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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0005—Vertebrate antigens
  • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
• • A—HUMAN NECESSITIES
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  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
• • A—HUMAN NECESSITIES
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  • A61K40/00—Cellular immunotherapy
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  • A61K40/22—Immunosuppressive or immunotolerising
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/416—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
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• • A—HUMAN NECESSITIES
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0636—T lymphocytes

Definitions

• the present invention relates generally to the field of treatment of autoimmune disease, such as multiple sclerosis (MS). More particularly, it concerns a CD8 + T cell vaccine prepared by using immunogenic fragments of Myelin Basic Protein (MBP).
• MBP Myelin Basic Protein
• MS Multiple sclerosis
• CNS central nervous system
• EAE experimental autoimmune encephalomyelitis
• CD4 + T cells recognizing MBP have been found to induce CNS pathology characterized by extensive inflammation and mild demyelmation (Zamvil et al, Nature 1985; 317: 355).
• CD8 + T cells recognizing short pep tides of MBP can induce EAE with distinct CNS pathology.
• CD8 T cells are cytotoxic toward target cells, recognize endogenously processed MBP and induced severe EAE upon adoptive transfer (Huseby et al, J. Exp. Med. 2001; 194: 669; Steinman, J. Exp. Med., 2001; 194: 27).
• the present invention is directed to the isolation of CD8 + cytotoxic T cells that recognize multiple sclerosis related antigens including but not limited to antigens such as Myelin Basic Protein (MBP), and/or fragments thereof.
• MBP Myelin Basic Protein
• the MBP fragments may be peptides that comprise 8 or more amino acids of any of the sequences set forth in SEQ ID NOS: 1-4.
• the MS associated antigen may also be proteolipid protein (PLP)or myelin oligodendrocyte glycoprotein (MOG).
• PBP proteolipid protein
• MOG myelin oligodendrocyte glycoprotein
• the present invention is directed to fragments of MBP, which bind HLA-A2 and HLA-A24 receptors with high affinity.
• the fragments of MBP encompassed by the present invention are those set out as SEQ ID NOS: 1-4.
• the fragments may also be homologs having conservative amino acids at one or more position of the fragment but which still bind to the HCA-A2 and HCA-A4 receptors.
• the present invention describes a method for preparing a vaccine useful in the treatment or prevention of MS comprising obtaining a population of peripheral blood mononuclear cells (PBMCs) comprising T cells from a patient to be treated; enriching said population for CD8 + T cells preferably by reducing or depleting the number of CD4 + cells in the population; and incubating said CD8 T cell enriched population with one or more peptides corresponding to MBP-fragments capable of binding to HLA-A2 and HLA-A24 so as to increase the number of CD8 + T cell clones in the population specific for said MBP polypeptides.
• the population of CD8 + T cells specifically responsive to the MBP's may be further expanded by, for example, alternately stimulating said cells with the corresponding MBP peptides and a mitogen, for example, in the presence of antigen presenting cells (APCs).
• APCs antigen presenting cells
• Yet another embodiment of the present invention discloses methods of testing CD8 T cell vaccines for their cytotoxicity against autologous cells primed with MBP-fragments, including but not limited to peptides having an amino acid sequence corresponding to SEQ ID NOS: 1, 2, 3 or 4.
• the present invention is concerned with a method of treating MS by administering to a patient in need of the treatment with autologous CD8 + T cells responsive to the MBP fragments and preferably capable of binding to HLA-A2 and HLA-A24.
• the present invention provides a method for producing an autologous CD8 + T cell vaccine by means of isolating or generating CD 8 + T cells that have cytotoxic activity against MPB-reactive CD8 + T cells of the patient. Under these methods, an autologus T cell memory clones are selected for their reactivity against MBP-reactive CD8 + T cells of the patient.
