WO2007091078A2 - Polypeptide conjugate comprising hla-g and uses thereof, stem cells transfected with hla-g - Google Patents

Polypeptide conjugate comprising hla-g and uses thereof, stem cells transfected with hla-g Download PDF

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WO2007091078A2
WO2007091078A2 PCT/GB2007/000448 GB2007000448W WO2007091078A2 WO 2007091078 A2 WO2007091078 A2 WO 2007091078A2 GB 2007000448 W GB2007000448 W GB 2007000448W WO 2007091078 A2 WO2007091078 A2 WO 2007091078A2
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nucleic acid
stem cell
cell
cells
acid molecule
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PCT/GB2007/000448
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French (fr)
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WO2007091078A3 (en
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James Walsh
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Axordia Limited
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Priority claimed from GB0602688A external-priority patent/GB0602688D0/en
Priority claimed from GB0605180A external-priority patent/GB0605180D0/en
Application filed by Axordia Limited filed Critical Axordia Limited
Publication of WO2007091078A2 publication Critical patent/WO2007091078A2/en
Publication of WO2007091078A3 publication Critical patent/WO2007091078A3/en

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to a polypeptide conjugate wherein the conjugate comprises a ligand binding moiety fused to a HLA G molecule, or part thereof and including oligomers and dimers of said conjugate and transgenic cells expressing HLA G.
  • EVT extra-villous trophoblast
  • HLA G molecules inhibit the natural killer cell-mediated cytotoxicity. HLA G molecules also down regulates both CD8 + and CD4 + T cell reactivity. T cell mediated responses have a major role in many autoimmune or inflammatory diseases that result in the destruction and damage of cells and tissues by the body's own immune system by recognition of self antigens by CD8 + T lymphocytes.
  • inflammatory joint diseases e.g., rheumatoid arthritis, osteoarthritis, polyarthritis and gout
  • chronic inflammatory connective tissue diseases e.g., lupus erythematosus, scleroderma, Sjorgen's syndrome, poly- and dermatomyositis, vasculitis, mixed connective tissue disease (MCTD), tendonitis, synovitis, bacterial endocarditis, osteomyelitis and psoriasis
  • chronic inflammatory lung diseases e.g., chronic respiratory disease, pneumonia, fibrosing alveolitis, chronic bronchitis, chronic obstructive pulmonary disease (COPD), bronchiectasis, emphysema, silicosis and other pneumoconiosis and tuberculosis
  • chronic inflammatory bowel and gastro-intestinal tract inflammatory diseases e.g., rheumatoid arthritis, osteoarthritis
  • autoimmune diseases such as; multiple sclerosis, type 1 diabetes, autoimmune thyroid disease, autoimmune hepatitis, rheumatoid arthritis, autoimmune colitis, Crohn's disease, celiac disease, autoimmune nephritis, autoimmune neuropathy, encephalopathy, fibrosing alveolitis.
  • HLA G HLA G.
  • conjugates comprising HLA G fused to ligands that enable the targeting of HLA G to specific cell-types and the use of the conjugates in the treatment of conditions that would benefit from the inhibition of T cell mediated responses.
  • a problem associated with gene therapy and/or tissue engineering is the provision of cell/tissue materials that do not induce a natural killer cell response in the recipient animal.
  • the provision of stem cell-lines that either constitutively express or developmentally express HLA G genes will facilitate procedures that involve gene and tissue replacement therapy.
  • a polypeptide conjugate comprising a first part wherein said first part is a ligand that binds a polypeptide expressed by a cell and a second part wherein said second part is a polypeptide encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i) a nucleic acid molecule consisting of at least one nucleic acid sequence as represented in Figure IA-T; ii) a nucleic acid molecule that hybridises to the nucleic acid molecule in Figure IA-T and that encodes a polypeptide with the activity associated with HLA G; iii) a nucleic acid molecule comprising a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequences as defined in (i) and (ii) above.
  • Hybridization 6x SSC at RT to 55°C for 16-20 hours
  • said first part is an antibody or active binding part thereof.
  • said antibody is a monoclonal antibody.
  • H and L chains consisting of two pairs of polypeptide chains, one pair of light (L) (low molecular weight) chain (K or ⁇ ), and one pair of heavy (H) chains ( ⁇ , ⁇ , ⁇ , ⁇ and ⁇ ), all four linked together by disulphide bonds.
  • Both H and L chains have regions that contribute to the binding of antigen and that are highly variable from one Ig molecule to another.
  • H and L chains contain regions that are non- variable or constant.
  • the L chains consist of two domains.
  • the carboxy-terminal domain is essentially identical among L chains of a given type and is referred to as the "constant” (C) region.
  • the amino terminal domain varies from L chain to L chain and contributes to the binding site of the antibody. Because of its variability, it is referred to as the "variable” (V) region.
  • the H chains of Ig molecules are of several classes, ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ (of which there are several sub-classes).
  • An assembled Ig molecule consisting of one or more units of two identical H and L chains derives its name from the H chain that it possesses.
  • Ig isotypes IgA, IgM, IgD, IgE and IgG (with four sub-classes based on the differences in the H chains, i.e., IgGl, IgG2, IgG3 and IgG4).
  • said Fab fragment is selected from the group consisting of: F (ab') 2 , Fab, Fv and Fd fragments, CDR3 regions, single chain antibody fragment or a domain antibody fragment.
  • a Fab fragment is a multimeric protein consisting of the immunologically active portions of an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, covalently coupled together and capable of specifically binding to an antigen.
  • Fab fragments are generated via proteolytic cleavage (with, for example, papain) of an intact immunoglobulin molecule.
  • a Fab 2 fragment comprises two joined Fab fragments. When these two fragments are joined by the immunoglobulin hinge region, a F(ab') 2 fragment results.
  • immunoglobulin or antibody fragments are within the scope of the invention and are described in standard immunology textbooks such as Paul, Fundamental Immunology or Janeway et al. Immunobiology (cited above). Molecular biology now allows direct synthesis (via expression in cells or chemically) of these fragments, as well as synthesis of combinations thereof. A fragment of an antibody or immunoglobulin can also have bispecific function as described above.
  • said antibody or fragment is a chimeric or humanised antibody.
  • Chimeric antibodies are recombinant antibodies in which all of the V-regions of a mouse or rat antibody are combined with human antibody C-regions.
  • Humanised antibodies are recombinant hybrid antibodies which fuse the complimentarity determining regions from a rodent antibody V-region with the framework regions from the human antibody V- regions. The C-regions from the human antibody are also used.
  • the complimentarity determining regions (CDRs) are the regions within the N-terminal domain of both the heavy and light chain of the antibody to where the majority of the variation of the V- region is restricted. These regions form loops at the surface of the antibody molecule. These loops provide the binding surface between the antibody and antigen. Antibodies from non-human animals provoke an immune response to the foreign antibody and its removal from the circulation.
  • said ligand comprises an aptamer.
  • Nucleic acids have both linear sequence structure and a three dimensional structure which in part is determined by the linear sequence and also the environment in which these molecules are located.
  • Conventional therapeutic molecules are small molecules, for example, peptides, polypeptides, or antibodies, which bind target molecules. It has become apparent that nucleic acid molecules also have potential with respect to providing agents with the requisite binding properties. These nucleic acid molecules are typically referred to as aptamers. Aptamers are small, usually stabilised, nucleic acid molecules, which comprise a binding domain for a target molecule. A screening method to identify aptamers is described in US 5,270,163, which is incorporated by reference.
  • modified nucleotides may also include 2' substituted sugars such as 2'-O-methyl-; 2-O-alkyl; 2-0- allyl; 2'-S-alkyl; 2'-S-allyl; T- fluoro-; 2'-halo or 2;azido-ribose, carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, and sedoheptulose.
  • 2' substituted sugars such as 2'-O-methyl-; 2-O-alkyl; 2-0- allyl; 2'-S-alkyl; 2'-S-allyl; T- fluoro-; 2'-halo or 2;azido-ribose, carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyx
  • the aptamers of the invention are synthesised using conventional phosphodiester linked nucleotides and synthesised using standard solid or solution phase synthesis techniques which are known in the art.
  • Linkages between nucleotides may use alternative linking molecules.
  • said ligand is a peptide.
  • said peptide is a modified peptide.
  • Modifications include, by example and not by way of limitation acetylation and amidation.
  • said peptide is acetylated.
  • said acetylation is to the amino terminus of said peptide.
  • said peptide is amidated.
  • said amidation is to the carboxyl-terminus of said peptide.
  • said peptide is modified by both acetylation and amidation.
  • said modification includes the use of modified amino acids in the production of recombinant or synthetic forms of peptides.
  • modified amino acids include, by way of example, 4-hydroxyproline, 5-hydroxylysine, N 6 -acetyllysine, N 6 -methyllysine, N 6 ,N 6 -dimethyllysine, N 6 ,N 6 ,N 6 - trimethyllysine, cyclohexyalanine, D-amino acids, ornithine.
  • Other modifications include amino acids with a C 2 , C 3 or C 4 alkyl R group optionally substituted by 1, 2 or 3 substituents selected from halo (e.g. F, Br 5 1), hydroxy or C 1 -C 4 alkoxy.
  • peptides could be modified by, for example, cyclisation.
  • Cyclisation is known in the art, (see Scott et al Chem Biol (2001), 8:801-815; Gellerman et al J. Peptide Res (2001), 57: 277-291; Dutta et al J. Peptide Res (2000), 8: 398-412; Ngoka and Gross J Amer Soc Mass Spec (1999), 10:360-363.
  • peptides according to the invention are modified by cyclisation.
  • polypeptide is expressed by an embryonic stem cell. In an alternative preferred embodiment of the invention said polypeptide is expressed by a stem cell.
  • ligands for example antibodies or preferably single chain antibody fragments
  • aptamers or peptides are specific for embryonic stem cell markers, for example ligands that bind Tra-1-60, Tra-1-81, SSEAl, SSEA3, SSEA4, A2B5 and ME311.
  • cell markers associated with pathological conditions such as Type 1 diabetes would be targeted, for example the antigen recognised by Anti-Human Pancreatic Islet Cells antibody clone 3D3 (cat#7099-1006, Biogenesis Ltd, Technology Road, Poole, BHl 7 7DA, England).
  • Rheumatoid arthritis has a number of disease associated markers, for example Advanced Glycation End-products (AGEs), receptors for AGEs (RAGE) and Collagen II.
  • AGEs Advanced Glycation End-products
  • RAGE receptors for AGEs
  • Collagen II A further example of an autoimmune disease treatable with the conjugate according to the invention is the targeting of sialoadhesin expressed by oligodendrocytes in the treatment of multiple sclerosis.
  • the treatment of asthma may be achieved by targeting epithelial cells that line the respiratory tract with ligands that bind, for example, the cell surface antigen recognised by Mouse Anti-Epithelial Cell Surface Monoclonal Antibody, Clone 5E11 (Cat# 1422, StemCell Technologies Inc., USA).
  • Further examples include RAGE polypeptides, CD antigens, mucins, collagens and cadherins.
  • a conjugate comprising a polypeptide encoded by a nucleic acid molecule as represented Figure IA-T.
  • polypeptide is a variant polypeptide and comprises the amino acid sequence represented in Figure 2A-O, which sequence has been modified by deletion, addition or substitution of at least one amino acid residue wherein said modification retains or modifies the activity of said polypeptide.
  • amino acids are considered conservative replacements (similar): a) alanine, serine, and threonine; b) glutamic acid and aspartic acid; c) asparagine and glutamine d) arginine and lysine; e) isoleucine, leucine, methionine and valine and f) phenylalanine, tyrosine and tryptophan. Most highly preferred are variants that retain or modify the same biological function and activity as the reference polypeptide from which it varies.
  • the invention features polypeptide sequences having at least 50% identity, over all or part of the length of the polypeptide, with the polypeptide sequences as herein disclosed, or fragments and functionally equivalent polypeptides thereof.
  • the polypeptides have at least 50% identity, more preferably at least 60% identity, even more preferably at least 70% identity, still more preferably at least 80% identity, and most preferably at least 90- 99% identity with the amino acid sequences illustrated herein.
  • said conjugate is an in-frame translational fusion between said first and second parts wherein said first and second parts are linked either directly or indirectly to each other.
  • said in-frame translational fusion is a direct fusion between first and second parts.
  • conjugate according to the invention for use as a pharmaceutical.
  • composition comprising a conjugate according to the invention.
  • composition is a pharmaceutical composition.
  • composition further comprises an adjuvant or carrier.
  • said adjuvant is an immunosuppressant.
  • the conjugate compositions of the present invention are administered in pharmaceutically acceptable preparations.
  • Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents.
  • the conjugates of the invention can be administered by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal.
  • a preferred route of administration is by pulmonary aerosol.
  • Techniques for preparing aerosol delivery systems containing antibodies are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the antibodies, such as the binding capacity (see, for example, Sciarra and Cutie, "Aerosols," in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712; incorporated by reference). Those of skill in the art can readily determine the various parameters and conditions for producing aerosols without resort to undue experimentation.
  • compositions of the invention are administered in effective amounts.
  • An "effective amount" is that amount of a composition that alone, or together with further doses, produces the desired response.
  • the desired response is inhibiting the progression of the disease. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine diagnostic methods.
  • Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.
  • compositions used in the foregoing methods preferably are sterile and contain an effective amount of conjugate for producing the desired response in a unit of weight or volume suitable for administration to a patient.
  • the doses of conjugate administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.
  • doses of conjugate are formulated and administered in doses between 1 ng and 1 mg, and preferably between 10 ng and 100 ⁇ g, according to any standard procedure in the art.
  • Other protocols for the administration of conjugate compositions will be known to one of ordinary skill in the art, in which the dose amount, schedule of injections, sites of injections, mode of administration and the like vary from the foregoing.
  • Administration of conjugate compositions to mammals other than humans, (e.g. for testing purposes or veterinary therapeutic purposes), is carried out under substantially the same conditions as described above.
  • a subject, as used herein, is a mammal, preferably a human, and including a non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent.
  • the pharmaceutical preparations of the invention When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents.
  • the salts When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention.
  • Such pharmacologically and pharmaceutically- acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like.
  • pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
  • Conjugate compositions may be combined, if desired, with a pharmaceutically-acceptable carrier.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
  • the pharmaceutical compositions may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
  • suitable buffering agents including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
  • suitable preservatives such as: benzalkonium chloride; chlorobutanol; and parabens.
  • compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active compound.
  • Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.
  • compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of conjugates, which is preferably isotonic with the blood of the recipient.
  • This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in I 5 3-butane diol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono-or di-glycerides.
  • fatty acids such as oleic acid may be used in the preparation of injectables.
  • Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
  • composition comprising a conjugate according to the invention and an embryonic stem cell.
  • said embryonic stem cell is a primate embryonic stem cell.
  • said embryonic stem cell is human and is not a totipotent embryonic stem cell.
  • said embryonic stem cell is a non- primate embryonic stem cell.
  • said non-primate embryonic stem cell is a murine embryonic stem cell.
  • said embryonic stem cell is a teratocarcinoma stem cell.
  • said embryonic stem cell is transfected with a nucleic acid molecule that encodes a polypeptide to which said ligand binds.
  • a method to treat an inflammatory disease or condition comprising administering an effective amount of a conjugate or a composition according to the invention to a subject in need of treatment.
  • a method to treat an autoimmune disease or condition comprising administering an effective amount of a conjugate or a composition according to the invention to a subject in need of treatment.
  • said autoimmune disease is selected from the group consisting of: multiple sclerosis, type 1 diabetes, autoimmune thyroid disease, autoimmune hepatitis, rheumatoid arthritis, autoimmune colitis, Crohns disease, celiac disease, autoimmune nephritis, autoimmune neuropathy, encephalopathy, fibrosing alveolitis.
  • a method to treat organ/tissue rejection in a recipient subject comprising administering an effective amount of a conjugate or composition according to the invention to a subject in need of treatment.
  • said autoimmune disease is selected from the group consisting of: multiple sclerosis, type 1 diabetes, autoimmune thyroid disease, autoimmune hepatitis, rheumatoid arthritis, autoimmune colitis, Crohns disease, celiac disease, autoimmune nephritis, autoimmune neuropathy, encephalopathy, fibrosing alveolitis.
  • said subject is human.
  • said conjugate is provided as multimeric complex of monomer conjugate subunits according to the invention.
  • said complex comprises at least two monomer subunits.
  • said complex consists of two monomer subunits. In a preferred embodiment of the invention said complex comprises identical monomer subunits.
  • said complex comprises dissimilar monomer subunits.
  • said complex consists of two monomer subunits forming a homodimer.
  • said complex consists of two dissimilar monomer subunits forming a heterodimer.
  • said complex is an in-frame translational fusion protein comprising monomer subunits.
  • a method to treat an inflammatory disease or condition comprising administering an effective amount of a complex or a composition comprising a complex according to the invention to a subject in need of treatment.
  • a method to treat an autoimmune disease or condition comprising administering an effective amount of a complex or a composition comprising a complex according to the invention to a subject in need of treatment.
  • said autoimmune disease is selected from the group consisting of: multiple sclerosis, type 1 diabetes, autoimmune thyroid disease, autoimmune hepatitis, rheumatoid arthritis, autoimmune colitis, Crohns disease, celiac disease, autoimmune nephritis, autoimmune neuropathy, encephalopathy, fibrosing alveolitis.
  • a method to treat organ/tissue rejection in a recipient subject comprising administering an effective amount of a complex or a composition comprising a complex according to the invention to a subject in need of treatment.
  • a stem cell wherein said cell is transfected with a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i) a nucleic acid molecule consisting of a nucleic acid sequence as represented in Figure IA-T ii) a nucleic acid molecule that hybridises to the nucleic acid molecule in Figure IA-T and that encodes a polypeptide with the activity associated with HLA-G; iii) a nucleic acid molecule comprising a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequences as defined in (i) and (ii) above.
  • said nucleic acid molecule hybridizes under stringent hybridization conditions.
  • said embryonic stem cell comprises the nucleic acid sequence as represented in Figure IA-T.
  • nucleic acid molecule consists of the nucleic acid sequence as represented in Figure IA-T.
  • said nucleic acid molecule is operably linked to a nucleic acid molecule comprising a transcription promoter sequence to form a transcription cassette.
  • said promoter sequence is a constitutive promoter; preferably said promoter sequence is a cell specific promoter sequence.
  • said promoter sequence is a developmentally regulated promoter sequence.
  • said nucleic acid molecule further comprises a repressor sequence.
  • nucleic acid molecule comprising said promoter sequence is part of a vector.
  • said vector is adapted for eukaryotic expression.
  • said adaptation includes the provision of transcription control sequences (promoter sequences) which mediate cell/tissue specific expression.
  • promoter sequences may be cell/tissue specific, inducible or constitutive.
  • Promoter elements also include so called TATA box and RNA polymerase initiation selection sequences which function to select a site of transcription initiation. These sequences also bind polypeptides which function, inter alia, to facilitate transcription initiation selection by RNA polymerase.
  • Adaptations also include the provision of selectable markers and autonomous replication sequences which facilitate the maintenance of said vector in either the eukaryotic cell or prokaryotic host.
  • Vectors which are maintained autonomously are referred to as episomal vectors.
  • Episomal vectors are desirable since these molecules can incorporate large DNA fragments (30-50kb DNA). Episomal vectors of this type are described in WO98/07876.
  • Adaptations which facilitate the expression of vector encoded genes include the provision of transcription termination/polyadenylation sequences. This also includes the provision of internal ribosome entry sites (IRES) which function to maximise expression of vector encoded genes arranged in bi-cistronic or multi-cistronic expression cassettes.
  • Expression control sequences also include so-called Locus Control Regions (LCRs). These are regulatory elements which confer position-independent, copy number-dependent expression to linked genes when assayed as transgenic constructs. LCRs include regulatory elements that insulate transgenes from the silencing effects of adjacent heterochromatin, Grosveld et al., Cell (1987), 51 : 975-985.
  • Expression control sequences also encompass, ubiquitous chromatin opening elements (UCOE's), see WO/GB00/05393.
  • UCOE's are nucleic acid elements that are responsible for establishing an open chromatin structure across a locus that consists exclusively of ubiquitously expressed, housekeeping genes. These elements are not derived from an LCR.
  • a UCOE is a polynucleotide which opens chromatin or maintains chromatin in an open state and facilitates reproducible expression of an operably-linked gene in cells of at least two different tissue types.
  • Vectors are typically viral based and may be derived from viruses including adenovirus; retrovirus; adeno-associated virus; herpesvirus; lentivirus; vaccinia virus; and baculovirus.
  • said vector is a viral based vector.
  • said viral vector is selected from the group consisting of: adenovirus; retrovirus; adeno- associated virus; herpesvirus; lentivirus; vaccinia virus; baculovirus.
  • said viral based vector is an adenovirus.
  • said viral based vector is a lentivirus.
  • said cell is transfected with a further nucleic acid molecule.
  • said further nucleic acid molecule encodes a therapeutic polypeptide.
  • the invention contemplates gene therapy.
  • Gene therapy involves the transfer and stable insertion of new genetic information into cells for the therapeutic treatment of disease.
  • Stem cells or pluripotent ES cells are the ideal target of gene transfer.
  • CaPO4 precipitation and DEAE dextran show the capability of integrating transferred genes stably in a wide variety of cell types.
  • Recombinant retrovirus vectors have been widely used experimentally to transduce hematopoietic stem and progenitor cells.
  • mice after transfer by retrovirus vectors include human hypoxanthine phosphoribosyl transferase (Miller A et al, 1984, Science 255:630). Bacterial genes have also been transferred into mammalian cells, in the form of bacterial drug resistance gene transfers in experimental models.
  • Transformation of hematopoietic progenitor cells to drug resistance by eukaryotic virus vectors has also accomplished with recombinant retrovirus based vector systems (Hock RA and Miller AD 1986, Nature 320:275-277; Joyner A et al, 1983, Nature 305:556-558; Williams DA et al 1984, Nature 310:476-480; Dick JE et al, 1985, Cell 42:71-79); Keller G et al 1985, Nature 318: 149-154; Eglitis M et al, 1985, Science 230: 1395-1398).
  • Adeno-associated virus vectors have been used successfully to transduce mammalian cell lines to neomycin resistance (Hermonat PL and Muzyczka N, 1984, supra; Tratschin JD et al, 1985, MoI. Cell. Biol. 5:3251).
  • Other viral vector systems that have been investigated for use in gene transfer include papovaviruses and vaccinia viruses (see Cline ML 1985, Pharmac. Ther. 29:69-92).
  • said vector is replication competent.
  • said vector is conditionally replication competent.
  • said vector is a replication incompetent vector.
  • said stem cell is a multipotent stem cell.
  • said multipotent stem cell is selected from the group consisting of: haemopoietic stem cell; neural stem cell; bone stem cell; muscle stem cell; mesenchymal stem cell; epithelial stem cell (derived from organs such as the skin, gastrointestinal mucosa, kidney, bladder, mammary glands, uterus, prostate and endocrine glands such as the pituitary); endodermal stem cell (derived from organs such as the liver, pancreas, lung and blood vessels).
  • said stem cell is a primate embryonic stem cell.
  • said embryonic stem cell is a human embryonic stem cell.
  • said embryonic stem cell is a non- primate stem cell.
  • said stem cell is a murine stem cell.
  • said embryonic stem cell is a teratocarcinoma stem cell.
  • said embryonic stem cell is not a totipotent cell.
  • a spheroid body comprising an embryonic stem cell according to the invention.
  • said body further comprises a collagen based cell support.
  • a cell derived from an embryonic stem cell or spheroid body according to the invention.
  • an in vitro cell culture comprising an embryonic stem cell or an embryoid body according to the invention.
  • a therapeutic vehicle for use in tissue engineering comprising a cell or spheroid body according to the invention.
  • said vehicle is selected from the group consisting of: prothesis; implant; matrix; stent; biodegradable matrix; or hydrogel.
  • composition comprising a cell according to the invention.
  • composition for use in therapeutic tissue engineering comprising a cell according to the invention.
  • a method to treat a condition that would benefit from tissue engineering comprising administering a cell or a therapeutic vehicle according to the invention to a subject in need of treatment.
  • ES cells according to the invention can be administered by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example, intravenous, intraperitoneal, intramuscular, intra-cavity, subcutaneous, or transdermal.
  • the cells of the invention are administered in effective amounts.
  • An "effective amount" is that amount of transfected ES cells that alone, or together with further doses, produces the desired response.
  • the desired response is inhibiting the progression of the disease. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently.
  • Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.
  • a non-human transgenic mammal wherein said mammal comprises a cell derived from a cell according to the invention.
  • Ware et al teaches an embryonic stem cell culture condition amenable for such animals as cattle, pigs and sheep ⁇ Society for the Study of Reproduction, 38:241 (1988)).
  • Figure IA-T illustrates the nucleic acid sequences of various HLA G molecules
  • Figure 2A-0 illustrates the amino acid sequence of various HLA G molecules.
  • Human embryonic stem cells can be grown in flasks with or without feeder cells; follow A or B, respectively:
  • RNA is isolated from hybridoma cells with Tri-reagent and subjected to DNase I treatment, and then mRNA is isolated. First-strand cDNA synthesis is prepared with random primers using reverse transcriptase.
