Patent Document

CROSS REFERENCE TO RELATED APPLICATION 
       [0001]    The present application is a continuation-in-part application to U.S. patent application Ser. No. 11/227,374, filed Sep. 15, 2005, which claims priority to U.S. Provisional Patent Application 60/610,822 filed Sep. 17, 2004, both of which are incorporated herein by reference. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    1. Field of the Invention 
         [0003]    The present invention relates to a tumor therapy that includes the injection of immature dendritic cells and adjuvant directly into the patient&#39;s (a human or an animal) tumor tissue, which presents antigenicity as a vaccine antigen at the injection sight. Conjugation of these elements within the tumor tissue rapidly induce and activate the patient&#39;s immune system to dramatically reduce and/or eliminate tumor cells. Most adjuvants, which augment the immune response, can be directly injected with immature dendritic cells to the tumor tissue to achieve the reduction or elimination of tumor tissues. 
         [0004]    2. Description of the Prior Art 
         [0005]    Immunological adjuvants are used in combination with vaccines to augment the immune response to the antigen. One way in which immunological adjuvants function is by attracting macrophages to the antigen, so that the macrophages can present the antigen to the regional lymph nodes and initiate an effective antigenic response. Adjuvants may also act as carriers themselves for the antigen, or may influence the immune response by other mechanisms such as depot effect, cytokine induction, complement activation, recruiting of different cell populations of the immunological system, antigen delivery to different antigen presenting cells, regulation of the expression of HLA class I or class II molecules and the stimulation to produce different antibody subtypes. Many of the newer vaccines are only weakly immunogenic and thus require the presence of adjuvants. 
         [0006]    Materials having adjuvant activity are well known. Alum (Al(OH) 3 ), and similar aluminum gels are adjuvants licensed for human use. The adjuvant activity of alum was first discovered in 1926 by Glenny (Chemistry and Industry, Jun. 15, 1926; J. Path. Bacteriol, 34, 267). Aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines. The efficacy of alum in increasing antibody responses to diphtheria and tetanus toxoids is well established and, more recently, a HBsAg vaccine has been adjuvanted with alum. 
         [0007]    One line of research in the development of adjuvants has been directed to the study of dendritic cells. Dendritic cells (DC) are professional antigen presenting cells (APC) that have the unique capacity to initiate primary immune responses in vivo and in vitro. They are derived from myeloid (DC1) or lymphoid (DC2) precursors and are distributed in their immature form throughout the body in tissues that commonly encounter environmental pathogens (skin, mucus membranes, gut epithelia, etc.). Whereas DC1 and DC2 comprise a small percentage of the total number of mononuclear cells in the peripheral circulation, DC1 precursors in the form of CD14+/CD11c+/HLA-DR+ monocytes are relatively abundant, constituting about 10% to 15% of mononuclear blood cells. 
         [0008]    Immature DC express a host of surface structures that are involved in antigen acquisition, DC activation/maturation, and antigen presentation. Once DC encounter antigen, they undergo a maturation process characterized by the up-regulation of HLA class I and II molecules as well as co-stimulatory molecules and interact with cognate receptors on T and B lymphocytes, resulting in the generation of antigen specific cellular and humoral immune responses. 
         [0009]    DC are considered to be the primary APC in the immune system. The ability to isolate these cells and/or their precursors and to study them in vitro has added considerable dimension to knowledge of their role in innate and acquired immunity. The classic means of generating human DC in vitro is to isolate and enrich CD14+-monocytes from peripheral blood and culture them for various periods of time in GM-CSF and IL-4 followed by final maturation with a number of cytokines, including IL-2, IL-6, IL-7, IL-13, IL-15, TNFα, IL-10, or with various other agents including lipopolysaccharides, PGE2, type 1 interferons, or double-stranded RNA. 
         [0010]    Numerous investigators have shown that these in vitro generated monocyte-derived DC are potent antigen presenting cells (APC) capable of initiating primary and recall antigen-specific CD4 +  and CD8 +  T cell responses. Recent in vitro studies have generated a rather extensive body of information regarding the biology of DC1 and shed light on the processes whereby antigen specific immune responses are generated in vivo. In the peripheral tissues, immature DC acquire antigenic materials in the context of danger signals initiating a complex cytokine/chemokine milieu that is generated by DC and other cell types in the vicinity. 
         [0011]    Soluble mediators produced by DC may act in an autocrine or paracrine fashion. T cells produce additional cytokines and chemokines following interaction with antigen armed DC, as do other immune cells that are activated by the cytokines released. This complex network of interactions may in turn create an environment that promotes the generation of DC from their monocyte precursors. 
         [0012]    It is thought that those adjuvants which promote that maturation of dendritic cells, when administered in combination with a vaccine antigen, will result in more antigen presenting cells presenting the vaccine antigen to T lymphocytes and B cells, thus bolstering the immune response to the vaccine antigen. However, isolation of the most effective vaccine antigen has been extremely difficult since antigenicity of APC has always been subject to its evolution with antigenic drift and/or shift, and therefore many of the newer vaccines are only weakly immunogenic even though dendritic cells and adjuvant are present. The most effective vaccine antigen against the live tumor cells should be used with dendritic cells and adjuvant during a course of treatment to promote and to induce a rather strong immunogenicity. 
       SUMMARY OF THE INVENTION 
       [0013]    The present invention solves the above need by providing the most effective antigenic vaccine antigen with dendritic cells and adjuvant to increase the amount and quality of the immune response against tumor cells. 
         [0014]    In an aspect of the present invention, there is provided a method of reduction of tumor cells in tumor tissue of a patient, comprising collecting monocyte cells from the patient, culturing the monocyte cells with IL-4 and GM-CFS to form immature dendritic cells from the monocyte cells, and administering a therapeutically effective amount of the immature dendritic cells with a leukocyte cultured medium (LCM) adjuvant to the patient. The LCM adjuvant comprises at least three, preferably at least six and more preferably at least ten cytokines selected from eotaxin, FGF, G-CSF, GM-CSF, IFNγ, IP10, ILβ, IL1ra, IL2, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL13, IL15, IL17, MCP1, MIP1α, MIP1β, PDGFbb, RANTES, TNFα and VEGF. 
         [0015]    The immature dendritic cells and LCM adjuvant are administered intratumorally, i.e., directly into the site of the tumor. 
         [0016]    Optionally, this method provides treating the patient with chemotherapy, radiation or anti T-cell antibodies prior to the administration of the immature dendritic cells and LCM adjuvant. 
         [0017]    In another aspect of the present invention, there is provided a method of reduction of tumor cells in tumor tissue comprising treating a tumor of a patient, with a chemotherapy regimen, collecting monocyte cells from the patient, culturing the monocyte cells with IL-4 and GM-CFS to form immature dendritic cells from the monocyte cells and administering a therapeutically effective amount of the immature dendritic cells with a leukocyte cultured medium (LCM) adjuvant to the patient. The LCM adjuvant comprises at least three, preferably at least six and more preferably at least ten cytokines selected from eotaxin, FGF, G-CSF, GM-CSF, IFNγ, IP10, IL1β, IL1ra, IL2, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL13, IL15, IL17, MCP1, MIP1α, MIP1β, PDGFbb, RANTES, TNFα, and VEGF. 
         [0018]    Optionally, this method provides treating the patient with radiation prior to the administration of the immature dendritic cells and LCM adjuvant. 
         [0019]    In a further aspect of the present invention, there is provided a method of reduction of tumor cells in tumor tissue comprising treating a tumor of a patient with a radiation therapy regimen, collecting monocyte cells from the patient, culturing the monocyte cells with IL-4 and GM-CFS to form immature dendritic cells from the monocyte cells, and administering a therapeutically effective amount of the immature dendritic cells with a leukocyte cultured medium (LCM) adjuvant into the tumor tissue of the patient. The LCM adjuvant comprises at least three, preferably at least six and more preferably at least ten cytokines selected from eotaxin, FGF, G-CSF, GM-CSF, IFNγ, IP10, IL1β, IL1ra, IL2, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL13, IL15, IL17, MCP1, MIP1α, MIP1β, PDGFbb, RANTES, TNFα, and VEGF. 
         [0020]    Optionally, this method provides treating the patient with chemotherapy prior to the administration of the immature dendritic cells and LCM adjuvant. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0021]    A full understanding of the invention can be gained from the following description of the preferred embodiments when read in conjunction with the accompanying drawings in which: 
           [0022]      FIG. 1  shows two protocols for treating patients with tumors according to the methods of the present invention; 
           [0023]      FIG. 2  shows a computerized tomography (CT) image of a patient with gastric cancer and liver metastasis before and after treatment according to the methods of the present invention; 
           [0024]      FIG. 3  shows a CT image of a patient with upper pharyngeal cancer before and after treatment according to the methods of the present invention; 
           [0025]      FIG. 4  shows a CT image of a patient with sigmoid cancer and liver metastasis before and after treatment according to the methods of the present invention; 
           [0026]      FIG. 5  shows a CT image of a patient with rectal cancer and lung, pelvic and left cervical metastasis before and after treatment according to the methods of the present invention; 
           [0027]      FIG. 6  shows a CT image of a patient with right breast cancer and left chest wall and mediastinal metastasis before and after treatment according to the methods of the present invention; 
           [0028]      FIG. 7  shows the effect of LCM on surface marker expression, in which monocytes in PBMCs differentiate to a DC-like phenotype following exposure to LCM; 
           [0029]      FIG. 8  shows the effect of LCM on surface marker expression, in which immature monocyte-derived DCs differentiate to a mature-phenotype following exposure to LCM; 
           [0030]      FIG. 9  shows that LCM augments CpG-induced maturation and IFNα production by CpG treated plasmacytoid DCs (pDCs); 
           [0031]      FIG. 10  shows the effect of LCM treatment on T cell responses in vitro; 
           [0032]      FIG. 11  shows T cell responses to vaccines are enhanced following treatment with LCM (ELISPOT); 
           [0033]      FIG. 12  shows antibody responses to vaccines are enhanced following treatment with LCM (ELISA); 
           [0034]      FIG. 13  provides an outline of an elutriation study; 
           [0035]      FIG. 14  shows percent viability following incubation with LCM; 
           [0036]      FIGS. 15  A, B provides recall responses and shows that LCM augments response to antigens (CMV, n=2); A: Aph082305 and Aph011006; B: Aph082305 and Aph011006; 
           [0037]      FIGS. 16  A, B shows tumor cell lysates (n=2); A: Aph062805; B: Aph011006; 
           [0038]      FIGS. 17  A, B shows responses of LCM-treated ‘naïve IL7-IL15-treated’ cells (N=2); A: Aph062805; B: Aph01106; and 
           [0039]      FIG. 18  shows a proposed culture system for lymphocytes. 
       
