Patent Description:
This patent application claims the benefit of <CIT>.

The primary cause of some cancer types such as, for example, uterine cervical cancer, is human papillomavirus (HPV) infection. A phase II study of lymphodepletion followed by autologous tumor-infiltrating lymphocytes (TILs) and high dose adesleukin for HPV-associated cancers has been described (ClinicalTrials. gov, NCT01585428, <NUM> April <NUM>). <NPL> describe systemic and local HPV <NUM>-specific T-cell immunity in patients with head and neck cancer. Isolation and characterization of tumor-infiltrating lymphocytes from cervical cancer has been described by <NPL>). <CIT> describes adoptive cell therapy with young T cells. Furthermore, Rosenberg et al. reported durable complete responses in heavily pretreated patients with metastatic melanoma using T cell transfer immunotherapy (<NPL>).

Despite advances in treatments such as chemotherapy, the prognosis for many cancers, including HPV-associated cancers, may be poor. Accordingly, there exists an unmet need for additional treatments for cancer, particularly HPV-associated cancers.

The invention provides a population of HPV-specific T-cells prepared according to a method comprising:.

for use in treating or preventing cancer in a mammal, wherein the population of HPV-specific T-cells is administered to the mammal in an amount effective to treat or prevent the cancer in the mammal.

In an embodiment, the cancer is head and neck squamous-cell carcinoma (HNSCC).

In an embodiment, the cancer is HPV-positive cancer, optionally HPV-positive oropharyngeal cancer.

In an embodiment, the cancer is selected from the group consisting of laryngeal cancer, hypopharyngeal cancer, nasal cavity cancer, nasopharyngeal cancer, cancer of the oral cavity, oropharyngeal cancer, and salivary gland cancer.

In an embodiment, a second culturing step is added after step (c) to further expand the number of selected T-cells.

In an embodiment, the expanded population of cells obtained in (d) secretes at least about <NUM> pg/mL of interferon-γ.

In an embodiment, the expanded population of cells obtained in (d) comprises multiple T-cells each having different HPV specificities.

In an embodiment, the population of cells obtained in step (d) is enriched for HPV-specific T-cells.

In an embodiment, the population of HPV-specific T-cells recognize HPV <NUM>-positive cancer cells.

In an embodiment, the population of HPV-specific T cells recognize an HPV antigen selected from the group consisting of HPV <NUM> E6 and HPV <NUM> E7.

In an embodiment, the population of HPV-specific cells recognize HPV <NUM>-positive cancer cells, optionally wherein the population of HPV-specific T cells recognize an HPV antigen selected from the group consisting of HPV <NUM> E6 and HPV <NUM> E7.

In an embodiment, the mammal is a female subject.

In an embodiment, the cancer is HPV+ cervical cancer.

In an embodiment, the method further comprises administering to the mammal nonmyleoablative lymphodepleting chemotherapy prior to the administration of HPV-specific T cells.

In an embodiment, the cancer is selected from the group consisting of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, cancer of the oropharynx, ovarian cancer, cancer of the penis, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, cancer of the uterus, ureter cancer, and urinary bladder cancer.

It has been discovered that populations of human papillomavirus (HPV)-specific T cells can be prepared for a variety of applications, for example, adoptive cell therapy. The non-claimed methods described herein may generate cells that are useful for treating a variety of conditions, e.g., cancer.

The non-claimed methods described herein provide numerous advantages. For example, the methods may, advantageously, generate T cells from HPV-positive cancers at a grade and scale suitable for clinical use. Additionally, the methods may, advantageously, generate T cells that recognize the HPV antigens E6 and E7, which are constitutively and specifically expressed by cancer cells and are not expressed by normal cells. Therefore, without being bound to a particular theory or mechanism, it is believed that T cells generated by the methods advantageously target the destruction of cancer cells while minimizing or eliminating the destruction of normal, non-cancerous cells, thereby reducing, for example, by minimizing or eliminating, toxicity. In addition, because an embodiment of the inventive population of HPV-specific T cells for use includes nonmyeloablative chemotherapy, the inventive population can advantageously be used to treat patients that would not be eligible for treatments that involve total body irradiation (TBI) such as, for example, patients that have already undergone myeloablative therapy, e.g., radiotherapy, prior to treatment; patients with comorbid conditions; and patients with less than <NUM> x <NUM><NUM> CD34+ cells/kg. Moreover, the inventive population of HPV-specific T cells for use in treating cancer may, advantageously, successfully treat or prevent HPV-positive cancers that do not respond to other types of treatment such as, for example, chemotherapy alone, surgery, or radiation.

A non-claimed embodiment comprises obtaining an HPV-positive tumor sample from a mammal. The tumor sample may be obtained from a mammal in any suitable manner such, for example, biopsy or surgical resection.

In an embodiment, the method by which the inventive population is prepared may comprise testing the tumor sample for HPV infection. The HPV may be any HPV subtype. Preferably, the HPV subtype is HPV <NUM> or HPV <NUM>. The testing may comprise testing for the expression of any protein (e.g., an antigen) specifically expressed by HPV-infected cells such as, for example, any one or more of HPV <NUM> E6, HPV <NUM> E7, HPV <NUM> E6, and HPV <NUM> E7, expression of any RNA encoding the HPV-specific protein, or a combination thereof. Testing for HPV infection may be carried out in any suitable manner known in the art. Exemplary HPV tests may include any one or more of reverse transcriptase (RT) polymerase chain reaction (PCR)-based genotyping and Western blots. The tumor sample may be positive for any subtype of HPV infection such as, for example, HPV <NUM> or HPV <NUM> infection.

The method by which the invention is prepared comprises dividing the HPV-positive tumor sample into multiple fragments. The tumor sample may be divided into any suitable number of fragments such as, for example, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or more fragments. Preferably, the tumor sample is divided into <NUM> fragments. The tumor sample may be divided in any suitable manner e.g., mechanically (disaggregating the tumor using, e.g., a gentleMACS™ Dissociator, Miltenyi Biotec, Auburn, CA) or enzymatically (e.g., collagenase or DNase).

An embodiment of the method by which the population of the invention is prepared comprises separately culturing the multiple fragments. In this regard, the fragments may be cultured in separate containers, e.g., separate plates or separate wells of a plate. The multiple fragments may be cultured in any suitable manner. For example, the fragments may be cultured in a gas permeable container as described in <CIT>. In an embodiment of the method by which the population of the invention is prepared, the tumor fragments are cultured in the presence of a combination of two or more cytokines. In a preferred embodiment, however, the method by which the population of the invention is prepared comprises culturing the tumor fragments in the presence of only one cytokine. The cytokine may be any suitable cytokine such as, for example, interleukin (IL)-<NUM>, IL-<NUM>, IL-<NUM>, or IL-<NUM>. Preferably, the cytokine is IL-<NUM>. The tumor fragments may be cultured in any suitable amount of cytokine (e.g., from about <NUM> IU/mL to about <NUM>,<NUM> IU/mL, preferably about <NUM>,<NUM> IU/mL). Preferably, the method comprises culturing tumor fragments in about <NUM>,<NUM> IU/mL IL-<NUM>.

The method by which the population is prepared comprises obtaining T cells from the cultured multiple fragments. The method by which the population is prepared may comprise culturing the T cells until confluence (e.g., about <NUM> x <NUM><NUM> lymphocytes per mL in a <NUM>-well plate), e.g., from about <NUM> to about <NUM> days.

