbance levels were beyond the linear range of the assay. Although the levels of CCL 17/TARC were lower in the matured dendritic cells generated in CellGenix DC media, it is difficult to determine if this observation is statistically significant considering that values were outside the linear range of the assay (Table 9).

Table 9. ELISA Results for CCL 17/TARC

Pg/mL TARC/CCL17

DC Media Average s.d.

Control 6924.18 1 13.14

AIMV C 1 6805.09 6.43

C 2 6639.64 50.14

Control 6867.82 100.28

Cell Genix CK1 5794.18 122.14

CK2 5262.36 33.43

[208| The results of this experiment indicate that maturation of dendritic cells generated in AIM-V DC media in CK2 resulted in the greatest secretion of IL-12 as detected by ELISA, suggesting that toll-like receptor engagement is optimal over the standard cytokine cocktail in inducing IL- 12 secretion. Differences in the maturation of dendritic cells were not detectable via flow cytometry as IL-12 was not detectable under the protocol employed.

[209] The results of the CCL 17/TARC ELISA were inconclusive because the values were outside of the linear range of the assay. However, it was apparent that large quantities of CCL 17/TARC were secreted in all conditions tested.

[210| Second, induction of normal donor dendritic cell maturation was performed in AIM-V dendritic cell media followed by sequential treatment with toll-like receptor ligand and maturation cytokines. Dendritic cells were exposed to 1 Lj.g/mL LPS or 30 )ig/mL poly IC in 5%FBS/AIM-V for 24 hours and then 0.5 |xg/mL CD40L and 1000 lU/mL IFNgamma were added for an additional 24 hours followed by 4 hours incubation with Golgi-stop (brefeldin-A). Cell culture supematants were collected at the end of the incubation period. The control consisted of A1M-V plus 5% FBS only.

[211] Induction of dendritic cell maturation as measured by an increase in the expression of CD54, CD40 and MHC class II were comparable in each of the conditions tested (Figure 20). No noticeable change in the expression of CD83 in response to maturation was detected.

[212] Levels of IL-12 as measured by ELISA in response to sequential maturation increased significantly where CD40L/gIFN and LPS were present (Table 10).

Table 10. ELISA Results for IL- 12

Pg/mL IL 12

Maturation Conditions Average s.d.

Control 2.00 0.37

LPS 2.74 1.07

Poly IC 2.80 0.80

Control->CD40L/gIFN 8.10 2.53

LPS->CD40L/gIFN 1 1.17 1 .44

Polv IC->CD40L/glFN 2.67 0.88

[213| Levels of CCL17/TARC as measured by ELISA were noticeably high in all conditions tested, regardless of the maturation condition used. The levels represented in Table 1 1 were extrapolated because the absorbance levels were beyond the linear range of the assay.

Table 1 1. ELISA Results for CCL 17/TARC

Pg/mL TARC/CCL 17

Maturation Conditions Average s.d.

Control 6759.64 1 16.99

LPS 6822.36 54.00

Poly IC 6902.36 12.86

Control->CD40L/gIFN 6893.27 43.71

LPS->CD40L/gIFN 6756.91 0.00

Polv IC->CD40L/gIFN 6846.91 47.57 [214] The results of this experiment indicate that out of all the conditions tested, sequential maturation of dendritic cells with CD40L and interferon-gamma followed by lipopolysacaride (toll-like receptor ligand) resulted in the greatest maturation as determined by the secretion of 1L- 12 by ELISA. Differences in maturation of dendritic cells were not detectable via flow cytometry, as IL- 12 was not detectable under the protocol employed. The results of the CCL17/TARC ELISA were inconclusive because values were outside of the linear range of the assay. However, it was apparent that large quantities of CCL 17/TARC were secreted in all conditions tested.

|215] Third, 3 separate autologous antigen-loaded dendritic cells were incubated in either

1 .g/mL LPS, 30 |j.g/mL poly IC, 0.5 ti.g/mL CD40L+1000 lU/mL IFNgammato induce maturation, or in combination where indicated in 5% FBS/AIM-V for 24 hours. Cell culture supematants were collected at the end of the incubation period.

[216] Induction of dendritic cell maturation was comparable in each of the conditions tested as measured by CD54 and to a lesser extent with CD40 and MHC class II (Figure 21 /Table 12). However, no noticeable change in the expression of CD83 in response to maturation was detected (Figure 21/Table 12). It was noted that Sample # 1940 had appreciable lower levels of dendritic cell markers such as CD40, CD54 and CD83 than the other two samples (Table 12).