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0040]    As used herein, the term “leukocyte cultured medium (LCM)” is synonymous and interchangeable with the term “activated leukocyte medium (ALM).” 
         [0041]    As used herein, “patients” in clude mammals, which include humans. 
         [0042]    As used herein, the term “therapeutically effective amount” refers to that amount of immature dendritic cells and lymphocyte cultured medium (LCM) adjuvant required to bring about a desired effect in a human or other mammal. In all instances, at its most basic level, the desired effect is a reduction of tumor cells in tumor tissue of the patient when compared to the tumor cells in the tumor tissue of the patient prior to employing the methods of the present invention. 
         [0043]    The present invention provides treatment tumor tissue using full antigenic elements, which include antigenicity of both known and unknown antigen presenting cells, by locating them within the live tumor tissue in the human body (or alternatively, the body of an animal). This is in contrast to prior art cultured antigens obtained from tumor cell lines or any process added antigen, which have limited antigencity and outdated antigenic data or potency as a vaccine antigen for the patient&#39;s tumor cells. In particular, the present invention relates to a therapy that includes the injection of immature dendritic cells and adjuvant directly into the patient&#39;s tumor tissue, which presents antigenic elements as the vaccine antigen at the injection sight. The conjugation of these elements within the tumor tissue rapidly induce and activate the patient&#39;s immune system to dramatically reduce and/or eliminate tumor cells. Most adjuvants, which augment the immune response, can be directly injected with immature dendritic cells into the tumor tissue to achieve the reduction or elimination of tumor cells. Such adjuvants may include, without limitation, lipid-based, protein-based and polysaccharides-based adjuvants, such as lymphocyte cultured medium, Marignase, Agaricus, OK432, BCG, Lentinan (shiitake), Reishi, Sarunokoshikake, TNF Meshimakobu, Froint&#39;s complete or incomplete adjuvant, LPS, fatty acids, phospholipids, cytokines or a virus. 
         [0044]    The present invention provides rapid reduction and/or elimination of tumor cells, which can be visually detected by MRI and/or CT and/or Echo scan within two weeks after the injection. The therapy according to a preferred embodiment of the invention includes the following steps: Step 1: Colleting peripheral blood monocyte cells (PBMC) from a patient; Step 2: Culturing these PBMC with GM-CFS and IL-4 to immature dendritic cells; Step 3: Injecting the cultured immature dendritic cells and an adjuvant into the tumor; and Step 4: Evaluating the tumor in two weeks. 
         [0045]    In one particular embodiment, the effectiveness (immuno-response) of this method of treatment can be enhanced by pre-treating the tumor cells using known chemotherapy and/or radiation therapy techniques, which diminish the existing immune system, prior to steps 1-4 described above. In addition, the effectiveness (immuno-response) of this method of treatment can also be enhanced by injecting the tumors cells with an anti T-cell monoclonal antibody prior to steps 1-4 described above (either alone or in addition to the chemotherapy and/or radiation therapy described above). 
       EXAMPLES 
       [0046]    The present invention is more particularly described in the following non-limiting examples, which are intended to be illustrative only, as numerous modifications and variations therein will be apparent to those skilled in the art. 
       Example 1 
     Treatment with Immature Dendritic Cells and Lymphocyte Cultured Medium Adjuvant 
       [0047]    Six patients, four with stomach cancer and two with colon cancer, were used in this clinical investigation to assess the effect of intratumoral administration of immature dendritic cells (imDCs) with a lymphocyte cultured medium adjuvant (LCMadj). All patients were self-referred, had advanced cancers and progressive disease that had not responded to conventional standard therapies. 
       1. Methods 
       [0048]    Four weeks prior to administration of the imDC and LCMadj, leukapheresis was performed on each patient to collect monocyte cells from the patient. The monocyte cells were cultured with IL4 and GM-CFS. This resulted in the production of imDCs. Four weeks later, a cocktail was prepared containing between about 10 7  to 10 8  imDCs and between about 1.0 to 2.0 mg of LCMadj to make up a 10% concentration in normal saline. Depending on the size of the tumor, between 2.0 to 50 cc of normal saline was injected into the tumor site of each patient. Four weeks after injection of the cocktail, the patients were evaluated by CT image analysis and measurement of serum tumor markers. 
       2. Results 
       [0049]    Of the six patients in this clinical study, three of the tumors of the patients showed stable disease (SD); defined as showing less than a 20% increase in tumor size and less than a 30% reduction in tumor size, with no increase in serum tumor markers. The tumors of the other three patients showed progressive disease (PD); defined as a 20% or greater increase in tumor size, new metastatic lesions and an increase in serum markers. 
       Example 2 
     Pretreatment with Chemotherapy Prior to Injection of Immature Dendritic Cells and Lymphocyte Cultured Medium Adjuvant 
       [0050]    Four patients, three with rectal cancer and one with colon cancer, were used in this clinical investigation to assess the effect of chemotherapy prior to intratumoral administration of imDCs with a LCMadj. All patients were self-referred, had advanced cancers and progressive disease that had not responded to conventional standard therapies. 
       1. Methods 
       [0051]    As shown in  FIG. 1 , Four weeks prior to administration of the imDC and LCMadj, leukapheresis was performed on each patient to collect monocyte cells from the patient. The monocyte cells were cultured with IL4 and GM-CFS. This resulted in the production of imDCs. Three weeks later, all patients were administered cytoxan intratumorally. One week later, a cocktail was prepared containing between about 10 7  to 10 8  imDCs and between about 1.0 to 2.0 mg of LCMadj to make up a 10% concentration in normal saline. Depending on the size of the tumor, between 2.0 to 50 cc of normal saline was injected into the tumor site of each patient. Four weeks after injection of the cocktail, the patients were evaluated by CT image analysis and measurement of serum tumor markers. 
       2. Results 
       [0052]    Of the four patients in this clinical study, two of the tumors of the patients showed a partial response (PR); defined as a 30% reduction in the size of the injected tumor, decline in serum markers, no increase in tumor size at other metastatic sites or appearance of new metastasis. The tumor from the third patient showed stable disease (SD), as defined above; and the tumor from the fourth patient showed progressive disease (PD), as defined above. 
       Example 3 
     Injection of Immature Dendritic Cells and Lymphocyte Cultured Medium Adjuvant or Pretreatment with Chemotherapy or Radiation Therapy Prior to Injection of Immature Dendritic Cells and Lymphocyte Cultured Medium Adjuvant 
       [0053]    Twenty patients with advanced malignancies of different types were used in this clinical study to assess the effect of intratumoral administration of imDCs with an LCMadj, chemotherapy prior to imDCs and LCMadj administration or radiation therapy prior to imDCs and LCMadj administration. All patients were self-referred, had advanced cancers and progressive disease that had not responded to conventional standard therapies. 1. 
         [0054]    Methods 
         [0055]    Four weeks prior to administration of the imDC and LCMadj, leukapheresis was performed on each patient to collect monocyte cells from the patient. The monocyte cells were cultured with IL4 and GM-CFS. This resulted in the production of imDCs. Three weeks later, three patients received radiation therapy and 11 patients were given chemotherapy (see Table 1) by administering the chemotherapeutic agent intratumorally. One week later, a cocktail was prepared containing between about 10 7  to 10 8  imDCs and between about 1.0 to 2.0 mg of LCMadj to make up a 10% concentration in normal saline. Depending on the size of the tumor, between 2.0 to 50 cc of normal saline was injected into the tumor site of each patient. Four weeks after injection of the cocktail, the patients were evaluated by CT image analysis and measurement of serum tumor markers. 
       2. Results 
       [0056]    As shown in Table 1, of the six patients that did not receive any prior treatment before administration of the imDCs and LCMadj cocktail, the tumors of two patients showed a partial response (PR) (see, for example,  FIG. 2 ); the tumors of two other patients showed no change (NC) from their previous condition (see, for example,  FIG. 3 ); and the tumors from two other patients showed progressive disease (PD) (see, for example,  FIG. 4 ). Of the three patients that had radiation therapy prior to administration of the imDCs and LCMadj cocktail, the tumor from one patient showed no change (NC) from its previous status. The other patient dropped out before they could be evaluated. Of the eleven patients that received chemotherapy prior to administration of the imDCs and LCMadj cocktail, the tumors from three of the patients showed a partial response (PR) (see for example  FIG. 5 ); the tumors from six of the patients showed no change (NC) from their previous condition (see, for example,  FIG. 6 ); and the tumors from two patients showed progressive disease (PD).  FIGS. 2-7  show CT images of various cancers and their response to the treatment protocol. 
         [0000]    
       
         
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                   
                   
                   
                   
                 Pre- 
                 Eval- 
               
               
                 Sex 
                 ID 
                 Diagnosis 
                 Stage 
                 treatment 
                 uation 
               
               
                   
               
             
             
               
                 F 
                 030593 
                 Gastric Ca Op 
                 Rec 
                 No 
                 PR 
               
               
                   
                   
                 Liver Meta 
               
               
                 M 
                 011077 
                 Epi pharyngeal Ca Op 
                 Rec 
                 No 
                 NC 
               
               
                 M 
                 040231 
                 Sigmoid Ca Op Liver, 
                 Rec 
                 No 
                 PD 
               
               
                   
                   
                 Lung &amp; Urinary Bladder 
               
               
                   
                   
                 Meta 
               
               
                 M 
                 040265 
                 Gastric Ca Op Peritoneal 
                 Rec 
                 No 
                 NC 
               
               
                   
                   
                 Meta 
               
               
                 M 
                 051585 
                 Gastric Ca Op Mult, 
                 Rec 
                 No 
                 PR 
               
               
                   
                   
                 Lever Meta 
               
               
                 M 
                 040402 
                 Rt. Lung Ca Op Rt Chest 
                 Rec 
                 Radiation 
                 Drop 
               
               
                   
                   
                 Wall Meta 
                   
                   
                 Out 
               
               
                 M 
                 040465 
                 Gastric Ca Op Liver 
                 Rec 
                 Radiation 
                 NC 
               
               
                   
                   
                 Meta 
               
               
                 M 
                 040865 
                 Malig. Melanoma of 
                 Rec 
                 Endoxan 
                 PR 
               
               
                   
                   
                 Gingiva Op Cervical LN 
               
               
                   
                   
                 Meta 
               
               
                 F 
                 031180 
                 Rectal Ca Op., Lung 
                 Rec 
                 Endoxan 
                 PR 
               
               
                   
                   
                 Meta Pelvic &amp; Lt. 
               
