Patent application title: IMMUNOSTIMULATORY COMPOSITIONS, PARTICLES, AND USES RELATED THERETO
Inventors:
Periasamy Selvaraj (Atlanta, GA, US)
Assignees:
EMORY UNIVERSITY
IPC8 Class: AA61K39385FI
USPC Class:
424450
Class name: Drug, bio-affecting and body treating compositions preparations characterized by special physical form liposomes
Publication date: 2015-03-12
Patent application number: 20150071987
Abstract:
In some embodiments, described herein is a method of tumor treatment or
tumor vaccination. The method generally comprises applying to a human
being in need thereof a tumor therapeutic composition or tumor vaccine
defined herein. The tumor therapeutic composition or tumor vaccine can be
produced by protein transfer of glycosyl-phosphatidylinositol
(GPI)-anchored immunostimulatory or costimulatory molecules.Claims:
1. A non-naturally occurring particle comprising, a lipid membrane; a
B7-1 or B7-2 molecule anchored to the lipid membrane on the exterior of
the particle; and an antigen molecule anchored to the lipid membrane on
the exterior of the particle.
2. The particle of claim 1 further comprising an adjuvant molecule anchored to the lipid membrane on the exterior of the particle wherein the adjuvant molecule and antigen molecule are not the same molecule.
3. The particle of claim 2, wherein the adjuvant molecule is selected from molecules comprising IL-2, IL-12, ICAM1 GM-CSF, flagellin, unmethylated, CpG oligonucleotide, lipopolysaccharides, lipid A, and heat stable antigen (HSA).
4. The particle of claim 1, wherein the lipid membrane is a phospholipid monolayer or phospholipid bilayer.
5. The particle of claim 1, wherein the particle is a cell, allogeneic or autologous cancer cell or its membrane fragments or vesicles, liposome, virosome, micelle, polymer, or virus like particle.
6. The particle of claim 1, wherein the B7-1 molecule is anchored to the lipid membrane on the exterior of the particle through a conjugated glycosyl-phosphatidylinositol, phospholipid, glycolipid, triglyceride, saturated or unsaturated fatty acid, or other lipophilic molecule.
7. The particle of claim 1, wherein the antigen molecule is anchored to the lipid membrane on the exterior of the particle through a conjugated glycosyl-phosphatidylinositol, phospholipid, glycolipid, triglyceride, saturated or unsaturated fatty acid, or other lipophilic molecule.
8. The particle of claim 1, wherein the adjuvant molecule is anchored to the lipid membrane on the exterior of the particle through a conjugated glycosyl-phosphatidylinositol, phospholipid, glycolipid, triglyceride, saturated or unsaturated fatty acid, or other lipophilic molecule.
9. The particle of claim 1, wherein antigen is a cancer marker molecule selected from HER-2, MUC-1, mucin antigens TF, Tn, STn, glycolipid globo H antigen, prostatic acid phosphatase (PAP), prostate-specific antigen, prostate-specific membrane antigen, early prostate cancer antigen-2 (EPCA-2), bcl-2, G-protein coupled estrogen receptor 1, CA15-3, CA19-9, CA 72-4, CA-125, carcinoembryonic antigen, CD20, CD31, CD34, PTPRC (CD45), CD99, CD117, melanoma-associated antigen (TA-90), peripheral myelin protein 22 (PMP22), epithelial membrane proteins (EMP-1, -2, and -3), HMB-45 antigen, MART-1 (Melan-A), S100A1, and S100B.
10. The particle of claim 1, wherein the antigen is contained in the interior of the particle.
11. The particle of claim 1, wherein the B7-1 molecule is a B7-1 and heat stable antigen (HSA) hybrid chimera.
12. The particle of claim 1, wherein the antigen is HER-2 and the adjuvant is flagellin or GM-CSF.
13. The particle of claim 1, wherein the antigen is HER-2 and the B7-1 molecule is a B7-1 and heat stable antigen (HSA) hybrid chimera.
14. The particle of claim 1, wherein the antigen is HER-2, the adjuvant is flagellin or GM-CSF, the B7-1 molecule is a B7-1 and heat stable antigen (HSA) hybrid chimera.
15. The particle of claim 1, wherein the antigen is HER-2 and the adjuvant is IL-12.
16. The particle of claim 1, wherein the antigen is HER-2, the adjuvant is IL-12, the B7-1 molecule is a B7-1 and heat stable antigen (HSA) hybrid chimera.
17. The particle of claim 1, wherein the antigen is PSA or PAP and the adjuvant is flagellin or GM-CSF.
18. The particle of claim 1, wherein the antigen is PSA or PAP and the B7-1 molecule is a B7-1 and heat stable antigen (HSA) hybrid chimera.
19. The particle of claim 1, wherein the antigen is PSA or PAP, the adjuvant is flagellin, the B7-1 molecule is a B7-1 and heat stable antigen (HSA) hybrid chimera.
20. The particle of claim 1, wherein the antigen is PSA or PAP and the adjuvant is IL-12.
21-50. (canceled)
Description:
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application No. 61/594,754 filed Feb. 3, 2012 hereby incorporated by reference in its entirety.
BACKGROUND
[0003] Provenge® is a recently FDA-approved autologous cellular immunotherapy treatment. Peripheral blood leukocytes of a subject are harvested via leukapheresis. These enriched monocytes are incubated with prostatic acid phosphatase (PAP) conjugated to cytokine granulocyte macrophage colony stimulating factor (GM-CSF). GM-CSF is thought to direct the target antigen to receptors on DC precursors, which then present PAP on their cell surface in a context sufficient to activate T cells for the cells that express PAP. Activated, PAP presenting DCs are administered to the subject to elicit an immune response retarding cancer growth. This strategy requires isolation and expansion of cells of the subject, and typically treatment does not entirely clear the subject of cancer or tumors. Thus, there is a need to identify improved methods.
[0004] B7-1 (also known as CD80) is a T cell costimulatory molecule that can be anchored in to autologous cancer cells to stimulate immune responses. McHugh et al., report the construction, purification and functional reconstitution of a glycolipid anchored form of B7-1 (CD80) on tumor cell membranes. Proc. Natl. Acad. Sci. USA 1995; 92:8059-8063. See also U.S. Pat. No. 6,491,925. Glycosyl phosphatidylinositol anchored B7-1 (GPI-B7-1) molecules have been incorporated onto tumor cells and isolated tumor cell membranes to provide costimulation for allogenic T cell proliferation. See Nagarajan & Selvaraj, Vaccine, 2006, 24(13):2264-74, U.S. Published Patent Application No. US 2007/0243159, Bozeman et al., Front Biosci. 2010; 15:309-320. Bumgarner et al., report surface engineering of microparticles by novel protein transfer for targeted antigen/drug delivery. J Control Release. 2009; 137:90-97.
[0005] Cubas et al., report virus-like particle (VLP) lymphatic trafficking and immune response generation after immunization by different routes. J Immunotherapy, 2009, 32(2):118-128. Kueng et al., report a general strategy for decoration of envelope viruses with functionally active lipid-modified cytokines, J Virology, 2007, 81, 8666-8676.
SUMMARY
[0006] In some embodiments, described herein is a method of tumor treatment or tumor vaccination. The method generally comprises applying to a human being in need thereof a tumor therapeutic composition or tumor vaccine defined herein. The tumor therapeutic composition or tumor vaccine can be produced by protein transfer of glycosyl-phosphatidylinositol (GPI)-anchored immunostimulatory or costimulatory molecules.
[0007] In one embodiment, the tumor therapeutic composition or tumor vaccine comprises a live tumor cell or tumor cell membranes that is or are modified by protein transfer to express one or more GPI-anchored immunostimulatory or costimulatory molecules. The tumor therapeutic composition or tumor vaccine can be prepared by a method that comprises obtaining one or more GPI-anchored immunostimulatory or costimulatory molecules, and transferring the GPI-anchored immunostimulatory or costimulatory molecules onto a tumor cell or isolated tumor cell membranes by protein transfer.
[0008] In certain embodiments, the disclosure relates to non-naturally occurring particle comprising, a lipid membrane; a B7-1 and/or B7-2 molecule anchored to the lipid membrane on the exterior of the particle; and an antigen molecule such as a tumor specific antigen or cancer marker anchored to the lipid membrane on the exterior of the particle. Typically, the particle further comprises an adjuvant molecule anchored to the lipid membrane on the exterior of the particle wherein the adjuvant molecule and antigen molecule are not the same molecule. In certain embodiments, the adjuvant molecule is selected from IL-2, IL-12, ICAM1 GM-CSF, flagellin, unmethylated, CpG oligonucleotide, lipopolysaccharides, lipid A, and heat stable antigen (HSA). The lipid membrane may be a phospholipid monolayer or phospholipid bilayer. Typically, the particle is selected from a cell, allogeneic or autologous cancer cell or its membrane fragments or vesicles, liposome, virosome, micelle, polymer, and virus like particle.
[0009] In certain embodiments, the B7-1 molecule is anchored to the lipid membrane on the exterior of the particle through a conjugated glycosyl-phosphatidylinositol, phospholipid, glycolipid, triglyceride, saturated or unsaturated fatty acid, or other lipophilic molecule.
[0010] In certain embodiments, the antigen molecule such as a tumor associated antigen or cancer marker is anchored to the lipid membrane on the exterior of the particle through a conjugated glycosyl-phosphatidylinositol, phospholipid, glycolipid, triglyceride, saturated or unsaturated fatty acid, or other lipophilic molecule.
[0011] In certain embodiments, the adjuvant molecule is anchored to the lipid membrane on the exterior of the particle through a conjugated glycosyl-phosphatidylinositol, phospholipid, glycolipid, triglyceride, saturated or unsaturated fatty acid, or other lipophilic molecule.
[0012] Particles comprising membranes such as tumor membranes carrying tumor antigens and immunostimulatory stimulatory molecules can be modified by incubating with lipophilic adjuvants such as lipopolysaccharides or an immunostimulatory unmethylated CpG oligonucleotides lipid conjugate.
[0013] In certain embodiments, antigen is a cancer marker molecule selected from HER-2, MUC-1, mucin antigens TF, Tn, STn, glycolipid globo H antigen, prostate-specific antigen, prostate-specific membrane antigen, early prostate cancer antigen-2 (EPCA-2), BCL-2, MAGE antigens such as CT7, MAGE-A3 and MAGE-A4, G-protein coupled estrogen receptor 1, CA15-3, CA19-9, CA 72-4, CA-125, carcinoembryonic antigen, CD20, CD31, CD34, PTPRC (CD45), CD99, CD117, melanoma-associated antigen (TA-90), peripheral myelin protein 22 (PMP22), epithelial membrane proteins (EMP-1, -2, and -3), HMB-45 antigen, MART-1 (Melan-A), S100A1, S100B and gp100:209-217(210M).
[0014] In certain embodiments, the disclosure relates to virus like particles comprising B7-1 and/or B7-2 molecule anchored to a lipid membrane on the exterior of the particle and an antigen molecule anchored to the lipid membrane on the exterior of the particle. Typically, the antigen molecule is a cancer marker or tumor associated antigen or tumor-specific antigen selected from HER-2, MKI67, prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), prostate-specific membrane antigen, early prostate cancer antigen-2 (EPCA-2), BCL-2, MAGE antigens, antigens comprising a Mage Homology Domain (MHD), MAGE-1, CT7, MAGE-A3 and MAGE-A4, ERK5, G-protein coupled estrogen receptor 1, CA15-3, CA19-9, CA 72-4, CA-125, carcinoembryonic antigen, CD20, CD31, CD34, PTPRC (CD45), CD99, CD117, melanoma-associated antigen (TA-90), peripheral myelin protein 22 (PMP22), epithelial membrane proteins (EMP-1, -2, and -3), HMB-45 antigen, MART-1 (Melan-A), S100A1, S100B and gp100:209-217(210M). Typically, the virus like particle further comprising an adjuvant molecule anchored to a lipid membrane on the exterior of the particle wherein the adjuvant molecule and the antigen molecule are not the same molecule. In certain embodiments, the adjuvant molecule is selected from is IL-2, IL-12, ICAM1 GM-CSF, flagellin, unmethylated, CpG oligonucleotide, lipopolysaccharides, lipid A, and heat stable antigen (HSA).
[0015] In certain embodiments, the disclosure relates to methods of treating cancer comprising administering an effective amount of a particle or a virus like particle as disclosed herein to a subject at risk of or diagnosed with cancer or a tumor optionally in combination with anti-CTLA-4 antibodies such as abatacept, belatacept, ipilimumab, tremelimumab, anti-PD-1 and PDL1 antibodies such as nivolumab, unmethylated CpG oligonucleotide, methyl jasmonate, cyclophosphamide, gemcitabine or other immunosuppression blocker or other anticancer agent. Typically, the subject is a human subject and the virus like particle comprises a B7-1 and/or B7-2 molecule anchored to a lipid membrane on the exterior of the particle and an antigen molecule wherein the antigen molecule is a viral protein.
[0016] Other anticancer agents contemplated include gefitinib, erlotinib, docetaxel, cis-platin, 5-fluorouracil, gemcitabine, tegafur, raltitrexed, methotrexate, cytosine arabinoside, hydroxyurea, adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin, vincristine, vinblastine, vindesine, vinorelbine taxol, taxotere, etoposide, teniposide, amsacrine, topotecan, camptothecin bortezomib anegrilide, tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene fulvestrant, bicalutamide, flutamide, nilutamide, cyproterone, goserelin, leuprorelin, buserelin, megestrol anastrozole, letrozole, vorazole, exemestane, finasteride, marimastat, trastuzumab, cetuximab, dasatinib, imatinib, bevacizumab, combretastatin, thalidomide, and/or lenalidomide or combinations thereof.
