WO2012091756A1 - Non-natural mic proteins - Google Patents
Non-natural mic proteins Download PDFInfo
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- WO2012091756A1 WO2012091756A1 PCT/US2011/042955 US2011042955W WO2012091756A1 WO 2012091756 A1 WO2012091756 A1 WO 2012091756A1 US 2011042955 W US2011042955 W US 2011042955W WO 2012091756 A1 WO2012091756 A1 WO 2012091756A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/473—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used alpha-Glycoproteins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70539—MHC-molecules, e.g. HLA-molecules
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K2318/00—Antibody mimetics or scaffolds
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the instant invention relates generally to non-natural protein molecules that can recruit and activate NK cells, and more specifically to non-natural, monomeric, soluble, mammalian MHC class I chain-related (MIC) molecules modified within the a3 domain to contain a heterologous peptide that binds a target molecule on target cell.
- MIC mammalian MHC class I chain-related
- NK cells and certain T-cells of the immunity system have important roles in humans and other mammals as first-line, innate defense against neoplastic and virus-infected cells (Cerwenka, A., and L.L. Lanier. 2001. NK cells, viruses and cancer. Nat. Rev. Immunol. 1 :41-49).
- NK cells and certain T-cells exhibit on their surfaces NKG2D, a prominent, homodimeric, surface immunoreceptor responsible for recognizing a target cell and activating the innate defense against the pathologic cell (Lanier, LL, 1998. NK cell receptors. Ann. Rev. Immunol.
- the human NKG2D molecule possesses a C-type lectin-like extracellular domain that binds to its cognate ligands, the 84% sequence identical or homologous, monomeric MICA [soluble form of MICA set forth in SEQ ID NOs: 1-6 and 13] and MICB [full protein of MICB set forth in SEQ ID NOs: 7-12], polymorphic analogs of the Major Histocompatibility Complex (MHC) Class I chain-related glycoproteins (MIC) (Weis et al. 1998. The C-type lectin superfamily of the immune system. Immunol. Rev. 163: 19-34; Bahram et al. 1994. A second lineage of mammalian MHC class I genes.
- MHC Major Histocompatibility Complex
- Viral infection is a common inducer of MIC protein expression and identifies the viral-infected cell for NK or T-cell attack (Groh et al. 1998; Groh et al. 2001. Co- stimulation of CD8+ ⁇ -cells by NKG2D via engagement by MIC induced on virus- infected cells. Nat. Immunol. 2: 255-260; Cerwenka, A., and L.L. Lanier. 2001).
- cytomegalovirus and other viruses have evolved mechanisms that prevent the expression of MIC proteins on the surface of the cell they infect in order to escape the wrath of the innate immunity system (Lodoen, M., K.
- cytomegalovirus expressing a ligand for the NKG2D receptor is attenuated and has improved vaccine properties. J. Clin. Invest. 120: 4532-4545).
- malignant cells such as those of lung cancer and glioblastoma brain cancer, also avoid the expression of MIC proteins and as a result may be particularly aggressive as they too escape the innate immunity system (Busche, A et al. 2006, NK cell mediated rejection of experimental human lung cancer by genetic over expression of MHC class I chain-related gene A.
- This invention describes soluble, monomeric, non-natural protein molecules that can recruit and activate NK cells and certain T-cells to attack specific cellular target cells by, after administration to a mammal, attaching the NKG2D-binding portions of MICA or MICB protein, i.e., their al-a2 platform domain, specifically to the intended target molecule or molecules on the cellular target via a molecular targeting motif of the non-natural protein molecules of the invention.
- MIC mammalian MHC class I chain-related
- the heterologous peptide or peptides are inserted into the MIC a3 domain within one or more sites selected from loop 1, loop 2, and loop 3.
- loop 1 corresponds to amino acids numbers 190-199
- loop 2 corresponds to amino acid residues 221-228
- loop 3 corresponds to amino acid residues 250-258 of the a3 domain of a MIC protein selected from the group consisting of SEQ ID NOs: l-13.
- the MIC molecule is glycosylated.
- non-natural MIC proteins the l-a2 platform domain and the a3 domain are from a human MIC protein.
- the l-a2 platform domain and the a3 domain are from a human MIC protein.
- the al-a2 platform domain and the a3 domain are from a human MICA protein selected from the group consisting of SEQ ID NOs: 1-6, and 13.
- a human MICA protein selected from the group consisting of SEQ ID NOs: 1-6, and 13.
- the al-a2 platform domain and the a3 domain are from a human MICB protein selected from the group consisting of SEQ ID NOs:7-12.
- the MICA or MICB protein is lacking its transmembrane domain.
- the a3 domain of the non-natural MIC molecule is a complete native a3 domain without a deletion.
- the a3 domain is a native a3 domain, wherein a portion of the domain has been deleted.
- the portion deleted from the a3 domain is adjacent to the insertion site of the heterologous peptide.
- the portion deleted is within 10 amino acid residues of the insertion site.
- the portion deleted is within 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue of the insertion site.
- the a3 domain comprises a deletion, insertion, amino acid substitution, mutation, or combination thereof at site different from the insertion site.
- the insertion of the heterologous peptides are within one or more solvent-exposed loops of the a3 domain.
- a solvent-exposed loop corresponds to amino acids numbers 190-199, 208-211, 221-228, 231-240, 250-258, or 264-266 of the a3 domain within a MIC protein selected from the group consisting of SEQ ID NOs: 1-13.
- the insertion is in a solvent-exposed loop corresponding to amino acids numbers 190-199, 221- 228, or 250-258 of the a3 domain within a MIC protein selected from the group consisting of SEQ ID NOs: 1-13.
- all or a portion of one or more of loop 1, loop 2, or loop 3 is deleted and replaced with the heterologous peptide.
- all of one or more of loop 1, loop 2, or loop 3 is deleted, and wherein further one, two, three, four, or five additional amino acids of the a3 domain adjacent to one or both sides of the deleted loop are deleted.
- all of one or more of loop 1 , loop 2, or loop 3 is deleted, and wherein further one, two, or three additional amino acids of the a3 domain adjacent to one or both sides of the deleted loop are deleted.
- a loop and two additional amino acids from both sides of the deleted loop are deleted, resulting in a deletion corresponding to amino acids residues 188-201, 219-230, or 248-260 of an a3 domain of a MIC protein selected from the group consisting of SEQ ID NOs: 1-13.
- loop 1 is deleted and two additional amino acids from both sides of the deleted loop are deleted, corresponding to amino acids numbers 188-201 of an a3 domain of a MIC protein selected from the group consisting of SEQ ID NOs: 1-13.
- more than one of loop 1, loop 2, or loop 3 of a MIC molecule contains a heterologous peptide.
- the heterologous peptides bind to the same target molecule.
- the heterologous peptides contain the same amino acid sequence.
- the heterologous peptides bind different target molecules.
- the target molecule is a cell-surface molecule.
- the cell-surface molecule is on the surface of a malignant cell or a virus infected cell.
- the target molecule is a human epidermal growth factor receptor 2 (HER2), NK- 1R, epidermal growth factor receptor (EGFR), Erb2 or melanoma antigen; antigens of LNcaP and PC-3 cancer cells; a growth factor receptor, an angiogenic factor receptor, an integrin, CD3, CD 19, CD20, CD113, CD271, or an oncogene-encoded protein product, or a fragment thereof.
- the target molecule is selected from the group consisting of an integrin, ErbB2, FGF1 Receptor, FGF2 Receptor, FGF3 Receptor, IGF1 Receptor, IGF2 Receptor, VEGF1 Receptor, VEGF2 Receptor, CD 19, CD20, CD113, CD271, or an oncogene-encoded protein product, or a fragment thereof.