  • VH and VL variable chains are performed using separate reaction mixtures with primers specific for each chain. Purification of PCR products is performed by gel electrophoresis in agarose gel. DNA bands corresponding to VH and VL chains respectively are excised and the DNA was purified (e.g. by Sephaglas Bandprep Kit (Amersham Biosciences)). The VH and VL chains are assembled with a linker by two rounds of PCR amplification as described in the Amersham Recombinant Phage Antibody System (RPAS) kit.
  • RPAS Amersham Recombinant Phage Antibody System
  • the entire coding sequence or a fragment thereof of the HLA-G gene is inserted into the multiple cloning site of the pCANTAB5E phagemid vector (Amersham Biosciences).
  • the 750 bp DNA band corresponding to the ScFv fragment is purified and cloned in-frame with the HLA-G sequence, either 5 ' or 3' to it.
  • Recombinant phage antibody production is accomplished using a kit such as RPAS (Amersham Biosciences), according to the manufacturer's instructions. Competent E. coli TGl cells were transformed with pCANTAB5E containing the ScFv fragment. Individual transformed colonies were infected with M13KO7 phage to rescue the phagemid. Recombinant phage antibodies arising from each clone are analysed for target binding activity in an ELISA assay or cell binding assay. Phage binding is detected with a peroxidase-labelled sheep anti-M13 antibody (Amersham Biosciences). The recombinant phage clone which gives the strongest positive signal in the assay is used to infect E. coli HB2151 cells.
  • individual HB2151 colonies are transferred to 5 ml of 2" 1 YT medium containing 100 lg ⁇ ml ampicillin and 1% (w ⁇ v) glucose and incubated overnight at 37 °C. Aliquots of 100 11 of cell suspension are grown in 2 ml of 2- 1 YT medium containing 100 lg ⁇ ml ampicillin and 0.1% (w ⁇ v) glucose until cell culture reaches an attenuance of 0.5 or 1 at 600 run.
  • Kits available from companies such as Pierce Biotechnology, Inc., Rockford, IL. 61105 U.S.A, are available to perform cross linking to conjugate the HLA-G molecule to an antibody of interest, using the reactive and functional species in the table above.
  • An HPLC-purified oligonucleotide library is prepared, consisting of base sequences in which 10-150 nucleotides are randomly arranged between primer binding sequences designated "F” (5') and “R” (3'), each consisting of 10-25 nucleotides and including an RNA polymerase initiation site immediately 3' of the F primer.
  • the randomized oligonucleotides are transcribed into RNA using an appropriate polymerase e.g. T7 polymerase, and purified by phenol chloroform extraction
  • the RNA aptamer library is incubated with both target and control cells in normal physiological conditions. Following incubation for between 1 minute and 6 hours, the cells are washed by repeated centrifugation and aspiration of the suspension solution.
  • the washed cell-nucleic acid complexes are subjected to RT-PCR using primers that recognize the primer binding sites. This amplifies a DNA pool complementary to those RNA molecules that bind the cells.
  • Steps 2 through 4 are repeated between 1 and 5 times to produce a highly enriched DNA pool.
  • An HPLC-purified oligonucleotide library is prepared, consisting of base sequences in which 10-150 nucleotides are randomly arranged between primer binding sequences designated "F” (5') and "R” (3'), each consisting of 10-25 nucleotides
  • the DNA aptamer library is incubated with both target and control cells in normal physiological conditions. Following incubation for between 1 minute and 6 hours, the cells are washed by repeated centrifugation and aspiration of the suspension solution.
  • the washed cell-nucleic acid complexes are subjected to PCR using primers that recognize the primer binding sites. This amplifies a DNA pool corresponding to those aptamers that show greatest binding affinity for the cells.
  • Steps 2 through 4 are repeated between 1 and 5 times to produce a highly enriched
  • the enriched DNA pool is cloned into a plasmid vector and the cell type specific aptamers selected by subtractive hybridization between the target and control cell libraries
  • a suitable aptamer Once a suitable aptamer has been identified that specifically binds the desired target population, it is conjugated to HLA-G.
  • a number of approaches are available to achieve this, including ready made reagents in kit form, available from companies such as Pierce Biotechnology, Inc., Rockford, IL. 61105 U.S.A:
  • a convenient functional group that can be chemically modified to allow the coupling of protein molecules on oligonucleotides is the 5 '-phosphate group.
  • Using the 5' end of the oligonucleotide as the conjugation point has an advantage of keeping the rest of the nucleic acid sequence unmodified and free so it can easily hybridize to a complementary target.
  • a 5 '-phosphate group may be put on the end of the molecule to facilitate this type of conjugation.
  • the alkyl phosphate is reactive with the water-soluble carbodiiniide EDC (Product # 22980, 22981), which forms a phosphate ester similar to the reaction of EDC with a carboxylate group. Subsequent coupling to an amine-containing molecule (i.e., nearly any protein or unmodified peptide) can be done to form a stable phosphoramidate linkage(Figurel ?????).
  • EDC water-soluble carbodiiniide EDC
  • the resultant amine- modified oligonucleotide can be coupled to enzyme molecules using a heterobifunctional reagent.
  • a diamine compound that contains a disulfide e.g., cystamine
  • reducing the disulfide group results in a sulfhydryl that may be conjugated with proteins rendered sulfhydryl-reactive using the heterobifunctional reagent Sulfo-SMCC (Product #
  • RNA molecules may be chemically modified to allow coupling with amine-containing molecules or proteins.
  • the diol on the 3'-ribose residue may be oxidized to result in two aldehyde groups using Sodium meta -Periodate (Product
  • the aldehydes then can be conjugated to the amine groups on a protein using reductive amination with Sodium Cyanoborohydride (Product # 44892).
  • the aldehyde and the amine first form a Schiff base that is reduced to secondary amine linkage with the cyanoborohydride reductant.
  • biotinylation of oligonucleotides can be done using photoreactive reagents.
  • photoreactive reagents There are two main options commonly used to add one or more biotin residues to nucleic acid probes.
  • Photoactivatable Biotin contains a phenyl azide group at the end of a spacer arm with the biotin group at the other end. Photolyzing a solution of the biotin compound together with an oligonucleotide in solution results in biotin being non-selectively inserted into the nucleic acid structure.
  • Psoralen-PEO- Biotin can be used to label double-stranded DNA or RNA.
  • the psoralen ring structure effectively intercalates into the double-stranded portions, and exposure to UV light causes a cyclo-addition product to be formed with the 5, 6-double bond in thymine residues.
  • the poly(ethylene oxide) spacer in Psoralen-PEO-Biotin contributes excellent water solubility, thus assuring that the resultant derivative will have accessibility to streptavidin-containing detection reagents.
  • the phage library is incubated with the human control (i.e. non target) cell population in normal physiological conditions
  • control cells are removed by centrifugation and the phage-containing supernatant transferred to a fresh tube
  • the target cell population is added to the collected supernatant and incubated for between 1 minute and 6 hours 4.
  • the target cells are pelleted by centrifugation and the supernatant discarded
  • the target cells are washed 3 times in PBS
  • Phage bound to target cells are eluted (e.g. by altering pH, salt concentration etc.) 7. Recovered phage are used to infect a permissive E.coli strain and the resulting phage-carrying bacteria grown in suitable medium
  • 8Phage are prepared from the infected bacteria and incubated with a human control population of cells, as in 1. 9Steps 2-8 are repeated between 1 and 4 times to enrich for phage carrying target cell specific peptide/ scFv encoding DNA lO.Phage DNA is sequenced to determine the targeting peptide
  • a suitable targeting sequence is determined, it is cloned either 5' or 3' of the HLA-G gene coding sequence in a suitable expression system to produce recombinant protein.
  • CDIb T645 IgSF MHC-like molecule
  • Group 1 CDl Cortical thymocytes Langerhans cells
  • DC Antigen presentation associates with b2m lq22-23
  • CD3d T3 21 IgSF T cells CD3d T3 21 IgSF T cells, thymocyte subset Associates with T cell antigen receptor, required for TCR cell surface expression and signal transduction 1 Iq23
  • CD4 T4 L3T455 IgSF Thymocyte subset, T subset (helper), monocytes, macrophages MHC class II coreceptor, receptor for HIV, thymic differentiation, T cell activation 12pter-pl2
  • CD6 T12 100-130 Scavenger receptor superfamily Thymocytes, T cells, B subset CD166 counter-receptor, T cell differentiation and costimulation 11
  • CD8a T8 Leu-2, Lyt2,3 32-34 IgSF Thymocyte subset, T subset (cytotoxic), NK cells MHC class I coreceptor, receptor for some mutated HIV-I, thymic differentiation, T cell activation 2p 12
  • CD9 p24 MRP-I 22-27 TM4SF Pre-B cells, eosinophils, basophils, platelets, activated T cells Cellular adhesion and migration 12pl3
  • CDwI 7 Neutrophils, monocytes, platelets Cell surface glycosphingolipid, lactosyl ceramide
  • CD22 BL-CAM CD22 BL-CAM, Siglec-2 150 IgSF, sialoadhesins B cells Adhesion, B-monocytes and B- T interactions 19ql3
  • CD24 BA-I 35-45 GPI-linked membrane protein B cells, granulocytes, epithelial cells B cell differentiation and proliferation 6q21
  • CD25 Tac CD25 Tac, p55 55 Tpe I transmembrane protein Activated T and activated B cells IL-2 receptor a chain, associates with IL-2 receptor b and g chains to form high affinity IL-2R complex 10pl4- ⁇ l5
  • DPP IV Dipeptidyl peptidase IV 110 Type II transmembrane protein Thymocyte subset, activated T cells, B cells, NK cells, macrophages, epithelial cells, soluble form A dipeptidyl peptidase, T cell costimulation, HIV entry into cells 2q24.3
  • T4444 IgSF Most T cells, thymocytes, plasma cells CD80 (B7-1) and B7-2 (CD86) counter-receptor, T cell costimulation 2q33 00448
  • CD34 105-120 Sialomucin, type I transmembrane protein Hematopoietic precursors, capillary endothelial cells, embryonic fibroblasts CD62L counter-receptor, adhesion, stem cell marker Iq32
  • CD35 CRl 250 CCRSF Erythrocytes, B cells, monocytes, neutrophils, eosinophils, FDC, T subset Complement receptor 1, binds C3b and C4b, adhesion, phagocytosis Iq32
  • CD36 GPPV 88 Platelets, monocytes, macrophage, endothelial cells, early erythrocytes Receptor for ECM, adhesion, phagocytosis 7ql 1.2
  • CD3740-52 TM4SFB cells T cells (low), granulocytes (low) Signal transduction 19pl3-ql3
  • CD38 TlO 45 ADP-ribosyl cyclase Variable levels on majority of hematopoietic cells, high expression on plasma cells, B cells and activated T cells An ecto-ADP-ribosyl cyclase, cell activation 4pl5
  • CD39 78 Activated B cells, NK cells, macrophages, Langerhans cells, DC B cell adhesion, protects activated cells from lysis, an ecto-apyrase 10q23.1-24.1
  • CD4048 TNFRSF B cells monocytes, macrophages, DC, endothelial cells, fibroblasts CDl 54 counter-receptor, B cell differentiation and costimulation, isotype-switchmg, rescues B cells from apoptosis 20ql2-ql3.2
  • CD42a GPIX 22 LRRF Platelets megakaryocytes Complex with CD42b, c and d, receptor to von Willebrand factor and thrombin, platelet adhesion to subendothelial matrices 17pter-pl2
  • CD44 H-CAM Pgp-1 80-95 Hyaladherin family Hematopoietic and non-hematopoietic cells, except platelets, hepatocytes, testis Binds hyaluronic acid, leukocyte adhesion Ilpter-pl3
  • CD44R CD44v Variants of CD44 Variants show heterogeneous expression, constitutively on epithelial cells, monocytes, upregulated on activated leukocytes Adhesion, metastasis
  • CD45RB 190-220 T subset B cells, monocytes, macrophages, granulocytes Exon B isoforms of CD45 Iq31-q32
  • CD45RO 180 Activated T cells, memory T cells, B subset, monocytes, macrophages, granulocytes Isoform of CD45 containing none of the A, B and C exons Iq31-q32
  • CD46 MCP 56/66 CCRSF Hematopoietic and non-hematopoietic nucleated cells
  • Membrane cofactor protein binds C3b and C4b to permit their degradation by Factor I, measles virus receptor Iq32
  • CD47 IAP 47-52 IgSF Hematopoietic cells, epithelial and endothelial cells, fibroblasts, other tissues Leukocyte adhesion, migration, activation 3ql3.1-13.2 Table 2 CD antigens
  • CD47R New designation for CDw 149, similar distribution as CD47 but dimmer CD48 Blast- 1 45 IgSF Broad, all leukocytes Cell adhesion lq21.3-22
  • VLA-4 150 Integrin family T cells, B cells, thymocytes, monocytes, eosinophils, NK cells, mast cells, DC Integrin a4, adhesion, CD49d/CD29 hinds fibronectin, VCAM- 1, MAdCAM-I 2q31-q32
  • ICAM-3 130 IgSF Thymocytes, T cells, B cells, monocytes, granulocytes, endothelial cells Adhesion, costimulation 19pl3.3-13.2
  • CD51 Vitronectin receptor 125 24 Integrin family Platelets, megakaryocytes, endothelial cells, osteoblasts, melanoma cells Integrin av, adhesion, CD51/CD61 dimer binds vitronectin, von Willebrand factor, fibrinogen and thrombospondin 2q3 l-q32
  • Thymocytes T cells, B cells (not plasma cells), monocytes, macrophages Unknown 1
  • CD55 Decay Accelerating Factor DAF 60-70 CCRSF
  • GPI-linked membrane protein Hematopoietic and non-hematopoietic cells Binds C3b, complement regulation Iq32
  • CD56 Neural Cell Adhesion Molecule, NCAM 175-185 IgSF NK cells, T subset, neural tissue, some large granular lymphocyte leukemias, myeloid leukemias Adhesion 11 q23.1 Table 2 CD antigens
  • CD59 Protectin GPI-linked membrane protein Hematopoietic and non-hematopoietic cells Binds complement components C8 and C9, blocks assembly of membrane attack complex (MAC) 1 Ipl3
  • CD60a GD3 CHO T subset CD60a GD3 CHO T subset, platelets, thymic epithelial cells, astrocytes Costimulation CD60b 9-O-sialyl GD3 CHO T subset, activated B cells CD60c 7-0-sialyl GD3 CHO T subset
  • CD61 GPIIIa 105 Integrin family Platelets, megakaryocytes, macrophages, endothelial cells Integrin b3, adhesion, CD41/CD61 or CD51/CD61 complexes mediate adhesion to ECM 17q21.3
  • CD62P P-selectin, PADGEM 140 Selectin family Activated platelets, endothelial cells CD 162 and sialyl Lewis x counter-receptor, adhesion, neutrophil rolling, platelet- neutrophil and platelet-monocyte interactions lq21-24
  • CD63 LIMP LAMP-3 53 TM4SF Activated platelets, monocytes, macrophages A lysosomal membrane protein, translocates to cell surface after activation 12ql2-ql3
  • CD65 Granulocytes CD65 Granulocytes, monocyte subset, myeloid leukemia cells
  • VIM2 Granulocytes, monocytes, myeloid leukemia cells Phagocytosis
  • CD66a Biliary glycoprotein- 1 (BGP-I), NCA-160 160-180 IgSF, CEA family Neutrophils, epithelial cells Cell adhesion, neutrophil activation 19ql3.1-2 Table 2 CD antigens
  • CD66b Previously CD67, CGM6, NCA-95 95-100 IgSF, CEA family Granulocytes Cell adhesion, neutrophil activation 19ql3.1-2
  • CD66e CarcinoEmbryonic Antigen 180-200 IgSF, CEA family Colon epithelium, colon cancer Cell adhesion 19ql3.1-2
  • CD66fPSG Sp-I 54-72 IgSF 5 CEA family Pregnancy specific glycoprotein, expressed on placental syncytiotrophoblasts, fetal liver Immune regulation, protects fetus from maternal immune system
  • CD68 Macrosialin, gpl 10 110 Sialomucin Expressed intracellularly in monocytes, macrophages, neutrophils, basophils, large lymphocytes, mast cells, DC, myeloid progenitors, liver 17pl3
  • CD69 Activation Inducer Molecule (AIM) 28 32 C-type lectin Activated T, B, NK cells and granulocytes, thymocytes, platelets, Langerhans cells Signal transduction 12pl2.3- pl3.2
  • CD74 Ii invariant chain 33,35,41,43
  • B cells macrophages, monocytes, Langerhans cells, DC, activated T cells MHC class II traffic and function 5q32
  • CD75 CHO Sialoglycan family B subset, epithelial cells Lactosamines Table 2 CD antigens
  • CD75s CHO Sialoglycan family B subset, T subset Alpha-2,6-sialylated lactosamines (previously CDw75 and CDw76)
  • Gb3 CD77 Globotiiaocylceramide
  • Pk blood group Germinal center B cells Burkitt's lymphomas (high), follicular center lymphomas (low) Apoptosis
  • CD79b Igb 37 IgSF B cells Component of BCR, required for cell surface expression and signal transduction 17q23
  • CD81 Target of Antiproliferative Antibody (TAPA-I) 26 TM4SF T, B, andNK cells, thymocytes, DC, endothelial cells, fibroblast, neuroblastomas, melanomas Complex with CD19 and CD21, signal transduction, T cell costimulation 1 Ipl5.5
  • CD83 HB 15 43 IgSF Activated B cells, activated T cells, circulating DC (veil cells),
  • CDw8473 Monocytes, platelets, B cells, T subset, macrophage subset
  • CD89 FcaR IgA receptor 55-75 IgSF Monocytes, macrophages, granulocytes, neutrophils, B subset, T subset IgA receptor, phagocytosis, degranulation, respiratory burst 19ql3.4 Table 2 CD antigens
  • CD93 120 Neutrophils, monocytes, endothelial cells
  • CD95 Apo-1 Fas 45 TNFRSF Lymphocytes (high level expression after activation), monocytes, neutrophils CD95L(CD178) counter-receptor, induction of apoptosis 10q24.1
  • CDlOO 150 Most hematopoietic cells except immature bone marrow cells, RBC and platelets Cell adhesion, cellular activation
  • CDlOl V7 CDlOl V7, ⁇ l26 120 IgSF Monocytes, granulocytes, DC, activated T cells T cell activation Ip 13 Table 2 CD antigens
  • CDl 03 HML-I a6, aE integrin 150,25 Integrin family Intraepithelial lymphocytes, peripheral blood lymphocyte subset, activated lymphocytes Associates with integrin b7, binds E-cadherin, lymphocyte homing and retention
  • CD 105 Endoglin 95 Homodimer Endothelial cells, bone marrow cell subset, activated macrophages Cellular response to TGF-bl 9q34.1
  • VCAM-I 110 IgSF Activated endothelial cells CDl 06 VCAM-I 110 IgSF Activated endothelial cells, FDC VLA-4 (CD49d/CD29) counter-receptor, leukocyte adhesion, transmigration, costimulation Ip31-p32
  • Activated platelets activated T cells, activated endothelial cells, metastatic tumors lysosomal membrane protein 13q34
  • Activated platelets activated T cells, activated endothelial cells, metastatic tumors lysosomal membrane protein Xq24-25
  • CD109 170/50 Activated T cells, activated platelets, CD34+ subset, endothelial cells
  • CDIl 1 PRRl Nectin-1 64-72 IgSF Stem cell subset, macrophages, neutrophils
  • CDl 12 PRR2 Nectin-2 64-72 IgSF Monocytes, neutrophils, CD34+ subset, megakaryocytes, endothelial cells, epithelial cells Intercellular adhesion 19ql3.2-ql3.4
  • CSF receptor a chain associates with common b chain, myeloid hematopoietic cell proliferation and differentiation Xp22.32, YpI 1.3
  • CD 120a TNFR-1 50-60 TNFRSF Hematopoietic and non-hematopoietic cells TNF receptor, binds both TNFa and TNFb 12pl3.2
  • CD 120b TNFR-II 75-85 TNFRSF Hematopoietic and non-hematopoietic cells TNF receptor, binds both TNFa and TNFb Ip36.3-p36.2
  • CDwl21b IL-IR type II 60-70 IgSF B cells, macrophages, monocytes, T subset Type II interleukin 1 receptor, binds IL-Ia and IL-Ib 1 negative signals 2ql2-q22
  • CD124 IL-4R 140 CRSF fibronectin type III superfamily Lymphocytes (low), monocytes, hematopoietic precursor cells, fibroblast, epithelial cells IL-4 receptor a chain, associates withCD132 or IL-13Rachain, T cell growth and differentiation 16pll.2-pl2.1
  • IL-8RA binds IL-8 only
  • IL-8RB binds IL-8, GRO, NAP-2 and ENA78 2q35
  • CDl 30 IL-6Rb, gpl30 130 CRSF Activated B cells and plasma cells, most leukocytes (weak), endothelial cells IL-6 receptor b subunit, signaling biological activities of IL-6, IL-11, LIF, CNF 5ql l
  • CDwl31 IL-3R common beta chain 95-120 CRSF Monocytes, granulocytes, early B cells Associates with the a subunits of IL-3, IL-5, and GM-CSF receptors, signal transduction 22ql2.2-ql3.1
  • CD132 Common gamma chain 64 CRSF T cells, B cells, NK cells, monocytes, granulocytes Subunit of IL-2, IL-4, IL-7, IL-9, and IL-15 receptors, signal transduction Xql3
  • CDl 34 OX-4048-50 TNFRSF Activated T cells OX-40L counter-receptor, T cell activation, differentiation, apoptosis Ip3 ⁇
  • CD 135 Flt3/Flk2 130-150 RTK family Multipotent myelomonocytic and primitive B progenitor cells Receptor tyrosine kinase, binds FLT3 ligand, early lymphocyte development 13ql2
  • CDwl36 MSP-R RON 180 RTK family Epithelial cells, CNS, PNS, hematopoietic subset Migration, morphological change, and proliferation in different target cells 3p21.1-22
  • CDwl374-1BB 30 TNFRSF Activated T cells T cell costimulation Ip36
  • CD 139 228 B cells monocytes, granulocytes, erythrocytes (low)
  • CD141 Thrombomodulin 100 C-type lectin Monocytes, neutrophils, endothelial cells, smooth muscle cells Activation of protein C and initiation of protein C anticoagulant Table 2 CD antigens
  • Endothelial cells Endothelial cells, epithelial cells, neuronal cells, fibroblats, activated macrophages a
  • CD 144 VE-Cadherin, Cadherin-5 130 Cadherin family Endothelial cells Adhesion, cell- cell interaction
  • CDwl45 110 90 Endothelial cells, some stromal cells
  • CD 146 MUCl 8 S-endo 113-118 IgSF Endothelial cells, melanomas, FDC, activated T cells Adhesion
  • CD150 SLAM surface lymphocyte activation marker
  • IgSF T subset upregulated after activation
  • B cells DC Costimulation, proliferation, Ig production
  • CD152 CTLA-4 33 IgSF Activated T cells, activated B cells CD80 and CD86 counter- receptor, negative regulation of T cell costimulation 2q33
  • Poliovirus receptor 80-90 IgSF Monocytes, macrophages, CD34+ thymocytes Polio virus receptor (PVR) 19ql3.1-13.2
  • CD 164 MGC-24 80 Epithelial cells, monocytes, lymphocytes (low), bone marrow stromal cells, CD34+ erythroid progenitor cells Hematopoietic progenitor cell-stromal cell interaction 6q21
  • CD 165 AD2 gp3742 Lymphocyte subset, monocytes, immature thymocytes, platelets Adhesion, thymocytes-thymic epithelium interaction
  • CD171 Ll cell adhesion molecule 200-210 IgSF CNS, PNS, glial cells, monocytes, T subset, B cells, DC, several human tumor cells Kidney morphogenesis, lymph node architecture, T cell costimulation, neurohistoge ⁇ esis, homotypic interaction, binds to CD9, CD24, CD56, CD 142, CD 166, integrins, X-linked neurological disorder CRASH Xq28
  • CD 173 Blood group H type 2 CHO Erythrocytes, stem cell subset, platelets
  • CDl 80 RP- 105 95-105 LRRF, TLR family B subset, monocytes, DC B cell recognition/signaling of LPS, associated with MD-I 5ql2
  • CDl 83 CXCR3 40 TM7SF(rhodopsin family) Activated T cells and NK cells, eosinophils, GM-CSF activated CD34+ hematopoietic progenitor cells IP-10 , Mig and I- TAC receptor, recruitment of T cells to inflammatory sites, enhancement of ThI response XqI 3
  • CD195 CCR5 45 Monocytes, T subset MIP-Ia, MIP-Ib and RANTES receptor, R5 HIV- 1 coreceptor 3p21
  • CD202b Tie2 (Tek) 150 RTK family Stem cells, endothelial cells Angiopoietin-1 receptor 9p21
  • CD203c NPP3/PDNP3, ENppl PD-Ib 130-150 Type II transmembrane protein Basophils, mast cells, glioma cells, megakaryocytes Ectoenzyme, binding to and clearance of extracellular nucleotides 6q22
  • B cells monocytes, fibroblasts, endothelial cells Binds IL- 13 with low affinity, associates with CD124 X
  • CD213a2 IL-13-R alpha2 65 B cells monocytes Binds IL-13 with high affinity XqI 3.1- 28 Table 2 CD antigens
  • CD222 IGF-II R mannose-6 phosphate-R250 Type I transmembrane protein Broad, 90- 95% intracellular Adhesion, tumor growth regulation, a receptor for TGFb-LAP, plasminogen, proliferin, truncated form (22OkD) found in serum 6q26
  • CD227 MUCl epithelial membrane antigen (EMA) 300 Mucin family, type I transmembrane protein Epithelial cells, stem cell subset, FDC, monocytes, B subset, some myelomas Adhesion, signal transduction, binds to CD 169, CD54, and selectins Iq21
