EP3273975A1 - In vitro artificial lymph node for sensitization and expansion of t cells for therapy and epitope mapping - Google Patents
In vitro artificial lymph node for sensitization and expansion of t cells for therapy and epitope mappingInfo
- Publication number
- EP3273975A1 EP3273975A1 EP16769745.7A EP16769745A EP3273975A1 EP 3273975 A1 EP3273975 A1 EP 3273975A1 EP 16769745 A EP16769745 A EP 16769745A EP 3273975 A1 EP3273975 A1 EP 3273975A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- cell
- cells
- antigen
- specific
- her2
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Definitions
- HER2+ invasive breast cancer (IBC) patients with residual disease following neoadjuvant chemotherapy have an anti-HER2 Type 1 T helper (Thl) cell immune deficit and a significant risk of recurrent disease. It has been shown that anti-HER2 CD4+ T-cell responses incrementally decrease along the breast cancer continuum - a robust response in healthy donors and patients with benign disease, a depressed response in patients with HER2+ ductal carcinoma in situ, and a nearly absent response in patients with HER2+ IBC.
- HER2 oncogene is a molecular driver that is overexpressed in a significant number of breast, ovarian, gastric esophageal, lung, pancreatic, prostate and other solid tumors.
- HER2 overexpression (“HER2 pos "), a molecular oncodriver in several tumor types including approximately 20-25% of breast cancers (Meric, F., et al., J. Am Coll. Surg. 194:488-501 (2002)), is associated with an aggressive clinical course, resistance to chemotherapy, and a poor overall prognosis in BC. See, Henson, E.S., Clin. Can. Res. 12:845-53 (2006) (“Henson, et al”) and Wang, G.S., Mol. Med. Rep. 6:779-82 (2012). In incipient BC, HER2
- HER2 neg -DCIS ductal carcinoma in situ
- HER2 neg -IBC invasive breast
- HER2 P0S DCISaHER2 P ° s -IBC (invasive breast cancer) in HER2P° s breast
- the depressed anti-HER2 Thl responses in HER2 pos -invasive breast cancer were differentially restored after HER2 -pulsed type-1 polarized dendritic cell ("DC1") vaccinations, but the depressed responses were not restored following HER2-targeted therapy with trastuzumab and chemotherapy ("T/C”) or by other standard therapies such as surgical resection or radiation. Id.
- the restored anti-HER2 Thl responses also appear to be durable for at least about six months or longer.
- T lymphocyte subsets CD4 + or CD8 +
- the expansion of T lymphocyte subsets is an essential step to gain enough T cells to perform adoptive therapy, or to identify epitopes on target antigens for peptide-based vaccines.
- Expansion of T cells in principle is a simple process.
- many technical problems exist including poor levels of expansion, premature activation-induced cell death (apoptosis), or loss of antigen specificity and/or function.
- Part of the problem lies in the inability to replicate, in vitro, the environment inside the body where antigen-specific T cell expansion occurs, which is the lymph node.
- Each of these cell types plays a different role (both currently defined and as yet incompletely characterized) by providing contact-dependent signals (surface receptors) and soluble signals (cytokines) important for T cell growth and maintenance of cell function.
- the present invention provides a method of expanding a T cell.
- the method comprises contacting the T cell with one or more of a dendritic cell, at least two cytokines, and a T cell growth factor.
- the T cell is contacted with an antigen thereby generating an antigen specific T cell.
- the dendritic is type I dendritic cell.
- the at least two cytokines is interleukin-7 (IL-7) and interleukin-15 (IL-15).
- the T cell growth factor is interleukin-2 (IL-2).
- the method comprises: a) contacting the T cell with autologous type I dendritic cells DCs in combination with an antigen in vitro thereby generating an antigen specific T cell; b) contacting the antigen specific T cells with IL-7 and IL-5 to generate a stimulated antigen specific T cell; c) contacting the stimulated antigen specific T cell with IL-2 thereby generating an expanded antigen specific T cell population that maintains antigen specificity and cellular function.
- the invention provides a T cell generated by a method of the invention.
- the invention provides a population of cultured expanded antigen specific T cell exhibiting antitumor activity generated by a method according to claims 1 -6, wherein the cells are expanded to a number sufficient for effective therapy in a mammal. In one embodiment, the invention provides a population of cultured expanded antigen specific T cell exhibiting antitumor activity generated by a method of the invention, wherein the cells are expanded to a number sufficient for effective epitope mapping.
- the invention provides an isolated polynucleotide encoding a T cell receptor (TCR) that is derived from an antigen specific T cell, wherein the antigen specific T cell is generated by a method of the invention.
- TCR T cell receptor
- the invention provides a method of immunotherapy comprising administering a T cell to a subject in need thereof, wherein the T cell is generated by a method of the invention.
- the invention provides a method of expanding a T cell population which comprises at least one T cell obtained from a blood sample from a subject who has been vaccinated against an antigen, comprising the step of: contacting the T cell with one or more of a dendritic cell or a precursor thereof, at least two cytokines, and a T cell growth factor.
- the blood sample contains at least one T cell of the population specific for the vaccine antigen and at least one DC precursor.
- the DC precursor is pulsed with the antigen and activated to an antigen-specific type I dendritic cell (DCl) and then co-cultured with the T cell to generate an antigen-specific DC l .
- DCl antigen-specific type I dendritic cell
- the at least two cytokines is interleukin-7 (IL-7) and interleukin-15 (IL-15).
- the T cell growth factor is interleukin-2 (IL-2).
- the method comprises: a) co-culturing the T cell from the patient sample with the antigen-specific T cell autologous type I dendritic cell (DC l) in vitro; b) contacting the cells from step a) with IL-7 and IL-5 to generate a stimulated antigen- specific T cell; c) subsequently contacting the stimulated antigen specific T cell with IL-2, thereby generating an expanded antigen specific T cell population that maintains antigen specificity and cellular function.
- DC l antigen-specific T cell autologous type I dendritic cell
- the method further comprises repeating step a) through c) from one to at least three additional times to generate further expanded antigen-specific T cell populations.
- T cell is CD4+.
- the antigen is HER2.
- the invention provides a method of expanding a CD4+ T cell population which comprises at least one CD4+ T cell obtained from a blood sample from a breast cancer patient who has been vaccinated against HER2, comprising the step of:
- At least one DC precursor in the sample is pulsed with MHC class II HER2 peptide and is contacted with the CD4+ T cell.
- the method comprises: a) co-culturing the T cell from claiml 1 with the HER2-pulsed DC1 ; b) contacting the cells from step a) with IL-7 and IL-5 to generate a stimulated antigen-specific T cell; c) subsequently contacting the stimulated antigen specific T cell with IL-2, thereby generating an expanded antigen specific T cell population that maintains antigen specificity and cellular function.
- the method further comprises repeating step a) through c) from one to at least three additional times to generate further expanded antigen-specific T cell populations.
- Figure 1 shows anti-HER2 Thl response repertoire of four HER2 + IBC patients with residual disease following neoadjuvant therapy who received adjuvant HER2- pulsed DC1 vaccines. Each patient is depicted in a different color and shows number of reactive peptides (n) (also referred to as "response repertoire”) pre-vaccine, 3-months-post vaccine, and 6-months post vaccine.
- n reactive peptides
- FIG. 3 and Figure 4 show a direct comparison between CD4 + T cells co- cultured with HER2-specific DCl's from patients vaccinated with HER2 peptidepulsed DC1 vaccines stimulated with IL-2 versus those stimulated with IL-2/7/15 for two different patients, respectively.
- Immature DCs (“iDC's") from the respective patients were pulsed with the following MHC class II peptides: peptide 42-56 (SEQ ID NO: 1), peptide 98-114 (SEQ ID NO: 2), peptide 328-345 (SEQ ID NO: 3), and peptide 776-790 (SEQ ID NO: 4) and matured to DCl 's.
- the resulting HER2-pulsed DCl's were then co-cultured with CD4 + T cells and stimulated with IL-2 alone or with IL-2/7/15 as indicated.
- the red outline boxes indicate the specific peptide and stimulation protocol for which specificity is shown (greater than 2: 1 ratio of specific antigen: control antigen IFN- ⁇ production).
- Control antigen shows non-specific iDC's co-cultured with control antigen
- Specific antigen represents anti-HER2 CD4+ T cells co-cultured with iDC's that were pulsed with HER2 antigen/peptide
- Tcell represents anti-HER2 CD4+ T cells in culture medium.
- Graphs showing fold expansion (defined as number of T cells post expansion/number of T cells pre expansion) are shown at right, respectively. Specificity was measured by antigen-specific IFN- ⁇ production by ELISA.
- Figure 5 and Figure 6 show specific responses followed by nonspecific immune responses: Figure 5 shows a specific response following a first
- CD3/CD28 The first stimulation of CD4 + T cells with HER2-specific DC Is resulted in multiple specific immune responses as shown by red outline boxes in Figure 5.
- Figure 6 shows the second stimulation of the HER2-specific CD4 + T cells with a non-specific anti- CD3/CD28 stimulus resulted in a four-fold expansion (side graph), but with a loss of specificity in three fourths of the peptide groups.
- iDC's from patients were pulsed with the following MHC class II peptides: peptide 42-56 (SEQ ID NO: 1), peptide 98-114 (SEQ ID NO: 2), peptide 328-345 (SEQ ID NO: 3), and peptide 776-790 (SEQ ID NO: 4) and matured to DCl's.
- the resulting HER2-pulsed DCl 's were then co-cultured with CD4 + T cells and stimulated with IL-2 alone or with IL-2/7/15 as indicated.
- Figure 7 and Figure 8 show non-specific immune response followed by specific immune responses:
- Figure 7 shows non-specific expansion of CD4 + T cells.
- Figure 8 shows failure to obtain specificity following subsequent stimulation with HER2-specific DCl 's.
- the first stimulation of CD4 + T cells with non-specific anti CD3/CD28 resulted in a 3.8 fold expansion ( Figure 7).
- the second stimulation of the non-specific CD4 + T cells with HER2-specific DCl's failed to result in a specific immune response (Figure 8).
- iDC's from patients were pulsed with the following MHC class II peptides: peptide 42-56 (SEQ ID NO: 1), peptide 98-114 (SEQ ID NO: 2), peptide 328-345 (SEQ ID NO: 3), and peptide 776-790 (SEQ ID NO: 4) and matured to DCl's.
- the resulting HER2-pulsed DCl 's were then co- cultured with CD4 + T cells and stimulated with IL-2 alone or with IL-2/7/15 as indicated.
- FIGS 9A and 9B show in vitro primary/first expansion of HER2specific Thl cells comparing CD4 + T cells co-cultured with HER2-specific DCl's expanded with IL-2 versus those expanded with IL-2/7/15.
- Immature DCs (“iDC's”) were pulsed with the following MHC class II peptides: peptide 42-56 (SEQ ID NO: 1), peptide 98-114 (SEQ ID NO: 2), peptide 776-790 (SEQ ID NO: 4), and peptide 927-941 (SEQ ID NO: 5), and matured to DCl's.
- FIG. 9B The red outline boxes ( Figure 9B) indicate the specific peptide and stimulation protocol for which specificity is shown (greater than 2: 1 ratio of specific antigen: control antigen IFN- ⁇ production).
- Control Antigen shows non-specific iDC's cocultured with control antigen
- Specific Antigen represents anti-HER2 CD4+ T cells co-cultured with iDC's that were pulsed with HER2 antigen/peptide
- T cells represents anti-HER2 CD4+ T cells in culture medium.
- Figure 9A shows mean fold expansion (defined as number of T cells post expansion/number of T cells pre expansion) of Thl cells was significantly better when stimulated with IL-2, IL-7, and IL-15 than with IL-2 alone (2.6 ⁇ 0.75 vs
- Figure 9B shows specificity for the various peptide/expansion protocols as measured by antigen-specific IFN- ⁇ production by ELISA. Both stimulation with IL-2, IL- 7, and IL-15 and with IL-2 alone resulted in a specific Thl response in the same HER2 peptide 776-790.