- the target molecule is an integrin. There are 18 known a-chains and 8 known ⁇ -chains forming at least 24 distinct integrin heterodimers, many of which are involved in pathogenic cells such as cancer cells (Koistinen and Heino, 2011. Integrins in Cancer Cell Invasion. Austin
- Such integrins include ⁇ , ⁇ 2 ⁇ 1, ⁇ 3 ⁇ 1, ⁇ 4 ⁇ 1, ⁇ 4 ⁇ 7, ⁇ 5 ⁇ 1, ⁇ 6 ⁇ 1, ⁇ 6 ⁇ 4, ⁇ 7 ⁇ 1, ⁇ 8 ⁇ 1, ⁇ 9 ⁇ 1, ⁇ , ⁇ , ⁇ 3 ⁇ 4 ⁇ 3, ⁇ , ⁇ 3, ⁇ 5 , ⁇ 6, and ⁇ 8.
- the integrin is selected from the group consisting of ⁇ 3, ⁇ 5 and ⁇ 5 ⁇ 1.
- the target molecule is a growth factor receptor or a cell determinant (CD) protein.
- the growth factor receptor or CD protein is selected from the group consisting of ErbB2, FGF1-3 Receptors, IGF1 Receptor, IGF2 Receptor, VEGF1 Receptor, VEGF2 Receptor, CD 19, CD20, CD 113, and CD271.
- the target molecule on the target cell is a phosphotidylserine, or a phosphotidylserine with an accessory protein; or a surface glycoprotein encoded by a virus, an adenovirus, a human
- immunodeficiency virus a herpetic virus, a pox virus, a flavivirus, a filovirus, a hepatitis virus, a papilloma virus, cytomegalovirus, vaccinia, rotavirus, influenza, a parvo virus, West Nile virus, rabies, polyoma, rubella, distemper virus, or Japanese encephalitis virus.
- compositions containing the non-natural MIC molecules of the invention and a carrier or excipient are provided.
- nucleic acid molecules encoding the non-natural, soluble, monomeric MIC molecules of the invention.
- nucleic acid molecules encoding non-natural, monomeric, soluble, mammalian MHC class I chain-related (MIC) molecules containing an al-a2 platform domain attached to a targeting motif, wherein the targeting motif contains a MIC a3 domain and one or more heterologous peptides, wherein the heterologous peptide(s) is/are inserted into one or more loops of the MIC a3 domain at a non-carboxy-terminal site, and wherein the heterologous peptides direct the binding of the targeting motif to a target molecule on a target cell, thereby delivering the attached l-a2 platform domain to the target cell.
- MIC MHC class I chain-related
- a polynucleotide encoding heterologous peptides are inserted within the nucleic acid sequence encoding a solvent-exposed loop of the a3 domain corresponding to amino acids numbers 190-199, 208-211, 221-228, 231-240, 250-258, or 264-266 of the a3 domain within a MIC protein selected from the group consisting of SEQ ID NOs : 1-13.
- the heterologous peptide or peptides are inserted into the MIC a3 domain within one or more sites selected from loop 1, loop 2, and loop 3.
- loop 1 corresponds to amino acids numbers 190-199
- loop 2 corresponds to amino acid residues 221-228
- loop 3 corresponds to amino acid residues 250-258 of the a3 domain of a MIC protein selected from the group consisting of SEQ ID NOs: 1-13.
- libraries containing non- natural MIC molecules of the invention in which the members of a library have diverse individual target binding properties.
- libraries containing genes encoding the non-natural MIC molecules of the invention in which the members of a library have diverse individual target binding properties.
- the non-natural MIC molecule binds a NKG2D-bearing cell and a malignant cell or a NKG2D-bearing cell and a virus-infected cell, resulting in the adhesion of the NKG2D-bearing cell to the malignant cell or the virus-infected cell.
- the adhering NKG2D-bearing cell destroys the viability of the malignant cell or the virus-infected cell.
- Figure 1 shows the structure of the soluble form of human MICA.
- the human MICA structure represented as ribbons as solved by Pingwei Li, et al. (Nature Immunology 2, 443 - 451, 2001).
- the al and o2 domains provide the binding sites for the NKG2D homodimer.
- the a3 domain is a member of the Ig super-family and in this soluble form expressed in E. coli contains the C-terminus.
- Figure 2. shows a photograph of the SDS-PAGE analysis of the cytosolic
- cytosol cytoplasmic membrane
- Cytopl memb cytoplasmic membrane
- Outer memb outer membrane proteins from induced (lanes labeled “B") or un-induced (lanes labeled “A”) cultures of E. coli harboring pKK29 detected by Coomassie blue staining or Western blotting with an antibody against human MICA. The molecular weights are indicated.
- the fusion of MICA a3 domain to intimin (EaeA) is expressed on the outer membrane of the E. coli cells induced with arabinose.
- FIG. 3 A cartoon of the configuration of DNA encoding soluble MICA amino acids 1-276 in the mammalian expression vector, pc5DNA/FRT. The positions of CMV promoter, secretion signal, His-hexamer tag ("His6"; SEQ ID NO: 127), "tight" loop 1 (amino acids 188-201) and loop 3 (amino acids 250-258) of a3, and the polyA tail of bgh are shown.
- His6 His-hexamer tag
- loop 1 amino acids 188-201
- loop 3 amino acids 250-258
- FIG. 4 The configuration of MICA a3 -encoding DNA fused to DNA encoding a portion of M13 phage pill.
- the positions of the lac promoter, P lac , pill secretion signal, a FLAG tag and "tight" loop 1 (amino acids 188-201) and loop 3 (amino acids 250- 258) of a3 are shown.
- Figure 5 shows a schematic of a DGR-based approach for diversifying the a3 domain of human MICA.
- Figure 6. shows a schematic of the structures of EaeA and the EaeA-a3 fusion proteins displayed on the surface of E. coli.
- OM is outer membrane;
- D1-D3 are Ig superfamily motifs,
- Xa is a factor X cleavage site, and
- H6 is a hexa-histadine.
- Figure 7. provides the amino acid or nucleic acid sequences for SEQ ID NOs:
- This invention describes soluble, monomeric, non-natural MIC protein molecules that can recruit and activate NK cells and certain T-cells to attack specific cellular target cells by, after administration to a mammal, binding of the NKG2D-binding portions of MICA or MICB protein, i.e. their al-a2 platform domain (amino acids 1-85 and 86-178, for the al domain and the a2 domain, respectively), to the intended target molecule or molecules specifically via a targeting motif attached to l-a2 platform domain.
- the targeting motif includes an a3 domain of a MICA or MICB protein and inserted heterologous peptide or peptides that bind a target molecule.
- a "heterologous peptide” is a peptide that is not naturally or normally within the a3 domain.
- the heterologous peptide is integral to one of the solvent-exposed loops of the soluble MICA or MICB a3 domain.
- An integral heterologous peptide can be a non-terminal component of the MIC a3 domain and direct the binding of the MICa3 domain to a target molecule.
- a heterologous peptide may be inserted into a a3 domain loop between two adjacent residues of the loop without deleting any of the existing loop. Alternatively, all or a portion of the loop may be deleted and replaced with the heterologous peptide.
- all of the loop is deleted and replaced with the insert.
- all of the loop is deleted and one, two, three, four, or five additional amino acids of the a3 domain adjacent to one or both sides of the deletion site are also deleted.
- one, two or three residues from one or both sides of the deletion site are deleted.
- residues corresponding to 188-201 and/or residues 250-258 of SEQ ID NOs: 1-13 are deleted.
- a portion of the loop containing one or more residues is deleted and replaced with the heterologous peptide.
