US20030077248A1 - Cell therapy method for the treatment of tumors - Google Patents

Cell therapy method for the treatment of tumors Download PDF

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US20030077248A1
US20030077248A1 US10/080,013 US8001302A US2003077248A1 US 20030077248 A1 US20030077248 A1 US 20030077248A1 US 8001302 A US8001302 A US 8001302A US 2003077248 A1 US2003077248 A1 US 2003077248A1
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cells
peripheral blood
nnapc
peptides
peptide
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Ann Moriarty
Didier Leturqc
Juli Degraw
Michael Jackson
Per Peterson
Marja Heiskala
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Janssen Pharmaceuticals Inc
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Ortho McNeil Pharmaceutical Inc
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Priority to US10/080,013 priority Critical patent/US20030077248A1/en
Assigned to ORTHO-MCNEIL PHARMACEUTICAL, INC. reassignment ORTHO-MCNEIL PHARMACEUTICAL, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: JACKSON, MICHAEL R.
Assigned to ORTHO-MCNEIL PHARMACEUTICAL, INC. reassignment ORTHO-MCNEIL PHARMACEUTICAL, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DEGRAW, JULI, HEISKALA, MARJA, LETURCQ, DIDIER J., MORIARTY, ANN
Assigned to ORTHO-MCNEIL PHARMACEUTICAL, INC. reassignment ORTHO-MCNEIL PHARMACEUTICAL, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: PETERSON, PER A.
Priority to US10/289,566 priority patent/US20040071671A1/en
Publication of US20030077248A1 publication Critical patent/US20030077248A1/en
Priority to US11/782,264 priority patent/US9222070B2/en
Priority to US12/014,863 priority patent/US9222071B2/en
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Definitions

  • Cancer continues to be a major health problem, despite significant progress made in the area of treatment.
  • the standard treatment regimes of chemotherapy, radiation therapy, surgical intervention and combinations of the three often fail to produce a long lasting cure.
  • the cancer patient having undergone the treatment often relapses back to the disease condition after some period of time, further exacerbating the problem, is the severity of these treatment regimes to the patient.
  • cancers have been found to be caused not by a single biological agent or factor, but rather by a combination of agents and factors. Unlike most medical treatments where a single causative agent or event is the focus of the treatment, cancer therapy requires addressing a plurality of biological factors.
  • the present invention provides a non-naturally occurring antigen-presenting cell (nnAPC) capable of presenting up to ten or more different peptides simultaneously, methods of manufacturing nnACP, methods of using said nnACP for the treatment of cancer.
  • nnAPC non-naturally occurring antigen-presenting cell
  • FIG. 1 This figure is a graphic depiction of the interaction between CD8 + cells, also known as cytotoxic T lymphocytes with antigen-presenting cells or target cells, in this case tumor cells.
  • FIG. 2 Panels A and B:
  • This figure is a two panel graphical depiction of mechanisms of lymphocyte-mediated cytosis.
  • FIG. 3 This figure shows the result of an experiment where several different peptides were tested in a competition assay to identify peptide binders that could be used to load multiple peptides onto Drosophila cells expressing human empty class I molecules.
  • FIG. 4 Panels A, B and C:
  • This figure shows the result of an experiment where three melanoma peptides were tested for the ability to raise CTLs when added as single epitopes on Drosophila cells.
  • CTL activity was elicited to each of the peptides when added alone to three different Drosophila preparations.
  • the specificity of the response was compared with control HBc peptide, a high affinity binder.
  • FIG. 5 Panels A, B and C:
  • This figure shows the results of a series of experiments where up to four different peptides were added to single Drosophila cells. CTL activity in each of the represented peptides was seen after a three-week stimulation protocol and is graphically depicted in this figure.
  • FIG. 6 Panels A, B and C:
  • FIG. 7 Panels A and B:
  • This figure compares the ability of Drosophila cells versus dendritic cells to elicit CTL responses to a single peptide epitope following standard stimulation protocols.
  • FIG. 8 This figure shows that the dendritic cells displaying either mature or immature phenotype was not as efficient as Drosophila cells in eliciting specific CTL responses when defined peptides were used to pulse the cells.
  • FIG. 9 Panels A, B and C:
  • This figure shows CTL activity generated by a single donor to three different in vitro stimulation protocols presenting four peptides.
  • FIG. 10 This figure show CTL activity generated to ten (10) peptides loaded, in combination, to Drosophila cells.
  • FIG. 11 This figure shows the peptide binding capacity of the HER-2 peptides (826, 835, 861 and 863) on the Drosophila cells transfected with the human HLA-A2.1 class I molecule.
  • FIG. 12 This figure demonstrates the anti-peptide and anti-tumor response for MART-1 specific effector cells.
  • T2 cells were loaded with MART-1 peptide or a negative control (HBc).
  • Malme3M is a melanoma line
  • Malme3 is a non-tumor cell line.
  • FIG. 13 Panels A and B:
  • This figure shows the tetrameric staining of the HER-2 specific CD8 effector cells from two different donors.
  • FIG. 14 This figure reveals the anti-peptide response for the HER-2 effector cells evaluated on peptide-loaded T2 cells.
  • FIG. 15 Panels, A, B, C, D:
  • This figure demonstrates the enhanced killing of an ovarian tumor cell line (HTB-77) when transfected with HLA-A2.1.
  • FIG. 16 This figure shows the enhanced killing of a breast cancer cell line (HTB-133) when transfected with HLA-A2.1
  • FIG. 17 This figure shows that IFN ⁇ pre-treatment is required to demonstrate lysis of the tumor cell line HTB-77/A2.1.
  • FIG. 18 Panels A and B:
  • This graph demonstrates that the surface expression of HLA-A2 and HER-2 is unaffected by the IFN ⁇ induction in the two cell lines (HTB-77 and HTB-77/A2.1).
  • FIG. 19 This graph shows which protein mRNA levels are elevated in the HTB-77/A2.1 cells after an induction with IFN ⁇ .
  • the present invention provides a method for treating a subject with cancer comprising:
  • nnAPC non-naturally occurring antigen-presenting cell line
  • a cytokine such as, IL-2, IL-7 or conditioned growth medium (CGM), preferably, IL-2, or IL-2 and IL-7 in combination;
  • peripheral blood monocyte suspension irradiating said peripheral blood monocyte suspension with a sufficient dose of ⁇ -radiation necessary to prevent proliferation of these cells in the suspension, such as a dose in the range of about 3,000 to 7,000 rads, preferably about 5,000 rads, alternatively, the peripheral blood lymphocyte suspension may be treated with cytostatic agents including, but not limited to, mitomycin C;
  • Another embodiment of the present invention provides a method for treating a subject with cancer comprising:
  • nnAPC non-naturally occurring antigen-presenting cell line
  • Another embodiment of the present invention provides a method for treating a subject with melanoma comprising:
  • nnAPC non-naturally occurring antigen-presenting cell line
  • Another embodiment of the present invention is a method of treating melanoma wherein the nnAPC presents the following peptides, Tyrosinase 369-377 , Tyrosinase 207-216 , gp100 209-217 , gp100 154-162 , MART-1 27-35 , HER-2/neu 789-797 , HER-2/neu 369-377 , C-lectin 8-16 , Pec60 20-29 , Pec60 25-33 .
