JP2005139118A - Cell therapy method for treating tumor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for isolating, stimulating and expanding a naive cytotoxic T lymphocyte precursor cell (CTLp) to an antigen-specific effector capable of lysing a tumor cell in vivo, to solve the problem wherein T cell response is often diminished in human with a compromised immune system. <P>SOLUTION: The present ex vivo protocol produces a fully functional effector. Artificial antigen presenting cells (AAPCs; Drosophila melanogaster) transfected with human HLA class I and defined accessory molecules, are used to stimulate CD8<SP>+</SP>T cells from normal donors and cancer patients. The class I molecules expressed to a high density on the surface of the Drosophila cells are empty, enabling efficient loading of multiple peptides that results in the generation of polyclonal responses recognizing tumor cells endogenously expressing specific peptides. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

This application is US patent application Ser. No. 10/080, entitled “A CELL THERAPY METHOD FOR THE TREATMENT OF TUMORS,” to LeTurqq et al., Filed Feb. 19, 2002. No. 013 (which in turn claims priority from US Provisional Application No. 60 / 270,252), the contents of these applications are hereby incorporated by reference in their entirety. Incorporated.
The present invention relates to a cell therapy method for the treatment of tumors. In particular, the present invention relates to a treatment regimen for melanoma using ex vivo generated autologous T lymphocytes with specificity for melanoma-associated target antigens.

  Cancer continues to be a major health problem, despite considerable progress made in the area of treatment. Standard treatment regimens of chemotherapy, radiation therapy, surgical intervention and tripartite combinations often fail to produce permanent healing. In many cases, treated cancer patients often return to a disease state after some period of time, making the problem even worse.

  Another factor complicating the development of cancer therapy is that it has been found that cancer is not caused 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 treatment, cancer treatment requires the handling of multiple biological factors.

  In recent years, research has been directed to developing cancer treatments that utilize the patient's own immune system. One such approach is adoptive immunotherapy. Adoptive immunotherapy requires the use of the patient's own cells to generate cytotoxic T lymphocytes (CTLs) that treat tumor or cancer cells.

  Because melanoma is known for its potential to elicit an immune response and is resistant to currently used regimens of systemic treatments such as chemotherapy and hormonal therapy, most preclinical and clinical trials of immunotherapy regimens The test targets this malignant pathogen. Melanoma is a serious health problem. Over the past 40 years, the incidence of melanoma has increased at a greater rate than any other type of cancer. Although most melanomas are managed by conventional surgical resection, treatment options are limited for patients with malignant melanoma that cannot be easily removed surgically. Dacarbazine remains the drug of choice in disseminated melanoma, but remission usually does not last. Interleukins and biochemotherapy have yielded good results, but a small percentage of patients will benefit from this treatment. Although high-dose interferon increases survival in some patients, interferon remains a controversial drug that is not easily tolerated. Continuous chemotherapy has promise, but current treatment options for individuals suffering from metastatic melanoma are not satisfactory.

  Current immunotherapeutic approaches to treat metastatic melanoma are either alone or exogenous cytokines, genetically modified tumor cells, dendritic cells supplemented with defined peptides, or internally processed Administration of a melanoma-related peptide in combination with dendritic cells presenting a complete complement of antigenic epitopes derived from the purified protein. These approaches attempt to enhance T and / or B cell responses in an effort to cure the disease. These approaches remain largely unproven as a viable clinical treatment regimen for human patients. In addition to the problem of identifying the proper epitope for immunizing CTLs, current approaches are sufficient for APC to target multiple antigens and treat cancer effectively. Does not provide a method for presenting various epitopes.

  The present invention satisfies unmet needs by providing a cell therapy method for the treatment of tumors. In particular, the present invention relates to a treatment regimen for melanoma using ex vivo generated autologous T lymphocytes with specificity for a melanoma-associated target antigen. Concomitant administration of either IFN-α or IL-2, or both cytokines, at specific times and doses will prime tumor cells for lysis by antigen-specific T cells and in vivo persistence of CTLs There is a possibility of profit.

  The present invention provides a non-naturally occurring antigen presenting cell (nnAPC) capable of simultaneously presenting up to 10 or more different peptides, a method for producing nnACP, the nnACP for the treatment of cancer, especially malignant melanoma. Provide usage.

In a first aspect, the present invention provides:
Preparing a non-naturally occurring antigen-presenting cell line (nnAPC) (the nnAPC can simultaneously present up to about 15 different peptide molecules associated with viral infection, each of which is a length About 6 to 12 amino acids);
Collecting CD8 ⁺ cells from a subject or suitable donor;
Stimulating the CD8 ⁺ cells with the nnAPC cell line;
Adding the CD8 ⁺ cells to a conditioned growth medium (CGM) or a medium containing a cytokine selected from the group consisting of IL-2, IL-7 (the cytokines can be used individually or in combination);
About 1 to 50 μg / ml of one of the peptides that the nnAPC can simultaneously present unsuspended peripheral blood monocytes or CD-8 depleted peripheral blood monocytes collected from the subject or a suitable donor Mixing;
Irradiating the peripheral blood monocyte suspension with a sufficient dose of gamma radiation necessary to sterilize all components in the suspension except the desired peripheral blood monocytes;
Isolating adherent peripheral blood monocytes;
Adding about 1 μg / ml to 50 μg / ml of each peptide to the adherent peripheral blood monocytes;
Comprising introducing a CD8 ⁺ suspension in and subject; the CD8 ⁺ cells, 1 in a ratio of CD8 ⁺ cells from about 10 to peripheral blood monocytes that combined with adherent peripheral blood monocytes And a method for treating said viral infection in a subject.

  In one aspect of the method, the nnAPC can present up to about 10 peptide molecules, and preferably the peptide molecule is about 8-10 amino acids in length. Preferably, the molecule is in the concentration range of about 10 nM to 100 μM. Also preferably, the cytokine component is IL-2 or a combination of IL-2 and IL-7. In another embodiment of the method, the dose of gamma radiation is about 3,000 to 7,000 rads, and preferably about 5,000 rads.

The present invention also provides:
Administering to a subject with melanoma an effective amount of interferon-α capable of enhancing the expression of a tumor antigen on the surface of the tumor; and an effective amount with specificity for a melanoma-associated target antigen Inoculating said subject with autologous cytotoxic T lymphocytes,
It also relates to a method for treating said subject.

In a preferred embodiment of this method, the method subjects an effective amount of interleukin-2 that is capable of enhancing in vivo maintenance of autologous cytotoxic T lymphocytes with specificity for a melanoma-associated target antigen. And further comprising the step of administering to. Preferably, the interferon-α is selected from interferon-α-2a or interferon-α-2b, and the effective amount of interferon-α is about 10 MU / m ² / day and specific for melanoma associated target antigen A sex effective amount of autologous cytotoxic T lymphocytes is administered subcutaneously to the subject continuously from day 5 to day 1 prior to inoculation of the subject. Also preferably, an effective amount of autologous cytotoxic T lymphocytes with specificity for a melanoma-associated target antigen is about 1-10 × 10 ⁹ cells per injection. Also preferably, an autologous cytotoxic T lymphocyte with specificity for a melanoma-associated target antigen is: preparing a non-naturally occurring antigen presenting cell line (nnAPC), wherein nnAPC is associated with melanoma Up to about 15 different epitopes can be presented simultaneously, each epitope being a peptide of 8-10 amino acids in length; adding up to about 15 different epitopes associated with melanoma to the nnAPC; We collect the CD8 ⁺ cells from; the CD8 ⁺ cells, to obtain a CD8 ⁺ cells specific for stimulation to melanoma nnAPC cell line added epitope; melanoma-specific CD8 ⁺ cells, Growing in media containing IL-2 and IL-7; CD8-depleted peripheral blood monocytes collected from subjects were added to nnAPC Mixing with each epitope; irradiating CD8-depleted peripheral blood monocytes with gamma radiation; isolating adherent CD8-depleted peripheral blood monocytes; each adhering peripheral blood monocyte added to nnAPC Adding epitope; restimulating CD8 ⁺ cells specific for melanoma with adherent peripheral blood monocytes that have been epitopeed; re-stimulated CD8 ⁺ cells specific for melanoma are 2 and growing in a medium containing IL-7; and increasing melanoma-specific restimulated CD8 ⁺ cells by OKT3 antibody stimulation.

  Preferably, the step of restimulation can be repeated at least once more. In another preferred embodiment of the method, the non-naturally occurring antigen presenting cell line is supplemented with an epitope that is a peptide derived from tyrosinase, gp100 and MART-1, and preferably the non-naturally occurring antigen presenting cell. The system is added with an epitope comprising the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. Also preferably, the effective amount of interleukin-2 is about 3 MIU / day, and after inoculating the subject with an effective amount of autologous cytotoxic T lymphocytes having specificity for a melanoma-associated target antigen. Subjects are administered subcutaneously from day 0 to day 28 continuously. Also preferably, the method is repeated at intervals of about 2 months. In a preferred embodiment, the method further comprises the steps of being repeated for a minimum of two cycles and evaluating the response in the subject after each cycle.

In yet another preferred embodiment of the method, the method comprises: continuous from day 5 to day 1 prior to inoculating a subject with autologous cytotoxic T lymphocytes having specificity for a melanoma-associated target antigen; Subcutaneous administration of 10 MU / m ² / day interferon-α-2b to the subject; about 1-10 × 10 ⁹ cells per injection with autocytotoxicity with specificity for melanoma-related target antigen Injecting T lymphocytes; and about 3 MIU / day continuously from day 0 to day 28 after inoculating the subject with autologous cytotoxic T lymphocytes with specificity for a melanoma-associated target antigen Administering interleukin-2 to a subject subcutaneously.

The present invention is:
a. Preparing a non-naturally occurring antigen presenting cell line (nnAPC) wherein said nnAPC is capable of simultaneously presenting up to about 15 different peptide molecules associated with cancer, preferably about 10 different peptide molecules Each peptide is about 6 to 12 amino acids in length, preferably about 8 to 10 amino acids in length and in a concentration range of about 10 nM to 100 μM);
b. Collecting CD8 ⁺ cells from a subject with cancer or a suitable donor;
c. Stimulating the CD8 ⁺ cells with the nnAPC cell line;
d. Adding said CD8 ⁺ cells to conditioned growth medium (CGM) or medium containing cytokines such as IL-2, IL-7, preferably IL-2 or combinations of IL-2 and IL-7;
e. Mixing unsuspended peripheral blood monocytes collected from said subject or a suitable donor, or CD-8 depleted peripheral blood monocytes with about 5-50 μg / ml peptide;
f. With a sufficient dose of gamma radiation necessary to prevent the growth of these cells in suspension, such as a dose in the range of about 3,000 to 7,000 rads, preferably about 5,000 rads. Irradiating the peripheral blood monocyte suspension, or the peripheral blood monocyte suspension may be treated with a cytostatic agent, including but not limited to mitomycin C;
g. Isolating adherent peripheral blood monocytes;
h. Adding about 5 μg / ml to 50 μg / ml of each of said peptides to said adherent peripheral blood monocytes;
i. The CD8 ⁺ cells, in combination with said adherent peripheral blood monocytes for one of the peripheral blood monocytes at a ratio of about ten CD8 ⁺ cells;
j. Optionally stimulating said combined suspension of CD8 ⁺ cells and peripheral blood monocytes for about 6 to 9 days;
k. Optionally stimulating said suspension of CD8 ⁺ cells and peripheral blood monocytes with IL-2 and IL-7 in culture medium;
l. Optionally assaying the CD8 ⁺ suspension for suitable CTL activity and optionally assaying for CTL purity, sterility and endotoxin content; and m. There is provided a method of treating the subject comprising inoculating the subject with the CD8 ⁺ suspension.

Another aspect of the invention is:
a. Preparing a non-naturally occurring antigen presenting cell line (nnAPC), wherein said nnAPC is capable of simultaneously presenting up to about 15 different peptide molecules associated with cancer, preferably about 10 peptides; Where each peptide is 8 to 10 amino acids in length);
b. Collecting CD8 ⁺ cells from a subject with cancer;
c. Stimulating the CD8 ⁺ cells with the nnAPC cell line for about 6 to 9 days;
d. Stimulating the CD8 ⁺ cells with IL-2 and IL-7 in culture medium;
e. Mixing peripheral blood monocytes collected from the subject with about 10 μg / ml of each peptide;
f. Irradiating the CD8-depleted peripheral blood monocyte suspension with about 5,000 rads of gamma radiation;
g. Isolating adherent peripheral blood monocytes;
h. Adding about 10 μg / ml of said epitope to said adherent peripheral blood monocytes;
i. Combining the CD8 + cells with the adherent peripheral blood monocytes at a ratio of about 10 CD8 ⁺ cells to one peripheral blood monocyte;
j. Stimulating said combined suspension of CD8 ⁺ cells and peripheral blood monocytes for about 6 to 9 days;
k. Stimulating said suspension of CD8 ⁺ cells and peripheral blood monocytes with IL-2 and IL-7 in medium;
l. Assaying said CD8 ⁺ suspension for suitable CTL activity, purity, sterility and endotoxin content; and m. There is provided a method of treating a subject comprising inoculating the subject with a CD8 ⁺ suspension.

Another aspect of the invention is:
a. Preparing a non-naturally occurring antigen presenting cell line (nnAPC) wherein said nnAPC is capable of simultaneously presenting up to about 15 different peptide molecules associated with melanoma, preferably about 10 peptides Where each peptide is 8 to 10 amino acids in length);
b. Collecting CD8 ⁺ cells from a subject with melanoma;
c. Stimulating the CD8 ⁺ cells with the nnAPC cell line for about 6 to 9 days;
d. Stimulating the CD8 ⁺ cells with IL-2 and IL-7 in culture medium;
e. Mixing peripheral blood monocytes collected from the subject with about 20 μg / ml of each peptide capable of being presented by the nnAPC;
f. Irradiating the CD8-depleted peripheral blood monocyte suspension with about 5,000 rads of gamma radiation;
g. Isolating adherent peripheral blood monocytes;
h. Adding about 10 μg / ml of said epitope to said adherent peripheral blood monocytes;
i. Combining the CD8 + cells with the adherent peripheral blood monocytes at a ratio of about 10 CD8 ⁺ cells to one peripheral blood monocyte;
j. Stimulating said combined suspension of CD8 ⁺ cells and peripheral blood monocytes for about 6 to 9 days;
k. Stimulating said suspension of CD8 ⁺ cells and peripheral blood monocytes with IL-2 and IL-7 in medium;
l. Assaying CD8 ⁺ suspension for suitable CTL activity, purity, sterility and endotoxin content; and m. There is provided a method of treating a subject comprising inoculating the subject with a CD8 ⁺ suspension.

In another embodiment of the present invention, nnAPC has the following peptides: tyrosinase _369-377 , tyrosinase _207-216 , gp100 _209-217 , gp100 _154-162 , MART-1 _27-35 , HER-2 / neu _789-797 , It is a method for treating melanoma, presenting HER-2 / neu _369-377 , C-lectin _8-16 , Pec60 _20-29 and Pec60 _25-33 .

  Another aspect of the invention is a method of treating a disease or disease state that results in an insufficient or inadequate immune response normally associated with class I HLA molecules, wherein the treatment is infected Alternatively, the transformed cells are eliminated.