- the portion deleted is one to three residues of the loop, or one to five amino acid residues of the loop, or even one to seven residues of the loop.
- the portion deleted is within 10 amino acid residues of the insertion site.
- the portion deleted is within 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue of the insertion site.
- the heterologous peptide may include a spacer and a binding motif.
- spacers can be a short, flexible linker peptide used to position the binding motif of the heterologous peptide so that it may bind or improve its ability to bind its target molecule.
- the heterologous peptide can include a portion of a complement-determining region of a natural or recombinant antibody, another protein or peptide molecule or binding motif.
- the heterologous peptide is a complement-determining region of an antibody.
- the heterologous peptide further contains an attached polysaccharide or other carbohydrate, a nucleic acid molecule such as an aptamer or synthetic analog of a nucleic acid molecule.
- heterologous peptide or peptides results in an unnatural (or non-natural), modified or converted a3 domain of a MICA or MICB protein, which acquires the useful function of directing the targeting the al-a2 platform based on the binding properties (e.g., cognate binding partner) of the heterologous peptide or peptides.
- the non-natural, monovalent molecules of the invention have the distinct advantage of not being linked or restricted to a common presenting surface and thereby can be modified, formulated and administered to a mammal as traditional biopharmaceuticals.
- more than one of loop 1, loop 2, or loop3 of the non-natural, soluble, monomeric MIC proteins of the invention contain a heterologous peptide.
- the heterologous peptides bind to the same target molecule.
- the heterologous peptides contain the same amino acid sequence within the binding motif.
- the target molecules may be on the same cell or the same cell type. In other aspects, the target molecules may be on different cells or cell types. In other embodiments, the heterologous peptides bind different target molecules. In some aspects, the target molecules may be on the same cell or the same cell type. In other aspects, the target molecules may be on different cells or cell types.
- the modifications to the a3 domain desired include those that add or increase the specificity or sensitivity of the binding of the a3 domain to a target molecule, such as a molecule on the surface of a target cell, for example, a malignant cell or virus-infected cell.
- the al-a2 platform domain is tethered to the modified targeting a3 domain and is diffusible in the intercellular or intravascular space of the mammal.
- the al-a2 platform domains of the non-natural MIC proteins of the invention are at least 80% identical or homologous to a native or natural l-a2 domain of a human MICA or MICB protein and bind an NKG2D receptor.
- the al-a2 platform domain is 85% identical to a native or natural l-a2 platform domain of a human MICA or MICB protein and binds an NKG2D receptor. In other embodiments, the al-a2 platform domain is 90%, 95%), 96%o, 97%o, 98%o, or 99% identical to a native or natural al-a2 platform domain of a human MICA or MICB protein and binds an NKG2D receptor. In some embodiments, the a3 domain is 85% identical (not including any modified loops) to a native or natural a3 domain of a human MICA or MICB protein.
- the a3 domain is 90%, 95%, 96%o, 97%o, 98%o, or 99% identical (not including any modified loops) to a native or natural a3 domain of a human MICA or MICB protein.
- exemplary human MICA proteins soluble form
- exemplary human MICB proteins full protein
- SEQ ID NOs: 7-12 include SEQ ID NOs: 7-12.
- a heterologous peptide tag may be fused to the N- terminus or C-terminus of the soluble MIC protein to aid in the purification of the soluble MIC protein.
- Tag sequences include peptides such as a poly-histidine, myc-peptide or a FLAG tag. Such tags may be removed after isolation of the MIC molecule by methods known to one skilled in the art.
- MICB refers to a MIC protein containing the al, a2, and a3 domains of the MIC protein but without the transmembrane or intracellular domains.
- Exemplary soluble MIC proteins include amino acid residues 1-274 or 1-276 of SEQ ID NOs: 1-13.
- the "full MIC protein” refers to a MIC protein containing the al, a2, and a3 domains, the transmembrane domain, and the intracellular domain.
- Exemplary full MIC proteins are set forth in SEQ ID NOs:7-12.
- the invention further provides a library of MIC genes or resulting soluble
- MIC proteins wherein each member of the library has or exhibits a different property, such as its binding property for the target cell, resulting in a library of diverse molecules.
- the library can contain diverse individual target binding properties representing 10 or more different binding specificities.
- "diverse individual target binding properties" refers to a library of MIC proteins, in which the individual members of the library bind to a different target molecule or have different affinities or avidities for the same target molecule. In some libraries of MIC proteins, two or more members may bind the same target but may have different binding affinities or avidities.
- a mammal having a malignancy or a viral infection can be treated by administering an effective amount of the soluble MIC protein to affect the malignant or viral condition.
- the administration of the molecule to the mammal may result in the adhesion of NKG2D-bearing NK cells or T-cells to the target malignant or virus-infected cell, wherein the NK cell or T-cell destructively attacks or destroys the target malignant or virus-infected cell.
- the term "destroys" as used herein in the context of the invention methods means to destroy the viability of the target cell.
- malignancy or “malignant conditions” refer to cancer, a class of diseases in which a group of cells display uncontrolled growth, invasion that intrudes upon and destroys adjacent tissues, and sometimes metastasis (i.e., spreading to other locations in the body via lymph or blood).
- the malignancy is a leukemia, a lymphoma, or a myeloma.
- the leukemia is acute lymphoblastic (ALL) leukemia, acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), hairy cell leukemia, or acute monocytic leukemia (AMOL);
- ALL acute lymphoblastic
- AML acute myelogenous leukemia
- CLL chronic lymphocytic leukemia
- CML chronic myelogenous leukemia
- hairy cell leukemia or acute monocytic leukemia
- AOL acute monocytic leukemia
- the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma
- the myeloma is multiple myeloma.
- the malignancy is a malignant solid tumor, including breast cancer, ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, brain cancer, glioblastoma, head and neck cancer, colon cancer, esophageal cancer, liver cancer, stomach cancer, uterine cancer, endocrine cancer, renal cancer, bladder cancer, or cervical cancer.
- the malignancy is breast cancer, ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, brain cancer, glioblastoma, head and neck cancer, or colon cancer.
- the malignancy is breast cancer.
- the invention also includes the means of converting the a3 domain (for example amino acids 182-274, in SEQ ID NOs: 1-13) of a MIC protein into a specific targeting domain that can directly deliver from the intercellular space its tethered l-a2 domain to the target cell surface in order to attract, recruit or bind the NKG2D-bearing NK cell or T-cell.
- a3 domain for example amino acids 182-274, in SEQ ID NOs: 1-13
- a specific targeting domain that can directly deliver from the intercellular space its tethered l-a2 domain to the target cell surface in order to attract, recruit or bind the NKG2D-bearing NK cell or T-cell.
- the high resolution structure of human MICA bound to the NKG2D receptor has been solved and demonstrates that the a3 domain of MICA has no direct interaction with the NKG2D receptor (Li et al. 2001. Complex structure of the activating immunoreceptor NKG2D and its MHC class I-like ligand MICA. Nature Immunol. 2: 443-451; Protein Data Bank accession code 1HYR).
- the a3 domain of MICA like that of MICB, is connected to the al-a2 platform domain by a short, flexible linker peptide, amino acids 175-182 [of SEQ ID 1-13], and itself is positioned naturally as "spacer" between the platform and the surface of the MIC expressing cell.
- the 3-dimensional structures of the human MICA and MICB a3 domains are nearly identical (root-mean square distance ⁇ 1 A on 94 C-aa's) and functionally interchangeable (Holmes et al. 2001. Structural Studies of Allelic Diversity of the MHC Class I Homolog MICB, a Stress-Inducible Ligand for the Activating Immunoreceptor NKG2D. J Immunol. 169: 1395-1400).