  • Another embodiment of the present invention is a method of treating a disease or disease condition that results in an insufficient or inadequate immune response that is normally associated with Class I HLA molecules, wherein the treatment eliminates infected or transformed cells has been demonstrated to be achieved by CTLs.
  • Another embodiment of the present invention is a method of treating a disease or disease condition that results in an insufficient or inadequate immune response that is normally associated with Class I HLA molecules, wherein infected or transformed cells that have been shown to be susceptible to elimination by CTL are treated by the method comprising:
  • nnAPC non-naturally occurring antigen-presenting cell line
  • said nnAPC is capable of presenting up to about fifteen different peptide molecules that is associated with said disease or disease condition, preferably about ten different peptide molecules, simultaneously where each peptide is about six to twelve amino acids in length, preferably about eight to ten amino acids in length and in a concentration range of about 10 nM to 100 ⁇ M;
  • a cytokine such as, IL-2, IL-7 or CGM, preferably, IL-2, or IL-2 and IL-7 in combination;
  • peripheral blood monocyte suspension irradiating said peripheral blood monocyte suspension with a sufficient dose of ⁇ -radiation necessary to sterilize all components in the suspension, except the desired peripheral blood monocytes, such as a dose in the range of about 3,000 to 7,000 rads, preferably about 5,000 rads;
  • the present invention provides a non-naturally occurring antigen-presenting cell (nnAPC) derived from Drosophila melanogaster cells transfected with DNA encoding human class I HLA, binding, and co-stimulatory molecules for expression, wherein the nnAPC is capable of presenting up to fifteen different peptide molecules, preferably ten peptide molecules.
  • nnAPC non-naturally occurring antigen-presenting cell
  • nnAPC that presents peptides that are associated with various desired functions that enhance the treatment of the subject.
  • the nnAPC can present peptides associated with accessory molecules such as, lymphocyte function antigens (LFA-1, LFA-2 and LFA-3), intercellular adhesion molecule 1 (ICAM-1), T-cell co-stimulatory factors (CD2, CD28, B7) enhance cell-cell adhesion or transduce additional cell activation signals.
  • lymphocyte function antigens LFA-1, LFA-2 and LFA-3
  • IAM-1 intercellular adhesion molecule 1
  • CD2, CD28, B7 T-cell co-stimulatory factors
  • Another embodiment of the present invention provides a nnAPC that presents peptides that are associated with several types of cancers.
  • the peptides associated or derived from a breast cancer related polypeptide such as, HER-2/neu
  • Another embodiment of the present invention provides a method for manufacturing non-naturally occurring antigen-presenting cell (nnAPC) capable of presenting up to ten different peptide molecules simultaneously, said method comprising of the step:
  • pRmHa-3 plasmid from a pRmHa-1 expression vector, where said pRmHa-3 plasmid includes a metallothionein promoter, metal response consensus sequences and an alcohol dehydrogenase gene bearing a polyadenylation signal isolated from Drosophila melanogaster;
  • insect growth media are commercially available from a number of vendors, such as, SchneiderTM's Drosophila Medium, Grace's Insect Media, and TC-100 Insect Media.
  • insect growth media can be prepared by one of ordinary skill in the art.
  • the media will include components necessary to promote and sustain the growth of insects cells, such as, inorganic salts (for example, calcium chloride, magnesium sulfate, potassium chloride, potassium phosphate, sodium bicarbonate, sodium chloride, and sodium phosphate), amino acids various carbohydrate and chemical species (Imogene Schneider, Exp. Zool. (1964) 156(1): pg. 91).
  • the media can also include vitamins, minerals, and other components that aid in the growth of insect cells.
  • APC Antigen-presenting cells CD8+ CD8+ T cells
  • CTL Cytotoxic T lymphocyte E Effector Fas Also known as CD95, epitope on T cells
  • ICAM Intercellular adhesion molecule
  • LAK Lymphokine-activated killer cells LFA Lymphocyte function antigens MHC Major histocompatibility complex
  • nnAPC non-naturally occurring antigen-presenting cell NP Nuclear protein PBMC Peripheral blood mononuclear cell PBS Phosphate-buffered saline PCR Polymerase chain reaction RPMI Roswell Park Memorial Institute RWJPRI The R. W. Johnson Pharmaceutical Research Institute T Target TCR T cell antigen receptor TIL Tumor-infiltrating lymphocytes
  • tyrosinase 369-377 refers to the amino acid sequence YMNGTMSQV (SEQ ID NO: 1). Also included within this definition is the peptide of the sequence YMDGTMSQV (SEQ ID NO: 2), which results from a post-translational event that modifies the amino acid residue “N” of sequence YMNGTMSQV (SEQ ID NO: 1) to “D” resulting in the amino acid sequence of YMDGTMSQV (SEQ ID NO: 2) (Skipper et al., J. Exp. Med. (1996) 183:527-534).
  • tyrosinase 207-216 refers to the amino acid sequence FLPWHRLFLL (SEQ ID NO: 3).
  • gp100 209-217 or “gp100 209-217 ” refers to the amino acid sequence ITDQVPFSV (SEQ ID NO: 4).
  • gp100 154-162 or “gp100 154-162 ” refers to the amino acid sequence KTWGQYWQV (SEQ ID NO: 5).
  • MART-1 27-35 refers to the amino acid sequence AAGIGILTV (SEQ ID NO: 6).
  • HER-2/neu 789-797 or “HER-2/neu 789-797 ” refers to the amino acid sequence CLTSTVQLV (SEQ ID NO: 7).
  • HER-2/neu 369-377 or “HER-2/neu 369-377 ” refers to the amino acid sequence KIFGSLAFL (SEQ ID NO: 8).
  • C-lectin 8-16 refers to the amino acid sequence KMASRSMRL (SEQ ID NO: 9).
  • Pec60 20-29 or “Pec60 20-29 ” refers to the amino acid sequence ALALAALLVV (SEQ ID NO: 10).
  • Pec60 25-33 or “Pec60 25-33 ” refers to the amino acid sequence ALLVVDREV (SEQ ID NO: 11).
  • CD8 peptide 59-70 refers to the amino acid sequence of AAEGLDTQRFSG (SEQ ID NO: 12).
  • adoptive immunotherapy refers the administration of donor or autologous T lymphocytes for the treatment of a disease or disease condition, wherein the disease or disease condition results in an insufficient or inadequate immune response that is normally associated with Class I HLA molecules.
  • adoptive immunotherapy is an appropriate treatment for any disease or disease condition where the elimination of infected or transformed cells has been demonstrated to be achieved by CTLs.
  • disease or disease conditions include but are not limited to cancer and/or tumors, such as, melanoma, prostate, breast, colo-rectal, stomach, throat and neck, pancreatic, cervical, ovarian, bone, leukemia and lung cancer; viral infections, such as, hepatitis B, hepatitis C, human immunodeficiency virus; bacterial infections, such as tuberculosis, leprosy and listeriosis, and paracytic infections such as malaria.