- MHC class II peptides peptide 42-56 (SEQ ID NO: 1), peptide 98-114 (SEQ ID NO: 2), peptide 776-790 (SEQ ID NO: 4), and peptide 927-941 (SEQ ID NO: 5) were used.
- the red outline boxes ( Figure 10B) indicate the specific peptide and stimulation protocol for which specificity is shown (greater than 2: 1 ratio of specific antigen: control antigen IFN- ⁇ production) (i.e., DC1 restimulation of peptide 42-56-and peptide 776-790 -specific Thl cells.
- Control Antigen shows non-specific iDC's co-cultured with control antigen
- Specific Antigen represents anti-HER2 CD4+ T cells co-cultured with iDC's that were pulsed with HER2 antigen/peptide
- T cells represents anti-HER2 CD4+ T cells in culture medium.
- Figures 11A and 1 IB show tertiary/third expansion of the Thl cells with HER2-pulsed DC1 's ( peptide 42-56 (SEQ ID NO: 1), peptide 98-114 (SEQ ID NO: 2), peptide 776-790 (SEQ ID NO: 4), and peptide 927-941 (SEQ ID NO: 5) were used).
- FIGS 12 throughl5 show sequential results of repeated in vitro stimulation
- Figure 12 for the first stimulation shows specificity only for peptide 776-790-specific Thl cells
- Figure 13 for the second stimulation shows an increase, specificity for peptide 42-56-and peptide 776-790-specific Thl cells
- Figure 14 for the third expansion shows specificity for all four peptides
- Figure 15 for the fourth expansion shows loss of specificity for one of the peptides (peptide 927-941) leaving three remaining HER2-specific peptides.
- Figure 16 shows cumulative fold expansion of the four expansions shown in
- This invention relates to a method of creating a microenvironment in vitro for culture expansion of antigen-specific helper-T cells.
- the expanded antigen-specific T cells can be used for a variety of therapeutic and research purposes, for example adoptive T cell therapy for cancer or other conditions and or for the identification of epitopes on target antigens to foster the production of peptide-based vaccines.
- the invention comprises the use of autologous type I dendritic cells (DCs) in combination with a protein or peptide antigen to stimulate T cells in vitro.
- DCs autologous type I dendritic cells
- at least two soluble factors e.g., cytokines
- the at least two soluble factors are Interleukin-7 (IL-7) and Interleukin-15 (IL-15).
- IL-7 Interleukin-7
- IL-15 Interleukin-15
- a T cell growth factor is added.
- the T cell growth factor is Interleukin-2 (IL-2).
- the soluble factors in addition to those naturally produced by the Type I DCs, support the proliferation and acquisition/maintenance of T cell function. This process of stimulation can be repeated in weekly cycles until T cells are of sufficient numbers for therapy or epitope
- the T cells are expanded to a level necessary for adoptive therapy and epitope mapping studies while maintaining antigen specificity and cellular function.
- Standard techniques are used for nucleic acid and peptide synthesis.
- the techniques and procedures are generally performed according to conventional methods in the art and various general references (e.g., Sambrook and Russell, 2012, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY, and Ausubel et al, 2012, Current Protocols in Molecular Biology, John Wiley & Sons, NY), which are provided throughout this document.
- the nomenclature used herein and the laboratory procedures used in analytical chemistry and organic syntheses described below are those well-known and commonly employed in the art. Standard techniques or modifications thereof are used for chemical syntheses and chemical analyses.
- an element means one element or more than one element.
- abnormal when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the "normal"
- Characteristics which are normal or expected for one cell or tissue type might be abnormal for a different cell or tissue type.
- activation refers to the state of a cell following sufficient cell surface moiety ligation to induce a noticeable biochemical or morphological change. Within the context of T cells, such activation refers to the state of a T cell that has been sufficiently stimulated to induce cellular proliferation. Activation of a T cell may also induce cytokine production and performance of regulatory or cytolytic effector functions. Within the context of other cells, this term infers either up or down regulation of a particular physico-chemical process. The term “activated T cells” indicates T cells that are currently undergoing cell division, cytokine production, performance of regulatory or cytolytic effector functions, and/or has recently undergone the process of "activation.”
- Adjuvant therapy for breast cancer as used herein refers to any treatment given after primary therapy (i.e., surgery) to increase the chance of long-term survival.
- Neoadjuvant therapy is treatment given before primary therapy.
- antigen or "ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.
- antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein.
- an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response.
- an antigen need not be encoded by a "gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.
- agent refers to a molecule that binds to a defined population of cells.
- the agent may bind any cell surface moiety, such as a receptor, an antigenic determinant, or other binding site present on the target cell population.
- the agent may be a protein, peptide, antibody and antibody fragments thereof, fusion proteins, synthetic molecule, an organic molecule (e.g., a small molecule), a carbohydrate, or the like.
- antibodies and natural ligands are used as prototypical examples of such agents.
- agent that binds a cell surface moiety and "cell surface moiety”, as used herein, are used in the context of a ligand/anti -ligand pair. Accordingly, these molecules should be viewed as a complementary/anti-complementary set of molecules that demonstrate specific binding, generally of relatively high affinity.
- APC antigen presenting cell
- T cells includes, but is not limited to, monocytes/macrophages, B cells and dendritic cells (DCs).
- Antigen-loaded APC or an “antigen-pulsed APC” includes an APC, which has been exposed to an antigen and activated by the antigen.
- an APC may become Ag-loaded in vitro, e.g., during culture in the presence of an antigen.
- the APC may also be loaded in vivo by exposure to an antigen.
- An "antigen-loaded APC” is traditionally prepared in one of two ways: (1) small peptide fragments, known as antigenic peptides, are "pulsed” directly onto the outside of the APCs; or (2) the APC is incubated with whole proteins or protein particles which are then ingested by the APC.
- the antigen-loaded APC can also be generated by introducing a polynucleotide encoding an antigen into the cell.
- Anti-HER2 response is the immune response specifically against HER2 protein.
- anti-tumor effect refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition.
- An "anti -tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention in prevention of the occurrence of tumor in the first place.
- autoimmune disease as used herein is defined as a disorder that results from an autoimmune response. An autoimmune disease is the result of an
- autoimmune diseases include but are not limited to, Addision's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, among
- autologous is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.
- Allogeneic refers to a graft derived from a different animal of the same species.
- B cell as used herein is defined as a cell derived from the bone marrow and/or spleen. B cells can develop into plasma cells which produce antibodies.
- CD4 + Thl cells “Thl cells,” “CD4 + T-helper type 1 cells,” “CD4 + T cells,” and the like are defined as a subtype of T-helper cells that express the surface protein CD4 and produce high levels of the cytokine IFN- ⁇ . See also, “T-helper cells.”
- cancer as used herein is defined as a hyperproliferation of cells whose unique trait-loss of normal control—results in unregulated growth, lack of differentiation, local tissue invasion, and/or metastasis. Examples include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, germ-cell tumors, and the like.
- a “co-stimulatory signal”, as used herein, refers to a signal, which in combination with a primary signal, such as TCR/CD3 ligation, leads to T cell proliferation and/or upregulation or downregulation of key molecules.
- a “co-stimulatory signal”, as used herein, refers to a signal, which in combination with a primary signal, such as TCR/CD3 ligation, leads to T cell proliferation and/or upregulation or downregulation of key molecules.
- a "co-stimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation.
- Co-stimulatory ligand includes a molecule on an antigen presenting cell (e.g. , a dendritic cell, B cell, and the like) that specifically binds a cognate co-stimulatory molecule on a T cell, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like.
- an antigen presenting cell e.g. , a dendritic cell, B cell, and the like
- a co-stimulatory ligand can include, but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L,
- ILT3, ILT4, HVEM an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3.
- “Cumulative response” means the combined immune response of a patient group expressed as the total sum of reactive spots (spot-forming cells “SFC” per 10 6 cells from IFN- ⁇ ELISPOT analysis) from all 6 MHC class II binding peptides from a given patient group.
- dendritic cell is an antigen presenting cell existing in vivo, in vitro, ex vivo, or in a host or subject, or which can be derived from a hematopoietic stem cell or a monocyte.
- Dendritic cells and their precursors can be isolated from a variety of lymphoid organs, e.g., spleen, lymph nodes, as well as from bone marrow and peripheral blood.
- the DC has a characteristic morphology with thin sheets (lamellipodia) extending in multiple directions away from the dendritic cell body.
- dendritic cells express high levels of MHC and costimulatory (e.g., B7-1 and B7-2) molecules. Dendritic cells can induce antigen specific differentiation of T cells in vitro, and are able to initiate primary T cell responses in vitro and in vivo.
- DC vaccination refers to a strategy using autologous dendritic cells to harness the immune system to recognize specific molecules and mount specific responses against them.
- DC-1 polarized dendritic cells refer to mature DCs that secrete Thl-driving cytokines, such as IL-12, IL-18, and IL-23.
- DCls are fully capable of promoting cell-mediated immunity.
- DCls are pulsed with HER2 MHC class II-binding peptides in preferred embodiments herein.
- an “activated DC” is a DC that has been exposed to a Toll-like receptor agonist.
- the activated DC may or may not be loaded with an antigen.
- mature DC is defined as a dendritic cell that expresses molecules, including high levels of MHC class II, CD80 (B7.1) and CD86 (B7.2). In contrast, immature dendritic cells express low levels of MHC class II, CD80 (B7.1) and CD86 (B7.2) molecules, yet can still take up an antigen.
- “Mature DC” also refers to an antigen presenting cell existing in vivo, in vitro, ex vivo, or in a host or subject that is DC1- polarized (i.e., fully capable of promoting cell-mediated immunity).
- a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
- a “disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.
- a disease or disorder is "alleviated” if the severity or frequency of at least one sign or symptom of the disease or disorder experienced by a patient is reduced.
- Effective amount or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result. Such results may include, but are not limited to, the inhibition of virus infection as determined by any means suitable in the art.
- endogenous refers to any material from or produced inside an organism, cell, tissue or system.
- exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
- epitope is defined as a small chemical molecule on an antigen that can elicit an immune response, inducing B and/or T cell responses.
- An antigen can have one or more epitopes. Most antigens have many epitopes; i.e., they are multivalent. In general, an epitope is roughly five amino acids and/or sugars in size.
- an epitope is roughly five amino acids and/or sugars in size.
- HER2 is a member of the human epidermal growth factor receptor
- EGFR EGFR family.
- HER2 is overexpressed in approximately 20-25% of human breast cancer and is expressed in many other cancers.
- HER2 binding peptides refer to MHC Class II peptides derived from or based on the sequence of the HER2/neu protein, a target found on approximately 2025% of all human breast cancers and their equivalents.
- HER2 extracellular domain refers to a domain of HER2 that is outside of a cell, either anchored to a cell membrane, or in circulation, including fragments thereof.
- HER2 intracellular domain "ICD” refers to a domain of the HER2/neu protein within the cytoplasm of a cell.
- HER2 epitopes or otherwise binding peptides comprise 6 HER2 binding peptides which include 3 HER2 ECD peptides and 3 HER2 ICD peptides.
- Preferred HER2 ECD peptides comprise: Peptide 42-56:
- HLDMLRHLYQGCQVV (SEQ ID NO: 1); Peptide 98-114: RLRIVRGTQLFEDNYAL (SEQ ID NO: 2); and Peptide 328-345: TQRCEKCSKPCARVCYGL (SEQ ID NO: 3);
- Preferred HER2 ICD peptides comprise: Peptide 776-790: GVGSPYVSRLLGICL (SEQ ID NO: 4); Peptide 927-941 : PAREIPDLLEKGERL (SEQ ID NO: 5); and Peptide 1166-1180: TLERPKTLSPGKNGV (SEQ ID NO: 6).
- HER2 pos is the classification or molecular subtype of a type of breast cancer as well as numerous other types of cancer. HER2 positivity is currently defined by gene amplification by FISH (fluorescent in situ hybridization) assay and 2+ or 3+ on intensity of pathological staining.
