Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0005—Vertebrate antigens
  • A61K39/0011—Cancer antigens
  • A61K39/001193—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; PAP or PSGR
  • A61K39/001195—Prostate specific membrane antigen [PSMA]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/19—Dendritic cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
  • A61K40/24—Antigen-presenting cells [APC]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/4274—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; Prostatic acid phosphatase [PAP]; Prostate-specific G-protein-coupled receptor [PSGR]
  • A61K40/4276—Prostate specific membrane antigen [PSMA]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/08—Drugs for disorders of the urinary system of the prostate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
  • A61K2239/58—Prostate

Definitions

• the present invention relates to compositions and methods of isolating and/or preserving and using human dendritic cells for immunotherapy for cancer.
• the invention relates to methods for the use of dendritic cells for the activation and expansion of large numbers of prostate antigen specific T cells for use in adoptive cellular immunotherapy against prostate cancer, as well as methods for the use of prostate antigen pulsed dendritic cells as vaccines and/or immunotherapeutics to slow or inhibit the growth of primary or metastatic prostate cancer.
• Prostate cancer is the second leading cause of death from cancer among men.
• prostate cancer is the most common (noncutaneous) cancer diagnosed in the American male and is steadily increasing, as a result of the increasing population of older men as well as greater awareness and earlier diagnosis of the disease. See, Wright et al., 1995, Urol. Oncol. 1: 18-28. In 1995, it is projected that over 244,000 men will be diagnosed with prostate cancer in this year. There will be 40,400 deaths.
• the life time risk for men, to suffer prostate cancer is now 1 in 8 for Caucasians, and an estimated 1 in 7 for African Americans. High risk groups are those with a positive family history or African Americans. Over a lifetime, above 25% of the men diagnosed with prostate cancer will die of the disease.
• prostate cancer is also a major cause of suffering and of health care expenditures.
• Catalona W.J., 1994, New Eng. J. Med. 331 :996-1004.
• Cytotoxic chemotherapy is largely ineffective in treating prostate cancer.
• a combination of agents is no more effective than a single agent, and the addition of chemotherapy to hormonal therapy does not improve survival.
• T H ,CD4 + ) and cytotoxic (CD8 + ) T cell subsets through their interaction with antigen presenting cells (APC) that express major histocompatibility (MHC)-class I or class II molecules associated with antigenic fragments (i.e. , specific amino acid sequences derived from the antigen which bind to MHC I and MHC II for presentation on the cell surface).
• APC antigen presenting cells
• MHC major histocompatibility-class I or class II molecules associated with antigenic fragments (i.e. , specific amino acid sequences derived from the antigen which bind to MHC I and MHC II for presentation on the cell surface).
• MHC major histocompatibility
• the sensitized or primed CD4 + T cells produce lymphokines that participate in the activation of B cells as well as various T cell subsets.
• the sensitized CD8 + T cells increase in numbers in response to lymphokines and are capable of destroying any cells that express the specific antigenic fragments associated with matching MHC-encoded class I molecules.
• CTL eradicate cells expressing cancer associated or cancer specific antigens, thereby limiting the progression of tumor spread and disease development.
• Antigen presenting cells are particularly important in eliciting an effective immune response.
• APC not only can present antigens to T cells with antigen- specific receptors, but can provide all the signals necessary for T cell activation. Such signals are incompletely defined, but probably involve a variety of cell surface molecules as well as cytokines or growth factors. Further, the factors necessary for the activation of naive or unprimed T cells may be different from those required for the re-activation of previously primed memory T cells.
• the ability of APC to both present antigens and deliver signals for T cell activation is commonly referred to as an accessory cell function.
• monocytes and B cells have been shown to be competent APC, their antigen presenting capacities in vitro appear to be limited to the re-activation of previously sensitized T cells. Hence, they are not capable of directly activating functionally naive or unprimed T cell populations.
• lymphoid cells refers to a diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissues (Steinman, 1991, Ann. Rev. Immunol. 9:271-296). These cells include lymphoid DC of the spleen, Langerhans cells of the epidermis, and veiled cells in the blood circulation. Although they are collectively classified as a group based on their morphology, high levels of surface MHC-class II expression, and absence of certain other surface markers expressed on T cells, B cells, monocytes, and natural killer cells, it is presently not known whether they derive from a common precursor or can all function as APC in the same manner. It should be noted that a putative novel antigenic marker of human DC recognized by monoclonal antibody CMRF-44 has been reported (Hock et al., 1994 Immunol. 85(4):P573-581).
• the present invention provides methods, and compositions, for use of dendritic cells to activate T cells for immunotherapeutic responses against primary or metastatic prostate cancer.
• the DCs obtained from human donors, after exposure to a prostate cancer antigen or antigenic fragment, are administered to a prostate cancer patient to activate the relevant T cell responses in vivo.
• the DCs are exposed to a prostate cancer antigen in vitro and incubated with primed or unprimed T cells to activate the relevant T cell responses in vitro.
• the activated T cells are then administered to a prostate cancer patient.
• the DCs are advantageously used to elicit an immunotherapeutic growth inhibiting response against a primary or metastatic prostate tumor.
• the invention provides a method for producing a cancer growth inhibiting response, which comprises administering, to a prostate cancer patient in need thereof, an effective amount of activated T cells, in which the T cells were activated in vitro by exposure to human dendritic cells exposed to a prostate cancer antigen.
• the invention provides a method for producing a cancer growth inhibiting response, which comprises administering, to a prostate cancer patient in need thereof, an effective amount of human dendritic cells, exposed in vitro to a prostate cancer antigen, such that after administration the human dendritic cells elicit an immune response or augment an existing immune response against the prostate cancer.