  • cancer and/or tumors such as, melanoma, prostate, breast, colo-rectal, stomach, throat and neck, pancreatic, cervical, ovarian, bone, leukemia and lung cancer
  • viral infections such as, hepatitis B, hepatitis C, human immunodeficiency virus
  • bacterial infections such as tuberculosis, leprosy and listeriosis
  • paracytic infections such as malaria.
  • B7.1 refers to a co-stimulatory molecule associated with antigen-presenting cells.
  • BCNU refers to carmustine, also known as, 1,3-bis (2chloroethyl)-1-nitrosourea.
  • BSE bovine spongiform encephalitis
  • CD refers to clusters of differentiation, T lymphocytes (originally), B lymphocytes, monocytes, macrophages, and granulocytes grouped by antigen epitopes and function.
  • DTIC refers to dacarbazine, 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide.
  • ex vivo refers to a therapy where biological materials, typically cells, are obtained from a patient or a suitable alternate source, such as, a suitable donor, and are modified, such that the modified cells can be used to treat a pathological condition which will be improved by the long-term or constant delivery of the therapeutic benefit produced by the modified cells.
  • Treatment includes the re-introduction of the modified biological materials, obtained from either the patient or from the alternate source, into the patient.
  • a benefit of ex vivo therapy is the ability to provide the patient the benefit of the treatment, without exposing the patient to undesired collateral effects from the treatment.
  • cytokines are often administered to patients with cancer or viral infections to stimulate expansion of the patient's CTLs.
  • cytokines often cause the onset of flu like symptoms in the patients.
  • cytokines are used to stimulate expansion of the CTLs outside of the patient's body, and the patient is spared the exposure and the consequent side effects of the cytokines.
  • the subject can be treated concurrently with low level dosages of ⁇ interferon, ⁇ interferon and/or IL-2.
  • the expected effect of the interferons is to possibly sensitize the tumor cells to lysis by antigen specific CTL, and the effect of the IL-2 is to possibly enhance antigen specific CTL persistence.
  • HEPES N-2-hydroxyethylpiperazine-N′2-ethanesulfonic acid buffer.
  • HLA-A2.1 refers to a HLA Class I molecule found in approximately 45% of Caucasians.
  • MART-1 or “(melanoma antigen recognized by T-Cells-1” refers to a melanoma-associated antigen.
  • the amino acid and nucleic acid sequences, as well as various characteristics of this antigen are disclosed in U.S. Pat. No. 5,994,523, issued Nov. 30, 1999 entitled “Melanoma Antigens and Their Use in Diagnostic and Therapeutic Methods ”; U.S. Pat. No. 5,874,560, issued Feb. 23, 1999 entitled “Melanoma Antigens and Their Use in Diagnostic and Therapeutic Methods” ; and U.S. Pat. No. 5,844,075, issued Dec.
  • MAGE refers to a melanoma-associated antigen.
  • the amino acid and nucleic acid sequences, as well as various characteristics of this antigen are disclosed in U.S. Pat. No. 6,140,050, issued Oct. 31, 2000 entitled “Methods for Determining Breast Cancer and Melanoma by Assaying for a Plurality of Antigens Associated Therewith ”; U.S. Pat. No. 5,759,783, issued Jun. 2, 1998 entitled “Method of Screening for Cancer by Detecting Messenger RNA for a MAGE-XP Gene ”; and U.S. Pat. No. 5,662,907, issued Sep. 2, 1997 entitled “Induction of Anti-Tumor Cytotoxic T Lymphocytes in Humans Using Synthetic Peptide Epitopes.”
  • MPC-10 refers to a magnetic particle concentrator
  • NK cells refers to natural killer cells.
  • OKT3 refers to ORTHOCLONE OKT3, muromonab-CD3, anti-CD3 monoclonal antibody.
  • TEP-1,2 refers to Transporter Associated with Antigen Processing-1,2.
  • Th cells refers to Helper T cells, CD4 + .
  • tyrosinase refers to a protein associated with melanoma (Brichard et al., J. Exp. Med. (1993) 178:489-495; Robbins et al., Cancer Res. (1994) 54: 3124-3126).
  • U.S. Pat. No. 5,843,648, issued Dec. 1, 1998 entitled “ P 15 and Tyrosinase Melanoma Antigens and Their Use in Diagnostic and Therapeutic Methods ” discloses antigenic peptides and associated polynucleic acids related to tyrosinase in FIG. 7, Panels A to D, the aforementioned figure incorporated herein by reference.
  • gp100 refers to a melanoma antigen recognized by tumor infiltrating lymphocytes (TIL).
  • TIL tumor infiltrating lymphocytes
  • the TIL which recognize gp100 is associated with in vivo tumor rejection (Bakker et al., J. Exp. Med. (1994) 179:1005-1009; Kawakami et al., J. Immunol . (1995) 154:3961-3968).
  • Antigenic peptides related to gp100 are disclosed in U.S. Pat. No. 5,994,523, issued Nov. 30, 1999 entitled “ Melanoma Antigens and Their Use in Diagnostic and Therapeutic Methods ”; U.S. Pat. No. 5,874,560, issued Feb.
  • U.S. Pat. No. 5,994,523 discloses nucleic acid and amino acid sequences related to GP100 in FIGS. 4 and 5, respectively. Also disclosed are antigenic peptides derived from the amino acid sequences, including those identified as SEQ ID NOs: 27, 33, 34, 35, 36, 37, 38, 39, 40, and 41. All of the aforementioned FIGS. 4 and 5, and the peptides identified by SEQ ID NOs are herein incorporated by referenced.
  • melanoma refers to, but is not limited to, melanomas, metastatic melanomas, melanomas derived from either melanocytes or melanocytes related nevus cells, melanosarcomas, melanocarcinomas, melanoepitheliomas, melanoma in situ superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma, invasive melanoma or familial atypical mole and melanoma (FAM-M) syndrome.
  • melanomas melanomas
  • metastatic melanomas melanomas derived from either melanocytes or melanocytes related nevus cells
  • melanosarcomas melanocarcinomas
  • melanoepitheliomas melanoepitheliomas
  • melanoma in situ superficial spreading melanoma nodular melanoma
  • Such melanomas in mammals may be caused by, chromosomal abnormalities, degenerative growth and developmental disorders, mitogenic agents, ultraviolet radiation (UV), viral infections, inappropriate tissue expression of a gene, alterations in expression of a gene, and presentation on a cell, or carcinogenic agents.
  • UV ultraviolet radiation
  • the aforementioned melanomas can be diagnosed, assessed or treated by methods described in the present application.
  • C-lectin refers to a peptide of the sequence that has been found to be associated with ovarian cancer.
  • MHC major histocompatibility complex
  • epitope refers to a peptide derived from an antigen capable of causing a cellular immune response in a mammal. Such peptides may also be reactive with antibodies from an animal immunized with the peptides. Such peptides may be about five to twenty amino acid in length preferably about eight to fifteen amino acids in length, and most preferably about nine to ten amino acids in length.
  • Pec60 refers to a peptide of the sequence that has been found to be associated with ovarian and breast cancer.