The method by which the population is prepared may comprise testing the T cells for one or both of specific autologous HPV-positive tumor recognition and HPV antigen recognition. Specific autologous HPV-positive tumor recognition can be tested by any method known in the art, e.g., by measuring cytokine release (e.g., interferon (IFN)-γ) following co-culture with autologous HPV-positive tumor cells. T cells may be considered to recognize HPV-positive tumor if, for example, co-culture with autologous HPV-positive tumor cells results in IFN-γ release that is one or more of (i) twice the amount of IFN-γ that is measured when the T cells are cultured alone (background); (ii) at least about <NUM> pg/mL or more (e.g., <NUM> pg/mL or more, <NUM> pg/mL or more, <NUM> pg/mL or more, <NUM> pg/mL or more, <NUM> pg/mL or more, <NUM> pg/mL or more, <NUM> pg/mL or more, <NUM>,<NUM> pg/mL or more, <NUM>,<NUM> pg/mL or more, <NUM>,<NUM> pg/mL or more, or <NUM>,<NUM> pg/mL or more); and (iii) blocked by MHC Class I antibody by greater than about <NUM>%, greater than about <NUM>%, or greater than about <NUM>%.

Specific HPV antigen recognition can be tested by any method known in the art, e.g., by measuring cytokine release (e.g., IFN-γ) following co-culture with antigen-negative antigen presenting cells (e.g., dendritic cells) that have been pulsed with a peptide of an HPV antigen. T cells may be considered to recognize HPV antigen if, for example, IFN-γ release is one or both of (i) twice the amount of IFN-γ that is measured when the T cells are cultured with antigen presenting cells that are pulsed with a negative control peptide and (ii) at least about <NUM> pg/mL or more (e.g., <NUM> pg/mL or more, <NUM> pg/mL or more, <NUM> pg/mL or more, <NUM> pg/mL or more, <NUM> pg/mL or more, <NUM> pg/mL or more, <NUM> pg/mL or more, <NUM>,<NUM> pg/mL or more, <NUM>,<NUM> pg/mL or more, <NUM>,<NUM> pg/mL or more, or <NUM>,<NUM> pg/mL or more) of IFN-γ upon co-culture with antigen-negative antigen presenting cells pulsed with a low concentration of HPV <NUM> or HPV <NUM> peptide (e.g., about <NUM> ng/mL to about <NUM> ng/mL, <NUM> ng/mL, <NUM> ng/mL, <NUM> ng/mL, <NUM> ng/mL, or <NUM> ng/mL). The T cells may also secrete IFN-γ upon co-culture with antigen-negative antigen presenting cells pulsed with higher concentrations of HPV peptide.

The HPV antigen may be any HPV antigen. For example, the HPV antigen may be any one or more of HPV <NUM> E6, HPV <NUM> E7, HPV <NUM> E6, and HPV <NUM> E7. While in some embodiments, the population of T cells may specifically recognize only one HPV antigen, in some embodiments, the population of T cells may specifically recognize more than one HPV antigen. In this regard, the population of T cells may comprise multiple T cells each having different HPV specificities. For example, the population of T cells may include some T cells that specifically recognize HPV <NUM> E6 and other T cells that specifically recognize HPV <NUM> E7, or the population may include some T cells that specifically recognize HPV <NUM> E6 and other T cells that specifically recognize HPV <NUM> E7.

The method by which the population is prepared may comprise selecting the T cells that exhibit one or both of specific autologous HPV-positive tumor recognition and HPV antigen recognition. In an embodiment of the invention, while testing the T cells for one or both of specific autologous HPV-positive tumor recognition and HPV antigen recognition may identify those cultures that contain T cells that recognize HPV, those cultures that contain the HPV-reactive T cells may also contain additional T cells that are reactive against other, non-HPV tumor antigens. Accordingly, the selected population of T cells may include polyclonal T cells with multiple specificities. In another embodiment of the invention, the testing identifies cultures that only contain T cells that recognize HPV. In this regard, the selected population of T cells may include T cells with only HPV specificity.

The method by which the population is prepared further comprises expanding the number of selected T cells to produce a population of HPV-specific T cells. Rapid expansion provides an increase in the number of antigen-specific T-cells of at least about <NUM>-fold (or <NUM>-, <NUM>-, <NUM>-, <NUM>-, <NUM>-, <NUM>-, <NUM>-, <NUM>-fold, or greater) over a period of about <NUM> to about <NUM> days, preferably about <NUM> days. More preferably, rapid expansion provides an increase of at least about <NUM>-fold (or <NUM>-, <NUM>-, <NUM>-, <NUM>-fold, or greater) over a period of about <NUM> to about <NUM> days, preferably about <NUM> days. Most preferably, rapid expansion provides an increase of at least about <NUM>-fold to about <NUM>-fold over a period of about <NUM> to about <NUM> days, preferably about <NUM> days.

Expansion of the numbers of T cells can be accomplished by any of a number of methods as are known in the art as described in, for example, <CIT>; <CIT>; <CIT>; <NPL>); and <NPL>). For example, the numbers of T cells can be rapidly expanded using non-specific T-cell receptor stimulation in the presence of feeder lymphocytes and either interleukin-<NUM> (IL-<NUM>) or interleukin-<NUM> (IL-<NUM>), with IL-<NUM> being preferred. The non-specific T-cell receptor stimulus can include around <NUM> ng/mL of OKT3, a mouse monoclonal anti-CD3 antibody (available from Ortho-McNeil, Raritan, N. Alternatively, the number of T cells can be rapidly expanded by stimulation in vitro with an antigen (one or more, including antigenic portions thereof, such as epitope(s), or a cell) of the cancer, which can be optionally expressed from a vector, e.g., <NUM> MART-<NUM>:<NUM>-<NUM> (<NUM>) or gp100:<NUM>-<NUM> (<NUM>), in the presence of a T-cell growth factor, such as <NUM> IU/mL IL-<NUM> or IL-<NUM>, with IL-<NUM> being preferred. The numbers of in vitro-induced T-cells may be rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto antigen-presenting cells. In an embodiment, the numbers of T cells are expanded in a gas permeable container as described in <CIT>.

The method by which the population is prepared comprises expanding the number of T cells using one or both of (i) irradiated allogeneic feeder cells and (ii) irradiated autologous feeder cells and both of (iii) OKT3 antibody and (iv) interleukin (IL)-<NUM>. In an especially preferred embodiment by which the population is prepared, the method comprises expanding the number of T cells using (i) irradiated allogeneic feeder cells, (ii) OKT3 antibody and (iii) interleukin (IL)-<NUM>.

In a non-claimed embodiment, the method comprises expanding the number of selected T cells using one or both of (i) OKT3 antibody and (ii) interleukin (IL)-<NUM> to produce a population of HPV-specific T cells, optionally in combination with one or both of irradiated allogeneic feeder cells and irradiated autologous feeder cells. In this regard, a non-claimed embodiment provides a method of preparing a population of HPV-specific T cells, the method comprising: dividing an HPV-positive tumor sample into multiple fragments; separately culturing the multiple fragments; obtaining T cells from the cultured multiple fragments; testing the T cells for one or both of specific autologous HPV-positive tumor recognition and HPV antigen recognition; selecting the T cells that exhibit one or both of specific autologous HPV-positive tumor recognition and HPV antigen recognition; and expanding the number of selected T cells using one or both of (i) OKT3 antibody and (ii) interleukin (IL)-<NUM> to produce a population of HPV-specific T cells. Dividing the tumor sample, culturing the tumor fragments, obtaining T cells, testing the T cells, selecting the T cells, and expanding the numbers of selected T cells may be carried out as described herein with respect to other aspects of the invention.