• the invention is also directed to T cell vaccines such as those described in copending U.S. Patent Application Nos. 09/952,532; PCT/US02/28874; 60/402,521; PCT/US03/24548 (all of which are incorporated herein by reference in their entirety), which are further modified by the addition of the CD8 + T cells reactive to MS-related antigens peptide produced according to the methods of the present invention.
• Figure 1A and IB illustrates percentage of CD4 T cells and CD8 + T cells before and after T cell depletion.
• PBMC derived from an MS patient (MS-4) were analyzed for percentage of CD4 + T cells and CD8 + T cells before (left panel) and after (right panel) magnetic bead- depletion of CD4 + T cells.
• the rate of CD4 + T cell depletion was always greater than 98% in all 30 experiments.
• the average percentage of CD8 + T cells in PBMC depleted for CD4 + T cells was 72 ⁇ 8%.
• FIG. 2 shows the estimated precursor frequency of CD8 T cells reactive to MBP- derived peptides in patients with MS and normal subjects (NS).
• the CD8 + T cell frequency analysis was performed by the split-well method in which responder PBMC fractions pre- depleted for CD4 + T cells were cultured with irradiated autologous PBMC that were not fractionated in the presence of the indicated MBP-derived peptides, respectively.
• a synthetic peptide corresponding to an irnmunod ⁇ minant epitope of tetanus toxoid was used as a control.
• Each open circle represents the frequency of CD8 + T cells in each individual.
• the data are expressed as the estimated frequency of CD8 + T cells recognizing the MBP-derived peptides in CD4-depleted fractions of PBMC.
• FIG. 3 shows phenotypic expression of CD8 + T cells reactive to MBP-derived peptides.
• CD8 + T cell lines El 1, D10, B9 and F12
• FIG 4 shows analysis of MHC Class I tetramer for binding to cloned CD8 + T cell lines by flow cytometry.
• Two A2-restricted CD8 T cell lines that recognized MBPj ⁇ - ⁇ 9 peptide (El 1) and MBP 87- 5 peptide (D10) were analyzed by flow cytometry using an HLA-A2- MBPi i j. 119 tetramer.
• the open profiles represent staining of T cells with a PE-conjugated control antibody.
• the solid profiles indicate staining of T cells with the tetramer in the same representative experiment.
• Figure 5 illustrates the cytokme profile of CD8 + T cells recognizing MBP-derived peptides.
• the T cell lines were challenged with the corresponding peptide, respectively, and the supernatants were tested after 48 hours for concentrations of the indicated cytokines.
• the bars indicate the mean concentration (pg/ml) ⁇ SEM.
• the detection limit of the assays for all cytokines was less than 25 pg/ml.
• Figure 6A and 6B shows cytotoxic activity of CD8 + T cell lines recognizing MBP- derived peptides against autologous target cells.
• CD8 + T cell lines reactive to MBP-derived peptides El 1 for MBP 111-119 , D10 for MBP 87-95 , B9 for MBP 134-142 and F12 for MBP 1 -22 were examined for cytotoxicity in LDH-release assays.
• Panel A CD8 + T cell lines were tested for cytotoxic activity against autologous target cells pulsed with corresponding peptides at the indicated effector (CD8 + T cells) to target (autologous EBN-transformed B cells) ratio.
• a synthetic 9-mer peptide corresponding to a unrelated TCR CDR3 sequence (STRQGPQET) (SEQ ID NO: 5) was used as a control.
• Panel B The same CD8 + T cell lines were analyzed for cytotoxicity against autologous target cells pulsed with different peptides of MBP. The same autologous target cells pulsed with the irrelevant TCR peptide served as a control peptide. The effector to target ratio was 10.
• Figure 7 illustrates MHC restriction of CD8 + cytotoxic T cell lines.
• the selected CD8 + cytotoxic T cell lines recognizing MBP-derived peptides were tested for specific cytotoxicity against autologous target cells in the presence and absence of two monoclonal antibodies to MHC class I (W6/32) and class II (HB55) used at a concentration of 20 ⁇ g/ml.