- FISH fluorescent in situ hybridization
- HER2 neg is defined by the lack of gene amplification by FISH, and can encompass a range of pathologic staining from 0 to 2+ in most cases.
- "Homologous” as used herein refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are completely or 100% homologous at that position.
- the percent homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50% identical, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology.
- the DNA sequences 5'ATTGCC3' and 5 ATGGC3' share 50% homology.
- inhibitor means to suppress or block an activity or function, for example, about ten percent relative to a control value. Preferably, the activity is suppressed or blocked by 50% compared to a control value, more preferably by 75%, and even more preferably by 95%. "Inhibit,” as used herein, also means to reduce a molecule, a reaction, an interaction, a gene, an mRNA, and/or a protein's expression, stability, function or activity by a measurable amount or to prevent entirely.
- Inhibitors are compounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate a protein, a gene, and an mRNA stability, expression, function and activity, e.g., antagonists.
- an "instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the invention.
- the instructional material of the kit of the invention may, for example, be affixed to a container which contains the nucleic acid, peptide, and/or composition of the invention or be shipped together with a container which contains the nucleic acid, peptide, and/or composition.
- the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.
- Interleukin 2 (“IL-2” or “IL2”) is an interleukin, a type of cytokine signaling molecule in the immune system. IL2 is the principal T cell growth and proliferation factor. Interleukin 7 (“IL-7” or “IL2”) is a hematapoietic growth factor produced by stromal epithelial cells in lymph nodes. IL-7 is essential for lymphocyte proliferation and survival.
- Interleukin 15 (“IL-15” or “IL2”) is a T cell growth activation and survival factor. IL-15 is produced by fibroblasts, dendritic cells and macrophages.
- isolated means altered or removed from the natural state.
- a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
- An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
- “Loaded” with a peptide refers to presentation of an antigen in the context of an MHC molecule.
- MHC major histocompatibility complex
- Class I MHC, or MHC class I function mainly in antigen presentation to CD8 T lymphocytes.
- Class II MHC, or MHC class II function mainly in antigen presentation to CD4 + T lymphocytes (T-helper cells).
- “Mature DC” as used herein means a dendritic cell that expresses molecules, including high levels of MHC class II, CD80 (B7.1) and CD86 (B7.2) molecules. In contrast, immature DCs (“iDCs”) express low levels of MHC class II, CD80 (B7.1) and CD86 (B7.2) molecules, yet can still take up an antigen. “Mature DC” also refers to an antigen presenting cell existing in vivo, in vitro, ex ⁇ , ⁇ ina host or subject that may also be DC 1 -polarized (i.e., fully capable of promoting cell-mediated immunity.)
- Methods of CD4 + Thl responses are defined for each subject group analyzed for anti-HER2 CD4 + Thl immune response: (a) overall anti-HER2 responsivity (expressed as percent of subjects responding to >1 reactive peptide); (b) response repertoire (expressed as mean number of reactive peptides (n) recognized by each subject group); and (c) cumulative response (expressed as total sum of reactive spots (spot- forming cells "SFC" per 10 6 cells from IFN- ⁇ ELISPOT analysis) from 6 MHC Class II binding peptides from each subject group.
- moduleating mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject.
- the term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.
- Non-equivocal HER2 neg is defined as non-gene amplified and 0 or 1+ on pathologic staining.
- Equivocal HER2 neg is defined as non-gene amplified but 2+ on pathologic staining.
- a "population” includes reference to an isolated culture comprising a homogenous, a substantially homogenous, or a heterogeneous culture of cells. Generally, a “population” may also be regarded as an "isolated” culture of cells.
- a "recombinant cell” is a host cell that comprises a recombinant polynucleotide.
- Responsivity or “anti-HER2 responsivity” are used interchangeably herein to mean the percentage of subjects responding to at least 1 of 6 binding peptides.
- Response repertoire is defined as the mean number ("n") of reactive peptides recognized by each subject group.
- sample or “biological sample” as used herein means a biological material from a subject, including but is not limited to organ, tissue, exosome, blood, plasma, saliva, urine and other body fluid.
- a sample can be any source of material obtained from a subject.
- stimulation is meant a primary response induced by binding of a stimulatory molecule with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex.
- Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF- ⁇ , and/or reorganization of cytoskeletal structures, and the like.
- a “stimulatory ligand,” as used herein, means a ligand that when present on an antigen presenting cell (e.g. , a dendritic cell, a B-cell, and the like) can specifically bind with a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like.
- an antigen presenting cell e.g. , a dendritic cell, a B-cell, and the like
- a cognate binding partner referred to herein as a "stimulatory molecule”
- a “stimulatory molecule,” as the term is used herein, means a molecule on a T cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell.
- the terms "subject,” “patient,” “individual,” and the like are used
- the patient, subject or individual is a human.
- substantially purified cell is a cell that is essentially free of other cell types.
- a substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state.
- a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state.
- the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.
- target therapies refers to cancer treatments that use drugs or other substances that interfere with specific target molecules involved in cancer cell growth usually while doing little damage to normal cells to achieve an anti-tumor effect.
- Traditional cytotoxic chemotherapy drugs by contrast, act against all actively dividing cells.
- monoclonal antibodies specifically trastuzumab/Herceptin ® , targets the HER2/neu receptor.
- T/C is defined as trastuzumab and chemotherapy. This refers to patients that receive both trastuzumab and chemotherapy before/after surgery for breast cancer.
- T cell as used herein is defined as a thymus-derived cell that participates in a variety of cell-mediated immune reactions.
- T-helper cells are used herein with reference to cells indicates a sub-group of lymphocytes (a type of white blood cell or leukocyte) including different cell types identifiable by a skilled person in the art.
- T-helper cells are effector T-cells whose primary function is to promote the activation and functions of other B and T lymphocytes and/or macrophages.
- Helper T cells differentiate into two major subtypes of cells known as “Thl” or “Type 1" and “Th2" or “Type 2" phenotypes. These Th cells secrete cytokines, proteins, or peptides that stimulate or interact with other leukocytes.
- Thl cell “CD4 + Thl cell,” “CD4 + T-helper type 1 cell,” “CD4 + T cell” and the like as used herein refer to a mature T-cell that has expressed the surface glycoprotein CD4.
- CD4 + T-helper cells become activated when they are presented with peptide antigens by MHC class II molecules which are expressed on the surface of antigen-presenting peptides ("APCs") such as dendritic cells.
- APCs antigen-presenting peptides
- APCs antigen-presenting peptides
- IFN- ⁇ interferon- ⁇
- cytotoxic T cell or "CD8+ T cell or “killer T cell” is a T lymphocyte that kills target cells such as cancer cells, cells that are infected, or cells that are damaged in other ways.
- Treg "Treg” and “regulatory T-cells” are used herein to refer to cells which are the policemen of the immune system, and which act to regulate the anticancer activities of the immune system. They are increased in some cancers, and are mediators in resistance to immunotherapy in these cancer types.
- terapéutica as used herein means a treatment and/or prophylaxis.
- a therapeutic effect is obtained by suppression, remission, or eradication of a disease state.
- “Therapeutically effective amount” is an amount of a compound of the invention, that when administered to a patient, ameliorates a symptom of the disease.
- the amount of a compound of the invention which constitutes a “therapeutically effective amount” will vary depending on the compound, the disease state and its severity, the age of the patient to be treated, and the like.
- the therapeutically effective amount can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.
- treat refers to therapeutic or preventative measures described herein.
- the methods of “treatment” employ administration to a subject, in need of such treatment, a composition of the present invention, for example, a subject afflicted a disease or disorder, or a subject who ultimately may acquire such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
- the term "vaccine” as used herein is defined as a material used to provoke an immune response after administration of the material to an animal, preferably a mammal, and more preferably a human. Upon introduction into a subject, the vaccine is able to provoke an immune response including, but not limited to, the production of antibodies, cytokines and/or other cellular responses.
- ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
- the invention relates to replicating the environment of the lymph node for generating a therapeutic amount of T cells for adoptive therapy.
- the T cells can also be used for the identification of epitopes on target antigens to foster the production of peptide-based vaccines.
- the invention provides an in vitro environment that replicates the environment of the lymph node.
- replication of the lymph node comprises supplying one or more of the following elements to the culture conditions: type 1 dendritic cells, IL-15, IL-7, and IL-2.
- Type 1 dendritic cells process and present peptide antigens to T cells and supply so-called "costimulatory molecules" including surface-expressed CD80 and CD86 (which bind to CD28 counter-receptor on T cells), as well as CD40 (which interacts with CD40L on T cells).
- costimulatory molecules including surface-expressed CD80 and CD86 (which bind to CD28 counter-receptor on T cells), as well as CD40 (which interacts with CD40L on T cells).
- the DCs produce soluble factors such as Interleukin-12 (IL- 12) which supports long life (anti-apoptotic factor) as well as Interferon-gamma production (T cell function).
- IL-12 Interleukin-12
- T cell function Interferon-gamma production
- DCs produce a number of other factors critical to T cell development. DCs are normally found in lymph nodes and are of known importance to T cell activation and expansion.
- IL-15 is a T cell growth activation and survival factor. IL-15 is produced by fibroblasts, dendritic cells and macrophages.
- IL-7 is a factor produced by stromal epithelial cells in lymph nodes. IL-7 is essential for lymphocyte proliferation and survival.
- IL2 is the principal T cell growth and proliferation factor.
- the invention provides compositions and methods for combining the cytokines and type of dendritic cells to generate desirable T cells.
- the T cells are expanded to a level necessary for adoptive therapy and epitope mapping studies while maintaining antigen specificity and cellular function.
- the invention relates to the discovery that HER2 + invasive breast cancer ("IBC") patients with residual disease following neoadjuvant chemotherapy have an anti-HER2 Type 1 T helper (Thl) cell immune deficit and a significant risk of recurrent disease. It was shown in Datta, et al.
- the present embodiments also relate to a method of creating a microenvironment in vitro for culture expansion of antigen-specific CD4 + or CD8 + T cells.
- the expanded antigen-specific T cells can be used for a variety of therapeutic and research purposes, for example adoptive T cell therapy for cancer or infectious disease such as chronic viral infections other conditions and/or for the identification of epitopes on target antigens to foster the production of peptide-based vaccines.
- the invention comprises the use of autologous type I dendritic cells ("DC Is") in combination with a protein or peptide antigen to stimulate T cells in vitro.
- DC Is autologous type I dendritic cells
- at least two soluble factors e.g., cytokines
- the at least two soluble factors are Interleukin-7 (“IL-7") and Interleukin-15 (“IL-15”).
- IL-7 Interleukin-7
- IL-15 Interleukin-15
- T cell growth factor is added.
- the T cell growth factor is Interleukin-2 ("IL-2").
- the soluble factors in addition to those naturally produced by the DC Is, support the proliferation and acquisition/maintenance of T cell function.
- This process of stimulation can be repeated in weekly cycles until T cells are of sufficient numbers for therapy or epitope scanning/mapping.
- the T cells are expanded to a level necessary for adoptive therapy and epitope mapping studies while maintaining antigen specificity and cellular function.
- the depressed anti-HER2 Thl responses in HER2 pos -invasive breast cancer were differentially restored after HER2 -pulsed type- 1 polarized dendritic cell ("DC1") vaccinations.
- the depressed responses were not restored following HER2targeted therapy with trastuzumab and chemotherapy ("T/C”) or by other standard therapies such as surgical resection or radiation.
- T/C trastuzumab and chemotherapy
- the restored anti-HER2 Thl responses appear to be durable for at least about six months or considerably longer.
- HER2 + IBC patients with residual disease following neoadjuvant therapy received adjuvant HER2-pulsed DC1 vaccines Immune responses were generated from PBMCs pulsed with HER2 Class II peptides by measuring IFN- ⁇ production via ELISPOT. Responses were evaluated on the three metrics of CD4 + Thl response: (1) the overall anti- HER2 responsivity (responding to >1 peptide), (2) the number of reactive peptides (response repertoire), and (3) the cumulative response across the 6 HER2 peptides. Pre-vaccination Thl responses were compared with 3-month and 6month post-vaccination responses.