[0017] In certain embodiments, the viral like particle has an hemagglutinin selected from influenza H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16 optionally in combination with or individually influenza N1, N2, N3, N4, N5, N6, N7, and N8.
[0018] In certain embodiments, the virus protein is an HIV envelope protein selected from gp 41, gp 120, and gp 160.
[0019] In certain embodiments, the disclosure relates to methods of treating or preventing a viral infection comprising administering an effective amount of a virus like particle disclosed herein to a subject at risk of, exhibiting symptoms of, or diagnosed with a viral infection.
[0020] In certain embodiments, the disclosure relates to particles comprising a cancer marker made by the process of mixing a cancer marker conjugated to a lipophilic moiety and a particle comprising a lipid membrane. Typically, the cancer marker is HER-2 or PSA or PAP.
[0021] In certain embodiments, the disclosure relates to particles comprising a cancer marker and B7-1 and/or B7-2 made by the process of mixing a B7-1 and/or B7-2 conjugated to a lipophilic moiety and a particle comprising a lipid membrane and a cancer marker.
[0022] In certain embodiments, the disclosure relates to methods of treating or preventing breast cancer comprising administering an effective amount of a particle comprising B7-1 and/or B7-2, GM-CSF, and HER-2 to a subject in need thereof.
[0023] In certain embodiments, the method further comprises analyzing the subject for overexpression of HER-2, by measuring, detecting, sequencing, hybridizing with a probe, HER-2 polypeptide or a nucleic acid indicative of HER-2 expression, or sequencing a nucleic acid associated with HER-2, on a cancer cell or tumor cell isolated from the subject.
[0024] In certain embodiments, the disclosure relates to methods of treating or preventing prostate cancer comprising administering an effective amount of a particle comprising B7-1 and/or B7-2, GM-CSF, and PSA or PAP to a subject in need thereof.
[0025] In certain embodiments, the disclosure relates to methods of treating or preventing prostate cancer comprising administering an effective amount of a particle comprising B7-1 and/or B7-2, GM-CSF, IL-12, and PSA or PAP to a subject in need thereof.
[0026] In certain embodiments, the compositions and method further comprises administering an immunostimulatory amount of particles disclosed herein in combination with an anticancer agent, individually as single agents and/or in a single pharmaceutical composition.
[0027] In the case of breast cancer the anticancer agent may be estradiol, tamoxifen, cetuximab and a HER-2 antibody, humanized antibody, or human chimera such as trastuzumab, pertuzumab. The HER-2 antibodies may be administered before or after immune stimulation with particle.
[0028] In the case of prostate cancer, the anticancer agent may be docetaxel, cabazitaxel, bevacizumab, alpharadin thalidomide, prednisone, abiraterone, finasteride and dutasteride, MDV3100, orteronel (TAK-700), omega-3 fatty acids such as ethyl esters of eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) or combinations thereof such as bevacizumab, docetaxel, thalidomide, and prednisone or abiraterone acetate in combination with prednisone.
[0029] In another embodiment, the tumor therapeutic composition or tumor vaccine comprises a microparticle with a lipid membrane encapsulating tumor antigens or peptides and one or more anchored immunostimulatory or costimulatory molecules expressed on the surface of the particle. The tumor therapeutic composition or tumor vaccine can be prepared by a method that comprises obtaining one or more anchored immunostimulatory or costimulatory molecules, and transferring the anchored immunostimulatory or costimulatory molecules onto a particle encapsulating at least one tumor antigen or peptide, tumor lysate, tumor membranes, or combinations thereof by protein transfer.
[0030] The microparticles can be formed of any biocompatible polymer capable of incorporating GPI-anchored immunostimulatory or costimulatory molecules. For example, representative useful biocompatible polymers include, but are not limited to, polyvinyl alcohols, polyvinyl ethers, polyamides, polyvinyl esters, polyvinylpyrrolidone, polyglycolides, polyurethanes, allyl celluloses, cellulose esters, hydroxypropyl derivatives of celluloses and cellulose esters, preformed polymers of poly alkyl acrylates, polyethylene, polystyrene, polyactic acid, polyglycolic acid, poly(lactide-co-glycolide), polycaprolactones, polybutyric acids, polyvaleric acid and copolymers thereof, alginates, chitosans, gelatin, albumin, zein and combinations thereof.
[0031] Anchored immunostimulatory or costimulatory molecules can be obtained by expressing the GPI-anchored immunostimulatory or costimulatory molecules in a cell, and isolating the GPI-anchored immunostimulatory or costimulatory molecules. The anchored immunostimulatory or costimulatory molecules can be any substance that stimulates or costimulates immune reaction against a tumor cell that is capable of being expressed in a cell. For example, the immunostimulatory or costimulatory molecules useful here can be a cytokine molecule. In one embodiment, a useful cytokine can be, for example, one or more of cytokines IL-2, IL-4, IL-6, IL-12, IL-15, IL-18, IL-19, granulocyte-macrophage colony stimulating factor (GM-CSF), and combinations thereof. In another embodiment, the immunostimulatory or costimulatory molecules can be, for example, the immunostimulatory or costimulatory molecules useful here can be a cytokine molecule. In another embodiment, the immunostimulatory or costimulatory molecules useful here can be, for example, B7-1, B7-2 and an intercellular adhesion molecule such as CD40L, ICAM-1, ICAM-2, and ICAM-3.
[0032] In any of the embodiments, particle may be a wild type cell, cancer cell or immortalized cell.
[0033] The immunostimulatory or costimulatory molecules can be used alone or together and can be used in conjunction with antibody fusion proteins.
[0034] The tumor therapeutic composition or tumor vaccine described herein can be used therapeutically or prophylactically for the treatment or prevention of a tumor. Representative tumors can be treated or prevented include, but are not limited to, breast cancer, prostate cancer, lung cancer, melanoma, liver cancer, leukemia, lymphoma, myeloma, colorectal cancer, gastric cancer, bladder carcinoma, esophageal carcinoma, head & neck squamous-cell carcinoma, sarcomas, kidney cancers, ovarian and uterus cancers, adenocarcinoma, glioma, and plasmacytoma, and combinations thereof.
[0035] In one embodiment, the vaccine or therapeutic composition described herein can be GPI-anchored cytokine such as GPI-IL-2 and GPI-IL-12 alone or in combination with GPI-anchored costimulatory molecules such as GPI-B7-1, GPI-B7-2, GPI-ICAM-1, GPI-ICAM-2 and GPI-ICAM-3. Such a vaccine or therapeutic composition can be used for the treatment of tumor and other diseases such as viral, bacterial and parasitic diseases.
[0036] In another embodiment, the vaccine and therapeutic composition can be biocompatible microparticles such as biodegradable microparticles modified with GPI-anchored immunostimulatory molecules such as IL-2, IL-4, IL-6, IL-12, ICAM-1, ICAM-2, ICAM-3, B7-1, B7-2, CD40L, IL-15, IL-18, IL-19, granulocyte-macrophage colony stimulating factor (GM-CSF), and combinations thereof.
[0037] In yet another embodiment, the vaccine or therapeutic compositions described herein can be tumor cells or membranes modified by protein transfer with GPI-anchored cytokines alone or/and in combination with other cytokines or/and other costimulatory molecules. One such embodiment can be, for example, tumor membranes modified with purified GPI-IL-12.
[0038] In a further embodiment, particles like inactivated or partially attenuated virus, bacteria and virus-like particles can be modified to express immunostimulatory molecules by protein transfer with GPI-anchored cytokines and immunostimulatory molecules. Vaccines and therapeutic compositions prepared in this manner can be used for preventing or treating viral, bacterial, or parasitic diseases or disorders.
[0039] In some other embodiments, the vaccine and therapeutic compositions described herein can be used for treating autoimmune disorders. For example, membrane anchored cytokines such as IL-10 and TGF-beta can also be used to induce tolerance or to suppress immunity which can be used in treating autoimmune diseases and transplant rejection.
BRIEF DESCRIPTION OF THE FIGURES
[0040] FIG. 1 illustrates the expression tumor associated antigens and immunostimulatory molecules onto particles containing a lipid membrane, e.g., CHO cells and envelope VLPs, using GPI anchoring for protein transfer.
[0041] FIG. 2 shows data on protein transfer of (A) GPI-ICAM1 or (B) GPI-IL-12 onto sheep RBCs. Red: Background control; Black: Protein transfer of GPI-ISMs.
[0042] FIG. 3 shows data on Concentration dependent protein transfer of (A) GPI-ICAM-1 or (B) GPI-IL-12 onto H5-VLPs.
[0043] FIG. 4 shows data on the kinetics of protein transfer of GPI-ICAM-1 onto H5 influenza VLPs.
[0044] FIG. 5 shows data on the specificity of protein transfer of GPI-ICAM1 onto VLPs.
[0045] FIG. 6 shows data on the inhibition of protein transfer of GPI-ICAM 1 via fatty acid binding proteins.
[0046] FIG. 7 shows data on the incorporation of two GPI-ISMs onto VLPs simultaneously.
[0047] FIG. 8 shows a EM of VLPs (A) before and (B) after protein transfer with GPI-ICAM1.
[0048] FIG. 9 shows data on the direct challenge with wild-type or GPI cytokine transfected 4T07 cells. BALB/C mice (n=5/group) were challenged s.c. in the hind flank with 2×105 cells in 100 μl PBS and were monitored every 2-3 days for tumor growth. Mean was calculated as the average of the tumor measurements from five mice per group. For the purpose of clarity, standard deviation was not included in the graph instead the values from individual mice in each group is given in FIG. 10.
[0049] FIG. 10 shows tumor size in individual mice post direct challenge with wild-type or
transfected 4T07 murine mammary tumor cells. BALB/C mice (n=5/group) were challenged s.c. in the hind flank with 2×105 cells in 100 μl PBS and were monitored every 2-3 days for tumor growth. Each data line represents an individual mouse per group.
[0050] FIG. 11 illustrates the production of extracellular portion of hHER-2 (hHER-2ECD).
Before the sequence, an optimized IL-2 Kozak sequence along with the restriction enzyme sites HindIII and KpnI have been added. Following the hHER2ECD sequence an EcoRI site is added. At base pair position 1365 of hHER2, a change in base pair from T was made to C in order to remove an EcoRI restriction enzyme site at this position, however, the final amino acid still remains as an isoleucine.
[0051] FIG. 12 shows flow cytometry analysis of CHO cells expressing GPI-human HER-2 (hHER-2-CD59) using TA1 mAb.
DETAILED DESCRIPTION
[0052] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.
[0053] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.
[0054] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
[0055] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.
[0056] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a support" includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.
[0057] Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.
[0058] As used herein, the term "combination with" when used to describe administration with an additional treatment means that the agent may be administered prior to, together with, or after the additional treatment, or a combination thereof.
[0059] As used herein, the terms "prevent" and "preventing" include the prevention of the recurrence, spread or onset. It is not intended that the present disclosure be limited to complete prevention. In some embodiments, the onset is delayed, or the severity is reduced.
[0060] As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e.g. patient) is cured and the disease is eradicated. Rather, embodiments of the present disclosure also contemplate treatment that merely reduces symptoms, and/or delays disease progression.
[0061] "Subject" refers any animal, preferably a human patient, livestock, rodent, monkey or domestic pet.
[0062] The terms "protein" and "polypeptide" refer to compounds comprising amino acids joined via peptide bonds and are used interchangeably.
[0063] As used herein, an "amino acid sequence" refers to an amino acid sequence of a protein molecule. The terms such as "polypeptide" or "protein" are not meant to limit the amino acid sequence to the deduced amino acid sequence, but such as amino acid deletions, additions, and modifications such as glycolsylations and addition of lipid moieties or other post-translational modifications.
[0064] With regard to any of the antigens or adjuvants disclosed herein, the protein generally refers to the most frequent human isoform, variant, mutated form, or protein with substantially identity to the full-length or portion thereof. Typically, an appropriate fragment is of the extracellular domain.
[0065] The term "portion" when used in reference to a protein (as in "a portion of a given protein") refers to fragments of that protein. The fragments may range in size from four amino acid residues or more than twenty or thirty or the entire amino sequence minus one amino acid.
[0066] The following terms are used to describe the sequence relationships between two or more proteins: "reference sequence", "sequence identity", "percentage of sequence identity", and "substantial identity". A "reference sequence" is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length amino acid sequence of a protein. Generally, a reference sequence is at least 20 amino acids in length, frequently at least 25 amino acids in length, and often at least 50 amino acids in length. Since two proteins may each (1) comprise a sequence (i.e., a portion of the complete amino acid sequence) that is similar between the two protein, and (2) may further comprise a sequence that is divergent between the two proteins, sequence comparisons between two (or more) proteins are typically performed by comparing sequences of the two proteins over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window", as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a sequence may be compared to a reference sequence of at least 20 contiguous amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math. 2: 482 (1981)) by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol. 48:443 (1970)), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. (U.S.) 85:2444 (1988)), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.