Table 12. Tabulated flow cytometry results for CD83, CD40, CD54 and MHC class II

Mean Fluorescence Intensity

CD83 CD40 CD54 MHC class II

1955 DC-TC Control 8.98 15.58 561.07 29

LPS 10.41 17.43 765.02 27.63

Poly IC 8.47 13.63 502.74 20.33

CD40L/gIFN 1 1.1 1 19. 14 913.87 34.07

LPS+CD40L/gIFN 10.54 14.03 629.46 22.82

Polv IC +CD40L/aIFN 8.93 13.59 699.91 21.26

1983 DC-TC Control 13.84 33.79 749.83 45.1

LPS 17.27 48.7 1908.62 45.27

Poly IC 16.54 55.18 1253.16 41.53

CD40L/gIFN 16.56 49.4 1757.21 53.42

LPS+CD40L/gIFN 18.97 49.64 1833.65 48.19

Polv IC +CD40L/gIFN 16.8 53.06 1684.65 42.31

1940 DC-TC* Control 3.71 2.59 650.98 1 15.28

LPS 3.56 2.58 612.43 1 1 1.92

Poly IC 3.63 2.58 634.73 1 19.19

CD40L/gIFN 3.5 2.58 680.67 1 12.52

LPS+CD40L/gIFN 2.58 2.38 631.03 86.17

Polv IC +CD40L/eIFN 3.91 2.5 616.15 1 10.74

[217] IL-12 detection by intracellular flow cytometry was not significantly increased in any of the conditions tested (Figure 22). Sample 1940 was determined later to be comprised of tumor cells only; no dendritic cells were present due to lack of CD40 and CD83 expression and the complete lack of TARC/CCL 17 (Table 13).

Table 13. Tabulated Flow Cytometry for IL-12

Percent Positive IL- 12

1 955 1983 1940

Maturation Conditions DC-TC DC-TC DC-TC*

Control 2.1 5 0.3 1.6

LPS 2.03 2.21 1 .36

Poly IC 1 .91 1 .42 1 .29

Control+CD40L/gIFN 1 .93 3.22 1 .1 3

LPS+CD40L/gIFN 1 .63 2.4 0.42

Polv IC+CD40L/elFN 1 .76 2.96 0.73

[218] IL-12 was consistently detectable by ELISA in conditions were dendritic cells were incubated with LPS and CD40L/gIFN. It was noted that 1940 failed to induce any IL- 12 under any condition (Table 14).

54

5. 1 Table 14. ELISA Results for IL-12

Pg/mL IL- 12

1955 DC-TC 1983 DC -TC 1940 DC-TC*

Maturation Conditions

Average s.d. Average s.d. Average s.d.

Control 1.80 0.76 2.1 1 0.93 2.1 1 0.22

LPS 3.78 1.68 4.29 1.56 1.89 1.07

Poly IC 287.26 51.13 2.66 0.16 1.86 0.76

Control+CD40L/glFN 10.15 0.96 3.96 1.10 2.52 1.65

LPS+CD40L/gIFN 529.85 0.00 283.43 30.17 2.04 0.32

Polv IC+CD40L/gIFN 529.85 0.00 8.57 1.99 1.68 0.82

|219| Levels of CCL 17/TARC as measured by ELISA were noticeably high in all conditions tested. The levels represented in Table 15 were extrapolated because absorbance levels were beyond the linear range of the assay. However, it was apparent that large quantities of CCL17/TARC were secreted in all conditions tested.

Table 15. ELISA Results for CCL 17/TARC

Pg/mL TARC/CCL 17

1955 DC-TC 1983 DC-TC 1940 DC-TC*

Maturation Conditions

Average s.d. Average s.d. Average s.d.

Control 5890.55 253.27 6872.36 129.85 0 0

LPS 6147.82 72.00 6846.91 127.28 0 0

Poly IC 6045.09 91.28 5161.45 2446.59 0 0

Control+CD40L/gIFN 6195.09 90.00 6831.45 79.71 0 0

LPS+CD40L/gIFN 5859.64 98.99 6954.18 45.00 0 0

Polv IC+CD40L/gIFN 5932.36 75.85 6978.73 41.14 0 0

[220] The results of this experiment indicate that out of all the conditions tested, maturation of dendritic cells with a combination of CD40L, interferon-gamma and lipopolysacaride resulted in the greatest maturation as determined by the secretion of IL-12 by ELISA. Differences in maturation of dendritic cells were not detectable via flow cytometry as IL-12 was not detectable under the protocol employed. The results of the CCL17/TARC ELISA were inconclusive because values were outside of the linear range of the assay. However, it was apparent that large quantities of CCL 17/TARC were secreted in all conditions tested.