Another aspect of the present invention is a method of treating a disease or disease state that results in an insufficient or inadequate immune response normally associated with class I HLA molecules, subject to exclusion by CTLs. Infected or transformed cells that have been shown to be susceptible:
a. Prepare a non-naturally occurring antigen presenting cell line (nnAPC), which can simultaneously present up to about 15 different peptide molecules associated with the disease or disease state, preferably about 10 different peptide molecules Each peptide is about 6-12 amino acids in length, preferably about 8-10 amino acids in length and in a concentration range of about 10 nM-100 μM);
b. Collecting CD8 ⁺ cells from the subject or a suitable donor;
c. Stimulating the CD8 ⁺ cells with the nnAPC cell line;
d. Adding said CD8 ⁺ cells to a medium containing cytokines such as IL-2, IL-7 or CGM, preferably IL-2, or a combination of IL-2 and IL-7;
e. Mixing unsuspended peripheral blood monocytes collected from said subject or a suitable donor, or CD-8 depleted peripheral blood monocytes with about 5-50 μg / ml peptide;
f. The peripheral blood monocyte suspension prevents proliferation while maintaining the ability to stimulate peripheral blood monocytes, such as a dose in the range of about 3,000 to 7,000 rads, preferably about 5,000 rads. Irradiation with a sufficient dose of gamma radiation necessary for
g. Isolating adherent peripheral blood monocytes;
h. Adding about 5 μg / ml to 50 μg / ml of each of said peptides to said adherent peripheral blood monocytes;
i. The CD8 ⁺ cells, relative to 1 peripheral blood monocytes at a ratio of about ten CD8 ⁺ cells, in combination with said adherent peripheral blood monocytes;
j. Optionally stimulating said combined suspension of CD8 ⁺ cells and peripheral blood monocytes for about 6 to 9 days;
k. Optionally stimulating said suspension of CD8 ⁺ cells and peripheral blood monocytes with IL-2 and IL-7 in culture medium;
l. Optionally assaying the CD8 ⁺ suspension for suitable CTL activity and optionally assaying for CTL purity, sterility and endotoxin content; and m. The subject is treated by a method comprising inoculating the CD8 ⁺ suspension.

  The present invention provides non-naturally occurring antigen presenting cells (nnAPC) derived from Drosophila melanogaster cells transfected with DNA encoding human class I HLA, binding and costimulatory molecules for expression, and nnAPC Can present up to 15 different peptide molecules, preferably 10 peptide molecules.

  Another aspect of the present invention provides nnAPCs that present peptides associated with a variety of desired functions that enhance the treatment of a subject. For example, in addition to peptides associated with the disease or disease state being treated, nnAPC enhances cell-cell adhesion or transmits additional cell activation signals (LFA-1, LFA- 2 and LFA-3), intercellular adhesion molecules 1 and 2 (ICAM-1, ICAM-2), proteins associated with accessory molecules (CD40, CD70, B7) Can be expressed.

  Another aspect of the invention provides nnAPCs that present peptides associated with several types of cancer. For example, a peptide associated with or derived from a breast cancer associated polypeptide such as HER-2 / neu may be presented with a peptide associated with or derived from a melanoma associated polypeptide such as MART-1 or MAGE. unknown.

Another aspect of the invention provides a method for producing a non-naturally occurring antigen presenting cell (nnAPC) capable of simultaneously presenting up to 10 different peptide molecules, said method comprising the steps of:
a. Preparing an insect cell line from Drosophila melanogaster eggs; or preparing an insect cell line to express human MHC class I molecules and costimulatory adhesion molecules;
b. Growing said insect cells in a medium suitable for growing insect cells, preferably Schneider ^™ Drosophila medium;
c. Creating a pRmHa-3 plasmid from a pRmHa-1 expression vector (the pRmHa-3 plasmid is an alcohol dehydration carrying a metallothionein promoter, a metal responsive consensus sequence, and a polyadenylation signal isolated from Drosophila melanogaster. Elemental enzyme gene);
d. Inserting the human class I HLA A2.1, B7.1, B7.2, ICAM-1, β-2 microglobulin and LFA-3 complementary DNA into the pRmHa-3 plasmid (A2.1 is either Of human class I DNA sequences);
e. Transfecting the insect cells with the phshneo plasmid and the pRmHa-3 plasmid containing the complementary DNA; and f. Creating nnAPC by contacting said insect cells with CuSO ₄ to induce expression of the transfected gene in said insect cells.

The insect cells of the present invention are grown in a medium suitable for growing insect cells (referred to below as “insect growth medium”). Insect growth media such as Schneider ^™ Drosophila media, Grace insect media, and TC-100 insect media are commercially available from a number of sources. Alternatively, insect growth media can be prepared by one skilled in the art. Typically, the medium is such as inorganic salts (eg, calcium chloride, magnesium sulfate, potassium chloride, potassium phosphate, sodium bicarbonate, sodium chloride and sodium phosphate), amino acids, various carbohydrates, and chemical species. Ingredients necessary to promote and sustain the growth of insect cells can be included (Imogene Schneider, Exp. Zool. (1964) 156 (1): pg. 91). Alternatively, the medium can also include vitamins, minerals, and other components that aid in the growth of insect cells.

The present invention further provides:
a) administering to a subject with cancer an effective amount of interferon-α capable of enhancing the expression of tumor antigens and HLA class I molecules on the surface of the tumor; and b) specificity for a cancer-related target antigen There is provided a method of treating a subject comprising the step of inoculating the subject with a sex effective amount of autologous cytotoxic T lymphocytes.

  In a preferred embodiment, the method of treatment further comprises an effective amount of interleukin-2 that is capable of enhancing in vivo maintenance of autologous cytotoxic T lymphocytes with specificity for a melanoma-associated target antigen. Comprising the steps of:

The following is a list of abbreviations and definitions used in this specification.
Abbreviation APC Antigen-presenting cell CD8 ⁺ CD8 ⁺ T cell CTL Cytotoxic T lymphocyte E Effector Fas CD95, also known as epitope ICAM intercellular adhesion molecule IL interleukin LAK lymphokine activated killer cell LFA lymph Sphere functional antigen MHC Major histocompatibility complex nnAPC Non-naturally occurring antigen-presenting cell NP Nucleoprotein PBMC Peripheral blood mononuclear cell PBS Phosphate buffered saline PCR Polymerase chain reaction RPMI Roswell Park Memorial Institute (Roswell Park Memorial Institute)
RWJPR R.I. W. Johnson Pharmaceutical Research Institute (The RW Johnson Pharmaceutical Research Institute)
T target TCP T cell antigen receptor TIL tumor infiltrating lymphocytes The following is a list of abbreviations used herein for various peptide epitopes. Individual amino acid residues are identified according to single letter symbols that are readily known and used by those skilled in the art.

Abbreviations for Peptide Epitopes As used herein, the term “tyrosinase 369-377” or “tyrosinase _369-377 ” refers to the amino acid sequence YMNTTMSQV (SEQ ID NO: 1). Within this definition, the sequence YMDGTMSQV (SEQ ID NO: 2) resulting from a post-translational event resulting from modifying the amino acid residue “N” of the sequence YMNTMSQV (SEQ ID NO: 1) to “D” resulting in the amino acid sequence of YMDGTMSQV (SEQ ID NO: 2) peptide are also encompassed (Skipper et al., J.Exp.Med (1996) 183:. 527-534).

The term “tyrosinase 207-216” or “tyrosinase _207-216 ” as used herein refers to the amino acid sequence FLPWHRLFLL (SEQ ID NO: 3).

The term “gp100 209-217” or “gp100 _209-217 ” as used herein refers to the amino acid sequence ITDQVPFSV (SEQ ID NO: 4).

As used herein, the term “gp100154-162” or “ _gp100154-162 ” refers to the amino acid sequence KTWGQYWQV (SEQ ID NO: 5).

The term “MART-1 27-35” or “MART-1 _27-35 ” as used herein refers to the amino acid sequence AAGIGILTV (SEQ ID NO: 6).

As used herein, the term "HER-2 / neu 789-797" or "HER-2 / neu _789-797 " refers to the amino acid sequence CLTSTVQLV (SEQ ID NO: 7).

The term “HER-2 / neu 369-377” or “HER-2 / neu _369-377 ” as used herein refers to the amino acid sequence KIFGSLAFL (SEQ ID NO: 8).

The term “C-lectin 8-16” or “C-lectin _8-16 ” as used herein refers to the amino acid sequence KMASRMRL (SEQ ID NO: 9).

The term “Pec60 20-29” or “Pec60 _20-29 ” as used herein refers to the amino acid sequence ALALAALLVV (SEQ ID NO: 10).

The term “Pec60 25-33” or “Pec60 _25-33 ” as used herein refers to the amino acid sequence ALLVVDREV (SEQ ID NO: 11).

The term “CD8 peptide 59-70” or “CD8 peptide _59-70 ” as used herein refers to the amino acid sequence of AAEGLDTQRFSG (SEQ ID NO: 12).
Terms and Definitions As used herein, the term “adoptive immunotherapy” refers to the administration of a donor or autologous T lymphocytes for the treatment of a disease or disease state, where the disease or disease state is It results in an insufficiency or inadequate immune response normally associated with class I HLA molecules. Adoptive immunotherapy is an appropriate treatment for any disease or condition in which the elimination of infected or transformed cells has been demonstrated to be achieved by CTL. For example, the disease or disease state includes, but is not limited to, cancers and / or tumors such as melanoma, prostate, breast, colorectal, stomach, throat, pancreas, cervix, ovary, bone, leukemia and lung cancer Mention may be made of viral infections such as hepatitis B, hepatitis C, human immunodeficiency virus; bacterial infections such as tuberculosis, leprosy and listeriosis, and parasitic infections such as malaria.

  As used herein, the terms “B7.1, B7.2” refer to costimulatory molecules associated with antigen presenting cells.

  The term “BCNU” as used herein refers to carmustine, also known as 1,3-bis (2chloroethyl) -1-nitrosourea.

  As used herein, the term “BSE” refers to bovine spongiform encephalopathy.

  As used herein, the term “CD” refers to differentiated antigen clusters or T lymphocytes (originally), B lymphocytes, monocytes, macrophages and granulocytes grouped by antigen epitope and function. .

  As used herein, the term “DTIC” refers to dacarbazine or 5- (3,3-dimethyl-1-triazeno) -imidazole-4-carboxamide.

  As used herein, the term “ex vivo” or “ex vivo therapy” refers to a pathological condition that can be ameliorated by long-term or constant delivery of a therapeutic benefit produced by the modified cells. Refers to a treatment in which biological material (typically cells) is obtained and modified from a suitable alternative source, such as a patient or a suitable donor, such that the modified cells can be used to treat. Treatment includes reintroduction of the modified biological material obtained from either the patient or an alternative source into the patient. One benefit of ex vivo therapy is the ability to provide treatment benefits to the patient without exposing the patient to unwanted side effects from the treatment. For example, high dose cytokines are often administered to patients with cancer or viral infections to stimulate expansion of the patient's CTL. However, cytokines often cause the development of flu-like symptoms in patients. In ex vivo procedures, cytokines are used to stimulate CTL expansion outside the patient's body, and patients are forgiven of cytokine exposure and the resulting side effects. Alternatively, the subject is treated simultaneously with a low level of dosage of gamma interferon, alpha interferon and / or IL-2 when appropriate and under the appropriate circumstances or conditions where the subject can benefit. Can do. The expected effect of interferon is probably to sensitize tumor cells to lysis by antigen-specific CTL, and the effect of IL-2 is probably to increase the persistence of antigen-specific CTL.

  As used herein, the term “HEPES” refers to N-2-hydroxyethylpiperazine-N′2-ethanesulfonic acid buffer.

  As used herein, the term “HLA-A2.1” refers to an HLA class I molecule found in approximately 45% of white people.

  As used herein, the term “MART-1” or “(melanoma antigen-1 recognized by T cells)” refers to a melanoma-associated antigen. The amino acid and nucleic acid sequences of this antigen, as well as various Characteristic is US Pat. No. 5,994,523 issued Nov. 30, 1999 entitled “Melanoma Antigens and Their Use in Diagnostic and Therapeutic Methods”. Description: 1999, entitled “Melanoma Antigens and Their Use in Diagnostic and Therapeutic Methods” 1998, entitled US Patent No. 5,874,560 issued February 23; and "Melanoma Antigens and Therapeutics and Therapeutic Methods". It is disclosed in US Pat. No. 5,844,075 issued on Dec. 1, 1980. In particular, US Pat. No. 5,994,523 discloses the full-length nucleic acid and amino acid sequences of MART-1 in FIG. 1 as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The foregoing FIG. 1 is incorporated herein by reference.

  The term “MAGE” as used herein refers to a melanoma associated antigen. The amino acid and nucleic acid sequence of this antigen, as well as various features, are described in “Methods for Determining Breast Cancer and Melanoma by Assuring for Plurality of Anti-Association of the Antigen.” U.S. Pat. No. 6,140,050 issued October 31, 2000; entitled “Method of Screening for Cancer by. Detection of Messenger RNA of MAGE-XP Gene” (Method of Screening for Cancer by Detecting Messenger RNA for a MAGE-XP Gene) US Pat. No. 5,759,783, issued Jun. 2, 1998, entitled; and “Induction of Anti-Tumor Cytotoxic T Lymphocytes in Humans Using Synthetic Peptide Epitopes (Induction of Anti-Tumor) U.S. Pat. No. 5,662,907 issued Sep. 2, 1997 entitled "Cytotoxic T Lymphophytes in Humans Using Peptides Epitopes".

  As used herein, the term “MPC-10” refers to a magnetic particle concentrator.

  The term “NK cell” as used herein refers to a natural killer cell.

  As used herein, the term “OKT3” refers to an orthoclone OKT3, muromonab-CD3, an anti-CD3 monoclonal antibody.

  As used herein, the term “TAP-1,2” refers to antigen processing-related transport proteins-1,2.

  The term “Th cell” as used herein refers to the helper T cell, CD4 +.

As used herein, the term “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). US Pat. No. 5, 1998, dated March 1998, entitled “P15 and Tyrosinase Melanoma Antigens and Their Use in Diagnostics and Therapeutic Methods”. 648 discloses the antigenic peptides and related polynucleic acids related to tyrosinase in FIG. 7, FIGS. AD, the aforementioned figures being incorporated herein by reference. “Method for Detecting Complexes Containing Human Leukocyte Anti-Antigen and A and B in Molecules and Tissues in the Tissues and the Tyrosinase Peptides” US Patent No. 5,487,974, issued January 30, 1996, entitled "Cells)" discloses additional peptides related to tyrosinase and melanoma in Table 3 in Example 9, The foregoing is incorporated herein by reference.

As used herein, the term “gp100” refers to a melanoma antigen recognized by tumor infiltrating lymphocytes (TIL). recognizes gplOO TIL 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 ). Antigen peptides related to gp100 are US Patent No. 5, issued November 30, 1999 entitled "Melanoma Antigens and Their Use in Diagnostic and Therapeutic Methods". U.S. Pat. No. 5, issued Feb. 23, 1999, entitled "Melanoma Antigens and Their Use in Diagnostic and Therapeutic Methods". , 874,560; and "Melanoma Antigens and their use in diagnosis and therapy." Their Use in Diagnostic and Therapeutic Methods) "and entitled December 1, 1998 is disclosed in US Pat. No. 5,844,075 of the grant. In particular, US 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 said amino acid sequence, including those identified as SEQ ID NOs: 27, 33, 34, 35, 36, 37, 38, 39, 40 and 41. All of the foregoing FIGS. 4 and 5 and the peptides identified by SEQ ID NOs are incorporated herein by reference.

  As used herein, the term “melanoma” includes, but is not limited to, melanoma, metastatic melanoma, melanoma from either melanocytes or melanocyte-related neurons, melanoma, melanoma, Melanoma, Melanoma in situ superficial enlarged melanoma, nodular melanoma, malignant melanoma, terminal melanoma, invasive melanoma or familial variant melanoma / melanoma (FAM-M) syndrome Point to. These melanomas in mammals are chromosomal abnormalities, degenerative growth and developmental disorders, mitogens, ultraviolet radiation (UV), viral infections, inappropriate tissue expression of genes, altered gene expression, and May be caused by presentation on cells or carcinogens. The aforementioned melanoma can be diagnosed, evaluated or treated by the methods described in this application.

  The term “C-lectin” as used herein refers to a peptide of a sequence that has been found to be associated with ovarian cancer.

  As used herein, the term “major histocompatibility complex” or “MHC” is intended to encompass histocompatibility antigen systems described in various species, including human leukocyte antigen (HLA). It is a generic name that is meant.

  As used herein, the terms “epitope”, “peptide epitope”, “antigenic peptide” and “immunogenic peptide” are derived from an antigen capable of eliciting a cellular immune response in a mammal. Refers to a peptide. Such peptides may also be reactive with antibodies from animals immunized with the peptide. Such peptides may be about 5-20 amino acids in length, preferably about 8-15 amino acids in length, and most preferably about 9-10 amino acids in length.