  • CD228 Melanotransferrin 80-95 Stem cells, melanomas 3q28-29 CD229 Ly-9 95, 110 IgSF T cells, B cells Adhesion lq21.3-22 CD230 Prion protein35 Broad 20pter-pl2
  • TALL T cell acute lymphoblastic leukemia
  • CD233 Band3 SLC4A1 90 Anion exchanger family Erythrocytes Anion exchanger pump, transport of C02, linking red cell membrane to cytoskeleton 17q2
  • CD234 Duffy (DARC) 35-45 Erythrocytes Duffy antigen chemokine receptor lq21-22 CD235a Glycophorin A 36 Erythrocytes 4q28.2-31.1 CD235ab Glycophorin A/B 20 Erythrocytes Table 2 CD antigens
  • CD236 Glycophorin C/D 32, 23 Erythrocytes, stem cell subset
  • CD236R Glycophorin C 32 Erythrocytes, stem cell subset 2ql4-21
  • CD240D Rhesus 3OD(Rh3OD) 30-32 TM12SF Erythrocytes Ip36.2
  • CD241 Rhesus 50 glycoprotein (RhAg) 50 TM12SF Erythrocytes Rh antigens complex with CD47, LW, and glycophorin B 6p21.1
  • CD243 MDR-I, pl70, P-gp 180 Stem cells, small intestine, kidney Ion Pump 7q21.1
  • CD246 Anaplastic lymphoma kinase (ALK), Ki-I 80 Anaplastic T cell leuketnias, small intestine, testis, brain, not on normal lymphoid cells Brain development, exerts its effects on specific neurons, implicated in ALK lymphomas 2p23
  • MUC-D Polymorphic epithelial mucin
  • PEMT Polymorphic epithelial mucin
  • Episialin Tuor-associated mucin
  • Carcinoma- associated mucin [Tumor-associated epithelial membrane antigen)
  • EMA Tumor-associated epithelial membrane antigen
  • PUM peanut-reactive urinary mucin
  • MUCl mucin isoform 4 precursor [Homo sapiens] gi
  • MUCl mucin isoform 3 precursor [Homo sapiens] gi I 67189069 I ref
  • MUCl mucin isoform 2 precursor [Homo sapiens] gi I 67189007
  • MUCl mucin isoform 1 precursor [Homo sapiens] gi I 65301117
  • NP_612155 mucin 4 isoform b [Homo sapiens] gi I 20143920 I ref
  • NP_477521 small breast epithelial mucin [Homo sapiens] gi J 17158005 I ref
  • NP_004742 glu ⁇ osaminyl (N-acetyl) transferase 3 mucin type [Homo sapiens] gi I 4758422
  • NP_14,9038 mucin 13 epithelial transmembrane [Homo sapiens] gi I 51593090 I ref
  • NP_689886 mucin 20 [Homo sapiens] gi 1230973311 ref JNPJ-89386.l
  • NP_563625 mucin 15 [Homo sapiens] gi
  • T cell immunoglobulin mucin 3 [Homo sapiens] gi I 49574534
  • NP_002548 oviductal glycoprotein 1 precursor [Homo sapiens] gi
  • NP_060187 mu-protocadherin isoform 2 [Homo sapiens] gi 1 12313881 j ref
  • NP_068743 mu-proto ⁇ adherin isoform 1 [Homo sapiens] gi 1 123138791 ref
  • NP_001006S25 lung type-I cell membrane-associated glycoprotein i soform c [Homo sapiens] gi I 54792060 I ref
  • NP__938203 lung ty ⁇ e- I cell membrane-associated glycoprotein isoform b [Homo sapiens] gi J 54792058 I ref
  • cystic fibrosis transmembrane conductance regulator, ATP-binding cassette [Homo sapiens] ' . gi
  • NP_1491S2 alpha 1 type II collagen isoform 2 preproprotein [Homo sapiens] gi J 151494791 ref
  • NP_001835 alpha 1 type II collagen isoform 1 [Homo sapiens] g ⁇ 1 13435125 J ref
  • NP_004360 alpha 3 type V3 collagen isoform 1 precursor [Homo sapiens] gi I 55743098 I ref
  • NP_47S507 alpha 3 type VI collagen isoform 4 precursor [Homo sapiens] gi I 55743104
  • NP_47650S alpha 3 type VI collagen isoform 3 precursor [Homo sapiens] gi I 55743102 J ref
  • NP_001840 alpha 2 type VI collagen isoform 2C2 precursor [Homo sapiens] gi
  • NPJDOl 83S alpha 1 type IV collagen preproprotein [Homo sapiens] gi I 76569851 ref
  • NP_478054 alpha 2 type VI collagen isoform 2C2a precursor [Homo sapiens] gi 1 17402877
  • NP_203700 alpha 5 type IV collagen isoform 3 precursor [Homo sapiens] gi 1 15890088 I ref JNP_203700. 1 1 [15890088]
  • NP_112734 alpha 3 type IV collagen isoform 5 precursor [Homo sapiens] gi 1 14165450
  • NP_112733 alpha 3 type IV collagen isoform 4 precursor [Homo sapiens] gi 1 14165448
  • NP_112731 alpha 3 type IV collagen isoform 3 precursor [Homo sapiens] gi ) 14165444
  • ref NP_112731.11 [14165444]
  • NP_569712 alpha 1 type XVIII collagen isoform 2 precursor [Homo sapiens] gi 118765748 j ref
  • NP_085059 alpha 1 type XVIII collagen isoform 1 precursor [Homo sapiens] gi 1 133856201 ref
  • NP_001837 alpha 2 type IV collagen preproprotein [Homo sapiens] gi 1179862771 ref
  • NP_000384 alpha 2 type V collagen preproprotein [Homo sapiens] gi 145029591 ref
  • NP_543005 alpha 1 type XIII collagen isoform 19 [Homo sapiens] gi J 22027609 J ref
  • NP_543002 alpha 1 type XIII collagen isoform 15 [Homo sapiens] gi I 22027603
  • NP_543000 alpha 1 type XIII collagen isoform 14 [Homo sapiens] gi I 22027599 j ref
  • NP_542999 alpha 1 type XIII collagen isoform 13 [Homo sapiens] gi 1 220275971 ref
  • NP_542998 alpha 1 type XIII collagen isoform 12 [Homo sapiens] gi I 22027595 1 ref
  • NP_542997 alpha 1 type XIII collagen isoform 11 [Homo sapiens] gi 1 22027593 j ref
  • NP_542995 alpha 1 type XIII collagen isoform 9 [Homo sapiens] gi I 220275891 ref
  • NP_542994 alpha 1 type XIII collagen isoform 8 [Homo sapiens] gi I 22027587 J ref
  • NP_542993 alpha 1 type XIII collagen isoform 7 [Homo sapiens] gi I 22027583 I ref
  • alpha 1 type XIII collagen isoform 6 [Homo sapiens] gi I 22027580 J ref
  • NP_542991 alpha 1 type XIII collagen isoform 5 [Homo sapiens] gi I 22027577 I ref
  • NP_110447 alpha 1 type XXI collagen precursor [Homo sapiens] gi 118780273
  • NP_542411 collagen, type XI, alpha 2 isoform 1 preproprotein [Homo sapiens] gi 118201917
  • NP_001841 alpha 1 type VIII collagen precursor [Homo sapiens] gi
  • NP_001032852 collagen type XXVIII precursor [Homo sapiens] gi
  • NPJ775736 collagen type XXIII, alpha 1 [Homo sapiens] gi I 29725624
  • NP_690848 collagen type XXII, alpha 1 [Homo sapiens] gi
  • NP_942014 collagen type XXV, alpha 1 isoform 1 [Homo sapiens] gi 138570075
  • NP_942014.11 38570075]
  • NP_115907 collagen, type XXV, alpha 1 isoform 2 [Homo sapiens] gi 138570073 I ref
  • NP_112729 alpha 3 type IV collagen binding protein isoform 2 [Homo sapiens] gi 114165452
  • NP_112729.11 [14165452]
  • NP_005704 alpha 3 type IV collagen binding protein isoform 1 [Homo sapiens] gi 15031717
  • NP_005704.11 [5031717]
  • NP_391988 integrin beta 1 isoform ID precursor [Homo sapiens] gi 119743819 j ref
  • NP_391987 integrin beta 1 isoform IC-I precursor [Homo sapiens] gi 1197438171 ref
  • NP_002202 integrin beta 1 isoform IA precursor [Homo sapiens] gi
  • NP_000543 von Willebrand factor precursor [Homo sapiens] gi I 45079071 ref
  • NP_997S47 fibronectin 1 isoform 1 preproprotein [Homo sapiens] gi I 471325571 ref
  • NP_997641 fibronectin 1 isoform 5 preproprotein [Homo sapiens] gi I 47132553 I ref
  • NP_997640 fibronectin 1 isoform 2 preproprotein [Homo sapiens] gi J 471325511 ref
  • NP_997639 fibronectin 1 isoform 6 preproprotein [Homo sapiens] gi J 471325491 ref
  • NP_473375 fibronectin 1 isoform 7 preproprotein [Homo sapiens] gi J 47132547
  • NP_852478 integrin alpha 1 precursor [Homo sapiens] gi J 31657142
  • NP_598014 decorin isoform e precursor [Homo sapiens] gi 1197438541 ref
  • NP_598012 decorin isoform c precursor [Homo sapiens] gi
  • NP_598011 decorin isoform b precursor [Homo sapiens] gi
  • NP_598010 decorin isoform a preproprotein [Homo sapiens] gi
  • NP_000651 transforming growth factor, beta 1 [Homo sapiens] gi I 63025222 J ref
  • NP_002412 matrix metalloproteinase 1 preproprotein [Homo sapiens] gi I 4505215 I ref
  • NP_054699 discoidin receptor tyrosine kinase isoform a [Homo sapiens] gi I 83977450 I ref
  • CD36 antigen [Homo sapiens] gi I 48375180 j ref
  • CD36 antigen [Homo sapiens] gi I 48375178 I ref
  • CD36 antigen [Homo sapiens] gi J 483751761 ref
  • NP_000293 lysyl hydroxylase precursor Homo sapiens
  • NP_002549 purinergi ⁇ receptor P2X1 [Homo sapiens] gi I 45055451 ref
  • NP_002308 lysyl oxidase preproprotein [Homo sapiens] gi I 20149540
  • NP_001191 bone morphogenetic protein 2 preproprotein [Homo sapiens] gi I 4557369
  • NP_057075 complement component 1, q subcomponent, alpha polypeptide precursor [Homo sapiens] gi I 7705753
  • NP_001017962 prolyl 4 -hydroxylase, alpha I subunit isoform 2 precursor [Homo sapiens] gi
  • NP_000908 prolyl 4 -hydroxylase, alpha I subunit isoform 1 precursor CHom ⁇ sapiens] gi
  • Homo sapiens gi I 62739166 I ref
  • NP_001014796 discoidin domain receptor family member 2 precursor [Homo sapiens] gi
  • NP_006173 discoidin domain receptor family member 2 precursor [Homo sapiens] gi I 62420884
  • NP_006030 mannose receptor C type 2 [Homo sapiens] gi I 5174485
  • NP_004985 matrix metalloproteinase 9 preproprotein [Homo sapiens] gi I 74272287
  • PTK2 protein tyrosine kinase 2 isoform b [Homo sapiens] gi I 27886593
  • PTK2 protein tyrosine kinase 2 isoform a [Homo sapiens] gi
  • NP_000909 prolyl 4 -hydroxylase, beta subunit [Homo sapiens] gi I 20070125

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Abstract

We describe a polypeptide conjugate wherein the conjugate comprises a ligand binding moiety fused to a HLA G molecule, or part thereof, and including oligomers and dimers of said conjugate and also including transgenic cells expressing HLA G.

Description

Molecule
The invention relates to a polypeptide conjugate wherein the conjugate comprises a ligand binding moiety fused to a HLA G molecule, or part thereof and including oligomers and dimers of said conjugate and transgenic cells expressing HLA G.
The foetus is considered a semi-allogeneic graft but during pregnancy avoids immunological rejection by the mother. In particular, extra-villous trophoblast (EVT) cells of the placenta invade the endometrium at the site of implantation and are found in direct contact with maternal immune cells. EVT cells and derivatives of these cells, specifically endovascular cells, have a number of unique features that modulate the immune system to prevent foetal rejection. For example, EVT and endovascular cells do not express the classical major class 1 molecules HLA A and B and therefore are mainly protected from T-cell mediated allorejection. EVT and endovascular cells express a unique non-classical HLA class 1 molecular HLA G as membrane-bound and soluble isoforms. Specifically HLA G molecules inhibit the natural killer cell-mediated cytotoxicity. HLA G molecules also down regulates both CD8+ and CD4+ T cell reactivity. T cell mediated responses have a major role in many autoimmune or inflammatory diseases that result in the destruction and damage of cells and tissues by the body's own immune system by recognition of self antigens by CD8+ T lymphocytes.
There are a large number of diseases exhibiting an inflammatory component. These include but are not limited to: inflammatory joint diseases (e.g., rheumatoid arthritis, osteoarthritis, polyarthritis and gout), chronic inflammatory connective tissue diseases (e.g., lupus erythematosus, scleroderma, Sjorgen's syndrome, poly- and dermatomyositis, vasculitis, mixed connective tissue disease (MCTD), tendonitis, synovitis, bacterial endocarditis, osteomyelitis and psoriasis, chronic inflammatory lung diseases (e.g., chronic respiratory disease, pneumonia, fibrosing alveolitis, chronic bronchitis, chronic obstructive pulmonary disease (COPD), bronchiectasis, emphysema, silicosis and other pneumoconiosis and tuberculosis), chronic inflammatory bowel and gastro-intestinal tract inflammatory diseases (e.g., ulcerative colitis and Crohn's disease), chronic neural inflammatory diseases (e.g., chronic inflammatory demyelinating polyradiculoneuropathy, chronic inflammatory demyelinating polyneuropathy, multiple sclerosis, Guillan-Barre Syndrome and myasthemia gravis), other inflammatory diseases (e.g., mastitis, laminitis, laryngitis, chronic cholecystitis, Hashimoto's thyroiditis, inflammatory breast disease). Amongst these diseases are autoimmune diseases such as; multiple sclerosis, type 1 diabetes, autoimmune thyroid disease, autoimmune hepatitis, rheumatoid arthritis, autoimmune colitis, Crohn's disease, celiac disease, autoimmune nephritis, autoimmune neuropathy, encephalopathy, fibrosing alveolitis.
In our co-pending application WO2006/111706 Al we disclose the isolation of unique human cytotrophoblast stem cell lines from human embryonic stem cells that express
HLA G. We herein disclose conjugates comprising HLA G fused to ligands that enable the targeting of HLA G to specific cell-types and the use of the conjugates in the treatment of conditions that would benefit from the inhibition of T cell mediated responses. We also disclose the ectopic expression of HLA G nucleic acid molecules in stem cells, in particular embryonic stem cells and the use of these transfected cells in inducing immune tolerance of said stem cells. A problem associated with gene therapy and/or tissue engineering is the provision of cell/tissue materials that do not induce a natural killer cell response in the recipient animal. The provision of stem cell-lines that either constitutively express or developmentally express HLA G genes will facilitate procedures that involve gene and tissue replacement therapy.
According to an aspect of the invention there is provided a polypeptide conjugate comprising a first part wherein said first part is a ligand that binds a polypeptide expressed by a cell and a second part wherein said second part is a polypeptide encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i) a nucleic acid molecule consisting of at least one nucleic acid sequence as represented in Figure IA-T; ii) a nucleic acid molecule that hybridises to the nucleic acid molecule in Figure IA-T and that encodes a polypeptide with the activity associated with HLA G; iii) a nucleic acid molecule comprising a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequences as defined in (i) and (ii) above.
Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other. The stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993). The Tm is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. The following is an exemplary set of hybridization conditions and is not limiting:
Very High Stringency (allows sequences that share at least 90% identity to hybridize")
Hybridization: 5x SSC at 650C for 16 hours Wash twice: 2x SSC at room temperature (RT) for 15 minutes each
Wash twice: 0.5x SSC at 65°C for 20 minutes each
High Stringency (allows sequences that share at least 80% identity to hybridize)
Hybridization: 5x-6x SSC at 65°C-70°C for 16-20 hours Wash twice: 2x SSC at RT for 5-20 minutes each
Wash twice: Ix SSC at 55°C-70°C for 30 minutes each
Low Stringency (allows sequences that share at least 50% identity to hybridize) Hybridization: 6x SSC at RT to 55°C for 16-20 hours
Wash at least twice: 2x-3x SSC at RT to 550C for 20-30 minutes each. In a preferred embodiment of the invention said first part is an antibody or active binding part thereof. Preferably said antibody is a monoclonal antibody.
Antibodies, also known as immunoglobulins, are protein molecules which have specificity for foreign molecules (antigens). Immunoglobulins (Ig) are a class of structurally related
' proteins consisting of two pairs of polypeptide chains, one pair of light (L) (low molecular weight) chain (K or λ), and one pair of heavy (H) chains (γ, α, μ, δ and ε), all four linked together by disulphide bonds. Both H and L chains have regions that contribute to the binding of antigen and that are highly variable from one Ig molecule to another. In addition, H and L chains contain regions that are non- variable or constant.
The L chains consist of two domains. The carboxy-terminal domain is essentially identical among L chains of a given type and is referred to as the "constant" (C) region. The amino terminal domain varies from L chain to L chain and contributes to the binding site of the antibody. Because of its variability, it is referred to as the "variable" (V) region.
The H chains of Ig molecules are of several classes, α, μ, σ, α, and γ (of which there are several sub-classes). An assembled Ig molecule consisting of one or more units of two identical H and L chains derives its name from the H chain that it possesses. Thus, there are five Ig isotypes: IgA, IgM, IgD, IgE and IgG (with four sub-classes based on the differences in the H chains, i.e., IgGl, IgG2, IgG3 and IgG4). Further detail regarding antibody structure and their various functions can be found in, Using Antibodies: A laboratory manual, Cold Spring Harbour Laboratory Press.
In a preferred embodiment of the invention said fragment is a Fab fragment.
Preferably said Fab fragment is selected from the group consisting of: F (ab')2, Fab, Fv and Fd fragments, CDR3 regions, single chain antibody fragment or a domain antibody fragment.
Various fragments of immunoglobulin or antibodies are known in the art, i.e., Fab, Fab2, F(ab')2, Fv, Fc, Fd, scFvs, etc. A Fab fragment is a multimeric protein consisting of the immunologically active portions of an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, covalently coupled together and capable of specifically binding to an antigen. Fab fragments are generated via proteolytic cleavage (with, for example, papain) of an intact immunoglobulin molecule. A Fab2 fragment comprises two joined Fab fragments. When these two fragments are joined by the immunoglobulin hinge region, a F(ab')2 fragment results. An Fv fragment is multimeric protein consisting of the immunologically active portions of an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region covalently coupled together and capable of specifically binding to an antigen. A fragment could also be a single chain polypeptide containing only one light chain variable region, or a fragment thereof that contains the three CDRs of the light chain variable region, without an associated heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multi specific antibodies formed from antibody fragments, this has for example been described in US patent No 6,248,516. Fv fragments or single region (domain) fragments are typically generated by expression in host cell lines of the relevant identified regions. These and other immunoglobulin or antibody fragments are within the scope of the invention and are described in standard immunology textbooks such as Paul, Fundamental Immunology or Janeway et al. Immunobiology (cited above). Molecular biology now allows direct synthesis (via expression in cells or chemically) of these fragments, as well as synthesis of combinations thereof. A fragment of an antibody or immunoglobulin can also have bispecific function as described above.
In a preferred embodiment of the invention said antibody or fragment is a chimeric or humanised antibody.
Chimeric antibodies are recombinant antibodies in which all of the V-regions of a mouse or rat antibody are combined with human antibody C-regions. Humanised antibodies are recombinant hybrid antibodies which fuse the complimentarity determining regions from a rodent antibody V-region with the framework regions from the human antibody V- regions. The C-regions from the human antibody are also used. The complimentarity determining regions (CDRs) are the regions within the N-terminal domain of both the heavy and light chain of the antibody to where the majority of the variation of the V- region is restricted. These regions form loops at the surface of the antibody molecule. These loops provide the binding surface between the antibody and antigen. Antibodies from non-human animals provoke an immune response to the foreign antibody and its removal from the circulation. Both chimeric and humanised antibodies have reduced antigenicity when injected to a human subject because there is a reduced amount of rodent (i.e. foreign) antibody within the recombinant hybrid antibody, while the human antibody regions do not illicit an immune response. This results in a weaker immune response and a decrease in the clearance of the antibody. This is clearly desirable when using therapeutic antibodies in the treatment of human diseases. Humanised antibodies are designed to have less "foreign" antibody regions and are therefore thought to be less immunogenic than chimeric antibodies.
In a preferred embodiment of the invention said ligand comprises the binding domain of a ligand that binds a cell surface receptor polypeptide.
In an alternative preferred embodiment of the invention said ligand comprises an aptamer.
Nucleic acids have both linear sequence structure and a three dimensional structure which in part is determined by the linear sequence and also the environment in which these molecules are located. Conventional therapeutic molecules are small molecules, for example, peptides, polypeptides, or antibodies, which bind target molecules. It has become apparent that nucleic acid molecules also have potential with respect to providing agents with the requisite binding properties. These nucleic acid molecules are typically referred to as aptamers. Aptamers are small, usually stabilised, nucleic acid molecules, which comprise a binding domain for a target molecule. A screening method to identify aptamers is described in US 5,270,163, which is incorporated by reference. Aptamers are typically oligonucleotides which may be single stranded oligodeoxynucleotides, oligoribonucleotides, or modified oligodeoxynucleotide or oligoribonucleotides.
The term "modified" encompasses nucleotides with a covalently modified base and/or sugar. For example, modified nucleotides include nucleotides having sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position. Thus modified nucleotides may also include 2' substituted sugars such as 2'-O-methyl-; 2-O-alkyl; 2-0- allyl; 2'-S-alkyl; 2'-S-allyl; T- fluoro-; 2'-halo or 2;azido-ribose, carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, and sedoheptulose.
Modified nucleotides are known in the art and include, by example and not by way of limitation, alkylated purines and/or pyrimidines; acylated purines and/or pyrimidines; or other heterocycles. These classes of pyrimidines and purines are known in the art and include, pseudoisocytosine; N4, N4-ethanocytosine; 8-hydroxy-N6-methyladenine; 4- acetylcytosine, 5-(carboxyhydroxylmethyl) uracil; 5-fluorouracil; 5-bromouracil; 5- carboxymethylaminomethyl-2-thiouracil; 5-carboxymethylaminomethyl uracil; dihydrouracil; inosine; N6-isopentyl-adenine; 1-niethyladenine; 1-methylpseudouracil; 1- methylguanine; 2,2-dimethylguanine; 2-methyladenine; 2-methylguanine; 3- methylcytosine; 5-methylcytosine; N6-methyladenine; 7-methylguanine; 5- methylaminomethyl uracil; 5-methoxy amino methyl-2-thiouracil; β-D- mannosylqueosine; 5-methoxycarbonylmethyluracil; 5-methoxyuracil; 2 methylthio-N6- isopentenyladenine; uracil-5-oxyacetic acid methyl ester; psueouracil; 2-thiocytosine; 5- methyl-2 thiouracil, 2-thiouracil; 4-thiouracil; 5-methyluracil; N-uracil-5-oxyacetic acid methylester; uracil 5 — oxyacetic acid; queosine; 2-thiocytosine; 5-propyluracil; 5- propylcytosine; 5-ethyluracil; 5-ethylcytosine; 5-butyluracil; 5-pentyluracil; 5- pentylcytosine; and 2,6,-diaminopurine; methylpsuedouracil; 1-methylguanine; 1- methylcytosine.
The aptamers of the invention are synthesised using conventional phosphodiester linked nucleotides and synthesised using standard solid or solution phase synthesis techniques which are known in the art. Linkages between nucleotides may use alternative linking molecules. For example, linking groups of the formula P(O)S, (thioate); P(S)S,
(dithioate); P(O)NR'2; P(O)R'; P(O)OR6; CO; or CONR'2 wherein R is H (or a salt) or alkyl (1-12C) and R6 is alkyl (1-9C) is joined to adjacent nucleotides through -O- or -S-. The binding of aptamers to a target polypeptide is readily tested.
In a further alternative embodiment of the invention said ligand is a peptide. In a preferred embodiment of the invention said peptide is a modified peptide.
Modifications include, by example and not by way of limitation acetylation and amidation.
In a preferred method of the invention said peptide is acetylated. Preferably said acetylation is to the amino terminus of said peptide.
In a further preferred method of the invention said peptide is amidated. Preferably said amidation is to the carboxyl-terminus of said peptide.