[0067] The term "sequence identity" means that two sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acids occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms "substantial identity" as used herein denotes a characteristic of a sequence, wherein the protein comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 amino acid positions, frequently over a window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
Particle Anchored Immunostimulatory or Costimulatory Molecules
[0068] In certain embodiments, the disclosure relates to non-naturally occurring particle comprising, a B7-1 and/or B7-2 molecule anchored on the exterior of the particle; and an antigen molecule such as a tumor specific antigen or cancer marker anchored to the lipid membrane on the exterior of the particle. In certain embodiments, the B7-1 and or B7-2 or antigen, or protein may be anchored onto the membrane of the particle through a variety of linkages, such as lipid palmatic acid, biotin-avidin interaction, or a GPI-anchor.
[0069] In one example, a contemplated sequence of B7-1 is MGHTRRQGTS PSKCPYLNFF QLLVLAGLSH FCSGVIHVTK EVKEVATLSC GHNVSVEELAQTRIYWQKEK KMVLTMMSGD MNIWPEYKNR TIFDITNNLS IVILALRPSD EGTYECVVLK YEKDAFKREH LAEVTLSVKA DFPTPSISDF EIPTSNIRRI ICSTSGGFPE PHLSWLENGE ELNAINTTVS QDPETELYAV SSKLDFNMTT NHSFMCLIKY GHLRVNQTFN WNTTKQEHFP DNLLPSWAIT LISVNGIFVI CCLTYCFAPR CRERRRNERL RRESVRPV (SEQ ID NO: 1) or fragment thereof.
[0070] In another example, a contemplated sequence is VIHVTKEVKE VATLSCGHNV SVEELAQTRI YWQKEKKMVL TMMSGDMNIW PEYKNRTIFD ITNNLSIVIL ALRPSDEGTY ECVVLKYEKD AFKREHLAEV TLSVKADFPT PSISDFEIPT SNIRRIICST SGGFPEPHLS WLENGEELNA INTTVSQDPE TELYAVSSKL DFNMTTNHSF MCLIKYGHLR VNQTFNWNTT KQEHFPDN (SEQ ID NO:2) or fragment thereof. See Stamper et al., Crystal structure of the b7-1/ctla-4 complex that inhibits human immune responses. Nature (2001) 410:608.
[0071] In another example, a contemplated fragment is KAMHVAQPAV VLASSRGIAS FVCEYASPGK ATEVRVTVLR QADSQVTEVC AATYMMGNELTFLDDSICTG TSSGNQVNLT IQGLRAMDTG LYICKVELMY PPPYYLGIGN GAQIYVIDPE PCPDSD (SEQ ID NO: 3) or fragment thereof.
[0072] In certain embodiments, the disclosure relates to non-naturally occurring particle comprising, a B7-1 and/or B7-2 molecule anchored on a lipid membrane; a B7-1 and/or B7-2 molecule anchored to the lipid membrane on the exterior of the particle; and an antigen molecule such as a tumor specific antigen or cancer marker anchored to the lipid membrane on the exterior of the particle.
[0073] A number of proteins commonly expressed by cells are attached to the cell membrane via a GPI-anchor. These proteins are post-translationally modified at their carboxy terminus to express this glycosylated moiety which is synthesized in the endoplasmic reticulum. These naturally expressing GPI-anchored molecules are widely distributed in mammalian cells and serve a host of different cellular functions, such as cell adhesion, enzymatic activity, and complement cascade regulation. Naturally occurring GPI-anchored proteins lack a transmembrane and cytoplasmic domain that otherwise anchor membrane proteins. The GPI-anchor consists of a glycosylated moiety attached to phosphatidylinositol containing two fatty acids. The phosphatidylinositol portion, as well as an ethanolamine which is attached to the C-terminal of the extracellular domain of the membrane proteins, anchor the molecule to the cell membrane lipid bilayer.
[0074] In order to exploit this natural linkage using recombinant DNA techniques, the transmembrane and cytoplasmic domains of a transmembrane surface protein need only be replaced by the signal sequence for GPI-anchor attachment that is found at the hydrophobic C-terminus of GPI-anchored protein precursors. This method may be used to generate GPI-anchored proteins is not limited to membrane proteins; attaching a GPI-anchor signal sequence to secretory proteins would also convert them to a GPI-anchored form. The method of incorporating the GPI-anchored proteins onto isolated cell surfaces or lipid particles is referred to here as protein transfer.
[0075] GPI-anchored molecules can be incorporated onto lipid membranes spontaneously. These GPI-anchored proteins can be purified from one cell type and incorporated onto different cell membranes. GPI-anchored proteins are used to customize of the lipid membranes disclosed herein for uses as a cancer vaccine. One may incorporate multiple molecules simultaneously onto the same cell membrane. One can control the level of protein expression by simply varying the concentration of the GPI-anchored molecules to be incorporated. The most significant outcome of this technology will be the reduction of time in preparing cancer vaccines from months to hours. These features make the protein transfer approach a more viable choice for the development of cancer vaccines for clinical settings. The molecules incorporated by means of protein transfer retain their functions associated with the extracellular domain. Cells and isolated membranes can be modified to express immunostimulatory molecules. In certain embodiments, the disclosure contemplates that the GPI-anchored molecules are incorporated onto the surface of albumin microparticles by this protein transfer method. GPI-anchored proteins attached to the surface of microparticles are used to target and/or enhance the adjuvant activity of microparticles, thereby enhancing the capacity to function as a targeted antigen or drug delivery device for cancer treatment.
[0076] The GPI-B7-1 expression (by protein transfer) was stable up to 7 days on isolated membranes at 37° C. and frozen membranes can be used up to 3 years of storage at -80° C. which makes the stability and storage a nonissue. These studies suggest that the membrane vaccines are more suitable to stably express the GPI-anchored molecules than on intact cells, which lose the expression within 24 hr.
[0077] This approach for introducing proteins onto membranes provides advantages over other immunotherapies for cancer vaccine development. This approach allows a protein to be added either singularly or in a combinatory manner to the tumor membrane surface. This approach navigates around the necessity to establish tumor cells as is the case for gene transfer. This GPI-mediated approach by protein transfer may be used for the co-stimulatory molecules, B7-1 and B7-2, GM-CSF, IL-2, and IL-12. With these cytokines being attached to the tumor membrane via a GPI-anchor, it enables them to exert their effector functions locally at the vaccination site without the risk of systemic toxicity.
Virus Like Particles
[0078] In certain embodiments, the disclosure relates to virus like particles comprising B7-1 and/or B7-2 molecule anchored to a lipid membrane on the exterior of the particle and an antigen molecule anchored to the lipid membrane on the exterior of the particle for uses disclosed herein.
[0079] Influenza virus-like particles (VLPs) are particulate in nature and have shown to elicit robust immunity against antigens. Influenza VLPs have an outer lipid bilayer with properties similar to the cell membranes. Modification of influenza VLPs with a protein transfer method to incorporates tumor-associated antigens (TAAs) on the surface along with immunostimulatory molecules (ISMs) elicits enhanced immune responses directed against the TAAs. One contemplated protein transfer approach utilizes glycosyl phosphatidylinositol (GPI)- to anchor the TAA, which can spontaneously incorporate onto the surface of the VLPs that contain a lipid bilayer upon incubation at 37° C. (See FIG. 1).
[0080] Incorporation of GPI-anchored forms of TAAs onto the surface of VLPs is used to direct the immune response against cancerous cells whereas the incorporation of immunostimulatory molecules (ISMs), such as GPI-anchored cytokines, costimulatory molecules, and adhesion molecules, onto the surface of VLPs is used to enhance the interaction between VLPs and antigen presenting cells (APCs) as well as lead to activation of these APCs and other immune effector cells. The incorporation of GPI-TAAs and GPI-ISMs onto VLPs by protein transfer leads to an antitumor immune response and tumor regression.
[0081] VLPs consist of a virus' capsid protein shell that presents viral antigens in an authentic conformation without the viral genome that is required for replication. Thus, they provide a safe approach for human use. VLPs contain a multivalent repetitive structure that is particulate in nature, allowing for recognition by many pattern recognition receptors and the induction of an enhanced innate and adaptive immune response. The particulate nature of VLPs allows for them to be readily taken up and presented by APCs, and thus could provide a means for breaking the immunosuppressive barrier initiated by the tumor microenvironment.
[0082] In certain embodiments, influenza virus-like particles (VLPs) may be produced using a variety of platform systems, including recombinant baculovirus vectors, transient plasmid expression systems, stable cell-line transformants, and plant expression systems. Typically VLPs are non-replicating particles that spontaneously self-assemble from expressed influenza virus proteins. In some expression systems, the viral hemagglutinin (HA) protein is sufficient for particle assembly and release from the cell. Typically the VLP comprises neuraminidase (NA). HA may present with a different type of glycosylation depending on whether they are obtained from. For the production of VLPs containing HA in mammalian cells, co-expression of NA or exogenously added NA was required for the effective release of influenza VLPs into culture media, implying an important role of the NA activity in cleaving sialic acids bound to HA of budding particles. In contrast, VLPs containing HA can be produced in insect cells in the absence of NA expression. Insect cells do not add sialic acids to the N-glycans during the posttranslational modification, which explains how VLPs containing HA but not NA are effectively released from insect cell surfaces. See Kang et al., Virus Res. 2009c, 143 (2), 140-6.
[0083] In certain embodiments, VLPs used herein are recombinant influenza VLPs that have been generated in insect cells infected with rBVs expressing influenza genes HA, NA, M1, and M2.
[0084] In certain embodiments, VLPs used herein are recombinant influenza VLPs that have been generated in insect cells infected with rBVs expressing influenza genes HA, NA, and M1.
[0085] In certain embodiments, VLPs used herein are recombinant influenza VLPs that have been generated in insect cells infected with rBVs expressing influenza genes of HA and M1.
[0086] In some instances, the VLP is obtained from influenza VLPs expressed from recombinant baculovirus (rBV) produced by replication in an insect cell system, e.g., Spodoptera frugiperda SF9 cells.
[0087] In some instances, the VLP is obtained from a modified vaccinia virus Ankara (MVA) system expressing expressing influenza H5N1 HA, NA, and M proteins to generate influenza VLPs produced by replication in mammalian cells. See Schmeisser et al., Vaccine, 2012, 30(23):3413-3422.
Tumor Associate Antigens and Cancer Markers
[0088] In certain embodiments, the disclosure relates to particles such as cells or virus like particles comprising B7-1 and/or B7-2 molecule anchored to a lipid membrane on the exterior of the particle and an antigen molecule anchored to the lipid membrane on the exterior of the particle. Typically, the antigen molecule is a cancer marker selected from HER-2, MKI67, prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), prostate-specific membrane antigen, early prostate cancer antigen, early prostate cancer antigen-2 (EPCA-2), BCL-2, MAGE antigens such as CT7, MAGE-A3 and MAGE-A4, ERK5, G-protein coupled estrogen receptor 1, CA15-3, CA19-9, CA 72-4, CA-125, carcinoembryonic antigen, CD20, CD31, CD34, PTPRC (CD45), CD99, CD117, melanoma-associated antigen (TA-90), peripheral myelin protein 22 (PMP22), epithelial membrane proteins (EMP-1, -2, and -3), HMB-45 antigen, MART-1 (Melan-A), S100A1, S100B and gp100:209-217(210M), MUC-1, mucin antigens TF, Tn, STn, glycolipid globo H antigen. Typically, the antigen is the human form.
[0089] HER-2, or Human Epidermal Growth Factor Receptor 2, refers to the human protein encoded by the ERBB2 gene that has been referred to as Neu, ErbB-2, CD340 (cluster of differentiation 340) or p185. See Coussens et al., 1985, Science 230 (4730): 1132-9.
[0090] In certain embodiments, HER-2 is the extracellular domain or fragment thereof. In one contemplated example the protein comprises or consists essentially of the following sequence: TQVCTGTDMK LRLPASPETH LDMLRHLYQG CQVVQGNLEL TYLPTNASLS FLQDIQEVQG YVLIAHNQVR QVPLQRLRIV RGTQLFEDNY ALAVLDNGDP LNNTTPVTGA SPGGLRELQL RSLTEILKGG VLIQRNPQLC YQDTILWKDI FHKNNQLALT LIDTNRSRAC HPCSPMCKGS RCWGESSEDC QSLTRTVCAG GCARCKGPLP TDCCHEQCAA GCTGPKHSDC LACLHFNHSG ICELHCPALV TYNTDTFESM PNPEGRYTFG ASCVTACPYN YLSTDVGSCT LVCPLHN QEVTAEDGTQRCE KCSKPCARVC YGLGMEHLRE VRAVTSANIQ EFAGCKKIFG SLAFLPESFD GDPASNTAPL QPEQLQVFET LEEITGYLYI SAWPDSLPDL SVFQNLQVIR GRILHNGAYS LTLQGLGISW LGLRSLRELG SGLALIHHNT HLCFVHTVPW DQLFRNPHQA LLHTANRPED ECVGEGLACH QLCARGHCWG PGPTQCVNCS QFLRGQECVE ECRVLQGLPR EYVNARHCLP CHPECQPQNG SVTCFGPEAD QCVACAHYKD PPFCVARCPS GVKPDLSYMP IWKFPDEEGA CQPCPIN (SEQ ID NO: 4) or fragment thereof.
[0091] In one contemplated example, the protein comprises or consists essentially of the following sequence: DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC (SEQ ID NO: 5) or fragment thereof.