• Prostate cancer antigens useful for the methods and compositions of the invention include but are not limited to: a lysate of LNCaP cells, a membrane preparation of LNCaP cells, a lysate of prostate tumor cells of a prostate cancer patient, a membrane preparation of prostate tumor cells of a prostate cancer patient, purified prostate specific membrane antigen (PSM A a/k/a PSM), a peptide having the amino acid sequence LLHETDSAV (SEQ. ID. NO. 1), a peptide having the amino acid sequence ALFDIESKV (SEQ. ID. NO. 2), a peptide having the amino acid sequence XL(or M)XXXXXXV(or L) (SEQ. ID. NO. 3) where X represents any amino acid, purified prostate specific antigen (PSA), and a purified prostate mucin antigen recognized by monoclonal antibody PD41.
• PSM A purified prostate specific membrane antigen
• the present invention further provides compositions comprising isolated human dendritic cells exposed to a prostate cancer antigen(s), as well as cryopreserved isolated human dendritic cells and extended life span human dendritic cells which are useful for eliciting immunotherapeutic responses against primary and/or metastatic prostate cancer.
• FIGURE 1 represents histograms of results of flow cytometric analyses of cultured dendritic cells which illustrate the expressed cell surface antigens.
• the cells were cultured in the presence of GM-CSF and IL-4.
• the (topmost) upper left histogram represents background fluorescence staining using the secondary (2°) antibody, i.e. , goat anti-mouse Ig, in the absence of any primary (1 °) antibody.
• the rest of the histograms represent fluorescence staining in the presence of 2° antibody and each of the enumerated 1 ° antibodies, respectively, anti-CDla, anti-CD3, anti-CD4, anti-CDllc, anti-CD14, anti-CD19, anti-B7/BBl and anti-HLA-DR antibodies. See text Section 6 for details.
• FIGURE 2 graphically illustrates in vitro activation of T cells by tetanus toxoid (TT) presented by autologous DCs. See text Section 6 for details of the assays conducted in triplicate.
• the extent of T cell proliferation, represented by 3 HTdR incorporated (cpm), is presented along the y axis.
• Three different culture conditions for the T cells are represented along the x axis: tetanus toxoid alone (+TT); dendritic cells alone (+DC); and tetanus toxoid with dendritic cells (+DC-TT). Individual standard deviations are shown.
• FIGURE 3 graphically illustrates in vitro activation of T cells from four prostate cancer patients by presentation of prostate cancer antigen by autologous dendritic cells. Data from each individual patient is shown by different bar graph patterns as indicated. The extent of T cell proliferation, represented by 3 H-Thymidine HTdR) incorporated (cpm), is presented along the y axis. Three different culture conditions for the T cells are represented along the x axis: a prostate cancer antigen alone (+ LNCaP lysate); dendritic cells alone (+DC); and a prostate cancer antigen with dendritic cells (+DC + LNCaP lysate). See text Section 6.4. for details.
• FIGURE 4 is a histogram illustrating fluorescence flow cytometric analysis of the population of T cells proliferated in response to autologous DC presentation of LNCaP lysate as antigen.
• Background fluorescence represented by experiments with 2° antibody (goat anti-mouse Ig) alone is shown by the dotted line histogram.
• Fluorescence obtained in experiments using the 2° antibody with the 1° antibody (anti-CD8) antibody are shown by the solid line histogram. See text Section 6 for details.
• FIGURE 5 graphically illustrates cytolytic activity of T cells stimulated, in vitro, in response to autologous DC presentation of LNCaP lysate as antigen. Average percent specific lysis of target cells is represented (y axis) as a function of (x axis) Effector (activated T cells): Target Cells (autologous DCs presenting LNCaP + ⁇ or autologous DCs alone - Q - ). Experiments were performed in triplicate and the values presented represent the average; standard error of the mean for all experiments was ⁇ 10%. See text Section 6.4 for details
• FIGURE 6 illustrates in vitro activation of T cells (PBMC) by presentation of prostate cancer antigen by previously frozen autologous dendritic cells.
• the prostate cancer antigen used was purified prostate specific membrane antigen (PSMA).
• PSMA prostate specific membrane antigen
• the extent of T cell proliferation, represented by 3 H-TdR incorporated is shown along the y axis. Values are expressed as mean cpm + standard deviation (S.D.). Each experiment was conducted in 4 replicates.
• Three different culture conditions for the T cells are represented along the x axis: T cells plus dendritic cells (PBMC + DC); T cells plus PSMA alone (PBMC + PSMA); and T cells plus dendritic cells plus PSMA (PBMC + DC + PSMA). See text Section 7.1 for details.
• FIGURE 7 illustrates in vitro activation of T cells by presentation of antigens by previously frozen autologous extended life span dendritic cells.
• the extent of T cell proliferation, represented by 3 H-TdR incorporated is shown along the x axis. Values are expressed as mean value ⁇ S.D. after subtraction of average background (PBMC + medium only).
• T cells Five different cultures of T cells are represented along the y axis: T cells plus extended life span dendritic cells plus tetanus toxoid (+ EBV-cells + TT); T cells plus extended life span dendritic cells plus prostate antigen (+ EBV-cells + LNCaP); T cells plus extended life span dendritic cells plus no antigen (+ EBV- cells); T cells plus tetanus toxoid alone (+TT); and T cells plus prostate antigen alone (+LNCaP). See text Section 8 for details.
• FIGURE 8 illustrates that a peptide having amino acid sequence LLHETDSAV (comprising a portion of the amino acid sequence of PSMA) can stimulate proliferation of a mixed population of T cells (PBMC) obtained from peripheral blood of a prostate cancer patient.
• PBMC mixed population of T cells
• FIGURE 9 is a histogram illustrating fluorescence flow cytometric analysis of the population of T cells proliferated in response to the peptide LLHETDSAV as illustrated in FIGURE 8.
• FIGURE 9A illustrates background fluorescence obtained in experiments using 2° antibody only.
• FIGURE 9B illustrates bound fluorescence obtained using the 2° antibody and anti-CD4 as the 1 ° antibody.