  As used herein, the term “Pec60” refers to a peptide of a sequence that has been found to be associated with ovary and breast cancer.

  As used herein, the term “analog” refers to, inter alia, one or more residues that are conservatively substituted with functionally similar residues and are described herein. Any polypeptide having an amino acid residue sequence that is substantially identical to a sequence of the invention set forth herein that represents a functional aspect of the invention is encompassed. Examples of conservative substitutions include substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine, between arginine and lysine, between glutamine and asparagine, Substitution of one other in place of one polar (hydrophilic) residue such as between glycine and serine, substitution of another in place of one basic residue such as lysine, arginine or histidine, Or the substitution of one acidic residue such as aspartic acid or glutamic acid or another.

  As used herein, the term “conservative substitution” also encompasses the use of chemically derivatized residues in place of non-derivatized residues.

  The term “chemical derivative” as used herein refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Examples of such derivatized molecules are derivatives such that, for example, the free amino group forms an amine hydrochloride, p-toluenesulfonyl group, carbobenzoxy group, t-butyloxycarbonyl group, chloroacetyl group or formyl group. Including conjugated molecules. 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 the histidine may be derivatized to form N-in-benzylhistidine. Also included as chemical derivatives are proteins or peptides containing one or more naturally occurring amino acid derivatives of the 20 standard amino acids. For example: 4-hydroxyproline may be used instead of proline; 5-hydroxylysine may be used instead of lysine; 3-methylhistidine may be used instead of histidine; homoserine may be used instead of serine And ornithine may be used in place of lysine. A protein or polypeptide of the present invention has one or more additions with respect to the sequence of the polypeptide whose sequence is encoded by the corresponding nuclear sequence of the present invention, as long as the essential activity is maintained. Also encompassed are any polypeptides having / or deletions or residues.

The term “HER-2 / neu” as used herein refers to an oncogene that expresses or overexpresses one or more membrane-associated receptor-like oncogene proteins. Cancers found to be associated with HER-2 / neu expression or overexpression include 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 epidermal growth factor receptor (EGFR). HER-2 / neu has been shown to play a role in cell growth and / or differentiation. HER-2 / neu appears to induce malignant lesions by a quantitative mechanism resulting from increased or unregulated expression of essentially normal gene products. "Immune Reactivity to HER-2 / neu Protein for Diagnostics and Treatment of Malignancy in Whith the HER-2 / neu Oncogene-Related Malignant Disease Diagnosis and Treatment US Pat. No. 6,075,122, issued June 13, 2000, entitled “-2 / neu Oncogene is Associated”), line 12 line 31 to line 13 line 7, ⁺ Disclose peptides that elicit T cell responses, and those identified by SEQ ID NO are incorporated herein by reference.

  HER-2 / neu (p185) is the protein product of the HER-2 / neu oncogene. In a variety of cancers, including breast, ovary, colon, lung and prostate cancer, the HER-2 / neu gene is amplified and the HER-2 / neu protein is overexpressed. HER-2 / neu is associated with malignant transformation. It is found in 50% to 60% of non-invasive ductal cancer and 20% to 40% of all breast cancers and a significant portion of adenocarcinoma that occurs in the ovary, prostate, colon and lung. HER-2 / neu is closely associated with not only the malignant phenotype, but also the aggressiveness of malignant lesions found in 1/4 of all invasive breast cancers. HER-2 / neu overexpression correlates with poor prognosis in both breast and ovarian cancers. HER-2 / neu is a transmembrane protein with a relative molecular weight of 185 kd that is approximately 1255 amino acids (aa) in length. It has an extracellular binding domain (ECD) of approximately 645aa with 40% homology to epidermal growth factor receptor (EGFR), a highly hydrophobic transmembrane anchoring domain (TMD), and 80% homology to EGFR It has a carboxy-terminal cytoplasmic domain (CD) of approximately 580 amino acids with

As used herein, the term “interferon-α (INF-α)” refers to a family of highly homologous species-specific proteins that inhibit viral replication and cell proliferation and modulate the immune response. Point to. Suitable interferon-α typical for the methods of the invention include, but are not limited to, Intron-A [ ^R ] interferon available from Schering Corporation, Kenilworth, NJ Recombinant interferon alpha-2b, recombinant interferon alpha-2a such as Hoffmann-La Roche, Roferon Interferon available from Nutley, NJ, Boehringer Ingelheim Pharmaceutical Ink (Boehringer Ingelheim) (Pharmaceutical, Inc.), a velophore available from Ridgefield, Connecticut (B erofor) Recombinant interferon α-2c such as α2 interferon, interferon α-n1, a purified blend of natural α interferon such as Sumiferon available from Sumitomo, Japan, or Glaxo-Welcome Limited ( Glaxo-Wellcom Ltd.), Wellferon interferon α-n1 (INS) available from London, UK, or US Pat. Nos. 4,897,471 and 4,695,623 (among others) Consensus alpha interferons such as those described in Example 7, 8 or 9), and certain products available from Amgen, Inc., Newbury Park, California Lists the interferon alpha-n3 mixture of natural alpha interferon produced by Interferon Sciences and available from Purdue Frederick Co., Norwalk, Connecticut, under the trade name Alferon be able to. Use of interferon α-2a or α-2b is preferred. Interferon alpha-2b is most preferred because it has the broadest approval in the world for treating chronic hepatitis C infection among all interferons. The production of interferon α-2b is described in US Pat. No. 4,530,901.

As used herein, the term “interleukin 2 (IL-2)” refers to its biological effects after stimulating the immune system and binding to specific receptors on the surface of target cells. Refers to a cytokine that exerts IL-2 has many biological effects such as activated B and T cells (including cytotoxic T cells), natural killer (NK) cells and lymphokine activated killer ( LAK) is known to induce cell stimulation. IL-2 may be obtained as a prescription drug, eg, Proleukin ^(R) manufactured by Chiron Corporation (Emeryville, Calif. ⁾ . IL-2 may be produced from a variety of sources and by a variety of methods, as disclosed in a number of US patents. These patents include, but are not limited to, hybrid mice as disclosed in US Pat. Nos. 4,407,945, 4,473,642, and 4,401,756, respectively. IL-2 production from T cells, such as T cell lines or malignant human T cell lines, and US Pat. Nos. 4,992,367, 4,407,945 and 4,473,642 Mention may be made of the production of recombinant human IL-2 as disclosed in the specification.

  Ongoing studies involving oncogenes have identified a minimum of 40 oncogenes that are effective in and associated with or associated with transformation in malignant cells. Oncogenes are classified into diverse groups based on the putative function or location of their gene products (such as proteins expressed by oncogenes). Oncogenes are thought to be essential for certain aspects of normal cell physiology.

  Cancer continues to be a major health problem, despite considerable progress made in the area of treatment. Standard treatment regimens of chemotherapy, radiation therapy, surgical intervention and tripartite combinations often fail to produce permanent healing. In many cases, treated cancer patients often return to a disease state after some period of time. Further exacerbating the condition is the severity of these treatment regimens for the patient. In the case of melanoma, healing of metastatic melanoma has not been achieved using conventional chemotherapy. Response rates of 35% to 50% have been reported with combination chemotherapy Dartmouth regimens (DTIC, cisplatin, BCNU, and tamoxifen), but survival has been limited to 6-10 months. High rates of recurrence have been reported for aggressive “high dose intensity” chemotherapy, as well as hematopoietic polycythemia with autologous bone marrow transplantation. The median survival in these treated patients was short (approximately 4 months).

  Rosenberg et al. Attempted to use activated lymphocyte injection as a treatment for various cancers. Initially, lymphokine-activated killer cells (LAK) and tumor-infiltrating lymphocytes (TIL) that were later activated ex vivo with IL-2 were used, but evidence of efficacy is uncertain. In fact, control clinical trials failed to show the benefits of using ex vivo activated cells over direct administration of IL-2 to patients. Thus, the benefits of LAK and TIL therapy are modest and the side effects are typically so severe that many trials were discontinued early.

Studies in mouse tumor models have demonstrated that adoptive immunotherapy, ie in vivo immunization of T cells specific for tumor antigen (s), is very effective with minimal toxicity. A major obstacle to applying this strategy to the treatment of human tumors is the identification of immunogenic antigens that sensitize tumor cells to cytotoxic T lymphocyte (CTL) -mediated destruction. Isolation of tumor-reactive T cells from melanoma patients has led to the identification of several tumor antigens (epitopes) to which CTLs are directed. These include tyrosinase ( Brichard et al., J. Exp. Med. (1993) 178: 489-495; Robbins et al., Cancer Res. (1994) 54: 3124-3126), MART 1 / Melan A ( Kawakami et al., J. Biol. Exp. Med. (1994) 180: 347-352), gp 100 ( Bakker et al., J. Exp. Med. (1994) 179: 1005-1007; and Kawakami et al., J. Immunol. (1995) 154: 3961- 3968), as well as MAGE ( Gaugler et al., J. Exp. Med. (1994) 179: 921-930). Of these, tyrosinase and MART-1 are almost universally expressed in melanoma and are therefore a logical choice for adoptive immunotherapy.

  In recent years, a significant improvement in survival over the course of several years has been shown in a small proportion of melanoma patients receiving immunological therapy. This involves active specific immunotherapy with “cancer vaccines” and the use of non-specific boosters of the immune system such as cytokines such as IL-2, α-interferon and γ-interferon. Include. However, the benefits of cytokines are diminished by the side effects often associated with their use, such as nausea, fever and flu-like symptoms.

Cytolytic T cells (CD8 ⁺ ) are the main line of defense against viral infections. CD8 ⁺ lymphocytes specifically recognize and kill host cells infected by the virus. In theory, it should be possible to utilize the immune system to combat other types of diseases, including cancer. However, a few in vitro / ex vivo procedures were available to specifically activate CTL. The identification of the important melanoma antigens shown above, and the specific in vitro activation methods of CTL described below, now allow testing of the concept of adoptive immunotherapy of metastatic melanoma.

All naive T cells require two signals for activation to elicit an immune response. For CD8 ⁺ lymphocytes (CTL), the first signal that confers specificity is the immunogenic peptide fragment of the antigen bound to the class I MHC (HLA) complex present on the surface of the antigen presenting cell (APC) It consists of presentation of (epitope) to CD8 ⁺ cells. This complex is specifically recognized by the T cell antigen receptor (TCR), which transmits signals within the cell.

Binding to the T cell receptor is necessary but not sufficient to induce T cell activation and usually cannot lead to cell proliferation or cytokine secretion. Full activation requires a second costimulatory signal (s), which serve to further enhance the activation cascade. Among costimulatory molecules on antigen presenting cells, cell adhesion molecules (integrins) such as B7 and ICAM-1 assist in this process by binding to CD28 and LFA-1 on T cells, respectively. CD8 ⁺ If cells, which interact with antigen presenting cells bearing class I binding immunogenic peptides by MHC molecules (epitopes) in the presence of the interaction of a suitable co-stimulatory molecules, CD8 ⁺ cells completely Activated cytolytic T cells.

Lymphocyte-mediated cell killing requires a series of biological events that begin with the binding of CD8 ⁺ CTL to antigen-bearing target (tumor) cells by the recognition process described above for T cell activation. And

The interaction between CD8 ⁺ cells and antigen presenting cells or target (tumor) cells as described above is depicted in FIG. The interaction begins with the binding of the antigen to the T cell antigen receptor (TCR) in conjunction with APC or MHC class I molecules on the target cell. Adjunct molecules such as lymphocyte functional antigens (LFA-1, LFA-2 and LFA-3), intercellular adhesion molecule 1 (ICAM-1, ICAM-2) and T cell costimulatory factors (CD2, CD28, B7) Increases cell-cell adhesion or transmits additional cell activation signals. However, the requirement for lymphocyte-mediated cell killing can occur in the presence of MHC / peptide alone (a situation common to most tumor cells).

  Following cell-cell interaction, CTL kills target cells by the action of soluble cytolytic mediators (perforin and granzyme stored in cytoplasmic granules of T cells) and CTL surface molecules (Fas ligand). After a cytolytic attack, target cells die by necrosis (membrane perforation and organelle disruption) or apoptosis (chromatin condensation, DNA fragmentation and membrane bubble formation).

  The mechanism of lysis mediated by lymphocytes is graphically depicted in FIG. In FIG. 2A, after binding to the target cells, the cytoplasmic granules in the CTL are rapidly redirected to the target cells for the release of granules containing perforin and granzyme into the intercellular space. These proteolytic enzymes form pores in the plasma membrane of the target cell, eventually leading to cell necrosis. In FIG. B, the level of Fas ligand expression on CTL increases after binding to target cells. The interaction of the 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) are involved in the induction of apoptosis.

Naturally occurring antigen presenting cells such as dendritic cells, macrophages and autologous tumor cells can be used for in vitro CD8 ⁺ activation. However, the efficiency of activation after this approach is low. This is because native APC class I molecules contain many other types of peptide epitopes in addition to tumor epitopes. Most of the peptides are derived from normal innocuous cellular proteins, resulting in a dilution of the number of active natural APCs that appear to be effective against tumors ( Allison et al., Curr. Op. Immunol. (1995) 7: 682-686).

One more direct and efficient approach to this problem is with only those epitopes associated with fighting specific diseases (such as cancer) or tumor-specific antigens (such as melanoma-specific antigens). To specifically activate CD8 ⁺ cells. For this purpose, artificial antigen-presenting cells are created by expressing MHC class I molecules in Drosophila melanogaster cells. Since Drosophila does not have an immune system, there are no TAP-1,2 peptide transport proteins involved in adding peptide epitopes on class I molecules. As a result, class I molecules appear on the Drosophila cell surface as an empty vessel. These transfected Drosophila cells are incubated with an exogenous peptide that binds to a class I molecule, such as, but not limited to, a cancer or tumor specific epitope that can include melanoma specific epitopes. Thus, it is possible to occupy all class I molecules with the same peptide. High density expression of class I molecules containing a single peptide in these Drosophila APCs allows the generation of cytotoxic CD8 ⁺ T cells in vitro that are completely specific for antigenic peptides. Methods and procedures for preparing Drosophila cells are described in “In Vitro Activation of Cytotoxic T-cells Using Insect Cells Expressing Human Class I MHC and β2-microglobulin (In Vitro Activation of Cytotoxic T- U.S. Pat. No. 5,529,921, issued June 25, 1996, entitled “Cells Using Insect Cells Expressing Human Class I MHC and β2-Microglobulin)” and “MHC Class I Antigens and β2-Microglobulin” A Drosophila cell line capable of expressing a gene coding for and assembling an empty complex, and a method for producing said cell line (Drosophila Cell L ines Expressing Genes Encoding MHC Class I Antigens And β2-Microglobulin and Capable of Assembling Empty Complexes and Methods of Met 94 Is taught. In particular, US Pat. No. 5,529,921 discloses various methods for separating and / or concentrating cultures of progenitor cells at line 26, line 56 to line 28, line 22.

  In addition, this feature eliminates the need for in vivo stimulation of the immune system with high doses of various cytokines, thereby providing a treatment that eliminates the side effects caused by cytokines. Alternatively, subjects are treated simultaneously with low levels of dosages of alpha interferon, gamma-interferon and / or IL-2 when appropriate and under circumstances and conditions where the subject can benefit. be able to.

  Eliminating the need for in vivo stimulation with cytokines provides improved patient treatment quality. Treatment regimes that include the administration of cytokines to patients often result in patients developing flu-like symptoms such as nausea, vomiting and fever. These side reactions are generally not life-threatening, although particularly severe reactions that occur in patients who are already weakened can lead to life-threatening situations. Another consideration is the detrimental effect of these side reactions on the patient's acceptability and compliance of otherwise beneficial treatment regimens. Eliminating the need for in vivo stimulation with cytokines results in a treatment regimen that improves patient comfort and also provides the clinician with an effective treatment that is more likely to be followed by his or her patient. provide.