The population of expanded numbers of T cells produced by the described methods specifically recognize HPV-positive cells, e.g., HPV-positive cancer cells. The cells that are recognized by the T cells may be positive for any subtype of HPV such as, for example, HPV <NUM> or HPV <NUM>. Alternatively or additionally, the population of T cells produced by the described methods may specifically recognize any HPV antigen such as, for example, any one or more of HPV <NUM> E6, HPV <NUM> E7, HPV <NUM> E6, and HPV <NUM> E7. While in some embodiments, the population of T cells may specifically recognize only one HPV antigen, in some embodiments, the population of T cells may specifically recognize more than one HPV antigen. In this regard, the population of expanded numbers of T cells may comprise multiple T cells each having different HPV specificities. For example, the population of expanded numbers of T cells may include some T cells that specifically recognize HPV <NUM> E6 and other T cells that specifically recognize HPV <NUM> E7, or the population may include some T cells that specifically recognize HPV <NUM> E6 and other T cells that specifically recognize HPV <NUM> E7. The ability of the population of expanded numbers of T cells produced by the described methods to specifically recognize HPV-positive cells and to specifically recognize a HPV antigen may be measured as described herein with respect to other aspects of the invention.

The population of T cells produced by the methods described herein may be useful for treating or preventing HPV-associated conditions, e.g., cancer. Accordingly, the invention provides a population of HPV-specific T cells for use in treating or preventing cancer in a mammal, the method comprising preparing a population of HPV-specific T cells according to any of the methods described herein and administering the population of T cells to the mammal in an amount effective to treat or prevent cancer in the mammal.

Another embodiment of the invention provides a population of HPV-specific T cells for use in treating or preventing cancer in a mammal, wherein the population of HPV-specific T cells is prepared according to a method comprising: dividing an HPV-positive tumor sample into multiple fragments; separately culturing the multiple fragments in the presence of at least one cytokine; obtaining T cells from the cultured multiple fragments; testing the T cells for one or both of specific autologous HPV-positive tumor recognition and HPV antigen recognition; selecting the T cells that exhibit one or both of specific autologous HPV-positive tumor recognition and HPV antigen recognition; expanding the number of selected T cells to produce a population of HPV-specific T cells using one or both of (i) irradiated allogenic feeder cells and (ii) irradiated autologous feeder cells; and both of (iii) OKT3 antibody and (iv) interleukin-<NUM> for adoptive cell therapy wherein the T-cells have not been depleted of CD4+ cells; and administering the expanded number of T cells to the mammal in an amount effective to treat or prevent cancer in the mammal. Dividing the tumor sample, culturing the tumor fragments, obtaining T cells, testing the T cells, selecting the T cells, and expanding the numbers of selected T cells may be carried out as described herein with respect to other aspects of the invention.

In an embodiment of the invention, the inventive population of HPV-specific T cells for use in treating or preventing cancer in a mammal further comprises administering to the mammal nonmyeloablative lymphodepleting chemotherapy. The nonmyeloablative lymphodepleting chemotherapy can be any suitable such therapy, which can be administered by any suitable route. The nonmyeloablative lymphodepleting chemotherapy can comprise the administration of cyclophosphamide and fludarabine, particularly if the cancer is an HPV-positive cancer, which can be metastatic. A preferred route of administering cyclophosphamide and fludarabine is intravenously. Likewise, any suitable dose of cyclophosphamide and fludarabine can be administered. Preferably, around <NUM>/kg of cyclophosphamide is administered for two days after which around <NUM>/m<NUM> fludarabine is administered for five days, particularly if the cancer is an HPV-positive cancer. In an embodiment of the invention, the nonmyeloablative lymphodepleting chemotherapy is administered prior to administering the T cells.

In an embodiment of the invention, the inventive population of HPV-specific T cells for use comprises, after administering the nonmyeloablative lymphodepleting chemotherapy, administering to the mammal the population of HPV-specific T cells prepared by any of the methods described herein.

The T-cells can be administered by any suitable route as known in the art. Preferably, the T-cells are administered as an intra-arterial or intravenous infusion, which preferably lasts about <NUM> to about <NUM> minutes. Other examples of routes of administration include intraperitoneal, intrathecal and intralymphatic.

Likewise, any suitable dose of T-cells can be administered. Preferably, from about <NUM> x <NUM><NUM> T-cells to about <NUM> x <NUM><NUM> T-cells are administered, with an average of around <NUM> x <NUM><NUM> T-cells, particularly if the cancer is an HPV-positive cancer. Alternatively, from about <NUM> x <NUM><NUM> to about <NUM> x <NUM><NUM> T-cells are administered.

In an embodiment of the invention, any of the populations of T cells for use described herein may further comprise combining the population of HPV-specific T cells with a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any pharmaceutically acceptable carrier that is suitable for adoptive cell therapy. For example, the pharmaceutically acceptable carrier may include any isotonic carrier such as, for example, normal saline (about <NUM>% w/v of NaCl in water, about <NUM> mOsm/L NaCl in water, or about <NUM> NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about <NUM>% dextrose in water, or Ringer's lactate. In an embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumen.

A non-claimed embodiment comprises enriching cultured T cells for CD8+ T cells prior to rapid expansion of the cells. Following culture of the T cells, the T cells are depleted of CD4+ cells and enriched for CD8+ cells using, for example, a CD8 microbead separation (e.g., using a CliniMACS plus CD8 microbead system (Miltenyi Biotec)). Without being bound to a particular theory, it is believed that CD8+ enrichment of some T cell cultures reveals in vitro tumor recognition that may not be evident in the bulk culture, and improved in vitro recognition of tumor in other cultures. Additionally, the enriched CD8+ T cells are believed to behave more reliably and predictably in clinical scale rapid expansions than the bulk T cells.

In an embodiment of the invention, the method by which the population of HPV-specific T cells for use is prepared comprises enriching cultured T cells for CD4+ T cells prior to rapid expansion of the cells. Following culture of the T cells, the T cells are depleted of CD8+ cells and enriched for CD4+ cells using, for example, a CD4 microbead separation (e.g., using a CliniMACS plus CD8 microbead system (Miltenyi Biotec)).

In an embodiment, the invention provides a population of HPV-specific T cells for use as described herein, wherein a T-cell growth factor that promotes the growth and activation of the autologous T cells is administered to the mammal either concomitantly with the autologous T cells or subsequently to the autologous T cells. The T-cell growth factor can be any suitable growth factor that promotes the growth and activation of the autologous T-cells. Examples of suitable T-cell growth factors include interleukin (IL)-<NUM>, IL-<NUM>, IL-<NUM>, and IL-<NUM>, which can be used alone or in various combinations, such as IL-<NUM> and IL-<NUM>, IL-<NUM> and IL-<NUM>, IL-<NUM> and IL-<NUM>, IL-<NUM>, IL-<NUM> and IL-<NUM>, IL-<NUM> and IL-<NUM>, IL-<NUM> and IL-<NUM>, or IL-<NUM> and IL2. IL-<NUM> is a preferred T-cell growth factor.

In an embodiment, the invention provides a population of HPV-specific T cells for use as described herein, wherein the autologous T-cells are modified to express a T-cell growth factor that promotes the growth and activation of the autologous T-cells. Suitable T-cell growth factors include, for example, any of those described above. Suitable methods of modification are known in the art. See, for instance, <NPL>; and <NPL>. Desirably, modified autologous T-cells express the T-cell growth factor at high levels. T-cell growth factor coding sequences, such as that of IL-<NUM>, are readily available in the art, as are promoters, the operable linkage of which to a T-cell growth factor coding sequence promote high-level expression.

The T-cell growth factor can be administered by any suitable route. If more than one T-cell growth factor is administered, they can be administered simultaneously or sequentially, in any order, and by the same route or different routes. Preferably, the T-cell growth factor, such as IL-<NUM>, is administered intravenously as a bolus injection. The dosage of the T-cell growth factor may be chosen based on patient tolerance. For example, the T-cell growth factor may be administered until one or more limiting adverse events occur. Desirably, the dosage of the T-cell growth factor, such as IL-<NUM>, is what is considered by those of ordinary skill in the art to be high. Preferably, a dose of about <NUM>,<NUM> IU/kg of IL-<NUM> is administered three times daily until tolerance, particularly when the cancer is an HPV-positive cancer. Preferably, about <NUM> to about <NUM> doses of IL-<NUM> are administered, with an average of around <NUM> doses.