               
                   
                   
                 Cervical LN Meta 
               
               
                 F 
                 040764 
                 Sigmoid Ca Op Mult. 
                 Rec 
                 Chemo 
                 NC 
               
               
                   
                   
                 Liver Meta 
                   
                 (TAI) 
               
               
                 F 
                 010863 
                 Rec. of Rectal &amp; Caecal 
                 Rec 
                 None 
                 PD 
               
               
                   
                   
                 Cancer Op., Lt. Cervical 
               
               
                   
                   
                 LN Meta 
               
               
                 F 
                 041095 
                 Rectal Ca Op. Lung Meta 
                 Rec 
                 Radiation 
                 NC 
               
               
                   
                   
                 Pelvic LN Meta 
               
               
                 F 
                 040924 
                 Breast Ca Op Skin Meta 
                 Rec 
                 Endoxan 
                 PR 
               
               
                 F 
                 040520 
                 Breast Ca Op Skin Meta 
                 Rec 
                 Endoxan 
                 NC 
               
               
                 M 
                 031119 
                 Lt. Pylvic Tumor Op. Lt. 
                 Rec 
                 Endoxan 
                 PD 
               
               
                   
                   
                 Cervical &amp; Axilla LN 
               
               
                   
                   
                 Meta 
               
               
                 F 
                 040558 
                 Rec. of Rt. Breast Cancer 
                 Rec. 
                 CDDP 
                 NC 
               
               
                   
                   
                 Op., Liver Metastasis 
               
               
                 M 
                 040325 
                 Malig. Mesothelioma 
                 IV 
                 Endoxan 
                 NC 
               
               
                 M 
                 041266 
                 Rectal Ca Op Liver 
                 Rec 
                 Endoxan 
                 PD 
               
               
                   
                   
                 Metastasis 
               
               
                 F 
                 900182 
                 Rt. Breast Ca Op., Lt. 
                 Rec 
                 CDDP 
                 NC 
               
               
                   
                   
                 Chest Wall &amp; Medistinal 
               
               
                   
                   
                 LN Meta 
               
               
                 F 
                 041264 
                 Rec. of Endometrial 
                 Rec 
                 CDDP 
                 NC 
               
               
                   
                   
                 Cancer op., Pelvic LN 
               
               
                   
                   
                 Metastasis 
               
               
                   
               
             
          
         
       
     
       3. Discussion 
       [0057]    Approximately 80% of the patients showed some degree of tumor regression. Moreover, none of the patients had any adverse reaction to the treatment protocol they were given. In those patients showing tumor regression, this was evident within one month after completion of the treatment protocol and effectiveness of the treatment was observed after over 3 months. The number of cases and percentage effectiveness of the treatment protocols were as follows: 
         [0058]    Complete response (CR); defined as a decrease in serum markers to normal levels, complete disappearance of all measurable lesions: 0 (0%) 
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 PR 
                 5 (26%) 
               
               
                   
                 NC 
                 10 (53%)  
               
               
                   
                 PD 
                 4 (21%) 
               
               
                   
                   
               
             
          
         
       
     
       Example 4 
     Preparation of Lymphocyte Cultured Medium (LCM) for Clinical Application 
     Objective 
       [0059]    To develop a clinically acceptable method for the production of LCM from elutriated cell fractions obtained from mononuclear cells (MNC) and generate preliminary data in support of a potential IND submission. 
       BACKGROUND 
       [0060]    A variety of cytokines are known to induce the differentiation and maturation of monocyte-derived dendritic cells (DC). Soluble factors found in cell-free supernatants from monocyte and anti-CD3-activated T cells have been found to increase the expression of activation and maturation markers. In this laboratory, earlier studies showed that activation of ficolled PBMC with anti-CD3/CD28 beads results in a product that could mature APCs and augment T cell responses. The activated lymphocyte medium contained a mix of cytokines and chemokines known to be important for the development and migration of DC including GM-CSF, TNFα, IFNγ, IL8, MCP-1 and MIP1. When cultured in LCM, purified monocytes and monocytes in whole PBMC preparations developed a DC-like phenotype characterized by the loss of CD14 and upregulation of costimulatory molecules. Immature DC exposed to LCM underwent maturation within 48 h marked by an increase in surface expression of CD40, CD80, CD86, CD83 and HLADR. LCM-treated DC stimulated potent allogeneic PBMC responses and boosted antigen-specific T cell responses to antigens. Enhanced T cell and antibody responses were observed when LCM was co-administered with a variety of vaccines in macaques. LCM represents a potential ‘physiologic’ product for the generation of DCs in vitro as well as vaccine adjuvant; providing a cytokine milieu for DC generation and immune activation in vivo. Data using activated PBMCs as well as activation products developed from elutriated lymphocyte fractions are included in this study. 
         [0061]    The cytokine composition of LCM is shown in Table 2. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Quantities of Cytokines or Chemokines (PBMCs) 
               
             
          
           
               
                   
                 Quantity (pg/ml) 
               
               
                   
                   
               
             
          
           
               
                   
                 GM-CSF 
                 23000 
               
               
                   
                 IFNα 
                 0 
               
               
                   
                 IFNγ 
                 31000 
               
               
                   
                 IL1β 
                 70 
               
               
                   
                 IL2 
                 5900 
               
               
                   
                 IL3 
                 1000 
               
               
                   
                 IL4 
                 280 
               
               
                   
                 IL6 
                 2170 
               
               
                   
                 IL8 
                 47970 
               
               
                   
                 IL10 
                 660 
               
               
                   
                 IL12 
                 10 
               
               
                   
                 IL15 
                 0 
               
               
                   
                 MCP1 
                 110040 
               
               
                   
                 M-CSF 
                 8690 
               
               
                   
                 MIP1α 
                 127200 
               
               
                   
                 MIP1β 
                 157890 
               
               
                   
                 PGE2 
                 1540 
               
               
                   
                 RANTES 
                 20640 
               
               
                   
                 CD40L 
                 1270 
               
               
                   
                 SDF1α 
                 0 
               
               
                   
                 TGFβ 
                 0 
               
               
                   
                 TNFα 
                 6430 
               
               
                   
                   
               
             
          
         
       
     
         [0062]      FIGS. 7 and 8  show the effect of LCM on surface marker expression. Regarding  FIG. 7 , monocytes in PBMCs differentiated to a DC-like phenotype following exposure to LCM. Expression of CD14, HLA-DR, CD40, CD80, and CD86 was analyzed at 0, 3 and 5 days following exposure to LCM. Data represent mean±SEM of 11 experiments and ** indicates p&lt;0.005. Regarding  FIG. 8 , immature monocyte-derived DCs differentiated to a mature-phenotype following exposure to LCM. Elutriated monocytes were cultured with GM-CSF/IL-4 for 3-4 days followed by addition of media alone, LCM or Maturation Cocktail for 48 hours. Monocytes cultured in cRPMI only were used as a negative control. CD11c+DCs were examined for surface expression of CD14, HLA-DR, CD40, CD83, CD80, and CD86 by flow cytometry. Open histograms represent staining of DC with isotype control mAb, and shaded histograms represent staining of DC with specific mAb. 
         [0063]      FIG. 9  shows that LCM augments CpG-induced maturation and IFNα production by CpG treated plasmacytoid DCs (pDCs). Human pDCs (91-96% purity assessed by surface expression of CD123) were isolated using positive BDCA-4 immunomagnetic selection (Miltenyi Biotech, Auburn, Calif.). Typically, 1×10 8  monocytes yielded 3-4×10 5  pDCs. The pDCs were adjusted to 0.5×10 6  cells/ml in DMEM (Life Technologies, Rockville, Md.) containing 10% fetal bovine serum (BioWhittaker, Walkersville, Md.) and cultured at 1×10 5  cells per well in 96 well round bottom plates. Freshly isolated pDCs expressed an immature phenotype (CD83 − , low MHC and co-stimulatory molecules). pDCs were matured with CpG2006 (20 μg/ml) for 24 to 48 h. LCM was added at a 25% dilution. 
         [0064]      FIG. 10  shows the effect of LCM treatment on T cell responses in vitro. PBMCs were cultured for 24 h with or without antigen and/or LCM (25%), washed to remove LCM and plated for: (A) Recall Responses (re-plated on ELISPOT for 24 hours; (B) Primary Responses (culture for 7 days with media containing IL7 and IL15, cells washed, then replated on ELISPOT with antigen for 24 hours). CMV=cytomegalovirus lysate; cancer cell lines: K=gastric cancer, P=pancreatic cancer, N=renal cell carcinoma, col=colon cancer. 
         [0000]    Effect of LCM Immunization with Vaccines on T Cell and Antibody Responses-In Vivo. 
         [0065]    Total solubilized protein was measured in pooled LCM samples (BioRad protein assay based on the method of Bradford, absorbance at 595 nm). To determine adjuvant activity of LCM in vivo, 0.3 ml LCM (97.5 ng) was mixed with individual vaccines (hepatitis A=HepA; tetanus diphtheria toxoid=TDT; rabies or prostate specific antigen=PSA) and each vaccine/LCM mixture was injected IM in macaques at four separate sites (right and left arms and thighs). Selected cytokine levels are calculated in Table 3. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Cytokine/Chemokine Concentrations of Pooled LCM Injected into 
               