In a further preferred method of the invention said peptide is modified by both acetylation and amidation.
Alternatively or preferably, said modification includes the use of modified amino acids in the production of recombinant or synthetic forms of peptides. It will be apparent to one skilled in the art that modified amino acids include, by way of example, 4-hydroxyproline, 5-hydroxylysine, N6-acetyllysine, N6-methyllysine, N6,N6-dimethyllysine, N6,N6,N6- trimethyllysine, cyclohexyalanine, D-amino acids, ornithine. Other modifications include amino acids with a C2, C3 or C4 alkyl R group optionally substituted by 1, 2 or 3 substituents selected from halo (e.g. F, Br5 1), hydroxy or C1-C4 alkoxy.
Alternatively, peptides could be modified by, for example, cyclisation. Cyclisation is known in the art, (see Scott et al Chem Biol (2001), 8:801-815; Gellerman et al J. Peptide Res (2001), 57: 277-291; Dutta et al J. Peptide Res (2000), 8: 398-412; Ngoka and Gross J Amer Soc Mass Spec (1999), 10:360-363.
In a preferred embodiment of the invention peptides according to the invention are modified by cyclisation.
In a preferred embodiment of the invention said polypeptide is expressed by an embryonic stem cell. In an alternative preferred embodiment of the invention said polypeptide is expressed by a stem cell.
Preferably said polypeptide is expressed by a differentiated cell or a cell undergoing differentiation.
Typically, ligands, for example antibodies or preferably single chain antibody fragments, aptamers or peptides are specific for embryonic stem cell markers, for example ligands that bind Tra-1-60, Tra-1-81, SSEAl, SSEA3, SSEA4, A2B5 and ME311. Similarly where autoimmune diseases are treated with the conjugate according to the invention, cell markers associated with pathological conditions such as Type 1 diabetes would be targeted, for example the antigen recognised by Anti-Human Pancreatic Islet Cells antibody clone 3D3 (cat#7099-1006, Biogenesis Ltd, Technology Road, Poole, BHl 7 7DA, England). Rheumatoid arthritis has a number of disease associated markers, for example Advanced Glycation End-products (AGEs), receptors for AGEs (RAGE) and Collagen II. A further example of an autoimmune disease treatable with the conjugate according to the invention is the targeting of sialoadhesin expressed by oligodendrocytes in the treatment of multiple sclerosis. The treatment of asthma may be achieved by targeting epithelial cells that line the respiratory tract with ligands that bind, for example, the cell surface antigen recognised by Mouse Anti-Epithelial Cell Surface Monoclonal Antibody, Clone 5E11 (Cat# 1422, StemCell Technologies Inc., USA). Further examples include RAGE polypeptides, CD antigens, mucins, collagens and cadherins.
Preferably said ligand binds at least one mucin expressed by pathological cells in the suppression of inflammatory conditions.
In a preferred embodiment of the invention there is provided a conjugate comprising a polypeptide encoded by a nucleic acid molecule as represented Figure IA-T.
In a preferred embodiment of the invention said conjugate comprises an amino acid sequence as represented in Figure 2A-O. In addition, the invention features nucleic acid molecules comprising nucleic acid sequences having at least 50% identity with the nucleic acid sequences as herein disclosed. In one embodiment, the nucleic acids have at least 60% identity, more preferably at least 70% identity, even more preferably at least 80% identity, still more preferably at least 90%, more preferably 95% identity, and most preferably at least 99% identity with the nucleic acid sequences illustrated herein.
In a further preferred embodiment of the invention said polypeptide is a variant polypeptide and comprises the amino acid sequence represented in Figure 2A-O, which sequence has been modified by deletion, addition or substitution of at least one amino acid residue wherein said modification retains or modifies the activity of said polypeptide.
A variant polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, truncations that may be present in any combination. Among preferred variants are those that vary from a reference polypeptide by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid by another amino acid of like characteristics. The following non-limiting list of amino acids are considered conservative replacements (similar): a) alanine, serine, and threonine; b) glutamic acid and aspartic acid; c) asparagine and glutamine d) arginine and lysine; e) isoleucine, leucine, methionine and valine and f) phenylalanine, tyrosine and tryptophan. Most highly preferred are variants that retain or modify the same biological function and activity as the reference polypeptide from which it varies.
In addition, the invention features polypeptide sequences having at least 50% identity, over all or part of the length of the polypeptide, with the polypeptide sequences as herein disclosed, or fragments and functionally equivalent polypeptides thereof. In one embodiment, the polypeptides have at least 50% identity, more preferably at least 60% identity, even more preferably at least 70% identity, still more preferably at least 80% identity, and most preferably at least 90- 99% identity with the amino acid sequences illustrated herein. In a preferred embodiment of the invention said conjugate is an in-frame translational fusion between said first and second parts wherein said first and second parts are linked either directly or indirectly to each other.
Preferably said in-frame translational fusion is a direct fusion between first and second parts.
According to a further aspect of the invention there is provided a conjugate according to the invention for use as a pharmaceutical.
According to a further aspect of the invention there is provided a composition comprising a conjugate according to the invention.
In a preferred embodiment of the invention said composition is a pharmaceutical composition. Preferably said composition further comprises an adjuvant or carrier.
In a preferred embodiment of the invention said adjuvant is an immunosuppressant.
When administered, the conjugate compositions of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents.
The conjugates of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal. A preferred route of administration is by pulmonary aerosol. Techniques for preparing aerosol delivery systems containing antibodies are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the antibodies, such as the binding capacity (see, for example, Sciarra and Cutie, "Aerosols," in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712; incorporated by reference). Those of skill in the art can readily determine the various parameters and conditions for producing aerosols without resort to undue experimentation.
The compositions of the invention are administered in effective amounts. An "effective amount" is that amount of a composition that alone, or together with further doses, produces the desired response. In the case of treating a particular disease the desired response is inhibiting the progression of the disease. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine diagnostic methods.
Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.
The pharmaceutical compositions used in the foregoing methods preferably are sterile and contain an effective amount of conjugate for producing the desired response in a unit of weight or volume suitable for administration to a patient.
The doses of conjugate administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.
In general, doses of conjugate are formulated and administered in doses between 1 ng and 1 mg, and preferably between 10 ng and 100 μg, according to any standard procedure in the art. Other protocols for the administration of conjugate compositions will be known to one of ordinary skill in the art, in which the dose amount, schedule of injections, sites of injections, mode of administration and the like vary from the foregoing. Administration of conjugate compositions to mammals other than humans, (e.g. for testing purposes or veterinary therapeutic purposes), is carried out under substantially the same conditions as described above. A subject, as used herein, is a mammal, preferably a human, and including a non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent.
When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically- acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
Conjugate compositions may be combined, if desired, with a pharmaceutically-acceptable carrier. The term "pharmaceutically-acceptable carrier" as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy. The pharmaceutical compositions may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt. The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; and parabens.
The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active compound. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.
Compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of conjugates, which is preferably isotonic with the blood of the recipient. This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in I5 3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables. Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
According to a further aspect of the invention there is provided a composition comprising a conjugate according to the invention and an embryonic stem cell. In a preferred embodiment of the invention said embryonic stem cell is a primate embryonic stem cell.
In a preferred embodiment of the invention said embryonic stem cell is human and is not a totipotent embryonic stem cell.
In an alternative preferred embodiment of the invention said embryonic stem cell is a non- primate embryonic stem cell. Preferably said non-primate embryonic stem cell is a murine embryonic stem cell.
In an alternative preferred embodiment of the invention said embryonic stem cell is a teratocarcinoma stem cell.
In a preferred embodiment of the invention said embryonic stem cell is transfected with a nucleic acid molecule that encodes a polypeptide to which said ligand binds.
According to a further aspect of the invention there is provided a method to treat an inflammatory disease or condition comprising administering an effective amount of a conjugate or a composition according to the invention to a subject in need of treatment.
According to a further aspect of the invention there is provided a method to treat an autoimmune disease or condition comprising administering an effective amount of a conjugate or a composition according to the invention to a subject in need of treatment.
In a preferred method of the invention said autoimmune disease is selected from the group consisting of: multiple sclerosis, type 1 diabetes, autoimmune thyroid disease, autoimmune hepatitis, rheumatoid arthritis, autoimmune colitis, Crohns disease, celiac disease, autoimmune nephritis, autoimmune neuropathy, encephalopathy, fibrosing alveolitis. According to a further aspect of the invention there is provided a method to treat organ/tissue rejection in a recipient subject comprising administering an effective amount of a conjugate or composition according to the invention to a subject in need of treatment.
According to a further aspect of the invention there is provided the use of an effective amount of a conjugate or a composition according to the invention to treat an inflammatory disease or condition.
According to a further aspect of the invention there is provided the use of an effective amount of a conjugate or a composition according to the invention for the treatment of an autoimmune disease.
In a preferred embodiment of the invention said autoimmune disease is selected from the group consisting of: multiple sclerosis, type 1 diabetes, autoimmune thyroid disease, autoimmune hepatitis, rheumatoid arthritis, autoimmune colitis, Crohns disease, celiac disease, autoimmune nephritis, autoimmune neuropathy, encephalopathy, fibrosing alveolitis.
According to a further aspect of the invention there is provided the use of an effective amount of a conjugate or composition according to the invention for the treatment of organ/tissue rejection in a recipient subject.
In a preferred embodiment of the invention said subject is human.
According to a further aspect of the invention said conjugate is provided as multimeric complex of monomer conjugate subunits according to the invention.
In a preferred embodiment of the invention said complex comprises at least two monomer subunits.
In a preferred embodiment of the invention said complex consists of two monomer subunits. In a preferred embodiment of the invention said complex comprises identical monomer subunits.
In an alternative embodiment of the invention said complex comprises dissimilar monomer subunits.
In a preferred embodiment of the invention said complex consists of two monomer subunits forming a homodimer.
In an alternative preferred embodiment of the invention said complex consists of two dissimilar monomer subunits forming a heterodimer.
In a preferred embodiment of the invention said complex is an in-frame translational fusion protein comprising monomer subunits.
According to a further aspect of the invention there is provided a composition comprising a complex according to the invention. Preferably said composition is a pharmaceutical composition.
According to a further aspect of the invention there is provided a method to treat an inflammatory disease or condition comprising administering an effective amount of a complex or a composition comprising a complex according to the invention to a subject in need of treatment.
According to a further aspect of the invention there is provided a method to treat an autoimmune disease or condition comprising administering an effective amount of a complex or a composition comprising a complex according to the invention to a subject in need of treatment.
In a preferred method of the invention said autoimmune disease is selected from the group consisting of: multiple sclerosis, type 1 diabetes, autoimmune thyroid disease, autoimmune hepatitis, rheumatoid arthritis, autoimmune colitis, Crohns disease, celiac disease, autoimmune nephritis, autoimmune neuropathy, encephalopathy, fibrosing alveolitis.
According to a further aspect of the invention there is provided a method to treat organ/tissue rejection in a recipient subject comprising administering an effective amount of a complex or a composition comprising a complex according to the invention to a subject in need of treatment.
According to a further aspect of the invention there is provided the use of an effective amount of a complex or a composition comprising a complex according to the invention to treat an inflammatory disease or condition.
According to a further aspect of the invention there is provided the use of an effective amount of a complex or a composition comprising a complex according to the invention for the treatment of an autoimmune disease.
According to a further aspect of the invention there is provided a stem cell wherein said cell is transfected with a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i) a nucleic acid molecule consisting of a nucleic acid sequence as represented in Figure IA-T ii) a nucleic acid molecule that hybridises to the nucleic acid molecule in Figure IA-T and that encodes a polypeptide with the activity associated with HLA-G; iii) a nucleic acid molecule comprising a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequences as defined in (i) and (ii) above.
In a preferred embodiment of the invention said nucleic acid molecule hybridizes under stringent hybridization conditions. In a preferred embodiment of the invention said embryonic stem cell comprises the nucleic acid sequence as represented in Figure IA-T.
In a further preferred embodiment of the invention said nucleic acid molecule consists of the nucleic acid sequence as represented in Figure IA-T.
In a preferred embodiment of the invention said nucleic acid molecule is operably linked to a nucleic acid molecule comprising a transcription promoter sequence to form a transcription cassette.
In a preferred embodiment of the invention said promoter sequence is a constitutive promoter; preferably said promoter sequence is a cell specific promoter sequence.
In a preferred embodiment of the invention said promoter sequence is a developmentally regulated promoter sequence.
In a preferred embodiment of the invention said nucleic acid molecule further comprises a repressor sequence.
In a preferred embodiment of the invention said nucleic acid molecule comprising said promoter sequence is part of a vector.
Preferably said vector is adapted for eukaryotic expression. Typically said adaptation includes the provision of transcription control sequences (promoter sequences) which mediate cell/tissue specific expression. These promoter sequences may be cell/tissue specific, inducible or constitutive.
"Promoter" is an art recognised term and, for the sake of clarity, includes the following features which are provided by example. Enhancer elements are cis acting nucleic acid sequences often found 5' to the transcription initiation site of a gene (enhancers can also be found 3' to a gene sequence or even located in intronic sequences). Enhancers function to increase the rate of transcription of the gene to which the enhancer is linked. Enhancer activity is responsive to trans acting transcription factors (polypeptides) which have been shown to bind specifically to enhancer elements. The binding/activity of transcription factors (please see Eukaryotic Transcription Factors, by David S Latchman, Academic Press Ltd, San Diego) is responsive to a number of physiological/environmental cues.
Promoter elements also include so called TATA box and RNA polymerase initiation selection sequences which function to select a site of transcription initiation. These sequences also bind polypeptides which function, inter alia, to facilitate transcription initiation selection by RNA polymerase.
Adaptations also include the provision of selectable markers and autonomous replication sequences which facilitate the maintenance of said vector in either the eukaryotic cell or prokaryotic host. Vectors which are maintained autonomously are referred to as episomal vectors. Episomal vectors are desirable since these molecules can incorporate large DNA fragments (30-50kb DNA). Episomal vectors of this type are described in WO98/07876.
Adaptations which facilitate the expression of vector encoded genes include the provision of transcription termination/polyadenylation sequences. This also includes the provision of internal ribosome entry sites (IRES) which function to maximise expression of vector encoded genes arranged in bi-cistronic or multi-cistronic expression cassettes. Expression control sequences also include so-called Locus Control Regions (LCRs). These are regulatory elements which confer position-independent, copy number-dependent expression to linked genes when assayed as transgenic constructs. LCRs include regulatory elements that insulate transgenes from the silencing effects of adjacent heterochromatin, Grosveld et al., Cell (1987), 51 : 975-985.
Expression control sequences also encompass, ubiquitous chromatin opening elements (UCOE's), see WO/GB00/05393. UCOE's are nucleic acid elements that are responsible for establishing an open chromatin structure across a locus that consists exclusively of ubiquitously expressed, housekeeping genes. These elements are not derived from an LCR. A UCOE is a polynucleotide which opens chromatin or maintains chromatin in an open state and facilitates reproducible expression of an operably-linked gene in cells of at least two different tissue types.
These adaptations are well known in the art. There is a significant amount of published literature with respect to expression vector construction and recombinant DNA techniques in general. Please see, Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, Cold Spring Harbour, NY and references therein; Marston, F (1987) DNA Cloning Techniques: A Practical Approach VoI III IRL Press, Oxford UK; DNA Cloning: F M Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994). Vectors are typically viral based and may be derived from viruses including adenovirus; retrovirus; adeno-associated virus; herpesvirus; lentivirus; vaccinia virus; and baculovirus.
In a preferred embodiment of the invention said vector is a viral based vector. Preferably, said viral vector is selected from the group consisting of: adenovirus; retrovirus; adeno- associated virus; herpesvirus; lentivirus; vaccinia virus; baculovirus.
In a preferred embodiment of the invention said viral based vector is an adenovirus. Alternatively said viral based vector is a lentivirus.
In a further alternative preferred embodiment of the invention said vector is a plasmid or phagemid.
In a preferred embodiment of the invention said cell is transfected with a further nucleic acid molecule.
Preferably said further nucleic acid molecule encodes a therapeutic polypeptide.
The invention contemplates gene therapy. Gene therapy involves the transfer and stable insertion of new genetic information into cells for the therapeutic treatment of disease. Stem cells or pluripotent ES cells are the ideal target of gene transfer.
Most studies in gene therapy have focused on the use of haematopoietic stem cells. High efficiency gene transfer systems for hematopoietic progenitor cell transformation have been investigated for use (Morrow, JF, 1976, Ann. NY Acad. Sci 265:13; Salzar, W et al, 1981 in Organization and Expression of Globin Genes, AR Liss, Inc, New York, p313; Bernstein A 1985 in Genetic Engineering: Principles and Methods, Plenum Press, New York, p 235; Dick JE et al 1986, Trends in Genetics 2:165). Viral vector systems show a higher efficiency of transformation than DNA-mediated gene transfer procedures (e.g. CaPO4 precipitation and DEAE dextran) and show the capability of integrating transferred genes stably in a wide variety of cell types. Recombinant retrovirus vectors have been widely used experimentally to transduce hematopoietic stem and progenitor cells.
Genes that have been successfully expressed in mice after transfer by retrovirus vectors include human hypoxanthine phosphoribosyl transferase (Miller A et al, 1984, Science 255:630). Bacterial genes have also been transferred into mammalian cells, in the form of bacterial drug resistance gene transfers in experimental models. Transformation of hematopoietic progenitor cells to drug resistance by eukaryotic virus vectors has also accomplished with recombinant retrovirus based vector systems (Hock RA and Miller AD 1986, Nature 320:275-277; Joyner A et al, 1983, Nature 305:556-558; Williams DA et al 1984, Nature 310:476-480; Dick JE et al, 1985, Cell 42:71-79); Keller G et al 1985, Nature 318: 149-154; Eglitis M et al, 1985, Science 230: 1395-1398). Adeno-associated virus vectors have been used successfully to transduce mammalian cell lines to neomycin resistance (Hermonat PL and Muzyczka N, 1984, supra; Tratschin JD et al, 1985, MoI. Cell. Biol. 5:3251). Other viral vector systems that have been investigated for use in gene transfer include papovaviruses and vaccinia viruses (see Cline ML 1985, Pharmac. Ther. 29:69-92).
Alternative methods of gene transfer include microinjection, electroporation, liposomes and chromosome transfer.
Gene therapy has also been investigated in murine models with the goal of enzyme replacement therapy. Thus, normal stem cells from a donor mouse have been used to reconstitute the hematopoietic cell system of mice lacking beta-glucuronidase (Yatziv S et al, 1982, J. Lab. Clin. Med 90:792-797).
In a preferred embodiment of the invention said vector is replication competent. Preferably said vector is conditionally replication competent.
In an alternative preferred embodiment of the invention said vector is a replication incompetent vector. In a preferred embodiment of the invention said stem cell is a multipotent stem cell. Preferably said multipotent stem cell is selected from the group consisting of: haemopoietic stem cell; neural stem cell; bone stem cell; muscle stem cell; mesenchymal stem cell; epithelial stem cell (derived from organs such as the skin, gastrointestinal mucosa, kidney, bladder, mammary glands, uterus, prostate and endocrine glands such as the pituitary); endodermal stem cell (derived from organs such as the liver, pancreas, lung and blood vessels).
In a preferred embodiment of the invention said stem cell is a primate embryonic stem cell. Preferably said embryonic stem cell is a human embryonic stem cell.
In an alternative preferred embodiment of the invention said embryonic stem cell is a non- primate stem cell. Preferably said stem cell is a murine stem cell.
In a preferred embodiment of the invention said embryonic stem cell is a teratocarcinoma stem cell.
In a preferred embodiment of the invention said embryonic stem cell is not a totipotent cell.
According to a further aspect of the invention there is provided a spheroid body comprising an embryonic stem cell according to the invention.
In a preferred embodiment of the invention said body further comprises a collagen based cell support.
According to a further aspect of the invention there is provided a cell derived from an embryonic stem cell or spheroid body according to the invention.
According to a further aspect of the invention there is provided an in vitro cell culture comprising an embryonic stem cell or an embryoid body according to the invention. According to a further aspect of the invention there is provided a therapeutic vehicle for use in tissue engineering comprising a cell or spheroid body according to the invention.
In a preferred embodiment of the invention said vehicle is selected from the group consisting of: prothesis; implant; matrix; stent; biodegradable matrix; or hydrogel.
According to a further aspect of the invention there is provided a composition comprising a cell according to the invention.
According to a further aspect of the invention there is provided a composition for use in therapeutic tissue engineering comprising a cell according to the invention.
According to a further aspect of the invention there is provided a method to treat a condition that would benefit from tissue engineering comprising administering a cell or a therapeutic vehicle according to the invention to a subject in need of treatment.
According to a further aspect of the invention there is provided the use of a cell or a therapeutic vehicle according to the invention in the manufacture of a medicament for use in tissue engineering.
ES cells according to the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, intravenous, intraperitoneal, intramuscular, intra-cavity, subcutaneous, or transdermal. The cells of the invention are administered in effective amounts. An "effective amount" is that amount of transfected ES cells that alone, or together with further doses, produces the desired response. In the case of treating a particular disease the desired response is inhibiting the progression of the disease. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.
According to a further aspect of the invention there is provided a non-human transgenic mammal wherein said mammal comprises a cell derived from a cell according to the invention.
Methods to generate transgenic animals utilising the embryonic stem cells of the invention are known in the art. For example, Ware et al teaches an embryonic stem cell culture condition amenable for such animals as cattle, pigs and sheep {Society for the Study of Reproduction, 38:241 (1988)).
Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.
Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.
An embodiment of the invention is described, by example only and with reference to the following figures: Figure IA-T illustrates the nucleic acid sequences of various HLA G molecules; and
Figure 2A-0 illustrates the amino acid sequence of various HLA G molecules.
Materials and Methods
Human embryonic stem cells can be grown in flasks with or without feeder cells; follow A or B, respectively:
PREPARATION OF T25 VESSELS FOR CULTURE OF HUMAN EMBRYONIC STEM CELLS
A: Feeder free methods
1. Remove screw-cap from fresh T25 culture vessel
2. Add XmI of coating solution (e.g. Matrigel in DMEM at 1 : 15)
3. Replace screw-cap
4. Incubate at room temperature for 2 hours or >24hr at 4 degC (can be stored at 4 deg C for up to 7 days) Immediately prior to use:
5. Remove screw-cap
6. Aspirate coating solution
B: Fibroblast feeders
1. Remove screw-cap from dividing culture of feeder cells in T75 flask
2. Aspirate medium and discard
3. Add 3ml mitomycin-c solution (lOμg/ml in medium)
4. Replace screw-cap 5. Return to incubator for 3 hours
6. Remove screw-cap
7. Aspirate mitomycin-c solution and discard
8. Add 5ml medium 9. Replace screw-cap
10. Gently agitate flask (left-right and front-back rocking)
11. Remove screw cap
12. Aspirate medium and discard 13. Add ImI trypsin solution
14. Replace screw cap
15. Incubate at room temperature for 5 minutes
16. Vigorously agitate culture vessel for 10 seconds (jarring blows)
17. Remove screw cap 18. Add 10ml medium
19. Collect/ pool cell suspension into fresh vessel
20. Count cells to determine density of suspension
21. Seed cells at X per cm2 in gelatin-coated T25 culture vessels
22. Return to incubator for =/>24 hours 23. Use within 7 days
PASSAGE PROTOCOL FOR HUMAN EMBRYONIC STEM CELLS IN T25CULTURE VESSELS
1. Remove screw-cap from expanded culture of human embryonic stem cells
2. Aspirate culture medium and discard
3. Add Im collagenase Type IV solution (lmg/ml in medium)
4. Replace screw-cap
5. Incubate at 37degC for 10 minutes 6. Remove screw-cap
7. Add glass beads (ready prepared in sterile vials)
8. Replace screw-cap
9. Agitate gently for 30 seconds to dislodge colonies of cells (horizontal shake and rocking) 10. Remove screw-cap
11. Pipette cells repeatedly to fragment colonies into multicellular clumps (requires optimisation, as clump size is critical to successful passage) 12. Remove screw-caps from fresh culture vessels containing 5ml of temp/gas equilibrated medium (preparation can be inserted between steps 5 and 6)
13. Withdraw cell suspension from original culture vessel
14. Seed cell suspension at a 1:3 passage ratio into fresh culture vessels 15. Replace screw-caps
16. Agitate gently, with caps uppermost, to evenly distribute cells throughout suspension
17. Slowly lower flasks to horizontal position
18. Place in incubator
FEEDING PROTOCOL FOR HUMAN EMBRYONIC STEM CELLS IN T25 FLASK
1. Remove screw cap from culture vessel 2. Aspirate culture medium and discard
3. Add 5ml of temp/gas equilibrated medium
4. Replace screw cap
5. Return to incubator
hES MEDIUM PREPARATION
Solution 1
Add 10ml PBS (-Ca2+, Mg2+) to 0.146g L-glutamine Add 7μl β-metcaptoethanol to the PBS/L-Glutamine solution and mix well
Medium (filter sterilise before use) Add together:
160ml Knockout DMEM
40ml Knockout SR 2ml solution 1
2ml 10Ox non essential amino acids
400μl of a 2μg/μl bFGF stock
Antibiotics (if required) Formulation of medium and component sources:
Final concentration Manufacturer/ cat. No. 80% Knockout DMEM Gibco 10829-018
20% GIBCO Knockout SR Gibco 10828-028
1% Non essential amino acids Gibco 11140-035
1 mM L-Glutamine Gibco 21051 -016
0. IMm β-mercaptoethanol Sigma M-7154 4ng/ml human bFGF Gibco 13256-029
MANUFACTURE OF SINGLE CHAIN VARIABLE FRAGMENT TsCfV) ANTIBODY DERIVATIVES FUSED TO HLA-G TO TARGET IMMUNE SUPPRESSIVE/ TOLEROGENIC EFFECTS TO A SPECIFIC CELL POPULATION
Construction of an ScFv-HLA-G fusion expression vector
Total RNA is isolated from hybridoma cells with Tri-reagent and subjected to DNase I treatment, and then mRNA is isolated. First-strand cDNA synthesis is prepared with random primers using reverse transcriptase.