In an embodiment, the autologous T-cells may be modified to express a T cell receptor (TCR) having antigenic specificity for an HPV antigen, e.g., any of the HPV antigens described herein. Suitable methods of modification are known in the art. See, for instance, Green and Sambrook and Ausubel, supra. For example, the T cells may be transduced to express a T cell receptor (TCR) having antigenic specificity for an HPV antigen using transduction techniques described in <NPL>) and <NPL>).

With respect to the inventive population of HPV-specific T cells, the cancer can be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, cancer of the oropharynx, ovarian cancer, cancer of the penis, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, cancer of the uterus, ureter cancer, and urinary bladder cancer. A preferred cancer is cancer is cancer of the uterine cervix, oropharynx, anus, anal canal, anorectum, vagina, vulva, or penis. A particularly preferred cancer is HPV-positive cancer. The HPV-positive cancer may be, for example, HPV <NUM>-positive or HPV <NUM>-positive cancer. While the cancers most commonly associated with HPV infection include cancer of the uterine cervix, oropharynx, anus, anal canal, anorectum, vagina, vulva, and penis, the inventive population of HPV-specific T cells may be used to treat any HPV-positive cancer, including those that occur at other anatomical areas.

As used herein, the term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.

The terms "treat," and "prevent" as well as words stemming therefrom, as used herein, do not necessarily imply <NUM>% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive populations of HPV-specific T cells can provide any amount of any level of treatment or prevention of cancer in a mammal. Furthermore, the treatment or prevention provided by the inventive population of HPV-specific T cells can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented. For example, the treatment or prevention provided by the inventive population of HPV-specific T cells can include promoting the regression of a tumor. Also, for purposes herein, "prevention" can encompass delaying the onset of the disease, or a symptom or condition thereof.

This example demonstrates a method of preparing HPV-positive tumor-infiltrating lymphocytes (TIL) for adoptive cell therapy.

Patients were entered into clinical protocols and signed informed consents that were approved by the Institutional Review Board of the National Cancer Institute prior to tumor resection. Tumors were excised from patients. Tumors were tested for HPV <NUM> E6, HPV <NUM> E7, HPV <NUM> E6, and HPV <NUM> E7 expression using reverse transcriptase (RT) polymerase chain reaction (PCR) genotyping.

Multiple (<NUM>) independent cultures of HPV <NUM> E6 positive, HPV E7 positive, HPV <NUM> E6 positive, and HPV E7 positive TIL were set up using enzymatic digests and tumor fragments (<NUM>-<NUM><NUM>) procured by sharp dissection. TIL from tumor digests were generated by culturing single-cell suspensions (<NUM> x <NUM><NUM>/mL) obtained by overnight enzymatic digestion of tumor fragments in media containing collagenase, hyaluronidase, and DNAse. Cultures of tumor fragments and digests were initiated in <NUM> wells of complete medium (CM) and IL-<NUM> (<NUM> IU/mL, Chiron Corp. , Emeryville, CA) in a humidified <NUM> incubator with <NUM>% CO<NUM>. CM included RPMI1640 with glutamine, plus <NUM>% human AB serum, <NUM> HEPES, <NUM>µg/mL gentamicin, and <NUM> x <NUM>-<NUM>M <NUM>-mercaptoethanol. Five days after initiation, one half of the media was aspirated from the wells and replaced with fresh CM and IL-<NUM>, and media was replaced every two to three days thereafter as needed. Under these conditions, lymphocytes will first lyse the adherent cells in the well, and then begin to multiply and grow.

TIL cultures achieved confluent growth of the original <NUM>-mL well and eliminated adherent tumor cells, typically about <NUM>-<NUM> days after initiation. In practice, this was about <NUM> x <NUM><NUM> lymphocytes from each original tumor fragment or digest well. By pooling all the wells in a single <NUM> well plate, approximately <NUM> x <NUM><NUM> TIL cells would be obtained.

When cultures designated for TIL generation expanded to confluence in <NUM>-mL wells, they were tested for HPV specific reactivity. Because the TIL were set up in large numbers (typically groups of <NUM> per tumor) it was not feasible to count each TIL culture individually. The TIL specificity assay measures activity per volume rather than activity per cell. Each well was mixed thoroughly, and one hundred microliters of lymphocytes (estimated 1x10<NUM> cells) were washed and cocultured overnight with autologous tumor digest or autologous monocyte-derived dendritic cells (DCs) pulsed with HPV <NUM> and HPV <NUM> E6 and E7 MACS PEPTIVATOR peptide pools. The peptide pools included <NUM>-mer peptides with <NUM>-amino-acid overlaps that covered the complete sequence of E6 or E7 (HPV <NUM> or HPV <NUM>). The peptide pools were over <NUM>% pure and low in endotoxin. IFN-γ release was then measured with enzyme-linked immunosorbent assay (ELISA). The results are shown in Table A.

Rapid expansion of the numbers of HPV reactive TIL were performed using the Rapid Expansion Protocol (REP) as previously described (<NPL>) and <NPL>)). Briefly, TIL cells were cultured in gas permeable, G-REX flasks with a <NUM> fold excess of irradiated (<NUM> Gy) allogeneic peripheral blood mononuclear "feeder" cells in complete medium (CM) with <NUM> ng/mL anti-CD3 antibody and <NUM> IU/mL IL-<NUM>. Half of the media was exchanged on day <NUM> using CM with <NUM> IU/mL IL-<NUM>, and cells were split as needed thereafter. TIL expanded an average of more than <NUM> fold.

This example demonstrates the reactivity of the TIL from Patient <NUM>.

TIL were generated as described in Example <NUM> from <NUM> different tumor fragments (F1-F22) from Patient <NUM>. The TIL from Patient <NUM> or melanoma TIL (control) were co-cultured with dendritic cells pulsed with the HPV <NUM> E7 peptide pool or a gp100 peptide pool (control) and IFN-γ was measured. The results are shown in <FIG>. As shown in <FIG>, the TIL from tumor fragment <NUM> of Patient <NUM> recognized an autologous tumor line but not HPV <NUM> E7 peptides.

The TIL from tumor fragments F16, F17, or F22 of Patient <NUM> or cells given to the patient for treatment ("infusion bag") were co-cultured with autologous tumor, peripheral blood mononuclear cells (PBMC) from autologous tissue, tumor cells matched at all class I loci, HeLa cells (HLA mismatched), or CaSki cells (HLA mismatched). IFN-γ was measured. The results are shown in <FIG>. As shown in <FIG>, TIL from tumor fragments F16 and F22 showed autologous tumor recognition.

Autologous tumor cells were transfected with silencing RNA against HLA-A, HLA-B, HLA-C, or irrelevant RNA (non-targeting) and were co-cultured with TIL from Patient <NUM>. IFN-γ was measured. The results are shown in <FIG>. As shown in <FIG>, recognition of TIL from Patient <NUM> was diminished by HLA-A silencing.

Effector/target cells (Patient <NUM> TIL/autologous tumor cells; DMF5/<NUM> cells; or Patient <NUM> (P12) F15/HPV18E6<NUM>-<NUM>) were cultured alone or co-cultured without antibody, with anti-HLA-A2 antibody, or anti-Class II antibody. DMF5 cells are T cells transduced to express a MHC class I-restricted TCR against MART-<NUM>. The results are shown in <FIG>. As shown in <FIG>, recognition of TIL from Patient <NUM> was not inhibited by HLA-A*<NUM> blocking, which suggested HLA-A*<NUM> restricted tumor recognition. Patient <NUM>'s haplotype was HLA-A*<NUM>, HLA-A*<NUM>.