- the 3 -dimensional structures of the MIC proteins' Ig-like a3 domains resemble that of Tendamistat, and in a sequence inverted form, that of the human tenth fibronectin domain III; both structures have served as scaffolds for engineering protein binding motifs (Pflugrath, JW, G Wiegand, R Huber, L Vertesy (1986) Crystal structure determination, refinement and the molecular model of the a-amylase inhibitor Hoe-467A. J. Molec. Biol. 189: 383-386; Koide A, Bailey CW, Huang X, Koide S. 1998. The fibronectin type III domain as a scaffold for novel binding proteins. J. Mol. Biol.
- One aspect of the invention contemplates engineering specific binding properties into 1 or more of the 6 solvent-exposed loops of the a3 domain of MICA or MICB, a soluble, non-natural MIC molecule is created that after administration to a mammal can diffuse in the intravascular or intercellular space and subsequently attach with high sensitivity and specificity to a target molecule on an intended target cell and, thereby promote binding and subsequent destructive attack of the particular target cell by NKG2D-bearing NK and/or T-cells.
- Examples of surface accessible molecules on target malignant cells include integrins, oncogene products or fragments thereof, such as NK-1R, human epidermal growth factor 2 (Her2 or ErbB2), growth factor receptors such as Epidermal Growth Factor Receptor
- EGFR vascular endothelial growth factor
- FGF FGF Receptor3
- CD30 CD19
- CD20 angiogenic factor receptors such as those for vascular endothelial growth factor (VEGF) receptor and VEGF-related molecules, melanoma antigens, and antigens of LNcaP and PC-3 prostate cancer cells.
- VEGF vascular endothelial growth factor
- the surface accessible molecules on target virus-infected cells include "inside -out" phosphotidylserine with or without accessory proteins such as apolipoprotein H, Gas6, MFG-E8; virus-encoded antigens, virus-encoded antigens of hepatitis viruses; adenoviruses; cytomegalovirus; other herpetic viruses; HIV especially pi 7; vaccinia; pox viruses; rotavirus; influenza; parvo viruses; West Nile virus; rabies; polyoma; papilloma viruses; rubella; distemper virus; and Japanese encephalitis virus (Balasubramanian, K and Schroit, AJ. 2003. Ann. Rev. Physiol.
- compositions can be produced by introducing specific binding motifs into the a3 domain of MICA or MICB deploying synthetic DNA, bacteriophage display or yeast or bacterial surface display technology, several of which have been deployed to create specific binding properties in Tendamistat and the human tenth fibronectin domain III (McConnell, SJ and RH Hoess, (1995) Tendamistat as a Scaffold for Conformationally Constrained Phage Peptide Libraries. J. Molec.
- the diversity generating retroelements (DGR) of Miller et al. is an example of a method of generating diversity at desired amino acid positions within the loops (Medhekar, B. & J.F. Miller. 2007. Diversity-Generating Retroelements. Current Opinion in Microbiol. 10: 388-395 and US Patent No. 7,585,957).
- a3 domains of human MICA and MICB are comprised of about 95 amino acids (182-276) of the 276 amino acid water-soluble form
- all solvent-exposed loops for example amino acids 190-199, 208-211 , 221-228, 231- 240, 250-258, or 264-266 of SEQ ID NOs: 1-13
- TR synthetic Template Repeat
- DGRs generate diversity in defined segments of protein-encoding DNA sequences, designated as variable repeats (VRs).
- VRs variable repeats
- IMH sequences serve as cz ' s-acting sites that direct mutagenic homing and determine the 3' boundary of sequence diversification.
- the 5' boundary of VR diversification may be determined by the extent of homology between VR and its cognate TR. Only partial homology is required and mismatches are tolerated.
- specific sites in VR which are subject to diversification may be determined by the location of adenine residues in TR. By inserting adenine residues at appropriate locations within
- “synthetic" TRs specific VR-encoded amino acid residues can be diversified.
- the atd protein, the TR-encoded RNA intermediate, and the RT reverse transcriptase efficiently function in trans when expressed on a plasmid vector, pDGR, under the control of a heterologous promoter, for example, P te tA or P bad -
- a heterologous promoter for example, P te tA or P bad -
- FIG. 5 A general outline of the DGR-based approach for diversifying the a3 domain is shown in Fig. 5.
- the sequences to be diversified correspond to the loops of a3 domain.
- IMH sequence is positioned immediately downstream from the stop codon (about AV277) of the gene encoding a3 domain, creating a "synthetic VR" which will be subject to
- TR on plasmid pDGR (Fig. 5).
- This TR element includes an IMH* and upstream sequences that are homologous to VR.
- the specific VR residues that will be subject to mutagenesis are precisely programmed by the placement of adenines in TR, and high densities of adenine residues can be tolerated by the system.
- the pDGR also includes loci which encode Atd and the RT reverse transcriptase. Atd, TR and rt are expressed from the tightly regulated tetA promoter/operator (P te tA), which allows precise control over the diversification process by the addition or removal of anhydotetracycline.
- P te tA tightly regulated tetA promoter/operator
- a3 domain is fused to a filamentous phage coat protein encoded on a phagemid vector in E. coli.
- VR would include solvent-exposed loops of the a3 domain, and pDGR would be designed to efficiently diversify VR at specified locations within those loops (Guo et al. 2008).
- Activating atd, TR, and rt expression would mutagenize VR sequences present on phagemid genomes. This would result in the creation of a library of phage, each of which presents a diversified binding protein on its surface and packages the encoding DNA.
- Desired specificities would be selected by binding phage to the immobilized target molecule, for example the surface exposed protein product of oncogene Her2, washing to remove nonbinding phage, and reamplification and enrichment. Further rounds of optimization of the selected phenotype could be efficiently accomplished by simply infecting E. coli containing pDGR with the selected or panned phage and repeating the steps described above. This system is capable of generating library sizes that are several orders of magnitude greater than those achieved by conventional approaches. Of equal advantage is the extraordinary ease with which successive rounds of optimization may be achieved with cumulative improvements, but without compromise of the integrity of the a3 domain scaffold.
- a3 domains of MICA or MICB will be expressed on the surface of E. coli as fusion proteins consisting of, as a non-limiting example, the outer membrane localization and anchor domains of the EaeA intimin protein encoded by enteropathogenic E. coli (Luo Y, Frey EA, Pfuetzner RA, Creagh AL, Knoechel DG, Haynes CA, Finlay BB, Strynadka NC. (2000) Crystal structure of enteropathogenic Escherichia coli intimin-receptor complex. Nature. 405: 1073-7).
- EaeA consists of an N-terminal segment of approximately 500 amino acids that anchors the protein to the outer membrane and is believed to form an anti-parallel ⁇ -barrel with a porin-like structure that facilitates translocation (Touze T, Hayward RD, Eswaran J, Leong JM, Koronakis V. (2004) Self-association of EPEC intimin mediated by the beta-barrel-containing anchor domain: a role in clustering of the Tir receptor. Mol Microbiol. 51 :73-87). This translocation domain is followed by a series of Ig-like motifs and a C-terminal C-type lectin domain responsible for binding to the intestinal epithelial surface (Fig. 6).
- the natural orientation of MICA and MICB is such that the C-terminus is anchored to the cell membrane (type I membrane protein).
- the a3 domain resides between the N-terminal al-a2 platform and the cell membrane.
- the opposite orientation e.g. type II membrane protein
- the opposite orientation is desired, that is, to attach the N-terminus the linker portion of the a3 domain in Figure 1 to EaeA so that those loops such as those located at amino acid positions 190-199, 221-228, 250-258 of SEQ ID NOs: 1-13 are readily available for binding target molecules.
- Such a type II membrane protein orientation is precisely that of EaeA, Figure 6.