The utility of this method for adoptive immunotherapy of tumors is using Drosophila cells transfected as APCs, and CD8 ⁺ cells from the 2C line of T cell receptor (TCR) transgenic mice. Proven with a mouse. In this system, purified CD8 ⁺ 2C cells are in vitro peptides presented by MHC class I (L ^d ) transfected Drosophila cells that also carry costimulatory molecules B7-1 and ICAM-1. Highly responsive. Transfected Drosophila cells ( Cai et al., P.N.A.S. USA (1996) 93: 14736-14741 as probes to define minimal requirements for stimulating unprimed CD8 + T cells ). Alternatively, if non-isolated mouse splenocytes are used as responders instead of purified 2C cells, the need for costimulatory molecules does not apply. In this example, CD8 ⁺ cells in the spleen population receive “bystander” costimulation from activated B cells. Using this finding, MHC class I (L ^d ) transfected Drosophila cells respond in vitro to syngeneic P815 mastocytoma tumor-specific peptides in the absence of added lymphokines. It was possible to show that normal DBA / 2 mouse splenocytes can be induced. Injection of these CTLs into DBA / 2 mice bearing P815 mastocytoma led to rapid tumor regression ( Sun et al., Immunity (1996) 4: 555-564).

Procedurally, normal DBA / 2 mouse splenocytes were isolated from the tumor-specific epitope P1A.P from the DBA / 2-derived P815 mastocytoma cell line. Cultured in vitro with MHC class I (L ^d ) transfected Drosophila cells supplemented with 35-43 peptides. Lymphocytes collected from the cultures after 5 days displayed strong cytotoxic T lymphocyte (CTL) activity against P815 tumor cells in vitro, however, as shown in FIG. Failed to lyse the P815 mutant cell line P1024 that does not express 35-43. When these CTLs were injected 3 days ago into DBA / 2 mice previously inoculated with P815 cells, the tumors grew undisturbed during the first week, but thereafter, FIG. As shown in B, it was eliminated within the next week. As shown in FIGS. 3 and B, when CTL were immunized in vitro against an irrelevant antigen such as a viral nucleoprotein peptide, specificity was demonstrated by the absence of any effect on P815 growth. In summary, major histocompatibility complex complex class I (Ld) transfected Drosophila cells in vitro to syngeneic P815 mastocytoma tumor-specific peptides in the absence of added lymphokine. Normal DBA / 2 mouse splenocytes were induced to respond. Injection of these CTLs into DBA / 2 mice bearing P815 mastocytoma led to rapid tumor regression ( Wolfel et al., J. Exp. Med. (1993) 178: 489-495).
In vitro human studies Human CTLs from healthy subjects were separately added on Drosophila cells against tyrosinase, MART-1 and gp100, immunized in vitro, and evaluated for lysis against JY cells (Figure 4). The same peptide can be added together to a single Drosophila APC to produce a multispecific bulk CD8 preparation. Melanoma-specific CTLs from healthy subjects were induced using a complete stimulation / restimulation protocol and tested for their ability to lyse Jurkat cells supplemented with each of the peptides used in stimulation. (FIG. 5). Cell line. FIG. 6 shows CTL activity after two different in vitro stimulation protocols. Multiple peptides added on individual Drosophila cells and mixed before primary stimulation (combo mix), or multiple peptides mixed and then added on Drosophila APC (combo load) . Figure A represents the results of the combo mix protocol on target cells spiked with peptides. FIG. B represents the results of the combo load protocol on target cells spiked with peptide. Figure C represents killing on a melanoma target generated from both protocols.

  The use of any natural or artificial antigen-presenting cell (APC) system to generate cytotoxic T lymphocytes in vitro is limited by the antigen specificity that these systems are capable of generating.

  The following APC system was utilized to generate antigen-specific CTL for a single epitope. 1) human dendritic cells (DC) pulsed with defined peptides; 2) peripheral blood mononuclear cells (PBMC) driven by lymphoblasts and peptide applied; 3) natural A lymphoblastoid cell line (LCL) in which the peptide is acid-stripped and supplemented with the peptide 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 (JB Latouche and M. Sadelain, Nature Biotech (2000) 18: 405-409).

  Dendritic cells (DCs) are considered the primary antigen presenting cell line in humans due to their broad application in the presentation of primary antigen cells. Self or foreign proteins are processed in DC. The resulting peptide epitope is presented by the HLA molecule and transported to the surface of the DC. However, it was found that DC could not be generated consistently in vitro and CTL directed against four different peptides. This would have provided CTLs with activity corresponding to each of the four peptides. In addition, it was also found that the DC phenotype (mature or immature) at the time of peptide application did not achieve results.

  Alternatively, stimulation of Drosophila cells usually resulted in CTLs directed against up to 10 different types of peptides. This provides a CTL that is active against each of the 10 peptides.

The ability of Drosophila cells and DCs to elicit CTL responses was first evaluated against a single peptide epitope according to each standard stimulation protocol. To compare DCs and transfected Drosophila cells, immature DCs were generated by culturing autologous monocytes for 1 week in the presence of IL-4 and GM-CSF. Mature DCs were obtained from immature DCs by the addition of TNFα to the medium 24 hours before collection. DC (immature and mature) were collected, peptide applied and mixed with purified CD8 cells according to the procedure used to stimulate CD8 cells and Drosophila cells to which the peptide was applied. Drosophila cells were found to be generally better stimulators than DC when assessed for tyrosinase peptide epitope 689, as shown in FIG. Furthermore, DCs representing either immature or mature phenotypes (Figure 8) are not as efficient as Drosophila cells in deriving specific CTL responses when applied to APC using defined peptides. It was. This is particularly surprising because of the dominant role played by DCs in the immune system. A comparative study with one donor was performed as shown in FIG. Specific killing was generated for four different peptides when using fly cells as stimulators, while immature DC resulted in meaningless specific killing, and mature DC was used for stimulation It resulted in specific killing against only one of the four peptides.
Preparation of cytotoxic lymphocytes CD8 ⁺ cells isolated from leukapheresis samples by positive selection with anti-CD8 antibody were transformed into human class I molecules (HLA-A2.1), B7.1, ICAM. Stimulates against four different melanoma-related peptides presented by Drosophila cells expressing -1, LFA-3 and B7.2. CD8 ⁺ cells are restimulated twice with autologous monocytes supplemented with peptide epitopes in the presence of IL-2 and IL-7. CTL is non-specifically expanded with OKT3 and IL-2. CTL activity is measured against Malme 3M cells and the purity of CD8 ⁺ T cells is assessed by flow cytometry.

  The manufacturing process and protocol are performed according to Good Laboratory Practice and Good Manufacturing Practice. “Excellent Laboratory Code” and “Good Manufacturing Code” are laboratory and manufacturing standards established by the United States Food and Drug Administration and are readily known to those skilled in the art. It is done. CTL are monitored for identity, viability, CTL activity, sterility and endotoxin content.

A list of peptide epitopes suitable for use in the methods of the invention for treating breast and ovarian cancer is shown in Table 1 below. In addition to those listed in Table 1 below, a wide range of peptide epitopes can also be suitable for use in the methods of the invention for treating breast and ovarian cancer, provided that such peptides are T cell epitopes. It will be readily apparent to those skilled in the art.

Treatment regimen for malignant melanoma using ex vivo generated autologous T lymphocytes with specificity for melanoma-related target antigen Clinical trials, ex vivo generated self cytotoxicity with specificity for melanoma-related target antigen Performed in patients with advanced metastatic melanoma using T lymphocytes (CTL). At least a single infusion of ex vivo generated CTL was administered to the patient per treatment cycle. Concomitant administration of either IFN-α or IL-2 to patients at specific times and doses was beneficial for priming tumor cells against lysis by antigen-specific CTL and in vivo persistence of CTL It has been shown.
Interferon and CTL Combination Therapy Interferon-α (IFNα) has a broad spectrum of immunomodulatory and antiproliferative effects in a variety of malignant lesions. Several clinical trials over the past decade have provided clear evidence that IFNα has antitumor activity in melanoma (Legha, Cancer (1986) 57: 1675-1777). When used as monotherapy, rIFN-α-2a and rIFNα-2b have an average response of 15% (range, 6% -27%) with response durations ranging from 1 to over 60 months Yielded rate. A summary of several single agent IFN-α studies in metastatic melanoma is listed in Tables 2 and 3.

  One mechanism of action of IFN-α appears to be upregulation of tumor antigen expression on melanoma cells. That is, it has the ability to enhance the expression of immunologically important molecules on the surface of the tumor. Such immunologically important molecules include, but are not limited to, histocompatibility (mouse H-2 or human HLA) antigens, accessory molecules such as intercellular adhesion molecule-1 (ICAM-1 / CD54), and Tumor-associated antigens (Robincon et al., Immunobiology (1986) 172: 275-282; Borden et al., Mitchell, MS (eds): Biological Approaches to Cancer Treatment; Biomodulation. Pp. 440-476) among 1992). The immunomodulatory effects of IFN-α can potentially improve the ability of the immune system, including both antibodies and lymphocytes, to recognize and attack tumors in vivo.

  Active specific immunotherapy has demonstrated significant clinical activity in the treatment of disseminated melanoma. The results of immune function studies have shown that melanoma vaccine treatment increases the frequency of anti-melanoma CTL (Carrel et al., Eur J Immunol (1985) 15: 118-123). Combination immunotherapy regimens (IFN-α and melanoma vaccine) are currently used in the treatment of disseminated melanoma (Agarwala et al., BioDrugs (1999) 12: 193-208). It is thought that these two modes of immunotherapy may act synergistically.

The Eastern Cooperative Oncology Group (ECOG) is a prospective randomized controlled trial of interferon α-2b (IFN-α-2b) for observation as surgical adjunct therapy in 287 patients with melanoma (Kirkwood et al., J Clin Oncol (1996) 14: 7-17). The dose used in the study was the maximum tolerated dose, ie 20 MU / m ² / day intravenously (IV) 5 days per week for 4 weeks, then 10 MU / m ² subcutaneously 3 times per week ( SC) 48 weeks. The results indicated that there was a significant prolongation of survival without recurrence and overall survival in the group receiving IFN-α-2b. However, toxicity was considerable; 67% of patients had severe (grade 3) toxicity and 9% had life-threatening toxicity. Dose changes were required by the majority of patients. The treatment was also expensive. Based on the ECOG study, the FDA approved interferon as a surgical adjunct treatment for melanoma in 1996.

In a large prospective, randomized study in high-risk melanoma patients, 642 participants had 3 arms; high-dose interferon α-2a, ie 20 MU / m ² per week, 3 times 12 Those who received low-dose interferon for a week or were randomized to observation. Time to progression (TIP) was prolonged only in the high dose group, although overall survival was identical for all three groups (Kirkwood et al., J Clin Oncol (2000) 18: 2444-2458). The World Health Organization (WHO) received a low-dose interferon alpha-2a for observation, 3 MU / m ² SC 3 times per week as an adjunct therapy in 427 patients with high-risk melanoma. Weekly testing was performed (Cascinelli, Proc Am Soc Clin Oncol, (1995) pp410 (abstr)). There was no difference in disease-free or survival in patients receiving interferon.

The present invention is:
a) administering to a subject with melanoma an effective amount of interferon-α capable of enhancing the expression of a tumor antigen on the surface of the tumor; and b) having specificity for a melanoma-associated target antigen. There is provided a method of treating a subject comprising the step of inoculating the subject with an effective amount of autologous cytotoxic T lymphocytes.

Either INF-α-2a or INF-α-2b can be used in this treatment method. INF-α can be administered to the patient via SC, IM or IV. An effective amount of interferon-α in this method is to up-regulate both HLA (class I) expression and melanoma-associated antigen expression (two important requirements for recognition and lysis by antigen-specific T cells). The amount of INF-α that is sufficient. This effective amount can be determined by one skilled in the art. Example 5 (below) describes some considerations in determining the effective amount of INF-α to be used in the treatment method. Preferably, to reduce the side effects caused by INF-α, the effective amount of INF-α-2a is about 5-20 MU / m 2 / day, and the effective amount of INF-α-2b is about 5 15 MU / m2 / day. Also preferably, an effective amount of INF-α is a short period of time immediately prior to inoculating a subject with an effective amount of autologous cytotoxic T lymphocytes with specificity for a melanoma-associated target antigen, such as Administer to patients for 3-7 days only. In a preferred embodiment, the subject is subcutaneously administered continuously from day 5 to day 1 before inoculating the subject with an effective amount of autologous cytotoxic T lymphocytes having specificity for a melanoma-associated target antigen. The effective amount of interferon-α that is done is about 10 MU / m ² / day.

An effective amount of autologous cytotoxic T lymphocytes with specificity for a melanoma-associated target antigen either stops the growth of the melanoma lesion or causes a reduction in its size when the subject is inoculated with CTL. Is the amount of CTL generated ex vivo with specificity for a melanoma-associated target antigen. Preferably, an effective amount of autologous cytotoxic T lymphocytes with specificity for a melanoma-associated target antigen is about 1-10 × 10 ⁹ cells per injection.

Autologous cytotoxic T lymphocytes with specificity for melanoma-associated target antigens are:
a) Preparing a non-naturally occurring antigen presenting cell line (nnAPC), which can simultaneously present up to about 15 different epitopes associated with melanoma, each epitope being 8-10 An amino acid peptide);
b) adding up to about 15 different epitopes associated with said melanoma to nnAPC;
c) collecting CD8 ⁺ cells from said subject;
d) stimulating said CD8 ⁺ cells with an nnAPC cell line supplemented with epitopes to obtain CD8 ⁺ cells specific for melanoma;
e) growing CD8 ⁺ cells specific for melanoma in a medium containing IL-2 and IL-7;
f) mixing CD8-depleted peripheral blood monocytes collected from the subject with each epitope added to the nnAPC;
g) irradiating said CD8-depleted peripheral blood monocytes with about gamma radiation;
h) isolating adherent CD8-depleted peripheral blood monocytes;
i) adding each epitope added to the nnAPC to the adherent peripheral blood monocytes;
j) restimulating the CD8 ⁺ cells specific for melanoma with gamma-irradiated epitope-attached peripheral blood monocytes;
k) growing restimulated CD8 ⁺ cells specific for melanoma in a medium containing IL-2 and IL-7;
l) Expanding restimulated CD8 ⁺ cells specific for melanoma with OKT3 antibody stimulation:
m) obtained by a method comprising assaying expanded CD8 ⁺ cells for activity, purity, sterility and endotoxin content of suitable cytotoxic T lymphocytes;
Here step (j) can be repeated at least once more.

Preferably, the non-naturally occurring antigen presenting cell line is tyrosinase, gp100 and MART-1, such as those specifically set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. An epitope that is a peptide of origin is added.
Interferon, CTL and Interleukin-2 Combination Therapy Human recombinant interleukin-2 is a lymphokine produced by recombinant DNA technology that has been shown to exhibit diverse biological activities. In vitro, it has been shown to enhance lymphokine mitogenesis, increase lymphocyte cytotoxicity, induce killer activity of both lymphokine activation and natural killer cells, and it is interferon-γ Production is also induced.

  It has been examined in clinical trials for the treatment of metastatic renal cell carcinoma, Kaposi's sarcoma, colorectal cancer, non-Hodgkin's lymphoma, and metastatic melanoma. It has been used alone or in combination with biologics such as chemotherapy, lymphokine-activated killer (LAK) cells or tumor infiltrating lymphocytes (TIL) or interferon. It was administered at both high and low doses and with various infusion schedules (bolus and continuous). Response rates ranged from 15 to 20%.

A 1997 Phillip review looked at 540 patients in 15 trials conducted in the 1990s using single agent IL-2 (Phillip et al., Semin Oncol (1997) 14: Suppl 4: 32- 38). Dosing varied from 3 12 MIU / m ² boluses per week to 3 to 7 MIU / m ² / day continuous infusion. The overall response rate in the tour was 15% with a range of 3-50% and 2% complete remission. In a 1994 study of 134 patients reported by Rosenberg et al. (JAMA (1994) 271: 907-913), high dose bolus IL-2 was 7% with 9 complete remissions (CR). Resulted in a remission rate. Of the 9 cases with CR, 8 cases had no disease for more than 9-91 months. The Cytokine Working Group performed a retrospective analysis of 266 patients treated with high dose IL-2 bolus therapy, which resulted in CR in 16 patients (6%) 69% survived and did not progress at 5 years (Atkins et al., Proc Am Soc Clin Oncol (1997) 16: 494a).