- HER2 MHC class II binding peptides identified by SEQ ID NOS: 1-6, and then interferon (“IFN”)-y and lipopolysaccharide (“LPS”) are added to complete the maturing and activation process to achieve full DC activation to DC Is before injecting back into the patient.
- IFN interferon
- LPS lipopolysaccharide
- Datta, et al. also describe blood tests/assays which generate a circulating anticancer CD4 + Thl response (i.e., IFN-y-secreting) and the resulting IFN- ⁇ production is detected and measured.
- Such blood tests were performed on patients pre-DCl vaccination, and 3-months and 6 months post-vaccination.
- subject blood samples containing CD4 + Thl cells and antigen-presenting cells or precursors thereof are pulsed with MHC class II immunogenic peptides based on the type of cancer the subject is afflicted with and which are capable of inducing an immune response in said subject.
- the antigen-presenting cells or precursors thereof are mature or immature dendritic cells or monocyte precursors thereof.
- the cancer is preferably HER2-expressing and the mammalian subject is preferably a human, and more preferably the cancer is HER2 pos breast cancer and the human subject is a female.
- a preferred embodiment is provided for generating a circulating antiHER2
- CD4 + Thl response in a mammalian subject by isolating unexpanded peripheral blood mononuclear cells ("PBMCs") from a subject and pulsing the PBMCs with a composition comprising HER2-derived MHC class II antigenic binding peptides capable of generating an immune response in the subject.
- PBMCs peripheral blood mononuclear cells
- HER2-derived MHC class II antigenic binding peptides capable of generating an immune response in the subject.
- DCls type-1 polarized dendritic cells derived from precursor pluripotent monocytes contained in the subject's PBMC sample are antigen-presenting cells ("APCs") which upon exposure to the binding peptides become antigen-loaded APCs which present the MHC class II antigen binding peptides to the subject's CD4 + Thl cells in the sample thereby activating the CD4 + Thl cells to produce/secrete IFN- ⁇ .
- APCs antigen-presenting cells
- each patient's PBMCs were pulsed with 6 HER2-specific MHC class II peptides, in particular, those having sequences identified by SEQ ID NOs: 1-6.
- IFN- ⁇ produced by anti-HER2 CD4 + Thl cells was detected and measured via IFN- ⁇ enzyme-linked immunospot ("ELISPOT") assay.
- DCs, immature or type-1 polarized DCls are pulsed with a composition comprising 6 MHC class II binding peptides derived from or based on HER2 that are capable of generating an immune response in a patient.
- HER2 MHC class II binding peptides or epitopes include:
- Peptide 42-56 HLDMLRHLYQGCQVV (SEQ ID NO: 1);
- Peptide 98-114 RLRIVRGTQLFEDNYAL (SEQ ID NO: 2);
- Peptide 328-345 TQRCEKCSKPCARVCYGL (SEQ ID NO: 3);
- Peptide 776-790 GVGSPYVSRLLGICL (SEQ ID NO: 4);
- Peptide 927-941 PAREIPDLLEKGERL (SEQ ID NO: 5);
- Peptide 1166-1180 TLERPKTLSPGKNGV (SEQ ID NO: 6).
- donors have A2.1 blood type HER2 MHC class I peptides or epitopes include:
- Peptide 369-377 KIFGSLAFL (SEQ ID NO: 7); and Peptide 689-697: RLLQETELV (SEQ ID NO: 8).
- Datta, et al. also describe an alternate preferred embodiment, wherein a circulating anti HER2 CD4 + Thl response is generated in a mammalian subject by co- culturing previously unstimulated purified CD4 + T-cells from a subject blood sample with autologous immature or mature dendritic cells ("iDCs" or mature “DCs") pulsed with a composition comprising HER2-derived MHC class II antigenic binding peptides capable of generating an immune response in the subject.
- iDCs autologous immature or mature dendritic cells
- a composition comprising HER2-derived MHC class II antigenic binding peptides capable of generating an immune response in the subject.
- the immature DCs are matured to DCl 's, which present the MHC class II binding peptides to the subject's CD4 + Thl cells that are present in the sample thereby activating the CD4 + Thl cells to produce IFN- ⁇ , which is subsequently measured for analysis.
- IFN- ⁇ produced by anti-HER2 CD4 + Thl cells is detected and measured via IFN- ⁇ enzyme-linked immunospot ("ELISPOT") assay, although it should be understood by one skilled in the art that other detection methods may be used.
- ELISPOT enzyme-linked immunospot
- flow cytometry, enzyme-linked immunosorbent assay (“ELISA”), and immunofluorescence (“IF”) can be used for monitoring immune response.
- ELISA enzyme-linked immunosorbent assay
- IF immunofluorescence
- immunofluorescence provides other ways to measure and visualize immune response via use of ELISPOT readers that read results by fluorescence.
- the results can be arranged to show 2, 3, or more cytokines/other secreted immune molecules, each showed in a different color, in the same patient sample.
- IFN- ⁇ ELISPOT was used.
- HER2 MHC class II binding peptides/epi topes other possible MHC class II HER2 peptides can be used in the present embodiments in that any components of the entire HER2 molecule can be used as a source for other binding peptides so long as they are sufficiently immunologically active in patients.
- Pre-vaccination only one IBC patient produced an immune response, defined as >20 SFC/10 6 cells in an experimental well after subtracting unstimulated background. Compared with pre-vaccination results, all vaccinated IBC patients produced an immune response, defined as >2-fold increase in anti-HER2 IFN-Y pos Thl responses.
- HER2 pos solid cancers in addition to breast cancer, such as, for example, brain, bladder, esophagus, lung, pancreas, liver, prostate, ovarian, colorectal, and gastric, and others, for which the materials and methods of the embodiments described herein can be used for diagnosis and treatment. Therefore the six anti-HER2 binding peptides described above may be used in accordance with the herein embodiments to generate immune responses capable of detection and useful for diagnostics for these and other HER2- expressing cancers.
- Vaccines can be developed to target HER2-expressing tumors using the same anti-HER2 binding peptides described above or may employ any composition of HER2 that is immunogenic such as, for example, DNA, RNA, peptides, or proteins or components thereof such as the ICD and ECD domains.
- HER2 that is immunogenic
- subjects can be vaccinated against the whole HER2 protein and the six above-referenced binding peptides can be used to monitor the patient's immune response.
- vaccines can be developed for other types of cancer such as other members of the HER2 family which includes HER1, HER3, and c- MET.
- present preferred embodiments are directed to treating and diagnosing HER2 pos breast cancer in women it should be readily appreciated by the skilled artisan that the present embodiments are not limited to female humans.
- the presently preferred embodiments includes male humans, for example, HER2expressing prostate cancer, as well as other mammalian subjects
- the identified anti-HER2 CD4 + Thl response decrement allows the detected immune response generated in such blood tests to be used as a cancer diagnostic/response predictor alone or, as in the example here, in tandem with the use of specialized vaccines to restore a patient's immune response.
- the preferred embodiments described herein thus shift the focus of cancer diagnosis and therapy to patient immunity and use of blood tests to determine and/or predict the immune response against a cancer, including patients at risk for recurrence, as opposed to diagnosis and treatment methods that rely on identification of tumor cells.
- HER2-specific Thl cells were generated by co-culture with HER2- peptide pulsed DCls and expanded using IL-2 alone or IL-2, IL-7, and IL-15. Thl cells were subsequently expanded either by repeat HER2-peptide pulsed DC1 co-culture or via anti- CD3/CD28 stimulation. Fold expansion was defined as: (#Tcells post expansion / #T-cells pre expansion); specificity was measured by antigen specific IFN- ⁇ production by ELISA.
- CD4 + T cells are CD4 + T cells.
- CART cells chimeric antigen receptor T cells
- CD8 + 's cytotoxic T lymphocytes
- all other kinds of T cells See, for example, Datta, J., et al, "CD4 + T-helper Type 1 Cytokines and
- Embodiments relate to replicating the environment of the lymph node for generating a therapeutic amount of antigen-specific T cells, either helper (CD4 + ) or cytotoxic (CD8 +) - for adoptive therapy for cancer or other conditions.
- the expanded antigen-specific T lymphocytes can also be used for the identification of epitopes on target antigens to foster the production of peptide-based vaccines.
- a present embodiment provides an in vitro environment that replicates the environment of the lymph node.
- replication of the lymph node comprises supplying one or more of the following elements to the culture conditions: type 1 dendritic cells, IL-15, IL-7, and IL-2.
- Type 1 dendritic cells process and present peptide antigens to T cells and supply so-called "costimulatory molecules” including surface-expressed CD80 and CD86 (which bind to CD28 counter-receptor on T cells), as well as CD40 (which interacts with CD40L on T cells).
- costimulatory molecules including surface-expressed CD80 and CD86 (which bind to CD28 counter-receptor on T cells), as well as CD40 (which interacts with CD40L on T cells).
- the DCs produce soluble factors such as Interleukin-12 (“IL- 12”) which supports long life (anti-apoptotic factor) as well as IFN- ⁇ production (T cell function).
- IL-12 Interleukin-12
- T cell function IFN- ⁇ production
- IL-15 is a T cell growth activation and survival factor.
- IL-15 is produced by fibroblasts, dendritic cells and macrophages.
- IL-7 is a factor produced by stromal epithelial cells in lymph nodes. IL7 is essential for lymphocyte proliferation and survival.
- IL2 is the principal T cell growth and proliferation factor.
- the embodiments provide compositions and methods for combining the particular cytokines and type of dendritic cells while also using particular timing and sequence of lymphocyte addition to generate desirable T cells.
- T cells are expanded to a level necessary for adoptive therapy and epitope mapping studies while maintaining antigen specificity and cellular function.
- a source of T cells is obtained from a subject.
- subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
- the subject is a human.
- T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, and tumors. In certain embodiments of the present invention, any number of T cell lines available in the art, may be used.
- T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as ficoll separation. In one preferred embodiment, cells from the circulating blood of an individual are obtained by apheresis or leukapheresis.
- the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
- the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
- the cells are washed with phosphate buffered saline (PBS).
- PBS phosphate buffered saline
- the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations.
- the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS.
- the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
- T cells are isolated from peripheral blood by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient.
- T cells can be isolated from umbilical cord.
- a specific subpopulation of T cells can be further isolated by positive or negative selection techniques.
- Enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells.
- a preferred method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
- a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8.
- the concentration of cells and surface can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells/ml is used. In one embodiment, a concentration of 1 billion cells/ml is used. In a further embodiment, greater than 100 million cells/ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
- a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further embodiments, concentrations of 125 or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion.
- T cells for stimulation can also be frozen after the washing step, which does not require the monocyte-removal step.
- the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population.
- the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, in a non- limiting example, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media. The cells are then frozen to -80°C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at - 20°C or in liquid nitrogen.
- T cells of the invention are expanded under conditions that replicate the lymph node.
- replication of the lymph node comprises supplying one or more of the following elements to the culture conditions: type 1 dendritic cells, IL-15, IL-7, and IL-2.
- antigen specific T cells can be expanded in the presence of one or more of type 1 dendritic cells, IL-15, IL-7, and IL-2.
- the T cells may be stimulated as described herein, such as by contacting with a DC.
- the DC is able to provide supply a costimulatory molecule to the T cell.
- the T cells are cultured in the presence of IL- 15, IL-7, and IL-2.
- the T cells are cultured with a mixture comprising one or more of DCs, IL-15, IL-7, and IL-2.
- the mixture may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between.
- the mixture may be cultured for 21 days.
- the T cells are cultured for about eight days.
- the T cells are cultured together for 2-3 days. Several cycles of stimulation may also be desired such that culture time of T cells can be 60 days or more.
- Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN- ⁇ , IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF , and TNF-a or any other additives for the growth of cells known to the skilled artisan.
- Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol.
- Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X- Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells.
- Antibiotics e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject.
- the target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g. , 37° C) and atmosphere (e.g., air plus 5% CO2).