- the a3 domain like EaeA, has an Ig-like motif, so that EaeA will translocate a3 domains to the E. coli surface (Li et al. 1999. Crystal structure of the MHC class I homolog MICA, a ⁇ cell ligand. Immunity 10: 577-584). Indeed, the ability of EaeA to translocate heterologous passenger polypeptides has been documented in the literature (Wentzel et al. 2001; Adams et al, 2005).
- the EaeA-a3 fusion protein will be expressed from the araBAD promoter
- a diversification system e.g. the L. pneumophila atd TR rt sequences (pDGR, Figure 2), can be placed under control of the tightly regulated tetA promoter/operator on a multicopy plasmid.
- the expression of the atd TR rt sequences is induced by addition of anhydrotetracycline to the growth medium and will result in high frequency diversification of a3 VR sequences. Once diversification has been achieved, removal of inducer from the growth media will "lock" the system (a3-VR) into a stable state.
- Bacterial cells that display binding characteristics of interest can be enriched using standard methods such as Fluorescent Activated Cell Sorting (FACS) or magnetic bead separation techniques. Selected bacterial cells are amplified by growth in the presence of arabinose and the absence of FACS.
- FACS Fluorescent Activated Cell Sorting
- Selected bacterial cells are amplified by growth in the presence of arabinose and the absence of
- a3 domains that bind to targets that are undesirable for NK or T-cell attack can be depleted from the diversified library by panning against, for example, normal tissues prior to selection for the desired binding properties.
- the selected a3 proteins can be cleaved from the bacterial cell surface by the addition of Factor Xa protease and then purified by affinity purification of the 6xHis-tagged C-terminal domain for further characterization and use. This permits convenient biochemical and physical analyses of structure and function of the selected a3 domain.
- the desired, non-natural a3 domain can then in each case be reintroduced into the rest of the soluble MIC protein via its linker or tether (amino acids 177-182) to create the desired passive NK cell vaccine with the specificity and sensitivity of the isolated a3 domain.
- the selected genotype can be used to produce and isolate the non-natural or unnatural, soluble cognate MIC protein in bacteria, yeasts, insect or mammalian cells.
- the produced MICA can be purified to the required degree, formulated by available methods to stabilize it in vitro and in vivo, and administered parenterally or by other routes to humans or other mammals where it can diffuse to treat malignancies or viral diseases by promoting the targeted attack by the cellular components of the innate immunity system.
- a non-natural MIC molecule is formulated with a
- pharmaceutically acceptable excipient or carrier Such a component is one that is suitable for use with humans or animals without undue adverse side effects. Non-limiting examples of adverse side effects include toxicity, irritation, and/or allergic response.
- the excipient or carrier is typically one that is commensurate with a reasonable benefit/risk ratio.
- the carrier or excipient is suitable for topical or systemic administration.
- Non- limiting pharmaceutically carriers include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples include, but are not limited to, standard
- a phosphate buffered saline solution water, emulsions such as oil/water emulsion, and various types of wetting agents.
- non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyloleate.
- Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
- Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
- Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
- composition comprising a non-natural MIC molecule of the disclosure may also be lyophilized or spray dried using means well known in the art.
- compositions comprising the therapeutically- active compounds.
- Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure, or buffers for securing an adequate pH value may be included.
- a non-natural MIC molecule is typically used in an amount or concentration that is "safe and effective", which refers to a quantity that is sufficient to produce a desired therapeutic response without undue adverse side effects like those described above.
- a non- natural MIC molecule may be biochemically modified to alter its pharmacokinetic properties in vivo.
- Well-known methods to increase half-life of circulating protein molecules are to chemically attach polyethylene glycol (PEG) to the basic structure or by genetic engineering to add polymers of natural amino acids such as glycine and serine to the N-terminus, C- terminus, or internally such as in the tether between al-a2 and a3 domains, amino acids 179- 182 of SEQ ID NOS: 1-13, without affecting binding functions of the MIC protein.
- a non- natural MIC molecule may be used in an amount or concentration that is "therapeutically effective", which refers to an amount effective to yield a desired therapeutic response, such as, but not limited to, an amount effective to bind target cells in order to recruit sufficient NK or T-cells to kill the target cells.
- the safe and effective amount or therapeutically effective amount will vary with various factors but may be readily determined by the skilled practitioner without undue experimentation. Non-limiting examples of factors include the particular condition being treated, the physical condition of the subject, the type of subject being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed.
- compositions and methods contemplates particular embodiments in which the composition or method consists essentially of or consists of those elements or steps.
- the DNA encoding the a3 domain was fused to a portion of the E. coli eaeA (intimin) gene, and the resulting fusion protein was expressed on the surface of E. coli in such a manner that the a3 domain was oriented with its C-terminal portion distal to the bacterial surface.
- Oligonucleotides AVI 401 (SEQ ID NO: 15) and AVI 402 (SEQ ID NO: 16) were kinased, annealed, and ligated into an aliquot of plasmid pET30a (Novagen) which had been digested with Ndel and Ncol to create pSW249, containing a pelB secretion signal sequence.
- Plasmid pSW249 was digested with Ncol and Blpl and ligated together with kinased and annealed oligonucleotides AVI 445 (SEQ ID NO: 17) and AVI 446 (SEQ ID NO: 18) to create pSW263.
- This construct contained sequence encoding six histidine residues (SEQ ID NO: 127) following the pelB sequence.
- a human MICA cDNA comprising a portion of 5 ' untranslated sequence, signal sequence, and codons 1-276 of the mature coding sequence, followed by a stop codon, was amplified by PCR from human spleen first strand cDNA (acquired from Invitrogen) using primers AVI 466 (SEQ ID NO: 19) and AVI 448 (SEQ ID NO: 20).
- PCR fragment was digested with Nhel and Hindlll and ligated together with pCDNA5-FRT (Invitrogen) which had also been digested with Nhel and Hindlll to create pSW265.
- pCDNA5-FRT Invitrogen
- Three mutations the MICA coding region were corrected, G14W, A24T and E125K, by directed mutagenesis to create pSW271.
- PCR fragment consisted of a tev protease cleavage site, ENLYFQG (SEQ ID NO: 128), followed by codons 1-276 of human MICA. This PCR fragment was digested with Xhol and Hindlll and ligated together with an aliquot of plasmid pSW263 which had also been digested with Xhol and Hindlll to create plasmid pSW286.
- the eaeA gene was amplified from E. coli EDL933 genomic DNA using
- a portion of plasmid pSW284 was PCR amplified using primers AV1602
- pKK5 was PCR amplified with primers kk52 (SEQ ID NO : 28) and kk45
- the PCR fragment was digested with Ncol and Hindlll and ligated together with an aliquot of pBAD24 (from ATCC) which had been digested with Ncol and Hindlll to create pKK29.
- the plasmid pKK29 was transformed according to the
- E. coli "NEB 10-beta” catalog AV C3019H from New England BioLabs
- E. coli "NEB 10-beta” catalog AV C3019H from New England BioLabs
- Cytosolic proteins, inner membrane proteins, and outer membrane proteins of arabinose-induced and non-induced pKK29-transformed E. coli cells were each isolated and analyzed by SDS-PAGE. The SDS-PAGE gels were stained with Coomassie blue or western-blotted with antibody to human MICA protein.
- Cells were then centrifuged for 10 min at 4000rpm in an Eppendorf 581 OR tabletop centrifuge. Supematants were discarded and the cell pellets were gently resuspended in 6ml FP buffer (0.1 M sodium phosphate buffer pH 7.0, 0.1 M KC1, 5 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) and transferred to 15ml conical tubes. Cells were sonicated for 6X 15 sec bursts using a Biologies Inc model 300 V/T ultrasonic homogenizer. Samples were incubated on ice between bursts. After sonication the tubes were centrifuged in the Eppendorf 581 OR tabletop centrifuge at 4000rpm for 5 minutes to remove any unbroken cells.