  Meta-analysis of 911 patients treated with the combination IFN-α and IL-2 resulted in a 17% response rate (Allen et al., Proc Am Soc Clin Oncol (1997) 16: 494a (1781)). The median survival is 11 months, which is not significantly longer than monotherapy.

  Administration of IL-2 is used immediately after CTL infusion to increase lymphocyte mitogenesis, lymphocyte cytotoxicity and gamma interferon production in an effort to maintain antigen-specific T cells in vivo. can do.

In a preferred embodiment, the present invention provides:
a) administering to a subject with melanoma an effective amount of interferon-α capable of enhancing the expression of tumor antigens on the surface of the tumor;
b) inoculating said subject with an effective amount of autologous cytotoxic T lymphocytes with specificity for melanoma-related target antigen; and c) self-cytotoxicity with specificity for melanoma-related target antigen. A method of treating the subject is provided comprising the step of administering to the subject an effective amount of interleukin-2 capable of enhancing in vivo maintenance of T lymphocytes.

  Interleukin-2 can be administered to patients via SC, IM or IV. An effective amount of interleukin-2 is that amount of IL-2 that is sufficient to maintain ex vivo generated antigen-specific CTL in vivo. This effective amount can be determined by one skilled in the art. Example 5 (bottom) describes some considerations in determining the effective amount of IL-2 to be used in the treatment method. Preferably, to reduce the side effects caused by IL-2, an effective amount of IL-2 is about 2-10 MIU / day. Also preferably, an effective amount of IL-2 is administered to the patient immediately after inoculating the subject with an effective amount of autologous cytotoxic T lymphocytes with specificity for a melanoma-associated target antigen. In a more preferred embodiment, the subject is subcutaneously administered continuously from day 0 to day 27 after inoculating the subject with an effective amount of autologous cytotoxic T lymphocytes having specificity for a melanoma-associated target antigen. The effective amount of interleukin-2 administered is about 3 MIU / day.

In a most preferred embodiment, the present invention provides:
a) 10 MU / m ² / day continuously from day 5 to day 1 before inoculating subjects with melanoma with their own cytotoxic T lymphocytes with specificity for melanoma-associated target antigen Of interferon-α-2b of the subject subcutaneously;
b) inoculating the subject with autologous cytotoxic T lymphocytes having specificity for about 1-10 × 10 ⁹ cells melanoma-related target antigen per injection; and c) melanoma-related target antigen Subcutaneous administration of about 3 MIU / day of interleukin-2 continuously from day 0 to day 28 after inoculating the subject with autologous cytotoxic T lymphocytes having specificity for A method of treating the subject comprising the steps of:

  At the end of each cycle of treatment, complete or partial response in the patient can be assessed. The treatment cycle can be repeated at intervals of about 2 months.

  The present invention will be better understood by reference to the experimental details that follow, but those skilled in the art will recognize that these are more fully described in the claims that follow. It will be readily appreciated that this is only a specific description of the invention. In addition, various publications are cited throughout this application. The disclosures of these publications are hereby incorporated by reference into this application in order to more fully describe the prior art to which this invention pertains.

Example 1
Production of Drosophila antigen-presenting cells The Schneider S2 cell line was prepared from Drosophila melanogaster (Oregon-R) eggs according to published procedures and the American Type Culture Collection (American) Deposited at Culture Collection) (CRL 10974). S2 cells are grown in commercial Schneider Drosophila medium supplemented with 10% fetal calf serum.

The pRmHa-3 plasmid vector for expressing MHC class I and costimulatory proteins in S2 cells was derived from the pRmHa-1 expression vector constructed as described in the literature. It contains a metallothionein promoter, a metal responsive consensus sequence, and an alcohol dehydrogenase gene carrying a polyadenylation signal isolated from Drosophila melanogaster.
Complementary DNA for transfection was prepared as follows:
HLA-A2.1 and β-2 microglobulin: reverse transcription PCR from K562 cells using primers derived from published sequences
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 published sequences
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 published sequences
Complementary DNA was individually inserted into the pRmHa-3 vector. S2 cells were transfected with a mixture of HLA-A2.1, B7.1 and ICAM-1 plasmid DNA, and phshneo plasmid using the calcium phosphate precipitation method. Stably transfected cells were selected by culturing in Schneider medium containing geneticin. 24 hours before use, and the 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 more than 30% of cells is required for efficient in vitro activation of CD8 ⁺ lymphocytes.
Isolated CD8 ⁺ cells of human CD8 ⁺ cells are isolated from a sample leukapheresis by positive selection using Dynabeads ^{[Dynabeads] TM} isolation procedure (Dynal (Dynal)). Anti-human CD8 mouse monoclonal antibody (human γ-globulin [Gammagard ^{(Gammagard) (R)]} 1ml of 50 [mu] g), 1% human serum albumin (Baxter - Hyland (Baxter-Hyland)) and 0.2% sodium citrate To the washed cells in supplemented Dulbecco's PBS. After 45 minutes incubation at 4 ° C. with gentle mixing, 1: 1 beads in the same buffer containing Dynal magnetic beads (Dynabeads ^™ ) coated with sheep anti-mouse IgG Wash and resuspend at cell to cell ratio. Cells and beads are placed in a sterile tube and mixed gently at 4 ° C. for 45 minutes. The time at the end, the antibody bound cells, magnetically removed using a MPC-1 ^(R) separator according to the manufacturer's instructions (Dynal (Dynal)). Dissociation of the CD8 cell-bead complex is achieved by incubation for 45 minutes at 37 ° C. in the presence of CD8 peptide _59-70 (AAEGLDQRFSG; SEQ ID NO: 12). Free beads are removed magnetically and CD8 cells are counted and analyzed by flow cytometry to assess purity. CD8 ⁺ cell recovery is typically greater than 80%. Table 4 summarizes the cellular composition of 14 separate CD8 ⁺ preparations from normal human PBMC preparations by positive selection with anti-CD8 antibodies.

Primary stimulation of purified human CD8 ⁺ cells in vitro immunization Transfected Drosophila S2 cells were cultured in Schneider medium (10 ⁶ cells / cell supplemented with 10% fetal calf serum and CuSO _4. Incubate for 24 hours at 27 ° C. Cells are _harvested , washed and resuspended in Insect X-press medium (BioWhittaker) containing 100 μg / ml human tyrosinase _366-377 . After 3 hours incubation at 27 ° C., 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 at 37 ° C. for 4 days, during which time Drosophila cells die. On day 5, IL-2 (20 U / ml) and IL-7 (30 U / ml) are added to selectively expand the tyrosinase-specific CTL population.
_{Restimulation} Frozen autologous CD8 depleted PBMC obtained at the time of leukocyte component thawing, washing, and containing 10% autologous serum (as a source of β2 microglobulin) and 20 μg / ml tyrosinase _369-377 resuspended at 10 ⁶ cells / ml in RPMI medium. Cells are incubated for 2 hours at 37 ° C. after gamma irradiation (5,000 rads). Non-adherent cells are removed by washing with Dulbecco's PBS. Tyrosinase epitopes are added to adherent monocytes by incubation for 90 minutes in Hepes buffered RPMI medium containing 10% autologous serum and 10 μg / ml tyrosinase _369-377 . The supernatant was removed and Drosophila activated CD8 ⁺ cell suspension (3 × 10 ⁶ cells / ml in RPMI medium with 10% autologous serum) was transferred to one adherent monocyte. Add in a ratio of 10 CD8 ⁺ cells. After 3-4 days of culture at 37 ° C., IL-2 (20 U / ml) and IL-7 (30 U / ml) are added with medium changes to selectively expand the tyrosinase-specific CTL population.
Nonspecific extension effector cells, autologous serum, anti-CD3 monoclonal antibody ^{(OKT (R)} 3), by culturing them IL-2 and γ-ray irradiated autologous PBMC in supplemented RPMI medium, non Increase specifically.
Activity and Purity Assay CTL Assay Malme 3M cells are used as target cells in a ⁵¹ Cr release assay. 4% fetal calf serum, 5 × 10 ⁶ Malme 3M cells in RPMI medium containing 1% HEPES buffer and 0.25% gentamycin are labeled for one hour at 37 ° C. with ⁵¹ Cr for 0.1 mCi. Cells are washed 4 times and diluted to 10 ⁵ cells / ml in RPMI containing 10% fetal calf serum (HyClone). In 96-well microtiter plates, 100 μl of effector CTL and 100 μl of peptide-added ⁵¹ Cr-labeled Malme 3M target cells are combined in 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 background lysis of natural killer cells. Non-specific lysis is assessed using the non-neoplastic HLA-A2.1 fibroblast line Malme 3. Controls for measuring spontaneous release and maximal release of ⁵¹ Cr are included in duplicate. After 6 hours incubation at 37 ° C., the plates are centrifuged and the supernatant is counted to measure ⁵¹ Cr release.

  The percentage of specific lysis is the following equation:

Use to calculate.
Flow cytometry.

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 activity protocol using cells from healthy donors are shown in Table 5.

  In addition to activity and purity, CTL preparations can be assayed for sterility and endotoxin content.

Example 2
Objectives of the test study of cytotoxic T cell infusion against melanoma This example demonstrates the following factors:
1. Safety and tolerability of reinjected autologous CTL after in vitro immunization;
2. Kinetics of injected CTL in factoring of systemic circulation in limiting dilution analysis;
3. Whole body placement of CTL by radioscinography;
4). Treatment of melanoma as assessed according to cellular composition of biopsied nodules by immunohistology (CTL, TH, NK, B cells); and measurable injury regression over 5.2 months and duration of response Teaches the effectiveness of cytotoxic T cell infusions.
Patient population eligibility for treatment requires the patient to have histologically reported unresectable malignant melanoma that was measurable or otherwise evaluable, and an HLA-A2 haplotype did. Pre-treatment evaluation included radiological assessment of the brain by MRI or CT scan, CT scan of the chest and abdomen, and physical examination of the skin and lymph nodes, among others. The total number of patients treated was 15 (9 males and 6 females). Age ranged from 33 to 75 years with an average of 58 years. The average duration of metastatic disease was 1.5 years. A pre-treatment skin test to determine if an anergic condition was present was performed in 14/15 patients, and 5/14 test results were negative for all 7 universal antigens evaluated. Met. Patients were screened for HLA-A2 haplotype by FACS analysis using an HLA-A2-specific monoclonal antibody (BB7.2). Subtyping was performed by PCR analysis. All but one patient were HLA-A * 0201, and the exception (patient 08) was HLA-A * 0205.
Treatment with ex vivo generated autologous CTL Fifteen patients were treated under this clinical protocol. All patients received at least a single infusion of autologous CTL. The number of cycles and cell doses administered to each patient are summarized in FIG. The number of cells generated in vitro depended on patient-related factors such as the number of PBMCs isolated from aphaeresis treatment, and the number of CD8 ⁺ T cells present in each PBMC preparation. As all of the cells generated in vitro were reinjected into the donor, the dose administered to each patient inevitably varied. In an attempt to normalize doses between patients, the calculated “efficacy” score was recorded for each dose. The value was obtained by multiplying the total number of cells by the lytic activity obtained with peptide-added target cells. The dose of infused T cells ranged from a minimum of 4 × 10 ⁷ (patient 08) to a maximum of 3.2 × 10 ⁹ (patient 13). Patients entered a second, third or fourth treatment cycle based on their clinical status at the end of each cycle. The number of PBMCs obtained from the apheresis sample tended to be smaller in patients undergoing additional cycles, especially when the beginning of the subsequent cycle was close to the end of the previous one. This is attributed to persistent lymphopenia by IFNα-2b administered during the previous cycle. The total number of CD8 ⁺ T cells that had not received an isolated dose was dependent on their proportion in each of the PBMC preparations. The percentage of CD8 ⁺ T cells varied between patients between 8% and 31%. The resulting expansion factor also contributed to the final cell number and ranged from 0.1 to 6.0 times. The procedure for generating CTL ex vivo is taught herein and in Example 1 above.
Up-regulation of class I and melanoma-associated antigens in response to IFNα-2b In an attempt to increase the ability of antigen-specific CTLs to lyse melanoma cells in vivo, low-dose IFNα-2b was continuously administered prior to CTL infusion. For 3 days a week for 5 days and for an additional 4 weeks. One method of measuring the in vivo response to cytokines is to evaluate biopsies obtained at successive time points by immunohistochemical analysis for positive staining with specific antibodies. Serial biopsies were obtained in one patient (patient 04) with multiple skin injuries for assessment of both class I and antigen expression. Biopsy showed that class I and MART-1 expression was weakly positive before either treatment (biopsy A). A dramatic increase in these two markers was shown 5 days after subcutaneous injection of 10 MU / m ² (biopsy B). For tyrosinase and gp100, immunohistochemical staining was negative or weakly positive in the pre-treatment samples, respectively (biopsy A). After the first 5 days of IFNα administration and 13 additional treatments, the expression of these latter antigens increased in the stained tissue samples (biopsy C).
Antigen specificity of ex vivo generated CTLs Generated CTLs from all patients were added to peptide-added T2 target, HLA-A2 melanoma cells when biopsy material was available to establish the lineage Lines (Malme 3M) and autologous melanoma lines were evaluated on the day of release. Each prepared dose of cells was evaluated for its cytolytic activity. The specificity of the CTL response generated for each patient was determined using peptide-added T2 cells that present either each peptide alone or all four peptides simultaneously. The ability to lyse cells carrying endogenously expressed melanoma-associated antigens was evaluated in HLA-A2 matched lines or autologous tumor lines. In addition to cytolytic activity, antigen specificity was evaluated with established detection methods for intracellular gamma interferon production generated in response to specific peptide stimulation. The CTL generated at the end of the ex vivo protocol was evaluated by this method. The percentage 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 at each successive treatment cycle.
Detection of CD8 and CD4 cells infiltrating tumor biopsies after CTL treatment Biopsy samples from all patients before, 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 5 of 15 patients who entered the study. In two patients (patients 08 and 13), biopsy samples were available at 5 and 6 weeks after T cell therapy, respectively. Examination of tissue samples showed the presence of both infiltrating CD8 and CD4 cells. One tumor sample was taken from a skin injury in the occipital region of the scalp that increased in size by the time of follow-up (4 weeks after the second injection of T cells). The biopsy showed necrosis of tissue heavily infiltrated with lymphocytes. The other biopsy was from the femoral head removed during hip replacement surgery. Skin injury from patient 08 was strongly positive (4+) for both general class I and specific HLA-A2 markers. Tyrosinase and gp100 were weakly positive (1+ and 2+, respectively), while MART-1 was negative in this same sample. The area of the biopsy from patient 13 was also necrotic with more heterogeneous staining; a clear population of tumor cells lacking expression of the HLA-A2.1 molecule, and one or more of the MAAs. However, the intact tissue area showed strong class I (4+) and all of the melanoma-associated antigens. Lymphocyte infiltration in this latter sample appeared to surround the tumor nodules rather than deeply infiltrate them. However, the highest proportion of cells directly associated with the tumor was CD8 cells. The lack of pretreatment biopsy samples from both of these patients prevented the identification of similar types of infiltrating cells in the pretreatment tissue samples.
CT scan after T cell treatment confirms objective response CT scan was part of pre-treatment screening criteria and post-treatment follow-up. Patient 10 received a single infusion of 8 × 10 ⁸ CTL (July 27, 1999) five weeks after the pre-treatment scan (June 23, 1999). Repeated chest CT scans one month after injection (August 27, 1999) showed a dramatic decrease in the magnitude of lung injury. Similarly, patient 14 underwent a chest CT scan as part of the entry process 3.5 weeks prior to the first infusion with 6.6 × 10 ⁸ cells (October 5, 1999) ( September 10, 1999). A follow-up CT scan one month after the second injection with 11.5 × 10 ⁸ cells (January 7, 1999) showed dramatic contraction of three separate injuries. Patient 13 also had an objective response as measured by pre and post CT scans. Paratracheal adenopathy went from 7.8 cm ² (before the test) to 4.4 cm ² after cycle I and disappeared after cycle II.
The presence of an anergic condition did not exclude the ability to generate CTLs or prevent clinical response. The majority of patients treated under this protocol received prior medical intervention. A pre-treatment skin study was conducted to determine whether anergic responses to a panel of seven universal antigens correlated with the inability to either generate CTL ex vivo or prevent reported clinical responses Decided. The ability to generate CTL ex vivo did not correlate with the results of the patient's pretreatment skin test. It should be noted that patients 03 and 04 (both mixed responders) had repeated skin tests prior to the start of the second cycle and remained anergy.
Example 3
Generation of HER-2 / neu-specific CTLs capable of lysing breast and ovarian tumor cells. To determine whether we can use this approach to target all forms of cancer, We were interested in applying our CTL generation technology to other tumor types. HER-2 / neu is a proto-oncogene with homology to EGFR that is amplified and overexpressed in many human cancers, mainly adenocarcinoma of the breast, ovary and colon. It is often associated with aggressive disease and can be a poor prognostic indicator. It has been studied in several clinical trials as a possible target for these types of cancer.