PCR amplifications of the antibody heavy (VH) and light (VL) variable chains are performed using separate reaction mixtures with primers specific for each chain. Purification of PCR products is performed by gel electrophoresis in agarose gel. DNA bands corresponding to VH and VL chains respectively are excised and the DNA was purified (e.g. by Sephaglas Bandprep Kit (Amersham Biosciences)). The VH and VL chains are assembled with a linker by two rounds of PCR amplification as described in the Amersham Recombinant Phage Antibody System (RPAS) kit.
The entire coding sequence or a fragment thereof of the HLA-G gene is inserted into the multiple cloning site of the pCANTAB5E phagemid vector (Amersham Biosciences). The 750 bp DNA band corresponding to the ScFv fragment is purified and cloned in-frame with the HLA-G sequence, either 5 ' or 3' to it.
Selection of recombinant phage antibodies specific for target cell or protein.
Recombinant phage antibody production is accomplished using a kit such as RPAS (Amersham Biosciences), according to the manufacturer's instructions. Competent E. coli TGl cells were transformed with pCANTAB5E containing the ScFv fragment. Individual transformed colonies were infected with M13KO7 phage to rescue the phagemid. Recombinant phage antibodies arising from each clone are analysed for target binding activity in an ELISA assay or cell binding assay. Phage binding is detected with a peroxidase-labelled sheep anti-M13 antibody (Amersham Biosciences). The recombinant phage clone which gives the strongest positive signal in the assay is used to infect E. coli HB2151 cells.
Expression of the target-binding ScFv-HLA-G fusion in E. coli HB2151 cells
After infection with the desired recombinant phage antibodies, individual HB2151 colonies are transferred to 5 ml of 2"1YT medium containing 100 lg}ml ampicillin and 1% (w}v) glucose and incubated overnight at 37 °C. Aliquots of 100 11 of cell suspension are grown in 2 ml of 2-1YT medium containing 100 lg}ml ampicillin and 0.1% (w}v) glucose until cell culture reaches an attenuance of 0.5 or 1 at 600 run.
Cells are induced overnight with 0.2±l mM isopropyl b-dthiogalactoside (IPTG), in presence or absence of 0.1% glucose, at 16, 24 or 30 0C. Cell lysate and culture supernatant are analysed for the presence of soluble ScFv fragments by immunoblot assay using the anti-E tag mAb, specific to the peptide E tag located at the C-terminus of the ScFv molecule (Amersham Biosiences). CROSS LINKING OF ANTIBODIES TO HLA-G
A range of chemistries are available for cross linking (covalently joining) antibodies to specific proteins. Examples of the reactive and functional groups involved are shown in the table below:
Cross-linker Groups and Their Functional Group Targets
Reactive Group Functional Group
Aryl Azide Non-selective (or primary amine) Maleimide Sulfhydryl
Carbodiimide Amine/Carboxyl
NHS-ester Amine
Hydrazide Carbohydrate (oxidized)
PFP-ester Amine Hydroxymethyl Phosphine Amine
Psoralen Thymine (photoreactive intercalator)
Imidoester Amine
Pyridyl Disulfide Sulfhydryl
Isocyanate Hydroxyl (non-aqueous) Vinyl Sulfone Sulfhydryl, amine, hydroxyl
Carbonyl Hydrazine
Kits available from companies such as Pierce Biotechnology, Inc., Rockford, IL. 61105 U.S.A, are available to perform cross linking to conjugate the HLA-G molecule to an antibody of interest, using the reactive and functional species in the table above.
DEVELOPMENT OF CELL TYPE SPECIFIC NUCLEIC ACID APTAMERS
RNA aptamers
1. An HPLC-purified oligonucleotide library is prepared, consisting of base sequences in which 10-150 nucleotides are randomly arranged between primer binding sequences designated "F" (5') and "R" (3'), each consisting of 10-25 nucleotides and including an RNA polymerase initiation site immediately 3' of the F primer.
2. The randomized oligonucleotides are transcribed into RNA using an appropriate polymerase e.g. T7 polymerase, and purified by phenol chloroform extraction
3. In order to select aptamers that bind the desired cell population(s), the RNA aptamer library is incubated with both target and control cells in normal physiological conditions. Following incubation for between 1 minute and 6 hours, the cells are washed by repeated centrifugation and aspiration of the suspension solution.
4. The washed cell-nucleic acid complexes are subjected to RT-PCR using primers that recognize the primer binding sites. This amplifies a DNA pool complementary to those RNA molecules that bind the cells.
5. Steps 2 through 4 are repeated between 1 and 5 times to produce a highly enriched DNA pool.
ό.The enriched DNA pool is cloned into a plasmid vector and the cell type specific aptamers selected by subtractive hybridization between the target and control cell libraries
r DNA aptamers
1. An HPLC-purified oligonucleotide library is prepared, consisting of base sequences in which 10-150 nucleotides are randomly arranged between primer binding sequences designated "F" (5') and "R" (3'), each consisting of 10-25 nucleotides
2. In order to select aptamers that bind the desired cell population(s), the DNA aptamer library is incubated with both target and control cells in normal physiological conditions. Following incubation for between 1 minute and 6 hours, the cells are washed by repeated centrifugation and aspiration of the suspension solution.
3. In order to select aptamers that bind the desired cell population(s), the DNA aptamer library is incubated with both target and control cells in normal physiological conditions. Following incubation for between 1 minute and 6 hours, the cells are washed by repeated centrifugation and aspiration of the suspension solution.
4. The washed cell-nucleic acid complexes are subjected to PCR using primers that recognize the primer binding sites. This amplifies a DNA pool corresponding to those aptamers that show greatest binding affinity for the cells.
5. Steps 2 through 4 are repeated between 1 and 5 times to produce a highly enriched
DNA pool.
6. The enriched DNA pool is cloned into a plasmid vector and the cell type specific aptamers selected by subtractive hybridization between the target and control cell libraries
Once a suitable aptamer has been identified that specifically binds the desired target population, it is conjugated to HLA-G. A number of approaches are available to achieve this, including ready made reagents in kit form, available from companies such as Pierce Biotechnology, Inc., Rockford, IL. 61105 U.S.A:
CONJUGATION TO 5'-PHOSPHATE GROUPS
Using chemical reagents to effect the conjugation of nucleic acids to enzymes can be done using different strategies. A convenient functional group that can be chemically modified to allow the coupling of protein molecules on oligonucleotides is the 5 '-phosphate group. Using the 5' end of the oligonucleotide as the conjugation point has an advantage of keeping the rest of the nucleic acid sequence unmodified and free so it can easily hybridize to a complementary target. For oligonucleotides that have been synthesized, a 5 '-phosphate group may be put on the end of the molecule to facilitate this type of conjugation. The alkyl phosphate is reactive with the water-soluble carbodiiniide EDC (Product # 22980, 22981), which forms a phosphate ester similar to the reaction of EDC with a carboxylate group. Subsequent coupling to an amine-containing molecule (i.e., nearly any protein or unmodified peptide) can be done to form a stable phosphoramidate linkage(Figurel ?????).
If a diamine molecule is used to modify the DNA 5 '-phosphate, then the resultant amine- modified oligonucleotide can be coupled to enzyme molecules using a heterobifunctional reagent. Using a diamine compound that contains a disulfide (e.g., cystamine) and then reducing the disulfide group results in a sulfhydryl that may be conjugated with proteins rendered sulfhydryl-reactive using the heterobifunctional reagent Sulfo-SMCC (Product #
22322). Pierce offers HRP and AP enzymes that have been made sulfhydryl reactive (i.e., maleimide-activated) by this mechanism (Product # 31485 and # 31486, respectively).
CONJUGATION TO THE 3' END OF RNA
Alternatively, the 3' end of RNA molecules may be chemically modified to allow coupling with amine-containing molecules or proteins. The diol on the 3'-ribose residue may be oxidized to result in two aldehyde groups using Sodium meta -Periodate (Product
# 20504). The aldehydes then can be conjugated to the amine groups on a protein using reductive amination with Sodium Cyanoborohydride (Product # 44892). The aldehyde and the amine first form a Schiff base that is reduced to secondary amine linkage with the cyanoborohydride reductant.
In addition, biotinylation of oligonucleotides can be done using photoreactive reagents. There are two main options commonly used to add one or more biotin residues to nucleic acid probes. Photoactivatable Biotin (Product # 29987) contains a phenyl azide group at the end of a spacer arm with the biotin group at the other end. Photolyzing a solution of the biotin compound together with an oligonucleotide in solution results in biotin being non-selectively inserted into the nucleic acid structure. Alternatively, Psoralen-PEO- Biotin (Product # 29986) can be used to label double-stranded DNA or RNA. The psoralen ring structure effectively intercalates into the double-stranded portions, and exposure to UV light causes a cyclo-addition product to be formed with the 5, 6-double bond in thymine residues. The poly(ethylene oxide) spacer in Psoralen-PEO-Biotin contributes excellent water solubility, thus assuring that the resultant derivative will have accessibility to streptavidin-containing detection reagents.
PEPTIDE APTAMER AND ScFv TARGETING MOLECULE IDENTIFICATION BY PHAGE DISPLAY
Phage libraries of peptides and scFv are available commercially:
(e.g. from New England Biolabs 240 County Road, Ipswich, MA 01938-2723, USA):
Cat # E811 OS Ph.D.-12 Phage Display Library Kit
Cat # E8100S Ph.D.-7 Phage Display Library Kit Cat # E8120S PLD.-C7C Phage Display Library Kit
(e.g. from Geneservice Ltd, 2 Cambridge Science Park, Milton Road, Cambridge, CB4 OFE, UK)
Tomlinson I+J scFv phage library
1. The phage library is incubated with the human control (i.e. non target) cell population in normal physiological conditions
2. After between 1 minute and 6 hours of incubation, the control cells are removed by centrifugation and the phage-containing supernatant transferred to a fresh tube
3. The target cell population is added to the collected supernatant and incubated for between 1 minute and 6 hours 4. The target cells are pelleted by centrifugation and the supernatant discarded
5. The target cells are washed 3 times in PBS
6. Phage bound to target cells are eluted (e.g. by altering pH, salt concentration etc.) 7. Recovered phage are used to infect a permissive E.coli strain and the resulting phage-carrying bacteria grown in suitable medium
8Phage are prepared from the infected bacteria and incubated with a human control population of cells, as in 1. 9Steps 2-8 are repeated between 1 and 4 times to enrich for phage carrying target cell specific peptide/ scFv encoding DNA lO.Phage DNA is sequenced to determine the targeting peptide
Once a suitable targeting sequence is determined, it is cloned either 5' or 3' of the HLA-G gene coding sequence in a suitable expression system to produce recombinant protein.
Table 1 ' '
1: NP_751947 ' • • , ' . advanced glycosylation end product- specific receptor isoform 2 precursor [Homo sapiens] . ■ gi|26787962|ref |NP_751947.l| [26787962] ■ .
2: NP_001127 • . . ' '
•advanced glycosylation end product-specific receptor isoform 1 precursor [Homo sapiens] . gi 110835203 J ref | NP_001127.11 [10835203]
Table 2 CD antigens
Data derived from table at http://www.ebioscience.com/ebioscience/whatsnew/humancdchart.htm
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CDIa T649 IgSF, MHC-like molecule, Group 1 CDl Cortical thymocytes, Langerhans cells, DC Antigen presentation, associates with b2m lq22-23
CDIb T645 IgSF, MHC-like molecule, Group 1 CDl Cortical thymocytes, Langerhans cells, DC Antigen presentation, associates with b2m lq22-23
CDIc T643 IgSF, MHC-like molecule, Group 1 CDl Cortical thymocytes, Langerhans cells, DC, B subset Antigen presentation, associates with b2m lq22-23
CDId 49 IgSF, MHC-like molecule, Group 2 CDl Intestinal epithelial cells, B subset, monocytes (low), DC Antigen presentation, associates withb2m lq22-23
CDIe 28 IgSF, MHC-like molecule, Group 3 CDl DC Antigen presentation, associates withb2m lq22-23
CD2 Tl 1, LFA-2, SRBC-R 50 IgSF Thymocytes, T cells, NK cells CD58 counter- receptor, adhesion, T cell activation Ipl3.1
CD2RT11-3 50 IgSF Activated T cells Activation-dependent conformational form of CD2
CD3g T3 20-26 IgSFT cells, thymocyte subset Associates with T cell antigen receptor, required for TCR cell surface expression and signal transduction 1 Iq23
CD3d T3 21 IgSF T cells, thymocyte subset Associates with T cell antigen receptor, required for TCR cell surface expression and signal transduction 1 Iq23
CD3e T3 20 IgSF T cells, thymocyte subset Associates with T cell antigen receptor, required for TCR cell surface expression and signal transduction 1 Iq23
CD4 T4, L3T455 IgSF Thymocyte subset, T subset (helper), monocytes, macrophages MHC class II coreceptor, receptor for HIV, thymic differentiation, T cell activation 12pter-pl2
CD5 Tl, Tp67, LyI 67 Scavenger receptor superfamily Thymocytes, T cells, B subset, B- CLL CD72 counter-receptor, signaling through TCR or BCR, T-B interaction 1 Iql3 Table 2 CD antigens
CD6 T12 100-130 Scavenger receptor superfamily Thymocytes, T cells, B subset CD166 counter-receptor, T cell differentiation and costimulation 11
CD7
40 IgSF Pluripotent hematopoietic progenitor cells, thymocytes, T cells, NK cells T cell costimulation 17q25.2-25.3
CD8a T8, Leu-2, Lyt2,3 32-34 IgSF Thymocyte subset, T subset (cytotoxic), NK cells MHC class I coreceptor, receptor for some mutated HIV-I, thymic differentiation, T cell activation 2p 12
CD8b 30-32 Thymocyte subset, T subset (cytotoxic)
CD9 p24, MRP-I 22-27 TM4SF Pre-B cells, eosinophils, basophils, platelets, activated T cells Cellular adhesion and migration 12pl3
CDlO CALLA, NEP, gp 100 100 Type II transmembrane protein B cell precursors, T cell precursors, neutrophils Zinc-binding metalloproteinase, B cell development 3q21-q27
CDl Ia LFA-I, integrin aL subunit 180 Integrin family Lymphocytes, granulocytes, monocytes, macrophages CD 11 a/CD 18 counter-receptor for ICAM-I, ICAM-2 and ICAM-3, intercellular adhesion, T cell costimulation 16p 11-13.1
CDl Ib Mac-1, integrin aM subunit 170 Integrin family Myeloid cells, NK cells CDl lb/CD18 counter-receptor for CD54, adhesion to ECM and complement iC3b 16pll-13.1
CDlIc ρl50, 95, CR4, integrin aX subunit 150 Integrin family Myeloid cells, NK cells, B cells, T subset CDllc/CD18 counter-receptor for CD54, fibrinogen and. iC3b, adhesion 16pll-13.1
CDwl2 90-120 Monocytes, granulocytes, platelets Unknown
CD13 Aminopeptidase N, APN, gpl50 150-170 Type II transmembrane protein Myeloid cells Zinc-binding metalloproteinase, antigen processing 15q25-q26
CD 14 LPS-R 53-55 GPI-linked membrane protein Monocytes, macrophages, Langerhans cells, granulocytes (low) Receptor for LPS/LBP, LPS recognition 5q35
CD 15 Lewis-x (Lex) CHO Neutrophils, eosinophils, monocytes Adhesion
CD15S Sialyl Lewis X CHO Neutrophils, eosinophils, monocytes, memory helper T cells, activated T and B cells, NK cells, HEV CD62L and CD62P counter-receptor, adhesion Table 2 CD antigens
CD15u Sulfated Lewis X CHO Myeloid subset Adhesion
CD 16a FcgRIIIA 50-65 IgSF Neutrophils, NK cells, macrophages Component of low affinity Fc receptor, phagocytosis and ADCC Iq23
CD 16b FcgRIIIB 48 IgSF Neutrophils Component of low affinity Fc receptor; FcgRIII, phagocytosis and ADCC Iq23
CDwI 7 Neutrophils, monocytes, platelets Cell surface glycosphingolipid, lactosyl ceramide
CD 18 Integrinb2 subunit 95 Integrin family Broad, all leukocytes Associates with CDl Ia, b and c, adhesion 21q22.3
CD19 B495 IgSF B cells, FDC Forms complex with CD21 (CR2) and CD81 (TAPA-I), BCR coreceptor, B cell activation and differentiation 16pl 1.2
CD20 Bl, Bp35 33-37 TM4SF B cells, T subset B cell activation 1 Iql3
CD21 CR2, EBV-R 14-5, 110 CCRSF B cells, FDC, T subset Complement C3d receptor, EBV receptor, forms complex with CD19 and CD81, BCR coreceptor Iq32
CD22 BL-CAM, Siglec-2 150 IgSF, sialoadhesins B cells Adhesion, B-monocytes and B- T interactions 19ql3
CD23 FceRII 45 C-type lectin B cells, activated macrophages, eosinophils, FDC, platelets CD19-CD21-CD81 counter-receptor, low affinity receptor for IgE, signal transduction 19pl3
CD24 BA-I 35-45 GPI-linked membrane protein B cells, granulocytes, epithelial cells B cell differentiation and proliferation 6q21
CD25 Tac, p55 55 Tpe I transmembrane protein Activated T and activated B cells IL-2 receptor a chain, associates with IL-2 receptor b and g chains to form high affinity IL-2R complex 10pl4-ρl5
CD26 Dipeptidyl peptidase IV (DPP IV) 110 Type II transmembrane protein Thymocyte subset, activated T cells, B cells, NK cells, macrophages, epithelial cells, soluble form A dipeptidyl peptidase, T cell costimulation, HIV entry into cells 2q24.3
CD27 T1450-55 TNFRSF Medullary thymocytes, T cells, B subset, NK subset CD70 counter-receptor, T cell costimulation 12pl3
CD28 Tp44, T4444 IgSF Most T cells, thymocytes, plasma cells CD80 (B7-1) and B7-2 (CD86) counter-receptor, T cell costimulation 2q33 00448
Table 2 CD antigens
CD29 Integrin bl chain 130 Integrin family Lymphocytes, monocytes, granulocytes (low), platelets, mast cells, fibroblasts and endothelial cells CD29/CD49a ( VLA-I) counter-receptor for VCAM-I, MAdCAM-I and ECM lOpl 1.2
CD30 Ki-I 105-120 TNFRSF Activated B, T and NK cells, Reed-Sternberg cells, anaplastic large-cell lymphoma CDl 53 counter-receptor, lymphocyte proliferation and apoptosis Ip36
CD31 PECAM-I 130-140 IgSF Monocytes, platelets, granulocytes, endothelial cells, lymphocyte subset CD38 counter-receptor, adhesion 17q23-ter
CD32 FcgRII 40 IgSF Monocytes, granulocytes, B cells, platelets Low affinity Fc receptor for aggregated Ig and immune complexes lq23-24
CD33 p67, Siglec-3 67 IgSF, sialoadhesins Myeloid progenitor cells, monocytes, granulocytes, DC, mast cells Adhesion 19ql3.3
CD34 105-120 Sialomucin, type I transmembrane protein Hematopoietic precursors, capillary endothelial cells, embryonic fibroblasts CD62L counter-receptor, adhesion, stem cell marker Iq32
CD35 CRl 250 CCRSF Erythrocytes, B cells, monocytes, neutrophils, eosinophils, FDC, T subset Complement receptor 1, binds C3b and C4b, adhesion, phagocytosis Iq32
CD36 GPPV 88 Platelets, monocytes, macrophage, endothelial cells, early erythrocytes Receptor for ECM, adhesion, phagocytosis 7ql 1.2
CD3740-52 TM4SFB cells, T cells (low), granulocytes (low) Signal transduction 19pl3-ql3
CD38 TlO 45 ADP-ribosyl cyclase Variable levels on majority of hematopoietic cells, high expression on plasma cells, B cells and activated T cells An ecto-ADP-ribosyl cyclase, cell activation 4pl5
CD39 78 Activated B cells, NK cells, macrophages, Langerhans cells, DC B cell adhesion, protects activated cells from lysis, an ecto-apyrase 10q23.1-24.1
CD4048 TNFRSF B cells, monocytes, macrophages, DC, endothelial cells, fibroblasts CDl 54 counter-receptor, B cell differentiation and costimulation, isotype-switchmg, rescues B cells from apoptosis 20ql2-ql3.2
CD41 gpllb 125/22 dimer Integrin family Platelets, megakaryocytes Complex with CD61 to form GPIIb, binds fibrinogen, fibronectin, von Willebrand factor and thrombospondin, platelet activation and aggregation 17q21.32 Table 2 CD antigens
CD42a GPIX 22 LRRF Platelets, megakaryocytes Complex with CD42b, c and d, receptor to von Willebrand factor and thrombin, platelet adhesion to subendothelial matrices 17pter-pl2
CD42b GPIba 145 LRRP Platelets, megakaryocytes Complex with CD42a, c and d, binds to von Willebrand and thrombin, platelets adhesion and activation
CD42c GPIbb 24 LRRF Platelets, megakaryocytes Complex with CD42a, b, d
CD42d GPV 82 LRRF Platelets, megakaryocytes Complex with CD42a, b, c
CD43 Leukosialin, sialophorin 115-135 Sialomucin, type I transmembrane protein Leukocytes, except resting B cells, platelets (low) Inhibition of T cell interaction, CD54 counter-receptor, adhesion 16p 11.2