• the effector to target ratio was 10 for all experiments.
• Data are expressed as % specific cytotoxicity. The procedure used is the same as that described in the Figure 5 legend.
• Figure 8 shows cytotoxic activity of CD8 + T cell lines reactive to MBP-derived peptides against COS cells transfected with human MBP and HLA-A2 genes.
• the selected CD8 + cytotoxic T cell lines were tested for cytotoxic activity in LDH- release assays using COS cells transfected with human MBP and HLA-A2 genes. The effector to target ratio was 10. Non-transfected COS cells were used as a control.
• CD8 + cytotoxic T cells recognizing MBP-derived peptides directly contribute to severe CNS demyelination in EAE presumably through induction of injury of oligodendrocytes (Huseby et al, J. Exp. Med. 2001; 194:669).
• the distinct role of these CD8 cytotoxic T cells is of particular relevance to MS where demyelination represents the most significant CNS pathology associated with neurologic deficits.
• This invention discloses an approach for identifying CD8 + T cells that are reactive to MS associated antigens, preferably MBP and/or fragments thereof and the effective generation of CD8 T cell lines, which are useful in the treatment of MS, in monitoring the progression of the disease and the monitoring of therapeutic response to treating of the disease.
• the methods of the present invention are also useful for the diagnosis and monitoring of the progression of MS.
• the approach includes obtaining PBMCs from an MS patient in need of treatment; pre- depleting the population of PBMCs of CD4 + T cells resulting in a population of PBMCs enriched for CD8 + cells with MS associated antigens so as to increase the number of CD8 + T cells in the population and optionally repeating the stimulation cycle in the presence or absence of antigen presenting cells.
• T cell lines using alternating cycles of stimulation are described in U.S. Patent Application No. 09/952,532 and International Application Nos. PCT/US02/02887 (Published as WO 03/024393) and PCT/US03/24548 (Published as WO 04/15070 ), the contents of which are incorporated herein by reference.
• Methods of selecting CD8 + T cell lines with reactivity to particular antigens, including immunogemc fragments of MBP are also included.
• immunogenic means the ability to induce or sustain a T cell response including, but not limited to, a proliferative response, or for example, to stimulate the production of cotoxins by T cells.
• the methods of identifying MBP immunogenic fragments are also disclosed and amino acid sequences for four MBP immunogenic fragments with high binding affinity to HLA-A2 and HLA-A24 are provided.
• the estimated frequency of CD8 cytotoxic T cells recognizing the identified MHC class I peptides of MBP is in the range of 3.4 to 5.4 xlO "7 in PBMC derived from MS patients and 1.1 — 2.0 x10 " in the control group.
• the observed frequency of CD8 + cytotoxic T cells recognizing the identified regions of MBP in PBMC is relatively lower than that of CD4 + T cells recognizing immunodominant peptides of MBP in MS patients (1 - 2 xlO "6 in PBMC) under, the same experimental condition (Ota et al., . Nature 1990; 346: 183; Zhang et al, J. Exp. Med.
• CD8 + cytotoxic MBP- reactive T cells can also be detected in healthy individuals (Ota et al., Nature 1990; 346: 183; Martin et al, J. Exp. Med. 173: 19; Zhang et al, J. Exp. Med. 1994; 179: 973; Tejada-Simon et al, Inern. Immunol. 2000;. 12: 1641).
• the estimated T cell frequency is significantly . higher in MS patients than that in controls.
• CD4 + MBP-reactive T cells The differences appear to be more significant than those for CD4 + MBP-reactive T cells seen in MS patients and controls. It should also be noted that unlike CD4 + MBP-reactive T cells that are naive T cells expressing both CD45RA and CD45RO (Muraro et al, J. Immunol. 2000; 164: 5474), these CD8 + cytotoxic T cells identified here belong to antigen-experienced memory T cell subset expressing CD45RO but not CD45RA phenotype. Secondly, the finding suggests that these CD8 + cytotoxic T cells recognizing MBP- derived peptides may undergo in vivo activation in MS patients.