[0092] In one contemplated example, the protein comprises or consists essentially of the following sequence: GTSHLVKCAE KEKTFCVNGG ECFMVKDLSN PSRYLCKCPN EFTGDRCQNY VMASF (SEQ ID NO: 6) or fragment thereof.
[0093] MKI67, or antigen identified by monoclonal antibody Ki-67, refers to the human protein that is encoded by the MKI67 gene. See Bullwinkel et al., 2006, J. Cell. Physiol. 206 (3): 624-35.
[0094] PAP, or Prostatic acid phosphatase or prostatic specific acid phosphatase (PSAP), refers to the human enzyme produced by the prostate in males. See Ostrowski & Kuciel, 1994, Clin. Chim. Acta 226 (2): 121-9.
[0095] PSA, or Prostate-specific antigen or gamma-seminoprotein or kallikrein-3 (KLK3), refers to the human protein encoded by the KLK3 gene. See Menez et al., J Mol Biol. 2008, 376(4):1021-33.
[0096] PSMA, or Prostate-specific membrane antigen or Glutamate carboxypeptidase II, refers to a human type 2 integral membrane glycoprotein found in prostate tissues. See William et al., Reviews on Recent Clinical Trials, 2007, 2, 182-190.
[0097] Bcl-2, or B-cell lymphoma 2 refers to an protein encoded by the BCL2 gene. Bcl-2 has two isoforms that differ by two amino acids. Isoform 1 is known as 1G5M, and Isoform 2 is known as 1G50/1GJH. See Petros et al., 2001, PNAS, 98: 3012-3017. Both isoforms are contemplated antigens.
[0098] In certain embodiments, the antigen is the entire protein, polypeptide, or a substantial fragment, or a fragment with conserved substitutions. The fragment may contain 5, 10, 20, 50, 100, or halve of the amino acids in the full length antigen. The fragment may be sufficient to mimic or replicate the folding of the full length antigen. The conserved substitutions may be amino acids that are in the interior of the folded polypeptide. A fragment is sufficient produce antibody production to the polypeptide. The antigen may be a chimera containing the fragment. The antigen may contain 1, 2, or 3, or 5 to 10, or 10 to 20 or more conserved substitutions within the full length or polypeptide fragment which are typically outside of functional domains. In certain embodiments, the antigen may have 80%, 90%, 95% or greater sequence identity to the full length or polypeptide fragment. An antigen protein may or may not be glycosylated.
Adjuvant Molecules
[0099] In certain embodiments, the virus like particles disclosed herein comprise an adjuvant molecule anchored to a lipid membrane on the exterior of the particle wherein the adjuvant molecule and the antigen molecule are not the same molecule. In certain embodiments, the adjuvant molecule is selected from is IL-2, IL-12, ICAM1, GM-CSF, flagellin, unmethylated, CpG oligonucleotide, lipopolysaccharides, lipid A, and heat stable antigen (HSA).
[0100] It is contemplated that the co-stimulatory molecules, antigens, and adjuvant molecules may the individually conjugated to the lipophilic molecules or two or more or all of them may be conjugated together in a chimera and conjugated to a lipophilic molecule. For example, B7-1 may be conjugated to the adjuvant, HSA, in a chimera and the chimera is conjugated to a GPI.
[0101] One contemplated antigen is heat stable antigen (HSA). A hybrid B7-1-HSA molecule on the cell surface membrane can function as a co-stimulatory molecule to induce T cell proliferation. CHO cells and CHO transfectants expressing HSA, B7-1, and B7-1-HSA were used as stimulator cells in a T cell proliferation assay. See Wang et al., Immunology Letters, 2006, 105(2):185-192.
[0102] Contemplated TLR 9 ligands as adjuvants are contemplated such as immunostimmulatory unmethylated CpG oligonucleotides, the cytosine of the oligonucleotide sequence 5'-CG-3' is unmethylated and the oligonucleotide is greater than about 6 base pairs in length and is less than about 100 base pairs in length such as 5'-TGACTGTGAACGTTC GAGATGA-3' (SEQ ID NO:8). It is contemplated that lipophilic molecules may be conjugated to the oligonucleotide for incorporation to the exterior of particles disclosed herein.
[0103] In certain embodiments, the antigen is also contained in the interior of the particle.
[0104] In certain embodiments, the B7-1 molecule is a B7-1 and heat stable antigen (HSA) hybrid chimera.
[0105] In certain embodiments, the antigen is HER-2 and the adjuvant is flagellin and/or GM-CSF.
[0106] In certain embodiments, the antigen is HER-2 and the B7-1 molecule is a B7-1 and heat stable antigen (HSA) hybrid chimera.
[0107] In certain embodiments, the antigen is HER-2, the adjuvant is flagellin and/or GM-CSF, the B7-1 molecule is a B7-1 and heat stable antigen (HSA) hybrid chimera.
[0108] In certain embodiments, the antigen is HER-2 and the adjuvant is IL-12.
[0109] In certain embodiments, the antigen is HER-2, the adjuvant is IL-12, the B7-1 molecule is a B7-1 and heat stable antigen (HSA) hybrid chimera.
[0110] In certain embodiments, the antigen is PSA or PAP and the adjuvant is flagellin and/or or GM-CSF.
[0111] In certain embodiments, the antigen is PSA or PAP and the B7-1 molecule is a B7-1 and heat stable antigen (HSA) hybrid chimera.
[0112] In certain embodiments, the antigen is PSA or PAP, the adjuvant is flagellin, the B7-1 molecule is a B7-1 and heat stable antigen (HSA) hybrid chimera.
[0113] In certain embodiments, the antigen is PSA or PAP and the adjuvant is IL-12.
[0114] In certain embodiments, the antigen is PSA or PAP, the adjuvant is IL-12, the B7-1 molecule is a B7-1 and heat stable antigen (HSA) hybrid chimera.
[0115] In certain embodiments, the B7-1 molecule is a B7-1 and heat stable antigen (HSA) hybrid chimera.
[0116] In certain embodiments, the antigen is HER-2 and the adjuvant is flagellin and/or GM-CSF.
[0117] In certain embodiments, the antigen is HER-2 and the B7-1 molecule is a B7-1 and heat stable antigen (HSA) hybrid chimera.
[0118] In certain embodiments, the antigen is HER-2, the adjuvant is flagellin and/or GM-CSF, the B7-1 molecule is a B7-1 and heat stable antigen (HSA) hybrid chimera.
[0119] In certain embodiments, the antigen is HER-2 and the adjuvant is IL-12.
[0120] In certain embodiments, the antigen is HER-2, the adjuvant is IL-12, the B7-1 molecule is a B7-1 and heat stable antigen (HSA) hybrid chimera.
[0121] In certain embodiments, the antigen is PSA or PAP and the adjuvant is flagellin and/or GM-CSF.
[0122] In certain embodiments, the antigen is PSA or PAP and the B7-1 molecule is a B7-1 and heat stable antigen (HSA) hybrid chimera.
[0123] In certain embodiments, the antigen is PSA or PAP, the adjuvant is flagellin or GM-CSF, the B7-1 molecule is a B7-1 and heat stable antigen (HSA) hybrid chimera.
Cellular Particles
[0124] In any of the embodiments, particle may be a wild type cell, cancer cell or immortalized cell.
[0125] In certain embodiments, the particle is a cell such as ZR-75-1, ZR-75-30, 184A1, UACC-812, UACC-893, HCC38, HCC70, HCC202, HCC1187, HCC1395, HCC 1428, HCC1500, HCC1569, HCC1599, HCC1806, HCC1937, HCC1954, HCC2157, HCC1419, HCC2218, AU-565, 184B5, MCF 10A, MCF 10F, MCF-12A, BT-20, MDA-kb2, BT-474, CAMA-1, MCF7, MDA-MB-134-VI, MDA-MB-157, MDA-MB-175-VII, MDA-MB-231, MDA-MB-361, SK-BR-3, BT-483, BT-549, DU4475, Hs 578T, MDA-MB-415, MDA-MB-436, MDA-MB-453, MDA-MB-468, T-47D, EFM19, EFM192A, Hs 578Bst, SUM44PE, SUM52PE, SUM102PT, SUM149PT, SUM190PT, 4T1 (CRL-2539), or CAL51 for use in the treatment of cancer, breast cancer, breast adenocarcinoma, or breast carcinoma.
[0126] In certain embodiments, the particle is a cell such as Jurkat, Clone E6-1 (ATCC Number: TIB-152), RBL-2H3 (CRL-2256), MOLT-4 (CRL-1582), K-562 (CCL-243), CCRF-CEM (CCL-119), HL-60 (CCL-240), or KG-1 (CCL-246) for use in the treatment of cancer, leukemia, leukemia (AML), leukemia (CML), promyelocytic leukemia, basophilic leukemia, or acute T cell leukemia.
[0127] In certain embodiments, the particle is a cell such as NCI-H358 (CRL-5807), LL/2 (CRL-1642), Calu-3 (HTB-55), NCI-H441 (HTB-174), NCI-H1975 (CRL-5908), NCI-H23 (CRL-5800), NCI-H1299 (CRL-5803), NCI-H460 (HTB-177), NCI-H292 (CRL-1848), A-549 (CCL-185), A-549 (CCL-185), A-549 (CCL-185), IMR-90 (CCL-186), MRC-5 (CCL-171), or WI-38 (CCL-75) for use in the treatment of cancer, lung cancer, lung adenocarcinoma, lung carcinoma, lewis lung carcinoma, or bronchioalveolar lung cancer.
[0128] In certain embodiments, the particle is a cell such as Ramos (CRL-1596), Daudi (CCL-213), Raji (CCL-86), EL4 (TIB-39), or U-937 (CRL-1593.2) for use in the treatment of cancer, lymphoma, B-cell lymphomas, histiocytic lymphoma, or Burkitt's lymphoma.
[0129] In certain embodiments, the particle is a cell such as HeLa (CCL-2) or HeLa S3 (CCL-2.2) for use in the treatment of cancer, cervical cancer or cervical adenocarcinoma.
[0130] In certain embodiments, the particle is a cell such as COLO 205 (CCL-222), SW620 (CCL-227), SW480 (CCL-228), LoVo (CCL-229), LS 174T (CL-188), Caco-2 (HTB-37), HT-29 (HTB-38), DLD-1 (CCL-221), HCT 116 (CCL-247), T84 (CCL-248), CT26.WT (CRL-2638) for use in the treatment of cancer, colon cancer, colon carcinoma, or a colon adenocarcinoma.
[0131] In certain embodiments, the particle is a cell such as HCN-1A (CRL-10442), U-87 MG (HTB-14), C6 (CCL-107), bEnd.3 (CRL-2299), or T98G (CRL-1690) for use in the treatment of cancer, brain cancer, glioma, glioblastoma multiforme, glioblastoma-astrocytoma, or brain endothelioma cancer.
[0132] In certain embodiments, the particle is a cell such as 3197-3 (CRL-1568), 3T3-Swiss albino (CCL-92), BALB/3T3 clone A31 (CCL-163), NTERA-2 cl.D1 (CRL-1973), 3T3-L1 (CL-173), NIH/3T3 (CRL-1658), SK-OV-3 (HTB-77), CHO-Kl (CCL-61), or F-12K (30-2004) for use in the treatment of cancer, ovarian cancer, ovarian adenocarcinoma, or testicular cancer.
[0133] In certain embodiments, the particle is a cell such as 293T/17 (CRL-11268), 293 (CRL-1573), VERO C1008 (CRL-1568), Vero (CCL-81), MDCK (CCL-34), BHK-21 (CCL-10), Caki-1 (HTB-46), 786-0 (CRL-1932), or COS-7 (CRL-1651) for use in the treatment of cancer, renal cancer, or renal carcinoma.
[0134] In certain embodiments, the particle is a cell such as H9c2 (CRL-1446) for use in the treatment of cancer or cardiac tumors.
[0135] In certain embodiments, the particle is a cell such as A-431 (CRL-1555), Detroit 551 (CCL-110), BJ (CRL-2522), B16-F10 (CRL-6475), SK-MEL-28 (HTB-72), A375 (CRL-1619), NCTC clone 929 (CCL-1), IRR-MRC-5 (55-X), or IRR-STO (56-X) for use in the treatment of cancer, skin cancer, squamous-cell carcinoma, melanoma, areolar lesions, or epidermoid carcinoma.
[0136] In certain embodiments, the particle is a cell such as HT-1080 (CCL-121) for use in the treatment of cancer or fibrosarcoma.
[0137] In certain embodiments, the particle is a cell such as AGS (CRL-1739) or NCI-N87 (CRL-5822) for use in the treatment of cancer, stomach cancer, gastric carcinoma or gastric adenocarcinoma.
[0138] In certain embodiments, the particle is a cell such as HepG2/C3A (CRL-10741), Hep 3B2.1-7 (HB-8064), Hep G2 (HB-8065), or Hepa 1-6 (CRL-1830) for use in the treatment of cancer, liver cancer, heptoma, or hepatocellular carcinoma.
[0139] In certain embodiments, the particle is a cell such as U266B1 (TIB-196) for use in the treatment of cancer or multiple myeloma.
[0140] In certain embodiments, the particle is a cell such as IMR-32 (CCL-127), Neuro-2a (CCL-131), or SK-N-SH (HTB-11) for use in the treatment of cancer or neuroblastoma.