CD44 H-CAM, Pgp-1 80-95 Hyaladherin family Hematopoietic and non-hematopoietic cells, except platelets, hepatocytes, testis Binds hyaluronic acid, leukocyte adhesion Ilpter-pl3
CD44R CD44v Variants of CD44 Variants show heterogeneous expression, constitutively on epithelial cells, monocytes, upregulated on activated leukocytes Adhesion, metastasis
CD45 Leukocyte common antigen (LCA), T200, B220 180-240 Hematopoietic cells, multiple isoforms from alternative splicing Tyrosine phosphatase, augments signaling through TCR and BCR Iq3 l-q32
CD45RA 205-220 B cells, T subset (naive T cells), monocytes Exon A isoforms of CD45 Iq31-q32
CD45RB 190-220 T subset, B cells, monocytes, macrophages, granulocytes Exon B isoforms of CD45 Iq31-q32
CD45RO 180 Activated T cells, memory T cells, B subset, monocytes, macrophages, granulocytes Isoform of CD45 containing none of the A, B and C exons Iq31-q32
CD46 MCP 56/66 CCRSF Hematopoietic and non-hematopoietic nucleated cells Membrane cofactor protein, binds C3b and C4b to permit their degradation by Factor I, measles virus receptor Iq32
CD47 IAP 47-52 IgSF Hematopoietic cells, epithelial and endothelial cells, fibroblasts, other tissues Leukocyte adhesion, migration, activation 3ql3.1-13.2 Table 2 CD antigens
CD47R New designation for CDw 149, similar distribution as CD47 but dimmer CD48 Blast- 1 45 IgSF Broad, all leukocytes Cell adhesion lq21.3-22
CD49a VLA-I 210 Integrin family Activated T cells, monocytes Integrin al, adhesion, CD49a/CD29 binds collagen and laminin 5
CD49b VLA-2 165 Integrin family B cells, monocytes, platelets, activated T cells, megakaryocytes Integrin a2, adhesion, CD49b/CD29 binds collagen, laminin 5q23-31
CD49c VLA-3 125 Integrin family B cells and T cells (low), most adhesive cell lines Integrin a3, adhesion, CD49c/CD29 binds laminin, fibronectiπ, collagen 17q
CD49d VLA-4 150 Integrin family T cells, B cells, thymocytes, monocytes, eosinophils, NK cells, mast cells, DC Integrin a4, adhesion, CD49d/CD29 hinds fibronectin, VCAM- 1, MAdCAM-I 2q31-q32
CD49e VLA-5 135, 25 Integrin family Thymocytes, T cells, monocytes, platelets, early B and activated B cells Integrin a5, adhesion, CD49e/CD29 binds fibronectin 12ql l-ql3
CD49f VLA-6 125 Integrin family Memory T cells, thymocytes, monocytes, platelets, megakaryocytes, epithelial cells Integrin a6, adhesion, CD49f/CD29 binds laminin 2
CD50 ICAM-3 130 IgSF Thymocytes, T cells, B cells, monocytes, granulocytes, endothelial cells Adhesion, costimulation 19pl3.3-13.2
CD51 Vitronectin receptor 125, 24 Integrin family Platelets, megakaryocytes, endothelial cells, osteoblasts, melanoma cells Integrin av, adhesion, CD51/CD61 dimer binds vitronectin, von Willebrand factor, fibrinogen and thrombospondin 2q3 l-q32
CD52 CAMPATH-121-28
Thymocytes, T cells, B cells (not plasma cells), monocytes, macrophages Unknown 1
CD53 35-42 TM4SF Leukocytes, DC, osteoblasts and osteoclasts Signal transduction lP21-pl3.3
CD54 Intercellular Adhesion Molecule, ICAM-I 85-110 IgSF Endothelial cells, monocytes, resting lymphocytes (low), upregulated on activation CDl la/CD18 (LFA-I) and CDl lb/CD18 (Mac-1) counter-receptor, rhinovirus receptor 19ρl3.3-13.2
CD55 Decay Accelerating Factor, DAF 60-70 CCRSF, GPI-linked membrane protein Hematopoietic and non-hematopoietic cells Binds C3b, complement regulation Iq32
CD56 Neural Cell Adhesion Molecule, NCAM 175-185 IgSF NK cells, T subset, neural tissue, some large granular lymphocyte leukemias, myeloid leukemias Adhesion 11 q23.1 Table 2 CD antigens
CD57 HNK-I, Leu-7 110 NK subset, T subset
CD58 Leukocyte Function-associated Antigen-3, LFA-3 55-70 IgSF Hematopoietic and non-hematopoietic cells CD2 counter-receptor, adhesion Ip 13.1
CD59 Protectin, MAC- inhibitor 19 GPI-linked membrane protein Hematopoietic and non-hematopoietic cells Binds complement components C8 and C9, blocks assembly of membrane attack complex (MAC) 1 Ipl3
CD60a GD3 CHO T subset, platelets, thymic epithelial cells, astrocytes Costimulation CD60b 9-O-sialyl GD3 CHO T subset, activated B cells CD60c 7-0-sialyl GD3 CHO T subset
CD61 GPIIIa 105 Integrin family Platelets, megakaryocytes, macrophages, endothelial cells Integrin b3, adhesion, CD41/CD61 or CD51/CD61 complexes mediate adhesion to ECM 17q21.3
CD62E E-selectin, ELAM-I (endothelium leukocyte adhesion molecule) 97, 107-115 Selectin family Endothelial cell Binds sialyl-Lewis x (CD 15s), cell rolling, tumor metastasis lq23-25
CD62L LAM-I, L-selectin, LECAM-I 74, 95 Selectin family B cells, naϊve and memory T cells, monocytes, granulocytes, NK cells, thymocytes CD34, GIyCAM, and MAdCAM-I counter-receptor, leukocyte homing, leukocyte tethering and rolling Iq23- 25
CD62P P-selectin, PADGEM 140 Selectin family Activated platelets, endothelial cells CD 162 and sialyl Lewis x counter-receptor, adhesion, neutrophil rolling, platelet- neutrophil and platelet-monocyte interactions lq21-24
CD63 LIMP, LAMP-3 53 TM4SF Activated platelets, monocytes, macrophages A lysosomal membrane protein, translocates to cell surface after activation 12ql2-ql3
CD64 FcgRI 72 IgSF Monocytes, macrophages, DC, Interferon or G-CSF activated granulocytes High affinity receptor for IgG, phagocytosis and ADCC 1 q21.1
CD65 Granulocytes, monocyte subset, myeloid leukemia cells
CD65s VIM2 Granulocytes, monocytes, myeloid leukemia cells Phagocytosis
CD66a Biliary glycoprotein- 1 (BGP-I), NCA-160 160-180 IgSF, CEA family Neutrophils, epithelial cells Cell adhesion, neutrophil activation 19ql3.1-2 Table 2 CD antigens
CD66b Previously CD67, CGM6, NCA-95 95-100 IgSF, CEA family Granulocytes Cell adhesion, neutrophil activation 19ql3.1-2
CD66c NCA 90 IgSF, CEA family Neutrophils, colon carcinoma Cell adhesion 19ql3.1- 2
CD66d CGMl 35 IgSF, CEA family Neutrophils 19ql3.1-2
CD66e CarcinoEmbryonic Antigen (CEA) 180-200 IgSF, CEA family Colon epithelium, colon cancer Cell adhesion 19ql3.1-2
CD66fPSG, Sp-I 54-72 IgSF5 CEA family Pregnancy specific glycoprotein, expressed on placental syncytiotrophoblasts, fetal liver Immune regulation, protects fetus from maternal immune system
CD68 Macrosialin, gpl 10 110 Sialomucin Expressed intracellularly in monocytes, macrophages, neutrophils, basophils, large lymphocytes, mast cells, DC, myeloid progenitors, liver 17pl3
CD69 Activation Inducer Molecule (AIM) 28, 32 C-type lectin Activated T, B, NK cells and granulocytes, thymocytes, platelets, Langerhans cells Signal transduction 12pl2.3- pl3.2
CD70 Ki-2450, 70, 90, 160 TNFSF Activated B cells, activated T cells CD27 counter- receptor, T and B cell costimulation 19pl3.3
CD71 T9 95
Proliferating cells, reticulocytes, erythroid precursors Transferrin receptor, iron uptake
3q26.2-qter
CD72 Lyb-242 C-type lectin, homodimer B cells, FDC CD5 and CDlOO counter- receptor, B cell proliferation 9pl 1.1-24
CD73 69 GPI-linked membrane protein B subset, T subset, FDC, epithelial cells Ecto-5'- nucleotidase, dephosphorylates nucleotides to allow nucleoside uptake, T cell costimulation, lymphocytes adhesion 6ql4-q21
CD74 Ii, invariant chain 33,35,41,43
B cells, macrophages, monocytes, Langerhans cells, DC, activated T cells MHC class II traffic and function 5q32
CD75 CHO Sialoglycan family B subset, epithelial cells Lactosamines Table 2 CD antigens
CD75s CHO Sialoglycan family B subset, T subset Alpha-2,6-sialylated lactosamines (previously CDw75 and CDw76)
CD77 Globotiiaocylceramide (Gb3), Pk blood group Germinal center B cells, Burkitt's lymphomas (high), follicular center lymphomas (low) Apoptosis
CD79a Iga 33, 45 IgSF B cells Component of BCR5 required for cell surface expression and signal transduction 19ql3.2
CD79b Igb 37 IgSF B cells Component of BCR, required for cell surface expression and signal transduction 17q23
CD80 B7,B7-1, BBl 60 IgSF Activated B cells, activated T cells, macrophages, DC CD28 and CD 152 counter-receptor, T cell costimulation 3q21
CD81 Target of Antiproliferative Antibody (TAPA-I) 26 TM4SF T, B, andNK cells, thymocytes, DC, endothelial cells, fibroblast, neuroblastomas, melanomas Complex with CD19 and CD21, signal transduction, T cell costimulation 1 Ipl5.5
CD82 R2 50-53 TM4SF Leukocytes Signal transduction llpll.2
CD83 HB 15 43 IgSF Activated B cells, activated T cells, circulating DC (veil cells),
Langerhans cells
6p23-p21.3
CDw8473 Monocytes, platelets, B cells, T subset, macrophage subset
CD85 110 IgSF, ILT/LIR family DC, monocytes, B cells, NK cells, T subset Inhibition of NK and T cell cytolytic function 19ql3.4
CD86 B70, B7-2 80 IgSF Monocytes, activated B cells, activated T cells, DC CD28 and CD 152 counter-receptor, T cell costimulation 3ql3-q23
CD87 UPA-R 39-66 GPI-linked membrane protein Granulocytes, monocytes, NK cells, activated T cells, endothelial cells, fibroblasts Urokinase plasminogen activator receptor, inflammatory cell invasion and tumor metastasis 19ql3
CD88 C5aR 40 TM7SF (rhodopsin family) Granulocytes, monocytes, DC Receptor for complement component C5a, granulocyte activation 19ql3.3-13.4
CD89 FcaR, IgA receptor 55-75 IgSF Monocytes, macrophages, granulocytes, neutrophils, B subset, T subset IgA receptor, phagocytosis, degranulation, respiratory burst 19ql3.4 Table 2 CD antigens
CD90 Thy-1 25-35 IgSF, GPI-linked membrane protein CD34+ prothymocytes, CD34+ hematopoietic subset, neurons Hematopoietic stem cell and neuron differentiation Ilq23.3
CD91
600 LDL receptor family Monocytes, macrophages, neurons, fibroblasts Receptor for a- 2-macroglobulin 12ql3.1-ql3.3
CDw9270 Neutrophils, monocytes, platelets, endothelial cells, fibroblasts
CD93 120 Neutrophils, monocytes, endothelial cells
CD94 KP43 43 C-type lectin NK cells, T subset Complex with NKG2, inhibits NK cell function 12pl2.3-pl3.1
CD95 Apo-1, Fas 45 TNFRSF Lymphocytes (high level expression after activation), monocytes, neutrophils CD95L(CD178) counter-receptor, induction of apoptosis 10q24.1
CD96 T cell ACTivation, Increased Late Expression (TACTILE) 160 IgSF Activated T cells, NK cells Adhesion of activated T and NK cells
CD97 74, 80, 90 TM7SF (rhodopsin family), EGF Activated B and T cells, monocytes, granulocytes
19ρl3.12-13.2
CD98 4F2 80, 40
T cells, B cells, NK cells, granulocytes, all human cell lines Cellular activation 1 Iq
CD99 MIC2> E2 32
Leukocytes T cell activation, adhesion Xp22.32-pter, Ypll.2-pter
CD99RE2 32
T cells, NK cells, myeloid cells Isoform of CD99
CDlOO 150 Most hematopoietic cells except immature bone marrow cells, RBC and platelets Cell adhesion, cellular activation
CDlOl V7, ρl26 120 IgSF Monocytes, granulocytes, DC, activated T cells T cell activation Ip 13 Table 2 CD antigens
CDl 02 ICAM-2 55-65 IgSF Lymphocytes, monocytes, platelets, endothelial cells Binds CDl la/CD18 (LFA-I), costimulation 17q23-25
CDl 03 HML-I, a6, aE integrin 150,25 Integrin family Intraepithelial lymphocytes, peripheral blood lymphocyte subset, activated lymphocytes Associates with integrin b7, binds E-cadherin, lymphocyte homing and retention
B-Ly7 PU, F, PE CD104 b4 integrin 220 Integrin family Epithelial cells, Schwann cells, keratinocytes, some tumor cells Associates with integrin a6 (CD49f), cell adhesion, migration, differentiation and tumor metastasis 17ql 1-pter
CD 105 Endoglin 95 Homodimer Endothelial cells, bone marrow cell subset, activated macrophages Cellular response to TGF-bl 9q34.1
CDl 06 VCAM-I 110 IgSF Activated endothelial cells, FDC VLA-4 (CD49d/CD29) counter-receptor, leukocyte adhesion, transmigration, costimulation Ip31-p32
CDl 07a Lysosomal Associated Membrane Protein- 1 (LAMP-I) 110
Activated platelets, activated T cells, activated endothelial cells, metastatic tumors lysosomal membrane protein 13q34
CD107b Lysosomal Associated Membrane Protein-2 (LAMP-2) 120
Activated platelets, activated T cells, activated endothelial cells, metastatic tumors lysosomal membrane protein Xq24-25
CD 108 80 Erythrocytes, lymphoblasts, resting lymphocytes (low)
CD109 170/50 Activated T cells, activated platelets, CD34+ subset, endothelial cells
CDl 10 MPL, TPO-R 82-84 CRSF Megakaryocytes, platelets, some CD34+ stem cells (weak) Thrombopoietin receptor, megakaryocyte progenitor cell growth and differentiation Ip34
CDIl 1 PRRl, Nectin-1 64-72 IgSF Stem cell subset, macrophages, neutrophils
CDl 12 PRR2, Nectin-2 64-72 IgSF Monocytes, neutrophils, CD34+ subset, megakaryocytes, endothelial cells, epithelial cells Intercellular adhesion 19ql3.2-ql3.4
CD 114 G-CSF R 95, 139 CRSF Myeloid progenitor cells, endothelial cells G-CSF receptor, myeloid cell proliferation and differentiation lp35-34.3
CDl 15 M-CSFR, c-fins 150 IgSF, RTK family Monocytes, macrophages, monocytic progenitor cells M-CSF (CSF-I) receptor, monocytic lineage proliferation and differentiation 5q33.2-33.3
CD 116 GM-CSFRa 70-85 CRSF Monocytes, granulocytes, DC, endothelial cells GM- Table 2 CD antigens
CSF receptor a chain, associates with common b chain, myeloid hematopoietic cell proliferation and differentiation Xp22.32, YpI 1.3
CDl 17 c-kit, SCFR 145 IgSF, RTK family Hematopoietic stem/progenitor cells, mast cells Stem Cell Factor (SCF) receptor, hematopoietic progenitor development and differentiation 12q22
CDwI 19 IFNgR 90-100 Macrophages, monocytes, B cells, T cells, NK cells, neutrophils, endothelial cells Interferon g receptor a chain, associates with IFNg AF-I, host defense 6q23-24
CD 120a TNFR-1 50-60 TNFRSF Hematopoietic and non-hematopoietic cells TNF receptor, binds both TNFa and TNFb 12pl3.2
CD 120b TNFR-II 75-85 TNFRSF Hematopoietic and non-hematopoietic cells TNF receptor, binds both TNFa and TNFb Ip36.3-p36.2
CD 121 a IL- 1 R type 180 IgSF Dimly expressed on many cells Type I interleukin 1 receptor, binds IL-Ia and IL-Ib, IL-I signal transduction 2ql2
CDwl21b IL-IR, type II 60-70 IgSF B cells, macrophages, monocytes, T subset Type II interleukin 1 receptor, binds IL-Ia and IL-Ib1 negative signals 2ql2-q22
CD122 IL-2Rb 75 CRSF NK cells, T cells, B cells, monocytes IL-2b and IL- 15b recepto, signal transduction 22qll.2-ql3
CDwl23 IL-3R 70 CR.SF, fϊbronectin type III superfamily Lymphocyte subset, basophils, hematopoietic progenitor cells, macrophages, DC, megakaryocytes IL-3 receptor a chain, associates with CDwl31, binds IL-3 Xp22.3, Ypl3.3
CD124 IL-4R 140 CRSF, fibronectin type III superfamily Lymphocytes (low), monocytes, hematopoietic precursor cells, fibroblast, epithelial cells IL-4 receptor a chain, associates withCD132 or IL-13Rachain, T cell growth and differentiation 16pll.2-pl2.1
CDw 125 IL-5R 60 CRSF, fibronectin type III superfamily Eosinophils, basophils IL-5 receptor a chain, associates with CDwI 31 3p26
CD 126 IL-6R 80 IgSF, CRSF, fibronectin type III superfamily Activated B cells and plsama cells, most leukocytes (weak), fibroblasts IL-6 receptor a subunit, binds IL-6, associates with CD 130 Iq21
CD 127 IL-7R 65-75 CRSF pro-B cells, T cells IL-7 receptor a chain, associates with CD 132, B and T cell development 5pl3
CDwl28 IL-8RA, CXCRl and IL-8RB, CXCR2 67-70 CRSF, GPI-linked membrane Table 2 CD antigens
protein Neutrophils, basophils, NK cells, T subset, monocytes Neutrophil chemotaxis/activation, IL-8RA binds IL-8 only, IL-8RB binds IL-8, GRO, NAP-2 and ENA78 2q35
CDl 30 IL-6Rb, gpl30 130 CRSF Activated B cells and plasma cells, most leukocytes (weak), endothelial cells IL-6 receptor b subunit, signaling biological activities of IL-6, IL-11, LIF, CNF 5ql l
CDwl31 IL-3R common beta chain 95-120 CRSF Monocytes, granulocytes, early B cells Associates with the a subunits of IL-3, IL-5, and GM-CSF receptors, signal transduction 22ql2.2-ql3.1
CD132 Common gamma chain 64 CRSF T cells, B cells, NK cells, monocytes, granulocytes Subunit of IL-2, IL-4, IL-7, IL-9, and IL-15 receptors, signal transduction Xql3
CD133 AC133 120 TM5SF Hematopoietic stem cell subset, epithelial cells, endothelial cells
CDl 34 OX-4048-50 TNFRSF Activated T cells OX-40L counter-receptor, T cell activation, differentiation, apoptosis Ip3δ
CD 135 Flt3/Flk2 130-150 RTK family Multipotent myelomonocytic and primitive B progenitor cells Receptor tyrosine kinase, binds FLT3 ligand, early lymphocyte development 13ql2
CDwl36 MSP-R, RON 180 RTK family Epithelial cells, CNS, PNS, hematopoietic subset Migration, morphological change, and proliferation in different target cells 3p21.1-22
CDwl374-1BB 30 TNFRSF Activated T cells T cell costimulation Ip36
CD 138 syndecan-1 80-150 Syndecan family Plasma cells, pre-B cells, basolateral surface of epithelial cells, neuronal cells Receptor for ECM, maintenance of cell morphology 2p23
CD 139 228 B cells, monocytes, granulocytes, erythrocytes (low)
CD 140a PDGF receptor a 180 RTK family Fibroblasts, smooth muscle cells, glial cells, chondrocytes Binds PDGF A and B chain 4ql 1-12
CD 140b PDGF receptor b 180 RTK family Fibroblasts, smooth muscle cells, glial cells, chondrocytes Binds PDGF B chain 5q31-32
CD141 Thrombomodulin 100 C-type lectin Monocytes, neutrophils, endothelial cells, smooth muscle cells Activation of protein C and initiation of protein C anticoagulant Table 2 CD antigens
pathway 20pl 1.2
CD 142 Tissue Factor 45
Monocytes, endothelial cells, keratinocytes, epithelial cells Binds clotting factor Vila, major initiator of clotting lp21-22
CD 143 Angiotensin converting enzyme (ACE) 170
Endothelial cells, epithelial cells, neuronal cells, fibroblats, activated macrophages a
Peptidyl-peptidase A, angiotensin II and bradykinin metabolism 17q23
CD 144 VE-Cadherin, Cadherin-5 130 Cadherin family Endothelial cells Adhesion, cell- cell interaction
CDwl45 110, 90 Endothelial cells, some stromal cells
CD 146 MUCl 8, S-endo 113-118 IgSF Endothelial cells, melanomas, FDC, activated T cells Adhesion
CD 147 Neurothelin, basoglin
IgSF Leukocytes, erythrocytes, platelets, endothelial cells Adhesion 19pl3.3
CD148 HPTP-eta, p260240-260 Granulocytes, monocytes, DC, T cells (upregulated after activation) Tyrosine phosphatase R Type III 1 IpI 1.2
CD150 SLAM (surface lymphocyte activation marker) 75-95 IgSF T subset (upregulated after activation), B cells, DC Costimulation, proliferation, Ig production
CD151 PETA-3 32 Endothelial cells, megakaryocytes, platelets, epithelial cells Cell adhesion
CD152 CTLA-4 33 IgSF Activated T cells, activated B cells CD80 and CD86 counter- receptor, negative regulation of T cell costimulation 2q33
CD153 CD30L 40 TNFSF Activated T cells, activated macrophages, neutrophils, B cells CD30 counter-receptor, T cell costimulation 9q33
CD154 CD40L, T-BAM, gp39, TRAP 32-39 TNFSF Activated T cells CD40 counter- receptor, costimulatory signal for B cells and DC Xq26.3-27.1
CD155 Poliovirus receptor (PVR) 80-90 IgSF Monocytes, macrophages, CD34+ thymocytes Polio virus receptor (PVR) 19ql3.1-13.2
CD156aADAM8 69
Neutrophils, monocytes Leukocyte extravasation 10q26.3
CD156b TACE/ADAM 17 100 Table 2 CD antigens
Broad Cleavage of membrane proteins (TNF, TGFa) to generate soluble forms 2p25
CD 157 BST-I 42-45
Granulocytes, monocytes, B cell progenitors, endothelial cells ADP-ribosyl-cyclic ADP- ribose hydrolase activities, pre-B cell growth promotion 4pl5
CD 158a p58.1 50-58 IgSF, KIR family NK subset, T subset Inhibition of NK cell cytolytic activity, MHC class-I specific NK-receptor 19
CD 158b p58.250-58 IgSF, KIR family NK subset, T subset Inhibition of NK cell cytolytic activity, MHC class-I specific NK-receptor 19
CD 159a NKG2A 43 NK cells, T subset Associates with CD94, NK cell receptor 12pl2.3-pl3.1
CD 160 BY55 27 IgSFNK subset, T subset Costimulation Iq42.3
CD 161 NKR-P IA 40 C-type lectin NK cells, T subset NK cell-mediated cytotoxicity 12pl2.3-pl3.1
CD 162 P-selectin glycoprotein ligand-1 (PSGL-I) 120 Mucin family Monocytes, granulocytes, most lymphocytes CD62P and CD62L counter-receptor, adhesion, leukocyte rolling 12q24
CD162R PEN-5 CHONK subset
CD163 130 Scavenger receptor superfamily Monocytes, macrophages
CD 164 MGC-24 80 Epithelial cells, monocytes, lymphocytes (low), bone marrow stromal cells, CD34+ erythroid progenitor cells Hematopoietic progenitor cell-stromal cell interaction 6q21
CD 165 AD2, gp3742 Lymphocyte subset, monocytes, immature thymocytes, platelets Adhesion, thymocytes-thymic epithelium interaction
CD 166 ALCAM 105 IgSF Neurons, activated T cells, monocytes, epithelial cells, fibroblasts CD6 counter-receptor, adhesion 3ql3.1-13.2
CD167a DDRl(discoidin domain R) 120 RTK family Epithelial cells, myoblasts A tyrosine kinase, adhesion to collagen 6p21.3
CD 168 RHAMM 84-88 Monocytes, T subset, thymocyte subset, intracellularly in breast cancer cells Adhesion, tumor migration and metastasis 5q33.2
CD 169 sialoadhesin, Siglec-1 185 IgSF, sialoadhesins Tissue macrophage subset Adhesion, cell-cell and cell-matrix interactions, binds to breast cancer cell CD227 and T Table 2 CD antigens