TIL from tumor fragment F16 or F22 of Patient <NUM>; cells given to the patient for treatment ("infusion bag"); melanoma TIL <NUM>, <NUM>, or <NUM> (TIL cultured from melanoma tumors); mE7 TCR (T cells from PBMC that were transduced to express a TCR against HPV <NUM> E7<NUM>-<NUM>); or F15 TIL (TIL from another patient that were reactive against HPV <NUM> E6<NUM>-<NUM>, class II-restricted, and therefore blockable with HB145) were co-cultured with a gp100 peptide pool, OKT3 antibody, or DCs pulsed with an HPV <NUM> E7 peptide pool, HPV <NUM> E7 peptide pool and W6/<NUM>, HPV <NUM> E7<NUM>-<NUM>, HPV <NUM> E7<NUM>-<NUM> and W6/<NUM>, HPV <NUM> E7<NUM>-<NUM> and HB145, HPV <NUM> E6<NUM>-<NUM>, HPV <NUM> E6<NUM>-<NUM> and W6/<NUM>, or HPV <NUM> E6<NUM>-<NUM> and HB <NUM>. The results are shown in <FIG>. As shown in <FIG>, TIL from tumor fragment F16 of Patient <NUM> showed class I restricted recognition of HPV <NUM> E7 peptides.

This example demonstrates the cloning of TIL from tumor fragment <NUM> of Patient <NUM> to isolate HPV <NUM> E7 reactive CD8 positive T cells.

DCs were loaded with HPV <NUM> E7 and co-cultured with TIL from tumor fragment <NUM> (F16) of Patient <NUM>. The TIL were sorted for <NUM>-1BB positive cells using fluorescence activated cell sorting (FACS). The sorted cells were cultured in <NUM>-well plates with two cells per well. The clones were screened for tumor reactivity against a gp100 peptide pool or a HPV <NUM> E7 peptide pool. The results are shown in <FIG>. As shown in <FIG>, CD8 positive T cell cloning from tumor fragment F16 using <NUM>-1BB-based FACS sorting resulted in the isolation of two clones (<NUM> and <NUM>) with E7 peptide pool reactivity.

This example demonstrates the reactivity of the TIL generated in Example <NUM> from Patient <NUM>.

TIL were generated as described in Example <NUM> from <NUM> different tumor fragments (F1-F36) from Patient <NUM>. The TIL from Patient <NUM> or melanoma TIL (control) were co-cultured with dendritic cells pulsed with the HPV <NUM> E6 peptide pool, the HPV <NUM> E7 peptide pool or a gp100 peptide pool (control) and IFN-γ was measured. The results are shown in <FIG>. As shown in <FIG>, the TIL from the F1 and F15 tumor fragments from Patient <NUM> showed the highest levels of IFN-γ production.

Autologous DCs were transduced with an HPV <NUM> E6 lentiviral vector or a green fluorescent protein (GFP) lentiviral vector. Other autologous cells were pulsed with a gp100 peptide pool or a HPV <NUM> E6 peptide pool. Transduced cells were co-cultured with TIL from tumor fragment F1 of Patient <NUM>, or melanoma TIL <NUM>, <NUM>, or <NUM>. The results are shown in <FIG>. As shown in <FIG>, TIL generated from tumor fragment F1 of Patient <NUM> recognized DCs transduced with HPV <NUM> E6, suggesting that the TIL target a naturally processed and presented antigen.

This example demonstrates the reactivity of TIL clones from tumor fragments <NUM> and <NUM> of Patient <NUM> to isolate HPV <NUM> E6 reactive CD8 positive T cells.

DCs were loaded with HPV <NUM> E6 and co-cultured with TIL from tumor fragments <NUM> and <NUM> of Patient <NUM>. The TIL were sorted for <NUM>-1BB positive cells using FACS. Cells were further sorted into CD4 positive and CD <NUM> positive populations. The sorted cells were cultured in <NUM>-well plates with two cells per well. The clones were screened for tumor reactivity against a HPV <NUM> E6 peptide pool. Out of <NUM> wells of CD4 positive cells from F1, <NUM> grew and <NUM> were reactive. Out of <NUM> wells of CD8 positive cells from F1, <NUM> grew and none were reactive. Out of <NUM> wells of CD4 positive cells from F15, <NUM> grew and <NUM> were reactive. Out of <NUM> wells of CD8 positive cells from F15, <NUM> grew and none were reactive.

The CD4 sorted cells were also tested for reactivity as measured by tumor necrosis factor (TNF) α secretion upon co-culture with a HPV <NUM> E6 peptide pool (a pool spanning the entire E6 protein), no peptide, subpools of the HPV <NUM> E6 protein, or peptides <NUM>-<NUM> of the HPV <NUM> E6 peptide pool. Each subpool contained a portion of the initial peptide pool. The results are shown in <FIG>. As shown in <FIG>, clones <NUM>, <NUM>, and <NUM> of tumor fragment F1 from Patient <NUM> were reactive against HPV <NUM> E6. The CD4 positive T cell clones that were generated recognized two sequential <NUM>-mers with an <NUM> amino acid overlap. The peptides shared the epitope HPV <NUM> E6<NUM>-<NUM>.

This example demonstrates that the clones generated from the F15 tumor fragment of Patient <NUM> recognize HPV <NUM> E6<NUM>-<NUM> in an HLA-DRB1*<NUM> restricted manner.

Clones <NUM>, <NUM>, and <NUM> were co-cultured with donor PBMC with the haplotypes set forth in Table B. The donor PBMC were pulsed with HPV <NUM> E6<NUM>-<NUM>.

TNFα secretion was measured. The results are shown in <FIG>. As shown in <FIG>, the clones generated from the F15 fragment of Patient <NUM> recognized HPV <NUM> E6<NUM>-<NUM> pulsed onto PBMC that were matched at both HLA-DRB1*<NUM> and HLA-DQB1*<NUM>, but not PBMC that were matched at only HLA-DQB1*<NUM>, suggesting HLA-DRB1*<NUM> restriction. The phenotypic allele frequency of HLA-DRB1*<NUM> is <NUM> percent.

The TIL from clone <NUM> of tumor fragment F15 of Patient <NUM> were co-cultured with autologous PBMC or donor PBMC pulsed with HPV <NUM> E6<NUM>-<NUM> or HPV <NUM> E6<NUM>-<NUM>. The results are shown in <FIG>. As shown in <FIG>, the TIL from clone <NUM> of tumor fragment F15 of Patient <NUM> recognized PBMC matched only at DRB1*<NUM> but not at only DRB1*<NUM>.

The TIL from clone <NUM> of tumor fragment F15 of Patient <NUM> were co-cultured with HPV <NUM> E6<NUM>-<NUM> in the presence of antibodies against HLA-DR, HLA-DQ, HLA-DP, pan-class I antibodies, or pan-class II antibodies. Pan-class I and II antibodies block T cell binding to MHC Class I or Class II molecules, respectively. The results are shown in <FIG>. As shown in <FIG>, the recognition of cognate peptide by TIL from clone <NUM> of tumor fragment F15 of Patient <NUM> was inhibited by blocking antibodies against HLA-DR. As shown in <FIG>, the TIL from tumor fragment F15 of Patient <NUM> recognize HPV <NUM> E6<NUM>-<NUM> in a DRB1*<NUM> restricted manner.

This example demonstrates the reactivity of TIL from Patients <NUM> and <NUM>.

TIL were generated as described in Example <NUM> from <NUM> different tumor fragments (F1-F24) from Patient <NUM> or Patient <NUM>. The TIL from Patient <NUM> or melanoma TIL (control) were co-cultured with autologous DCs pulsed with the HPV <NUM> E6 peptide pool, the HPV <NUM> E7 peptide pool or a gp100 peptide pool (control) and IFN-γ was measured. The results are shown in <FIG>. As shown in <FIG>, the TIL from F4, F5, F14, F19, and F22 tumor fragments were among those tumor fragments that showed reactivity against autologous DCs pulsed with HPV <NUM> E6 and E7 peptide pools.