[0141] In certain embodiments, the particle is a cell such as Saos-2 (HTB-85), U-2 OS (HTB-96), or MG-63 (CRL-1427) for use in the treatment of cancer, bone cancer, or osteosarcoma.
[0142] In certain embodiments, the particle is a cell such as Beta-TC-6 (CRL-11506), AsPC-1 (CRL-1682), BxPC-3 (CRL-1687), MIA PaCa-2 (CRL-1420), PANC-1 (CRL-1469), Capan-1 (HTB-79), or AR42J (CRL-1492) for use in the treatment of cancer, pancreatic cancer, or pancreatic carcinoma.
[0143] In certain embodiments, the particle is a cell such as PC-12 (CRL-1721) for use in the treatment of cancer or pheochromocytoma.
[0144] In certain embodiments, the particle is a cell such as RPMI 8226 (CCL-155) for use in the treatment of cancer or plasmacytoma.
[0145] In certain embodiments, the particle is a cell such as PC-3 (CRL-1435), VCaP (CRL-2876), DU 145 (HTB-81), LNCaP clone FGC (CRL-1740), or 22Rv1 (CRL-2505) for use in the treatment of cancer, prostate cancer, prostate adenocarcinoma.
[0146] In certain embodiments, the particle is a cell such as ARPE-19 (CRL-2302) for use in the treatment of cancer, eye cancer, or retinal cancer.
[0147] In certain embodiments, the particle is a cell such as RD (CCL-136) for use in the treatment of cancer, sarcoma, or rhabdomyosarcoma.
[0148] In certain embodiments, the particle is a cell such as a stem cells, mesenchymal stromal/stem, pluripotent stem cell, embryo, myoblast, hybridoma or macrophage, examples include RAW 264.7 (TIB-71), J774A.1 (TIB-67), C2C12 (CRL-1772), L6 (CRL-1458), Sp2/0-Ag14 (CRL-1581) for use in the treatment of cancer.
Combination Strategies for Cancer Treatment:
[0149] In some embodiments, In certain embodiments, the disclosure contemplates compositions disclosed herein and using any of the compositions in combination with the administration of dendritic cell (DC)-based cancer vaccines, systemic administration of cytokines, targeted therapy using Abs or other anti-cancer agents.
[0150] In certain embodiments, the disclosure contemplates compositions disclosed herein and using any of the compositions in combination with the administration of dendritic cell (DC)-based cancer vaccines. DCs have the unique ability to take up and process antigens, move into secondary lymphoid tissues, and activate both helper and cytotoxic T cells. Preparation of DC-based cancer vaccines involves loading DCs with known tumor-specific antigens, antigenic peptides, cDNA, or RNA isolated from tumor cells. In certain embodiments, an object of this disclosure is to develop more effective methods to deliver tumor antigens to DCs. One strategy is making hybrid cells by fusing tumor cells, tumor antigens, or conjugates with DCs and using the hybrid cells as vaccines. Combination therapies with DC-based cancer vaccines may be used to treat melanoma, breast cancer, multiple myeloma, NHL, lymphatic leukemia, prostatic adenocarcinoma, lung cancer, and hepatocarcinoma
[0151] In certain embodiments, the disclosure contemplates compositions disclosed herein and using any of the compositions in combination with antigen activated DCs for cancer treatments. In one example, the compositions are used in combination with DCs fused with granulocyte macrophage colony-stimulating factor (GM-CSF) and prostatic acid phosphatase (PAP) conjugate for cancer treatments.
[0152] Provenge, an autologous DC-based vaccine, was approved by the FDA for the treatment of men with advanced prostate cancer. Provenge consists of patient-derived DCs pulsed ex vivo with a recombinant fusion protein (PA 2024) containing granulocyte macrophage colony-stimulating factor (GM-CSF) and prostatic acid phosphatase (PAP), an antigen found in 90-95% of prostate cancers.
[0153] Another cell-based approach involves using irradiated whole tumor cells as potential cancer vaccines. This strategy allows the induction of a more polyclonal immune response through the presentation of a wide array of tumor antigens. In certain embodiments, the disclosure contemplates compositions disclosed herein and using any of the compositions in combination with irradiated tumor cells for cancer treatments.
[0154] The presence of immunosuppressive cytokines in the tumor microenvironment is an important factor in the establishment of tumors. Through the secretion of immunosuppressive cytokines, such as TGF-β and IL-10, the innate and adaptive immune responses are inhibited during tumor development. In order to overcome this immunosuppression, the systemic administration of certain immunostimulatory cytokines, such as IL-2, IL-12, and IFN-α, has been used to alter the tumor microenvironment to mediate tumor recognition by immune cells. In certain embodiments, the disclosure contemplates compositions disclosed herein and using any of the compositions in combination with cytokines such as IL-2, IL-12, and INF-α for cancer treatments.
[0155] Cytokines activate immune cells, such as NK and CD8+ T cells, and can also inhibit tumor angiogenesis. In certain embodiments, the disclosure contemplates compositions disclosed herein and using any of the compositions in combination with IL-2, IL-12, and INF-α for the treatment of metastatic melanoma and renal cell carcinoma (RCC).
[0156] T-cell growth cytokine, IL-15, promotes the activation of a variety of immune cells, namely NK, NKT, and memory CD8+ T cells, and can overcome activation-induced cell death (AICD) caused by IL-2. In certain embodiments, the disclosure contemplates compositions disclosed herein and using any of the compositions in combination with IL-15 as a potential cancer immunotherapeutic agent.
[0157] In certain embodiments, the disclosure contemplates compositions disclosed herein and using any of the compositions in combination intra-tumoral administration of cytokines, modification of tumor cells to secrete cytokines, and fusion of cytokines with antibodies for cancer treatments. In one embodiment, the cytokine is TNF-α. In one embodiment the cancer is melanoma.
[0158] In certain embodiments, the disclosure contemplates compositions disclosed herein and using any of the compositions in combination with administration of soluble GM-CSF and optionally a cytokine for cancer treatments.
[0159] In certain embodiments, the disclosure contemplates compositions disclosed herein and using any of the compositions in combination with an antibody therapy for cancer treatment. In certain embodiments, the contemplated anti-bodies are directed to epidermal growth factor receptor (EGFR), human EGFR-2 (HER-2), CD20 (an unglycosylated transmembrane phosphoprotein expressed on B and T cells), CD33 (a transmembrane protein expressed on cells of myeloid lineage and also on some lymphoid cells), CD52 (a highly glycosylated 12 amino acid membrane-anchored glycosylphosphatidylinositol (GPI) protein which is expressed on all circulating lymphocytes), and VEGF. In certain embodiments the antibody may be humanized, chimeric, a radiolabeled mouse antibody for targeted radiation.
[0160] In certain embodiments, the disclosure contemplates compositions disclosed herein and using any of the compositions in combination with rituximab for the treatment of B-cell non-Hodgkin's lymphoma or chronic lymphocytic leukemia.
[0161] In certain embodiments, the disclosure contemplates compositions disclosed herein and using any of the compositions in combination with ofatumumab for the treatment of B-cell non-Hodgkin's lymphoma or chronic lymphocytic leukemia.
[0162] In certain embodiments, the disclosure contemplates compositions disclosed herein and using any of the compositions in combination with ibritumomab (tiuxetan) for the treatment of B-cell non-Hodgkin's lymphoma.
[0163] In certain embodiments, the disclosure contemplates compositions disclosed herein and using any of the compositions in combination with tositumomab for the treatment of B-cell non-Hodgkin's lymphoma.
[0164] In certain embodiments, the disclosure contemplates compositions disclosed herein and using any of the compositions in combination with gemtuzumab ozogamicin for the treatment of acute myeloid leukemia.
[0165] In certain embodiments, the disclosure contemplates compositions disclosed herein and using any of the compositions in combination with alemtuzumab for the treatment of B-cell non-Hodgkin's lymphoma or chronic lymphocytic leukemia.
[0166] In certain embodiments, the disclosure contemplates compositions disclosed herein and using any of the compositions in combination with trastuzumab for the treatment of breast cancer.
[0167] In certain embodiments, the disclosure contemplates compositions disclosed herein and using any of the compositions in combination with bevacizumab for the treatment of breast, lung, or colon cancer.
[0168] In certain embodiments, the disclosure contemplates compositions disclosed herein and using any of the compositions in combination with cetuximab for the treatment of brain and neck, or colon cancer.
[0169] In certain embodiments, the disclosure contemplates compositions disclosed herein and using any of the compositions in combination with panitumomab for the treatment of colon cancer.
[0170] In certain embodiments, the disclosure relates to methods of treating cancer comprising administering an effective amount of a particle as disclosed herein to a subject at risk of or diagnosed with cancer or a tumor optionally in combination with another anticancer agent. Other anticancer agents contemplated include gefitinib, erlotinib, docetaxel, cis-platin, 5-fluorouracil, gemcitabine, tegafur, raltitrexed, methotrexate, cytosine arabinoside, hydroxyurea, adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin, vincristine, vinblastine, vindesine, vinorelbine taxol, taxotere, etoposide, teniposide, amsacrine, topotecan, camptothecin bortezomib anegrilide, tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene fulvestrant, bicalutamide, flutamide, nilutamide, cyproterone, goserelin, leuprorelin, buserelin, megestrol, anastrozole, letrozole, vorazole, exemestane, finasteride, marimastat, trastuzumab, cetuximab, dasatinib, imatinib, bevacizumab, combretastatin, thalidomide, and/or lenalidomide or combinations thereof
Examples
Construct, Express and Purify GPI-TAAs
[0171] HER-2/neu, a surface glycoprotein, is overexpressed on many aggressive forms of breast cancer. One constructs a GPI-HER-2 by attaching extracellular domain of human HER-2 with a GPI-signal sequence and expresses it on CHOK1 cells by gene transfection. One grows CHO cells, lyses, and purifies GPI-HER-2 by affinity chromatography.
[0172] One constructs a pCDNA3.1 plasmid expression vector containing the DNA encoding the GPI-anchored form of human HER-2 attached with the GPI-anchor signal sequence from CD59 to the extracellular domain of HER-2 using PCR and ligation into the vector as described for making GPI-GM-CSF. See Poloso et al., Mol Immunol 38:803-816. One transfects CHOK1 cells with the plasmids encoding GPI-HER-2 and confirms the GPI-anchoring by PI-PLC treatment. One grows cells using roller bottles and lyses the collected cell pellets using the detergent octyl glucoside.
Purification and Incorporation of GPI-ICAM1, -IL-12, and -GM-CSF from CHOKI Transfectants
[0173] CHOK1 cells were transfected to express GPI-ICAM1 or GPI-IL-12. Expression of the GPI-ISMs was assessed by flow cytometry and verification of the GPI-anchor was confirmed by a phospholipase (PIPLC) treatment. The transfectants were grown in large quantities, lysed, and the GPI-ISMs were purified by affinity chromatography. To determine if the purified GPI-ISMs still retained the GPI-anchor and could incorporate onto lipid bilayers by protein transfer, sheep red blood cells (RBCs) were used. The GPI-ISMs were individually incubated with the RBCs at 37° C. for 2 hours, washed and then analyzed by flow cytometry. FIG. 2 demonstrates that the purified GPI-ISMs were able to incorporate onto sheep RBCs.
Optimization of Incorporation of GPI-ISMs onto Influenza H5 VLPs Using Protein Transfer
[0174] These VLPs are constructed by the rBV system through the expression of the hemagluttinin and matrix 1 protein in Sf9 insect cells. See Song et al., J Proteome Res. 2011, 10(8):3450-9.
[0175] To determine the optimal conditions for incorporation of GPI-ISMs onto influenza H5 VLPs, protein transfer was conducted at different concentrations of GPI-ISMs and at different temperatures. As the concentration of GPI-ISMs was increased, the amount of incorporation, as detected by western blot, also increased (FIG. 3). Optimal incorporation occurred at 37° C. Blotting against the H5 VLPs by using serum from mice injected with H5 VLPs showed that the VLP protein expression was not altered by incorporation of the ISMs. The kinetics of GPI ICAM1 incorporation was also determined to show that maximum incorporation occurs after only 2 hrs of incubation (FIG. 4).
Incorporation of GPI-ISMs onto H5 VLPs by Protein Transfer is GPI-Anchor Dependent.
[0176] To determine if incorporation of GPI-ISMs onto VLPs occurred via the GPI-anchor or via non-specific binding, PI-PLC treatment to cleave the GPI-anchor of GPI-ICAM1 either before incorporation (FIG. 5A) or after (FIG. 5B) incorporation was carried out. PI-PLC treatment of GPI-ICAM1 before incorporation and PI-PLC treatment of VLPs that have been
incorporated with GPI-ICAM1 both led to decreased expression of ICAM1 on VLPs as detected by Western blotting to ICAM1, whereas when the PI-PLC inhibitors, ZnCl2, or 1,10-phenanthroline, were included, expression was retained. To further confirm that incorporation occurs via the GPI-anchor, GPI-ICAM1 was incubated with 1% fatty-acid-free bovine serum albumin (BSA) or 1% orosomucoid that bind to the GPI-anchor before protein transfer in order to competitively inhibit incorporation of GPI-ICAM1 onto VLP membranes. FIG. 6 shows that GPI-ICAM1 incubated first with 1% BSA or 1% orosomucoid showed decreased incorporation onto VLPs compared to those incorporated without prior incubation with BSA or orosomucoid. Incorporation of More than One GPI-Protein Simultaneously on the Same VLPs by Protein Transfer
[0177] To determine if more than one GPI-protein could incorporate simultaneously onto the surface of VLPs by protein transfer, VLPs were incubated with GPI-ICAM1 and GPI-IL-12 simultaneously at 37° C. for 2 h. FIG. 7 shows that influenza VLPs can incorporate both GPI-ISMs on their surface and the expression of the first GPI-protein is not affected by the expression of the second GPI-protein.