               
                 Macaques 
               
             
          
           
               
                   
                   
                 ng/ml 
                 ng/injection site 
                 total ng/injection 
               
               
                   
                   
               
             
          
           
               
                   
                 GM-CSF 
                 310 
                 93 
                 372 
               
               
                   
                 IL-4 
                 2.5 
                 0.75 
                 3 
               
               
                   
                 IL-5 
                 1.5 
                 0.45 
                 1.8 
               
               
                   
                 IL-8 
                 4.3 
                 1.29 
                 5.2 
               
               
                   
                 IL-10 
                 3.2 
                 0.96 
                 3.8 
               
               
                   
                 MCP-1 
                 3.7 
                 1.11 
                 4.4 
               
               
                   
                 IL-1α 
                 0.228 
                 0.07 
                 0.274 
               
               
                   
                 IL-1β 
                 0.364 
                 0.11 
                 0.437 
               
               
                   
                 IL-12p40 
                 0.313 
                 0.09 
                 0.376 
               
               
                   
                   
               
             
          
         
       
     
         [0066]    Animals were injected with vaccines alone or vaccines plus LCM and cell and serum samples removed for testing according to the following timeline, shown in Table 4. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Treatment timeline for animals receiving vaccine or vaccine plus LCM 
               
             
          
           
               
                 Treatment 
                 0 
                 7 
                 14 
                 21 
                 28 
                 35 
                 42 
                 49 
                 56 
               
               
                   
               
             
          
           
               
                   
                 Days of injection 
               
             
          
           
               
                 Vaccine 
                 HepA 
                 + 
                 + 
                 + 
                   
                   
                   
                   
                   
                   
               
               
                 alone 
               
               
                   
                 TDT 
                 + 
                 + 
                 + 
               
               
                   
                 Rabies 
                 + 
                 + 
                 + 
               
               
                   
                 PSA 
                 + 
                 + 
                 + 
               
             
          
           
               
                   
                 Days samples collected for testing 
               
             
          
           
               
                   
                 Cells (ELISPOT) 
                 + 
                   
                   
                   
                   
                 + 
                 + 
                 + 
                 + 
               
               
                   
                 Serum (ELISA: IgG 
                 + 
                   
                   
                   
                   
                 + 
                 + 
                 + 
                 + 
               
               
                   
                 antibodies to HLA class I 
               
               
                   
                 and HLA class II antigens 
               
               
                   
                 and vaccines) 
               
               
                 Vaccine + LCM 
                 HepA 
                 + 
                 + 
                 + 
               
               
                   
                 TDT 
                 + 
                 + 
                 + 
               
               
                   
                 Rabies 
                 + 
                 + 
                 + 
               
               
                   
                 PSA 
                 + 
                   
                 + 
               
             
          
           
               
                   
                 Cells (ELISPOT) 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                   
                 Serum (ELISA: IgG 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                   
                 antibodies to HLA class I 
               
               
                   
                 and HLA class II antigens 
               
               
                   
                 and vaccines) 
               
               
                   
                   
               
               
                   
                 + = procedure done on indicated day 
               
             
          
         
       
     
         [0067]      FIG. 11  shows that T cell responses to vaccines were enhanced following treatment with LCM (ELISPOT).  FIG. 12  shows that antibody responses to vaccines were enhanced following treatment with LCM (ELISA). 
         [0068]    Table 5 shows detection of HLA Ab in Macaque serum using solid phase ELISA. 
         [0000]                                                                                                                                                                                                                                                                                                TABLE 5                           Class I   Class II            Monkey ID   Day of serum collection            code   0   7   14   21   28   35   42   49   56   0   7   14   21   28   35   42   49   56                    Vaccine only            CC8A   −                   −   −   −   −   −                   −   −   −   −       CG33   −                   −   −   −   −   −                   −   −   −   −       98021   −                   −   −   −   −   −                   −   −   −   −       99E030   −                   −   −   −   −   −                   −   −   −   −       99061   −                   −   −   −   −   −                   −   −   −   −            Vaccine and LCM            LCM-98023   −   −   −   −   +   +   +   +   +   −   −   −   −   −   −   −   −   −       LCM-99E145   −   −   −   −   −   −   −   −   −   −   −   −   −   −   −   −   −   −       LCM-99E107   −   −   +   +   +   +   +   +   +   −   −   −   −   −   −   −   −   −               *GTI, Waukesha, WI;       + = positive detection,       − not detected            
Summary: Media from Anti-CD3/CD28 Activated PBMCs:
 
         [0069]    Contain cytokines and chemokines that are known to influence the generation of immune responses; induces maturation and differentiation of monocyte-derived DCs and pDCs; augments primary and recall antigen specific T cell responses in vitro; and augments antibody and T cell responses to vaccines in non-human primates. 
         [0000]    Data Generated from ‘Purified’ Elutriated Lymphocyte-Derived LCM 
         [0070]    To determine if LCM production could be adapted to a larger scale process potentially better defined and more easily amenable to FDA guidelines than the use of ficolled whole blood PBMCs, a study on apheresed cells with autologous testing was initiated. MNC were fractionated into different cell types from healthy individuals utilizing a programmable semi-closed cell separation device (Elutra, Gambro BCT) that allows the collection of cells based primarily on size. This system offers obvious advantages including the automated removal of platelets and red blood cells, collection of a large number of enriched cell populations for autologous treatment including monocytes for generation of DCs, and lymphocytes for activation of T cells and LCM. Using a program developed for monocyte collection; we were able to collect upstream fractionated products containing predominantly lymphocytes. Designated as Fractions 2 and 3, these cells were cryopreserved for LCM preparation and testing. Cell profiles of each fraction of each donor were generated by flow cytometry. Cells were activated with either anti-CD3 antibody+ionomycin or anti-CD3/CD28 beads. The media was tested for cytokine composition and its capacity to ‘mature’ dendritic cells (DCs) and augment T cell responses. 
         [0071]    Because this study involved the injection to humans of activated cell products, prior to any laboratory studies, the acceptability of culture materials was first determined by enquiry with FDA. It was recommended that GMP-produced serum-free media filed in previous IND&#39;s be used; and all media ‘components’ (including cytokines) be well-defined. 
       Data: Characterization of Apheresis Products Pre- and Post-Elutriation. 
       [0072]    The cell number in healthy donor leukapheresis products and lymphocyte recoveries is shown in Table 6. 
         [0000]    
       
         
               
               
             
               
               
             
           
               
                   
                 TABLE 6 
               
               
                   
                   
               
               
                   
                 mean ± SD* 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 Pre-elutriation 
                   
               
               
                 Total MNC in product (×10 9 ) 
                 7.2 ± 3   
               
               
                 Total RBC in product (×10 10 ) 
                 4.3 ± 1.1 
               
               
                 Total lymphocytes in product (×10 9 ) 
                 5.6 ± 2.2 
               
               
                 Total monocytes in product (×10 9 ) 
                  1.2 ± 0.38 
               
               
                 HCT (%) 
                 2.2 ± 0.5 
               
               
                 Total PLT in product (×10 11 ) 
                 2.7 ± 0.9 
               
               
                 Percentage of lymphocytes in product 
                 79.3 ± 2.9  
               
               
                 Post-elutriation 
               
               
                 Cell recovery in lymphocyte-rich fraction a   
                 Fraction 2: ~63%; Fraction 3: 
               
               
                   
                 ~42% 
               
               
                 Lymphocyte purity b   
                 81-86 ± 3%  
               
               
                   
               
               
                 *n = 9; 
               
               
                   a Percentage of cells recovered in lymphocyte-rich fractions 2 and 3 with respect to cell counts in starting material (manual count) 
               
               
                   b Percentage of lymphocytes in lymphocyte-rich fraction determined by CD3+ labling 
               
             
          
         
       
     
       Phenotype of Fraction 2 and 3 Cells 
       [0073]    To verify that the majority of cells in fractions 2 and 3 were lymphocytes, fresh and cryopreserved fractionated cells were phenotyped by labeling with fluorochrome-conjugated monoclonal antibodies against leukocyte cell surface markers. Profiles of cryopreserved cells are shown in Table 7 as in practice stored cells will be used to generate the batches of clinical product. 
         [0000]                                                                                              TABLE 7                   Phenotype of Thawed elutriated fractions                Fraction 2*       Fraction 3**                        AVE   SD   AVE   SD                            Viability   87   7   88   15           CD45 +     97   2   97   3           CD3 +     86   3   81   3           CD4 +     41   5   48   10           CD8 +     29   6   21.8   7           CD4 + DR +     8   2   10   2           CD4 + CD25 +     5   0.5   6   1           CD25 +     7   1   9   3           CD3 + CD56 +     16   6   19   9           CD3 − CD56 +     7   5   8   5           CD56 +     23   10   24   16           CD19 +     3   1   4   2                       FACScan analysis,           *n = 87 (storage time 9-547 days);           **n = 45 (storage time = 9-399 days)            
Cytokine Composition of LCM Derived from Fractions 2 and 3
 
         [0074]    Culture conditions based on historical data in flasks and plates (Table 8) were tested with fractions 2 and 3 to select the ‘best’ conditions for further clinical process development. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Culture conditions tested (37° C., humidified, 5% CO 2 ) 
               
               
                 Table 6: 
               
               
                 Culture 
               
             
          
           
               
                   
                 Fraction 2 
                   
                 Fraction 3 
                   
               
             
          
           
               
                   
                 incubation time 
                   
               
             
          
           
               
                   
                 48 h 
                 72 h 
                 48 h 
                 72 h 
               
               
                   
                   
               
             
          
           
               
                   
                 CD3-CD28 beads 
                 X 
                 X 
                 X 
                 X 
               
               
                   
                 No beads 
                 X 
                 — 
                 X 
                 — 
               
               
                   
                 CD3-CD28 beads + 
                 — 
                 X 
                 — 
                 X 
               
               
                   
                 IL2 
               
               
                   
                 No beads + IL2 
                 — 
                 X 
                 — 
                 — 
               
               
                   
                 Anti-CD3 coating 
                 — 
                 X 
                 — 
                 — 
               
