EP1425421A2 - Polymorphismes de pd-1 - Google Patents
Polymorphismes de pd-1Info
- Publication number
- EP1425421A2 EP1425421A2 EP02797957A EP02797957A EP1425421A2 EP 1425421 A2 EP1425421 A2 EP 1425421A2 EP 02797957 A EP02797957 A EP 02797957A EP 02797957 A EP02797957 A EP 02797957A EP 1425421 A2 EP1425421 A2 EP 1425421A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- gene
- nucleic acid
- loss
- seq
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
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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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to PD-1 polymorphisms. More particularly the present invention relates to nucleic acids encoding PD-1 polymorphs, and their use for the diagnosis and treatment of diseases and conditions associated with autoimmune disorders.
- autoimmune disorders such as SLE, myasthenia gravis, multiple sclerosis, Type 1 diabetes, rheumatoid arthritis, Sj ⁇ grens syndrome, atopy and allergy are major health risks through the industrialised and developing world.
- An autoimmune disorder is a condition in which the body creates antibodies against its own tissues.
- SLE Systemic lupus erythematosus
- a person with SLE produces antibodies against many of their own tissues. This autoimmune reaction can damage many parts of the body for example the brain and nervous system, digestive system, eyes, heart, joints and muscles, kidney, lung and skin. SLE can manifests as a single symptom or many disparate symptoms.
- SLE autoimmunogenic disorder
- SLE tends to run in families and tends to be hereditary.
- Research suggests that autoimmune disorders may be triggered by a transfer of cells between the foetus and the mother during pregnancy.
- women with scleroderma an autoimmune disorder involving the skin, it was shown that these women have more foetal cells in their blood decades after a pregnancy than women who don't have scleroderma. While further research is needed to substantiate these findings, the study does offer an expJanation for the much higher incidence of autoimmune disorders in women especially mothers or women who have been pregnant, than in men.
- Certain medications are also known to cause systemic lupus erythematosus. These include procainamide, hydralazine, isoniazid, and chlorpromazine. Events which may trigger the disease include infection, stress, exposure to toxins, and sunlight.
- SLE Women account for 80% to 90% of cases of SLE. It is more common in black women than in white women. SLE is also more common in Asian, Hispanic, and Native American women. Most cases of SLE cannot be prevented.
- SLE can be fatal, often as a result of kidney failure, infections, or heart attack.
- a number of medications are used to treat SLE, for example, the following:
- antimalarial medications such as quinacrine and hydroxychloroquine. These are used to treat skin problems and arthritis,
- corticosteroids such as prednisone and methylprednisolone.
- nonsteroidal anti-inflammatory drugs such as ibuprofen and naproxen
- these medications reduce fever and treat pain
- kidney dialysis or a kidney transplant Individuals with end-stage kidney disease may benefit from kidney dialysis or a kidney transplant.
- the medications used to treat lupus have significant side effects. Unfortunately, some of these side effects can mimic the symptoms of the disease itself.
- the human PD-1 gene has been mapped to 2q37.3 (references 11 and 12). This gene codes for a membrane molecule containing a tyrosine-based inhibitory motif and is of importance in T-cell development and tolerance (reference 13).
- the cDNA encoding PD-1 has been disclosed in Patents US 5,629,204 and US 5,698,520. PD-1 is expressed in humans during B and T cell development and is induced by lymphocyte activation. Cross-linking of mouse PD-1 with the ligand PD-1L1 and PD-1 L2 induce inhibition of T-cell activation (references 15 and 16) and mice made deficient for PD-1 develop autoantibodies.
- PD-1 in humans is a factor in the development of autoimmune disease and conditions such as SLE.
- polymorphs of PD-1 are factors in the development of autoimmune diseases, especially human autoimmune diseases such as SLE.
- the inventors have found that the prevalence or susceptibility of an individual or population of individuals to autoimmune diseases such as SLE is in part determined by which of the PD-1 nucleic acid polymorph sequences an individual or population of individuals possess.