Structural Integrity of VLPs Remains Intact after Protein Transfer.
[0178] To determine if the structural integrity of the VLPs remains intact after incorporation, electron microscopy of VLPs before and after incorporation was conducted. FIG. 8 shows that even after incorporation, the VLP membranes remain intact suggesting that the protein transfer method is not detrimental to the VLP structural integrity. This data show that purified GPI-proteins are able to incorporate onto influenza VLPs within 2 h at 37° C. via the GPI-anchor without disturbing the structural integrity of the VLPs.
Study Tumor Regression and Immune Responses Induced by Vaccination with VLPs Modified with GPI-HER-2 and GPI-ISMs by Protein Transfer in Mice with Established Tumors
[0179] Protein transferred-VLPs that express the GPI-HER-2 in combination with GPIISMs, such as GPI-IL-2, GPI-IL-12, GPI-B7-1, and GPI-ICAM-1, leads to tumor regression in mice with established tumors that express HER-2. Although it is not intended that the disclosure be limited by any particular mechanism, the incorporation of cytokines onto the surface of VLPs allows for a slow release depot of the cytokines into the administered microenvironment, leading to increased activation of immune effector cells at the vaccination site while decreasing chances of systemic toxicity. Furthermore, the receptors of the ISMs, IL-2, IL-12, and ICAM-1 are found on APCs allowing for enhanced adhesion and activation of the APCs by the VLPs, thus leading to enhanced uptake and presentation. The receptors for IL-12 and B7-1 are also found on other immune cells such as NK cells and mast cells, allowing for the activation of a wide variety of immune effector cells to be elicited by the association of these ISMs onto the surface of VLPs. Since the immune response is directed against the antigens found on the VLPs, incorporating TAAs along with ISMs onto the surface of VLPs will direct the immune response towards the TAAs that are overexpressed on tumor cells as well.
[0180] To determine the efficacy of VLPs incorporated with GPI-TAAs and GPI-ISMs in regressing established tumors in vivo, one inoculates BALB/c mice with 4T07 tumor cells that expressing HER-2 and then start treatment a few days later (Table 1).
TABLE-US-00001 TABLE 1 Vaccination groups (n = 9) Group Vaccination groups 1 PBS 2 VLP 3 VLP-GPI-HER-2 4 VLP-GPI-HER-2 + GPI-IL-12 + GPI-IL-2 5 VLP-GPI-HER-2 + GPI-IL-12 + GPI-IL-2 + GPI-B7-1 6 VLP-GPI-HER-2 + GPI-IL-12 + GPI-IL-2 + GPI-B7-1 + GPI-ICAM-1 7 VLP-GPI-IL-12 + GPI-IL-2 + GPI-B7-1 + GPI-ICAM-1
[0181] One injects live 4T07 tumor cells s.c. into the left flank of the mice and injects VLP in the right flank starting on days 4, 8, and 12 after tumor inoculation. If tumors do not regress, one uses a more vigorous 2-day interval immunization schedule. One monitors the mice daily and measures the size of the tumor. One screens for the production of antibodies to HER2/neu using flow cytometry or cell ELISA.
Expression of Human Breast Cancer Antigens in 4T07-WT Cells
[0182] Using the 4T07 murine breast cancer model the effects of expressing GPI-anchored immune stimulatory molecules (GPI-ISMs), namely cytokines (IL-2, IL-12) and the costimulatory protein B7-1, were investigated on the surface of the tumor cells. BALB/c mice were challenged subcutaneously (s.c.) with either wild-type 4T07 cells (4T07-WT) or 4T07 cells expressing GPI-ISMs. Significant splenomegaly was observed in the mice challenged with 4T07-WT cells relative to the mice challenged with 4T07 cells expressing GPI-ISMs. This observed splenomegaly correlated with tumor size and a 4-5 fold increase in the percentage of splenic CD11b+Gr1+MDSCs indicating the role of active immune suppression in the tumorigenicity of 4T07 breast cancer cells. Studies were conducted to analyze the effect of GPI-ISMs on infiltrating cells into the tumor microenvironment as well as in the spleen and draining lymph nodes (dLNs). Three groups of mice were challenged (s.c.) with the following cells mixed in a 1:1 ratio with BD Matrigel® (a solubilized basement membrane preparation derived from a mouse sarcoma): 4T07-WT, 4T07-B7/IL-12 or PBS (control). Seven days post challenge, the Matrigel/tumor, spleen and dLNs were harvested from the mice, digested and analyzed for cellular infiltrates by flow cytometry. The expression of GPI-ISMs on the surface of tumor cells led to reduced angiogenesis as evidenced by a reduced level of blood vessels and decreased presence of CD4+CD25+FOXP3+ regulatory T cells and CD11b+Gr1+MDSCs locally at the tumor site and dLNs as well as systemically in the spleen. Additionally, there was a decrease in CD8+PD1+ exhausted T cells at the tumor site. Along with the inhibition of immune suppressive cell populations, the GPI-ISMs increased the presence of CD4+ and CD8+ T cells as well as dendritic cells and B cells. These observations suggest that components of the active immune suppression evident in this model can be inhibited by expressing GPI-ISMs on the surface of the 4T07 tumor cells and could be effective in a therapeutic setting.
[0183] BALB/C female mice (five per group) were challenged subcutaneously (s.c.) with wild-type 4T07 or transfected 4T07-B7, GPI-IL-2, GPI-IL-12, B7/GPI-IL-2 or B7/GPI-IL-12 cells (all 2×105 cells in 100 μl PBS). Mice were injected s.c. in the rear flank and were monitored daily. Tumor size was measured using Vernier calipers every 2nd-3rd day by taking 2×2 perpendicular measurements, and tumor size (mm2) was calculated by multiplying the two diameters. Mice were euthanized when the tumor size reached close to 2 cm2. After 33 days of the initial challenge, tumor-free mice in the experimental groups were rechallenged on the opposite hind flank with wild-type 4T07 cells (2×105 in 100 μl PBS). Mice in each group were marked individually by ear punch and tumor growth was measured and recorded for each mouse separately. The wild-type and transfected tumor cell lines all began to grow tumors in vivo, but while the wild-type tumors continued to increase in size, the tumors from the modified cell lines all regressed (See FIGS. 9 and 10).
Preparation and Evaluation of hHER-2(ECD)-CD59 GPI
[0184] HER-2ECD is the extracellular portion of hHER-2. The hHER-2 extracellular domain with CD59 GPI signal sequence were join and introduced by a EcoRI site, i.e., joining region: g/aattc introduced EcoRV site (gat/atc) before sequence and Apal (gggcc/c) site after sequence at the joining region as illustrated in FIG. 11. Before the sequence, an optimized IL-2 Kozak sequence along with the restriction enzyme sites HindIII and KpnI were added. Following the hHER2ECD sequence an EcoRI site is added. At base pair position 1365 of hHER2, a change in base pair from T was made to C in order to remove an EcoRI restriction enzyme site at this position, however, the final amino acid still remains as an isoleucine. (2015 bp). FIG. 12 shows flow cytometry analysis of CHO cells expressing GPI-human HER-2 (hHER-2-CD59) using TA1 mAb. Testing shows that HER-2 expressed in CHO cells is GPI-anchored. PIPLC is an enzyme which cleaves GPI anchor, reduces the level of expression. PI-PLC treated CHOK1-hHER-2ECD-CD59 cells reduced hHER-2 cell surface expression by 98.4%. PIPLC will not have any effect on normal HER-2.
[0185] Nucleic acid encoding the hHER-2 extracellular domain E (Amino Acids 22-652) and GPI-anchor signal sequence (SEQ ID NO: 7) AAGGGGAGGT AACCCTGGCC CCTTTGGTCG GGGCCCCGGG CAGCCGCGCG CCCCTTCCCA CGGGGCCCTT TACTGCGCCG CGCGCCCGGC CCCCACCCCT CGCAGCACCC CGCGCCCCGC GCCCTCCCAG CCGGGTCCAG CCGGAGCCAT GGGGCCGGAGGATATC CCGCAGTGAG CACCATGGAG CTGGCGGCCT TGTGCCGCTG GGGGCTCCTC CTCGCCCTCT TGCCCCCCGG AGCCGCGAGC ACCCAAGTGT GCACCGGCAC AGACATGAAG CTGCGGCTCC CTGCCAGTCC CGAGACCCACCTGGACATGC TCCGCCACCT CTACCAGGGC TGCCAGGTGG TGCAGGGAAA CCTGGAACTC ACCTACCTGC CCACCAATGC CAGCCTGTCC TTCCTGCAGG ATATCCAGGA GGTGCAGGGC TACGTGCTCA TCGCTCACAA CCAAGTGAGG CAGGTCCCAC TGCAGAGGCT GCGGATTGTG CGAGGCACCC AGCTCTTTGA GGACAACTAT GCCCTGGCCG TGCTAGACAA TGGAGACCCG CTGAACAATA CCACCCCTGT CACAGGGGCC TCCCCAGGAG GCCTGCGGGA GCTGCAGCTT CGAAGCCTCA CAGAGATCTT GAAAGGAGGG GTCTTGATCC AGCGGAACCC CCAGCTCTGC TACCAGGACA CGATTTTGTG GAAGGACATC TTCCACAAGA ACAACCAGCT GGCTCTCACACTGATAGACA CCAACCGCTC TCGGGCCTGC CACCCCTGTT CTCCGATGTG TAAGGGCTCC CGCTGCTGGG GAGAGAGTTC TGAGGATTGT CAGAGCCTGA CGCGCACTGT CTGTGCCGGT GGCTGTGCCC GCTGCAAGGG GCCACTGCCC ACTGACTGCT GCCATGAGCA GTGTGCTGCC GGCTGCACGG GCCCCAAGCA CTCTGACTGC CTGGCCTGCC TCCACTTCAA CCACAGTGGC ATCTGTGAGC TGCACTGCCC AGCCCTGGTC ACCTACAACA CAGACACGTT TGAGTCCATG CCCAATCCCG AGGGCCGGTA TACATTCGGC GCCAGCTGTG TGACTGCCTG TCCCTACAAC TACCTTTCTA CGGACGTGGG ATCCTGCACC CTCGTCTGCC CCCTGCACAA CCAAGAGGTG ACAGCAGAGG ATGGAACACA GCGGTGTGAG AAGTGCAGCA AGCCCTGTGC CCGAGTGTGC TATGGTCTGG GCATGGAGCA CTTGCGAGAG GTGAGGGCAG TTACCAGTGC CAATATCCAG GAGTTTGCTG GCTGCAAGAA GATCTTTGGG AGCCTGGCAT TTCTGCCGGA GAGCTTTGAT GGGGACCCAG CCTCCAACAC TGCCCCGCTC CAGCCAGAGC AGCTCCAAGT GTTTGAGACT CTGGAAGAGA TCACAGGTTA CCTATACATC TCAGCATGGC CGGACAGCCT GCCTGACCTC AGCGTCTTCC AGAACCTGCA AGTAATCCGG GGACGAATTC TGCACAATGG CGCCTACTCG CTGACCCTGC AAGGGCTGGG CATCAGCTGG CTGGGGCTGC GCTCACTGAG GGAACTGGGC AGTGGACTGG CCCTCATCCA CCATAACACC CACCTCTGCT TCGTGCACAC GGTGCCCTGG GACCAGCTCT TTCGGAACCC GCACCAAGCT CTGCTCCACA CTGCCAACCG GCCAGAGGAC GAGTGTGTGG GCGAGGGCCT GGCCTGCCAC CAGCTGTGCG CCCGAGGGCA CTGCTGGGGT CCAGGGCCCA CCCAGTGTGT CAACTGCAGC CAGTTCCTTC GGGGCCAGGA GTGCGTGGAGGAATGCCGAG TACTGCAGGG GCTCCCCAGG GAGTATGTGA ATGCCAGGCA CTGTTTGCCGTGCCACCCTG AGTGTCAGCC CCAGAATGGC TCAGTGACCT GTTTTGGACC GGAGGCTGACCAGTGTGTGG CCTGTGCCCA CTATAAGGAC CCTCCCTTCT GCGTGGCCCG CTGCCCCAGC GGTGTGAAAC CTGACCTCTC CTACATGCCC ATCTGGAAGT TTCCAGATGA GGAGGGCGCA TGCCAGCCTT GCCCCATCAA CTGCACCCAC TCCTGTGTGG ACCTGGATGA CAAGGGCTGC CCCGCCGAGC AGAGAGCCAG CCCTCTGACGGAATTC CTTGAAAATGGTGGGACATCCTTATCAGAGAAAACAGTTCTTCTGCTGGT GACTCCATTTCTGGCAGCAGCCTGGAGCCTTCATCCCTAACAGAAG GCCAAGGGGCCCTCCG