  In the early 1990s, either HER-2 / neu HLA-A2.1 restricted peptide epitopes were mapped either by computer assisted peptide binding algorithms or by mapping CTL isolated from ascites of ovarian cancer patients Identified (Table 6).

  All of the peptides were synthesized, given an identification number (PRI #) and evaluated for their ability to generate CTL ex vivo using the same method we used for melanoma-related T cell peptide epitopes. CD8 cells were isolated from normal donors to determine their ability to routinely generate CTL ex vivo using Drosophila cells supplemented with known CTL peptide epitopes. Peptides 826, 835, 861 and 863 had the highest frequency of CTL generation (Table 7).

  Transfected Drosophila cells have the unique ability to display up to 10 different peptide epitopes (FIG. 10), while we have 4 types depending on the frequency of generating CTL for these peptides ex vivo. HER-2 peptides, 826, 835, 861 and 863 were selected. These four different HER-2 peptides represent weak to moderate binders to the HLA-A2.1 molecule presented on the surface of transfected Drosophila cells. We show that our experiments with melanoma-related peptides generally generate potent CTLs that recognize tumor cells when weak class I conjugates actually represent natural T cell epitopes. As suggested, it tends to include weak A2 conjugates. The majority of the tumor-associated proteins we target are autoantigens and can themselves be expected to have a high affinity for class I molecules found with viral peptides. Low to moderate conjugates generally produce CTLs that lyse tumor cells very efficiently. This is a low affinity binder to Drosophila cells (Figure 3) but can still lyse both target cells (T2) loaded with peptides or more importantly melanoma cells (Malme3M) This was demonstrated with the MART-1 peptide, which represents an epitope that routinely produces a strong CTL (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 many normal tissue epithelial cells. In normal cells, the HER-2 gene exists as a single copy. Amplification of the gene and / or overexpression of related proteins has been identified in many human cancers, including breast, ovary, uterus, stomach, and adenocarcinoma of the lung. The sequence differences between HER-2 and the EGF-R receptor are shown in Table 8. Three of the four HER-2 peptides we evaluated have three or more amino acid changes between the two proteins. A single amino acid change is sufficient to distinguish between the two proteins.

After CTLs were generated after a 4 week ex vivo stimulation protocol, we used HLA-A2.1 tetramer molecules prepared with immunized peptides to determine whether peptide-specific cells were present. evaluated. As demonstrated in FIG. 13, the ability to generate peptide-specific CTL was donor dependent. In Figure A (donor 261), the donor produced a strong CTL response to peptide 835 (37.55%). In Figure B (donor 262), peptide-specific CTL can be detected with both 835 and 861 tetramer molecules (3.6% and 15.1%, respectively). This supports the use of multiple peptides to ensure peptide-specific CTL at the end of the stimulation protocol. This ex vivo protocol makes it possible to generate multispecific CTL relatively easily.
Anti-peptide and anti-tumor responses After completion of the complete ex vivo protocol, the generated CTLs were evaluated for antigen specificity. Four HER-2 peptide combinations were added to Drosophila cells on day 0 to generate CTL. At the end of the 4 week ex vivo stimulation protocol, large numbers of CD8 cultures were evaluated for antigen specificity. T2 cells supplemented with each of the immunizing peptides were used as target cells. A typical response is depicted in FIG. Large cultures contain specificity for each of the four HER-2 peptides. The anti-tumor response was evaluated with an ovarian tumor cell line (ATCC; HTB-77). If the target cell line was not HLA-A2.1 restricted, we transfected the cell line to have a +/− assay system. When HLA-A2.1 was transfected into the HTB-77 line, enhanced killing by CD8 effector cells was shown (FIG. 15, FIGS. AD). HER-2 specific effectors representing individual peptides were evaluated to confirm the presentation of each peptide epitope on this tumor cell line.

A breast adenocarcinoma cell line (ATCC; HTB-131) transfected with HLA-A2.1 was also evaluated for its ability to demonstrate oncolysis with HER-2 specific peptide effectors. CTL specific for peptide 861 was able to lyse this tumor cell line when transfected with HLA-A2.1 (FIG. 16).
IFNγ treatment required for tumor cell lysis The HTB-77 / A2.1 cell line requires pretreatment with IFNγ to demonstrate peptide-specific lysis. Cells were treated with 500 U / ml IFNγ (25 ng / ml specific activity) for 24 hours prior to the start of the ⁵¹ Cr release assay. In FIG. 17, the addition of IFNγ resulted in enhanced lysis of cell lines transfected with HLA-A2.1. To determine the effect of this dose of IFNγ on the surface expression of both HLA-A2.1 and HER-2, FACS analysis was performed to measure the levels of these molecules both 24 and 48 hours after induction. . Figures 18, A and B depict the results of FACS analysis. In Figure A, there was no promotion of HER-2 molecules on the surface of HTB-77 cells at 24 and 48 hours after induction with IFNγ. In cells transfected with HLA-A2.1, neither HER-2 nor HLA-A2.1 demonstrated an increase in surface expression levels after a similar treatment protocol. What was shown was the expression of TAP-1 and the increased levels of HLA-DM and -DR, cathepsin S and D and caspase 5 when mRNA levels were assessed by microarray DNA chip analysis (Figure 19). . This appears to explain why there is accelerated killing of HTB-77 / A2.1 cells in the presence of IFNγ. This particular molecular up-regulation appears to result in more efficient processing of the HER-2 molecule and allow better presentation of the peptide of interest.
Peptides 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. Melanoma-related peptides are: peptide 819 is MART-1 specific (AAGIGILTV SEQ ID NO: 6), 817 and 853 are both gp100 peptides (ITDQVPFSV SEQ ID NO: 4 and KTWGQYWQV SEQ ID NO: 5 respectively), tyrosinase specific peptide Were 689 and 792, and 792 included a post-translationally modified version (YMDGTMSQV SEQ ID NO: 2) of the native sequence represented by peptide 689 (YMNTTMSQV SEQ ID NO: 1). 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) was an overlapping sequence representing a myxin protein detected in ovarian tumor lines. C-lectin is also a protein detected in ovarian tumor cell lines, and the peptide from that sequence (C-lectin ₈ ) is represented by KMASRMRL SEQ ID NO: 9.
The in vitro cytotoxicity assay standard ^{a 51 Cr} release assay was performed to measure the CTL effector cell recognition of melanoma-associated peptide epitopes added to T2 cells. Collected 3 × 10 ⁶ T2 cells were grown in RPMI + 10% FBS (medium). 0.1 mCi of ⁵¹ Cr was added and incubated at 37 ° C. in a water bath. Labeled cells are added to 10 ml of 4% wash solution (RPMI + 4% FBS) and pellet, washed an additional 2 times, and 0.2 × 10 6 to record the radioactivity of cells dissolved in spontaneous versus detergent. Resuspended in medium to a final concentration of ⁶ / mL. 20 μg / mL of the appropriate peptide (s) was applied to the cells for 30 minutes. 50 μL is added to each 96-well plate containing 10, 2, 0.4 and 0.08 × 10 ⁶ CD8 effector cells, respectively, which is incubated at 37 ° C. for 6 hours, spun and supernatant Collected about.
Flow cytometry and tetramer staining Cells were either bound to FITC or PE by incubation in FACS buffer (1% BSA in PBS, 0.02% NaN ₃ ) for 30 minutes at 4 ° C. followed by washing with the same buffer. Labeled with monoclonal antibody. Cells were fixed in 0.5% formaldehyde prior to data acquisition and analysis on a FACScan flow cytometer (Becton Dickinson) using its CellQuest software. Non-specific staining was measured using the same secondary antibody used to label the purified primary antibody, or an isotype matched control if the primary antibody was directly labeled. Tetramer staining was performed using an HLA-A2.1 specific HIV gag tetramer molecule (Beckman Coulter) having the sequence SLYVTVATL SEQ ID NO: 43 as a negative control. A HER-2 specific tetramer was made using the sequence CLTSTVQLV (826 SEQ ID NO: 7), KIFGSLAFL (835 SEQ ID NO: 8) or VMAGVGFSPYV (861 SEQ ID NO: 16) peptide. The PE-labeled tetrameric HLA-A2.1-peptide complex was used with a fluorescein isothiocyanate (FITC) labeled anti-human CD8a (BD PharMagin) monoclonal antibody to generate epitope-specific CD8 + T cells. Stained as described on the package insert. Samples were analyzed by two-color flow cytometry on a Becton Dickinson FACScan and gated CD8 + T cells were examined for staining with tetrameric HLA-A2.1-peptide complexes. .
Example 4
Generation of additional breast and ovary specific CTL using this ex vivo stimulation protocol We generate CTL responses against all known HLA-A2.1 restricted peptide epitopes of several tumor antigens of diverse tumor origin Proven ability. Our initial study focused on melanoma, where we were treated with CTL specific for four different peptide epitopes specific for MART-1, gp100 and tyrosinase melanoma-related proteins It was possible to establish an objective clinical response in patients [ Richards et al., Amer. Soc. Clin. Oncol. San Francisco, California (May 2001)].

  In order to expand the ability to generate CTLs against other tumor antigens present in a wide range of other cancers, we have chosen published and novel sequences for tumor antigens common to several different tumor types. These include AES, MUC-1, CEA, FBP, C-lectin, NY-ESO-1, Pec60, CA-125, MAGE-3, telomerase and G250. Table 10 describes these antigens, the frequency of expression and the cancers that express them. The frequency of response to these peptides using our ex vivo stimulation protocol is listed in Table 9.

Example 5
Objectives of treatment test for malignant melanoma using CTL infusion linked to interferon and interleukin-2 This example demonstrates the following factors:
1. Establishing the efficacy of self-CTL reinjected after in vitro immunization with Drosophila cells transfected with human class I and costimulatory molecules and supplemented with melanoma-related peptide epitopes;
2. Of autologous CTL generated ex vivo by immunization with interferon-α-2b (IFN-α), interleukin-2 (IL-2), and Drosophila cells at the prescribed dose and schedule Establish safety and tolerability of administration;
3. Determining whether tumor-infiltrating T cells are present in the tumor biopsy after treatment;
4). Demonstrates the presence and persistence of antigen-specific CD8 cells in the peripheral blood of treated patients;
5). As assessed by confirming that five consecutive daily subcutaneous injections of IFNα (10 MU / m ² ) can up-regulate class I and melanoma-associated antigens on the surface of melanoma in vivo Teaches the effectiveness of cytotoxic T cell injection in the treatment of melanoma.

An overview of the clinical trial CTL-03 is presented in FIG.
Patient population Eligibility for treatment required patients to have histologically reported unresectable malignant melanoma that was measurable and easily evaluated. In addition, patients were required to have an HLA-A2 haplotype. Pre-treatment evaluation included radiological assessment of the brain by MRI or CT scan, CT scan of the chest and abdomen, and physical examination of the skin and lymph nodes, among others. The total number of patients to be entered is 42. Twenty-one patients are in the control cytokine-only arm and 21 patients are in the cytokine + T cell therapy arm. 31 patients have entered to date (17 have entered the control arm and 14 have entered the T cell therapy arm). Patients progressing in the control arm provide crossing to the T cell arm if they wish (11 patients crossed). The total number of patients treated to date is 31 (16 men, 15 women). Age ranged from 27 to 80 years with an average of 52 years. Patients were screened for HLA-A2 haplotype by FACS analysis using HLA-A2 specific monoclonal antibody (ATCC: BB7.2). Subtype classification was performed by PCR analysis. All patients except 2 were positive for HLA-A * 0201. The other HLA-A2 subtypes were HLA-A2 * 0202 and HAL-A2 * 0205.
Administration of Drug Interferon-α-2b (Intron-A [Intron-A] ^(R) ; Recombinant Interferon α-2b, Schering Corporation, Kenilworth, NJ) for 5 consecutive days prior to CTL injection, The patient was administered subcutaneously at 10 MU / m ² . Ex vivo autologous lymphocytes (1-10 × 10 ⁹ ) activated in Drosophila cells were injected into the patient the day after INF-α administration. Interleukin-2 (pro Liu Kin ^{[PROLEUKIN] (R);} Al des Liu Kin (Aldesleukin), recombinant IL-2, Chiron Corporation (Chiron Corporation), Emeryville, CA) subcutaneously, to a patient in daily 3MIU immediately CTL infusion And continued for another 27 consecutive days.

  Table 11 uses what is used in FDA-approved protocols for a single dose of IFN-α or high dose IL-2 in an auxiliary setting (after surgical removal of all detectable tumors) 3 shows a comparison of cytokine doses used in test CTL-03.

Primary Stimulation of Purified CD8 Cells In Vitro Immunization Transfected Drosophila S2 cells were placed in Schneider medium supplemented with 10% fetal bovine serum and copper sulfate (10 ⁶ cells / mL). ), Incubate at 27-28 ° C. for 24-72 hours. S2 cells were collected, washed, and 0.1 μg / mL of each peptide; human tyrosinase 369-377 (SEQ ID NO: 1 YMNGTMSQV and SEQ ID NO: 2 YMDGTMSQV), gp100 209-217 (SEQ ID NO: 4 ITDQVPFSV), gp100 154 Insect X-press medium (BioWhittaker) containing -162 (SEQ ID NO: 5 KTWGQYWQV) and MART-1 27-35 (SEQ ID NO: 6 AAGIGILTV) and 5 μg / mL human β2 microglobulin Resuspend in 3). After incubation at room temperature (23-25 ° C.) for 3-4 hours, S2 cells were transformed into Roswell Park Memorial Institute (RPMI) medium (synthetic medium, Gibco) supplemented with 5-10% autologous serum. Mix with CD8 + cells at a ratio of 1:10 in Gibco)). The cell mixture is incubated at 37 ° C. for 4 days, during which time Drosophila cells die. On day 4 or 5, IL-2 (20 U / mL) and IL-7 (30 U / mL) were added with medium change to specificity for melanoma associated antigens (MRRT-1, gp100 and tyrosinase) Selectively expand the antigen-specific CTL population of cells with
Restimulation Autologous CD8-depleted PBMCS obtained at the time of leukocyte component fractionation and frozen for future use are thawed, washed, and 10% autologous serum, 5 μg / mL recombinant human β2 microglobulin, and 5 ~20μg / mL of (depending on the total number of peptides to be added), tyrosinase was used in the above stimulation, gplOO and ¹⁰ in RPMI medium containing MART-1 6 ~10 ⁷ cells / mL ( (Depending on the number of CD8-depleted PBMC collected at the time of the CD8 isolation step). After gamma irradiation (5,000 rads), the cells are incubated for 2 hours at 37 ° C. Non-adherent cells are removed by washing with Dulbecco's PBS. Adherent monocytes contain 5 μg / mL human β2 microglobulin and 5-10 μg in 1% HSA (to avoid the possibility of introducing potential proteases that may be present in the serum instead of autoserum). The five peptide epitopes described above are added by incubation for 90 minutes in Leibowitz medium containing each of the five peptide epitopes / mL. The supernatant was removed and the Drosophila activated CD8 cell suspension (2.5 × 10 ⁶ cells / mL in RPMI medium containing 10% autologous serum) Add at a ratio of 10 CD8 cells to monocytes. After 3-4 days of culture at 37 ° C., IL-2 (20 U / mL) and IL-7 (30 U / mL) are added with medium change to selectively increase the melanoma-specific CTL population. Let A total of two such peptide-specific restimulation phases of adherent cells occur (one about 1 week after primary stimulation and the second about 1 week after). The non-specific growth phase occurs at the beginning of the 4 week ex vivo protocol.
Non-specific augmentation CD8 + effector cells that have undergone two restimulations are expanded in cell culture bags with feeder cells (irradiated, CD8 + unselected cells) after stimulation with OKT3 antibody. Frozen CD8 + unselected cells are thawed, washed and then gamma irradiated (3500 rads). A 4: 1 (feeder: effector) ratio is placed in a T-225 flask coated with OKT3 antibody. OKT3 stimulation is performed in complete RPMI medium containing 10% autologous serum supplemented with 20 U / mL IL-2. Two days later, stimulated T cells are diluted with fresh media and transferred to cell culture bags for expansion. Replenish fresh media and IL-2 approximately every 2 days to feed rapidly expanding T cells.
Phenotypic analysis of CD8 cells after the ex vivo stimulation protocol The phenotypic analysis as measured by flow cytometry was performed on the day of isolation from the leukocyte sample and on the day the cells were released for injection back into the patient. Performed on purified CD8 + cells. Statistical analysis was performed using samples from patients and normal donors who received the same ex vivo stimulation protocol. A statistically significant difference is seen between CD8 + samples that have not been dosed and CD8 + effector cells obtained at the end of the ex vivo stimulation protocol, regardless of whether the sample was from a patient or a normal donor. It was issued.