               
                   
                   
               
             
          
         
       
     
         [0075]    LCM supernatants were collected by centrifugation and stored at 4° C. until assayed. Cytokines were assessed within a single assay for direct comparison using flow cytometry-based technology (BioRad, BD Biosciences) (see Table 7). 
         [0076]    Comment: Data suggest that anti-CD3/CD28 stimulation provide a ‘manufacturing’ system which is easy to execute and yields fairly consistent cytokine patterns. The use of beads compared to flask/bag surface coating with antibody may be preferred as beads can be systematically measured, their use subject to less operator error, and ‘generally’ similar cytokine patterns are observed. 
         [0077]    Tables 9A and 9B show survey assay on cultures in traditional polystyrene plates or flasks. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 9A 
               
             
             
               
                   
               
               
                 Activation of FRACTION 2 cells 
               
               
                 Cytokines Produced from Cells Stimulated under Different Conditions (27-Bioplex) 
               
               
                 (pg/ml) 
               
             
          
           
               
                   
                   
                   
                   
                   
                 No 
                 Anti-CD3 
               
               
                   
                 CD3-CD28 
                 No 
                 CD3-CD28 
                 CD3-CD28 
                 beads + 
                 Ab 
               
               
                   
                 beads 
                 beads 
                 beads 
                 beads + IL2 
                 IL2 
                 coating 
               
             
          
           
               
                   
                 48 h stimulation (n = 2) 
                 72 h stimulation (n = 2) 
               
               
                   
                   
               
             
          
           
               
                 Eotaxin 
                 145 ± 22  
                  7 ± 10 
                 138 ± 60  
                 487 ± 260 
                 18 ± 24 
                 147 ± 94  
               
               
                 FGF 
                 39 ± 2  
                 0 ± 0 
                 76 ± 43 
                 162 ± 132 
                 0 ± 0 
                 23 ± 32 
               
               
                 G-CSF 
                 16.7 ± 3   
                 0 ± 0 
                 27 ± 11 
                 89 ± 51 
                 1 ± 1 
                 26 ± 16 
               
               
                 GM-CSF 
                 1124 ± 140  
                 20 ± 23 
                 3551 ± 2115 
                 5944 ± 2657 
                 60 ± 52 
                 1373 ± 961  
               
               
                 IFNγ 
                 42512 ± 13867 
                 0 ± 0 
                 50335 ± 56228 
                 42997 ± 24322 
                 227 ± 60  
                 55600 ± 49000 
               
               
                 IP10 
                 86034 ± 39358 
                 254 ± 360 
                 170543 ± 400405 
                 13066 ± 2666  
                 694 ± 327 
                 1183 ± 1006 
               
               
                 IL1β 
                 24 ± 2  
                 0.6 ± 0.1 
                 21 ± 15 
                 130 ± 81  
                 14 ± 24 
                 34 ± 10 
               
               
                 IL1ra 
                 164 ± 109 
                  92 ± 118 
                 232 ± 195 
                 320 ± 143 
                 140 ± 76.  
                 45.5 ± 10   
               
               
                 IL2 
                 7944 ± 1549 
                 0 ± 0 
                 6750 ± 4760 
                 24724 ± 9572  
                 10867 ± 1961  
                 5836 ± 3686 
               
               
                 IL4 
                 155 ± 39  
                 0 ± 0 
                 292 ± 320 
                 251 ± 123 
                  8.2 ± 11.3 
                 61 ± 47 
               
               
                 IL5 
                 236 ± 225 
                 0 ± 0 
                 423 ± 403 
                 493 ± 188 
                 1.5 ± 0.6 
                 183 ± 103 
               
               
                 IL6 
                 1646 ± 526  
                 12.9 ± 18.2 
                 2797 ± 3566 
                 2032 ± 670  
                 18 ± 21 
                 226 ± 155 
               
               
                 IL7 
                 0.8 ± 0.8 
                 0.0 ± 0   
                 4.20 ± 2.8  
                 5.00 ± 4.8  
                 0 ± 0 
                 0.50 ± 0.7  
               
               
                 IL8 
                 2205 ± 1700 
                 283 ± 350 
                 4892 ± 8372 
                 2197 ± 561  
                 815 ± 37  
                 1408 ± 1092 
               
               
                 IL9 
                 1590 ± 1601 
                 15 ± 18 
                 1918 ± 2235 
                 2523 ± 1007 
                 0 ± 0 
                 118 ± 167 
               
               
                 IL10 
                 7298 ± 2236 
                 0 ± 0 
                 2122 ± 2349 
                 2289 ± 629  
                 0 ± 0 
                 1385 ± 1033 
               
               
                 IL12 
                 3 ± 1 
                 0 ± 0 
                 16 ± 14 
                 0 ± 0 
                 0 ± 0 
                 0 ± 0 
               
               
                 IL13 
                 874 ± 534 
                 0 ± 0 
                 1479 ± 772  
                 1574 ± 370  
                 3 ± 3 
                 530 ± 160 
               
               
                 IL15 
                   3 ± 0.4 
                   2 ± 0.1 
                 3 ± 2 
                 10 ± 21 
                 1.5 ± 3   
                 0 ± 0 
               
               
                 IL17 
                 481 ± 70  
                 0 ± 0 
                 1087 ± 1860 
                 386 ± 293 
                 0 ± 0 
                 25 ± 35 
               
               
                 MCP1 
                 36 ± 36 
                 26 ± 37 
                 69 ± 74 
                 70 ± 80 
                 0 ± 0 
                 6 ± 8 
               
               
                 MIP1α 
                 21945 ± 0   
                 15 ± 15 
                 17781 ± 6324  
                 19153 ± 0   
                 413 ± 98  
                 6146 ± 6832 
               
               
                 MIP1β 
                 13684 ± 13363 
                 812 ± 646 
                 18277 ± 7656  
                 48070 ± 49740 
                 7079 ± 2396 
                 17893 ± 7504  
               
               
                 PDGFbb 
                 267 ± 41  
                 0 ± 0 
                 189 ± 169 
                 779 ± 586 
                 28 ± 33 
                 122 ± 172 
               
               
                 RANTES 
                 15101 ± 182  
                 322 ± 82  
                 34432 ± 29650 
                  7969 ± 11890 
                 781 ± 33  
                 1338 ± 709  
               
               
                 TNFα 
                 3457 ± 1540 
                 0 ± 0 
                 7540 ± 8717 
                 7142 ± 2016 
                 0 ± 0 
                 2631 ± 1052 
               
               
                 VEGF 
                 136 ± 81  
                 0 ± 0 
                 242 ± 173 
                 582 ± 478 
                 22 ± 0  
                 117 ± 132 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 9B 
               
             
             
               
                   
               
               
                 Activation of FRACTION 3 cells 
               
               
                 Cytokines Produced under Different Culture Conditions 
               
               
                 (pg/ml) 
               
             
          
           
               
                   
                   
                   
                   
                 CD3-CD28 
               
               
                   
                 CD3-CD28 
                 No 
                 CD3-CD28 
                 beads + 
               
               
                   
                 beads 
                 beads 
                 beads 
                 IL2 
               
             
          
           
               
                   
                 48 h stimulation 
                 72 h stimulation 
               
               
                   
                 (n = 2) 
                 (n = 2) 
               
               
                   
                   
               
             
          
           
               
                 Eotaxin 
                 173 ± 21  
                 12 ± 17 
                 225 ± 9  
                 0 ± 0 
               
               
                 FGF 
                 38 ± 1  
                 3 ± 4 
                 56 ± 15 
                 0 ± 0 
               
               
                 G-CSF 
                   22 ± 0.52 
                 0 ± 0 
                 33 ± 5  
                 0 ± 0 
               
               
                 GM-CSF 
                 2279 ± 746  
                 22 ± 1  
                 5577 ± 1278 
                 4 ± 6 
               
               
                 IFNγ 
                 223663 ± 20006  
                 14 ± 21 
                 327345 ± 41111  
                 0 ± 0 
               
               
                 IP10 
                 21221 ± 5076  
                 884 ± 459 
                 31794 ± 684  
                 1680 ± 1073 
               
               
                 IL1β 
                 45 ± 3  
                   1 ± 0.97 
                 67 ± 24 
                   1 ± 0.94 
               
               
                 IL1ra 
                 1969 ± 180  
                 1397 ± 172  
                 2715 ± 1086 
                 1545 ± 495  
               
               
                 IL2 
                 5518 ± 1387 
                 0 ± 0 
                 2655 ± 1569 
                 0 ± 0 
               
               
                 IL4 
                 267 ± 124 
                 0 ± 0 
                 252 ± 50  
                 0 ± 0 
               
               
                 IL5 
                 411 ± 322 
                 0 ± 0 
                 542 ± 273 
                 0 ± 0 
               
               
                 IL6 
                 1840 ± 394  
                 40 ± 50 
                 2378 ± 75  
                 35 ± 45 
               
               
                 IL7 
                 0.9 ± 0.9 
                 0.05 ± 0.07 
                   2 ± 0.08 
                 0 ± 0 
               
               
                 IL8 
                 26859 ± 13919 
                 9081 ± 883  
                 36702 ± 0   
                 17269 ± 11218 
               
               
                 IL9 
                 3437 ± 2136 
                 43 ± 3  
                 9363 ± 4575 
                 43 ± 25 
               
               
                 IL10 
                 8554 ± 973  
                 0 ± 0 
                 10940 ± 2529  
                 0 ± 0 
               
               
                 IL12 
                 27 ± 11 
                 0 ± 0 
                 23 ± 5  
                 0 ± 0 
               
               
                 IL13 
                 977 ± 574 
                 0 ± 0 
                 1938 ± 697  
                 0 ± 0 
               
               
                 IL15 
                   4 ± 0.8 
                   2 ± 0.2 
                   6 ± 0.9 
                   2 ± 0.01 
               
               
                 IL17 
                  1928 ± 200.8 
                 0 ± 0 
                 2860 ± 1255 
                 0 ± 0 
               
               
                 MCP1 
                  689 ± 94.6 
                 243 ± 87  
                 936 ± 308 
                 222 ± 177 
               
               
                 MIP1α 
                 12242 ± 13722 
                 48 ± 65 
                 21945 ± 0   
                 3 ± 4 
               