Sequence CWU
1
1
81288PRTHomo sapiens 1Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys
Cys Pro Tyr 1 5 10 15
Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys
20 25 30 Ser Gly Val Ile
His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu 35
40 45 Ser Cys Gly His Asn Val Ser Val Glu
Glu Leu Ala Gln Thr Arg Ile 50 55
60 Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met
Ser Gly Asp 65 70 75
80 Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr
85 90 95 Asn Asn Leu Ser
Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly 100
105 110 Thr Tyr Glu Cys Val Val Leu Lys Tyr
Glu Lys Asp Ala Phe Lys Arg 115 120
125 Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe
Pro Thr 130 135 140
Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile 145
150 155 160 Ile Cys Ser Thr Ser
Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu 165
170 175 Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn
Thr Thr Val Ser Gln Asp 180 185
190 Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn
Met 195 200 205 Thr
Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg 210
215 220 Val Asn Gln Thr Phe Asn
Trp Asn Thr Thr Lys Gln Glu His Phe Pro 225 230
235 240 Asp Asn Leu Leu Pro Ser Trp Ala Ile Thr Leu
Ile Ser Val Asn Gly 245 250
255 Ile Phe Val Ile Cys Cys Leu Thr Tyr Cys Phe Ala Pro Arg Cys Arg
260 265 270 Glu Arg
Arg Arg Asn Glu Arg Leu Arg Arg Glu Ser Val Arg Pro Val 275
280 285 2208PRTHomo sapiens 2Val
Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys 1
5 10 15 Gly His Asn Val Ser Val
Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp 20
25 30 Gln Lys Glu Lys Lys Met Val Leu Thr Met
Met Ser Gly Asp Met Asn 35 40
45 Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr
Asn Asn 50 55 60
Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Tyr 65
70 75 80 Glu Cys Val Val Leu
Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His 85
90 95 Leu Ala Glu Val Thr Leu Ser Val Lys Ala
Asp Phe Pro Thr Pro Ser 100 105
110 Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile
Cys 115 120 125 Ser
Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn 130
135 140 Gly Glu Glu Leu Asn Ala
Ile Asn Thr Thr Val Ser Gln Asp Pro Glu 145 150
155 160 Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp
Phe Asn Met Thr Thr 165 170
175 Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn
180 185 190 Gln Thr
Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn 195
200 205 3126PRTHomo sapiens 3Lys
Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg 1
5 10 15 Gly Ile Ala Ser Phe Val
Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr 20
25 30 Glu Val Arg Val Thr Val Leu Arg Gln Ala
Asp Ser Gln Val Thr Glu 35 40
45 Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe
Leu Asp 50 55 60
Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr 65
70 75 80 Ile Gln Gly Leu Arg
Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val 85
90 95 Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu
Gly Ile Gly Asn Gly Ala 100 105
110 Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp
115 120 125 4607PRTHomo sapiens
4Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser 1
5 10 15 Pro Glu Thr His
Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln 20
25 30 Val Val Gln Gly Asn Leu Glu Leu Thr
Tyr Leu Pro Thr Asn Ala Ser 35 40
45 Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val
Leu Ile 50 55 60
Ala His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val 65
70 75 80 Arg Gly Thr Gln Leu
Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp 85
90 95 Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro
Val Thr Gly Ala Ser Pro 100 105
110 Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu
Lys 115 120 125 Gly
Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr 130
135 140 Ile Leu Trp Lys Asp Ile
Phe His Lys Asn Asn Gln Leu Ala Leu Thr 145 150
155 160 Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His
Pro Cys Ser Pro Met 165 170
175 Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser
180 185 190 Leu Thr
Arg Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro 195
200 205 Leu Pro Thr Asp Cys Cys His
Glu Gln Cys Ala Ala Gly Cys Thr Gly 210 215
220 Pro Lys His Ser Asp Cys Leu Ala Cys Leu His Phe
Asn His Ser Gly 225 230 235
240 Ile Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr
245 250 255 Phe Glu Ser
Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser 260
265 270 Cys Val Thr Ala Cys Pro Tyr Asn
Tyr Leu Ser Thr Asp Val Gly Ser 275 280
285 Cys Thr Leu Val Cys Pro Leu His Asn Gln Glu Val Thr
Ala Glu Asp 290 295 300
Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val Cys 305
310 315 320 Tyr Gly Leu Gly
Met Glu His Leu Arg Glu Val Arg Ala Val Thr Ser 325
330 335 Ala Asn Ile Gln Glu Phe Ala Gly Cys
Lys Lys Ile Phe Gly Ser Leu 340 345
350 Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn
Thr Ala 355 360 365
Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu Ile 370
375 380 Thr Gly Tyr Leu Tyr
Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp Leu 385 390
395 400 Ser Val Phe Gln Asn Leu Gln Val Ile Arg
Gly Arg Ile Leu His Asn 405 410
415 Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu
Gly 420 425 430 Leu
Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His His 435
440 445 Asn Thr His Leu Cys Phe
Val His Thr Val Pro Trp Asp Gln Leu Phe 450 455
460 Arg Asn Pro His Gln Ala Leu Leu His Thr Ala
Asn Arg Pro Glu Asp 465 470 475
480 Glu Cys Val Gly Glu Gly Leu Ala Cys His Gln Leu Cys Ala Arg Gly
485 490 495 His Cys
Trp Gly Pro Gly Pro Thr Gln Cys Val Asn Cys Ser Gln Phe 500
505 510 Leu Arg Gly Gln Glu Cys Val
Glu Glu Cys Arg Val Leu Gln Gly Leu 515 520
525 Pro Arg Glu Tyr Val Asn Ala Arg His Cys Leu Pro
Cys His Pro Glu 530 535 540
Cys Gln Pro Gln Asn Gly Ser Val Thr Cys Phe Gly Pro Glu Ala Asp 545
550 555 560 Gln Cys Val
Ala Cys Ala His Tyr Lys Asp Pro Pro Phe Cys Val Ala 565
570 575 Arg Cys Pro Ser Gly Val Lys Pro
Asp Leu Ser Tyr Met Pro Ile Trp 580 585
590 Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys Pro
Ile Asn 595 600 605
5214PRTHomo sapiens 5Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30 Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95 Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100
105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser Gly 115 120
125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145
150 155 160 Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165
170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 180 185
190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
Ser 195 200 205 Phe
Asn Arg Gly Glu Cys 210 655PRTHomo sapiens 6Gly Thr
Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys 1 5
10 15 Val Asn Gly Gly Glu Cys Phe
Met Val Lys Asp Leu Ser Asn Pro Ser 20 25
30 Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly
Asp Arg Cys Gln 35 40 45
Asn Tyr Val Met Ala Ser Phe 50 55
72254PRTHomo sapiens 7Ala Ala Gly Gly Gly Gly Ala Gly Gly Thr Ala Ala Cys
Cys Cys Thr 1 5 10 15
Gly Gly Cys Cys Cys Cys Thr Thr Thr Gly Gly Thr Cys Gly Gly Gly
20 25 30 Gly Cys Cys Cys
Cys Gly Gly Gly Cys Ala Gly Cys Cys Gly Cys Gly 35
40 45 Cys Gly Cys Cys Cys Cys Thr Thr Cys
Cys Cys Ala Cys Gly Gly Gly 50 55
60 Gly Cys Cys Cys Thr Thr Thr Ala Cys Thr Gly Cys Gly
Cys Cys Gly 65 70 75
80 Cys Gly Cys Gly Cys Cys Cys Gly Gly Cys Cys Cys Cys Cys Ala Cys
85 90 95 Cys Cys Cys Thr
Cys Gly Cys Ala Gly Cys Ala Cys Cys Cys Cys Gly 100
105 110 Cys Gly Cys Cys Cys Cys Gly Cys Gly
Cys Cys Cys Thr Cys Cys Cys 115 120
125 Ala Gly Cys Cys Gly Gly Gly Thr Cys Cys Ala Gly Cys Cys
Gly Gly 130 135 140
Ala Gly Cys Cys Ala Thr Gly Gly Gly Gly Cys Cys Gly Gly Ala Gly 145
150 155 160 Gly Ala Thr Ala Thr
Cys Cys Cys Gly Cys Ala Gly Thr Gly Ala Gly 165
170 175 Cys Ala Cys Cys Ala Thr Gly Gly Ala Gly
Cys Thr Gly Gly Cys Gly 180 185
190 Gly Cys Cys Thr Thr Gly Thr Gly Cys Cys Gly Cys Thr Gly Gly
Gly 195 200 205 Gly
Gly Cys Thr Cys Cys Thr Cys Cys Thr Cys Gly Cys Cys Cys Thr 210
215 220 Cys Thr Thr Gly Cys Cys
Cys Cys Cys Cys Gly Gly Ala Gly Cys Cys 225 230
235 240 Gly Cys Gly Ala Gly Cys Ala Cys Cys Cys Ala
Ala Gly Thr Gly Thr 245 250
255 Gly Cys Ala Cys Cys Gly Gly Cys Ala Cys Ala Gly Ala Cys Ala Thr
260 265 270 Gly Ala
Ala Gly Cys Thr Gly Cys Gly Gly Cys Thr Cys Cys Cys Thr 275
280 285 Gly Cys Cys Ala Gly Thr Cys
Cys Cys Gly Ala Gly Ala Cys Cys Cys 290 295
300 Ala Cys Cys Thr Gly Gly Ala Cys Ala Thr Gly Cys
Thr Cys Cys Gly 305 310 315
320 Cys Cys Ala Cys Cys Thr Cys Thr Ala Cys Cys Ala Gly Gly Gly Cys
325 330 335 Thr Gly Cys
Cys Ala Gly Gly Thr Gly Gly Thr Gly Cys Ala Gly Gly 340
345 350 Gly Ala Ala Ala Cys Cys Thr Gly
Gly Ala Ala Cys Thr Cys Ala Cys 355 360
365 Cys Thr Ala Cys Cys Thr Gly Cys Cys Cys Ala Cys Cys
Ala Ala Thr 370 375 380
Gly Cys Cys Ala Gly Cys Cys Thr Gly Thr Cys Cys Thr Thr Cys Cys 385