Example 4: Determination of CCL17/TARC as a Marker for Dendritic Cell Potency

[221] In this study, monocytes, "unloaded" dendritic cells (DC only) and/or antigen "loaded" dendritic cells (DC-TC) generated from a randomized phase II clinical trial were thawed and prepared for assaying for potency by incubating the cells in AIM-V media overnight under unstimulated and cytokine maturation conditions. Cells and cell culture supernatants were collected and analyzed by flow cytometry and ELISA as described above in order to determine specifications for markers associated with dendritic maturation state.

[222] At the end of a phase II randomized study, 1 1 cryopreserved tumor antigen- loaded dendritic cell patient samples were available for analysis (out of total of 18 patients treated in the dendritic cell-tumor cell arm of the study). However, 5/1 1 of those patient samples also had monocytes and unloaded dendritic cells available for analysis and were analyzed as paired samples.

[223] Samples were thawed, washed in AIM-V and the cells were counted by hemocytometer (data not shown). Immediately following thawing and cell counting, a sample was stained for CD 14/CD45/7-AAD/CD 1 lc to determine the viability and identity of the prepared samples. Samples sets included paired samples in which three types of samples were available from each patient;

monocytes, unloaded dendritic cells (DC only) and tumor antigen-loaded dendritic cells (DC-TC) and single sample sets where only DC-TC available.

[224] In the paired samples, dendritic cells had sufficiently differentiated from monocytes as measured by the reduction in the expression of CD 14 (Table 16). Comparison of the determination of viability by trypan-blue dye exclusion and 7-actinomycin D demonstrated comparable values for each sample type (trypan-blue dye: 83.0 ± 3.4%, 43.0 ± 14.5%, 62.8 ± 4.1% versus 7-actinomycin D: 83.2 ± 2.7%, 40.7 ± 17.2%, 62.8 ± 4.1% for monocytes, DC only and DC-TC, respectively). Seven out of 21 of the thawed samples had insufficient viable cell numbers to achieve enough cells at 1 x 10⁶ cells per mL in the three media conditions studied. In these instances, the cells were incubated in 1 mL of AIM- V and noted for the decreased number of cells per mL for calculation purposes.

Table 16. Results from Thawing and Preparation of Samples Collected During Randomized

Phase II Trial

Paired sample set Sample ID

Sample Tvpe Parameter 2206 2243 2254 2270 23 19

Total Cel ls x 10^b 67 75 78 140 50

%Viability 72 87 85 79 92

%CD 14/CD45 82.5 78.4 94.3 68. 1 88.8

%7AAD- 78.7 80.4 91.2 77.4 88.2

Total Cells 15 6.9 12 1 .8 13

DC Only % Viability 71 85 17. 1 1 8 24

%CD 14/CD45 1 .1 19.4 3.3 0.2 0.5

%7AAD- 88.5 76.5 7.9 14.9 15.6

Total Cells 8 8.4 14 13 7.8

Viabi lity 75 83 66 71 62

DC-TC %Recovery 50.6 67.2 103 .7 60.7 Monocytes^ ^

%CD 14/CD45 1 .44 1 1 .52 2.09 0.9 2.13

%7AAD- 85.6 62.4 51 .3 70.8 54.9

DC-TC Samples Samples ID

Sample Type 2219 2284 2333 2350 2404 2432

Total Cells 14 4.3 8.1 8.4 2.1 1 1

%Viability 58 54 70 65 63 56

DC-TC % Recovery 70 21.8 54 57.1 75 66.7

%CD 14/CD45 1 .2 0.8 0.5 6.8 2.1 1 .6

%7AAD- 52.4 60.9 71 .9 66.3 2.7 51.9

[225] Cells obtained from the randomized Phase II trial were placed into unstimulated and cytokine stimulated conditions as described above, in order to determine potential for maturation.

[226] The results of this experiment indicated that expression of CD40 trended higher than monocytes in the unstimulated state (15.8 ± 5.7% vs. 5.2 ± 1.8% p value 0.233, respectively), indicating that the dendritic cells were primed for interaction with CD40L expressing cells after differentiation from monocytes (Figure 23). The expression of CD83 in DC-TC significantly increased from 6.1 1 ± 0.5% to 8.38 ± 0.93% (p value 0.021) in response to CD40L/gIFN maturation cytokines indicating the dendritic cells were functional and responsive. CD83 expression was maximally induced in the CD40L/gIFN/LPS in agreement with published reports on the effect of LPS on dendritic cell maturation (Figure 23)

[227] Supernatants from the cells placed into unstimulated and cytokine stimulated conditions were collected at the end of the incubation. Samples were diluted neat (no dilution), 1 :2, 1 :4, 1 :8 and 1 : 16 in triplicate to determine the concentration of CCL 17/TARC secreted by monocytes, DCs only, or antigen-loaded DCs (DC-TC).