• MBP-reactive T cells can be activated by a variety of microbial antigens through the mechanism known as molecular mimicry (Oldstone, Curr. Topics Microbiol. Immunol. 1989; 145: 127; Oldstone, FASEB J. 1998; 12: 1255; Hafler, J. Clin. Invest. 1999; 104: 527; Tejada-Simon et al, Annals of Neurology 2003; 53: 189).
• the actual precursor frequency of CD8 + cytotoxic T cells may be under-estimated using the method.
• a number of studies on the precursor frequency analysis of CD4 + specific T cells have provided some indications. It has been reported that the frequency of CD4 + MBP-reactive T cells in MS is in the range of 4 xlO "5 by ELISPOT based on ex vivo secretion of cytokines in response to antigenic stimulation, which is higher than that measured in 1-2 xlO "6 by the split- well method employed here.
• ELISPOT is not applicable to quantitative detection of CD8 + T cells because the source of ex vivo secretion of ⁇ -IFN could not be distinguished between CD8 + T cells and CD4 + T cells, even though they were irradiated.
• further characterization has confirmed that these CD8 + T cells recognizing MBP-derived peptides are cytotoxic in nature. They recognize and are cytotoxic toward both autologous target cells pulsed with the MBP peptides and_endogenously processed MBP in the context of MHC class I molecules as evidenced in a series of experiments involving COS cells doubly transfected with HLA-A2 and human MBP genes.
• CD8 + cytotoxic T cells reactive to MBP-derived peptides as described here are reminiscent of CD8 + T cells of similar functional properties in EAE, which are able to induce extensive CNS demyelination potentially through specific recognition and cytotoxic activity toward oligodendrocytes (Huseby et al, J. Exp. Med. 2001; 194: 669).
• TEPITOPE (Vaccinome website) an application that allows the identification of HLA class I ligand binding epitopes (Schroers et al., Cancer Res. 2002; 62: 2600; Engelhard Annu. Rev. Immunol. 1994; 12: 181; Manici et al., J. Exp. Med. 1999; 189: 871), was used to screen amino acid sequence of human myelin basic protein (MBP) for fragments capable of binding to HLA-A2 and HLA-A24. Two fragments with predicted HLA-A2 binding sequences (1% threshold) and two fragments with predicted HLA-A24 binding sequences (1% threshold) were identified. The amino acid sequences of the fragments are as follows:
• Peptides with SEQ ID NOS: 1-4 were then synthesized using the Mayfield method and were purified using HPLC (MD Anderson Cancer Center Peptide Core, Houston, TX). The purity of the peptides was greater than 90%.
• Example 2 Precursor frequency analysis of CD8 + T cells recognizing MBP-derived peptides in MS patients and healthy controls
• Fifteen patients with relapsing-remitting or secondary progressive MS (Poser et al., Ann. Neurol., 13(3):227-31 , 1983) were included in the study. Patients had not been treated with irnmunosuppressive or immunomodulatory drugs (e.g., Azathioprine, Cyclophosphamide. Beta Jnterferons or Glatiramer Acetate) at least 3 months before entering the study.
• the protocol was approved by the Institutional Review Board at Baylor College of Medicine. A group of 15 healthy subjects matched for age and sex with the MS group was included as controls.
• the clinical characteristic/demographic data and HLA-A2 and -A24 genotypes of MS patients and control subjects are shown in Table 1.
• the precursor frequency of T cells recognizing the selected peptides of MBP with SEQ ID NOS: 1-4 was estimated in MS patients and controls using the split-well method (Ota et al., Nature 1990; 346: 183; Zhang et al., J. Exp. Med. 1994; 179: 973).