cell CD43 20pl3
CD170 Siglec-5, CD33-like2 140 IgSF, sialoadhesins Macrophage subset, neutrophils Adhesion 19ql3.3
CD171 Ll, cell adhesion molecule 200-210 IgSF CNS, PNS, glial cells, monocytes, T subset, B cells, DC, several human tumor cells Kidney morphogenesis, lymph node architecture, T cell costimulation, neurohistogeπesis, homotypic interaction, binds to CD9, CD24, CD56, CD 142, CD 166, integrins, X-linked neurological disorder CRASH Xq28
CD 172a SIRP alpha 110 Monocytes, T subset, stem cells Adhesion, complex with CD47 20pl3
CD 173 Blood group H type 2 CHO Erythrocytes, stem cell subset, platelets
CD 174 Lewis Y CHO Stem cells subset, epithelial cells
CD 175 Tn CHO Stem cells subset
CD 175s Sialyl-Tn CHO Erythroblasts
CD 176 Thomson-Friedrenreich antigen, TF CHO Stem cell subset
CD177 NBl 56-62 Neutrophil subset
CD178 Fas Ligand, CD95L, TNFSF638-42 TNFSF Activated T cells, testis Induction of apoptosis of CD95+ cells, immune privilege, soluble form in serum of Toxic epidermal necrolysis (TEN) patient Iq23
CD 179a V pre B 16-18 IgSF Pro- and Pre-B cells Early B cell differentiation, signaling, complex with IgM 22ql 1.2
CD 179b Lambda 5 22 IgSF Pro- and Pre-B cells Early B cell differentiation, signaling, complex with IgM 22ql 1.23
CDl 80 RP- 105 95-105 LRRF, TLR family B subset, monocytes, DC B cell recognition/signaling of LPS, associated with MD-I 5ql2
CDl 83 CXCR3 40 TM7SF(rhodopsin family) Activated T cells and NK cells, eosinophils, GM-CSF activated CD34+ hematopoietic progenitor cells IP-10 , Mig and I- TAC receptor, recruitment of T cells to inflammatory sites, enhancement of ThI response XqI 3
CD 184 CXCR4 45 TM7SF(rhodopsin family) T subset, B cells, DC, monocytes, endothelial cells SDF-I receptor, X4 HIV-I coreceptor 2q21 Table 2 CD antigens
CD195 CCR5 45 Monocytes, T subset MIP-Ia, MIP-Ib and RANTES receptor, R5 HIV- 1 coreceptor 3p21
GD197 CCR7 45 T subset, DC subset 6Ckine and MIP-2b receptor 17ql2-21.2
CD200 OX-245-50 Thymocytes, B cells, activated T cells, endothelial cells Inhibition of immune response 3ql2-13
CD201 EPC-R 50 Endothelial cell subset Activated protein C receptor 20ql 1.2
CD202b Tie2 (Tek) 150 RTK family Stem cells, endothelial cells Angiopoietin-1 receptor 9p21
CD203c NPP3/PDNP3, ENppl, PD-Ib 130-150 Type II transmembrane protein Basophils, mast cells, glioma cells, megakaryocytes Ectoenzyme, binding to and clearance of extracellular nucleotides 6q22
CD204 Macrophage scavenger-R 220 Macrophages Endocytosis of macromolecules 8p22
CD205 DEC-205 205 DC5 thymic epithelial cells Antigen uptake, inhibition of immune response 2q24
CD206 macrophage mannose-R 180
DC subset, macrophages, monocytes Phagocytosis and pinocytosis of marrnose containing molecules 10pl3
CD207 Langerin 40 Langerhans cells Eαdocytic receptor 2pl3
CD208 DC-LAMP 70-90 Activated DC, interdigitating DC 3q26.3-27
CD209 DC-SIGN 44 DC subset ICAM-3 receptor, HIV-I binding protein 19pl3
CDw210 IL-10-R 90-110 T cells, B cells, NK cells, monocytes, macrophages IL-IO receptor, signal transduction Hq
CD212 IL-12-R beta 1 100 Activated T cells and NK cells Binds IL-12 with high affinity, associates with. IL-12 receptor b2 19pl3.1
CD213al IL-13-R alphal 65
B cells, monocytes, fibroblasts, endothelial cells Binds IL- 13 with low affinity, associates with CD124 X
CD213a2 IL-13-R alpha2 65 B cells, monocytes Binds IL-13 with high affinity XqI 3.1- 28 Table 2 CD antigens
CDw217 IL-17-R 120 Broad IL-17 receptor 2q31
CD220 Insulin-R 140, 70 Broad Insulin receptor 19pl3.2-13.3
CD221 IGF-I R 140, 70 Broad Binds IGF with high affinity, signaling, cell proliferation and differentiation 15q25-26
CD222 IGF-II R, mannose-6 phosphate-R250 Type I transmembrane protein Broad, 90- 95% intracellular Adhesion, tumor growth regulation, a receptor for TGFb-LAP, plasminogen, proliferin, truncated form (22OkD) found in serum 6q26
CD223 LAG-3 70 IgSF Activated T cells and NK cells An MHC class Iligand 12pl3.32 CD224 GGT (gamma-glutamyl transferase) 27, 68 Leukocytes, stem cells 22qll.23 CD225 Leu-13 17 Broad 11
CD226 DNAM-I, PTA-I, TLiSAl 65 IgSF T cells, NK cells, monocytes, platelets, activated HUVEC Activation of cell-mediated cytotoxicity, soluble form (5OBGDa) found in normal serum 18q22.3
CD227 MUCl, epithelial membrane antigen (EMA) 300 Mucin family, type I transmembrane protein Epithelial cells, stem cell subset, FDC, monocytes, B subset, some myelomas Adhesion, signal transduction, binds to CD 169, CD54, and selectins Iq21
CD228 Melanotransferrin 80-95 Stem cells, melanomas 3q28-29 CD229 Ly-9 95, 110 IgSF T cells, B cells Adhesion lq21.3-22 CD230 Prion protein35 Broad 20pter-pl2
CD231 TALLA-I, Al 530-45 TM4SF T leukemias, neuroblastomas, normal brain neurons A marker foi T cell acute lymphoblastic leukemia (TALL) XqI 1
CD232 VESP-R 200 Broad 12
CD233 Band3, SLC4A1 90 Anion exchanger family Erythrocytes Anion exchanger pump, transport of C02, linking red cell membrane to cytoskeleton 17q2
CD234 Duffy (DARC) 35-45 Erythrocytes Duffy antigen chemokine receptor lq21-22 CD235a Glycophorin A 36 Erythrocytes 4q28.2-31.1 CD235ab Glycophorin A/B 20 Erythrocytes Table 2 CD antigens
CD235b Glycophorin B 20 Erythrocytes 4q28-31
CD236 Glycophorin C/D 32, 23 Erythrocytes, stem cell subset
CD236R Glycophorin C 32 Erythrocytes, stem cell subset 2ql4-21
CD238 KeIl 93 Erythrocytes, stem cell subset 7q33
CD239 B-CAM 78-85 Erythrocytes, stem cell subset 19ql3.2
CD240CE Rhesus 30CE (Rh30CE) 30-32 Erythrocytes Iρ36.2
CD240D Rhesus 3OD(Rh3OD) 30-32 TM12SF Erythrocytes Ip36.2
CD241 Rhesus 50 glycoprotein (RhAg) 50 TM12SF Erythrocytes Rh antigens complex with CD47, LW, and glycophorin B 6p21.1
CD242 ICAM-442 IgSF Erythrocytes Adhesion, Landsteiner- Wiener blood group 19ρl3.2
CD243 MDR-I, pl70, P-gp 180 Stem cells, small intestine, kidney Ion Pump 7q21.1
CD244 2B470 Type II transmembrane protein NK cells, T subset, monocytes, basophils NK cell activation receptor, CD48 counter-receptor
CD245 p220/240220-240 T subset
CD246 Anaplastic lymphoma kinase (ALK), Ki-I 80 Anaplastic T cell leuketnias, small intestine, testis, brain, not on normal lymphoid cells Brain development, exerts its effects on specific neurons, implicated in ALK lymphomas 2p23
CD247 TCR zeta 16 RTK family T cells, NK cells TCR complex subunit, coupling of antigen recognition to signaling, low expression results in impaired immune function Iq22-q23
Table 3
1: XP_493263
PREDICTED: similar to Mucin 1 precursor (MUC-D (Polymorphic epithelial mucin) (PEM) (PEMT) (Episialin) (Tumor-associated mucin) (Carcinoma- associated mucin) [Tumor-associated epithelial membrane antigen) (EMA) (H23AG) (Peanut-reactive urinary mucin) (PUM) (B... [Homo sapiens] gi J 51466822 | ref |XP_498263.11 [51466822]
2: NP_001018021
MUCl mucin isoform 4 precursor [Homo sapiens] gi|67189113 |ref |NP_001018021.l| [67189113]
3: NP_001018017
MUCl mucin isoform 3 precursor [Homo sapiens] gi I 67189069 I ref |NP_001018017.l| [67189069]
4: NP_001018016
MUCl mucin isoform 2 precursor [Homo sapiens] gi I 67189007 | ref |NP_001018016. lj [67189007]
5: NP_002447
MUCl mucin isoform 1 precursor [Homo sapiens] gi I 65301117 | ref |NP_002447.4 | [65301117]
6: XP_377538
PREDICTED: similar to Mucin 4 (Tracheobronchial mucin) [Homo sapiens] gi I 42661757 | ref | XP_377538.11 [42661757]
7: NP_002448 mucin 2 [Homo sapiens] gi 14505285 I ref |NP_002448.l| [4505285]
8 : NP_612154 mucin 4 isoform e [Homo sapiens] gi I 201439411 ref |NP_612154.11 [20143941]
9: NP_004523 mucin 4 isoform d [Homo sapiens] gi I 20143939 I ref |NP_004523.2 | [20143939]
10: NP_060876 mucin 4 isoform a [Homo sapiens] gi| 20143937 I ref |NP_060876.2 | [20143937]
11: NP_612156 mucin 4 isoform c [Homo sapiens] gi I 20143924 I ref |NP_612156. l| [20143924] Table 3
12: NP_612155 mucin 4 isoform b [Homo sapiens] gi I 20143920 I ref |NP_612155.11 [20143920]
13: NP_001004430 mucin 17 [Homo sapiens] gi| 52138723 |re£|NP_001004430.l| [52138723]
14: NP_689504 mucin 7, salivary [Homo sapiens] gi)22748SS5|ref |NP_689504.1| [227486S5]
IS: NP_07896S mucin 16 [Homo sapiens] gi|83367077|ref |NP_0789Sδ.2| [83367077]
16: NP_477521 small breast epithelial mucin [Homo sapiens] gi J 17158005 I ref | NP__477521.11 [17158005]
17: NP_612388
T-cell immunoglobulin and mucin domain containing 4 [Homo sapiens] gi 119923905 | ref | NP_612388.11 [19923905]
18: NP_004742 gluσosaminyl (N-acetyl) transferase 3, mucin type [Homo sapiens] gi I 4758422 | ref | NP_004742.11 [4758422]
19: NP_14,9038 mucin 13, epithelial transmembrane [Homo sapiens] gi I 51593090 I ref |NP_149038.2| [51593090]
20: NP_689886 mucin 20 [Homo sapiens] gi 1230973311 ref JNPJ-89386.l| [23097331]
21: NP_563625 mucin 15 [Homo sapiens] gi|40255098 | ref |NP_663625.2 j [40255098]
22: NP_116171
T cell immunoglobulin mucin 3 [Homo sapiens] gi I 49574534 | ref |UP_116171.3 | [49574534] Table 3
23: XP_495859
PREDICTED: similar to mucin 5 [Homo sapiens] gi| 51493056 I ref |XP_495859.l| [51493055]
24: XP_290540
PREDICTED: mucin 6, gastric [Homo sapiens] gi| 51493054 |ref|XP_290540.5| [51493054]
25: XP_379904
PREDICTED: mucin 12 [Homo sapiens] gi I 51492917 | ref | XP_379904.2 | [51492917]
26: XP_168585
PREDICTED: similar to mucin 11 [Homo sapiens] gi J 51492915 j ref | XP_168585.4 | [51492915]
27: XP_1S8578
PREDICTED: mucin 3B [Homo sapiens] gi|51492913 |ref |XP_168578.5 | [51492913]
28: XP_495921
PREDICTED: mucin 19 [Homo sapiens] gi I 51471037 J ref |XP_495921.11 [51471037]
29: XP_039877
PREDICTED: mucin 5, subtype B, tracheobronchial [Homo sapiens] gi I 51470790 | ref | XP_039877.91 [51470790]
30: XP_499351
PREDICTED: similar to transmembrane mucin 12 [Homo sapiens] gi I 51466453 | ref | XP_499351.11 [51466453]
31: XP_499350
PREDICTED: similar to mucin 11 [Homo sapiens] gi J 514564511 ref |XP_499350.11 [51466451]
32: XP_374501
PREDICTED: similar to intestinal mucin 3 [Homo sapiens] gi I 514664111 ref I XP_374501.2 I [514S6411]
33: XP_374502
PREDICTED: similar to intestinal mucin [Homo sapiens] gi I 41143068 I ref |XP_374502.l| [41148068]
34 t NP_000567 interleukin 1, beta proprotein [Homo sapiens] Table 3
gi 1 10835145 | ref |NP_000567.1 1 [10835145]
35 : NP_003174
ADAM metallopeptidase domain 17 preproprotein [Homo sapiens] gi I 73747889 | ref |NP_003174 .3 | [73747B 89]
36 : NP_002548 oviductal glycoprotein 1 precursor [Homo sapiens] gi | 58386720 | ref | NP_0Q2548 .3 | [5838S720]
37 : MP_001963 elastase 2 , neutrophil preproprotein [Homo sapiens] gi I 45035491 ref |NP_001963 .11 [4503549]
38 : NP_112555 mu-protoσadherin isoform 4 [Homo sapiens] gi 1 13929445 I ref |NP_112555 . l | [13929445]
39 : NP_112554 mu-protocadherin isoform 3 [Homo sapiens] gi 1 13929443 j ref | NP_112554 .11 [13929443]
40 : NP_060187 mu-protocadherin isoform 2 [Homo sapiens] gi 1 12313881 j ref |NP_060187.2 | [12313881]
41 : NP_068743 mu-protoσadherin isoform 1 [Homo sapiens] gi 1 123138791 ref |NP_068743 .1 j [12313879]
42 : KP_001006626 lung tyρe- I cell membrane-associated glycoprotein isoform d [Homo sapiens] gi | 54792062 | ref |NP_001006626. l | [54792052]
43 : NP_001006S25 lung type-I cell membrane-associated glycoprotein i soform c [Homo sapiens] gi I 54792060 I ref |NP_00100S625 .1 | [54792060]
44 : NP__938203 lung tyρe- I cell membrane-associated glycoprotein isoform b [Homo sapiens] gi J 54792058 I ref |NP_938203 .2 | [54792058]
45 : NP 006465 .Table 3 ■' • : ■ • '-' ' ■ •. ■ • ' ■ '• ' . '• ■.• '. ■. ' • • • ■' .
lung tyρe-I cell membrane -associated glycoprotein isoform a [Homo sapiens] ■ . , gi|54792056|ref|NP__005465.3| [54792056] .' ' . ;■ ..
46: NP_000483 . " cystic fibrosis transmembrane conductance regulator, ATP-binding cassette (sub-family C, member 7) [Homo sapiens] '. gi|6995996|ref |NP_000483.2| [6995995] .• ■ . •.'•
,47: NP_693S34 \ ■ • • ' ■ ' \ , ' • ' • egf-like module-containing mucin-like receptor 3 isoform b [Homo sapiens] • .' •'.' ' ' . •, • . '■ • ' ••' .. • gi|23397639|ref |NP_693β34.l| [23397639] . . . ' . . '■.. .
■48: NP_115960 • •. '. ■ . •.', ' '. . ' "■■ ' ■ ■' ' , .' -' •• ' ■'.'. ' .-' • ' v. .'.. .. ' egf-like module- containing muσin-like receptor. '3 isoform a. [Homo .sapiens] •'• •'• ■• • • ■ '• ': • ■•• •'• '.' '. , ■ ' •: . gi|l4211883|ref|NP_115960.l| [14211883] .. ;. ' ■• : ■..■ ..' • . : '
Table 4
I i NP_000079 alpha 1 type I collagen preproprotein [Homo sapiens] gi J 4502945 I ref I NP_OOO079.11 [4502945]
2 : XP_295195
PREDICTED : similar to alpha 2 type VI collagen isoform 2C2 precursor,- collagen VI , alρha-2 polypeptide ; human mRNA for collagen VI alpha-2 C- terminal globular domain [Homo sapiens] gi I 5147577S | ref | XP_295195.51 [51475776]
3 : NP_000080 alpha 2 type I collagen [Homo sapiens] gi I 48762934 | ref | NP__000080 .2 | [48762934]
4 : NP_1491S2 alpha 1 type II collagen isoform 2 , preproprotein [Homo sapiens] gi J 151494791 ref | NP_1491S2 .1 1 [15149479]
5 : NP_001835 alpha 1 type II collagen isoform 1 [Homo sapiens] gα 1 13435125 J ref | NP_001835.2 | [13435125]
6 : XP_496659
PREDICTED : similar to alpha 3 type VI collagen isoform 2 precursor; collagen VI , alpha-3 polypeptide [Homo sapiens] gi I 51492S22 | ref | XP_496659.1 1 [51492S22]
7 : NP_004360 alpha 3 type V3 collagen isoform 1 precursor [Homo sapiens] gi I 55743098 I ref | NP_004360.2 | [55743098]
8 : NP_47S508 alpha 3 type VI collagen isoform 5 precursor [Homo sapiens] gi I 55743106 | ref | ΪTP_476508.2 | [55743106]
9 : NP_47S507 alpha 3 type VI collagen isoform 4 precursor [Homo sapiens] gi I 55743104 | ref | NP__476507.2 | [55743104]
10 : NP_47650S alpha 3 type VI collagen isoform 3 precursor [Homo sapiens] gi I 55743102 J ref | NP_476506.2 | [55743102]
11 : NP_476505 alpha 3 type VI collagen isoform 2 precursor [Homo sapiens] gi 1 55743100 | ref | NP_476505 .2 | [55743100] Table 4
12 : NP_000085 alpha 1 type VII collagen precursor [Homo sapiens] gi I 4502961 ] ref | NP_000085 . l | [4502961]
13 : NP_001840 alpha 2 type VI collagen isoform 2C2 precursor [Homo sapiens] gi | 17402875 I ref | NP_001840 . 2 j [17402875]
14 : NPJDOl 83S alpha 1 type IV collagen preproprotein [Homo sapiens] gi I 76569851 ref |NP_001836.1 1 [7656985]
15 : NP_478055 alpha 2 type VI collagen isoform 2C2a precursor [Homo sapiens] gi 1 17402879 I ref | NP_478055. l | [17402879]
16 : NP_478054 alpha 2 type VI collagen isoform 2C2a precursor [Homo sapiens] gi 1 17402877 | ref | NP_478054 . 1 1 [17402877]
17 : NP_203700 alpha 5 type IV collagen isoform 3 , precursor [Homo sapiens] gi 1 15890088 I ref JNP_203700. 1 1 [15890088]
18 : NP_203699 alpha 5 type IV collagen isoform 2 , precursor [Homo sapiens] gi 1 15890086 I ref |NP_203699. 11 [15890085]
19 : NP_000486 alpha 5 type IV collagen isoform 1, precursor [Homo sapiens] gi 14502955 | ref |NP_000486.11 [4502955]
20 : NP_112734 alpha 3 type IV collagen isoform 5, precursor [Homo sapiens] gi 1 14165450 | ref | NP_112734. 11 [14155450]
21 : NP_112733 alpha 3 type IV collagen isoform 4, precursor [Homo sapiens] gi 1 14165448 | ref | NP_112733 . 1 1 [14155448]
22 : NP_112732 alpha 3 type IV collagen isoform 3 , precursor [Homo sapiens] gi J 14165446 | ref | NP_112732 .1 1 [14165446] Table 4
23 : NP_112731 alpha 3 type IV collagen isoform 3 , precursor [Homo sapiens] gi ) 14165444 | ref ) NP_112731.11 [14165444]
24 : NP_112730 alpha 3 type IV collagen isoform 2, precursor [Homo sapiens] gi J 14165442 | ref | NPJL12730. 11 [14165442]
25 : NP_000082 alpha 3 type IV collagen isoform 1, precursor [Homo sapiens] gi 110835113 I ref |NP_000082. l | [10835113]
26 : NP_001839 collagen, type VI, alpha 1 precursor [Homo sapiens] gi 115011913 J ref | NP_001839. l | [15011913]
27: NP_000083 alpha 4 type IV collagen precursor [Homo sapiens] gi J 629524991 ref | NP_00O083.2 | [62952499]
28 : NP_569712 alpha 1 type XVIII collagen isoform 2 precursor [Homo sapiens] gi 118765748 j ref |NP_569712.11 [18765748]
29 : NP_569711 alpha 1 type XVIII collagen isoform 3 precursor [Homo sapiens] gi J 18765746 | ref |NP_569711.11 [18765746]
30 : NP_085059 alpha 1 type XVIII collagen isoform 1 precursor [Homo sapiens] gi 1 133856201 ref |NP_085059. l | [13385620]
31 : NP_378667 type IV alpha 6 collagen isoform B precursor [Homo sapiens] gi j 16357503 j ref JNP_378667.l | [16357503]
32 : NP_001838 type IV alpha 6 collagen isoform A precursor [Homo sapiens] gi 1 163575011 ref | NP_001838 . l | [16357501]
33 : NP_001837 alpha 2 type IV collagen preproprotein [Homo sapiens] gi 1179862771 ref | NP_001837.11 [17986277]
34 : NP_542197 alpha 1 type XI collagen isoform C preproprotein [Homo sapiens] Table 4
gi 118375522 | ref JNP_542197.11 [18375522]
35 s NP_542196 alpha 1 type XI collagen isoforra B preproprσtein [Homo sapiens] gi J 18375520 | ref |NP_542196. l | [18375520]
36 : MP_001845 alpha 1 type XI collagen isoform A preproprσtein [Homo sapiens] gi J 18375518 | ref JNP_001845.2 | [18375518]
37 : NP_000484 collagen, type X, alpha 1 precursor [Homo sapiens] gi 118105032 I ref |NP_000484.2 | [18105032]
38 : NP_004361 alpha 1 type XII collagen long isoform precursor [Homo sapiens] gi 1 18201923 I ref | NP_0043βl .2 | [18201923]
39: NP_570134 alpha 1 type XVII collagen [Homo sapiens] gi J 18641356 | ref JNP_570134.11 [18641356]
40 : NP_000485 alpha 1 type XVII collagen [Homo sapiens] gi 118641354 | ref | NP_000485.21 [18641354]
41 : NP_001842 alpha 1 type IX collagen isoforra 1 precursor [Homo sapiens] gi J 734866661 ref |NP_001842.3 | [73486666]
42 : NP_001849 alpha 1 type XIX collagen precursor [Homo sapiens] gi j 477789211 ref | NP_001849.21 [47778921]
43 : NP_000384 alpha 2 type V collagen preproprotein [Homo sapiens] gi 145029591 ref | NP_O0O384.11 [4502959]
44 : NP_543005 alpha 1 type XIII collagen isoform 19 [Homo sapiens] gi J 22027609 J ref |NP_543005.2 | [22027609]
45 : NP_543004 alpha 1 type XIII collagen isoform 18 [Homo sapiens] gi J 22027607 | ref |NP_543004.2 | [22027607] Table 4
46 : NP_543003 alpha 1 type XIII collagen isoform 17 [Homo sapiens] gi I 220276051 ref j NP_543003 . 2 | [22027605]
47 : NP_543002 alpha 1 type XIII collagen isoform 15 [Homo sapiens] gi I 22027603 | ref | NP_543002 . 2 | [22027603]
48 : NP_543001 alpha l type XIII collagen isoform 15 [Homo sapiens] gi I 22027601 1 ref j NP_543001 . 2 | [22027601]
49 : NP_543000 alpha 1 type XIII collagen isoform 14 [Homo sapiens] gi I 22027599 j ref | NP_543000 .2 j [22027599]
50 : NP_542999 alpha 1 type XIII collagen isoform 13 [Homo sapiens] gi 1 220275971 ref | NP_542999 .2 | [22027597]
51 : NP_542998 alpha 1 type XIII collagen isoform 12 [Homo sapiens] gi I 22027595 1 ref | NP_542998 .2 | [22027595]
52 : NP_542997 alpha 1 type XIII collagen isoform 11 [Homo sapiens] gi 1 22027593 j ref | NP_542997 .2 | [22027593]
53 : NP_542996 alpha 1 type XIII collagen isoform 10 [Homo sapiens] gi ] 22027591 J ref | NP_54299S .2 | [22027591]
54 : NP_542995 alpha 1 type XIII collagen isoform 9 [Homo sapiens] gi I 220275891 ref | NP_542995. 2 | [22027589]
55 : NP_542994 alpha 1 type XIII collagen isoform 8 [Homo sapiens] gi I 22027587 J ref | NP_542994 . 2 | [22027587]
56 : NP_542993 alpha 1 type XIII collagen isoform 7 [Homo sapiens] gi I 22027583 I ref | NP_542993 .2 | [22027583 ]
57 : NP 542992 Table 4 • ' ■ - ■ ■ ;'■ • . ' ' ' . " • '. . • •'■
alpha 1 type XIII collagen isoform 6 [Homo sapiens] gi I 22027580 J ref|NP_542992.2 | [22027580] ' ' • ■ .
58: NP_542991 alpha 1 type XIII collagen isoform 5 [Homo sapiens] gi I 22027577 I ref |NP_542991.2 I [22027577]
59: NP_542990 ■ ■ - . ' ■ alpha 1 type XIII collagen isoform 4 [Homo sapiens] gi I 22027574 I ref |NP_542990.2 I [22027574]
60: NP_542989 . ' . v .• " . • . ' ■• • alpha 1 type XIII collagen isoform 3 [Homo sapiens] gi I 220275711 ref |NP_542989.2 | [22027571] ' ;
61: . NP_542988 . • ,- ' "'.V '. • '' '. . .• ' /" ■ / ..'. ' :' . alpha 1 type XIII collagen isoform 2 [Homo sapiens] gi I 22027568 I ref 1 NP_542988.2 I [22027568]
62: NP_005194 • " •; • ,.• ■. ' :.• .-.'' • '.'. '. "' ' ' alpha 1 type XIII collagen isoform 1 [Homo sapiens] gi I 22027565 J ref |NP_005194.3 J [22027565]
63: NP_511040 . • . . •• ■ '• • alpha 1 type IX collagen isoform 2 precursor [Homo sapiens] gi|73486664|ref |HP_511040.2| [73486664] • Z -.
64: NP_542376 . . ' / '■ ' '. ' : /'■ '' ■ ' '' ': '■ '■■ '■ : : ' •' "■ . ■ ' ; '.. •':. ■'/ alpha 1 type XII collagen short isoform precursor [Homo sapiens] gi|l820192l|refJNP_542376.l| [18201921] •. Z; . ^.; ■
65: • NP_000084 , ■ . ■. • .; =.• ■ ;■ . . ' . •■ alpha 1 type V collagen preproprotein [Homo sapiens] gi|l6554579|ref|NP_000084.2J [16554579] ■■ '■ ; ■.