  • analog includes any polypeptide having an amino acid residue sequence substantially identical to the sequences of the present invention, specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the functional aspects of the present invention as described herein.
  • conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid or another.
  • the term “conservative substitution” also includes the use of a chemically derivatized residue in place of a non-derivatized residue.
  • the term “chemical derivative” refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group.
  • derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
  • Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides.
  • Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives.
  • the imidazole nitrogen of histidine may be derivatized to form N-imbenzylhistidine.
  • chemical derivatives those proteins or peptides which contain one or more naturally-occurring amino acid derivatives of the twenty standard amino acids. For examples: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.
  • Proteins or polypeptides of the present invention also include any polypeptide having one or more additions and/or deletions or residues relative to the sequence of a polypeptide whose sequence is encoded is the corresponding nucleic sequence of the present invention, so long as the requisite activity is maintained.
  • HER-2/neu refers to an oncogene, which express or over-express, one or more membrane-associated, receptor-like oncogene proteins.
  • cancers which have been found to be associated with expression or over-expression of HER-2/neu are certain breast, stomach, ovarian colon and salivary gland cancers.
  • the HER-2/neu oncogene is a member of the tyrosine protein kinase family of oncogenes and shares a high degree of homology with the epidermal growth factor receptor (EGFR).
  • EGFR epidermal growth factor receptor
  • HER-2/neu appears to induce malignancies through quantitative mechanisms that result from increased or deregulated expression of an essentially normal gene product.
  • U.S. Pat. No. 6,075,122, issued Jun. 13, 2000 entitled “ Immune Reactivity to HER- 2/ neu Protein for Diagnosis and Treatment of Malignancies in Which the HER- 2/ neu Oncogene is Associated” discloses peptides that elicit CD8 + T cell responses at column 12, line 31 to column 13, line 7, identified according to SEQ ID numbers are herein incorporated by reference.
  • HER-2/neu is the protein product of the HER-2/neu oncogene.
  • the HER-2/neu gene is amplified and the HER-2/neu protein is over-expressed in a variety of cancers including breast, ovarian, colon, lung and prostate cancer.
  • HER-2/neu is related to malignant transformation. It is found in 50% to 60% of ductal in situ carcinoma and 20% to 40% of all breast cancers, as well as a substantial fraction of adenocarcinomas arising in the ovaries, prostate, colon and lung.
  • HER-2/neu is intimately associated not only with the malignant phenotype, but also with the aggressiveness of the malignancy, being found in one-fourth of all invasive breast cancers.
  • HER-2/neu over-expression is correlated with a poor prognosis in both breast and ovarian cancer.
  • HER-2/neu is a transmembrane protein with a relative molecular mass of 185 kd that is approximately 1255 amino acids (aa) in length. It has an extracellular binding domain (ECD) of approximately 645 aa, with 40% homology to epidermal growth factor receptor (EGFR), a highly hydrophobic transmembrane anchor domain (TMD), and a carboxyterminal cytoplasmic domain (CD) of approximately 580 amino acids with 80% homology to EGFR.
  • ECD extracellular binding domain
  • Ongoing research involving oncogenes has identified at least forty oncogenes operative in malignant cells and responsible for, or associated with, transformation. Oncogenes have been classified into different groups based on the putative function or location of their gene products (such as the protein expressed by the oncogene). Oncogenes are believed to be essential for certain aspects of normal cellular physiology.
  • Cancer continues to be a major health problem, despite significant progress made in the area of treatment.
  • the standard treatment regimes of chemotherapy, radiation therapy, surgical intervention and combinations of the three often fail to produce a long lasting cure.
  • the cancer patient having undergone the treatment often relapses back to the disease condition after some period of time, further exacerbating the problem, is the severity of these treatment regimes to the patient.
  • a cure for metastatic melanoma has not been achieved using conventional chemotherapy.
  • Response rates of 35% to 50% have been reported with the Dartmouth regimen of combination chemotherapy (DTIC, cis-platin, BCNU and tamoxifen), but the duration of survival has remained at six to ten months.
  • High rates of remission have been reported for aggressive “high dose intensity” chemotherapy and repletion of hematopoeisis with autologous bone marrow transplants. Nevertheless, the median duration of survival was short, approximately four months.
  • LAK lymphokine-activated killer cells
  • TIL tumor-infiltrating lymphocytes
  • cytokines like IL-2, ⁇ -interferon and ⁇ -interferon.
  • cytokines like IL-2, ⁇ -interferon and ⁇ -interferon.
  • side effects such as, nausea, and fever.
  • Cytolytic T cells are the main line of defense against viral infections. CD8 + lymphocytes specifically recognize and kill host cells that are infected by a virus. Theoretically, it should be possible to harness the immune system to combat other types of diseases including cancer. However, few in vitro/ex vivo procedures have been available for specifically activating CTLs. The identification of key melanoma antigens noted above and a method for specific in vitro activation CTLs described below now allow testing of the concept of adoptive immunotherapy of metastatic melanoma.
  • TTLs CD8 + lymphocytes
  • the first signal which imparts specificity, consists of presentation to the CD8 + cell of an immunogenic peptide fragment (epitope) of the antigen bound to the Class I MHC (HLA) complex present on the surface of antigen-presenting cells (APCs).
  • HLA Class I MHC
  • APCs antigen-presenting cells
  • Binding to the T cell receptor is necessary but not sufficient to induce T cell activation, and usually will not lead to cell proliferation or cytokine secretion. Complete activation requires a second co-stimulatory signal(s), these signals serve to further enhance the activation cascade.
  • B7 and cell adhesion molecules (integrins) such as ICAM-1 assist in this process by binding to CD28 and LFA-1, respectively, on the T cell.
  • CD8 + cell When a CD8 + cell interacts with an antigen-presenting cell bearing an immunogenic peptide (epitope) bound by a Class I MHC molecule in the presence of appropriate co-stimulatory molecule interactions, the CD8 + cell becomes a fully activated cytolytic T cell.
  • Lymphocyte-mediated cell killing involves a sequence of biological events beginning with the binding of the CD8 + CTL to an antigen-bearing target (tumor) cell by means of the recognition process described above for T cell activation.
  • FIG. 1 The interaction between CD8 + cells and antigen-presenting cells or target (tumor) cells as described above is depicted in FIG. 1.
  • the interaction begins with the binding of antigen in association with an MHC Class I molecule on the APC or target cell to the T cell antigen receptor (TCR).
  • Accessory molecules such as lymphocyte function antigens (LFA-1, LFA-2 and LFA-3), intercellular adhesion molecule 1 (ICAM-1), T cell co-stimulatory factors (CD2, CD28, B7) enhance cell-cell adhesion or transduce additional cell activation signals.
  • the CTL kills the target cell through the action of soluble cytolytic mediators (perforin and granzymes stored in cytoplasmic granules in the T cell) and a CTL surface molecule (Fas ligand).
  • soluble cytolytic mediators perforin and granzymes stored in cytoplasmic granules in the T cell
  • Fas ligand a CTL surface molecule
  • target cells die by necrosis (membrane perforation and organelle destruction) or apotosis (chromatin condensation, DNA fragmentation and membrane blebbing).