• the initial attempts to detect CD8 + T cell responses to the MBP peptides in unfractionated peripheral blood mononuclear cells yielded low frequencies of specific T cell isolates of mixed CD4 + and CD8 + phenotypes.
• PBMC Peripheral blood mononuclear cells
• CD4 + T cells were pre-depleted using magnetic beads coupled with an anti-CD4 antibody (Dynal ASA, Oslo, Norway). Briefly, PBMC were incubated with magnetic beads coated with the antibody at a bead to cell ratio of 10 for 30 min with gentle shaking. Unbound PBMC fractions were collected by magnetic separation. The depletion rate for CD4 + T cells was greater than 98% in all cases. The resulting CD4-depleted fractions typically contained 72 ⁇ 8% CD8 + T cells as determined by flow cytometric analysis. A representative experiment is shown in Figure 1.
• PBMC fractions were then seeded in 96-well U-bottomed microtiter plates at a density of 50,000 cells/well together with 10 5 of autologous unfractionated PBMC that were irradiated to provide "helper" function of CD4 + T cells while they themselves were unable to proliferate in response to the antigens.
• Peptides with SEQ ID NOS: 1 -4 were added at a final concentration of 20 ⁇ g/ml to cultures. A total of 32 wells were set for each peptide.
• a synthetic peptide corresponding to an immunodominant peptide of tetanus toxoid was included in the precursor frequency analysis as a negative control.
• a well/culture was defined as specific for the peptide when the CPM were greater than 1,500 and exceeded the reference CPM (in the absence of the peptide) by at least three times.
• the frequency of specific CD8 T cells was then estimated by dividing the number of positive wells by the total number of CD4-depleted PBMC seeded in the initial culture.
• a panel of 39 CD8 + T cell lines generated in Example 2 were characterized for phenotypic expression, cytokine profile and specific cytotoxic activity toward autologous target cells.
• the panel included 25 T cell lines from MS patients and 14 T cell lines from healthy controls and was representative for reactivity to all four MBP-derived peptides (Table 2).
• CD8 + T cell lines express TCR ⁇ /CD8 (>95% on average) but not CD4 ( ⁇ 5%) and CD45RO. but not CD45RA, regardless of their reactivity to the various . MBP-derived peptides.
• the findings indicate that the selected T cell lines belong to the CD8 + memory T cell subset.
• the cytokine profile of the resulting CD8 T cell lines was analyzed to determine whether they belonged to a Thl or a Th2 subset.
• the cytokine profile was determined quantitatively using ELISA kits (PharMingen, San Diego, CA). Microtiter plates (96-wells, NUNC Maxisorp) were coated overnight at 4 ° C with 1 ⁇ g/well of a purified mouse capturing monoclonal antibody to human cytokine (IL-4, IL-10. TNF- ⁇ , ⁇ -IFN) (PharMingen).
• Optical density was measured at 450 nm using an ELISA reader (Bio-Rad Laboratories, Hercules, CA) and cytokine concentrations were quantitated by Microplate computer software (Bio-Rad) using a double eight-point standard curve.
• the selected CD8 + T cell lines recognizing the MBP-derived peptides predominantly produced TNF- ⁇ and LFN- ⁇ but not IL-4 and LL-10, thus belonging to a Thl phenotype. No significant quantitative differences between the MS-derived T cell lines and the T cell lines derived from the control subjects could be discerned.
• T cell lines (El 1, D10, B9 and F12) were selected for their recognition of the four MBP peptides and further cloned by limiting dilution. Briefly, T cells were plated out at one cell per well in 96-well U-bottomed plates under limiting dilution conditions in the presence of irradiated PBMC (100,000 cells per well) and phytohemaglutinin- protein (PHA-P) at 2 ⁇ g/ml. Cells were cultured in IL-2 containing medium for 10-12 days with medium change every 3 to 4 days. Growth positive wells were confirmed for the phenotypic expression of CD 8 and for reactivity to the corresponding peptides. The obtained T cell clones were further expanded by alternate stimulation with the corresponding MBP peptides and PHA-P in the presence of autologous APC.