               
                 MIP1β 
                 13922 ± 16298 
                 822 ± 583 
                 15449 ± 14138 
                 501 ± 443 
               
               
                 PDGFbb 
                  227 ± 14.6 
                 0 ± 0 
                 581 ± 96  
                 0 ± 0 
               
               
                 RANTES 
                 8405 ± 944  
                 111 ± 22  
                 34085 ± 14533 
                  93 ± 2.4 
               
               
                 TNFα 
                 7015 ± 770  
                 0 ± 0 
                 18531 ± 6916  
                 0 ± 0 
               
               
                 VEGF 
                  176 ± 90.2 
                 0 ± 0 
                 277 ± 40  
                 0 ± 0 
               
               
                   
               
             
          
         
       
     
       Development of LCM Closed ‘Manufacturing’ Process 
       [0078]    There appeared to be no large differences in cellular composition between fractions 2 and 3; however, cell recovery was highest in fraction 2. Fraction 2 cells were selected for further analysis and development in a closed system. A 3-day culture period using anti-CD3-CD28 bead stimulation was selected. Closed FEP VueLife® bags (2 PF-0025, American Fluoroseal Corporation, Gaithersburg, Md.) were used (in part based on our previous DC culture IND work) as they: reduce risk of contamination while allowing easy access to cells; are transparent so cells can be easily monitored; are non-reactive, i.e., no plasticizers, leachables or extractables to affect cell culture; are manufactured to meet FDA approval; allow O 2 , CO 2 , and N 2  gas transfer. FEP is impermeable to water and allows incubation without water loss; and therefore, there is no need to use humidified chambers which often is a source of contamination; 
         [0079]    Five different aphereses from different donors were used to make LCM in a bag system. Cells were cultured in serum-free, phenol-red free XVIVO10 (BioWhittaker) media using syringe loading at 1×10 6  cells/ml in 15 ml media plus CD3-CD28 beads (Dynabeads, Dynal) at 3 beads to 1 cell. Bags were placed atop wire racks to ensure proper gas exchange and even cell distribution then incubated for 3 days at 37° C. 
         [0080]    Following culture, cells and LCM from individual units were collected by removing beads with a Dynal magnet followed by centrifugation (10 min at 400×g). Cells were phenotyped (Table 10) and collected supernatants were assayed for cytokines using 27 Bioplex flow-based analyses (Table 11 A, B). 
       Characterization of Activation Products Produced in Closed System 
       [0081]      
         [0000]                                                                              TABLE 10                   Phenotype of elutriated cells following activation*                CD3-CD28   Non-activated           activated   (i.e., no beads but cells in culture)                AVE   SD   AVE   SD                        Viability   80   4   91   6       CD3 +     76   7   73   6       CD4 +     42   6   36   9       CD8 +     39   18   32   9       HLA-DR +     20   16   14   5       CD25 +     71   9   1   1       CD19 +     7   7   10   4                    
Cytokines Released from Activated Cells
 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 11A 
               
             
             
               
                   
               
               
                 Cytokines found in supernatants from lymphocyte cultures 
               
               
                 in ‘bag’ system* 
               
               
                 CD3-CD28 bead activation 
               
             
          
           
               
                   
                 Apheresis unit (pg/ml) 
                 N = 5 
               
             
          
           
               
                   
                 APH062805 
                 APH082305 
                 APH112905 
                 APH011006 
                 APH112706 
                 Ave ± SD 
               
               
                   
                   
               
             
          
           
               
                 Eotaxin 
                 78 
                 86 
                 132 
                 160 
                 117 
                 115 ± 34  
               
               
                 FGF 
                 79 
                 95 
                 117 
                 134 
                 115 
                 108 ± 21  
               
               
                 G-CSF 
                 18 
                 21 
                 31 
                 41 
                 31 
                 29 ± 9  
               
               
                 GM-CSF 
                 3409 
                 3619 
                 3595 
                 8189 
                 3446 
                 4452 ± 2091 
               
               
                 IFNγ 
                 5240 
                 7474 
                 27957 
                 188081 
                 54453 
                 56641 ± 76098 
               
               
                 IP10 
                 6158 
                 9415 
                 54621 
                 87794 
                 1234754 
                 278548 ± 535605 
               
               
                 IL1β 
                 7 
                 6 
                 9 
                 18 
                 19 
                 12 ± 6  
               
               
                 IL1ra 
                 115 
                 117 
                 160 
                 659 
                 396 
                 289 ± 237 
               
               
                 IL2 
                 3161 
                 4510 
                 14896 
                 14896 
                 6676 
                 8828 ± 5680 
               
               
                 IL4 
                 241 
                 247 
                 351 
                 1108 
                 254 
                 440 ± 376 
               
               
                 IL5 
                 425 
                 358 
                 328 
                 1431 
                 247 
                 558 ± 492 
               
               
                 IL6 
                 977 
                 955 
                 3207 
                 11942 
                 2852 
                 3986 ± 4567 
               
               
                 IL7 
                 5 
                 6 
                 8 
                 7 
                 6 
                 6 ± 1 
               
               
                 IL8 
                 973 
                 759 
                 1216 
                 7404 
                 26397 
                  7350 ± 11006 
               
               
                 IL9 
                 531 
                 132 
                 2040 
                 6364 
                 1397 
                 2093 ± 2501 
               
               
                 IL10 
                 469 
                 302 
                 591 
                 2625 
                 1424 
                 1082 ± 965  
               
               
                 IL12 
                 29 
                 46 
                 12 
                 26 
                 20 
                 27 ± 12 
               
               
                 IL13 
                 1804 
                 2718 
                 737 
                 2309 
                 1148 
                 1743 ± 813  
               
               
                 IL15 
                 3 
                 3 
                 5 
                 7 
                 5 
                 5 ± 2 
               
               
                 IL17 
                 102 
                 118 
                 1855 
                 5808 
                 805 
                 1738 ± 2385 
               
               
                 MCP1 
                 37 
                 24 
                 44 
                 138 
                 242 
                 97 ± 93 
               
               
                 MIP1α 
                 19153 
                 19153 
                 19153 
                 19153 
                 19153 
                 19153 ± 0   
               
               
                 MIP1β 
                 23200 
                 23200 
                 23200 
                 23200 
                 9465 
                 20453 ± 6143  
               
               
                 PDGFbb 
                 57 
                 65 
                 70 
                 127 
                 165 
                 97 ± 47 
               
               
                 RANTES 
                 76360 
                 73239 
                 15223 
                 64659 
                 37225 
                 53341 ± 26299 
               
               
                 TNFα 
                 2026 
                 1801 
                 7344 
                 29476 
                 9507 
                 10031 ± 11373 
               
               
                 VEGF 
                 266 
                 577 
                 97 
                 133 
                 108 
                 236 ± 202 
               
               
                   
               
               
                 *72 h incubation 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 11B 
               
             
             
               
                   
               
               
                 Cytokines found in supernatants from lymphocyte cultures 
               
               
                 in ‘bag’ system* 
               
               
                 No Beads 
               
             
          
           
               
                   
                 Apheresis unit (pg/ml) 
                 N = 5 
               
             
          
           
               
                   
                 APH062805 
                 APH082305 
                 APH112905 
                 APH011006 
                 APH112706 
                 Ave ± SD 
               
               
                   
                   
               
             
          
           
               
                 Eotaxin 
                 78 
                 86 
                 132 
                 160 
                 117 
                 115 ± 34  
               
               
                 FGF 
                 79 
                 95 
                 117 
                 134 
                 115 
                 108 ± 21  
               
               
                 G-CSF 
                 18 
                 21 
                 31 
                 41 
                 31 
                 29 ± 9  
               
               
                 GM-CSF 
                 3409 
                 3619 
                 3595 
                 8189 
                 3446 
                 4452 ± 2091 
               
               
                 IFNγ 
                 5240 
                 7474 
                 27957 
                 188081 
                 54453 
                 56641 ± 76098 
               
               
                 IP10 
                 6158 
                 9415 
                 54621 
                 87794 
                 1234754 
                 278548 ± 535605 
               
               
                 IL1β 
                 7 
                 6 
                 9 
                 18 
                 19 
                 12 ± 6  
               
               
                 IL1ra 
                 115 
                 117 
                 160 
                 659 
                 396 
                 289 ± 237 
               
               
                 IL2 
                 3161 
                 4510 
                 14896 
                 14896 
                 6676 
                 8828 ± 5680 
               
               
                 IL4 
                 241 
                 247 
                 351 
                 1108 
                 254 
                 440 ± 376 
               
               
                 IL5 
                 425 
                 358 
                 328 
                 1431 
                 247 
                 558 ± 492 
               
               
                 IL6 
                 977 
                 955 
                 3207 
                 11942 
                 2852 
                 3986 ± 4567 
               
               
                 IL7 
                 5 
                 6 
                 8 
                 7 
                 6 
                 6 ± 1 
               
               
                 IL8 
                 973 
                 759 
                 1216 
                 7404 
                 26397 
                  7350 ± 11006 
               
               
                 IL9 
                 531 
                 132 
                 2040 
                 6364 
                 1397 
                 2093 ± 2501 
               
               
                 IL10 
                 469 
                 302 
                 591 
                 2625 
                 1424 
                 1082 ± 965  
               
               
                 IL12 
                 29 
                 46 
                 12 
                 26 
                 20 
                 27 ± 12 
               
               
                 IL13 
                 1804 
                 2718 
                 737 
                 2309 
                 1148 
                 1743 ± 813  
               
               
                 IL15 
                 3 
                 3 
                 5 
                 7 
                 5 
                 5 ± 2 
               
               
                 IL17 
                 102 
                 118 
                 1855 
                 5808 
                 805 
                 1738 ± 2385 
               
               
                 MCP1 
                 37 
                 24 
                 44 
                 138 
                 242 
                 97 ± 93 
               
               
                 MIP1α 
                 19153 
                 19153 
                 19153 
                 19153 
                 19153 
                 19153 ± 0   
               