The TIL from Patient <NUM> or melanoma TIL (control) were co-cultured with autologous DCs pulsed with the HPV <NUM> E6 peptide pool, the HPV <NUM> E7 peptide pool or a gp100 peptide pool (control) and IFN-γ was measured. The results are shown in <FIG>. As shown in <FIG>, TIL were generated that showed reactivity against autologous DCs pulsed with the HPV <NUM> E7 peptide pool.

This example demonstrates the cloning of TIL from tumor fragments of Patient <NUM> to isolate HPV reactive CD4 and CD8 positive T cells.

DCs were loaded with HPV <NUM> E6 or HPV <NUM> E7 and co-cultured with TIL from tumor fragments of Patient <NUM>. The HPV <NUM> E6 and HPV <NUM> E7 reactive TIL were separately sorted for <NUM>-1BB positive cells using FACS. The numbers of cells were expanded as described in Example <NUM>. Cells were further sorted into <NUM>-1BB positive cells by FACS. The sorted cells were cultured in <NUM>-well plates with two cells per well. Cells were further sorted into CD4 positive and CD8 positive populations. The clones were screened for tumor reactivity against a HPV <NUM> E6 or E7 peptide pool.

The results are shown in <FIG>. As shown in <FIG>, CD8 positive and CD4 positive T cell clones with reactivity against HPV <NUM> E6 and E7 were generated.

This example demonstrates that adoptive cell therapy using anti-HPV T cells treats cancer.

Inclusion Criteria for the study included (<NUM>) recurrent/refractory or metastatic cervical cancer or high-risk HPV-positive cancer from any site and (<NUM>) prior chemotherapy with platinum, including chemoradiation.

Tumors were resected from patients. TIL were obtained from the tumor, grown, the numbers of TIL were expanded, and the expanded numbers of TIL were screened for HPV reactivity as described in Example <NUM>.

Patients received a non-myeloablative, lymphodepleting preparative regimen of cyclophosphamide (<NUM>/kg/day) intravenously (IV) on days -<NUM> and -<NUM> and fludarabine (<NUM>/m2/day) IV on days -<NUM> through -<NUM>.

TIL were intravenously administered to the patients on Day <NUM>. A high dose of aldeskeukin (interleukin (IL)-<NUM>) (<NUM>,<NUM> IU/kg) was intravenously administered to the patients on Days <NUM> through <NUM>.

Patients underwent complete evaluation of tumor <NUM> to <NUM> weeks after the completion of the initial treatment regimen (defined as the last day of aldesleukin administration). If the patient had stable disease or tumor shrinkage, repeat complete evaluations were performed monthly for approximately <NUM>-<NUM> months, and then every <NUM>-<NUM> months until off study criteria are met. All measurable lesions up to a maximum of <NUM> lesions representative of all involved organs were identified as target lesions and recorded and measured at baseline. All other lesions (or sites of disease) were identified as non-target lesions and were also recorded at baseline. Lesions were evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST) guideline (version <NUM>) as set forth in Table C (target lesions) and Table D (non-target lesions).

Eleven patients were treated. The results are summarized in Table E.

As shown in Table E, out of the eight patients for which results were available, adoptive cell therapy with HPV reactive TIL resulted in three objective responders (OR), all of which were partial responders (PR). Two partial responses are ongoing at two months (Patients <NUM> and <NUM>) following treatment and one partial response (Patient <NUM>) is ongoing at nine months following treatment.

Computed tomography (CT) scans of the chest and pelvis of Patient <NUM> were carried out before treatment and nine months after treatment. The results are shown in <FIG>. As shown in <FIG>, the cancerous lesion in the paraaortic lymph node had shrunk by <NUM>% nine months after treatment. As shown in <FIG>, the cancerous lesion in the left lung hilar lymph node had also shrunk by <NUM>% nine months after treatment. As shown in <FIG>, the cancerous lesion in the common iliac lymph node had also shrunk by <NUM>% nine months after treatment.

Magnetic resonance imaging (MRI) scans of the liver of Patient <NUM> were carried out before treatment and two months after treatment. The results are shown in <FIG>. As shown in <FIG>, the cancerous mass on the liver shrunk by <NUM>% two months after treatment. CT scans of the abdomen and pelvis of Patient <NUM> were also carried out before treatment and two months after treatment. The results are shown in <FIG>. As shown in <FIG>, the cancerous lesion in the retroperitoneal lymph node had also shrunk by <NUM>%. As shown in <FIG>, the cancerous mass in the abdominal wall had also shrunk by <NUM>%. In addition, as shown in <FIG>, the cancerous left pericolic mass shrunk dramatically.

This example provides updated results of the clinical study described in Example <NUM> that were obtained nine months after the results described in Example <NUM> were obtained. This example demonstrates that adoptive cell therapy using anti-HPV T cells treats cancer.

Methods: A clinical trial to treat metastatic HPV+ cancers with tumor-infiltrating lymphocytes (TIL) selected for HPV E6- and E7-reactivity (HPV-TIL) was carried out as described in Example <NUM>. HPV-TIL infusion was preceded by non-myeloablative conditioning and followed by high-dose bolus aldesleukin as described in Example <NUM>. HPV-reactivity was assessed by ELISPOT, IFN-gamma production, and CD137 expression assays.

Results: Nine cervical cancer patients were treated on the study. They received a median of <NUM> x <NUM><NUM> T cells (range <NUM> to <NUM> x <NUM><NUM>) as a single infusion. The infused cells possessed reactivity against high-risk HPV E6 and/or E7 in <NUM>/<NUM> patients. The two patients with no HPV reactivity did not respond to treatment. Three out of six patients with HPV reactivity demonstrated objective tumor responses by RECIST (<NUM> PR and <NUM> CR). One patient had a <NUM>% best response. Two patients with widespread metastases had complete tumor responses that were ongoing <NUM> and <NUM> months after treatment. One patient with a complete response had a chemotherapy-refractory HPV-<NUM>+ squamous cell carcinoma (Patient <NUM> of Example <NUM>) and the other a chemoradiation-refractory HPV-<NUM>+ adenocarcinoma (Patient <NUM> of Example <NUM>). Both patients demonstrated prolonged repopulation with HPV-reactive T cells following treatment. Increased frequencies of HPV-specific T cells were detectable after <NUM> months in one patient and <NUM> months in the other. Two patients with HPV-reactive TIL that did not respond to treatment did not display repopulation with HPV-reactive T cells.

Six non-cervical cancer patients were also treated on the study. One patient experienced an objective clinical response, that is, a partial response of a metastatic tonsil cancer that was ongoing four months after treatment (<FIG>).

These data show that HPV-TIL can mediate durable, complete regression of metastatic cervical cancer and that cellular therapy can mediate complete regression of an epithelial malignancy. These data also show that HPV-TIL can mediate regression of a metastatic tonsil cancer.

This example further describes the complete tumor responses obtained in Example <NUM> with adoptive cell therapy using anti-HPV T cells.

HPV-TIL generation: Tumor-infiltrating lymphocytes (TIL) were grown from <NUM> fragments of excised tumors as described previously (<NPL>)). After two to three weeks of lymphocyte outgrowth, the cultures were assessed for cellular composition by flow cytometry and for reactivity against HPV type-specific E6 and E7 by interferon (IFN)-gamma production assay as described in the Assessment of HPV oncoprotein reactivity section below. Flow cytometric analysis was performed with fluorescent antibodies specific for CD3, CD4, CD8, and CD56 (BD Biosciences). Cultures were selected for additional expansion based on reactivity against the HPV oncoproteins, rapid growth, high T cell purity, and high frequency of CD8+ T cells. Expansion to the cell numbers used for treatment was accomplished with a rapid expansion protocol with G-REX gas permeable flasks (<NPL>); <NPL>)). Infusion products were certified for viable cell numbers, T cell purity (flow cytometry), potency (IFN-γ production), sterility (microbiological studies), and absence of tumor cells (cytopathology).