Activation markers (eg, CD69 not expressed on resting lymphocytes but rapidly induced upon activation; class II molecule HLA-DR expressed on activated T cells), memory markers (eg CD45RO expressed on activated cells and most memory cells), integrin markers (eg, CD49d / CD29 involved in lymphocyte migration from blood to tissue at the site of inflammation), accessory molecules ( For example, CD11a / CD18; assists in killing cytotoxic lymphocytes and mediates adhesion to many cells including the epithelium, as well as cytokine receptor markers (eg, functional high affinity IL-2R receptors) Is composed of non-covalently associated CD25 / CD122 / CD132 heterodimers) all of the ex vivo It was found to be upregulated on CD8 effector cells after completion of the super protocol. Table 12 summarizes the results of the analysis.

  The results of phenotypic analysis support that CD8 + effector cells are activated by the ex vivo protocol described herein. These activated CD8 + effector cells can move from blood to the tumor site in vivo, can kill tumor target cells with very strong sensitivity, and low doses It can potentially grow in the presence of IL-2 (because high affinity IL-2R is present on these cells). CD122 is constitutively expressed on a subpopulation of resting T cells. In Table 12, the white area is a statistically significant value that is up-regulated after completion of the CD8 + ex vivo stimulation protocol. The light gray area does not show significant up-regulation or down-regulation. The dark gray area indicates a statistically significant down-regulation of the reported molecule. The regulation of these markers is consistent with the induction of activated T cells that are fully capable of tracking and lysing tumor cells as measured by phenotypic analysis.

Flow cytometry Obtain approximately 1 × 10 ⁷ purified CD8 + cells from donor leukocyte component sorting. Each sample is placed in a sterile tube at 1 × 10 ⁶ per 100 μl wash buffer (PBS + 1% BSA + 0.02% NaN 3). To each tube containing 100 μl of cell suspension, add 10 μl of the appropriate antibody directly labeled with either FITC or PE, or if the primary antibody is unlabeled, continue with the labeled secondary antibody ( Add goat anti-mouse IgG). Each antibody step is incubated at 40 ° C. for 30 minutes. Wash with 1-2 mL wash buffer between each antibody step. Cells are pelleted at 600 × g for 7 minutes. Discard the supernatant. Each cell pellet is resuspended in 0.5 ml 0.5% formaldehyde / PBS fixative and read on an appropriately configured FACScan (Becton Dickinson) flow cytometer.
Up-regulation of class I and melanoma-associated antigens in response to the addition of IFN-α-2b From the clinical study described in Example 2 herein, 10 MU / m ^{2 on} days 17-21 of the clinical protocol The 5 day course of IFN-α-2b (Intron-A [Intron-A] ^(R) ) administered subcutaneously in HCV has two important requirements for recognition and lysis by antigen-specific T cells, HLA (class I) It was shown to be sufficient to up-regulate both expression and melanoma-associated antigen expression. This finding was recorded in serial biopsies obtained from a single patient (04-MJ) with multiple subcutaneous skin injuries available for analysis. Four samples were classified as (A) before treatment; (B) after initiation of IFNα treatment (5 consecutive doses); (C) after CTL infusion 1 and (D) after infusion 2. The results from immunohistochemical analysis for the expression of HLA (HLA-A2.1) and melanoma-associated antigen (MART-1) from four biopsy samples are summarized in Table 13, where the higher numbers are A higher level of expression of each protein is shown. The results indicate that there was no additional increase in expression of HLA (HLA-A2.1) and melanoma-associated antigen (MART-1) after the first consecutive 5 days of IFN-α treatment; It has been shown that interferon-α2b treatment over 5 consecutive days was not necessary to reach optimal levels of HLA-A2.1 and MART-1 expression.

Immunohistochemical staining Biopsy samples were shipped in RPMI medium at 4 ° C. overnight. Tissue, ^{OCT (R)} for the frozen tissue samples (tissue - Tech (Tissue-Tek)) were placed in embedding medium. Organization: The OCT matrix, were placed on filter paper before quick freezing step in the liquid NO _2. Frozen samples were stored at −80 ° C. before slicing. Cryostat sections (5 μm) were placed on Superfrost ^™ (Fisher) charged glass slides. Glass slides were fixed in cold acetone (−20 ° C.) and stored at −80 ° C. until stained. Endogenous peroxidase activity was sequestered by incubating in 0.3% H ₂ O ₂ in methanol for 10 minutes. Primary antibody was added for 1 hour in a humidified chamber. Biotinylated secondary antibody was added and incubated for 30 minutes at room temperature. Streptavidin-HRP was added under the same conditions. DAB substrate was added for up to 5 minutes and rinsed in water. The glass slides were hydrated with 4 cycles of alcohol (70-95-95-100%) followed by 3 cycles of xylene. Antibodies used in immunohistochemical analysis were from ATCC (pan HLA class I, W6 / 32 and HLA-A2 specific, BB7.2) or NeoMarker (MART-1, M2-7C10). there were.
Increasing the number of cells with repeated cycles of treatment In clinical trial CTL-03, the number of cells recorded on day 6 of the in vitro culture cycle was less than that detected with each cycle of treatment in CTL-02 Rather it was shown to have increased (FIG. 21). FIG. 21A represents a typical growth curve of ex vivo generated CD8 ⁺ cells in the CRL-02 test. The typical decline on day 6 reflected the death of nonspecific cells. CD8 cells from each cycle of treatment for patient 15-RT had a similar growth curve, suggesting that there were no memory cells remaining in the peripheral blood after each cycle of treatment. This was not true in test CTL-03. A typical drop in cell number is designated 01-KN-1 and reflects the drop seen in cells obtained during the first cycle. However, when cell counts were performed on cells obtained at the beginning of cycles 2 and 3 (FIG. 21B; 01-KN-2 and 01-KN-3), growth curves were typical due to the presence of memory cells . This was demonstrated in all patient samples that were from the second and third cycles of treatment in study CTL-03. This result can be attributed to the addition of IL-2 to the patient at the time of CTL infusion and 27 days after CTL infusion.
Cell Count A 0.2% solution of trypan blue was prepared. Cell counts were performed using a hemocytometer. The number of at least 100 viable (unstained cells) in at least one complete square (total 9.1 square mm) was recorded. The number of viable cells was recorded and the number of complete 1 mm squares was counted. Number of cells / square. The calculation to determine cells / mL is: (number of cells / 1 mm square) (dilution count) (1 × 10 ⁴ ) = cells / mL.
Tetramer analysis confirms the presence of antigen-specific T cells after CTL injection For test CTL-03, tetramer molecules containing either MART-1 or gp100 melanoma-related peptides are It was commercially available for cell analysis (Beckman Coulter; Immunomics). These tetramer molecules were not available for test CTL-02. As shown in FIG. 22, 6 patients who underwent a second cycle of treatment in study CTL-03, 6 of 6 patients were treated from the time of the first white blood cell component sampling. Had an increase in the level of MART-1-specific T cells by the start of the second cycle (generally a period of 2 months). When gp100 tetramer molecules were used to evaluate antigen-specific T cells, 5 of 6 patients had elevated gp100-specific T cells at the time of second leukocyte component sorting. (FIG. 22). The start of the leukocyte component sorting procedure indicates the start of the cell therapy cycle. A total of 6 cycles of T cell therapy were performed in one patient (15-DC). While each cycle was separated by approximately 2 months, the time frame between cycles 4 and 5 was 5 months because the patient wanted to take a break for vacation. Antigen-specific T cells for MART-1 and gp100 at the start of cycle 4 were 0.26% and 0.65%, respectively. When CD8 cells were evaluated at the beginning of cycle 5, antigen-specific cells for MART-1 were 0.29% and 0.46% for gp100. This can suggest that antigen-specific cells were maintained in vivo even after suspending cytokines and cell therapy.

Tetramer staining Approximately 1 × 10 ⁷ purified CD8 ⁺ cells were obtained from donors by leukocyte component sorting. Each sample was placed in a sterile tube at 1 × 10 ⁶ per 100 μl wash buffer (PBS + 1% BSA + 0.02% NaN 3). To each tube containing 100 μl of cell suspension, 5 μl of HLA-A2.1-tetramer-streptavidin-phycoerythrin (SA-PE) labeled molecule (with the peptide of interest added (MART-1, gp100 or Tyrosinase specific)) was added and incubated for 30 minutes at room temperature. Anti-CD8 ⁺ monoclonal antibody (FITC label) was added and incubation continued for an additional 30 minutes. Samples were washed at the end of the incubation period using 1-2 mL of wash buffer. Cells were pelleted at low speed (400 × g) for 10 minutes and the supernatant was discarded. Each cell pellet was resuspended in 0.5 ml 0.5% formaldehyde / PBS fixative and read on an appropriately configured FACScan (Becton Dickinson) flow cytometer.
Correlation between the three different in vitro assays used to measure cytolytic activity, antigen specificity and cell proliferation With the advent of tetramer technology that also evaluates antigen-specific cells, many publications are black A significant number of antigen-specific T cells have been reported in the peripheral blood of tumor patients (Lee et al., Nature Medicine (1999) 5: 677-685). There has been a great deal of failure when attempting to isolate these antigen-specific cells and expand the cells in an antigen-specific manner. While tetramer staining allows the presence of antigen-specific T cells to be detected, additional assays such as CTL lysis assays can determine whether the cells can properly kill peptide-specific targets. Useful for determining. An intracellular interferon gamma assay can be used to determine whether a cell can proliferate in response to an antigen. These three assays were performed on the final CD8 + T cell product (FIG. 23), and the number of antigen-specific T cells detected was able to lyse tumor cells in vivo and respond to antigen stimulation. To accurately reflect the number of T cells that can proliferate.
Interferon gamma measured in the cell supernatant after primary stimulation (day 6) increases with repeated cycles of T cell therapy IL-2 has a dramatic effect on T cell persistence in vivo Another indication of what had been shown was by measuring IFNγ production in the cell supernatant (FIG. 24). After 6 days of primary stimulation with Drosophila cells in each treatment cycle, cell supernatants are collected and evaluated for the level of cytokine, interferon gamma released by CD8 + cells in response to antigen-specific stimulation did. In repeated cycles of 4 patients, the level of IFNγ production was shown to increase with each round of T cell treatment in this time frame. IFNγ levels were low or undetectable in all cases. With each additional round of T cell therapy, higher levels of IFNγ were detected, indicating that more antigen-specific T cells were present in the isolated CD8 + population at the time of each leukocyte component sorting. Suggested.
Detection of T cells infiltrating tumor cells In one patient (04-AD) who received 3 cycles of cytokines and T cell therapy in the CTL-03 study, a biopsy of skin injury was performed 6 weeks after the end of the third cycle Got later. The presence of class I and melanoma-associated antigens MART-1 and gp100 was present by immunohistochemical staining analysis. The presence of infiltrating T cells (CD3 +) was also shown. Positive immunohistochemical staining of the tumor suggests that class I and melanoma associated antigens were maintained and / or upregulated by 5-day administration of IFNα. The presence of these markers on melanoma cells allows antigen-specific T cells to lyse tumor cells in an antigen-specific manner. The presence of infiltrating T cells within the tumor mass 6 weeks after T cell injection is persistent in vivo if the T cells represent a subset population of T cells that were actually injected with these T cells. To suggest.
And an in vitro cytotoxicity assay standard ^{a 51 Cr} release assay was used to determine the CTL effector cell recognition of added melanoma-associated peptide epitopes on T2 cells. 3 × 10 ⁶ T2 cells (TAP deficient, HLA-A2.1 positive) were grown and collected in RPMI + 10% PBS (medium). 100 mL ⁵¹ Cr per target was added and incubated at 37 ° C. in a water bath. Labeled cells were added to a volume of 10 mL 4% wash (RPMI with 4% FBS) and pelleted. Two additional washes were performed. Cells were resuspended in media to a final concentration of 0.2 × 10 ⁶ cells / mL and spontaneous vs. maximum (detergent lysed) cell radioactivity was recorded. The cells were applied with 10-20 μg / mL of the appropriate peptide (s) for 30 minutes. 96-well plate containing CD8 + effector cells at 50 μL / each different effector: target ratio. The mixture was incubated at 37 ° C. for 6 hours. Spin the plate and collect the supernatant. Radioactive supernatants were counted with a Cobra gamma counter. Specific ⁵¹ Cr release was calculated using the formula ( ⁵¹ Cr release−spontaneous release) / (maximum release−spontaneous release) × 100.

Intracellular gamma interferon assay Effector cells were stimulated for 5 hours by T2 cells to which peptides were applied (CD8: T2 ratio was 2: 1) in the presence of GolgiStop (PharMingen) To do. Stimulated cells were surface stained with anti-CD8-PE, fixed, and permeabilized with Cytofix / Cytoperm (PharMingen catalog number 2076KK) and then stained with anti-IFN □ -FITC did. Double labeled cells were analyzed by flow cytometry (Becton Dickson FACScan) and expressed as percent of maximum expression. Maximum expression of IFN □□ was measured as the amount expressed in response to OKT3 (Orthoclone) activation of the same effector cells.
Toxicity associated with biological response modifiers The goal of clinical research with biological response modifiers (eg, IFNα and IL-2) is to maintain therapeutic benefit while reducing toxicity to a minimal level It is to determine the optimum condition to do. Nonetheless, currently approved doses and dosing schedules of IFN-α-2b can significantly prolong the survival of melanoma patients. Administration can also be accompanied by severe toxicity. Although high doses of IL-2 appear to be more effective than low dose continuous infusions, high doses of IL-2 are still more toxic. The most common side effect is flu-like symptoms. The most serious side effects are hypotension, capillary leak syndrome and reduced organ perfusion. IL-2 has been used clinically for both renal cell carcinoma and malignant melanoma. A combination of IFN-α-2b and IL-2 in a low dose subcutaneous regimen has been described in other clinical settings (Pectasides et al., Oncology (1998) 55: 10-15; Piga et al., Cancer Immunol Immunotherapy (1997). ) 44: 348-351).