  • FIG. 2 The mechanisms of lymphocyte-mediated cytolysis is graphically depicted in FIG. 2.
  • Panel A of FIG. 2 after binding to the target cell, cytoplasmic granules in the CTL are rapidly reoriented toward the target cell for release of granules containing perforin and granzymes into the intercellular space. These proteolytic enzymes form pores in the plasma membrane of the target cell eventually leading to cell necrosis.
  • Panel B after binding to the target cell, the level of Fas ligand expression on the CTL increases. The interaction of Fas ligand and the Fas receptor on the target cell leads to apoptosis.
  • Proteases such as CPP32 and others related to IL-1b-converting enzyme (ICE) have been implicated in the induction of apoptosis. It is possible to use naturally-occurring antigen-presenting cells, for example, dendritic cells, macrophages, autologous tumor cells for in vitro CD8 + activation. However, the efficiency of activation following this approach is low. This is because the Class I molecules of native APCs contain many other types of peptide epitopes besides tumor epitopes. Most of the peptides are derived from normal innocuous cell proteins, resulting in a dilution of the number of active native APCs that would actually be effective against a tumor (Allison et al., Curr. Op. Immunol. (1995) 7:682-686).
  • ICE IL-1b-converting enzyme
  • a more direct and efficient approach to this problem is to specifically activate CD8 + cells only with those epitopes relevant to combating a specific disease, (such as, cancer) or tumor specific antigens (such as, melanoma-specific antigens).
  • a specific disease such as, cancer
  • tumor specific antigens such as, melanoma-specific antigens.
  • an artificial antigen presenting cell is created by expressing MHC Class I molecules in Drosophila melanogaster (fruit fly) cells. Since Drosophila does not have an immune system, the TAP-1,2 peptide transporters involved in loading peptide epitopes onto class I molecules are absent. As a result, the class I molecules appear on the Drosophila cell surface as empty vessels.
  • this feature eliminates the need for in vivo stimulation of the immune system with high doses of various cytokines. Thereby resulting in a treatment that fore goes the side effects caused by cytokines.
  • the subject can be treated concurrently with low level dosages of ⁇ interferon, ⁇ -interferon, and/or IL-2.
  • Eliminating the need for in vivo stimulation with cytokines provides an improvement to the quality of patient care.
  • Treatment regimes that include the administration of cytokines to patients often result in the patient developing flu-like symptoms, such as nausea, vomiting, and fever. These side reactions are generally not life threatening, although a particularly severe reaction occurring in a patient who is already in a weaken condition could result in a life endangering situation. Another consideration is the adverse impact such side reactions have on patient acceptance and compliance of an otherwise beneficial treatment regime.
  • Removing the need for in vivo stimulation with cytokines results in a treatment regime that improves the comfort of the patient, and provides the clinician with an effective method of treatment that his or her patient is more likely to comply with.
  • APC antigen presenting cell
  • APC systems have been utilized to generate antigen-specific CTL's to single epitopes: 1) human dendritic cells (DC) pulsed with defined peptides; 2) peripheral blood mononuclear cells (PBMCs) which have been driven to lymphoblasts and pulsed with peptides; 3) lymphoblastoid cell lines (LCL) where the natural peptides are acid-stripped and loaded with the peptides of interest; 4) Drosophila cells engineered to express empty class I molecules; and Mouse 3T3 cells transfected with human class I and co-stimulatory molecules (J. B. Latouche and M. Sadelain, Nature Biotech (2000) 18:405-409).
  • DC dendritic cells
  • PBMCs peripheral blood mononuclear cells
  • LCL lymphoblastoid cell lines
  • DCs Dendritic cells
  • HLA molecules HLA molecules
  • Drosophila cell stimulation usually resulted in CTLs directed against up to ten different types of peptides. This provides CTLs that are active to each of the ten peptides.
  • Immature DCs were generated by culturing for one week autologous monocytes in the presence of IL-4 and GM-CSF. Mature DCs were obtained from immature DCs by addition of TNF a to the culture medium twenty-four hours prior to harvesting. DCs (immature and mature) were harvested, pulsed with peptides and mixed with purified CD8 cells following the procedure used for the stimulation of CD8 cells and peptide-pulsed Drosophila cells.
  • Drosophila cells were found to be generally better stimulators than DC when evaluated for tyrosinase peptide epitope 689 , as shown in FIG. 7 . Further, DCs displaying either the immature or mature phenotype (FIG. 8) were not as efficient as Drosophila cells in eliciting specific CTL responses when defined peptides were used to pulse the APCs. This is particularly surprising, because of the dominant role played by DCs in the immune system. A comparison study with one donor was performed, as shown in FIG. 9. Specific killing was generated against four different peptides when using fly cells as stimulators whereas immature DCs resulted in marginal specific killing and mature DCs resulted in specific killing against only one of the four peptides used for stimulation.
  • CD8 + cells isolated from leukapheresis samples by positive selection with anti-CD8 antibody are stimulated against four different melanoma associated peptides presented by Drosophila cells expressing Human Class I molecules (HLA-A2.1), B7.1, ICAM-1, LFA-3 and B7.2.
  • CD8 + cells are re-stimulated for two rounds with autologous monocytes loaded with the peptide epitope in the presence of IL-2 and IL-7.
  • CTLs are non-specifically expanded with OKT3 and IL-2.
  • CTL activity is measured against Malme 3M cells and purity of CD8 + T cells is assessed by flow cytometry.
  • the manufacturing processes and protocols are done according to Good Laboratory Practices and Good Manufacturing Practices. “Good Laboratory Practices” and “Good Manufacturing Practices” are standards of laboratory and manufacturing practices which are set by United States Food and Drug Administration, and are readily known to those of skill in the art.
  • the CTLs are monitored for identity, viability, CTL activity, sterility, and endotoxin content.
  • peptide epitopes suitable for use in the methods of the present invention to treat breast and ovarian cancers are shown in the following Table 1. It is readily apparent to those of ordinary skill in the art that a wide variety of peptide epitopes in addition to those listed in the following Table 1 will also be suitable for use in the methods of the present invention to treat breast and ovarian cancers, provided that such peptides are T cell epitopes.
  • HLA Peptide Name PRI # AKA (SEQ ID NO:) Binding Prediction Her-2/neu 789-797 826 E90 CLTSTVQLV 160 (SEQ ID NO:7) 48-56 827 D113 HLYQGCQVV (SEQ ID NO:13) 369-377 835 E75 KIFGSLAFL 481 (SEQ ID NO:8) 654-662 837 GP2 IISAVVGIL (SEQ ID NO:14) 650-658 838 GP1 PLTSIISAV (SEQ ID NO:15) 773-782 861 VMAGVGSPYV (SEQ ID NO:16) 851-859 862 E89 VLVKSPNHN 118 (SEQ ID NO:17) 971-979 863 C85 ELVSEFSRM (SEQ ID NO:18) AES Amino enhance
  • the Schneider S2 cell line was prepared from Drosophila melanogaster (Oregon-R) eggs according to published procedures and has been deposited with the American Type Culture Collection (CRL 10974). S2 cells are grown in commercial Schneider's Drosophila medium supplemented with 10% fetal bovine serum.