• FIGURE 9C illustrates bound fluorescence obtained using 2° antibody and anti-CD8 as the 1° antibody. See text Section 9 for details.
• FIGURE 10 illustrates that a peptide having amino acid sequence ALFDIESKV can stimulate T cells (PBMC) obtained from purified blood of prostate cancer patients.
• PBMC T cells
• FIGURE 11 present results of a clinical trial demonstrating that administration of dendritic cells exposed to a peptide having amino acid sequence LLHETDSAV (PSM-Pl) or amino acid sequence ALFDIESKV (PSM-P2) induced an enhanced immune response in late stage prostate cancer patients as assessed by ratio of T cell proliferation pre- and post- administration of the DCs.
• PSM-Pl amino acid sequence LLHETDSAV
• PSM-P2 amino acid sequence ALFDIESKV
• FIGURE 11(A) illustrates results with patients infused with peptide PSM-Pl alone (Group I); FIGURE 11(B), with patients infused with peptide PSM-P2 alone (Group II); FIGURE 11(C), with patients infused with autologous dendritic cells alone (Group III); FIGURE 11(D), with patients infused with dendritic cells and PSM-Pl (Group IV); and FIGURE 11(E), with patients infused with dendritic cells and PSM-P2 (Group V). See text Section 10 for details.
• the present invention provides methods, and compositions, for use of dendritic cells to activate T cells for immunotherapeutic responses against primary or metastatic prostate cancer.
• the DCs obtained from human donors, once exposed to a prostate cancer antigen or specific antigenic peptide, are administered to a prostate cancer patient to activate the relevant T cell responses in vivo.
• the DCs are exposed to a prostate cancer antigen or specific antigenic peptide in vitro and incubated with primed or unprimed T cells to activate the relevant T cell responses in vitro.
• the activated T cells are then administered to a prostate cancer patient.
• the DCs are advantageously used to elicit an immunotherapeutic growth inhibiting response against a primary or metastatic prostate tumor.
• the description of the invention is divided into the following sections: (1) methods for obtaining or isolating dendritic cells, including DCs with extended lifespan or cryopreserved DCs; (2) prostate specific antigens for presentation by DCs; and (3) applications or methods of use of DCs to stimulate T cells against prostate cancer in vitro and in vivo.
• Human DCs are obtained from any tissue where they reside including non-lymphoid tissues such as the epidermis of the skin (Langerhans cells) and lymphoid tissues such as the spleen, bone marrow, lymph nodes and thymus as well as the circulatory system including blood (blood DCs) and lymph (veiled cells).
• Human peripheral blood is an easily accessible ready source of human DCs and is used as a source according to a preferred embodiment of the invention.
• Cord blood is another source of human DCs and in cases where a male is born into a family known to be at high risk for prostate cancer, cord blood can be used as a source of DCs which can be cryopreserved for later use, if needed.
• DCs occur in low numbers in any tissues in which they reside, including human peripheral blood, DCs must be enriched or isolated for use. Any of a number of procedures entailing repetitive density gradient separation, positive selection, negative selection or a combination thereof are used to obtain enriched populations or isolated DCs. Examples of such methods for isolating DCs from human peripheral blood include: O'Doherty et al, 1993, J. Exp. Med. 178: 1067-1078; Young and Steinman, 1990, J. Exp. Med. 171 : 1315-1332; Freudenthal and Steinman, 1990, PNAS USA 87:7698-7702; Macatonia et al. , 1989, Immunol.
• the DCs are cultured in appropriate culture medium to expand the cell population and/or maintain the DCs in a state for optimal antigen uptake, processing and presentation.
• Particularly advantageous for maintenance of the proper state of "maturity" of DCs in in vitro culture is the presence of both granulocyte/macrophage colony stimulating factor (GM-CSF) and interleukin 4 (IL-4).
• GM-CSF granulocyte/macrophage colony stimulating factor
• IL-4 interleukin 4
• DCs are obtained from a prostate cancer patient to be treated.
• the DCs are used to activate autologous T cells of the patient, either in vitro or in vivo, for cancer immunotherapy and/or tumor growth inhibition.
• DCs are obtained from a healthy individual known not to be suffering from prostate cancer.
• the relevant HLA antigens both class I and II, e.g. , HLA-A, B, C and DR
• DCs which match the prostate cancer patient, in terms of HLA antigens, are isolated and expanded as described above.
• HLA antigens both class I and II, e.g. , HLA-A, B, C and DR
• DCs which match the prostate cancer patient, in terms of HLA antigens, are isolated and expanded as described above.
• a late stage prostate cancer patient who has been treated with radiation and/or chemotherapy agents often are not able to provide sufficient or efficient DCs.
• DCs from healthy HLA-matched individuals can be obtained and expanded using any of the methods described above and incubated in vitro with a prostate cancer antigen to elicit activated T cells for immunotherapy and/or tumor growth inhibition in the HLA-matched prostate cancer patient.
• extended life span dendritic cells are used. Human cells have a finite life span in vitro usually limited to approximately 50-70 population doublings before undergoing apoptosis.
• extended life span dendritic cells is intended to mean DCs that have been genetically modified so that they can be expanded in in vitro cell culture medium for an extended period of time, including but not limited to at least about 100 additional population doublings.
• Extended life span DCs are obtained, for example, by EBV- transformation of DCs obtained from peripheral blood of prostate cancer patients, or by insertion into DCs, using techniques known to those skilled in the art, of a specific cell cycle regulatory gene including but not limited to a gene which encodes cyclin A, B, D or E or retinoblastoma protein.
• DCs can be preserved, e.g. , by cryopreservation either before exposure or following exposure to a prostate cancer antigen.