               
                 MIP1β 
                 23200 
                 23200 
                 23200 
                 23200 
                 9465 
                 20453 ± 6143  
               
               
                 PDGFbb 
                 57 
                 65 
                 70 
                 127 
                 165 
                 97 ± 47 
               
               
                 RANTES 
                 76360 
                 73239 
                 15223 
                 64659 
                 37225 
                 53341 ± 26299 
               
               
                 TNFα 
                 2026 
                 1801 
                 7344 
                 29476 
                 9507 
                 10031 ± 11373 
               
               
                 VEGF 
                 266 
                 577 
                 97 
                 133 
                 108 
                 236 ± 202 
               
               
                   
               
               
                 *72 h incubation 
               
             
          
         
       
     
         [0082]    Comment: Particularly IFNγ, IP10, IL6, IL9, IL10, TNFα and the chemoattractants appear to be produced at the highest concentrations following stimulation with some variability between units. Though fraction 2 is relatively pure, variation could be possibly due to cell types (e.g., NK cells) and their proportion in each fraction. 
         [0083]    A summary of the function of these cytokines for reference is given in Table 12. Awareness of the cytokine concentrations prior to experiments may be used to calculate actual cytokine amount in dilutions, enable matched comparisons between donors, and establish a dosing level for LCM application. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 12 
               
             
             
               
                   
               
               
                 Selected Cytokines and Their Activities* 
               
             
          
           
               
                 Cytokine 
                 Producing Cell 
                 Function 
               
               
                   
               
               
                 GM-CSF 
                 Th cells 
                 growth and differentiation of monocytes 
               
               
                   
                   
                 and DC 
               
               
                 IFNγ 
                 T cells, NK cells 
                 antiviral, anti-tumor activity, 
               
               
                   
                   
                 immunoregulation; activates APCs, 
               
               
                   
                   
                 promotes Th1 
               
               
                 IP10 (IFNγ inducible 
                 activated T cells 
                 mediates Ca+ mobilization, chemotaxis 
               
               
                 protein) 
               
               
                 IL-2 
                 Th1 cells 
                 growth, proliferation, activation 
               
               
                 IL-4 
                 Th2 cells 
                 proliferation and differentiation 
               
               
                   
                   
                 MHC Class II 
               
               
                   
                   
                 proliferation 
               
               
                 IL-5 
                 Th2 cells 
                 proliferation and differentiation 
               
               
                 IL-6 
                 monocytes, 
                 differentiation into plasma cells 
               
               
                   
                 macrophages, 
                 antibody secretion 
               
               
                   
                 Th2 cells 
                 differentiation 
               
               
                 IL-8 
                 macrophages, 
                 chemotaxis 
               
               
                   
                 endothelial cells 
               
               
                 IL-10 
                 Th2 cells 
                 cytokine production 
               
               
                   
                   
                 activation 
               
               
                 IL-12 
                 macrophages, B 
                 differentiation into CTL (with IL-2) 
               
               
                   
                 cells 
                 activation 
               
               
                   
                   
                 MHC expression 
               
               
                   
                   
                 proliferation 
               
               
                   
                   
                 pathogen elimination 
               
               
                 MIP-1α 
                 macrophages, 
                 chemotaxis 
               
               
                   
                 activated NK, 
               
               
                   
                 CD8+T, CD4+T 
               
               
                 MIP-1β 
                 activated NK, 
                 chemotaxis 
               
               
                   
                 CD8+T, CD4+T 
               
               
                 RANTES 
                 activated NK, 
                 chemotaxis 
               
               
                 (regulated on activation 
                 CD8+T, CD4+T 
               
               
                 normal T cell expressed 
               
               
                 and secreted) 
               
               
                 TNFα 
                 macrophages, 
                 CAM and cytokine expression, cellular 
               
               
                   
                 mast cells, NK 
                 proliferation, differentiation, 
               
               
                   
                 cells 
                 inflammation, 
               
               
                   
                   
                 cell death 
               
               
                   
               
               
                 *derived from sources in the literature 
               
             
          
         
       
     
       The Effects of LCM Produced in Closed System on Autologous Monocytes/DCs 
       [0084]    To assess their properties LCM, or activated T (AT) cells, were added to autologous DCs (for 2-3 days or overnight, respectively). The autologous setting was first tested as this would be the likely protocol ‘type’ for immunotherapeutic approval. Treated cells were examined for: (a) viability following culture measured by trypan blue exclusion ( FIG. 14 ); (b) changes in surface marker expression (e.g., CD14, CD40, CD80, CD83, CD86) measured by flow cytometry (Tables 13, 14); (c) effects on T cell responses measured in IFNγ ELISPOT following exposure to CMV and tumor lysates before and after IL7+IL15 expansion ( FIGS. 15 ,  16 ). 
         [0085]    Cell surface marker expression on DCs following exposure to autologous LCM is shown in Table 13. 
         [0000]    
       
         
               
               
             
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
             
           
               
                   
                 TABLE 13 
               
             
             
               
                   
                   
               
               
                   
                 % expression 
               
             
          
           
               
                   
                 CD14 
                 CD40 
                 CD80 
                 CD86 
                 CD83 
               
               
                   
               
             
          
           
               
                 pre-LCM exposure 
                 Day 0 
               
               
                   
               
             
          
           
               
                 DC (thawed, no additional 
                 20 
                 77 
                 74 
                 86 
                 2 
               
               
                 cytokines added) 
               
               
                   
               
             
          
           
               
                 post-LCM exposure 
                 Day 2→Day 3 
               
               
                   
               
             
          
           
               
                 DC alone 
                 20→7 
                 55→31 
                 58→22 
                 73→51 
                  4→5 
               
               
                 DC + LCM (50%) 
                  9→23 
                 47→54 
                 42→30 
                 54→70 
                 11→16 
               
               
                 DC + LCM (25%) 
                 21→22 
                 64→60 
                 63→39 
                 67→70 
                 12→13 
               
               
                 DC + LCM (10%) 
                 22→ND 
                 52→ND 
                 49→ND 
                 49→ND 
                 13→ND 
               
               
                 DC + non-activated (i.e., no bead) 
                  4→40 
                 42→44 
                 35→36 
                 19→51 
                  6→5 
               
               
                 medium 
               
               
                 DC + maturation cocktail (IL1β, 
                 43→56 
                 64→54 
                 56→52 
                 66→63 
                 27→15 
               
               
                 IL6, TNFα, PGE2) 
               
               
                   
               
               
                 n = 3 units; 
               
               
                 ND = not determined 
               
             
          
         
       
     
         [0086]    Table 14 shows cell surface marker expression on DCs* following overnight exposure to autologous activated T cells. 
         [0000]    
       
         
               
               
             
               
               
               
               
               
               
               
               
             
               
             
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 14 
               
               
                   
               
             
             
               
                 Culture 
                 Marker (% expression) 
               
             
          
           
               
                 Condition 
                 Donor 
                 Viability 
                 CD14 
                 CD40 
                 CD80 
                 CD83 
                 CD86 
               
               
                   
               
               
                 DCs 
                 APH112706 
                 93 
                 38 
                 79 
                 86 
                 1 
                 98 
               
               
                 pre- 
                 APH082305 
                 98 
                 2 
                 70 
                 80 
                 0 
                 42 
               
               
                 coculture 
               
               
                 DCs + 
                 APH112706 
                 57 
                 31 
                 92 
                 92 
                 78 
                 65 
               
               
                 non- 
               
               
                 activated 
               
               
                 T cells 
                 APH082305 
                 84 
                 12 
                 98 
                 94 
                 95 
                 94 
               
               
                 DCs + 
                 APH112706 
                 63 
                 5 
                 27 
                 12 
                 4 
                 43 
               
               
                 activated 
                 APH082305 
                 85 
                 0 
                 83 
                 94 
                 77 
                 94 
               
               
                 T cells 
               
               
                   
               
             
          
           
               
                 *Gating set on DCs 
               
               
                 Refer to cytokine profiles for activation of T cells from these 
               
               
                 units (Table 9). High 
               
               
                 IFNγ production was measured following stimulation of APH112706. 
               
               
                 NOTE: Cytokine release assessed following coculture of DC 
               
               
                 and non-activated T cells or AT cells** showed the following 
               
             
          
           
               
                   
                 Culture Condition 
               
             
          
           
               
                   
                 DC 
                   
                 DC + non- 
                   
               
               
                   
                 alone 
                 DC + AT cells 
                 activated T cells 
                 T cells alone 
               
               
                   
               
               
                 Eotaxin 
                 36 
                 63 
                 0 
                 1 
               
               
                 FGF 
                 38 
                 123 
                 41 
                 10 
               
               
                 G-CSF 
                 0 
                 19 
                 0 
                 0 
               
               
                 GM-CSF 
                 608 
                 536 
                 250 
                 0 
               
               
                 IFNγ 
                 23 
                 241 
                 16 
                 0 
               
               
                 IP10 
                 70 
                 54621 
                 5256 
                 71 
               
               
                 IL1β 
                 1 
                 18 
                 3 
                 1 
               
               
                 IL1ra 
                 386 
                 16394 
                 730 
                 0 
               
               
                 IL2 
                 0 
                 12 
                 1 
                 0 
               
               
                 IL4 
                 56 
                 45 
                 31 
                 0 
               
               
                 IL5 
                 1 
                 10 
                 1 
                 1 
               
               
                 IL6 
                 25 
                 421 
                 152 
                 45 
               
               
                 IL7 
                 4 
                 15 
                 0 
                 0 
               
               
                 IL8 
                 248 
                 18225 
                 2542 
                 137 
               
               
                 IL9 
                 5 
                 58 
                 6 
                 0 
               
               
                 IL10 
                 2 
                 11 
                 2 
                 0 
               
               
                 IL12 
                 5 
                 4 
                 0 
                 0 
               
               
                 IL13 
                 0 
                 329 
                 0 
                 0 
               
               
                 IL15 
                 1 
                 2 
                 1 
                 0 
               
               
                 IL17 
                 20 
                 82 
                 31 
                 23 
               
               
                 MCP1 
                 8 
                 288 
                 44 
                 0 
               
               
                 MIP1α 
                 4 
                 39 
                 9 
                 16 
               