- the T cells are expanded to a level necessary for adoptive therapy and epitope mapping studies while maintaining antigen specificity and cellular function. Accordingly, any cell number is within the context of the present invention.
- Cells stimulated by the methods of the present invention are activated as shown by the induction of signal transduction, expression of cell surface markers and/or proliferation.
- One such marker appropriate for T cells is IFNy production which is an important immunomodulating molecule. The production of IFNy is extremely beneficial in amplifying the immune response.
- the T cell populations resulting from the various expansion methodologies described herein may have a variety of specific phenotypic properties, depending on the conditions employed.
- phenotypic properties include enhanced expression of CD25, CD154, IFN- ⁇ and GM-CSF, as well as altered expression of CD 137, CD 134, CD62L, and CD49d.
- the ability to differentially control the expression of these moieties may be very important. For example, higher levels of surface expression of CD 154 on "tailored T cells," through contact with CD40 molecules expressed on antigen- presenting cells (such as dendritic cells, monocytes, and even leukemic B cells or lymphomas), will enhance antigen presentation and immune function.
- antigen- presenting cells such as dendritic cells, monocytes, and even leukemic B cells or lymphomas
- Such strategies are currently being employed by various companies to ligate CD40 via antibodies or recombinant CD40L.
- the approach described herein permits this same signal to be delivered in a more physiological manner, e.g., by the T cell.
- the ability to increase IFN- ⁇ secretion by tailoring the T cell activation process could help promote the generation of Thl-type immune responses, important for anti -tumor and anti -viral responses.
- increased expression of GM-CSF can serve to enhance APC function, particularly through its effect on promoting the maturation of APC progenitors into more functionally competent APC, such as dendritic cells.
- Altering the expression of CD137 and CD134 can affect a T cell's ability to resist or be susceptible to apoptotic signals. Controlling the expression of adhesion/homing receptors, such as CD62L and/or CD49d and/or CCR7 may determine the ability of infused T cells to home to lymphoid organs, sites of infection, or tumor sites.
- T cell populations of the present invention can be monitored by a variety of methods including standard flow cytometry methods and ELISA methods known by those skilled in the art.
- a bioreactor is also useful.
- devices that can be used to grow cells and be used in combination with the methods of the present invention. See for example, Celdyne Corp., Houston, TX; Unisyn Technologies, Hopkinton, MA; Synthecon, Inc., Houston, TX; Aastrom Biosciences, Inc., Ann Arbor, MI; Wave Biotech LLC, Bedminster, NJ.
- patents covering such bioreactors include U.S. Patent Nos: 6,096,532; 5,985,653; 5,888,807; 5,190,878, which are incorporated herein by reference.
- a bioreactor with a base rocker platform is used, for example such as "The Wave” (Wave Biotech LLC, Bedminster, NJ), that allows for varying rates of rocking and at a variety of different rocking angles.
- the skilled artisan will recognize that any platform that allows for the appropriate motion for optimal expansion of the cells is within the context of the present invention.
- the methods of stimulation and expansion of the present invention provide for rocking the culture container during the process of culturing at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 rocks per minute.
- the methods of stimulation and expansion of the present invention provide for the angle of the rocking platform to be set at 1.5°, 2°, 2.5°, 3°, 3.5°, 4°, 4.5°, 5°, 5.5°, 6°, 6.5°, 7°, 7.5°, 8°, 8.5°, or 9.0°.
- the capacity of the bioreactor container ranges from about 0.1 liter to about 200 liters of medium.
- the cells of the present invention such as T cells are seeded at an initial concentration of about 0.2 X 10 6 cells/ml to about 5 X 10 6 cells/ml, and any concentration therebetween.
- the cells may be cultured initially in a static environment and transferred to a bioreactor on a rocking platform after 1, 2, 3, 4, 5, 6, 7, 8, or more days of culture.
- the entire process of stimulation, activation, and expansion takes place in a bioreactor comprising a rocking platform and an integrated magnet, as described above.
- Illustrative bioreactors include, but are not limited to, "The Wave".
- the cell stimulation methods of the present invention are carried out in a closed system, such as a bioreactor, that allows for perfusion of medium at varying rates, such as from about 0.1 ml/minute to about 10 ml/minute.
- the container of such a closed system comprises an outlet filter, an inlet filter, and a sampling port for sterile transfer to and from the closed system.
- the container of such a closed system comprises a syringe pump and control for sterile transfer to and from the closed system.
- a mechanism such as a load cell, for controlling media in-put and out-put by continuous monitoring of the weight of the bioreactor container.
- the system comprises a gas manifold.
- the bioreactor of the present invention comprises a CO2 gas mix rack that supplies a mixture of ambient air and CO2 to the bioreactor container and maintains the container at positive pressure.
- the bioreactor of the present invention comprises a variable heating element.
- media is allowed to enter the container starting on day 2, 3, 4, 5, or 6 at about 0.5 to 5.0 liters per day until the desired final volume is achieved. In one preferred embodiment, media enters the container at 2 liters per day starting at day 4, until the volume reaches 10 liters.
- perfusion of media can be initiated. In certain embodiments, perfusion of media through the system is initiated on about day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of culture. In one embodiment, perfusion is initiated when the volume is at about 0.1 liter to about 200 liters of media. In one particular embodiment, perfusion is initiated when the final volume is at 4, 5, 6, 7, 8, 9, 10, or 20 liters or higher volume.
- the rate of perfusion can be from about .5 ml/minute to about 10 ml/minute. In certain embodiments, the perfusion rate is about 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8.0 mis/minute.
- the cells such as T cells
- the methodologies of the present invention provide for the expansion of cells, such as T cells, to a concentration of about between 6 X 10 6 cell/ml and about 90 X 10 6 cells/ml in less than about two weeks.
- the methodologies herein provide for the expansion of T cells to a concentration of about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85 X 10 6 cells/ml and all concentrations therein.
- the cells reach a desired concentration, such as any of those listed above, by about day 5, 6, 7, 8, 9, 10, 11, or 12 of culture.
- the T cells expand by at least about 1.5 fold in about 24 hours from about day 4 to about day 12 of culture.
- the cells expand from a starting number of cells of about 100 X 10 6 to a total of about 500 X 10 9 cells in less than about two weeks. In further embodiments, the T cells expand from a starting number of cells of about 500 X 10 6 to a total of about 500 X 10 9 cells in less than about two weeks. In related embodiments, the cells expand from a starting number of about 100 - 500 X 10 6 to a total of about 200, 300, or 400 X 10 9 cells in less than about two weeks. Therapy
- a population of T cells is first contacted with antigen, and then subjected to a mixture of the invention comprising one or more of DCs, IL-15, IL-7, and IL-2.
- the antigen-specific T cells are induced by vaccination of a patient with a particular antigen, either alone or in conjunction with an adjuvant or pulsed on dendritic cells.
- Antigen-specific cells for use in expansion using the stimulation method of the invention may also be generated in vitro.
- Another aspect of the present invention provides a method for expanding antigen specific T cells, comprising contacting a population of T cells with an antigen for a time sufficient to induce activation of T cells specific to said antigen; contacting said population of antigen-specific T cells ex vivo with a mixture comprising one or more of DCs, IL-15, IL-7, and IL-2 under conditions and for time sufficient to induce proliferation of T cells specific to said antigen, thereby expanding antigen-specific T cells.
- the antigen is a tumor antigen.
- the antigen is pulsed on or expressed by an antigen-presenting cell.
- the population of T cells is contacted with said antigen ex vivo.
- the method comprises at least one round of peptide-MHC tetramer sorting of said antigen-specific T cells. In certain embodiments, the method of the present invention further comprises at least one round of peptide-MHC tetramer magnetic selection of said antigen-specific T cells.
- Another aspect of the present invention provides a method for the treatment of cancer comprising administering to a cancer patient antigen-specific T cells expanded according to the methods provided herein.
- the cells generated according to the present invention can also be used to treat autoimmune diseases.
- autoimmune disease include but are not limited to,
- AIDS Acquired Immunodeficiency Syndrome
- alopecia areata ankylosing spondylitis
- antiphospholipid syndrome autoimmune Addison's disease
- autoimmune hemolytic anemia autoimmune hepatitis
- AIED autoimmune inner ear disease
- ALPS autoimmune lymphoproliferative syndrome
- ATP autoimmune thrombocytopenic purpura
- Behcet's disease cardiomyopathy, celiac sprue-dermatitis hepetiformis
- chronic fatigue immune dysfunction syndrome CIDS
- chronic inflammatory demyelinating polyneuropathy CIPD
- cicatricial pemphigoid cold agglutinin disease
- crest syndrome Crohn's disease
- Degos' disease dermatomyositis- juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guilla
- the cells generated according to the present invention can also be used to treat inflammatory disorders.
- inflammatory disorders include but are not limited to, chronic and acute inflammatory disorders.
- inflammatory disorders include Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft vs. host disease, hemolytic anemias, osteoarthritis, sepsis, stroke, transplantation of tissue and organs, vasculitis, diabetic retinopathy and ventilator induced lung injury.
- the present invention also provides methods for preventing, inhibiting, or reducing the presence of a cancer or malignant cells in an animal, which comprise administering to an animal an anti-cancer effective amount of the anti-tumor cells of the invention.
- the cancers contemplated by the present invention against which the immune response is induced, or which is to be prevented, inhibited, or reduced in presence, may include but are not limited to melanoma, non-Hodgkin's lymphoma, Hodgkin's disease, leukemia, plasmocytoma, sarcoma, glioma, thymoma, breast cancer, prostate cancer, colorectal cancer, kidney cancer, renal cell carcinoma, pancreatic cancer, esophageal cancer, brain cancer, lung cancer, ovarian cancer, cervical cancer, multiple myeloma, hepatocellular carcinoma, nasopharyngeal carcinoma, ALL, AML, CML, CLL, and other neoplasms known in the art.
- compositions as described herein can be used to induce or enhance responsiveness to pathogenic organisms, such as viruses, (e.g., single stranded RNA viruses, single stranded DNA viruses, double-stranded DNA viruses, HIV, hepatitis A, B, and C virus, HSV, CMV, EBV, HPV), parasites (e.g., protozoan and metazoan pathogens such as Plasmodia species, Leishmania species, Schistosoma species, Trypanosoma species), bacteria (e.g., Mycobacteria, Salmonella, Streptococci, E. coli, Staphylococci), fungi (e.g., Candida species, Aspergillus species) and Pneumocystis carinii.
- viruses e.g., single stranded RNA viruses, single stranded DNA viruses, double-stranded DNA viruses, HIV, hepatitis A, B, and C virus, HSV, CMV, EBV
- the immune response induced in the animal by administering the subject compositions of the present invention may include cellular immune responses mediated by CD8+ T cells, capable of killing tumor and infected cells, and CD4+ T cell responses.
- Humoral immune responses mediated primarily by B cells that produce antibodies following activation by CD4+ T cells, may also be induced.
- a variety of techniques may be used for analyzing the type of immune responses induced by the compositions of the present invention, which are well described in the art; e.g., Coligan et al, Current Protocols in Immunology, John Wiley & Sons Inc., 1994.
- an immunologically effective amount When “an immunologically effective amount,” “an anti-tumor effective amount,” “a tumor-inhibiting effective amount,” or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient. It can generally be stated that a pharmaceutical composition comprising the subject cells of the invention, may be administered at a dosage to be determined during appropriate clinical trials. Cells of the invention may also be administered multiple times at these dosages. The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
- Cells of the invention can be administered in dosages and routes and at times to be determined in appropriate clinical trials. Cell compositions may be administered multiple times at dosages within these ranges.
- the cells of the invention may be combined with other methods.
- the cells of the invention for administration may be autologous, allogeniec or xenogenic to the patient undergoing therapy.
- the treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and/or chemokines (e.g., GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIPl-a, etc.) as described herein to enhance induction of the immune response.
- mitogens e.g., PHA
- lymphokines e.g., cytokines, and/or chemokines (e.g., GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIPl-a, etc.) as described herein
- the administration of the cells of the invention may be carried out in any convenient manner.