390 395 400 Thr Gly Cys Ala
Gly Gly Ala Thr Ala Thr Cys Cys Ala Gly Gly Ala 405
410 415 Gly Gly Thr Gly Cys Ala Gly Gly Gly
Cys Thr Ala Cys Gly Thr Gly 420 425
430 Cys Thr Cys Ala Thr Cys Gly Cys Thr Cys Ala Cys Ala Ala
Cys Cys 435 440 445
Ala Ala Gly Thr Gly Ala Gly Gly Cys Ala Gly Gly Thr Cys Cys Cys 450
455 460 Ala Cys Thr Gly Cys
Ala Gly Ala Gly Gly Cys Thr Gly Cys Gly Gly 465 470
475 480 Ala Thr Thr Gly Thr Gly Cys Gly Ala Gly
Gly Cys Ala Cys Cys Cys 485 490
495 Ala Gly Cys Thr Cys Thr Thr Thr Gly Ala Gly Gly Ala Cys Ala
Ala 500 505 510 Cys
Thr Ala Thr Gly Cys Cys Cys Thr Gly Gly Cys Cys Gly Thr Gly 515
520 525 Cys Thr Ala Gly Ala Cys
Ala Ala Thr Gly Gly Ala Gly Ala Cys Cys 530 535
540 Cys Gly Cys Thr Gly Ala Ala Cys Ala Ala Thr
Ala Cys Cys Ala Cys 545 550 555
560 Cys Cys Cys Thr Gly Thr Cys Ala Cys Ala Gly Gly Gly Gly Cys Cys
565 570 575 Thr Cys
Cys Cys Cys Ala Gly Gly Ala Gly Gly Cys Cys Thr Gly Cys 580
585 590 Gly Gly Gly Ala Gly Cys Thr
Gly Cys Ala Gly Cys Thr Thr Cys Gly 595 600
605 Ala Ala Gly Cys Cys Thr Cys Ala Cys Ala Gly Ala
Gly Ala Thr Cys 610 615 620
Thr Thr Gly Ala Ala Ala Gly Gly Ala Gly Gly Gly Gly Thr Cys Thr 625
630 635 640 Thr Gly Ala
Thr Cys Cys Ala Gly Cys Gly Gly Ala Ala Cys Cys Cys 645
650 655 Cys Cys Ala Gly Cys Thr Cys Thr
Gly Cys Thr Ala Cys Cys Ala Gly 660 665
670 Gly Ala Cys Ala Cys Gly Ala Thr Thr Thr Thr Gly Thr
Gly Gly Ala 675 680 685
Ala Gly Gly Ala Cys Ala Thr Cys Thr Thr Cys Cys Ala Cys Ala Ala 690
695 700 Gly Ala Ala Cys
Ala Ala Cys Cys Ala Gly Cys Thr Gly Gly Cys Thr 705 710
715 720 Cys Thr Cys Ala Cys Ala Cys Thr Gly
Ala Thr Ala Gly Ala Cys Ala 725 730
735 Cys Cys Ala Ala Cys Cys Gly Cys Thr Cys Thr Cys Gly Gly
Gly Cys 740 745 750
Cys Thr Gly Cys Cys Ala Cys Cys Cys Cys Thr Gly Thr Thr Cys Thr
755 760 765 Cys Cys Gly Ala
Thr Gly Thr Gly Thr Ala Ala Gly Gly Gly Cys Thr 770
775 780 Cys Cys Cys Gly Cys Thr Gly Cys
Thr Gly Gly Gly Gly Ala Gly Ala 785 790
795 800 Gly Ala Gly Thr Thr Cys Thr Gly Ala Gly Gly Ala
Thr Thr Gly Thr 805 810
815 Cys Ala Gly Ala Gly Cys Cys Thr Gly Ala Cys Gly Cys Gly Cys Ala
820 825 830 Cys Thr Gly
Thr Cys Thr Gly Thr Gly Cys Cys Gly Gly Thr Gly Gly 835
840 845 Cys Thr Gly Thr Gly Cys Cys Cys
Gly Cys Thr Gly Cys Ala Ala Gly 850 855
860 Gly Gly Gly Cys Cys Ala Cys Thr Gly Cys Cys Cys Ala
Cys Thr Gly 865 870 875
880 Ala Cys Thr Gly Cys Thr Gly Cys Cys Ala Thr Gly Ala Gly Cys Ala
885 890 895 Gly Thr Gly Thr
Gly Cys Thr Gly Cys Cys Gly Gly Cys Thr Gly Cys 900
905 910 Ala Cys Gly Gly Gly Cys Cys Cys Cys
Ala Ala Gly Cys Ala Cys Thr 915 920
925 Cys Thr Gly Ala Cys Thr Gly Cys Cys Thr Gly Gly Cys Cys
Thr Gly 930 935 940
Cys Cys Thr Cys Cys Ala Cys Thr Thr Cys Ala Ala Cys Cys Ala Cys 945
950 955 960 Ala Gly Thr Gly Gly
Cys Ala Thr Cys Thr Gly Thr Gly Ala Gly Cys 965
970 975 Thr Gly Cys Ala Cys Thr Gly Cys Cys Cys
Ala Gly Cys Cys Cys Thr 980 985
990 Gly Gly Thr Cys Ala Cys Cys Thr Ala Cys Ala Ala Cys Ala
Cys Ala 995 1000 1005
Gly Ala Cys Ala Cys Gly Thr Thr Thr Gly Ala Gly Thr Cys Cys 1010
1015 1020 Ala Thr Gly Cys Cys
Cys Ala Ala Thr Cys Cys Cys Gly Ala Gly 1025 1030
1035 Gly Gly Cys Cys Gly Gly Thr Ala Thr Ala
Cys Ala Thr Thr Cys 1040 1045 1050
Gly Gly Cys Gly Cys Cys Ala Gly Cys Thr Gly Thr Gly Thr Gly
1055 1060 1065 Ala Cys
Thr Gly Cys Cys Thr Gly Thr Cys Cys Cys Thr Ala Cys 1070
1075 1080 Ala Ala Cys Thr Ala Cys Cys
Thr Thr Thr Cys Thr Ala Cys Gly 1085 1090
1095 Gly Ala Cys Gly Thr Gly Gly Gly Ala Thr Cys Cys
Thr Gly Cys 1100 1105 1110
Ala Cys Cys Cys Thr Cys Gly Thr Cys Thr Gly Cys Cys Cys Cys 1115
1120 1125 Cys Thr Gly Cys Ala
Cys Ala Ala Cys Cys Ala Ala Gly Ala Gly 1130 1135
1140 Gly Thr Gly Ala Cys Ala Gly Cys Ala Gly
Ala Gly Gly Ala Thr 1145 1150 1155
Gly Gly Ala Ala Cys Ala Cys Ala Gly Cys Gly Gly Thr Gly Thr
1160 1165 1170 Gly Ala
Gly Ala Ala Gly Thr Gly Cys Ala Gly Cys Ala Ala Gly 1175
1180 1185 Cys Cys Cys Thr Gly Thr Gly
Cys Cys Cys Gly Ala Gly Thr Gly 1190 1195
1200 Thr Gly Cys Thr Ala Thr Gly Gly Thr Cys Thr Gly
Gly Gly Cys 1205 1210 1215
Ala Thr Gly Gly Ala Gly Cys Ala Cys Thr Thr Gly Cys Gly Ala 1220
1225 1230 Gly Ala Gly Gly Thr
Gly Ala Gly Gly Gly Cys Ala Gly Thr Thr 1235 1240
1245 Ala Cys Cys Ala Gly Thr Gly Cys Cys Ala
Ala Thr Ala Thr Cys 1250 1255 1260
Cys Ala Gly Gly Ala Gly Thr Thr Thr Gly Cys Thr Gly Gly Cys
1265 1270 1275 Thr Gly
Cys Ala Ala Gly Ala Ala Gly Ala Thr Cys Thr Thr Thr 1280
1285 1290 Gly Gly Gly Ala Gly Cys Cys
Thr Gly Gly Cys Ala Thr Thr Thr 1295 1300
1305 Cys Thr Gly Cys Cys Gly Gly Ala Gly Ala Gly Cys
Thr Thr Thr 1310 1315 1320
Gly Ala Thr Gly Gly Gly Gly Ala Cys Cys Cys Ala Gly Cys Cys 1325
1330 1335 Thr Cys Cys Ala Ala
Cys Ala Cys Thr Gly Cys Cys Cys Cys Gly 1340 1345
1350 Cys Thr Cys Cys Ala Gly Cys Cys Ala Gly
Ala Gly Cys Ala Gly 1355 1360 1365
Cys Thr Cys Cys Ala Ala Gly Thr Gly Thr Thr Thr Gly Ala Gly
1370 1375 1380 Ala Cys
Thr Cys Thr Gly Gly Ala Ala Gly Ala Gly Ala Thr Cys 1385
1390 1395 Ala Cys Ala Gly Gly Thr Thr
Ala Cys Cys Thr Ala Thr Ala Cys 1400 1405
1410 Ala Thr Cys Thr Cys Ala Gly Cys Ala Thr Gly Gly
Cys Cys Gly 1415 1420 1425
Gly Ala Cys Ala Gly Cys Cys Thr Gly Cys Cys Thr Gly Ala Cys 1430
1435 1440 Cys Thr Cys Ala Gly
Cys Gly Thr Cys Thr Thr Cys Cys Ala Gly 1445 1450
1455 Ala Ala Cys Cys Thr Gly Cys Ala Ala Gly
Thr Ala Ala Thr Cys 1460 1465 1470
Cys Gly Gly Gly Gly Ala Cys Gly Ala Ala Thr Thr Cys Thr Gly
1475 1480 1485 Cys Ala
Cys Ala Ala Thr Gly Gly Cys Gly Cys Cys Thr Ala Cys 1490
1495 1500 Thr Cys Gly Cys Thr Gly Ala
Cys Cys Cys Thr Gly Cys Ala Ala 1505 1510
1515 Gly Gly Gly Cys Thr Gly Gly Gly Cys Ala Thr Cys
Ala Gly Cys 1520 1525 1530
Thr Gly Gly Cys Thr Gly Gly Gly Gly Cys Thr Gly Cys Gly Cys 1535
1540 1545 Thr Cys Ala Cys Thr
Gly Ala Gly Gly Gly Ala Ala Cys Thr Gly 1550 1555
1560 Gly Gly Cys Ala Gly Thr Gly Gly Ala Cys
Thr Gly Gly Cys Cys 1565 1570 1575
Cys Thr Cys Ala Thr Cys Cys Ala Cys Cys Ala Thr Ala Ala Cys
1580 1585 1590 Ala Cys
Cys Cys Ala Cys Cys Thr Cys Thr Gly Cys Thr Thr Cys 1595
1600 1605 Gly Thr Gly Cys Ala Cys Ala
Cys Gly Gly Thr Gly Cys Cys Cys 1610 1615
1620 Thr Gly Gly Gly Ala Cys Cys Ala Gly Cys Thr Cys
Thr Thr Thr 1625 1630 1635
Cys Gly Gly Ala Ala Cys Cys Cys Gly Cys Ala Cys Cys Ala Ala 1640
1645 1650 Gly Cys Thr Cys Thr
Gly Cys Thr Cys Cys Ala Cys Ala Cys Thr 1655 1660
1665 Gly Cys Cys Ala Ala Cys Cys Gly Gly Cys
Cys Ala Gly Ala Gly 1670 1675 1680
Gly Ala Cys Gly Ala Gly Thr Gly Thr Gly Thr Gly Gly Gly Cys
1685 1690 1695 Gly Ala
Gly Gly Gly Cys Cys Thr Gly Gly Cys Cys Thr Gly Cys 1700
1705 1710 Cys Ala Cys Cys Ala Gly Cys
Thr Gly Thr Gly Cys Gly Cys Cys 1715 1720
1725 Cys Gly Ala Gly Gly Gly Cys Ala Cys Thr Gly Cys
Thr Gly Gly 1730 1735 1740
Gly Gly Thr Cys Cys Ala Gly Gly Gly Cys Cys Cys Ala Cys Cys 1745
1750 1755 Cys Ala Gly Thr Gly
Thr Gly Thr Cys Ala Ala Cys Thr Gly Cys 1760 1765
1770 Ala Gly Cys Cys Ala Gly Thr Thr Cys Cys
Thr Thr Cys Gly Gly 1775 1780 1785
Gly Gly Cys Cys Ala Gly Gly Ala Gly Thr Gly Cys Gly Thr Gly
1790 1795 1800 Gly Ala
Gly Gly Ala Ala Thr Gly Cys Cys Gly Ala Gly Thr Ala 1805
1810 1815 Cys Thr Gly Cys Ala Gly Gly
Gly Gly Cys Thr Cys Cys Cys Cys 1820 1825
1830 Ala Gly Gly Gly Ala Gly Thr Ala Thr Gly Thr Gly
Ala Ala Thr 1835 1840 1845
Gly Cys Cys Ala Gly Gly Cys Ala Cys Thr Gly Thr Thr Thr Gly 1850
1855 1860 Cys Cys Gly Thr Gly
Cys Cys Ala Cys Cys Cys Thr Gly Ala Gly 1865 1870
1875 Thr Gly Thr Cys Ala Gly Cys Cys Cys Cys
Ala Gly Ala Ala Thr 1880 1885 1890
Gly Gly Cys Thr Cys Ala Gly Thr Gly Ala Cys Cys Thr Gly Thr
1895 1900 1905 Thr Thr
Thr Gly Gly Ala Cys Cys Gly Gly Ala Gly Gly Cys Thr 1910
1915 1920 Gly Ala Cys Cys Ala Gly Thr
Gly Thr Gly Thr Gly Gly Cys Cys 1925 1930
1935 Thr Gly Thr Gly Cys Cys Cys Ala Cys Thr Ala Thr
Ala Ala Gly 1940 1945 1950
Gly Ala Cys Cys Cys Thr Cys Cys Cys Thr Thr Cys Thr Gly Cys 1955
1960 1965 Gly Thr Gly Gly Cys
Cys Cys Gly Cys Thr Gly Cys Cys Cys Cys 1970 1975
1980 Ala Gly Cys Gly Gly Thr Gly Thr Gly Ala
Ala Ala Cys Cys Thr 1985 1990 1995
Gly Ala Cys Cys Thr Cys Thr Cys Cys Thr Ala Cys Ala Thr Gly
2000 2005 2010 Cys Cys
Cys Ala Thr Cys Thr Gly Gly Ala Ala Gly Thr Thr Thr 2015
2020 2025 Cys Cys Ala Gly Ala Thr Gly
Ala Gly Gly Ala Gly Gly Gly Cys 2030 2035
2040 Gly Cys Ala Thr Gly Cys Cys Ala Gly Cys Cys Thr
Thr Gly Cys 2045 2050 2055
Cys Cys Cys Ala Thr Cys Ala Ala Cys Thr Gly Cys Ala Cys Cys 2060
2065 2070 Cys Ala Cys Thr Cys
Cys Thr Gly Thr Gly Thr Gly Gly Ala Cys 2075 2080
2085 Cys Thr Gly Gly Ala Thr Gly Ala Cys Ala
Ala Gly Gly Gly Cys 2090 2095 2100
Thr Gly Cys Cys Cys Cys Gly Cys Cys Gly Ala Gly Cys Ala Gly
2105 2110 2115 Ala Gly
Ala Gly Cys Cys Ala Gly Cys Cys Cys Thr Cys Thr Gly 2120
2125 2130 Ala Cys Gly Gly Ala Ala Thr
Thr Cys Cys Thr Thr Gly Ala Ala 2135 2140
2145 Ala Ala Thr Gly Gly Thr Gly Gly Gly Ala Cys Ala
Thr Cys Cys 2150 2155 2160
Thr Thr Ala Thr Cys Ala Gly Ala Gly Ala Ala Ala Ala Cys Ala 2165
2170 2175 Gly Thr Thr Cys Thr
Thr Cys Thr Gly Cys Thr Gly Gly Thr Gly 2180 2185
2190 Ala Cys Thr Cys Cys Ala Thr Thr Thr Cys
Thr Gly Gly Cys Ala 2195 2200 2205
Gly Cys Ala Gly Cys Cys Thr Gly Gly Ala Gly Cys Cys Thr Thr
2210 2215 2220 Cys Ala
Thr Cys Cys Cys Thr Ala Ala Cys Ala Gly Ala Ala Gly 2225
2230 2235 Gly Cys Cys Ala Ala Gly Gly
Gly Gly Cys Cys Cys Thr Cys Cys 2240 2245
2250 Gly 822PRTHomo sapiens 8Thr Gly Ala Cys Thr Gly
Thr Gly Ala Ala Cys Gly Thr Thr Cys Gly 1 5
10 15 Ala Gly Ala Thr Gly Ala 20
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