               
                 MIP1β 
                 69 
                 821 
                 507 
                 261 
               
               
                 PDGFbb 
                 29 
                 849 
                 122 
                 34 
               
               
                 RANTES 
                 9 
                 1125 
                 319 
                 673 
               
               
                 TNFα 
                 16 
                 243 
                 23 
                 14 
               
               
                 VEGF 
                 85 
                 87 
                 20 
                 13 
               
               
                   
               
               
                 **APH082305; sups from other cocultures have been stored and are available to assay 
               
             
          
         
       
     
         [0087]    Comment: DCs incubated with LCM (for 2 or 3 days) demonstrate some upregulation in the maturation marker CD83, as well as changes in costimulatory molecule expression. When autologous activated or non-activated T cells are added (overnight) to DCs in another set of wells, as expected, upregulation of costimulatory markers is observed in both cell populations-except with AT cells from donor APH112706, which showed a negative change in costimulatory molecules. Though difficult to make sweeping statements with such low sample sizes, these changes could be attributable to a number of factors including level of stimulation, receptor activation on T cells, cytokines and or viable status. Viability may not be the issue here as non-activated T cell-DC samples demonstrated equal viability with maintained high DC marker expression. The ‘stimulatability’ of T cells from donor APH112706 shows that CD3-CD28-activation can produce high levels of IFNγ (see Table 9a) which is APC activating and our observation could be due to high activation and ‘spent’ status which occurred prior to our measurement point. 
         [0088]    Cytokines released from DC-T cell cocultures underscore the importance of activation levels (IFNγ and chemotactic cytokines). With the addition of antigen and expanded observation points, these measures may prove useful to further characterize and screen individual cells for activation status and potential clinical efficacy, particularly if indicative of differences between induction of immunity or tolerance. 
       Recall and Primary T Cell Responses-ELISPOT 
       [0089]    Description
       1. Supernatants and antigen were added to monocytes and DCs (designated as APC):
           i. Source of DCs: cryopreserved/cultured from monocytes (3 days, serum-free DC medium (CellGenix, Germany) GM-CSF (800 IU/ml)+IL4 (500 IU/ml) (CellGenix);   ii. Source of monocytes: cryopreserved elutriated rotor-off fraction;   iii. Cell supernatants tested: 50%, 25% and 10% of original strength from CD3-CD28 bead-activated or non-activated cells;   
           2. Cultures were incubated for 2 days at 37° C. and washed free or LCM or non-activated supernatants then placed in IFNγ ELISPOT assay (see schematic below):
           a. For recall responses:
               i. cells were counted; autologous lymphocytes (fraction 2) were added at 10 lymphocytes: 1 APC (total 1.5×10 5  cells/well) then   ii. plated on IFNγ antibody-coated ELISPOT plates, incubated for 3 days at 37° C. then plates developed and enumerated   
               b. For primary responses:
               i. Washed cells were cultured in IL7+IL15 (5 ng/ml each) for 7 days, then washed and plated on coated ELISPOT plates and developed as above.   
               
               
 
         [0100]    Table 15 is a schematic of the assay. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 15 
               
             
             
               
                   
               
               
                 Schematic of Assay 
               
             
          
           
               
                   
                   
                   
                   
                 Culture Conditions 
               
               
                 Day 0 
                 Day 2 
                 Day 8 
                 Day 9 
                 DCs and monocytes 
               
               
                   
               
               
                 Culture DCs 
                 Harvest APCs, 
                 Restimulate 
                 Harvest 
                 alone 
               
               
                 and 
                 wash and add T 
                 cells with 
                 cells and 
                 +GM-IL4 
               
               
                 monocytes: 
                 cells: 
                 antigen or 
                 assay in 
                 +25% LCM 
               
               
                 ±LCM 
                 RECALL: 
                 Add 
                 ELISPOT 
                 +10% LCM 
               
               
                 ±tumor 
                 Assay 
                 antigen to 
                 IFNγ 
                 +no bead sup 
               
               
                 (myeloma 
                 portion 
                 antigen- 
                   
                 +tumor lysate (d0 + d8 
               
               
                 8226)(3 × 10 4   
                 cells in 
                 naive cells 
                   
                 or d8) 
               
               
                 cell 
                 ELISPOT 
                 overnight 
                   
                 +GM-IL4 + tumor 
               
               
                 equivalent 
                 PRIMARY: 
                   
                   
                 lysate (d0 + d8 or d8) 
               
               
                 lysate per 
                 Expand 
                   
                   
                 +25% LCM + tumor 
               
               
                 well or 
                 portion of 
                   
                   
                 lysate (d0 + d8 or d8) 
               
               
                 CMV 
                 cells in IL7 + 
                   
                   
                 +10% LCM + tumor 
               
               
                 lysate 
                 IL15 for 7 
                   
                   
                 lysate (d0 + d8 or d8) 
               
               
                 (0.01 mg/well) 
                 days 
                   
                   
                 +no bead sup + tumor 
               
               
                   
                   
                   
                   
                 lysate (d0 + d8 or d8) 
               
               
                   
               
             
          
         
       
     
       Results 
       [0101]      FIGS. 15  A, B provides recall responses and shows that LCM augments response to antigens (CMV, n=2). 
         [0102]      FIGS. 16  A, B shows tumor cell lysates (n=2), in which Fig. A is Aph062805 and Fig. B is Aph-11006. 
         [0103]    Comment: Cocultures of either DC preparation with LCM and tumor cells show enhanced T cell responses; however, the response is larger in cultures from donor APH011006 compared to donor APH062805. It is interesting to refer to the cytokine table (Table 9) and compare the differences in the degree of the capacity for IFNγ production following activation between the donors. Though different levels in the number of spots in this type of assay are expected, in vivo potential may be predictable by determining a stimulation index for a particular cytokine. Such an index would prove useful for screening potential positive activity; however, to determine if this is a real response, a larger sample evaluation to include appropriate controls will be necessary. Interestingly, the monocyte-antigen cocultures in donor APH011006 also show a larger response than those in the APH062805 donor ( FIG. 16B ) possibly due to the capacity for detection of IFNγ in this donor or activity of other cytokines such as TNFγ. Higher TNFγ levels were also present in the LCM of this donor which could ‘push’ the monocyte to a DC. Unfortunately, the phenotype of these cells was not determined due to limited amount of material. 
         [0104]    These data warrant future study to determine the cell (maturation) status and how the cytokine levels should be manipulated to control and potentially predict function. 
       Primary Responses: IL7-IL15 T Cell Expansion 
       [0105]      FIG. 17  shows responses of LCM-treated ‘naïve IL7-IL15-treated’ cells (i.e., cells first exposed to tumor on day 8) were enhanced compared to cells exposed to antigen on days 0 and 8. 
         [0106]    Comment: LCM added to DCs and monocytes enhanced tumor antigen presentation to antigen-naïve T cells cultured in IL7 and IL15 for 7 days prior to antigen stimulation. The higher response levels compared to short recall responses ( FIG. 15 ) could be due to the cytokines that help to maintain viability of T or APCs (cell viability 78-100%). When IL7 and IL15 antigen-treated expanded cultures were restimulated with antigen, that is, pulsed with antigen both on days 0 and 8, there was a response in LCM-treated APCs above non-treated; however, the responses were lower than that of APCs that had been treated with GM-IL4. The LCM data may indicate the presence of suppressive factors or optimal levels of cytokine were present-absent and should be adjusted. This data is reported from two different donors. Expanded studies would be valuable to better characterize the responding cells functionally and phenotypically. 
         [0107]    Though it may appear that using a few cytokines would be ‘easiest’ to generate a desired immune response, it may be that the mix of cytokines found in LCM will be the most potent; mimicking a true physiological response and demonstrating that cytokine interactions are essential in optimizing functional activity. 
       SUMMARY 
       [0108]    In this protocol, elutriated fractions 2 or 3 may be used for activation. The greatest number of lymphocytes were collected in fraction 2 (Table 6). There were fairly consistent results between the two fractions (Table 9); however, purity in fraction 3 may be an issue if cell levels in the starting units do not meet optimal elutriation criteria. That is, if the starting total cell number (i.e., ≧5×10 9  cells) or monocyte count (i.e., ≧1×10 9 ) falls below the recommended level for the cell separator, cell fractionation patterns can shift and result in heterogeneous cell distribution in later fractions. 
         [0109]    Fractionated or lymphocyte-enriched cell populations permit ‘controlled’ activation as measured by the composition of cell products in the LCM. Cytokines, particularly GM-CSF, IFNγ, IP10, IL2, IL6, IL8, IL9, IL10, IL13, MIP1α, MIP1β, RANTES, TNFα, were most highly induced at fairly even distributions (Table 13); however, more samples should be evaluated for presentation to FDA. 
         [0110]    LCM enhanced the expression levels of costimulatory molecules (e.g., CD40, CD80, CD86, and CD83) on DCs, an indication of the maturation process important to antigen presentation (Table 13). 
         [0111]    LCM promoted an ‘adjuvant-like’ effect on DC function. DCs treated in vitro with 50-25% of the original LCM solution were able to stimulate responses to CMV and tumor antigens in recall assays ( FIG. 15 ). 
         [0112]    LCM may help APC function and expand antigen-specific T cells ( FIG. 16 ); however, optimal levels of cytokine are currently undefined (Note: compare to cells incubated with the ‘standard’ GM-CSF+IL4 formulation). 
         [0113]    Based on preliminary results, elutriated cells appear to be a good source for the preparation of LCM in the autologous setting. Note PBMC preparations and elutriated fractions were not directly compared from the same donors in “side-by-side” studies. Stimulated PBMCs, presumably due to the presence of monocytes or possibly platelets, do appear to express some cytokines (e.g., MCP1) not seen at high levels in the elutriated cells which could endow a more robust adjuvant effect. 
         [0114]    Further development of the production of LCM or cells is warranted, in which a closed system design illustrated in  FIG. 17  could be applied to clinical use. 
         [0115]      FIG. 18  shows a proposed culture system for lymphocytes. 
         [0116]    It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications that are within the spirit and scope of the invention, as defined by the appended claims.

Technology Category: 1