• Cryopreservation agents which can be used include but are not limited to dimethyl sulfoxide (DSO) (Lovelock and Bishop, 1959, Nature 183: 1394-1395; Ashwood-Smith, 1961 , Nature 190:1204-1205), glycerol, polyvinylpyrrolidone (Rinfret,
• a controlled slow cooling rate is critical.
• Different cryoprotective agents (Rapatz et al. , 1968, Cryobiology SQ): 18-25) and different cell types have different optimal cooling rates (see, e.g. , Rowe and Rinfret, 1962, Blood 20:636; Rowe, 1966, Cryobiology 3 ⁇ 1): 12-18; Lewis et al., 1967, Transfusion 7(1): 17-32; and Mazur, 1970, Science 168939-949 for effects of cooling velocity on survival of marrow-stem cells and on their transplantation potential).
• the heat of fusion phase where water turns to ice should be minimal.
• the cooling procedure can be carried out by use of, e.g. , a programmable freezing device or a methanol bath procedure.
• Programmable freezing apparatuses allow determination of optimal cooling rates and facilitate standard reproducible cooling.
• Programmable controlled-rate freezers such as Cryomed or Planar permit tuning of the freezing regimen to the desired cooling rate curve.
• samples can be cryogenically stored in liquid nitrogen (-196°C) or its vapor (-165°C).
• liquid nitrogen -196°C
• vapor -165°C
• cryopreservation of viable cells or modifications thereof, are available and envisioned for use (e.g. , cold metal-mirror techniques; Livesey and Linner, 1987, Nature 227:255; Linner et al. , 1986, J. Histochem. Cytochem. 34£9): 1123-1135; see also U.S. Patent No. 4,199,022 by Senken et al., U.S. Patent No. 3,753,357 by Schwartz, U.S. Patent No. 4,559,298 by Fahy.
• Frozen cells are preferably thawed quickly (e.g. , in a water bath maintained at 37-41 °C) and chilled immediately upon thawing.
• cryoprotective agent if toxic in humans, should be removed prior to therapeutic use of the thawed DCs.
• cryoprotective agent One way in which to remove the cryoprotective agent is by dilution to an insignificant concentration.
• a number of antigens or antigenic compositions are useful, according to the present invention, for presentation by the DCs to activate T cells for prostate immunotherapeutics.
• a lysate of LNCaP a prostate cancer cell line, first described by Horoszewicz et al., 1980, Prog. Clin. Biol. Res. 37: 115-132; 1983, Cancer Res. 43: 1809-1818, is used as antigen presented by the DCs.
• a crude cell lysate obtained simply by repetitive freezing and thawing of LNCaP cells can be used as antigen.
• An illustrative example of the use of this antigen is presented in Section 6, infra.
• a membrane preparation of LNCaP cells as described by Horoszewicz et al., 1987, Anticancer Res. 7:927-936, can be used.
• a prostate tumor cell lysate recovered from surgical specimens can be used as antigen.
• a sample of a cancer patient's own tumor, obtained at biopsy or at surgical resection, can be used to provide a cell lysate for antigen.
• a membrane preparation of tumor cells of a prostate cancer patient can be used as antigen.
• purified prostate specific membrane antigen (PSMA, also known as CYPP antigen and as PSM antigen), which specifically reacts with monoclonal antibody 7E11-C.5 [(see generally Horoszewicz et al., 1987, supra. U.S. Patent No. 5, 162,504; Feng et al. , 1991 , Proc. Am. Assoc. Cancer Res. 32:(Abs. 1418):238)] can be used as antigen.
• Cloning of the gene encoding the PSMA antigen has been described by Israeli et al. , 1994, Cancer Res. 54: 1807-1811. Expression of the cloned gene, e.g. , in yeast cells, in a baculovirus expression system or in a mammalian cell culture expression system, will provide a ready source of the PSMA antigen for use according to the present invention.
• an antigenic peptide having the amino acid sequence LLHETDSAV (SEQ. ID. NO. 1) (designated “PSM-Pl") which corresponds to amino acid residues 4-12 PSMA can be used as antigen.
• an antigenic peptide having the amino acid sequence ALFDIESKV (SEQ. ID. NO. 2) (designated "PSM-P2") which corresponds to amino acid residues 711-719 of PSMA can be used as antigen.
• an antigenic peptide having an amino acid sequence XL(or M)XXXXXV(or L) (SEQ. ID. NO. 3) (designated "PSM-X") where X represents any amino acid residue can be used as antigen.
• This peptide resembles the HLA-A0201 binding motif, i.e. , a binding motif of 9-10 amino acids with "anchor residues", leucine and valine found in HLA-A2+ patients. Grey et al., 1995, Cancer Surveys 22:37-49.
• This peptide is preferably used as antigen for HLA-A2+ patients.
• HLA-A2+ is expressed by a large proportion of patients. [See, Central Data Analysis Committee "Allele Frequencies", Section 6.3, Tsuji, K. et al. (eds.), Tokyo University Press, pp. 1066-1077].
• an antigenic peptide selected from the peptides listed in Table IA can be used as antigen.
• the peptides listed in Table IA have amino acid sequences corresponding to fragments of PSM and have been matched to a binding motif of a specific haplotype.
• the peptides are selected to be presented by DCs to activate T cells of a patient which matched the haplotype indicated for each peptide in Table IA.
• PSM peptides refers to peptides having an amino acid sequence corresponding to a fragment of PSMA (a/k/a PSM).
• Initial Amino Acid Residue refers to the residue number of the amino acid of PSM to which the first amino acid of the peptide corresponds.
• PSA prostate specific antigen
• an antigenic peptide selected from the peptides listed in Table IB can be used as antigen.
• the peptides listed in Table IB have amino acid sequences corresponding to fragments by PSA and [PLEASE CONFIRM] have been matched to a binding motif of a specific haplotype as indicated in Table IB.
• the peptide are presented by DCs to activate T cells of patients which match the haplotype indicated for each peptide in Table IB.