66: , NP_001846 ■ } ■ ■ ■ '■"■ -. \"- ■ . ■ \ ' ' : ■ ' ' . alpha 1 type XV collagen precursor [Homo sapiens] gi 118641350 I ref |NP_001846.2 | [18641350]
67: NP_001843 . . " ■ . .• ., alpha 2 type IX collagen [Homo sapiens] gi 1113861611 ref |NP_001843 ,l| [11386161]
68: NP_056534 . : . collagen, type V, alpha 3 preproprotein [Homo sapiens] Table 4
gi I 7656989 | ref | NP_056534.1 1 [7656989]
69 : NP_110447 alpha 1 type XXI collagen precursor [Homo sapiens] gi 118780273 | ref | NP_110447.2 | [18780273]
70 : NPJ)01847 alpha 1 type XVI collagen precursor [Homo sapiens] gi 118641352 | ref | NP__001847.2 | [18641352]
71s NP_542412 collagen, type XI, alpha 2 isoform 2 preproprotein [Homo sapiens] gi 118201919 I ref|NP_542412.l| [18201919]
72 : NP_542411 collagen, type XI, alpha 2 isoform 1 preproprotein [Homo sapiens] gi 118201917 | ref |NP_542411.11 [18201917]
73 : NP_542410 collagen, type XI, alpha 2 isoform 3 preproprotein [Homo sapiens] gi 1 18201915 I ref JNP_542410. l | tl8201915]
74 : NP_001844 alpha 3 type IX collagen [Homo sapiens] gi J 17921995 | ref |NP_001844 .21 [17921995]
75 : NP__066933 collagen, type XIV, alpha 1 [Homo sapiens] gi 155743096 | ref |NP_066933.11 [55743096]
76 : NP_065084 alpha 1 type VIII collagen precursor [Homo sapiens] gi j 32895368 J ref |MP_065084.2 | [32895368]
77 : NP_690850 collagen, type XXIV, alpha 1 [Homo sapiens] gi I 23308589 J ref |NP_690850. l | [23308589]
78 : NP_001841 alpha 1 type VIII collagen precursor [Homo sapiens] gi | l7738302 | ref |NP_001841.2 | [17738302]
79 : NP_005193 collagen, type VIII, alpha 2 [Homo sapiens] gi 132964830 J ref |NP_005193 . l | [32964830] Table 4
80: NP_116277 collagen, type XXVII, alpha 1 [Homo sapiens] gi J 32140760 J ref JNP_116277.2 | [32140760]
81: NP_001032852 collagen, type XXVIII precursor [Homo sapiens] gi|83S49733 |ref |NP_001032852.l| [83649733]
82: NP_612464 collagen triple helix repeat containing 1 [Homo sapiens] gi 119923989 | ref |NP__6124S4.1 ) [19923989]
83: NP_940898
A-domain containing protein similar to matrilin and collagen [Homo sapiens] gi| 38348304 I ref |NP_940898.1 ) [38348304]
84: NPJ775736 collagen, type XXIII, alpha 1 [Homo sapiens] gi I 29725624 | ref | NPJ775736.2 | [29725624]
85: NP_690848 collagen, type XXII, alpha 1 [Homo sapiens] gi|40805823 |ref |NP_690848.11 [40805823]
86: NP_942014 collagen, type XXV, alpha 1 isoform 1 [Homo sapiens] gi 138570075 | ref |NP_942014.11 [38570075]
87: NP_115907 collagen, type XXV, alpha 1 isoform 2 [Homo sapiens] gi 138570073 I ref |NP_1159O7.2 | [38570073]
88: NP_112729 alpha 3 type IV collagen binding protein isoform 2 [Homo sapiens] gi 114165452 | ref |NP_112729.11 [14165452]
89: NP_005704 alpha 3 type IV collagen binding protein isoform 1 [Homo sapiens] gi 15031717 | ref | NP_005704.11 [5031717]
90: NP_597716 collagen and calcium binding EGF domains 1 [Homo sapiens] gi I 39930511 ) ref | NP_597716.11 [39930511] Table 4
91: NPJ)OlO 14442 prostate collagen triple helix [Homo sapiens] gi j 62234340 j ref | NP_001014442.2 [ [S2234340]
92 : XP_497385
PREDICTED: similar to cuticular collagen [Homo sapiens] gi I 51471486 J ref | XP_497385.1 | [51471486]
93 : XP_497872
PREDICTED: similar to Collagen alpha 3 (IX) chain precursor [Homo sapiens] gi J 514604711 ref | XP_497872.11 [51460471]
94: XP_067585
PREDICTED: similar to RIKEN cDMA E330O26BO2 [Homo sapiens] gi I 51492624 | ref | XP_067585.9 | [51492624]
95: NP_000081 procollagen, type III, alpha 1 [Homo sapiens] gi j 45029511 ref | NP_000081 .1 | [4502951]
96: NP_002194 integrin alpha 2 precursor [Homo sapiens] gi J 4504743 J ref |NP_O02194.11 [4504743]
97 : NPJ557447 glycoprotein VI (platelet) [Homo sapiens] gi J 37574620 | ref | NP_057447.3 | [37574620]
98 : NP_596867 integrin beta 1 isoform IA precursor [Homo sapiens] gi 1 19743823 j ref |NP_596867.11 [19743823]
99 : NP_391989 integrin beta 1 isoform lC-2 precursor [Homo sapiens] gi 1 197438211 ref I NP_391989.11 [19743821]
100 : NP_391988 integrin beta 1 isoform ID precursor [Homo sapiens] gi 119743819 j ref |NP_391988.l | [19743819]
101 : NP_391987 integrin beta 1 isoform IC-I precursor [Homo sapiens] gi 1197438171 ref | NP_391987.11 [19743817]
102 : NP 389647 Table 4 integrin beta 1 isoform IB precursor [Homo sapiens] gi J 19743815 | ref |NP_389647.11 [19743815]
103 : NP_002202 integrin beta 1 isoform IA precursor [Homo sapiens] gi | 19743813 | ref | NP_002202 .2 | [19743813]
104 : NP_000543 von Willebrand factor precursor [Homo sapiens] gi I 45079071 ref |NP_000543 . 11 [4507907]
105 : NP_997S47 fibronectin 1 isoform 1 preproprotein [Homo sapiens] gi I 471325571 ref |NP_997647.11 [47132557]
106 : NP_997S43 fibronectin 1 isoform 4 preproprotein [Homo sapiens] gi I 471325551 ref |NP_997643 . l | [47132555]
107 : NP_997641 fibronectin 1 isoform 5 preproprotein [Homo sapiens] gi I 47132553 I ref |NP_997641. l | [47132553]
108 : NP_997640 fibronectin 1 isoform 2 preproprotein [Homo sapiens] gi J 471325511 ref | MP_997640.11 [47132551]
109 : NP_997639 fibronectin 1 isoform 6 preproprotein [Homo sapiens] gi J 471325491 ref |NP_997639.11 [47132549]
110 : NP_473375 fibronectin 1 isoform 7 preproprotein [Homo sapiens] gi J 47132547 | ref |NP_473375.2 | [47132547]
111 : NP_002017 fibronectin 1 isoform 3 preproprotein [Homo sapiens] gi 1 16933542 I ref | NP_0O2O17. l | [16933542]
112 : NP_004521 matrix metalloproteinase 2 preproprotein [Homo sapiens] gi J 11342666 | ref ] NP_004521.11 [11342666]
113 : NP_852478 integrin, alpha 1 precursor [Homo sapiens] gi J 31657142 | ref | NP_852478.11 [31657142] Table 4
114: NP_598014 decorin isoform e precursor [Homo sapiens] gi 1197438541 ref |NP_598014.1 | [19743854]
115: NP_598013 decorin isoform d precursor [Homo sapiens] gi) 19743852)ref |NP_598013.l| [19743852]
116: NP_598012 decorin isoform c precursor [Homo sapiens] gi| 197438S0|ref JNP_598012.1 j [19743850]
117: NP_598011 decorin isoform b precursor [Homo sapiens] gi|l9743848|ref |NP_598011.l| [19743848]
118: NP_598010 decorin isoform a preproprotein [Homo sapiens] gi| 19743846|ref |NP_598010.l| [19743846]
119: NP_001911 decorin isoform a preproprotein [Homo sapiens] gi I 45032711 ref | NP_001911.11 [4503271]
120: NP_000651 transforming growth factor, beta 1 [Homo sapiens] gi I 63025222 J ref |NP_000651.3 J [63025222]
121: NP_002412 matrix metalloproteinase 1 preproprotein [Homo sapiens] gi I 4505215 I ref |NP_O02412.11 [4505215]
122: NP_001226 serine (or cysteine) proteinase inhibitor, clade H, member 1 precursor [Homo sapiens] gi J 324547411 ref JNP_0O1226.21 [32454741]
123 : XP_497913
PREDICTED: similar to RIKEN cDNA E330026B02 [Homo sapiens] gi I 51464016 I ref |XP_497913.l| [51464016]
124: NP_054700 discoidin receptor tyrosine kinase isoform c [Homo sapiens] gi) 83977452 | ref |NP_0547OO.2 | [83977452] Table 4
125: NP_054699 discoidin receptor tyrosine kinase isoform a [Homo sapiens] gi I 83977450 I ref |NP_054699.2 | [83977450]
126: NP_001945 discoidin receptor tyrosine kinase isoform b [Homo sapiens] gi I 38327632 | ref )NP_OO1945.3 | [38327632]
127: NP_001001548
CD36 antigen [Homo sapiens] gi I 48375180 j ref |NP_001001548.11 [48375180]
128: NP_000063
CD36 antigen [Homo sapiens] gi I 48375178 I ref |NP_000063.2 | [48375178]
129: NP_001001547
CD36 antigen [Homo sapiens] gi J 483751761 ref |NP_001001547.11 [48375176]
130: NP_000293 lysyl hydroxylase precursor [Homo sapiens] gi I 32307144 | ref |NP__OOO293.2 | [32307144]
131: NP_002549 purinergiσ receptor P2X1 [Homo sapiens] gi I 45055451 ref | NP_O02549.11 [4505545]
132: NP_003109 secreted protein, acidic, cysteine- rich (osteonectin) [Homo sapiens] gi I 45071711 ref |NP_O03109.11 [4507171]
133: NP_002308 lysyl oxidase preproprotein [Homo sapiens] gi I 20149540 | ref |NP_0023O8.2 | [20149540]
134: NP_001191 bone morphogenetic protein 2 preproprotein [Homo sapiens] gi I 4557369 | ref | NP_001191.11 [4557369]
135: NP_057075 complement component 1, q subcomponent, alpha polypeptide precursor [Homo sapiens] gi I 7705753 | ref | NP_057075.11 [7705753] Table 4
136: NP_001017962 prolyl 4 -hydroxylase, alpha I subunit isoform 2 precursor [Homo sapiens] gi| 63252888 I ref|NP_001017962.l| [63252888]
137: NP_000908 prolyl 4 -hydroxylase, alpha I subunit isoform 1 precursor CHomσ sapiens] gi| 63252886|ref |NP_000908.2| [63252886]
138: NP_000926 procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 isoforra b precursor [Homo sapiens] gi I 62739166 I ref |NP_000926.2 | [62739166]
139: NP_001014796 discoidin domain receptor family, member 2 precursor [Homo sapiens] gi|62420886|ref |NP_001014796.l| [62420886]
140: NP_006173 discoidin domain receptor family, member 2 precursor [Homo sapiens] gi I 62420884 | ref | NP_006173.2 | [62420884]
141: NP_891988 procollagen- lysine, 2-oxoglutarate 5-dioxygenase 2 isoform a precursor [Homo sapiens] gi I 33636742 I ref |NP_891988.11 [33636742]
142: NP_006030 mannose receptor, C type 2 [Homo sapiens] gi I 5174485 | ref |NP_006030.11 [5174485]
143 : NP_004985 matrix metalloproteinase 9 preproprotein [Homo sapiens] gi I 74272287 | ref | NP_004985.2 | [74272287]
144: NP_0O5598
PTK2 protein tyrosine kinase 2 isoform b [Homo sapiens] gi I 27886593 | ref |NP_005598.3 | [27886593]
145: NP_722560
PTK2 protein tyrosine kinase 2 isoform a [Homo sapiens] gi|24476013 |ref |NP__722560.l| [24476013]
146: NP_000909 prolyl 4 -hydroxylase, beta subunit [Homo sapiens] gi I 20070125 | ref | NP_000909.2 | [20070125]

Claims

Claims
1. A polypeptide conjugate comprising a first part wherein said first part is a ligand that binds a polypeptide expressed by a cell and a second part wherein said second part is a polypeptide encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of i) a nucleic acid molecule consisting of at least one nucleic acid sequence as represented in Figure IA-T; ii) a nucleic acid molecule that hybridises to the nucleic acid molecule in Figure IA-T and that encodes a polypeptide with the activity associated with HLA-G; iii) a nucleic acid molecule comprising a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequences as defined in (i) and (ii) above.
2. A conjugate according to claim 1 wherein said first part is an antibody or active binding part thereof.
3. A conjugate according to claim 2 wherein said antibody is a monoclonal antibody.
4. A conjugate according to claim 2 or 3 wherein said binding part is a Fab fragment.
5. A conjugate according to claim 4 wherein said Fab fragment is selected from the group consisting of: F(ab')2, Fab, Fv and Fd fragments, CDR3 regions, single chain antibody fragment or a domain antibody fragment.
6. A conjugate according to any of claims 2-5 wherein said antibody or binding part is a chimeric or humanised antibody.
7. A conjugate according to any of claims 1-6 wherein said ligand comprises the binding domain of a ligand that binds a cell surface receptor polypeptide.
8. A conjugate according to any of claims 1-6 wherein said ligand comprises a peptide that binds a cell surface receptor polypeptide.
9. A conjugate according to any of claims 1-6 wherein said ligand is an aptatner that binds a cell surface receptor polypeptide.
10. A conjugate according to any of claims 1-9 wherein said polypeptide is expressed by an embryonic stem cell.
11. A conjugate according to any of claims 1-9 wherein said polypeptide is expressed by a stem cell.
12. A conjugate according to any of claims 1-11 wherein said polypeptide is expressed by a differentiated cell or a cell undergoing differentiation.
13. A conjugate according to any of claims 1-12 wherein said conjugate comprises a polypeptide encoded by a nucleic acid molecule as represented in Figure 1.
14. A conjugate according to any of claims 1-12 wherein said conjugate is an in-frame translational fusion between said first and second parts wherein said first and second parts are linked either directly or indirectly to each other.
15. A conjugate according to any of claims 1-8 or 10-14 wherein said in-frame translational fusion is a direct fusion between first and second parts.
16. A conjugate according to any of claims 1 -15 for use as a pharmaceutical.
17. A composition comprising a conjugate according to any of claims 1-15.
18. A composition according to claim 17 wherein said composition is a pharmaceutical composition.
19. A composition according to claim 18 wherein said composition further comprises an adjuvant or carrier.
20. A composition according to claim 18 or 19 wherein adjuvant is an immunosuppressant.
21. A composition comprising a conjugate according to any of claims 1-15 and an embryonic stem cell.
22. A composition according to claim 21 wherein said embryonic stem cell is a primate embryonic stem cell.
23. A composition according to claim 22 wherein said embryonic stem cell is human and is not a totipotent cell.
24. A composition according to claim 21 wherein said embryonic stem cell is a non- primate embryonic stem cell.
25. A composition according to claim 24 wherein said non-primate embryonic stem cell is a murine embryonic stem cell.
26. A composition according to any of claims 21-25 wherein said embryonic stem cell is transfected with a nucleic acid molecule that encodes a polypeptide to which said ligand binds.
27. A composition according to claim 21 wherein said embryonic stem cell is a teratocarcinoma stem cell.
28. A method to treat an inflammatory disease or condition comprising administering an effective amount of a conjugate or a composition according to any of claims 1-26 to a subject in need of treatment.
29. A method to treat an autoimmune disease or condition comprising administering an effective amount of a conjugate or a composition according to any of claims 1-26 to a subj ect in need of treatment.
30. A method according to claim 29 wherein said autoimmune disease is selected from the group consisting of: multiple sclerosis, type 1 diabetes, autoimmune thyroid disease, autoimmune hepatitis, rheumatoid arthritis, autoimmune colitis, Crohns disease, celiac disease, autoimmune nephritis, autoimmune neuropathy, encephalopathy, fibrosing alveolitis.
31. A method to treat organ/tissue rejection in a recipient subject comprising administering an effective amount of a conjugate or composition according to any of claims 1-26 to a subject in need of treatment.
32. The use of an effective amount of a conjugate or a composition according to any of claims 1 -26 to treat an inflammatory disease or condition.
33. The use of an effective amount of a conjugate or a composition according to any of claims 1-26 for the treatment of an autoimmune disease.
34. Use according to claim 33 wherein said autoimmune disease is selected from the group consisting of: multiple sclerosis, type 1 diabetes, autoimmune thyroid disease, autoimmune hepatitis, rheumatoid arthritis, autoimmune colitis, Crohns disease, celiac disease, autoimmune nephritis, autoimmune neuropathy, encephalopathy, fibrosing alveolitis.
35. The use of an effective amount of a conjugate or composition according to any of claims 1-26 for the treatment of organ/tissue rejection in a recipient subject.
36. A polypeptide conjugate according to claim 1 wherein the first part that binds a polypeptide expressed by a cell and is encoded by a nucleic acid molecule selected from the group consisting of: i) a nucleic acid molecule consisting of a nucleic acid sequence as identified by the accession number in Table 1; ii) a nucleic acid molecule that hybridises to the nucleic acid molecule in table 1 and that encodes a polypeptide with the activity associated with a RAGE polypeptide; iii) a nucleic acid molecule comprising a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequences a ass d deeffiinneedd i inn a anndd f (ini)} a ahbnovvee
37. A polypeptide conjugate according to claim 1 wherein the first part is a ligand that binds a CD antigen polypeptide as represented in Table 2
38. A polypeptide conjugate according to claim 1, wherein said first part is a ligand that binds a polypeptide expressed by a cell and is encoded by a nucleic acid molecule selected from the group consisting of: i) a nucleic acid molecule consisting of a nucleic acid sequence as identified by the accession number in Table 3; ii) a nucleic acid molecule that hybridises to the nucleic acid molecule in table 3 and that encodes a polypeptide with the activity associated with a mucin polypeptide; iii) a nucleic acid molecule comprising a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequences as defined in (i) and (ii) above.
39. A polypeptide conjugate according to claim 1, wherein the first part is a ligand that binds a polypeptide expressed by a cell and is encoded by a nucleic acid molecule selected from the group consisting of: i) a nucleic acid molecule consisting of a nucleic acid sequence as identified by the accession number in Table 4; ii) a nucleic acid molecule that hybridises to the nucleic acid molecule in table 4 and that encodes a polypeptide with the activity associated with a collagen polypeptide; iii) a nucleic acid molecule comprising a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequences as defined in (i) and (ii) above.
40. A polypeptide conjugate according to claim 1, wherein said first part is a ligand that binds a polypeptide expressed by a cell and is encoded by a nucleic acid molecule selected from the group consisting of: i) a nucleic acid molecule consisting of a nucleic acid sequence as identified by the accession number in Table 5; ii) a nucleic acid molecule that hybridises to the nucleic acid molecule in table 5 and that encodes a polypeptide with the activity associated with a cadhedrin polypeptide; iii) a nucleic acid molecule comprising a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequences as defined in (i) and (ii) above.
41. A conjugate wherein said conjugate is a multimeric complex of monomer conjugate subunits according to any of claims 1-16 or 36-40.
42. A conjugate according to claim 41 wherein said complex comprises at least two monomer subunits.
43. A conjugate according to claim 41 wherein said complex consists of two monomer subunits.
44. A conjugate according to any of claims 41-43 wherein said complex comprises identical monomer subunits.
45. A conjugate according to any of claims 41-43 wherein said complex comprises dissimilar monomer subunits.
46. A conjugate according to claim 41 wherein said complex consists of two monomer subunits forming a homodimer.
47. A conjugate according to claim 41 wherein said complex consists of two dissimilar monomer subunits forming a heterodimer.
48. A conjugate according to any of claims 41-47 wherein said complex is an in-frame translational fusion protein comprising monomer subunits.
49. A composition comprising a complex according to any of claims 41-48.
50. A composition according to claim 49 wherein said composition is a pharmaceutical composition.
51. A method to treat an inflammatory disease or condition comprising administering an effective amount of a complex or a composition comprising a complex according to any of claims 41-50 to a subject in need of treatment.
52. A method to treat an autoimmune disease or condition comprising administering an effective amount of a complex or a composition comprising a complex according to any of claims 41-50 to a subject in need of treatment.
53. A method according to claim 52 wherein said autoimmune disease is selected from the group consisting of: multiple sclerosis, type 1 diabetes, autoimmune thyroid disease, autoimmune hepatitis, rheumatoid arthritis, autoimmune colitis, Crohns disease, celiac disease, autoimmune nephritis, autoimmune neuropathy, encephalopathy, fibrosing alveolitis.
54. A method to treat organ/tissue rejection in a recipient subject comprising administering an effective amount of a complex or a composition comprising a complex according to any of claims 41-50 to a subject in need of treatment.
55. The use of an effective amount of a complex or a composition comprising a complex according to any of claims 41-50 to treat an inflammatory disease or condition.
56. The use of an effective amount of a complex or a composition comprising a complex according to any of claims 41-50 for the treatment of an autoimmune disease.
57. Use according to claim 56 wherein said autoimmune disease is selected from the group consisting of: multiple sclerosis, type 1 diabetes, autoimmune thyroid disease, autoimmune hepatitis, rheumatoid arthritis, autoimmune colitis, Crohns disease, celiac disease, autoimmune nephritis, autoimmune neuropathy, encephalopathy, fibrosing alveolitis.
58. A stem cell wherein said cell is transfected with a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i) a nucleic acid molecule consisting of a nucleic acid sequence as represented in Figure IA-T ii) a nucleic acid molecule that hybridises under stringent hybridization conditions to the nucleic acid molecule in Figure IA-T and that encodes a polypeptide with the activity associated with HLA-G; iii) a nucleic acid molecule comprising a nucleic acid sequence that is degenerate as a result of the genetic code to the nucleic acid sequences as defined in (i) and (ii) above.
59. A stem cell according to claim 58 wherein said stem cell comprises the nucleic acid sequence as represented in Figure IA-T.
60. A stem cell according to claim 58 wherein said nucleic acid molecule consists of the nucleic acid sequence as represented in Figure IA-T.
61. A stem cell according to any of claims 58-60 wherein said nucleic acid molecule is operably linked to a nucleic acid molecule comprising a transcription promoter sequence to form a transcription cassette.
62. A stem cell according to claim 61 wherein said promoter sequence is a constitutive promoter.
63. A stem cell according to claim 61 wherein said promoter sequence is a cell specific promoter sequence.
64. A stem cell according to claim 63 wherein said promoter sequence is a developmentally regulated promoter sequence.
65. A stem cell according to any of claims 61-64 wherein said nucleic acid molecule further comprises a repressor sequence.
66. A stem cell according to any of claims 61-65 wherein said nucleic acid molecule comprising said promoter sequence is part of a vector.
67. A stem cell according to claim 66 wherein said vector is a viral based vector.
68. A stem cell according to claim 67 wherein said viral vector is selected from the group consisting of: adenovirus; retrovirus; adeno-associated virus; herpesvirus; lentivirus; vaccinia virus; baculovirus.
69. A stem cell according to claim 68 wherein said viral based vector is an adenovirus.
70. A stem cell according to claim 68 wherein said viral based vector is a lentivirus.
71. A stem cell according to claim 66 wherein said vector is a plasmid or phagemid.
72. A stem cell according to any of claim 58-71 wherein said cell is transfected with a further nucleic acid molecule.
73. A stem cell according to claim 72 wherein said further nucleic acid molecule encodes a therapeutic polypeptide.
74. A stem cell according to any of claims 66-73 wherein said vector is replication competent.
75. A stem cell according to claim 74 wherein said vector is conditionally replication competent.
76. A stem cell according to any of claims 66-73 wherein said vector is a replication incompetent vector.
77. A stem cell according to any of claims 58-76 wherein said stem cell is a multipotent stem cell.
78. A stem cell according to claim 77 wherein said multipotent stem cell is selected from the group consisting of: haemopoietic stem cell; neural stem cell; bone stem cell; muscle stem cell; mesenchymal stem cell; epithelial stem cell; endodermal stem cell.
79. A stem cell according to any of claims 58-76 wherein said stem cell is a primate embryonic stem cell.
80. A stem cell according to claim 79 wherein said embryonic stem cell is a human embryonic stem cell.
81. A stem cell according to any of claims 58-76 wherein said stem cell is a non- primate embryonic stem cell.
82. A stem cell according to claim 81 wherein said stem cell is a murine stem cell.
83. A stem cell according to claim 81 wherein said embryonic stem cell is a teratocarcinoma stem cell.
84. A stem cell according to any of claims 58-76 or 80 wherein said embryonic stem cell is not a totipotent cell.
85. A spheroid body comprising an embryonic stem cell according to any of claims 79-84.
86. A spheroid body according to claim 85 wherein said body further comprises a collagen based cell support.
87. A cell derived from an embryonic stem cell or spheroid body according to any of claims 58-86.
88. An in vitro cell culture comprising an embryonic stem cell or an embryoid body according to any of claims 58-86.
89. A therapeutic vehicle for use in tissue engineering comprising a cell or spheroid body according to any of claims 58-86.
90. A vehicle according to claim 89 wherein said vehicle is selected from the group consisting of: prothesis; implant; matrix; stent; biodegradable matrix; or hydrogel.
91. A composition comprising a cell according to any of claims 58-84.
92. A composition for use in therapeutic tissue engineering comprising a cell according to any of claims 58-84.
93. A method to treat a condition that would benefit from tissue engineering comprising administering a cell according to any of claims 58-84 to a subject in need of treatment.
94. A method to treat a condition that would benefit from tissue engineering comprising administering a therapeutic vehicle according to claim 89 or 90 to a subject in need of treatment.
95. The use of a cell according to any of claims 58-84 in the manufacture of a medicament for use in tissue engineering.
96. A non-human transgenic mammal wherein said mammal comprises a cell derived from a cell according to any of claims 58-84.
PCT/GB2007/000448 2006-02-10 2007-02-08 Polypeptide conjugate comprising hla-g and uses thereof, stem cells transfected with hla-g WO2007091078A2 (en)

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