Patient treatments: Patients had metastatic cervical cancer and measurable disease. Prior treatment with a platinum agent in either the primary chemoradiation or metastatic setting was required. The conditioning regimen consisted of cyclophosphamide <NUM>/kg IV daily for two days followed by fludarabine <NUM>/m<NUM> IV daily for five days. Cells were administered IV over <NUM> to <NUM> minutes. Aldesleukin <NUM>,<NUM> IU/kg/dose IV was initiated within <NUM> hours of cell infusion and continued every eight hours until stopped for toxicity or for a maximum of <NUM> doses. Filgrastim was initiated the day after cell infusion and continued until neutrophil counts recovered.

Tumor responses: Baseline imaging studies were obtained within four weeks before initiating the conditioning regimen. Follow-up imaging was obtained six weeks after treatment, monthly for three assessments, every three months for three assessments, and then every <NUM> months for two assessments.

Assessment of HPV oncoprotein reactivity: HPV reactivity was determined by coculture of T cells (<NUM>,<NUM> to <NUM>,<NUM> cells) with autologous immature dendritic cells (<NUM>,<NUM> cells) loaded with <NUM> of peptide pools spanning E6, E7, gp100, or EBNA1 and BZLF1 (Miltenyi Biotec, Bergisch Gladbach, Germany). Peptide pools included <NUM>-mer peptides overlapping by <NUM> amino acids. Dendritic cells were generated from the adherent fraction of peripheral blood mononuclear cells (PBMC) or from CD14+ cells isolated from PBMC using magnetic bead isolation (Miltenyi Biotec) by culturing in DMEM supplemented with <NUM>% human serum and <NUM> IU/ml GM-CSF and <NUM> IU/ml IL-<NUM> for five to six days. Anti-EBV control T cells were generated before treatment by culturing PBMC with EBNA1 and BZLF1 peptide pools (<NUM>µg/mL) in AIM-V/RPMI media supplemented with <NUM>% human serum and <NUM> IU/ml IL-<NUM>. For IFN-γ production assays, the concentration of IFN-γ in the supernatants was determined after overnight coculture (R&D Systems (Minneapolis, MN) or Thermo Fisher Scientific (Waltham, MA)).

ELISPOT (Mabtech (Cincinnati, OH)) analysis was performed according to the manufacturer's instructions. Briefly, ELIIP plates (WAIPSWU from Millipore (Billerica, MA)) precoated with capture antibody (clone <NUM>-D1K, Mabtech) were seeded with <NUM>,<NUM> effector cells and <NUM>,<NUM> target cells. After <NUM> to <NUM> hours of incubation, IFN-γ secretion was detected by addition of a biotinylated anti-IFN-γ antibody (<NUM>-B6-<NUM> biotin, Mabtech) for two hours at room temperature. Following incubation with streptavidin-alkaline phosphatase (Mabtech) for one hour, substrate reagent (<NUM>-bromo-<NUM>-chloro-<NUM>'-indolyphosphate p-toluidine/ nitro-blue tetrazolium chloride, Kirkegaard & Perry Laboratories, Inc. (Gaithersburg, MD)) was added to allow spot formation. Spot formation was stopped by rinsing with tap water. Spots were counted using an IMMUNOSPOT automated reader (Cellular Technology, Ltd. (Shaker Heights, OH)). ELIPSOT responses against E6 or E7 were defined as positive if more than two times the negative control and greater than <NUM> spots/well.

CD137 upregulation assays were performed by flow cytometric analysis after <NUM> to <NUM> hour coculture (<NPL>)). Cells were labeled with fluorescent antibodies against CD137, CD4, CD8, and CD3 (BD Biosciences, San Jose, CA). They were counterstained with propidium iodide (BD Pharmingen, Franklin Lakes, New Jersey) prior to data acquisition with a BD FACSCANTO II cell analyzer (BD Biosciences). Data was analyzed with FLOWJO software, Mac version <NUM> (TreeStar, Ashland, OR).

Immunohistochemistry: Immunohistochemical stainings were performed in the Laboratory of Pathology, NCI, on <NUM> sections from formalin-fixed, paraffin-embedded metastatic tumors according to standard procedures. After deparaffinization, rehydration, and antigen retrieval, tumor sections were incubated with anti-human CD4 clone 1F6 (Novocastra, Wetzlar, Germany) at a <NUM>:<NUM> dilution for <NUM> hours, anti-human CD8 clone CD8/144B (Dako Corp. , Glostrup, Denmark) at a <NUM>:<NUM> dilution for <NUM> hours, or anti-human p16 clone JC8 (Santa Cruz, Dallas, Texas) at a <NUM>:<NUM> dilution for <NUM> minutes. The CD4 stained slides were stained on an AUTOSTAINER Link <NUM> (Dako Corp. ) and visualized with the ENVISION FLEX+ detection system (Dako Corp. The CD8 and p16 stained slides were stained on a VENTANA Benchmark XT (Ventana Medical Systems, Tucson, AZ) and visualized with the ULTRAVIEW detection system (Ventana Medical Systems). Images were captured with 10x microscopy.

Determination of lymphocyte subsets from peripheral blood: Complete blood counts with manual differential determination were performed by the Clinical Center Hematology Laboratory. Lymphocyte phenotyping for T, B, and NK cells was performed by the NIH Immunology Flow Cytometry Laboratory using standardized criteria.

Real-time reverse transcription polymerase chain reaction (RT-PCR): RNA was isolated from a <NUM> fragment of fresh tumor tissue using an RNEASY kit (Qiagen, Valencia, CA). Reverse transcription first-strand DNA synthesis was performed using QSCRIPT cDNA supermix (Quanta BioSciences, Gaithersburg, MD). Custom made TAQMAN primer and probe sequences (Applied Biosciences, Foster City, CA) were used for HPV16-E6, HPV16-E7, HPV18-E6, and HPV18-E7. Readily available glyceraldehyde-<NUM>-phosphate dehydrogenase (GAPDH) primer probe set was used to standardize oncoprotein expression levels (Hs02758991_g1, Applied Biosciences, Foster City, CA). RT-PCR was performed on a <NUM> FAST REAL-TIME PCR System (Applied Biosciences).

Analysis of serum cytokine levels: Levels of <NUM> cytokines (IL-1β IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-12p70, IL-<NUM>, IL-<NUM>, granulocyte-colony stimulating factor (G-CSF), granulocyte monocyte-colony stimulating factor (GM-CSF), IFN-γ, monocyte chemotactic protein (MCP)-<NUM>, macrophage inflammatory protein (MIP)-1β, and tumor necrosis factor (TNF)-α were measured in sera from patients collected before and after treatment with HPV-TIL using BIO-PLEX Pro Human Cytokine <NUM>-plex Assay (Bio-Rad Laboratories) according to the manufacturer's instructions. The cytokine levels were acquired by the BIO-PLEX <NUM> system (Bio-Rad).

Patient <NUM> was diagnosed with stage 3B poorly-differentiated, squamous cell cervical cancer fourteen months before treatment with HPV-TIL. The patient was initially treated with cisplatin, vincristine, and bleomycin followed by chemoradiotherapy with gemcitabine plus cisplatin, and brachytherapy. Two months later, metastatic cancer was detected in paratracheal (biopsy-confirmed), subcarinal, and bilateral hilar lymph nodes. She received four cycles of topotecan and paclitaxel before disease progression, and then was referred for the clinical trial described in Examples <NUM> and <NUM>. HPV-TIL was prepared from a resected paratracheal lymph node. The patient received lymphocyte-depleting chemotherapy followed by a single intravenous infusion of <NUM> x <NUM><NUM> HPV-TIL and two doses of aldesleukin. Aldesleukin dosing was stopped for patient fatigue. She was discharged from the hospital after hematological recovery, <NUM> days after cell infusion.