- 6ml FP buffer 0.1 M sodium phosphate buffer pH 7.0, 0.1 M KC1, 5 mM EDTA, 1 mM
- Tris-Glycine SDS 2X sample buffer (Invitrogen AVLC2676) and electrophoresed on 4-20% Tris-Glycine Gradient Gel (Invitrogen AVEC60285BOX).
- AVEC60285BOX Tris-Glycine Gradient Gel
- For western blotting the electrophoresed sample lanes in the slab gel were transferred to a nitrocellulose membrane (Invitrogen Nitrocellulose Membrane Filter Paper Sandwich AVLC2001) using an Invitrogen XCell II Blot Module (AVEI9051). The membrane filter was blocked overnight at 4°C in 5% milk-Phosphate Buffered Saline, Tween-20 (PBST).
- Human MICA is naturally glycosylated, although its unglycosylated form does bind its receptor, NKG2D, in vitro (Li et al, 2001). However, to be able to evaluate a human-like glycosylated form in vitro and eventually in vivo, we expressed MICA in cultured human cells. For expression, two common human alleles were inserted into the transient expression vector pcDNA5/FRT, which has a human CMV promoter and a bovine growth hormone (bgh) polyadenylation signal, Figure 3.
- pcDNA5/FRT which has a human CMV promoter and a bovine growth hormone (bgh) polyadenylation signal
- Human MHC class I-related protein mRNA were obtained by amplifying with a Polymerase Chain Reaction (PCR) the appropriate DNA sequence from human spleen first-strand cDNA (available from Life Technologies/Invitrogen) using primers AV1466 (SEQ ID NO: 30) and AVI 448 (SEQ ID NO: 31).
- the amplified DNA product was digested with Nhel and Hindlll restriction enzymes, and the resulting product was ligated into Nhel/Hindlll-digested pCDNA5/FRT (Invitrogen), to create pSW265.
- the DNA of the inserted PCR product of pSW265 was sequenced and verified to include an Nhel site followed by 26 bases of the 5 'untranslated (UT) sequence, followed by secretion signal sequence and codons 1-276 of mature HUMMHCREP, followed by a termination codon, followed by a Hindlll site.
- the codons were changed by site-directed mutagenesis (using New England BioLabs Phusion® site-directed mutagenesis kit and appropriate primers) so that the amino acid sequence matched the relevant portion (amino acids 1-276) of the sequence described as SEQ ID NO: 13.
- the corrected plasmid was designated pSW271 and contained the corrected
- Primers AV1490 (SEQ ID NO: 32) and AV1489 (SEQ ID NO: 33) and pSW271 were used to generate a PCR product which was subsequently digested with BamHI and BsmBI and ligated to -5259 bp BamHI/BsmBI fragment from pSW271.
- the resulting construct pSW275 lacks a BsmBI site.
- Plasmid pSW267 is the same as pSW271 except MICA codon 125 is GAG
- Glu instead of AAG (Lys). It was derived from pSW265 by site-directed mutagenesis (using New England BioLabs Phusion kit with primers AV1478 (SEQ ID NO:39) and AV1479 (SEQ ID NO:40). This mutagenesis changed MICA codon 14 from GGG (Gly) to TGG (Tip) and codon 24 from GCT (Ala) to ACT (Thr).
- Plasmid pSW273 was made from pSW267 by site-directed mutagenesis using primers AV1486 (SEQ ID NO:41) and AV1487 (SEQ ID NO:42). pSW273 contains six histidine codons between the signal peptide and residue 1 of the mature peptide.
- Plasmid pKK33 is identical to plasmid pSW273 except a BsmBI site has been deleted from the partial hph (hygromycin resistance) gene.
- a PCR fragment was obtained from plasmid pSW273 by amplifying with primers AV1490 (SEQ ID NO:32) and 1489 (SEQ ID NO:33). This -727 bp fragment was digested with BamHI and BsmBI and was ligated together with the -5277 bp BamHI/BsmBI-digested fragment from pSW273. This resulted in the removal of the BsmBI site and the creation of pKK34.
- loop 1 constructs were generated by insertion of the indicated heterologous peptide "insert" between T189 and V200 of MICA and replacing the residues at positions 190-199 with the indicated insert.
- loop 3.1 phosphorylated oligonucleotides AVI 830 (top strand) (SEQ ID NO:44) and AVI 831 (bottom strand) (SEQ ID NO:45) were ligated into BsmBI-digested pKK34.
- loop 3.2 inserted between T189 and V200 of MICA
- phosphorylated oligonucleotides AVI 832 (top strand) (SEQ ID NO:47) and AVI 833 (bottom strand) (SEQ ID NO:48) were ligated into BsmBI-digested pKK34.
- loop 3.3 phosphorylated oligonucleotides AVI 834 (top strand) (SEQ ID NO:50) and AVI 835 (bottom strand) (SEQ ID NO:51) were ligated into BsmBI-digested pKK34.
- pKK40 which has a sequence coding for VTRGDTFTQS (SEQ ID NO:52; referred to as loop 3.4) inserted between T189 and V200 of MICA, phosphorylated oligonucleotides AVI 836 (top strand) (SEQ ID NO:53) and AVI 837 (bottom strand) (SEQ ID NO:54) were ligated into BsmBI-digested pKK34. [0096] To create pKK40, which has a sequence coding for VTRGDTFTQS (SEQ ID NO:52; referred to as loop 3.4) inserted between T189 and V200 of MICA, phosphorylated oligonucleotides AVI 836 (top strand) (SEQ ID NO:53) and AVI 837 (bottom strand) (SEQ ID NO:54) were ligated into BsmBI-digested pKK34. [0096] To create pKK40, which has a sequence coding for VTRGDT
- loop 5.1 phosphorylated oligonucleotides AVI 838 (top strand) (SEQ ID NO:56) and AVI 839 (bottom strand) (SEQ ID NO:57) were ligated into BsmBI-digested pKK34.
- loop 5.2 phosphorylated oligonucleotides AV1840 (top strand) (SEQ ID NO:59) and AV1841 (bottom strand) (SEQ ID NO:60) were ligated into BsmBI-digested pKK34.
- loop 5.3 phosphorylated oligonucleotides AVI 842 (top strand) (SEQ ID NO:62) and AVI 843 (bottom strand) (SEQ ID NO:63) were ligated into BsmBI-digested pKK34.
- SGGSGGGSTSRGDHPRTQSGGSGGG (SEQ ID NO:64; referred to as extended loop 3.2sp) inserted between T189 and V200 of MICA, phosphorylated oligonucleotides AVI 854 (top strand) (SEQ ID NO:65) and AVI 855 (bottom strand) (SEQ ID NO:66) were ligated into BsmBI-digested pKK34.
- SGGSGGGSRVPRGDSDLTSGGSGGG (SEQ ID NO:67; referred to as extended loop 3.3sp) inserted between T189 and V200 of MICA, phosphorylated oligonucleotides AV1856 (top strand) (SEQ ID NO:68) and AVI 857 (bottom strand) (SEQ ID NO:69) were ligated into BsmBI-digested pKK34.
- SGGSGGGSVTRGDTFTQSSGGSGGG (SEQ ID NO:70; referred to as extended loop 5.1sp) inserted between T189 and V200 of MICA, phosphorylated oligonucleotides AVI 858 (top strand) (SEQ ID NO:71) and AV1859 (bottom strand) (SEQ ID NO:72) were ligated into BsmBI-digested pKK34.