  • the pRmHa-3 plasmid vector for expressing MHC Class I and co-stimulatory proteins in S2 cells was derived from the pRmHa-1 expression vector constructed as described in the literature. It contains a metallothionein promoter, metal response consensus sequences and an alcohol dehydrogenase gene bearing a polyadenylation signal isolated from Drosophila melanogaster.
  • HLA-A2.1 and ⁇ -2 microglobulin Reverse transcription-PCR from K562 cells using primers derived from the published sequence
  • B7.1 Reverse transcription-PCR from K562 cells using primers derived from the published sequence
  • ICAM-1 Reverse transcription-PCR from K562 cells using primers derived from the published sequence
  • B7.2 Reverse transcription-PCR from HL-60 cells (ATCC CCL-240) using primers derived from the published sequence
  • LFA-3 Reverse transcription-PCR from HL-60 cells (ATCC CCL-240) using primers derived from the published sequence
  • Complementary DNAs were individually inserted into the pRmHa-3 vector.
  • S2 cells were transfected with a mixture of HLA-A2.1, B7.1 and ICAM-1 plasmid DNAs and the phshneo plasmid using the calcium phosphate precipitation method.
  • Stably transfected cells were selected by culturing in Schneider's medium containing geneticin. Twenty-four hours before use, expression of the transfected genes was induced by addition of CuSO 4 . The level of expression was assessed by flow cytometry using anti-HLA-A2.1, anti-B7.1 and anti-ICAM-1 antibodies. HLA expression by greater than 30% of the cells is necessary for efficient in vitro activation of CD8 + lymphocytes.
  • CD8 + cells are isolated from leukapheresis samples by positive selection using the DynabeadsTM isolation procedure (Dynal).
  • An anti-human CD8 mouse monoclonal antibody (50 ⁇ g/ml in human gamma globulin [Gammagard®]) is added to washed cells in Dulbecco's PBS supplemented with 1% human serum albumin (Baxter-Hyland) and 0.2% Na citrate. After incubation at 4° C.
  • the cells are washed and re-suspended in the same buffer containing Dynal magnetic beads (DynabeadsTM) coated with sheep anti-mouse IgG at a bead to cell ratio of 1:1.
  • the cells and beads are placed into a sterile tube and gently mixed at 4° C. for forty-five minutes.
  • the antibody-bound cells are removed magnetically using the MPC-1® separator according to the manufacturer's instructions (Dynal).
  • Dissociation of the CD8 cell-bead complex is achieved by incubation at 37° C. for forty-five minutes in the presence of CD8 peptide 59-70 (AAEGLDTQRFSG; SEQ ID NO: 12).
  • CD8 + cells Free beads are removed magnetically and the CD8 cells are counted and analyzed by flow cytometry to evaluate purity. Recovery of CD8 + cells is typically greater than 80%.
  • Table 1 summarizes the cell composition of fourteen separate CD8 + preparations from normal human PBMC preparations by positive selection with anti-CD8 antibody.
  • Transfected Drosophila S2 cells are incubated in Schneider's medium (10 6 cells/ml) supplemented with 10% fetal calf serum and CuSO 4 at 27° C. for twenty-four hours. Cells are harvested, washed and re-suspended in Insect X-press medium (BioWhittaker) containing 100 ⁇ g/ml human tyrosinase 369-377 . Following incubation at 27° C. for three hours, the S2 cells are mixed with CD8 + cells at a ratio of 1:10 in RPMI medium (Gibco) supplemented with 10% autologous serum. The cell mixture is incubated for four days at 37° C. during which the Drosophila cells die off. On Day five, IL-2 (20 U/ml) and IL-7 (30 U/ml) are added to selectively expand the tyrosinase-specific CTL population.
  • Schneider's medium (10 6 cells/ml) supplemented with 10% fetal cal
  • Adherent monocytes are loaded with the tyrosinase epitope by incubation for 90 minutes in Hepes-buffered RPMI medium containing 10% autologous serum and 10 ⁇ g/ml tyrosinase 369-377 .
  • the supernatant is removed and the Drosophila-activated CD8 + cell suspension (3 ⁇ 10 6 cells/ml in RPMI medium with 10% autologous serum) is added at a ratio of 10 CD8 + cells to 1 adherent monocyte.
  • IL-2 (20 U/ml) and IL-7 (30 U/ml) are added with a medium change to selectively expand the tyrosinase-specific CTL population.
  • Effector cells are non-specifically expanded by culturing them in RPMI medium supplemented with autologous serum, anti-CD3 monoclonal antibody (OKT®3), IL-2 and ⁇ irradiated autologous PBMCs.
  • RPMI medium supplemented with autologous serum, anti-CD3 monoclonal antibody (OKT®3), IL-2 and ⁇ irradiated autologous PBMCs.
  • Malme 3M cells are used as target cells in a 51 Cr release assay. 5 ⁇ 10 6 Malme 3M cells in RPMI medium containing 4% fetal calf serum, 1% HEPES buffer and 0.25% gentamycin are labeled at 37° C. for one hour with 0.1 mCi 51 Cr. Cells are washed four times and diluted to 10 5 cells/ml in RPMI with 10% fetal bovine serum (HyClone). In a 96-well microtiter plate, 100 ⁇ l effector CTLs and 100 ⁇ l peptide-loaded, 51 Cr-labeled Malme 3M target cells are combined at ratios of 100:1, 20:1 and 4:1 (effector: target).
  • K562 cells are added at a ratio of 20:1 (K562:Malme 3M) to reduce natural killer cell background lysis.
  • Non-specific lysis is assessed using the non-tumor HLA-A2.1 fibroblast cell line, Malme 3. Controls to measure spontaneous release and maximum release of 51 Cr are included in duplicate. After incubation at 37° C. for six hours, the plates are centrifuged and the supernatants counted to measure 51 Cr release.
  • CD8 + cells before and after in vitro activation, were analyzed for a number of cell surface markers using fluorescent monoclonal antibodies and FACS analysis. Results from a typical activation protocol using cells from a healthy donor is shown in Table 2.
  • Table 2 TABLE 3 Flow Cytometry Analysis of In Vitro Activated CD8+ Cells PRE-ACTIVATION POST-ACTIVATION MARKER/CELL TYPE Mean % Mean % CD8 T cell 98 99 TCR ⁇ T cell receptor 98 92 CD 44 lymph node horming 91 99 receptor CD45RO memory T cell 58 88 CD45RA 41 31 CID62L HEV homing 24 38 receptor CD56 NK cell 1 11 CD25 activated T cell 0.1 29
  • CTL preparations will be assayed for sterility and endotoxin content.
  • Eligibility for treatment required patients to have histologically-documented, unresectable malignant melanoma that was measurable or evaluable, and the HLA-A2 haplotype.
  • Pretreatment evaluation included radiologic evaluation of the brain by MRI or CT scan, CT scanning of the chest and abdomen, and physical examination, especially of the skin and lymph nodes.
  • the total number of patients treated was fifteen (nine male and six female). The ages ranged from 33 to 75 years with an average of 58 years. The average duration of metastatic disease was 1.5 years.