- the cells of the present invention may be administered to a patient subcutaneously, intradermally, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
- the cells of the invention are administered to a patient by intradermal or subcutaneous injection.
- the cells of the invention are administered by i.v. injection.
- the cells of the invention are injected directly into a tumor or lymph node.
- the cells of the invention can also be administered using any number of matrices.
- the present invention utilizes such matrices within the novel context of acting as an artificial lymphoid organ to support, maintain, or modulate the immune system, typically through modulation of T cells. Accordingly, the present invention can utilize those matrix compositions and formulations which have demonstrated utility in tissue engineering.
- the type of matrix that may be used in the compositions, devices and methods of the invention is virtually limitless and may include both biological and synthetic matrices.
- Matrices comprise features commonly associated with being biocompatible when administered to a mammalian host. Matrices may be formed from natural and/or synthetic materials. The matrices may be non-biodegradable in instances where it is desirable to leave permanent structures or removable structures in the body of an animal, such as an implant; or biodegradable. The matrices may take the form of sponges, implants, tubes, telfa pads, fibers, hollow fibers, lyophilized components, gels, powders, porous compositions, or nanoparticles.
- matrices can be designed to allow for sustained release of seeded cells or produced cytokine or other active agent.
- the matrix of the present invention is flexible and elastic, and may be described as a semisolid scaffold that is permeable to substances such as inorganic salts, aqueous fluids and dissolved gaseous agents including oxygen.
- a matrix is used herein as an example of a biocompatible substance.
- the current invention is not limited to matrices and thus, wherever the term matrix or matrices appears these terms should be read to include devices and other substances which allow for cellular retention or cellular traversal, are biocompatible, and are capable of allowing traversal of macromolecules either directly through the substance such that the substance itself is a semi-permeable membrane or used in conjunction with a particular semi-permeable substance.
- the cells of the invention can be used in vivo as an adjuvant as described in U. S. Pat. No. 6,464,973.
- the cells of the invention can be used as a vaccine to induce an immune response in vivo against an antigen of interest such as those described herein (e.g., tumor antigens, viral antigens, autoantigens, etc).
- an antigen of interest such as those described herein (e.g., tumor antigens, viral antigens, autoantigens, etc).
- the cells of the invention can be used to generate an immune response in vivo, either administered alone or in combination with other immune regulators and in combination with other known therapies.
- the invention provides for compositions and methods to expand antigen-specific T-cells.
- the antigen-specific T-cells can be expanded according to the invention which comprises contacting the T cell with one or more of DCs, IL-15, IL-7, and IL-2.
- the expanded T cells can be used to identify antigen-specific T-cell receptors
- TCRs TCRs
- epitopes derived therefrom TCRs from the expanded T cells can be cloned.
- the cloned TCRs present a promising tool for the development of specific adoptive T-cell therapies to treat a desired disease or disorder.
- the cloned TCRs can be used to generate peptides/antigens useful for vaccines.
- T cells In addition to their role in combating infections, T cells have also been implicated in the destruction of cancerous cells. Autoimmune disorders have also been linked to antigen-specific T cell attack against various parts of the body. One of the major problems hampering the understanding of and intervention on the mechanisms involved in these disorders is the difficulty in identifying T cells specific for the antigen to be studied.
- TCRs are closely related to antibody molecules in structure, and they are involved in antigen binding although, unlike antibodies, they do not recognize free antigen; instead, they bind antigen fragments which are bound and presented by antigen-presenting molecules.
- An important group of antigen-presenting molecules are the MHC class I and class II molecules that present antigenic peptides and protein fragments to T cells.
- Variability in the antigen binding site of a TCR is created in a fashion similar to the antigen binding site of antibodies, and also provides specificity for a vast number of different antigens.
- Diversity occurs in the complementarity determining regions (CDRs) in the N-terminal domains of the disulfide-linked alpha (a) and beta ( ⁇ ), or gamma ( ⁇ ) and delta ( ⁇ ), polypeptides of the TCR.
- CDR loops are clustered together to form an MHC- antigen-binding site analogous to the antigen-binding site of antibodies, although in TCRs, the various chains each contain two additional hypervariable loops as compared to antibodies.
- TCR diversity for specific antigens is also directly related to the MHC molecule on the APC's surface to which the antigen is bound and presented to the TCR.
- a peptide of the invention can be located within the MHC molecule of a dendritic cell in order to generate suitable T-cells.
- the MHC molecule is loaded with the peptide extracellularly by incubating cells at 37°C, 5% CO2 for 4 hours with varying concentrations of peptide, then washed once in serum-free RPMI.
- the antigen presenting cells are transfected with a polynucleotide encoding a fusion protein comprising the peptide connected to at least an MHC Class I molecule alpha chain by a flexible linker peptide.
- the fusion protein results in the peptide occupying the MHC Class I binding groove.
- Suitable MHC Class I molecules and costimulatory molecules are available from public databases. Further details of the synthesis of such a fusion molecule may be found in Mottez et al, J Exp Med. 1995 Feb. 1; 181(2):493-502, which is incorporated herein by reference.
- the advantage of expressing a fusion protein of the peptide and the MHC molecule is that a much higher concentration of peptide is displayed on the surface of the antigen presenting cells.
- the antigen- presenting cells display an MHC molecule of an allele for which the donor of the T-cells is HLA positive. In some embodiments, this is achieved by obtaining the antigen presenting cells from a first individual and the T-cells from a second individual wherein the first and second individuals have an HLA match.
- the antigen presenting cells and the T- cells are obtained from the same individual but the antigen presenting cells are transfected with polynucleotides encoding the MHC molecule of a similar HLA allele.
- the polynucleotide encodes a protein which encodes the MHC molecule connected to the peptide via a linker.
- HLA Class I alleles there are numerous HLA Class I alleles in humans and the MHC molecule displayed by the antigen presenting cells, may, in principle, be of any of these alleles. However, since the HLA-A*0201 allele is particularly prevalent, it is preferred that the MHC molecule be of this allele. However, any HLA-A2 allele is usable or other alleles such as HLA-A1, HLA- A3, HLA-A 11 and HLA-A24 may be used instead.
- a method for preparing T-cells suitable for delivery to a patient suffering from cancer comprises providing dendritic cells expressing an HLA molecule of a first HLA allele and locating a peptide in the binding groove of the HLA molecule.
- the peptide may or may not be a peptide of the invention.
- T-cells are then primed with the dendritic cells, the T-cells being obtained or obtainable from an individual who is HLA matched for a first HLA allele.
- the dendritic cells may either be obtained from a first donor individual and the T-cells from a second donor individual wherein the first and second donor individuals are HLA matched.
- the peptide is a cell type specific peptide, that is to say a peptide that is obtained from a protein which is only expressed, or is expressed at a much higher level (e.g. at least 10X higher concentration) in specific cells than in other cell types.
- the T-cells prepared in accordance with the invention are administered to patients in order to treat cancer in the patients.
- the T-cells of the invention are capable of being used for the treatment of many different types of cancer including leukemia, lymphomas such as non-Hodgkin lymphoma, multiple myeloma and the like.
- compositions comprising a T-cell of the invention and a pharmaceutically acceptable carrier, diluent or excipient, further details of which may be found in
- the HLA allele of the MHC molecule used to present the peptide to the T-cells is an HLA allele also expressed by the patient and therefore when the T-cells are administered to the patient, they recognize the peptide displayed on MHC molecules of that HLA allele.
- multiple sets (e.g. 2 or 3 sets) of T-cells are provided, each T-cell being specific for a different peptide.
- the T-cells are allogeneic, as described elsewhere herein, that is to say the HLA allele of the MHC molecule on which the peptide is displayed during preparation of the T-cells is an HLA allele which is not expressed in the donor individual from whom the T-cells are obtained.
- the peptides may all be from the same cell specific protein or may be from different proteins but specific for the same cell type.
- the peptide may or may not be a peptide of the invention.
- the multiple sets of T-cells are administered simultaneously but in other embodiments they are administered sequentially.
- T-cell receptor TCR
- TCR T-cell receptor
- the T-cell receptors of T-cells specific for a certain peptide when complexed with an MHC molecule of a particular allele are harvested and sequenced.
- a cDNA sequence encoding the T-cell receptor is then generated and which can be used to express the T-cell receptor recombinantly in a T-cell (e.g.
- the cDNA may be incorporated into a vector such as a viral vector (e.g. a retroviral vector), lentiviral vector, adenoviral vector or a vaccinia vector.
- a viral vector e.g. a retroviral vector
- lentiviral vector e.g. a lentiviral vector
- adenoviral vector e.g. a vaccinia vector
- a non- viral approach such as using naked DNA or lipoplexes and polyplexes or mRNA in order to transfect a T-cell.
- a T-cell which is "obtainable” from a donor individual includes a T-cell which is obtained recombinantly in the manner described elsewhere herein because the recombinantly expressed TCR is naturally produced.
- the T-cells are pre-selected, prior to transfection, to eliminate T-cells that would give rise to graft-versus-host disease.
- the T-cells are pre-selected such that the specificity of their endogenous TCRs is known. For instance, T-cells are selected which are reactive with glypican-3.
- the T-cells are obtained from the patient and thus are naturally tolerized for the patient. This approach can only be adopted where the T- cells of the patient are healthy.
- the T-cell receptor as a whole, is not recombinantly expressed but rather the regions of the T-cell receptor which are responsible for its binding specificity are incorporated into a structure which maintains the confirmation of these regions. More specifically, complementarity determining regions (CDRs) 1 to 3 of the T-cell receptor are sequenced and these sequences are maintained in the same conformation in the recombinant protein.
- CDRs complementarity determining regions
- the expanded T cells provide a source for cloning TCRs and epitopes/antigens associated therewith.
- the epitopes/antigens identified can be used to generate a vaccine.
- the vaccination antigens can be constructed by modifying a polypeptide (e.g. the target antigen) at specific amino acid positions identified by epitope mapping.
- the method of the invention includes identifying relevant positions for modification in the target antigen by epitope mapping, modifying the target antigen at relevant positions to produce variants, and including the variants in separate candidate preparations.
- Vaccination antigen polypeptides may be epitope mapped by a number of methods, including those disclosed in detail in WO00/26230 and WO01/83559.
- these methods comprises a database of epitope patterns (determined from an input of peptide sequences, known to bind specifically to anti-protein antibodies) and an algorithm to analyze 3-D structure of a given protein against the epitope partem database. This will determine the possible epitopes on that protein, and the preference of each amino acid in the protein sequence to be part of epitopes.
- methods are provided for identifying candidate MHC class
- candidate epitopes can be identified using a computer- implemented algorithm for candidate epitope identification.
- Such computer programs include, for example, the TEPITOPE program (see, e.g., Hammer et al, Adv. Immunol 66:67-100 (1997); Sturniolo et al, Nat. Biotechnol. 17:555-61 (1999); Manici et al, J Exp. Med. 189:871-76 (1999); de Lalla et al, J. Immunol. 163: 1725-29 (1999); Cochlovius et al, J. Immunol. 165:4731-41 (2000); the disclosures of which are incorporated by reference herein), as well as other computer implemented algorithms.
- the computer-implemented algorithm for candidate epitope identification can identify candidate epitopes in, for example, a single protein, in a very large protein, in a group of related proteins (e.g., homologs, orthologs, or polymorphic variants), in a mixtures of unrelated proteins, in proteins of a tissue or organ, or in a proteome of an organism.
- a group of related proteins e.g., homologs, orthologs, or polymorphic variants
- peptides or pools of peptides can be formed that correspond to the candidate epitope(s). For example, once a candidate epitope is identified, overlapping peptides can be prepared that span the candidate epitope, or portions thereof, to confirm binding of the epitope by the MHC class II molecule, and, as necessary, to refine the identification of that epitope. Alternatively, pools of peptides can be prepared including a plurality of candidate epitopes identified using a computer-implemented algorithm for candidate epitope identification.