• PBMC 100,000 cells per well
• PHA-P phytohemaglutinin- protein
• CD8 + T cell lines recognized MBP 11 19 peptide (El 1) (SEQ ID NO: 2) and MBP 87-95 peptide (D10) (SEQ LD NO: 1), respectively, in the context of HLA-A2 while two other CD8 + T cell lines were A24 restricted and reacted with MBP 134-1 2 peptide (B9) (SEQ ID NO: 3) and MBP 14 . 22 peptide (F12) (SEQ LD NO: 4).
• Figure 3 illustrates the representative phenotypic expression of four cloned T cell lines described above. [0045] Specific binding of an HLA-A2 tetramer to selected CD8 + T cell lines was then examined.
• HLA-A2-MBP 111-119 tetramer was obtained from Immunomics (San Diego, CA). As shown in Figure 4, the HLA-A2/MBP 1 ⁇ 1 - ⁇ i 9 tetramer exhibited greater than 90% specific binding to a CD8 + T cell line (El 1) recognizing peptide MBP 111-119 but not to an A2 + CD8 + T cell line (D10) recognizing peptide MBP 87-95
• All 39 selected CD8 + T cell lines were analyzed for cytotoxic activity toward autologous target cells.
• a panel of autologous B cell lines was generated from patients and controls using EBV transformation on a procedure described previously (Zhang et al, J. Neuroimmunol. 1989; 23: 249; Tejada-Simon et al, Immunology 2002; 107:403).
• the generated cell lines were pulsed with corresponding peptides of BMP and used as autologous target cells. Pulsing of B cells was carried out by incubating cells with MBP-derived peptides or a control T cell receptor peptide (40 ⁇ g/ml), respectively, for 2 hrs followed by washing to remove free peptides.
• Cytotoxicity test was performed using a lactate dehydrogenase (LDH)-release assay (Promega Madison, WI). LDH release was measured in an enzymatic assay according to manufacturer's instruction. Briefly, CD8 + T cells (50,000 effector cells/well) were inqubated with autologous cells at an effector to target ratio of 10 and centrifuged once at 250 x g. Unpulsed autologous B cells and non-transfectants were used as controls. RPMI1640 without phenol red was used throughout the assay to avoid background absorbance. After incubation at 37°C and 5% CO 2 for 4 hr, the plates were centrifuged again.
• LDH lactate dehydrogenase
• %cytotoxicity (experimental release - spontaneous release) / (maximum release — spontaneous release) x 100.
• the stable transfectants were selected using selective medium containing Zeocin at 400 ⁇ g/ml (Invitrogen, San Diego, CA). Stable expression of MBP and HLA-A2 were evaluated by incubating the cells with conjugated monoclonal antibodies to MBP (Sigma, St. Louise, MO) or HLA-A2 (BD Pharmingen, San Diego, CA) and analyzed subsequently by flow cytometry. [0051] Selected MS-derived CD8 + T cell lines that were cytotoxic toward peptide-pulsed autologous target cells were analyzed in cytotoxicity experiments using transfected COS cells.
• the two HLA-A2 + CD8 + T cell lines (El l and D10) displayed specific cytotoxicity toward A2- MBP transfected COS cells but not non-transfectants while the other CD8 + cytotoxic T cell lines of A24- restriction (B9 and F12) had no cytotoxic effect on transfected COS cells.
• Results on four representative CD8 + T cell lines are shown in Figure 8, indicating specific recognition and cytotoxicity towards target cells expressing both human MBP and HLA-A2.
• RR relapsing-remitting MS
• SP secondary progressive MS
• EDSS expanded disability scale score.
• HLA typing was determined by RT-PCR using specific primers for A*0201 and A*2402.

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