- SGGSGGGSHLARGDDLTYSGGSGGG (SEQ ID NO:73; referred to as extended loop 5.3sp) inserted between T189 and V200 of MICA, phosphorylated oligonucleotides AV1860 (top strand) (SEQ ID NO:74) and AV1861 (bottom strand) (SEQ ID NO:75) were ligated into BsmBI-digested pKK34.
- the plasmid pSW276 was digested with BsmBI and ligated to kinased and annealed oligonucleotides AVI 826 (SEQ ID NO: 36) and AVI 827 (SEQ ID NO: 37) to create pKK35.
- This plasmid contained a sequence encoding
- SGGSGGGSTSRGDHPRTQSGGSGGG (SEQ ID NO:76; referred to as extended loop 3.2sp) inserted between 1249 and C259 of MICA, phosphorylated oligonucleotides AV1864 (top strand) (SEQ ID NO:77) and AVI 865 (bottom strand) (SEQ ID NO:78) were ligated into BsmBI-digested pSW276.
- SGGSGGGSRVPRGDSDLTSGGSGGG (SEQ ID NO:79; referred to as extended loop 3.3sp) inserted between 1249 and C259 of MICA, phosphorylated oligonucleotides AV1866 (top strand) (SEQ ID NO: 80) and AVI 867 (bottom strand)( SEQ ID NO:81) were ligated into BsmBI-digested pSW276.
- AVI 868 top strand SEQ ID NO: 83
- AVI 869 bottom strand( SEQ ID NO: 84) were ligated into BsmBI-digested pSW276.
- SGGSGGGSHLARGDDLTYSGGSGGG (SEQ ID NO: 85; referred to as extended loop 5.3sp) inserted between 1249 and C259 of MICA, phosphorylated oligonucleotides AV1870 (top strand) (SEQ ID NO: 86) and AVI 871 (bottom strand) (SEQ ID NO: 87) were ligated into BsmBI-digested pSW276.
- T loop 1 constructs were generated by insertion of the indicated heterologous peptide "insert" between N187 and C202 of MICA, that is, residues 188-201 of MICA were replaced with the indicated insert.
- the plasmid pKK34 was mutagenized with primers AV1873 (SEQ ID NO:88) and AVI 872 (SEQ ID NO: 89) to create pSW324, which contains convenient BsmBI sites suitable for cloning inserts between N187 and C202 of MICA.
- phosphorylated oligonucleotides AV1882 top strand
- AV1883 bottom strandX SEQ ID NO: 104
- pKK1208 which has extended 5.1sp in loop 1 and a sequence coding for SGGSGGGSTSRGDHPRTQSGGSGGG (SEQ ID NO:76) referred to as extended loop 3.2sp) inserted between 1249 and C259 of MICA, phosphorylated oligonucleotides AV1864 (top strand) (SEQ ID NO:77) and AVI 865 (bottom strand)( SEQ ID NO:78) were ligated into BsmBI-digested pKKl 15.
- pKK129 which has extended 5.1sp in loop 1 and a sequence coding for SGGSGGGSVTRGDTFTQSSGGSGGG (SEQ ID NO:82; referred to as extended loop 5.1sp) inserted between 1249 and C259 of MICA, phosphorylated oligonucleotides AV1868 (top strandX SEQ ID NO: 83) and AVI 869 (bottom strand)( SEQ ID NO: 84) were ligated into BsmBI-digested pKKl 15.
- pKK130 which has extended 5.1sp in loop 1 and a sequence coding for SGGSGGGSHLARGDDLTYSGGSGGG (SEQ ID NO: 85; referred to as extended loop 5.3sp) inserted between 1249 and C259 of MICA, phosphorylated oligonucleotides AV1870 (top strand) (SEQ ID NO: 86) and AVI 871 (bottom strand)( SEQ ID NO: 87) were ligated into BsmBI-digested pKKl 15.
- phosphorylated oligonucleotides AVI 908 (top strand)( SEQ ID NO: 110) and AVI 909 (bottom strand)( SEQ ID NO: 111) were ligated into BsmBI-digested pKKl 15.
- the concentrators were pre-rinsed with phosphate buffered saline (PBS). Each sample was added to the concentrator and then centrifuged for 30 min at 4000rpm in the Eppendorf 581 OR tabletop centrifuge. Each sample was washed and concentrated 3 times with 6ml PBS- each time spinning 4000 rpm 30 min in the Eppendorf 581 OR tabletop centrifuge. The concentration of soluble MICA in each resulting sample solution was estimated by an ELISA for soluble MICA.
- PBS phosphate buffered saline
- the capture agent was mouse anti-human MICA (R&D Systems part 841612), and the detection antibody was biotinylated goat anti-human MICA (R&D Systems part 841613) that was developed with Streptavidin-HRP and Ultra TMB.
- the quantity of soluble MICA in each sample was determined by the MICA-specific ELISA.
- the signals from the bound soluble MICA molecules (per ng of total MICA) to the specific integrins are shown.
- the ELISA signal from a non-binding, negative control MICA (generated by pSW273) was subtracted from each integrin binding signal.
- the results of the MICA products with single peptides inserts generated from pKK35-42 and pKK44-56 along with controls are shown in Table 1.
- the amino acid sequences and SEQ ID NOs of their specific inserts are tabulated in Table 2.
- the quantity of soluble MICA in each sample was determined by the MICA-specific ELISA.
- the signal of soluble MICA molecules with more than 1 insert bound to the specific integrins are shown, Table 3.
- the amino acid sequences and SEQ ID NOs of their specific inserts are tabulated in Table 2.
- Those soluble MICA molecules with binding peptides inserted into more than 1 internal loop exhibited greater binding than those with only a single internal binding peptide, indicating the avidity effect of more than 1 binding motif per molecule.
- soluble MICA molecule for its intended target, for example ⁇ 5
- soluble MICA molecule had more than one ⁇ 5 -specific, internal binding peptide per MICA molecule.
- avidity for its target.
- bivalent MICA binders were created, both duplicate binders and different binders, enabling the generation bi-specific MICA molecules and MICA molecules with avidity exceeding affinity for its target.
- Tendamistat is a bacterial protein with a 3-dimensional structure resembling that of the a3 domain of MICA (Pflugrath et al., 1986). Random peptides have been inserted into Tendamistat and selected by M13 phage display for binding to human integrins
- the M13 phage M13KE (obtained from New England BioLabs) was used as template in a PCR amplification reaction with primers AVI 887 (SEQ ID NO: l 12) and AV1888 (SEQ ID NO: 113).
- PCR product consisting of a C-terminal portion of M13KE gene III, was digested with EcoRI and Hindlll and li gated together with EcoRI/Hindlll-digested Ml 3 phage vector M 13mp 18 (GenBank X02513) to create phage vector pSW326.
- Phage vector pKK59 was created by inserting the synthesized sequence TEND
- Phage vector pKK60 was created by inserting the synthesized sequence
- TEND-3A (SEQ ID NO: l 15), digested with EcoRI and Avrll, into EcoRI/Avrll-digested pSW326.
- Phage vector pKK61 was created by inserting the synthesized sequence
- TEND-3B (SEQ ID NO: 116), digested with EcoRI and Avrll, into EcoRI/Avrll-digested pSW326.
- Phage vector pKK63 was created by inserting the synthesized sequence
- TEND-5A (SEQ ID NO: l 17), digested with EcoRI and Avrll, into EcoRI/Avrll-digested pSW326.
- Phage vector pKK64 was created by inserting the synthesized sequence
- TEND-5B (SEQ ID NO: 118), digested with EcoRI and Avrll, into EcoRI/Avrll-digested pSW326.