- T cell epitope peptide A T cell epitope of the invention is a short peptide that can be derived from a protein antigen.
- Antigen presenting cells can directly bind antigen via surface MHC molecules and/or internalize antigen and process it into short fragments which are capable of binding MHC molecules.
- the specificity of peptide binding to the MHC depends on specific interactions between the peptide and the peptide-binding groove of the particular MHC molecule.
- Peptides which bind to MHC class I molecules are usually between 6 and 30, more usually between 7 and 20 amino or between 8 and 15 amino acids in length.
- the amino- terminal amine group of the peptide makes contact with an invariant site at one end of the peptide groove, and the carboxylate group at the carboxy terminus binds to an invariant site at the other end of the groove.
- such peptides typically have a hydrophobic or basic carboxy terminus and an absence of proline in the extreme amino terminus.
- the peptide is in an extended confirmation along the groove with further contacts between main-chain atoms and conserved amino acid side chains that line the groove. Variations in peptide length are accommodated by a kinking in the peptide backbone, often at proline or glycine residues.
- Peptides which bind to MHC class II molecules are usually at least 10 amino acids, for example about 13-18 amino acids in length, and can be much longer. These peptides lie in an extended confirmation along the MHC II peptide-binding groove which is open at both ends. The peptide is held in place mainly by main-chain atom contacts with conserved residues that line the peptide-binding groove.
- the peptide of the present invention may be made using chemical methods.
- peptides can be synthesized by solid phase techniques (Roberge J Y et al (1995) Science 269: 202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography. Automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.
- the peptide may alternatively be made by recombinant means or by cleavage from a longer polypeptide.
- the peptide may be obtained by cleavage from full- length glypican-3 protein.
- the composition of a peptide may be confirmed by amino acid analysis or sequencing.
- the peptides of the invention can be tested in an antigen presentation system which comprises antigen presenting cells and T cells.
- the antigen presentation system may be a murine splenocyte preparation, a preparation of human cells from tonsil or PBMC.
- the antigen presentation system may comprise a particular T cell line/clone and/or a particular antigen presenting cell type.
- T cell activation may be measured via T cell proliferation (for example using H-thymidine incorporation) or cytokine production.
- Activation of THl-type CD4+ T cells can, for example be detected via IFNy production which may be detected by standard techniques, such as an ELISPOT assay.
- the present invention provides improved methods for the use of a peptide library in analysis of T cells in samples including diagnostic, prognostic and immune monitoring methods. Furthermore the use of a peptide library in anti-tumor therapy are described elsewhere herein, including isolation of antigen-specific T cells capable of inactivation or elimination of undesirable target cells or isolation of specific T cells capable of regulation of other immune cells.
- the present invention also relates to MHC multimers comprising one or more tumor derived peptides.
- identification of particular antigenic peptides provides new opportunities for the development of diagnostic and therapeutic strategies against cancer.
- identification of novel T cell epitopes enable the production of MHC class I and class II multimers, tetramers and pentamers, useful as analytical tools delivering both increased sensitivity of immuno-monitoring.
- detection of antigen specific CTL in the periphery of individuals at risk of disease recurrence is a useful diagnostic tool.
- the invention provides an MHC multimer, tetramer or a pentamer comprising at least one of the MHC class I or II glypican-3 peptide epitopes as described herein.
- the invention also provides compositions and methods for identifying peptides useful for cancer therapy.
- Peptide sequences from a candidate protein predicted to bind to HLA-A*0201 can be identified by a computer algorithm. Peptides are selected for synthesis according to predicted affinity with a cut-off value of 500 nM or less, but also higher values may be chosen. Peptides are synthesized and binding to HLA-A*0201 can be confirmed using biochemical assays. Peptide binding is compared with the binding achieved with a pass/fail control peptide, designated 100%, and with a positive control peptide.
- Corresponding HLA-A*0201 -peptide multimers are also synthesized for peptides with a binding affinity above the pass/fail control peptide. These peptides are tested for the ability to generate a T cell line specifically reacting with the specific peptide-HLA-A*0201 complex.
- the cell line can be referred to as multimer-, tetramer-, or pentamer-positive T cells.
- Multimer positive cells indicate a high immunogenicity for the corresponding peptide.
- Additional responses can be measured to assess production of the cytokine interferon- ⁇ , degranulation and killing of target cells.
- the peptides of the invention can be administered directly to a patient as a vaccine.
- the peptides of the invention are immunogenic epitopes of specific proteins and are used in order to elicit a T-cell response to their respective proteins.
- the polypeptide of the invention is administered directly to a patient as a vaccine.
- a polypeptide comprising a peptide from a hematopoietic cell specific protein is administered to the patient in order to elicit a T-cell response to the protein.
- the T-cell response leads to death of hematopoietic cells, including the cancerous cells, but is specific to these cells and does not result in an immune response to other cell types.
- the peptides of the invention are used to generate T-cells obtained, or obtainable, from an allogeneic donor individual. This individual should preferably be HLA negative for an HLA allele of which the patient is HLA positive.
- T-cells are obtained from an individual who is negative for HLA-A*0201. It is generally preferred that the donor individual is otherwise HLA-identical to the patient.
- Antigen presenting cells APCs
- the T-cells of the donor individual are then primed with the APCs and the resulting cells are allowed to proliferate.
- the proliferated T-cells which are capable of binding the peptide of the invention when in complex with the HLA-A*0201 antigen are then enriched using artificial structures which comprise a plurality of peptide-MHC molecules (e.g. pentamers or tetramers).
- artificial structures which comprise a plurality of peptide-MHC molecules (e.g. pentamers or tetramers).
- the T cells specific for the particular peptide-HLA-A*0201 complex within the mixture of T cells have the capacity to bind to these structures when mixed with them.
- the T cells are subsequently mixed with magnetic beads with the capacity to bind the artificial structures.
- the artificial structures and the T cells bound to them are then removed from the remainder of the mixture by magnetic attraction of the beads.
- the antigen specific T cell can be administered to an animal as frequently as several times daily or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less.
- the frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.
- An antigen specific T cell may be co-administered with the various other compounds (cytokines, chemotherapeutic and/or antiviral drugs, among many others).
- the compound(s) may be administered an hour, a day, a week, a month, or even more, in advance of an antigen specific T cell, or any permutation thereof. Further, the compound(s) may be administered an hour, a day, a week, or even more, after administration of an antigen specific T cell, or any permutation thereof.
- the frequency and administration regimen will be readily apparent to the skilled artisan and will depend upon any number of factors such as, but not limited to, the type and severity of the disease being treated, the age and health status of the animal, the identity of the compound or compounds being administered, the route of administration of the various compounds and the antigen specific T cell, and the like.
- the administration of the composition of the invention may be for either "prophylactic” or "therapeutic" purpose.
- the composition of the present invention is provided in advance of any symptom, although in particular embodiments the vaccine is provided following the onset of one or more symptoms to prevent further symptoms from developing or to prevent present symptoms from becoming worse.
- the prophylactic administration of composition serves to prevent or ameliorate any subsequent infection or disease.
- the pharmaceutical composition is provided at or after the onset of a symptom of infection or disease.
- the present invention may be provided either prior to the anticipated exposure to a disease-causing agent or disease state or after the initiation of the infection or disease.
- an effective amount of the composition would be the amount that achieves this selected result of enhancing the immune response, and such an amount could be determined as a matter of routine by a person skilled in the art.
- an effective amount of for treating an immune system deficiency against cancer or pathogen could be that amount necessary to cause activation of the immune system, resulting in the development of an antigen specific immune response upon exposure to antigen.
- the term is also synonymous with "sufficient amount.”
- the effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular composition being administered, the size of the subject, and/or the severity of the disease or condition.
- One of ordinary skill in the art can empirically determine the effective amount of a particular composition of the present invention without necessitating undue experimentation.
- DC1 polarized dendritic cell
- HER2 + IBC patients with residual disease following neoadjuvant therapy received adjuvant HER2-pulsed DC1 vaccines. Thl immune responses of each patient were determined pre-DCl vaccination, 3 -months post-DC vaccination, and 6-months post- vaccination and were generated from patient PBMCs pulsed with six HER2 Class II peptides (SEQ ID NOS: 1-6) by measuring IFNy production via ELISPOT as described above.
- Pre-vaccination only one IBC patient produced an immune response, defined as >20 SFC/10 6 cells in an experimental well after subtracting unstimulated background. Compared with pre-vaccination results, all vaccinated IBC patients produced an immune response, defined as >2-fold increase in anti-HER2 IFN-Y pos Thl responses.
- the anti-HER2 Thl immune responses increase in both breadth (response repertoire) and depth (cumulative response).
- T cells are expanded to a level necessary for adoptive therapy and epitope mapping studies while maintaining antigen specificity and cellular function.
- HER2-specific Thl cells were generated by coculture with HER2 -peptide pulsed DCls and expanded using IL-2 alone or IL-2, IL7, and IL-15. Thl cells were subsequently expanded either by repeat HER2-peptide pulsed DC1 co-culture or via anti-CD3/CD28 stimulation. Fold expansion was defined as: (#T-cells post expansion / #T- cells pre expansion); specificity was measured by antigen specific IFNy production by ELISA.
- a subject when vaccinated against a protein antigen (for example, a tumor target antigen), blood can be removed from the subject after vaccination and collected.
- the collected blood contains dendritic cell precursors as well as low levels of T cells specific for the tumor target antigen.
- DC precursors and T cells are separated from each other.
- DC precursors can be loaded/pulsed with tumor target protein/antigen and then activated to DC1 status.
- the antigen-specific DCls can then be co-cultured with the T cells, and cytokines (IL-15, IL-7 and IL-2) are added to the co-culture in appropriate sequence. This cycle can be repeated weekly until T cells grow to sufficient numbers (e.g. 1X10 9 ).
- the T cells can then be supplied to the original subject, infusing them with a large quantity of T cells that their body could not produce naturally. This large army of antigen-specific T cells can have strong antitumor activity.
- DC precursors were obtained from HER2 breast cancer patients (DCIS) who were vaccinated with HER2 peptide-pulsed DC1 vaccines, as described previously. DC precursors were obtained via tandem leukapheresis/countercurrent centrifugal elutriation. DCs were incubated at 3x10 6 cells in 1ml Macrophage Serum-free Medium (SFM-Gibco Life Technologies, Carlsbad, CA) with GM-CSF 50 ng/ml (Berlex, Richmond, CA) at 37° C.
- SFM-Gibco Life Technologies Carlsbad, CA
- DCs were pulsed with a single HER2 peptide antigen (42-56, 98-114, 328-345, 776-790, 927- 941, 1166-1180 (SEQ ID NOS 1-6)); 20 ⁇ g/ml) 48-72hrs after the cells were initially plated. For maturation, DCs were further activated 6 hours later with IFN- ⁇ (1000 U/ml) and the following day with lipopolysaccharide (“LPS”) (lOng/ml). HER2 specific DCls were harvested 6 hours after LPS administration at the point of maximum IL-12 production.
- HER2 peptide antigen 42-56, 98-114, 328-345, 776-790, 927- 941, 1166-1180 (SEQ ID NOS 1-6)
- IFN- ⁇ 1000 U/ml
- LPS lipopolysaccharide
- HER2 specific DCls were harvested 6 hours after LPS administration at the point of maximum IL-12 production.
- Lymphocytes were also obtained from previously vaccinated (HER2pulsed
- CD4 + T-cells were purified by negative selection using Human CD4+ T Cell Enrichment Kit (Stemcell Technologies; Vancouver BC, Canada). CD4+ T-cells were resuspended at 2xl0 6 cells/ml in culture medium (ISOCOVE's Medium, 1% L-Glutamine, 1% Pen/Strep, 1% Sodium Pyruvate, 1% non-essential amino acids, Mediatech; Manassas, VA and 5% heat inactivated human AB serum)
- DCls were plated with CD4 + T-cells at a 1 : 10 ratio (2xl0 5 DCls/ml with 2xl0 6 CD4 + T-cells/ml) in 24-well plates and incubated at 37°C.