  • a pretreatment skin test to determine whether a state of anergy existed was performed on 14/15 patients with 5/14 testing negative for all seven of the common antigens evaluated.
  • HLA-A2 haplotype Patients were screened for the HLA-A2 haplotype by FACS analysis with an HLA-A2 specific monoclonal antibody (BB7.2). Subtyping was performed by PCR analysis. All, but one of the patients, were HLA-A*0201, the exception (patient 08) was HLA-A*0205.
  • T cells infused ranged from a minimum of 4 ⁇ 10 7 (patient 08) to a maximum of 3.2 ⁇ 10 9 (patient 13).
  • Patients were entered into a second, third or fourth cycle of treatment based on their clinical status at the end of each cycle.
  • the number of PBMCs obtained from the aphaeresis samples tended to be lower in patients undergoing additional cycles, especially if the start of the subsequent cycle was close to the end of the previous one. This is attributed to persistent lymphopenia due to the IFN ⁇ -2b administered during the previous cycle.
  • the total number of naive CD8 + T cells isolated was dependent on its percentage in each of the PBMC preparations.
  • the percent of CD8 + T cells varied between 8% to 31% among the patients.
  • the obtained expansion factor also contributed to the final cell numbers and ranged from 0.1-6.0 fold.
  • the procedure for generating CTLs ex vivo is taught in the Specification and Example 1, above.
  • biopsy B Following five days of subcutaneous injections of 10 MU/m 2 , a dramatic increase in these two markers was noted (biopsy B). For tyrosinase and gp100, immunohistochemical staining was negative to weakly positive, respectively in the pretreatment samples (biopsy A). After the initial five-day IFN ⁇ dose, and thirteen additional treatments, expression of these later antigens was increased in the stained tissue samples (biopsy C).
  • CTLs generated from all patients were evaluated on the day of release against peptide-loaded T2 targets, an HLA-A2 melanoma cell line (Malme3M) and an autologous melanoma line, if biopsy material was available to establish a line.
  • HLA-A2 melanoma cell line Malme3M
  • An autologous melanoma line if biopsy material was available to establish a line.
  • Each prepared dose of cells was evaluated for its cytolytic activity.
  • Peptide-loaded T2 cells presenting either each peptide alone, or all four peptides simultaneously, were used to determine the specificity of the CTL response generated for each patient.
  • the ability to lyse endogenously-expressed, melanoma-associated antigen-bearing cells was assessed with an HLA-A2 matched line or an autologous tumor line.
  • antigen-specificity was evaluated with an established method for detecting intracellular gamma interferon production, made in response to a specific peptide stimulus.
  • the CTLs generated at the end of the ex vivo protocol were evaluated by this method.
  • the percent of cells specific for each of the peptides was recorded individually.
  • the total number of specific cells in each bulk CD8 culture from patient 13 was calculated by adding each of the peptide specificities detected in that population of T cells. An increase in the total number of specific cells could be detected with each successive treatment cycle.
  • Biopsy samples from all patients prior to, during and after treatment would have been ideal. However, the experimental conditions allowed for biopsy samples from only a limited number of patients. Tumor tissue was obtained from five of the fifteen patients enrolled in the study. In two patients (patients 08 and 13) biopsy samples were available at five and six weeks post T cell therapy, respectively. Examination of the tissue samples revealed the presence of both infiltrating CD8 and CD4 cells. One of the tumor samples was taken from a skin lesion in the occipital region of the scalp, which increased in size by the time of the follow up examination, four weeks after a second infusion of T cells. The biopsy revealed necrosis of the tissue that was heavily infiltrated with lymphocytes.
  • the other biopsy was from the head of a femur bone, removed during hip replacement surgery.
  • the skin lesion from patient 08 was strongly positive (4+) for both a general class I, and a specific HLA-A2 marker.
  • Tyrosinase and gp100 were weakly positive (1+ and 2+, respectively), while MART-1 was negative in this same sample.
  • Regions of the biopsy from patient 13 were also necrotic, with more heterogeneous staining; distinct populations of tumor cells lacking expression of the HLA-A2.1 molecule, and one or more of the MAAs.
  • intact tissue regions revealed strong class I (4+), and all of the melanoma-associated antigens.
  • lymphocytic infiltrations in this later sample appeared to surround the tumor nodules rather than to deeply infiltrate them.
  • the highest percentage of cells directly associated with the tumor were CD8 cells.
  • CT scans were part of the pretreatment screening criteria and the post treatment follow-up examination.
  • Patient 10 received a single infusion of 8 ⁇ 10 8 CTLs (Jul. 7, 1999) five weeks after the pretreatment scan (Jun. 23, 1999).
  • a CT scan of the chest was repeated one month after the infusion (Aug. 27, 1999)
  • a dramatic decrease in the size of a lung lesion was noted.
  • patient 14 underwent a chest CT scan as part of the enrollment process (Sep. 10, 1999), three and one-half weeks before a first infusion with 6.6 ⁇ 10 8 cells (Oct. 5, 1999).
  • a follow-up CT scan Jan.
  • HER-2/neu is a proto-oncogne with homology to EGFR that is amplified and over-expressed in many human cancers, largely adenocarcinomas of the breast, ovary and colon. It is often associated with aggressive disease and can be an indicator of a poor prognosis. It has been studied in several clinical trials as a possible target for these types of cancers.
  • transfected Drosophila cells have the unique ability to present up to ten different peptide epitopes (FIG. 10)
  • These four different HER-2 peptides represent weak to moderate binders to the HLA-A2.1 molecule presented on the surface of the transfected Drosophila cells.
  • the majority of the tumor-associated proteins that we target are self-antigens and such would be expected to have the high affinity for the class I molecule that is seen with viral peptides.
  • the low to moderate binders generally generate CTLs that lyse the tumor very efficiently. This was demonstrated with the MART-1 peptide which is a low affinity binder on the Drosophila cells (FIG. 3), yet represents an epitope that routinely generate potent CTLs capable of lysing both peptide-loaded target cells (T2), or more importantly, melanoma cells (Malme3M) (FIG. 12).
  • HER-2/neu is a member of the EGF-R family and functions as a growth factor receptor.
  • HER-2 protein is expressed during fetal development in humans. In adults, the protein is weakly detectable in epithelial cells of many normal tissues. In normal cells the HER-2 gene is present as a single copy. Amplification of the gene and/or over-expression of the associated protein has been identified in many human cancers including breast, ovarian, uterine, stomach and adenocarcinoma of the lung. Sequence differences between HER-2 and EGF-R receptor are noted in Table 5. Three of the four HER-2 peptides we have evaluated have three or more amino acids changes between the two proteins. A single amino acid change is sufficient to discriminate between the two proteins.
  • the CTLs generated were evaluated for antigen-specificity.
  • Drosophila cells were loaded with a combination of the four HER-2 peptides.
  • the bulk CD8 culture was evaluated for antigen-specificity.
  • T2 cells loaded with each of the immunizing peptides were used as target cells.
  • FIG. 14 a typical response is depicted.
  • the bulk culture contains specificity for each of the four HER-2 peptides.
  • the anti-tumor response was assessed on an ovarian tumor cell line (ATCC; HTB-77).