• PSA peptides refers to peptides having an amino acid sequence corresponding to a fragment of PSA.
• a prostate mucin antigen recognized by monoclonal antibody PD41 , described by Wright (U.S. Patent No. 5,227,471 and No. 5,314,996; Beckett et al. , 1991 , Cancer Res. 51: 1326-1333) can be used as antigen.
• a crude lysate of prostate tumor cells which bind to the antibody produced by the hybridoma cell line (ATCC HB 11094) and which express the PD41 mucin antigen could be used as antigen.
• DCs can be exposed to a desired prostate cancer antigen or antigenic composition by incubating the DCs with the antigen in in vitro culture medium.
• the antigen in aqueous soluble or aqueous suspension form, is added to cell culture medium at the same time as the DCs and T cells to be stimulated are added. See Section 6, infra, for an illustrative example of this method. As demonstrated in Section 6, the DCs advantageously took up antigen for successful presentation to T cells.
• antigens are introduced to the cytosol of the DCs by alternate methods, including but not limited to osmotic lysis of pinocytic vesicles and the use of pH sensitive liposomes, etc.
• isolated human DCs are used to activate T cells in vitro against prostate cancer.
• the DCs can be used immediately after exposure to antigen to stimulate T cells.
• the DCs can be maintained in the presence of a combination of GM-CS and IL-4 prior to simultaneous exposure to antigen and T cells.
• T cells or a subset of T cells can be obtained from various lymphoid tissues for use as responder cells. Such tissues include but are not limited to spleens, lymph nodes, and peripheral blood.
• the cells can be co-cultured with DC exposed to antigen as a mixed T cell population or as a purified T cell subset. For example, it may be desired to culture purified CD8 + T cells with antigen exposed DCs to elicit prostate specific CTL. In addition, early elimination of CD4 + T cells may prevent the overgrowth of CD4 + cells in a mixed culture of both CD8 + and CD4 + T cells.
• T cell purification may be achieved by positive, or negative selection, including but not limited to, the use of antibodies directed to CD2, CD3, CD4, CD5, and CD8.
• the T cells are obtained from the same prostate cancer patient from which the DCs were obtained. After stimulation or activation in vitro, the autologous T cells are administered to the patient to provoke and afford an immunoresponse which slows or inhibits prostate tumor growth.
• T cells are administered, by intravenous infusion, at doses of about 10 8 -10 9 cells/m 2 of body surface area (see, Ridell et al., 1992, Science 257: 238-241). Infusion can be repeated at desired intervals, for example, monthly. Recipients are monitored during and after T cell infusions for any evidence of adverse effects.
• the T cells are obtained from a prostate cancer patient and the DCs which are used to stimulate the cells are obtained from an HLA-matched healthy donor.
• both the T cells and the DCs are obtained from an HLA-matched healthy donor, e.g. , a sibling of the prostate cancer patient. This embodiment may be particularly advantageous, for example, when the patient is a late stage prostate cancer patient who has been treated with radiation and/or chemotherapy agents and may not be able to provide sufficient or efficient DCs.
• the T cells after stimulation, are administered as described above.
• DCs isolated from a prostate cancer patient are cultured, exposed in vitro to a prostate cancer antigen and after expansion and/or cryopreservation are administered back to the patient to stimulate an immune response, including T cell activation, against the patient's cancer cells in vivo.
• dendritic cells Using this approach with the patient's own dendritic cells provides the following advantages: (1) no foreign DNA is utilized; (2) infection of cells for pu ⁇ oses of cDNA expression using various viral vectors are eliminated; (3) antigen is presented to dendritic cells in the form of soluble protein which will be taken into the dendritic cells and processed for MHC/peptide presentation of the cell surface; (4) dendritic cells express B7's on their surface alleviating the necessity to transfect this cDNA into dendritic cells; (5) the use of endogenous B7's on dendritic cell surface eliminates the need to provide T cells with 11-2 or other cytokines either in the form of the cytokine itself or transfection of the cDNA into specific cells; (6) all procedures are carried out using the patient's own cells.
• DCs obtained as described above in Section 5.1 are exposed in vitro to a prostate cancer antigen, washed and administered to elicit an immune response or to augment an existing, albeit weak, response.
• the DCs constitute an anti-prostate cancer vaccine and/or immunotherapeutic agent.
• DCs presenting a prostate specific antigen are administered, via intravenous infusion, at a dose of about 10 6 -10 8 cells.
• the immune response of the patient can be monitored. Infusion can be repeated at desired intervals based upon the patient's immune response.
• LNCaP a prostate cancer cell line, Horoszewicz et al., 1983, LNCaP Model of Human Prostatic Carcinoma, Cancer Research, 43: 1809-1818, (CRL 1740, ATCC, Rockville, MD), was maintained in culture in RPMI 1640.
• Granulocyte/macrophage colony stimulating factor (GM-CSF), recombinant human interleukin 2 (IL2) and interleukin 4 (IL4) were generous gifts from Amgen (Thousand Oaks, CA).
• Monoclonal antibodies Leu-6 (anti-CDla), Leu-4 (anti-CD3), Leu-3a (anti- CD4), Leu-2a (anti-CD8), Leu-M3 (anti-CD 14), anti-HLA-DR (MHC Class II), and BBl (anti-B7/BBl) were purchased from Becton-Dickinson, San Jose, CA.
• Monoclonal antibodies S125-C1 (anti-CD19) and 3.9 (anti-CDl lc) were purchased from Sigma, St. Louis, MO.
• PBMC peripheral blood mononuclear cells
• Peripheral blood was drawn from prostate cancer patients and was subjected to Lymphoprep (GIBCO-BRL, Gaithersburg, MD) density gradient centrifugation.
• the peripheral blood mononuclear cells (PBMC) isolated were plated in 24 well plates (10 6 -10 7 cells/well) and were incubated in a humidified incubator (37°C, 5% Co 2 ) for 90 minutes.