  In one study (Pectasides, 1998, supra), IFN-α-2b and IL-2 were administered to patients with metastatic renal cell carcinoma (RCC) in a triple combination immunochemotherapy regimen. Phase II studies included IL-2 [4.5 mU × 2/24 hours, 3 times a week for 2 weeks] and IFN-α-2b [3 MU / 24 hours, 3 times a week (alternating days with IL-2) 2 weeks] as well as low dose subcutaneous activity of vinblastine (VLB) [4 mg / m 2 every 3 weeks]. The patient had a one week rest from cytokines at which time they received a VLB. Treatment was repeated every 3 weeks with a maximum duration of 1 year. Toxicity was mild to moderate (Grades I and II) and consisted of fever, loss of appetite, malaise and nausea-vomiting in> 80% of patients. None of the patients experienced major VLB-related toxicity. It was proposed that the triple combination may be a promising regimen (38.7% overall response rate) with modest toxicity with progressive RCC.

  In another study evaluating low-dose IFN-α-2b and IL-2 in RCC (Piga, 1997, supra), IFN-α-2b [3 MU / m2 daily, continued intramuscularly] and IL-2 (0.5 MU / m 2 x 2/24 hours on days 1-5 for the first week, subcutaneously and 1 MU / m 2 x 2/24 hours, 5 days in the following 3 weeks). Side effects were said to be low in most patients (grades I and II) and showed abnormalities in liver and kidney function tests. While the regimen is said to be moderately toxic, low cost, and does not require hospitalization, the flu-like symptoms observed in most patients may cause subsequent treatment in 1/3 of patients. It was enough and sufficient to make it reject.

Side effects from the IFNα and IL-2 regimens described for the CTL-03 study were considered to be mild to moderate effects similar to those detected in the RCC study listed above. However, our cytokine regimen lasts a total of 33 days (5 days with IFNα and 28 days with IL-2), making it more attractive if the desired results can be achieved in this shorter time frame. In clinical trial CTL-02, we were able to reach our desired effect with 5 consecutive days of dosing and no additional 11 doses were required. In study CTL-03, only 1 of 31 patients who entered the study refused to complete a single cycle of cytokine therapy. Overall, the IL-2 dose was better tolerated by most patients.
Amount of cytokine required to reach desired effect In a phase I study, in a combined immunotherapy protocol that administers granulocyte macrophage colony stimulating factor (GM-CSF) and IFN-α-2b in addition to IL-2 The minimum dose of IL-2 was 2-4 MIU / m2, subcutaneously every 3 weeks for 12 days. Immune activation was monitored and a significant increase in lymphocytes, activated CD4 + and CD8 + T cells, NK cells, and monocyte DR expression was found27. The administration of IL-2 in this study was similar to that we used in study CTL-03 (3 MIU administered subcutaneously daily for 4 consecutive weeks), however, we It has the added benefit of providing a patient with a significant number of antigen-specific T cells at the start of treatment.

  Another study evaluates the impact of concomitant administration of pineal hormone melatonin (MLT) and low dose IL-2 in cancer patients who had progressed during previous immunotherapy with IL-2 alone Started for. Progressive solid tumors included lung, kidney, stomach, liver and melanoma. IL-2 was administered at a daily dose of 3 MIU subcutaneously for 6 days per week for 4 weeks; almost identical to that used for CTL-03. MLT was administered orally at a daily dose of 40 mg / day, starting 7 days before IL-2. Objective tumor regression was shown in 3 of 14 patients (21%). Patients with stable disease or documented response are progressively more robust to IL-2 with a significantly greater increase in the average number of lymphocytes and eosinophils with respect to patients with disease progression Neoplasms have been shown to be responsive to IL-2 therapy with concomitant administration of MLT. It is possible that MLT increased the antitumor immune effect of IL-2 and / or increased the sensitivity of cancer cells to cytolysis mediated by IL-2 induced cytotoxic lymphocytes.

  Temporal regulation and administration of IFN-α-2b and IL-2 as defined in test CTL-03, by up-regulating important tumor markers (expression of class I and melanoma-associated antigens) It is likely that melanoma cells are prepared for lysis and provide for the persistence of antigen-specific T cells. The conclusions are: 1) the presence of tetramer positive cells detected at the beginning of each successive cell therapy treatment was detected; 2) a growth curve resembling a secondary or memory response was observed And 3) supported by the observation that interferon gamma was produced in the culture supernatant evaluated on day 6 of the ex vivo protocol for each cycle of cell therapy. Advances in understanding cellular and molecular biology of IL-2 and its receptor complex have led to theories to better utilize IL-2 to augment and activate immune effectors in patients with cancer Provided rationale. The ex vivo protocol described herein for generating antigen-specific T cells recognizes and lyses tumor cells with very strong selectivity, trafficking to tumor sites and expressing melanoma-associated antigens Resulting in a large quantity of CD8 + preparation with all of the necessary surface molecules to do. The presence of a high affinity IL-2 receptor (CD25 / CD122 / CD132) on the surface of CD8 + cells suggests that these cells are capable of responding to lower doses of IL-2 in vivo. .

  The combination of cytokine therapy with added T cell therapy represents a novel method for treating advanced metastatic melanoma.

  While the foregoing specification has taught the principles of the invention, examples have been provided for the purposes of illustration and the practice of the invention is generally within the scope of the following claims and their equivalents. It will be understood to encompass all of the variations, adaptations and / or modifications.

Figure ² is a graphical 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) according to the invention. 2 is a graphical depiction of two diagrams of the mechanism of lymphocyte-mediated cytocytosis according to the invention. Test several different peptides in a competitive assay to identify peptide conjugates that can be used to add multiple peptides on Drosophila cells expressing human empty class I molecules according to the invention The result of the experiment was shown. Figure 3 shows the results of an experiment tested for the ability to generate CTL when the three melanoma peptides according to the invention are added as a single epitope to Drosophila cells. In this donor (# 60), CTL activity was elicited for each of the peptides when added alone to three different Drosophila preparations. The specificity of the response was compared to the high affinity binder, control HBc peptide. Figure 3 shows the results of a series of experiments in which up to four different peptides according to the invention were added alone to Drosophila cells. CTL activity for each of the represented peptides is seen after a 3 week stimulation protocol and is graphed in this figure. Results from 3 different donors (# 93, 94, 95) are represented. Figure 2 shows CTL activity after two different primary in vitro stimulation protocols according to the invention. The invention compares the ability of Drosophila cells to dendritic cells to elicit CTL responses to a single peptide epitope after a standard stimulation protocol. Invented dendritic cells, representing either mature or immature phenotypes, are as efficient as Drosophila cells in deriving specific CTL responses when using defined peptides to apply to the cells. Indicates no. Figure 3 shows CTL activity generated by a single donor for three different in vitro stimulation protocols presenting four peptides according to the invention. Figure 2 shows the CTL activity generated for 10 peptides added in combination to Drosophila cells according to the invention. Figure 2 shows the peptide binding ability of HER-2 peptides (826, 835, 861 and 863) on Drosophila cells transfected with human HLA-A2.1 class I molecules according to the invention. The invention demonstrates anti-peptide and anti-tumor responses to MART-1 specific effector cells. MART-1 peptide or negative control (HBc) was added to T2 cells. Malme3M is a melanoma line and Malme3 is a non-tumor cell line. Figure 4 shows tetramer staining of HER-2 specific CD8 effector cells from two different donors according to the invention. Figure 3 shows the anti-peptide response for HER-2 effector cells evaluated on peptide-added T2 cells according to the invention. The invention demonstrates an enhanced killing of the ovarian tumor cell line (HTB-77) when transfected with HLA-A2.1. Figure 3 shows enhanced killing of a breast cancer cell line (HTB-133) when transfected with HLA-A2.1 according to the invention. FIG. 4 shows that IFNγ pretreatment is required to demonstrate lysis of the tumor cell line HTB-77 / A2.1 according to the invention. The invention demonstrates that HLA-A2 and HER-2 surface expression is not affected by IFNγ induction in two cell lines (HTB-77 and HTB-77 / A2.1). Inventive, shows which protein mRNA levels are elevated in HTB-77 / A2.1 cells after induction with IFNγ. The outline | summary of the experiment of clinical trial CTL-03 concerning an invention is shown. Figure 2 shows in vitro growth curves of CTL cells isolated from a patient (15-RT) under clinical study CTL-02, according to the invention. Cells are: 3 treatment cycles starting on September 28, 1999 (15-RT-1), November 22, 1999 (15-RT-2) and February 15, 1999 (15-RT-3) Isolated from the patient on each day 0 and cultured in vitro. B) In vitro growth curve of CTL cells isolated from one patient (01-KN) under clinical trial CTL-03. Cells are: 3 treatment cycles starting on October 30, 2000 (01-KN-1), January 30, 2001 (01-KN-2) and April 9, 2001 (01-KN-3) Isolated from the patient on each day 0 and cultured in vitro. FIG. 3 shows that the number of melanoma antigen-specific T cells according to the invention increased in patients after one cycle of treatment with test CTL-03. Antigen-specific T in purified CD8 + preparations obtained from white blood cell component samples using HLA-A2.1 tetramer molecules prepared using either gp100 or MART-1 peptides The presence of cells was monitored. The percentage (%) of tetramer positive cells was recorded at the time of the first (I) and second (II) leukocyte fraction samples, which could be about 2 months apart. Figure 2 shows the correlation between three different in vitro assays used to measure CTL cytolytic activity, antigen specificity and cell proliferation according to the invention. Three different assays were performed on all of the CD8 + CTL preparations at the end of the ex vivo stimulation protocol (CTL-03). All three assays are generally consistent in determining the final bulk culture composition. In this particular preparation, the majority of T cells in the product were directed to the gp100 peptide (817). CTL activity defines cell lysis and effector function. Tetramer staining determines the frequency of antigen-specific cells, and intracellular IFN-γ staining reflects the ability of T cells to respond to specific peptides. The invention shows an increase in the amount of interferon γ at the same time point of the cycle with each repeated cycle of treatment in test CTL-03, suggesting the presence of memory T cells at the same time point of the cycle. Interferon γ was measured in the supernatant of ex vivo cultures 6 days after primary stimulation with Drosophila cells. The number of cycles and cell doses administered to each patient in the clinical trial described in Example 2 according to the invention will be described specifically. The efficacy of each dose was calculated by multiplying the number of CD8 + cells by the lysis unit recorded for the lysis of the peptide combination added on the target cells.

Claims (21)

a. Preparing a non-naturally occurring antigen-presenting cell line (nnAPC) (the nnAPC can simultaneously present up to about 15 different peptide molecules associated with viral infection, each of which is a length About 6 to 12 amino acids);
b. Collecting CD8 ⁺ cells from a subject or suitable donor;
c. Stimulating the CD8 ⁺ cells with the nnAPC cell line;
d. Adding the CD8 ⁺ cells to a conditioned growth medium (CGM) or a medium containing a cytokine selected from the group consisting of IL-2, IL-7 (the cytokines can be used individually or in combination);
e. About 1 to 50 μg / ml of one of the peptides that the nnAPC can simultaneously present unsuspended peripheral blood monocytes or CD-8 depleted peripheral blood monocytes collected from the subject or a suitable donor Mixing with;
f. Irradiating the peripheral blood monocyte suspension with a sufficient dose of gamma radiation necessary to nullify all components in the suspension except the desired peripheral blood monocytes;
g. Isolating adherent peripheral blood monocytes;
h. Adding about 1 μg / ml to 50 μg / ml of each of said peptides to said adherent peripheral blood monocytes;
i. It said CD8 ⁺ cells, at a ratio of about ten CD8 ⁺ cells to one peripheral blood monocytes that combined with said adherent peripheral blood monocytes; and j. A method of treating the viral infection in a subject comprising inoculating the subject with a CD8 ⁺ suspension.   The method of claim 1, wherein the nnAPC is capable of presenting up to about 10 peptide molecules.   2. The method of claim 1, wherein the peptide molecule is about 8 to 10 amino acids in length.   The method of claim 1, wherein the peptide molecule is in a concentration range of about 10 nM to 100 μM.   The method of claim 1, wherein the cytokine component is IL-2.   The method of claim 1, wherein the cytokine component is a combination of IL-2 and IL-7.   The method of claim 1, wherein the dose of gamma radiation is about 3,000 to 7,000 rads.   The method of claim 1, wherein the dose of gamma radiation is about 5,000 rads. a. Administering to a subject with melanoma an effective amount of interferon-α capable of enhancing the expression of tumor antigens on the surface of the tumor; and b. A method of treating a subject comprising the step of inoculating the subject with an effective amount of autologous cytotoxic T lymphocytes having specificity for a melanoma-associated target antigen.   Further comprising administering to said subject an effective amount of interleukin-2 capable of enhancing in vivo maintenance of autologous cytotoxic T lymphocytes with specificity for a melanoma-associated target antigen. 10. The method of claim 9.   11. The method of claim 10, wherein the interferon-α is selected from interferon-α-2a or interferon-α-2b. Before inoculating the subject with an effective amount of autologous cytotoxic T lymphocytes having an effective amount of interferon-α of about 10 MU / m ² / day and having specificity for a melanoma-associated target antigen. 11. The method of claim 10, wherein the subject is administered subcutaneously from day to day 1 continuously. 11. The method of claim 10, wherein an effective amount of autologous cytotoxic T lymphocytes with specificity for a melanoma-associated target antigen is about 1-10 × 10 ⁹ cells per injection. Autologous cytotoxic T lymphocytes with specificity for melanoma-associated target antigens:
a) preparing a non-naturally occurring antigen presenting cell line (nnAPC) (the nnAPC can simultaneously present up to about 15 different epitopes associated with the melanoma, and each epitope is of length A peptide of 8 to 10 amino acids);
b) adding up to about 15 different epitopes associated with said melanoma to nnAPC;
c) collecting CD8 ⁺ cells from said subject;
d) stimulating said CD8 ⁺ cells with an nnAPC cell line supplemented with epitopes to obtain CD8 ⁺ cells specific for melanoma;
e) growing CD8 ⁺ cells specific for melanoma in a medium containing IL-2 and IL-7;
f) mixing CD8-depleted peripheral blood monocytes collected from said subject with each epitope used for nnAPC addition;
g) irradiating said CD8-depleted peripheral blood monocytes with gamma radiation;
h) isolating adherent CD8-depleted peripheral blood monocytes;
i) adding each epitope used for nnAPC addition to the adherent peripheral blood monocytes;
j) restimulating said CD8 ⁺ cells specific for melanoma with epitope-attached adherent peripheral blood monocytes;
k) a melanoma-specific restimulated CD8 ⁺ cells, IL-2 and IL-7 that are grown in a medium containing; and l) CD8 ⁺ cells restimulated specific for melanoma The method according to claim 10, which is obtained by a method comprising the step of increasing by stimulation with OKT3 antibody.   15. The method of claim 14, wherein step (j) can be repeated at least once more.   15. The method of claim 14, wherein the non-naturally occurring antigen presenting cell line is added with an epitope that is a peptide derived from tyrosinase, gp100 and MART-1.   17. The method of claim 16, wherein said non-naturally occurring antigen presenting cell line is added with an epitope comprising the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.   From day 0 after inoculating the subject with an effective amount of autologous cytotoxic T lymphocytes with an effective amount of interleukin-2 of about 3 MIU / day and specificity for a melanoma-associated target antigen 15. The method of claim 14, wherein the method is administered subcutaneously to the subject continuously until day 28.   15. The method of claim 14, wherein the method is repeated at an interval of about 2 months.   20. The method of claim 19, wherein the method is repeated for a minimum of two cycles and further comprising the step of assessing the response in the subject after each cycle. a. Continuously from day 5 to day 1 before inoculating the subject with autologous cytotoxic T lymphocytes with specificity for a melanoma-associated target antigen, the subject received 10 MU / m ² / day of interferon − administering α-2b subcutaneously;
b. Introducing about 1-10 × 10 ⁹ cells per injection of autologous cytotoxic T lymphocytes with specificity for a melanoma-associated target antigen; and c. 15. The method of claim 14, comprising the step of administering about 3 MIU / day of interleukin-2 subcutaneously to the subject continuously from day 0 to day 28 after the introduction step.

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