  • HER-2 specific effectors representing the individual peptides were evaluated to confirm the presentation of each of the peptide epitopes on this tumor cell line.
  • a breast adenocarcinoma cell line (ATCC; HTB-131), transfected with HLA-A2.1 was also evaluated for the ability to demonstrate tumor lysis with the HER-2 specific peptide effectors. CTLs specific for peptide 861 could lysis this tumor cell line when transfected with HLA-A2.1 (FIG. 16).
  • the HTB-77/A2.1 cell line requires a pretreatment with IFN ⁇ to demonstrate peptide-specific lysis.
  • the cells were treated with 500 U/ml of IFN ⁇ (specific activity of 25 ng/ml) for twenty-four hours prior to the initiation of the 51 Cr-release assay.
  • FIG. 17 the addition of the IFN ⁇ resulted in enhanced lysis of the HLA-A2.1 transfected cell line.
  • a FACS analysis was performed to determine the levels of these molecules after both twenty-four and forty-eight hours of induction.
  • FIG. 18, Panels A and B depict the FACS analysis results.
  • Synthetic peptides were made by standard Fmoc chemistry using a peptide synthesizer (Gilson Company, Inc.) All peptides were purified to >95% purity by reverse-phase HPLC on a C-8 column. Purity and identity were established using a mass spectrometer with electrospray ionization.
  • peptide 819 was MART-1 specific (AAGIGILTV SEQ ID NO:6), 817 and 853 were both gp100 peptides (ITDQVPFSV SEQ ID NO:4 and KTWGQYWQV SEQ ID NO:5, respectively), tyrosinase-specific peptides were 689 and 792, with 792 representing the post translational modified version (YMDGTMSQV SEQ ID NO:2) of the native sequence (YMNGTMSQV SEQ ID NO:1) represented by peptide 689.
  • Peptides 826 ( CLTSTVQLV SEQ ID NO:7) and 835 (KIFGSLAFL SEQ ID NO:8) represented HER-2/neu sequences from the intracellular and extracellular domains, respectively of the p185 protein.
  • Pec60 20 (ALALAALLVV SEQ ID NO:10)
  • Pec60 25 (ALLVVDREV SEQ ID NO:11) were overlapping sequences representing a mucinous protein detected in ovarian tumor lines.
  • C-lectin also was a protein detected in ovarian tumor cell lines and a peptide from its sequence (C-lectin 8 ) is represented by KMASRSMRL SEQ ID NO:9.
  • Standard 51 Cr-release assays were performed to determine CTL effector cell recognition of melanoma-associated peptide epitopes loaded onto T2 cells.
  • Harvest 3 ⁇ 106 T2 cells were grown in RPMI+10% FBS (media). 0.1 mCi of 51 Cr was added and incubated at 37° C. in a water bath. Labeled cells were added to 10 ml of 4% wash (RPMI+4% FBS) and pellet, washed two additional times, and re-suspended in media to a final concentration of 0.2 ⁇ 106/mL to record radioactivity of spontaneous versus detergent lysed cells. The cells were pulsed with the appropriate peptide(s) at 20 ⁇ g/mL for thirty minutes. 50 ⁇ L was added to each 96-well plate each containing CD8 effector cells at 10, 2, 0.4, and 0.08 ⁇ 10 6 /mL, which was incubated at 37° C. for six hours, spun and harvested for supernatant.
  • the cells were labeled with FITC- or PE conjugated monoclonal antibodies by incubation at 4° C. for 30 minutes in FACS buffer (1% BSA, 0.02% NaN 3 in PBS), followed by a wash in the same buffer. Cells were fixed in 0.5% formaldehyde prior to data acquisition and analysis on a FACScan flow cytometer (Becton Dickinson) with its CellQuest software. Nonspecific staining was measured with the same secondary antibody used to label purified primary antibodies, or an isotype-matched control when the primary antibodies were directly labeled.
  • Tetrameric staining was performed with HLA-A2.1 specific HIVgag tetrameric molecules (Beckman Coulter) harboring the sequence SLYVTVATL SEQ ID NO:43 as a negative control.
  • HER-2 specific tetramers were made with the sequences CLTSTVQLV (826 SEQ ID NO:7), KIFGSLAFL (835 SEQ ID NO:8), or VMAGVGFSPYV (861 SEQ ID NO:16) peptides.
  • PE-labeled tetrameric HLA-A2.1-peptide complexes were used in conjunction with fluorescein isothicyante (FITC)-labeled anti-human CD8a (BD PharMagin) monoclonal antibodies to stain epitope-specific CD8+ T cells as described in package insert. Samples were analyzed by two-color flow cytometry on a Becton Disckenson FACScan, and gated CD8+ T cells were examined for staining with tetrameric HLA-A2.1-peptide complexes.
  • FITC fluorescein isothicyante
  • BD PharMagin anti-human CD8a
  • Antigen 125 is an epithelial cell marker expressed by ovarian tumors and some ovarian cell lines. About 85% of ovarian cancer patients have an increased serum CA125 and is therefore commonly used as a serum tumor marker. (Cancer Letters (1999, Oct.) 145(1-2) pg. 133-141) MUC-1 Mucin is a transmembrane glycoprotein expressed on both normal and malignant epithelium. The underglycosylated form of MUC-1 over-expressed on the cell surface of many human adenocarcinomas such as breast and ovarian cancer, as well as hematological malignancies including multiple myeloma and B-cell Lymphoma.
  • HER-2/neu A proto-oncogene (HER-2) encoding a transmembrane protein similar in sequence and structure to EGF-R. HER-2/neu is over-expressed as much as 200 fold over normal tissues in breast and ovarian tumors. It has also been identified in renal cell and lung carcinomas.
  • NY-ESO-1 A cancer-testes antigen found in 30% of breast, prostate and ovarian cancers, lung cancer, bladder cancer, head and neck cancer and melanoma. Patients who have cancers with tumors expressing this antigen usually have circulating antibodies against it as well. (J. Immunology (2000) vol. 165 pg.
  • CEA Carcinoembryonic antigen is a tumor-associated antigen frequently expressed in epithelial tumors (colon, breast, lung). CEA levels in the serum can correlate with disease stage and is used to monitor treatment and reoccurrence of disease.
  • MAGE-3 A cancer-testis antigen expressed on 70-80% of metastatic melanoma lesions and cell lines. It is a member of the family of melanoma associated or MAGE proteins. In addition, MAGE-3 has been found in 20-60% of epithelial tumors (colon, breast, lung, gastric carcinomas).
  • AES The amino enhancer of split protein is part of a set of transcriptional repressors encoded by the Enhancer of split genes. This tumor antigen was identified in tumor- associated lymphocytes of ovarian and breast tumors.
  • HTR Telomerase(hTR) is a specialized type of reverse transcriptase (hTRT or hTERT) that catalyzes the synthesis and extension of telomeric DNA. The activity of this enzyme is elevated in about 90% of all human tumors including cancers of the breast, thyroid, bladder, cervix, prostate, colon, pancreas and stomach.
  • Cancer Research (2001, Dec) 61(23) pg. 8366-8370)

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