• Non-adherent cells were removed with the supernatant and the wells are washed gently with warm (37°C) OPTIMEM medium (GIBCO-BRL, Gaithersburg, MD) and 5% FCS.
• OPTIMEM medium Gibco-BRL, Gaithersburg, MD
• FCS Dendritic cell propagation medium
• DCPM OPTIMEM supplemented with 5% FCS, 500 units/ml GM-CSF and 500 units/ml IL-4
• DCPM OPTIMEM supplemented with 5% FCS, 500 units/ml GM-CSF and 500 units/ml IL-4
• the purity and identity of the isolated DC was confirmed by incubation with monoclonal antibodies anti-CDla, -CD3, -CD4, -CD8, -CDllc, -CD14, -CD19, - HLA-DR and -B7/BB1 for 30 minutes on ice followed by a fluorescein-isothiocyanate labeled goat-anti-mouse Ig antibody for 30 minutes on ice. Fluorescence binding was analyzed using a FACS SCAN flow cytometer (Becton Dickinson, San Jose, CA).
• the cultured DC cells were harvested by pipetting (leaving behind highly adherent, bound macrophages) and were subjected to flow cytometer analyses for surface expression of different protein markers for cells of hematopoietic origin to confirm their DC identity.
• CD14 " , CD3- and CD19 confirms identity of the DC population. 6.1.4.
• T cell media consisting of RPMI 1640, HEPES, 2-mercaptoethanol, L- glutamine and penicillin-streptomycin, supplemented with 10% human AB serum (Sigma, St. Louis, MO) and IU/ml recombinant human 11-2.
• TCM T cell media
• the antigens used in these assays were: (1) tetanus toxoid at 500 ng/ml (TT; Sigma, St. Louis, MO); (2) the lysate of LNCaP cells from an equivalent of 10 s LNCaP cells/ml; or (3) purified PSMA.
• Lysate was prepared as described previously Topalian et al., 1994, Melanoma-Specific CD4+ T Lymphocytes Recognize Human Melanoma Antigens Processed and Presented by Epstein-Barr Virus-Transformed B Cells, Int. J. Cancer 58:69-79. Briefly, IO 7 LNCaP in 1 ml phosphate buffer saline (PBS) was subjected to cycles of repeated freezing in liquid nitrogen and quick thawing in a 37°C water bath. Purified PSMA was prepared as follows:
• the pH of the 2 ml of 7E11-C5 monoclonal antibody solution was adjusted to pH 9.0 and the NaCl concentration adjusted to 3 M.
• Antibody solution and beads were mixed 1 hr at room temperature. After the incubation the beads were washed with 10X volume 3 M NaCl, 50 mM Sodium Borate.
• Dimethylpimeladate was added to a concentration to 20 mM.
• the mixture was allowed to mix 30 minutes at room temperature.
• the reaction was stopped by washing the beads with 0.2 M methanolamine pH 8.0.
• 0.2 M ethanolamine pH 8.0 was added at 10X volume and the mixture was allowed to incubate with mixing for 2 hours.
• T cell proliferation assays T cell proliferation were assessed by measurement of average 3 HTdR incorporation.
• DC were obtained from PBMC of prostate cancer patients as described in Section 6.1.3. above and cultured in DCPM as described. After 4-7 days in culture, clusters of dividing cells started to form and became less adherent to the tissue culture flask. These cells increased in size and showed a typical dendritic morphology (results not shown). In addition to these slightly adherent cells, tightly adherent macrophages were also present.
• the average number of cells with dendritic mo ⁇ hology obtained after a 7 day culture was 2-7 x 10° from 50 ml peripheral blood, representing 4-14% of the starting number of PBMC cultured (see Table 2).
• the cells were harvested by pipetting (leaving tightly bound macrophages behind) and were subjected to flow cytometric analyses for surface expression of different protein markers for cells of hematopoietic origin. Results are illustrated in FIGURE 1.
• the cultured cells do not express lineage specific markers for T cells (CD3), B cells (CD 19), or macrophages (CD 14).
• CD la a marker for Langerhans cells (e.g. , dendritic cells isolated from the skin), was expressed at a high level early in the culture, but the level decreased when the cells were maintained in culture for more than 14 days.
• CDl lc beta-2-integrin
• HLA-DR were expressed at high levels while B7/BB1 was expressed at moderate levels by these cells, confirming further the identify of the cultured cells as DCs.
• DCPM dendritic cell propagation medium
• T cell proliferation assays were conducted as described above in Section 6.1.4. in triplicate. Tetanus toxoid (TT) was chosen as a representative antigen in these experiments to determine whether patients' memory T cells could be activated in vitro. Results are presented in FIGURE 2.
• FIGURE 2 shows that autologous T cells cultured with the patients' DCs and TT proliferated at levels significantly higher than background levels (in the absence DC) and at levels significantly higher than T cells cultured with DC without TT, i.e. , showing an autologous mixed lymphocyte reaction.
• results demonstrate that the presentation of TT by DCs is useful for T cell proliferation. More particularly, the results demonstrate that DCs obtained from prostate cancer patients are useful to activate antigen specific T cell proliferation. 6.4. STIMULATION OF PROSTATE
• FIGURE 3 shows that significant increases in 3 HTdR incorporation were observed in 2 of 4 cases when both DCs and LNCaP lysates were included in the T cell cultures.
• presentation of prostate specific antigen stimulated autologous T cell proliferation in vitro. It is our belief that the 2 negative cases with LNCaP lysate as antigen reflect the limitations of using a crude cellular lysate with variable concentration of prostate antigen. Additional experiments using purified prostate-specific membrane antigen (PSMA) support this view (results not shown).