- Phage vector pKK65 was created by inserting the synthesized sequence
- TEND-His8 (SEQ ID NO: l 19; His8 is SEQ ID NO: 130), digested with EcoRI and Avrll, into EcoRI/Avrll-digested pSW326.
- a -320 bp fragment was amplified by PCR from pKK29 using primers AV1897 (SEQ ID NO: 120) and AV1898 (SEQ ID NO: 121). This fragment representing the wild type MICA a3 sequence (SEQ ID NOs: l-6, 13), was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- a -356 bp fragment was amplified by PCR from pKK35 using primers AV1897 (SEQ ID NO: 120) and AV1898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- a -305 bp fragment was amplified by PCR from pKK36 using primers AV1897 (SEQ ID NO: 120) and AV1898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- a -311 bp fragment was amplified by PCR from pKK37 using primers AV1897 (SEQ ID NO: 120) and AV1898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- a -308 bp fragment was amplified by PCR from pKK38 using primers AV1897 (SEQ ID NO: 120) and AV1898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- a -308 bp fragment was amplified by PCR from pKK39 using primers AV1897 (SEQ ID NO: 120) and AV1898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- phage vector pKK96 To create phage vector pKK96, a -308 bp fragment was amplified by PCR from pKK40 using primers AV1897 (SEQ ID NO: 120) and AV1898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- a -302 bp fragment was amplified by PCR from pKK41 using primers AV1897 (SEQ ID NO: 120) and AV1898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- phage vector pKK98 a -308 bp fragment was amplified by PCR from pKK42 using primers AV1897 (SEQ ID NO: 120) and AV1898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- a -353 bp fragment was amplified by PCR from pKK44 using primers AV1897 (SEQ ID NO: 120) and AV1898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- a -353 bp fragment was amplified by PCR from pKK45 using primers AV1897 (SEQ ID NO: 120) and AV1898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- a -353 bp fragment was amplified by PCR from pKK46 using primers AV1897 (SEQ ID NO: 120) and AV1898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- a -353 bp fragment was amplified by PCR from pKK47 using primers AV1897 (SEQ ID NO: 120) and AV1898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- a -356 bp fragment was amplified by PCR from pKK48 using primers AV1897 (SEQ ID NO: 120) and AV1898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- a -356 bp fragment was amplified by PCR from pKK49 using primers AV1897 (SEQ ID NO: 120) and AV1898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- a -344 bp fragment was amplified by PCR from pKK52 using primers KK69 (SEQ ID NO: 122) and AVI 898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- a -284 bp fragment was amplified by PCR from pKK53 using primers KK70 (SEQ ID NO: 123) and AV1898 SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- a -284 bp fragment was amplified by PCR from pKK54 using primers KK71 (SEQ ID NO: 124) and AVI 898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- a -284 bp fragment was amplified by PCR from pKK55 using primers KK72 (SEQ ID NO: 125) and AV1898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- a -284 bp fragment was amplified by PCR from pKK56 using primers KK73 (SEQ ID NO: 126) and AV1898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- a -278 bp fragment was amplified by PCR from pKK84 using primers AV1897 (SEQ ID NO: 120) and AV1898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- a -413 bp fragment was amplified by PCR from pKK128 using primers AV1897 (SEQ ID NO: 120) and AV1898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- a -413 bp fragment was amplified by PCR from pKK129 using primers AVI 897 (SEQ ID NO: 120) and AVI 898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- a -413 bp fragment was amplified by PCR from pKK130 using primers AV1897 (SEQ ID NO: 120) and AV1898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- a -413 bp fragment was amplified by PCR from pKK131 using primers AV1897 (SEQ ID NO: 120) and AV1898 (SEQ ID NO: 121). This fragment was then digested with EcoRI and BsmBI and ligated together with the -7911 bp Mfel/Avrll-digested fragment of pKK59.
- the Ml 3 phages titers ranged from 8 X 10
- the results of phages displaying a3 domains with binding peptides inserted into more than one loop generated from pKK136, 138, and 139 along with controls are shown in Table 5.
- the ELISA assays of the integrin target-binding of Ml 3 phages displaying a3 domains with binding peptide inserts grafted into more than one loop were performed as follows. After the respective integrins were adhered to the bottoms of the wells of the ELISA plate, the wells were washed and blocked. Each sample (100 ⁇ ) of phage preparation was added to wells containing ⁇ 3 or ⁇ 5, incubated and washed. The phages captured by the integrins were detected by HRP-conjugated antibody to the Ml 3 phage coat developed with Ultra TMB-ELISA substrate and the optical densities read.
- the M13 phages titers ranged
- Soluble targeted MICA acted as a specific adapter molecule to recruit NK cells to kill target cells.
- the LIVE/DEAD® cell viability assays were carried out essentially as described by Chromy et al, 2000 and by the manufacturer's protocol (Invitrogen, Carlsbad, CA). Briefly, human target cells (MCF7 and HeLa) were seeded at a density of 1 x 10 4 cells/well in 96-well flat-bottomed culture plates and reached 80% confluency in 2 days. The culture supernatants of 293T cells transiently transfected with pKK131 were concentrated approximately 100-fold by Pierce 9K MWCO Concentrators and the MICA concentrations determined by the ELISA specific for MICA.
- soluble, targeted MICA molecules To demonstrate the ability of soluble, targeted MICA molecules to recruit human NK cells to kill target cells, different concentrations of the concentrated soluble MICA protein were incubated for 16 hours in different wells containing the target cells. Unbound protein was then removed by washing the wells twice with phosphate buffered saline (PBS). The target cells exposed to the soluble MICA were then treated with calcein-AM (2 ⁇ ) for 30 minutes at 37°C to achieve green fluorescence in all living cells. Following incubation, cells were again washed twice with PBS and then exposed to live NK-92MI cells in a 10: 1 and 5: 1 ratio to target cells.
- PBS phosphate buffered saline
- NK-92MI cells were incubated with target cells for four hours, and then unbound NK-92MI cells were removed and target cells washed twice with PBS. Next, ethidium homodimer was added (2 ⁇ ) for 30 min at 37°C to determine the extent of NK cell killing. Cells were washed and analyzed on a fluorescent plate reader (SoftMaxPro). Live cells and dead cells were quantified using average of red (ethidium) and green (calcein-AM) fluorescence signals from wells in the absence of NK cells and at the 5: 1 and 10: 1 ratios and at 0, 64 pg and 128 pg of added soluble, targeted MICA.
- SoftMaxPro fluorescent plate reader
- red fluorescent signals from (dead) MCF cells changed from 19.9 + 2.4 to 27.0 ⁇ 1.0 to 31.0 ⁇ 1.6 as NK cells were added at a ratio of 0 to 5: 1 to 10: 1.
- red fluorescent signals from (dead) MCF cells changed from 25.6 + 2.2 to 41.0 + 6.7 as NK cells were added at a ratio of 5: 1 to 10: 1.
- the corresponding fluorescent (green) signals from live cells in the same wells were 5621 + 372, 5535 + 205 and 5721 + 335 as NK cells were added at a ratio of 0 to 5: 1 to 10: 1 in the absence of targeted MICA.
- green fluorescent signals from live MCF cells changed from 5028 + 177 to 5181 + 102 to 3697 + 591 as NK cells were added at a ratio of 0 to 5: 1 to 10: 1.
- targeted MICA was added at 128 pg
- green fluorescent signals from live MCF cells changed from 3459 + 394 to 2191 + 331 as NK cells were added at a ratio of 5 : 1 to 10: 1.
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AU2015357578B2 (en) * | 2014-12-05 | 2018-11-08 | Xyphos Biosciences Inc. | Insertable variable fragments of antibodies and modified a1-a2 domains of NKG2D ligands |
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