[228] The results of this experiment indicated that upon differentiation from monocytes, dendritic cells produce significant amounts of CCL 17/TARC over their monocyte counterparts (5541 pg/mL in DC only vs. undetectable amounts in monocytes) (Figure 24). For all conditions tested, monocytes did not produce any measurable quantities of CCL17/TARC (Figure 24).

Unstimulated DC-TCs had significantly higher levels of CCL 17/TARC than their unstimulated, unloaded DC counterparts, demonstrating that antigen-loading itself further increases secretion of CCL 17/TARC (p = 0.046) (Figure 24). Maturation of DC-TC with CD40/gIFN was associated with significantly higher levels of CCL 17/TARC secretion indicating that CCL 17/TARC is associated with the maturation status of DC-TC (p = 0.014) (Figure 24).

[229] Figure 25 shows the range of CCL 17/TARC secretion from samples normalized to time. We have previously established that a DC-TC product must contain at least 25% CD1 l c positive dendritic cells (data not shown). Thus, the lowest threshold of CCL 17/TARC secretion was calculated based on the lowest CCL 17/TARC value extrapolated to 25% CD 1 l c positive dendritic cells. For example, the lowest value for CCL 17/TARC secretion per day was 783.9 pg/mL from a sample containing 71.5% CD1 l c positive dendritic cells. Therefore, a final product containing 25% CD1 l c positive cells would secrete 274.3 pg/mL/day.

[230] Based on these results, CCL 17/TARC is a suitable marker for potency considering that increased levels of CCL17/TARC were correlated with antigen loading and maturation status. In addition, a lower limit of acceptable CCL 17/TARC secretion by a potent, antigen-loaded DC (DC- TC) product can be set at 274 pg/mL/day.

[231] While the present invention has been described with reference to the specific embodiments thereof it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adopt a particular situation, material, composition of matter, process, process step or steps, to the objective spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

WHAT IS CLAIMED IS:

1. A method for preparing a population of immunopotent dendritic cells activated in vitro with a tumor- specific antigen derived from a population of purified cultivated tumor cells derived from a patient comprising:

(a) obtaining peripheral blood mononuclear cells (PBMCs) by leukapheresis from the patient from whom the tumor cells were derived;

(b) optionally shipping the collected PBMCs from (a) to a manufacturing facility;

(c) purifying the PBMCs from (a) from other lymphocytes;

(d) incubating the purified PBMCs from (c) with GM-CSF and IL-4 for 6 days to generate dendritic cells; and

(e) contacting the dendritic cells from (d) with the purified cultivated tumor cells derived from the patient for 18-24 hours to form immunopotent dendritic cells,

wherein immunopotency of the dendritic cells formed in (e) is measured by an amount of a biomarker produced by the dendritic cells formed in (e), and

wherein the dendritic cells formed in (e) are effective to generate an effective immune response against the tumor-specific antigen comprising activation and proliferation of CD4+ T-cells, CD8+ T-cells, B-cells or a combination thereof.

2. The method according to claim 1 , wherein the amount of the biomarker is measured by an immunoassay.

3. The method according to claim 2, wherein, the immunoassay is selected from the group consisting of Western blot, ELISA and flow cytometry.

4. The method according to claim 1 , wherein the biomarker is CCL17/TARC.

5. The method according to claim 1 , wherein the amount of the biomarker is at least 274.3 pg/mL/day.

6. A method for treating a subject suffering from a cancer comprising:

(a) preparing for a cancer patient a patient-specific immunogenic composition comprising an immunopotent amount of an isolated population of dendritic cells contacted ex vivo with a cancer cell expressing a cancer-specific antigen by the method according to claim 1 ;

(b) administering the immunogenic composition to the cancer patient; and (c) generating an effective immune response against the cancer-specific antigen comprising activation and proliferation of CD4+ T-cells, CD8+ T-cells, B-cells or a combination thereof,

wherein the effective immune response is effective to improve a clinical parameter selected from the group consisting of progression-free survival, disease-free survival, time to progression, time to distant metastasis and overall survival of the subject when compared to a control.