• PSMA prostate-specific membrane antigen
• T cells proliferated as a result of DC presentation of LNCaP lysate were expanded in culture, for 2 weeks, and subjected to fluorescence flow cytometric analysis to determine the representation of the two T cell subtypes, i.e. , cytolytic T lymphocytes (CTL) and helper T cells (T H ) elicited.
• CTL cytolytic T lymphocytes
• T H helper T cells
• T cells proliferated as a result of DC presentation of LNCaP lysate were expanded, for two weeks, by culture in T cell propagation medium in the presence of mitomycin C- inactivated autologous DC and LNCaP lysate (equivalent to IO 4 LNCaP cell/ml).
• a 14 day old culture of T cells was harvested and analyzed for the expression of cytolytic T cell specific antigen; CD8, by fluorescence flow cytometry as described above. Results are illustrated in FIGURE 4.
• the CTL's (CD8+) represented about 40- 50% of the T cells elicited against LNCaP by the DC presentation of LNCaP lysate.
• presentation of prostate antigen by autologous DCs is useful to elicit both cytolic (CTL's) and helper (T H ) T cells specific for prostate cancer.
• T cells (both CD4 + and CD8 + ) activated as described above by DCs presenting LNCaP lysate as antigen demonstrated cytolytic activity using, as target cells, autologous DCs presenting LNCaP antigen.
• DCs were isolated from a prostate cancer patient, as described in Section 6.2 and were exposed to LNCaP lysate as described above.
• Autologous T cells were obtained as PBMCs and cultured in the presence of the LNCaP presenting DCs. T cells proliferated as a result of prostate antigen presentation by the DCs were expanded for a total of 21 days as described above.
• effector cells the 21 day T cells including both CD4 + and CD8 + cells
• target cell ratios 20: 1, 10: 1 and 3: 1 for 5 hours at 37°C in a CO 2 incubator.
• Target cells included: autologous DCs alone or autologous DCs exposed to LNCaP lysate [equivalent of 10 5 cells/ml (DC + LNCaP Lysate)] overnight.
• T cells proliferated as a result of prostate antigen presentation by DCs according to the present invention showed enhanced specific lytic activity against cells presenting the same prostate antigen.
• presentation of prostate antigen by DCs is useful to elicit T cells having specific cytolytic activity against cells expressing prostate antigen.
• Dendritic cells were isolated from PBMC of a prostate cancer patient as described above in Section 6 and cultured, as described above, in Section 6, for 7 days in the presence of 500 units/ml GM-CSF and IL-4.
• the isolated DCs were harvested and cryopreserved using 90% fetal calf serum and 10% dimethylsulfoxide.
• the cryopreserved DCs were stored frozen for a period of time, thawed in a 37°C water bath and transferred to a 15 ml polypropylene tube and centrifuged at 1200 rpm for 5 min. The thawed DCs were then resuspended in medium containing 10% heat-inactivated human serum and counted.
• T cells obtained from peripheral blood of the same prostate cancer patient were cultured in the presence or absence of 10 4 DCs together with 5 ⁇ l/well purified PSMA (see Section 6.1.4 above) in a total volume of 200 ⁇ l medium containing 10% heat-inactivated human serum and 1 unit/ml IL-2.
• PSMA serum-inactivated human serum
• 3 H-TdR inco ⁇ oration was counted in a liquid scintillation counter. Each experiment was done in 4 replicates. Results are presented in FIGURE 6. The average cpm and standard deviation are shown in the graph.
• FIGURE 6 demonstrates that a highly significant increase in 3 HTdR inco ⁇ oration was observed when both previously cryopreserved DCs and prostate specific antigen, i.e. , PSMA, were included in the T cell cultures.
• prostate specific antigen i.e. , PSMA
• PBMC are obtained from a normal or healthy individual known not to be suffering from prostate cancer.
• the PBMC are cross-typed and the relevant HLA antigens expressed noted.
• Relevant HLA antigens include such as HLA-A, B, C and DR.
• DC are isolated from the PBMCs as described in Section 6. The DCs are then cryopreserved as described in Section 7.1 above.
• cryopreserved DC are used according to the present invention to stimulate T cells in vitro, from a similarly matched HLA-typed individual or from a patient suffering from prostate cancer for use in the cancer patient.
• Dendritic cells can be isolated from a prostate cancer patient or from a healthy donor, cryopreserved and used according to the methods of the invention as follows.
• viable DC count e.g. , by trypan blue exclusion (Kucker, 1977, Biochemical Methods in Cell Culture and Virology, Dwoder, Hutchinsen & Ross, Stroudsburg, PA, pp. 18-19) and manual cell counting can be performed.
• the DC from the prostate cancer patient are cryopreserved using the following protocol:
• a cryogenic storage vessel which can be used is the LR 1000 refrigerator (Union Carbide Co ⁇ ., Indianapolis, Indiana) which accommodates up to 40,000 cryules. Following any desired length of time post-cry opreservation, the protocol below can be used to thaw the DC cells for use to stimulate autologous T cells specific to prostate cancer according to the present invention.
• Loss of cells due to clumping during the stepwise removal of DMSO can be diminished by including Dnase (20 U per 2xl0 6 cells) or low molecular weight dextran and citrate (to reduce the Ph to 6.5).
• Extended life span dendritic cells were prepared according to the present invention as follows:
• DCs were isolated from human peripheral blood and cultured for 4-6 days as described above in Section 6.1.3. After 4-6 days in culture, in the presence of GM-CSF and IL-2, the DCs were infected with Epstein Barr Virus (EBV) generally as described by Walls & Crawford, in Lymphocytes: A Practical Approach, GCB Klaus, ed., IRL Press, Oxford, England, pp. 149-162. Briefly, DCs were harvested ( ⁇ 3-5 x IO 6 cells) and resuspended in a 5 ml B95-8 (an EBV-producing marmoset cell line, American Type Culture Collection, Rockville, Maryland) culture supernatant.