- Recombinant Human IL-7 (lOng/ml) and IL-15 (lOng/ml) were added 48-72hrs after co- culture. Twenty-four hours after adding IL-7 and IL-15, Recombinant Human IL-2 (5U/ml) was added.
- each well was pulsed with a single peptide antigen (20ug/ml), and was considered as immature DCs ("iDCs").
- each well was pulsed with a single peptide antigen (20 ⁇ g/ml) and matured to DCls as described above.
- the HER2 specific CD4 + T-cells were harvested.
- the T-cells were co-cultured with iDCs for ELISA testing. Interferon gamma production was measured by ELISA assay according to manufacturer's recommendations and protocols.
- the T-cells were also co-cultured with DCls and stimulated with IL-7/15 and IL-2 as described above. The cycle was repeated with co-culture of CD4 + T-cells with HER2 specific DCls a total of 4 times.
- AM pulse with antigen (2C ⁇ g/ml).
- iDCs Pulse with antigen (2C ⁇ g/ml) (given at the same time to future iDCs and DCls)
- DCls AM: pulse with antigen (2( ⁇ g/ml).
- Figure 3 and Figure 4 show a direct comparison between CD4 + T cells co- cultured with HER2-specific DCl's stimulated with IL-2 versus those stimulated with IL- 2/7/15 for two different patients who had received HER2-pulsed DC1 vaccination, respectively.
- immature DCs from the respective patients were pulsed with the following MHC class II peptides: peptide 42-56 (SEQ ID NO: 1), peptide 98-114 (SEQ ID NO: 2), peptide 328-345 (SEQ ID NO: 3), and peptide 776790 (SEQ ID NO: 4) and matured to DC s.
- the resulting HER2-pulsed DCl's were then co-cultured with CD4 + T cells and stimulated with IL-2 alone or with IL-2/7/15 as indicated.
- the red outline boxes indicate the specific peptide and stimulation protocol for which specificity is shown (greater than 2: 1 ratio of specific antigen: control antigen IFN- ⁇ production).
- Figures 5 and Figure 6 show specific to non-specific immune responses: Figure 5 shows a specific response following a first stimulation/expansion with HER2- specific DCl's and Figure 6 shows the subsequent loss of that specific response after the second stimulation/expansion with non-specific anti CD3/CD28.
- FIG. 5 shows the first stimulation of CD4 + T cells with HER2-specific DC Is resulted in multiple specific immune responses as shown by red outline boxes in Figure 5: peptide 42- 56, IL2/7/15 protocol; peptide 98-114, both protocols, peptide 328-345, both protocols, and peptide 776-790, both protocols.
- Figure 6 shows the second stimulation of the HER2-specific CD4 + T cells with a non-specific anti CD3/CD28 stimulus resulted in a four-fold expansion (side graph), but with a loss of specificity in three fourths of the peptide groups (only peptide 328-345 showed specificity after CD3/28 expansion).
- iDC's from patients were pulsed with the following MHC class II peptides: peptide 42-56 (SEQ ID NO: 1), peptide 98-114 (SEQ ID NO: 2), peptide 328-345 (SEQ ID NO: 3), and peptide 776-790 (SEQ ID NO: 4) and matured to DCl 's as described above.
- the resulting HER2-pulsed DCl 's were then co- cultured with CD4 + T cells and stimulated with IL-2 alone or with IL2/7/15 as indicated.
- Figure 7 and Figure 8 show non-specific immune response followed by specific immune responses:
- Figure 7 shows non-specific expansion of CD4 + T cells.
- Figure 8 shows failure to obtain specificity following subsequent stimulation with HER2-specific DCl 's.
- the first stimulation of CD4 + T cells with non-specific anti CD3/CD28 resulted in a 3.8 fold expansion ( Figure 7).
- the second stimulation of the non-specific CD4 + T cells with HER2-specific DCl 's failed to result in a specific immune response (Figure 8).
- iDC's from patients were pulsed with the following MHC class II peptides: peptide 42-56 (SEQ ID NO: 1), peptide 98-114 (SEQ ID NO: 2), peptide 328-345 (SEQ ID NO: 3), and peptide 776-790 (SEQ ID NO: 4) and matured to DCl 's as described above.
- the resulting HER2pulsed DCl 's were then co-cultured with CD4 + T cells and stimulated with IL-2 alone or with IL-2/7/15 as indicated.
- Figures 9A and 9B show in vitro primary/first expansion of HER2-specific Thl cells comparing CD4 + T cells co-cultured with HER2-specific DC1 's expanded with IL-2 versus those expanded with IL-2/7/15.
- iDC's were pulsed with the following MHC class II peptides: peptide 42-56 (SEQ ID NO: 1), peptide 98-114 (SEQ ID NO: 2), peptide 776-790 (SEQ ID NO: 4) and peptide 927-941 (SEQ ID NO: 5), and) and matured to DCl 's.
- Figure 9B The red outline boxes ( Figure 9B) indicate the specific peptide and stimulation protocol for which specificity is shown (greater than 2: 1 ratio of specific antigen: control antigen IFN- ⁇ production).
- Figure 9B shows specificity for the various
- the red outline boxes indicate the specific peptide and stimulation protocol for which specificity is shown (greater than 2: 1 ratio of specific antigen: control antigen IFN- ⁇ production) (i.e., DC1 restimulation of peptide 42-56-specific ThlCells and peptide 776-790 -specific Thl cells.
- Figure 12 for the first stimulation shows specificity only for peptide 776-790-specific Thl cells
- Figure 13 for the second stimulation shows an increase, specificity for peptide 42-56 in addition to peptide 776-790specific Thl cells
- Figure 14 for the third expansion shows specificity for all four peptide-specific Thl cells (peptide 42-56, peptide 98-114, peptide 776-790, and peptide 927-941)
- Figure 15 for the fourth expansion shows loss of specificity for one of the peptides (peptide 927-941) leaving three remaining HER2-specific peptides (peptide 42-56, peptide 98-114, and peptide 776-790).
- Figure 16 shows cumulative fold expansion of the four expansions shown in Figures 12-15 for all the HER2-specific Thl cells, with the last bar of each group (dots) showing cumulative fold expansion. Average cumulative fold expansion was over 100-fold.
- Co-culture with peptide specific DCls and IL-2, IL-7, and IL-15 stimulation may mimic the lymph node environment and be used to significantly expand any population of antigen specific Thl cells.
- peptide specific DCls and IL-2, IL-7, and IL-15 stimulation may mimic the lymph node environment and be used to significantly expand any population of antigen specific Thl cells.
- present embodiments related to T cell expansion are in no way limited to CD4 + t cells.
- the present embodiments provide methods for growing CART cells, cytotoxic T lymphocytes (CD8 + 's) as well as all other kinds of T cells.
- CD8 + 's cytotoxic T lymphocytes
- the results presented herein demonstrate that HER2 -pulsed DC1 vaccination of HER2 + IBC patients with residual disease following treatment with neoadjuvant chemotherapy boosts anti-HER2 Thl immune responses.
- the antiHER2 Thl immune response increases in both breadth (response repertoire) and depth (cumulative response). Adoptive transfer of HER2-specific Thl cells may serve a role in resurrecting the CD4+ Thl immune response. Repeated co-culture with HER2-peptide pulsed DC Is stimulated with IL- 2, IL-7, and IL-15 can result in significant expansion of highly specific anti-HER2 Thl cells.
- T cell subsets are essential steps to gain enough T cells to perform adoptive therapy, or to identify epitopes on target antigens for peptide-based vaccines.
- Expansion of T cells in principle is a simple process. However, in practice, many technical problems exist including poor levels of expansion, premature activation-induced cell death (apoptosis) or loss of antigen specificity and/or function.
- T cell expansion Part of the problem lies in the inability to replicate, in vitro, the environment inside the body where antigen-specific T cell expansion occurs, which is the lymph node.
- T cells include antigen-presenting dendritic cells, stromal cell such as epithelial cells.
- stromal cell such as epithelial cells.
- T cells are expanded to a level necessary for adoptive therapy and epitope mapping studies while maintaining antigen specificity and cellular function.
- Freshly elutriated myeloid monocytes were cultured in 6 well microplates (12x10 6 cells/well).
- Culture medium consisted of Serum Free Medium (SFM Invitrogen Carlsbad CA).
- the final concentration of added GMCSF was 50ng/ml and of IL4 is 1000 U/ml.
- Cells were cultured overnight at 37° C in 5% CC . In some batches, the cells were pulsed with the adequate peptides after 16-20 hr and cultured for additional 6-8 hr, after which lOOOU/ml IFN- ⁇ was added.
- Dendritic cells were matured with TLR agonist LPS (TLR 4, lOng/ml) or R848 (TLR8, l g ml).
- the DCs are activated with combinations of the cytokine IFN- ⁇ , or the TLR agonists bacterial LPS and/or R848. This should induced T cells that produce IFN- ⁇ .
- the DCs can be activated with combinations of ATP, bacterial LTA, LPS and prostaglandin E2 (PGE2). This can cause IL-23, IL-6 and IL- ⁇ to be amplified, leading an immune response dominated by IL-17 and IL-22-secreting Thl7 cells.
- Immune responses were generated from PBMCs pulsed with HER2 Class II peptides by measuring IFNy production via ELISPOT. Responses were evaluated on: (1) the overall anti-HER2 responsivity (responding to >1 peptide), (2) the number of reactive peptides (response repertoire), and (3) the cumulative response across the six HER2 peptides.
- Pre-vaccination Thl responses were compared with 3 -month and 6-month responses. Production of HER2-specific Thl cells
- HER2-specific Thl cells were generated by co-culture with HER2- peptide pulsed DCls and expanded using IL-2 alone or IL-2, IL-7, and IL-15. Thl cells were subsequently expanded either by repeat HER2-peptide pulsed DC1 co-culture or via anti- CD3/CD28 stimulation. Fold expansion was defined as: (#T-cells post expansion / #T-cells pre expansion); specificity was measured by antigen specific IFNy production by ELISA.
- Pre-vaccination only one IBC patient produced an immune response, defined as >20 SFC/2*10 5 cells in an experimental well after subtracting unstimulated background. Compared with pre-vaccination results, all vaccinated IBC patients produced an immune response, defined as >2-fold increase in anti-HER2 IFNyposThl responses.
- HER2 -peptide pulsed DC1 vs. anti-CD3/CD28.
- Re- stimulation of the Thl cells with HER2-specific DCls enhanced the specific Thl response; whereas non-specific stimulation with anti-CD3/CD28 resulted in an overall loss of HER2- peptide specificity (Figure 10B).
- the T cells can be expanded for a fourth round of stimulation. It was observed that the T cells continued to expand and maintain T cell function (Figure 12).
- the results presented herein demonstrate that HER2 -pulsed DC1 vaccination of HER2+ IBC patients with residual disease following treatment with neoadjuvant chemotherapy boosts anti-HER2 Thl immune responses.
- the anti-HER2 Thl immune responses increase in both breadth (response repertoire) and depth (cumulative response).
- Adoptive transfer of HER2-specific Thl cells may serve a role in resurrecting the CD4+ Thl immune response.
- Repeated co-culture with HER2-peptide pulsed DCls stimulated with IL- 2, IL-7, and IL-15 can result in significant expansion of highly specific anti-HER2 Thl cells.
- a subject when vaccinated against a protein antigen (for example, a tumor target antigen), blood can be removed from the subject after vaccination.
- the blood containing dendritic cell precursors as well as low levels of T cells specific for the tumor target antigen can be collected.
- DC precursors and T cells is separated from each other.
- DC precursors can be loaded with tumor target protein and then activated. They can then be co-cultured with the T cells, and cytokines (IL-15, IL-7 and IL-2) added to the co-culture in appropriate sequence. This cycle can be repeated weekly until T cells grow to sufficient numbers (e.g. 1X10 9 ).
- the T cells can then be supplied to the original subject, infusing them with a large quantity of T cells that their body could not produce naturally. This large army of antigen-specific T cells can have strong anti -tumor activity.
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