Patient <NUM> was diagnosed with stage IB2 adenocarcinoma of the uterine cervix <NUM> months before treatment with HPV-TIL. Her primary tumor was treated with chemoradiation with cisplatin followed by brachytherapy. Five months later, she was noted to have a chemoradiation-refractory primary tumor (biopsy-confirmed). Salvage surgery identified paraaortic and iliac lymph node involvement and residual pelvic disease. Her cancer progressed to involve additional retroperitoneal lymph nodes and the liver surface, and she developed right hydroureteronephrosis and bilateral pulmonary emboli, which required a ureteral stent and anticoagulation therapy. The patient was then treated according to the protocol described in this Example using HPV-TIL generated from two peritoneal nodules. She received lymphocyte-depleting chemotherapy followed by <NUM> x <NUM><NUM> HPV-TIL cells and eight doses of aldesleukin. Aldesleukin dosing was stopped for hypoxia secondary to pulmonary edema, which required supplemental oxygen and resolved with diuresis. Discharge from the hospital was <NUM> days after cell infusion.

Both patients had disseminated progressive disease before treatment (<FIG>; <FIG>; <FIG>; <FIG>; and <FIG>). Patient <NUM> had metastatic tumors involving a paraaortic mediastinal lymph node, bilateral lung hila, subcarinal lymph nodes, and iliac lymph nodes (<FIG> A; <FIG>; and <FIG>). Patient <NUM> had metastatic cancer involving at least seven sites: two tumors on the liver surface, paraaortic and aortocaval lymph nodes, the abdominal wall, a pericolic mass in the left pelvis, and a nodule obstructing the right ureter (<FIG> B; <FIG>; <FIG>; and <FIG>). Each patient was treated with a single infusion of T cells, which resulted in tumor regression that occurred over months (<FIG>). Both patients experienced objective complete tumor responses, which were ongoing <NUM> and <NUM> months after treatment (<FIG> (Patient <NUM>) and <FIG> (Patient <NUM>). A previously placed ureteral stent was removed from Patient <NUM> following regression of the tumor obstructing her right ureter (<FIG>). Neither patient received additional therapy. Both patients have returned to full-time employment.

There were no acute toxicities related to cell infusion. No autoimmune adverse events occurred. Both patients displayed transient serum cytokine elevations (<FIG>) that were associated with fevers, but neither patient developed severe cytokine release syndrome. The levels of cytokines in cryopreserved serum were determined. Testing was for the following cytokines: IL-1β, IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-<NUM>(p70), IL-<NUM>, IL-<NUM>, G-CSF, Granulocyte macrophage colony-stimulating factor (GM-CSF), IFN-γ, MCP-<NUM>, Macrophage inflammatory protein <NUM> beta (MIP-1β), and TNF-α. Cytokines with levels greater than twofold baseline on two consecutive measurements are displayed. Aldesleukin was dosed every eight hours after cell infusion (Patient <NUM> received two doses and Patient <NUM> received eight doses). GCSF was administered daily beginning the day after cell infusion and continued until neutrophil counts recovered (Patient <NUM> received <NUM> doses and Patient <NUM> received nine doses).

Aldesleukin was dosed to tolerance by protocol design, stopping for fatigue in Patient <NUM> and dyspnea in Patient <NUM>. Grade <NUM> and grade <NUM> adverse events are listed in Table H. The most common toxicities were hematological and the expected effects of the lymphocyte-depleting conditioning regimen (cyclophosphamide and fludarabine).

Metastatic tumors excised for the generation of HPV-TIL were a squamous cell carcinoma from Patient <NUM> and an adenocarcinoma from Patient <NUM>. The malignant cells expressed p16INK4A, a sensitive indicator of high-risk HPV-infection. The HPV type and the expression levels of E6 and E7, the target antigens of HPV-TIL, were determined for each patient's tumor by real-time reverse transcription polymerase chain reaction (RT-PCR). Patient <NUM> had a HPV-<NUM>+ cancer and Patient <NUM> had a HPV-<NUM>+ cancer. The T cell infiltrate in tumors from both patients showed a mixed composition with predominantly CD8+ cells in Patient <NUM> and CD4+ cells in Patient <NUM>. Both CD4+ and CD8+ T cells grew from the excised tumors. The infused HPV-TIL were composed of <NUM>% CD4+ and <NUM>% CD8+ T cells for Patient <NUM>, and <NUM>% CD4+ and <NUM>% CD8+ T cells for Patient <NUM>.

The HPV-TIL administered to Patient <NUM> were reactive against both the E6 and E7 oncoproteins as demonstrated by interferon (IFN)-γ production and ELISPOT assays (<FIG>). Five percent and greater than seven percent of the infused cells showed responses to E6 or E7, respectively by ELISPOT assay (<FIG>). E6 responses were CD8+ T cell-mediated, and E7 responses were CD4+ and CD8+ T cell-mediated. In total, <NUM> percent of the infused cells displayed HPV reactivity as measured by CD137 upregulation assay. For Patient <NUM>, HPV-TIL were reactive against E7 (<FIG>), with four percent of T cells responding to the antigen by ELISPOT assay (<FIG>). This response was primarily mediated by CD4+ T cells.

HPV-TIL infusion was followed by rapid increases in peripheral blood CD4+ and CD8+ T cells but not NK and B cells (<FIG>). Expansion of the numbers of infused T cells was associated with establishment and persistence of peripheral blood T cell reactivity against the HPV oncoproteins as measured by IFN-γ production, ELISPOT, and CD137 upregulation assays (<FIG>). Both patients had little, if any, reactivity against E6 or E7 prior to treatment. Following treatment, Patient <NUM> acquired robust T cell recognition of E6 and E7. For Patient <NUM>, this recognition was weaker but nonetheless detectable and, consistent with the infused T cells, directed against only E7. One-month after treatment, <NUM> percent of Patient <NUM>'s peripheral blood T cells were oncoprotein reactive (seven percent against E6 and five percent against E7) (<FIG>). Reactivity against these antigens was sustained with one percent of peripheral blood T cells showing oncoprotein recognition four and <NUM> months after cell infusion (<FIG>). Patient <NUM> showed <NUM> percent HPV reactive T cells one-month after treatment (<FIG>). This reactivity was sustained, albeit at lower levels, three and six months after treatment (<FIG> D and F). Consistent with the reactivity of the T cell subsets in the infused HPV-TIL, the HPV specific T cells that repopulated the patients were primarily E6 and E7 reactive CD8+ T cells for Patient <NUM>, and E7 reactive CD4+ T cells for Patient <NUM>.

This example provides updated results of Patients <NUM> and <NUM> from the clinical study described in Examples <NUM> and <NUM> that were obtained four months after the results described in Examples <NUM> and <NUM> were obtained. This example demonstrates that adoptive cell therapy using anti-HPV T cells treats cancer.

The objective complete tumor responses of Patients <NUM> and <NUM>, who were treated as described in Examples <NUM> and <NUM>, were ongoing <NUM> and <NUM> months after treatment, respectively.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

Claim 1:
A population of HPV-specific T-cells prepared according to a method comprising:
(a) dividing an HPV-positive tumor sample into multiple fragments;
(b) culturing the multiple fragments in the presence of at least one cytokine;
(c) obtaining T-cells from the cultured fragments;
(d) expanding the number of T-cells to produce an expanded population of HPV-specific T-cells using
one or both of (i) irradiated allogenic feeder cells and (ii) irradiated autologous feeder cells; and
one or both of (iii) OKT3 antibody and (iv) interleukin-<NUM>,
wherein the T-cells have not been depleted of CD4+ cells;
(e) optionally, adding a second culturing step following (c);
for use in treating or preventing cancer in a mammal, wherein the population of HPV-specific T-cells is administered to the mammal in an amount effective to treat or prevent the cancer in the mammal.