• EBV Epstein Barr Virus
• the cell suspension was transferred to a 25 cm 2 flask (Nunc, Inc., Naperville, IL) and incubated in a 37°C CO 2 incubator for 24 hours. Five ml of fresh DCPM were added to the culture. Clusters of growing cells were observed 2-3 weeks post-infection. Extended life span DC cells were subcultured weekly, at 3-5 weeks post-infection.
• TT tetanus toxoid
• the autologous T cells used in the proliferation assay were obtained from peripheral blood and had been previously cryopreserved.
• One hundred thousand thawed T cells (designated "PBMC") were cultured in complete medium containing 10% heat ⁇ inactivated human serum and 1 unit/ml IL-2 as follows: in the presence of LNCaP antigen alone (+LNCaP); in the presence of TT antigen alone (+TT); in the presence of EBV-transformed DCs (+EBV cells); in the presence of EBV-transformed dendritic cells plus LNCaP antigen (+ EBV-cells + LNCaP); or in the presence of EBV- transformed dendritic cells plus TT (+ EBV-cells + TT). T cells in the presence of medium alone (PBMC + medium only) served as a background sample.
• FIGURE 7 demonstrates that extended life span human dendritic cells, exposed either to TT antigen or PSMA antigen, significantly increased 3 HTdR inco ⁇ oration in previously cryopreserved T cell cultures.
• extended life span DCs are useful to present prostate specific antigen to stimulate autologous T cell proliferation in vitro.
• LLHETDSAV SEQ. ID. NO. 1
• PSM-Pl amino acid sequence having the amino acid sequence having the amino acid sequence, LLHETDSAV (SEQ. ID. NO. 1) (designated PSM-Pl), which corresponds to amino acid residues 4-12 of the PSMA antigen as deduced from cDNA (of the PSMA gene) to stimulate T cells was assessed in order to determine whether presentation of such antigenic peptide would be useful using the methods of the present invention.
• PSM-Pl amino acid sequence having the amino acid sequence, LLHETDSAV (SEQ. ID. NO. 1) (designated PSM-Pl), which corresponds to amino acid residues 4-12 of the PSMA antigen as deduced from cDNA (of the PSMA gene) to stimulate T cells was assessed in order to determine whether presentation of such antigenic peptide would be useful using the methods of the present invention.
• the following data show that this antigenic peptide is suitable for use in the methods of the invention.
• a peptide having amino acid sequence LLHETDSAV (designated PSM-Pl peptide) was synthesized and purified by Genemed Biotechnologies (San Francisco, CA).
• PBMC previously frozen T cells
• the T cells proliferated as a result of the PSMA peptide were expanded in culture for 5 days, in complete medium containing 2 ⁇ M PSMA peptide and 1 unit/ml 112 for 5 days prior to harvest.
• One million cells were incubated for 30 minutes on ice with anti-CD4, anti-CD8 antibodies or medium alone, followed by another 30 minute incubation with fluorescein isothiocyanate labelled-2° antibody. Fluorescence-bound was analyzed in a FACScan (Becton Dickinson, San Jose, CA) flow cytometer as described above. The percent CD4+ and CD8+ cells were calculated. Results are illustrated in FIGURE 9.
• the CTL's represented about 41 % of the T cells stimulated by the PSMA peptide antigen.
• this peptide antigen should be useful to be presented by DCs to T cells to elicit prostate specific responses, both cytolytic (CTL's) and helper (T H ) cells against prostate cancer according to the methods of the invention.
• Peptide PSM-P2 was synthesized and purified.
• T cells i.e. PBMC obtained from HLA-A2(+) or HLA-A2(-) prostate cancer patients were cultured in the presence or absence (i.e. "medium control") of 20 ⁇ g PSM-P2 peptide for five (5) days in a humidified, 37 °C, 5% CO 2 incubator. On day 6, l ⁇ Ci 3 H-Thymidine was added to each well. After another 24 h incubation, cells were harvested and the 3 H-TdR inco ⁇ orated was counted in a liquid scintillation counter. Each experiment was done in triplicate. Results are presented in FIGURE 10 as average cpm and standard error of the mean.
• the peptide corresponding to amino acid residues 711-719 of PSMA elicited a greatly enhanced cell proliferation of HLA-A2( T cells compared to HLA-A2(+) T cells in the absence of this peptide.
• a less dramatic enhanced T cell proliferation was observed when HLA-A2 (-) T cells were exposed to this peptide.
• a phase 1 clinical trial has been established using five treatment groups of late stage prostate cancer patients.
• the groups were treated as follows: Group I, received peptide PSM-Pl having amino acid sequence LLHETDSAV (SEQ. ID. NO 1) only at 0.2, 2.0 and 20.0 ⁇ g; Group II, peptide PSM-P2 having amino acid sequence ALFDIESKV (SEQ. ID. NO. 2) only at 0.2, 2.0 and 20.0 ⁇ g; Group III, 1 x IO 6 (#3-4) or 5 x IO 6 (#1, 2) autologous dendritic cells only; Group IV, up to 2 x IO 7 autologous dendritic cells loaded with PSM-Pl (SEQ. ID. NO.
• results in FIGURES l l(D-E) demonstrate that administration of DCs exposed to and presenting either peptide PSM-Pl or PSM-P2 induced an enhanced T cell proliferation. More particularly, patients receiving dendritic cells containing PSM-Pl peptide showed an increased T cell proliferation in 3 of 5 HLA-A(2)(+)as illustrated in FIGURE 11(D). In addition, the sole HLA-A(2)(-) patient did not demonstrate an increased T cell proliferation.
• dendritic cells exposed either to PSM-Pl or to PSM-P2 peptide are useful to produce an immune response in prostate cancer patients and that the methods and compositions of this invention are advantageously useful for cancer immunotherapy.
• MOLECULE TYPE peptide
• xi SEQUENCE DESCRIPTION: SEQ ID N ⁇ :31:

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