- the present invention is derived from the discovery of the genomic structure of the human PD-1 gene (NCBI REF 986034, BanklT 392218 and GeneBank AF363458, unpublished) followed by the identification and sequencing of unexpected polymorphic regions within the gene, which are surprisingly associated with specific diseases, disorders or conditions, including autoimmune disorders such as myasthenia gravis, multiple sclerosis, Type 1 diabetes, rheumatoid arthritis, Sj ⁇ grens syndrome , atopy, allergy, systemic lupus erythematosus and diseases associated with SLE which include fatigue, fever, loss of appetite, nausea, weight loss, hives, loss of scalp hair, red "butterfly rash” and raised rash, sensitivity to sun, ulcers in mouth, nose; or vagina, arthritis, joint pain, loss of blood supply to bone, pain, infections within joints, decrease in kidney function including, blood, aberrant amounts of protein or white blood cells in urine, intracerebral haemorrhage, headaches, loss of coordination
- the human PD-1 gene contains a 5' promoter region, 5 coding exons, 4 non- coding introns and a 3'UTR region. This gene unexpectedly contains at least nine polymorphic regions. The structure of the gene and the position of the 5'promoter region, exons, introns, 3'UTR and polymorphic regions are shown in figure 1 and in SEQ ID N°1.
- the invention provides isolated or purified nucleic acids comprising an intronic sequence from a PD-1 gene that comprises the 5' promoter region, regions of coding nucleic acids, intronic regions and 3' UTR region of nucleic acid.
- the PD-1 gene is a human gene.
- the nucleic acid of the invention has a nucleotide sequence set forth in figure 1 and SEQ ID N°1, complements thereof, or homologues thereof.
- the sequence of the nucleic acid is capable of hybridising under appropriate stringency to a nucleic acid having a nucleotide sequence set forth in any one of the nucleic acid sequences of SEQ ID N°s1 to 12 or complements thereof.
- the nucleic acids of the invention have at least 80% sequence identity with any one of the nucleic acid sequences of SEQ ID N°s1 to 12.
- polymorphic regions have been identified in the human PD-1 gene by analysing the DNA of a specific population of individuals.
- One polymorphism found in the population is a change from a guanine to adenine at position 126, as shown in figure 1 and SEQ ID N°s1 , 6 or 15 and figure 1 and SEQ ID N°s1 , 6 or 16.
- This polymorphism is located in the 5" promotor region of the PD-1 gene, and results in an inhibition of binding of the Ikaros transcription factor to bind to its binding site when a guanine is replaced by an adenine.
- a second polymorphism, located intron 1 is a change from cytosine to thymine at position 6371 , as shown in figure 1 and SEQ ID N°1 or SEQ ID N°2.
- a third polymorphism located in intron 2 is a change of guanine to adenine at position 7101, as shown in figure 1 and SEQ ID N°s1, 3, 19 or 20.
- a fourth, fifth and six polymorphism are located in intron 4 at nucleotide positions 7809, 7872 and 8162, respectively.
- the substitution at position 7809 is of guanine with adenine, as shown in figure 1 and SEQ ID N°1 , 5 or SEQ ID N° 26 to 29.
- the substitution at position 7872 is of cytosine with thymine, as shown in figure 1 and SEQ ID N°1 , 5 or SEQ ID N°s26 to 29.
- the substitution at position 8162 is of guanine with adenine, as shown in figure 1 and SEQ ID N°1 and 5.
- This polymorphism at position 7809 in the PD-1 gene is an AML-1 transcription factor binding site, and results in an inhibition of binding of the AML- 1 transcription factor to bind to its binding site when a guanine is replaced by an adenine.
- a seventh polymorphism is located in exon 5 at position 8288 as shown in figure 1, and figure 19, SEQ ID N°1 and SEQ ID N°12. This polymorphism is a change from cytosine to thymine.
- An eighth polymorphism is located in exon 5 at position 8448 as shown in figure 1, SEQ ID N°1, SEQ ID N°12, SEQ ID N°30 or SEQ ID N°31. This polymorphism is a change from cytosine to thymine.
- a ninth polymorphism is to be found 3' to the PD-1 stop codon at position 9400, as shown in figure 1 and SEQ ID N°s1 , 7, 33 or 34. This polymorphism is a change from guanine to adenine.
- the invention provides isolated or purified expression products or fragments thereof of the PD-1 gene as shown in SEQ ID N° 1.
- the polypeptides of the invention have at least 90% sequence identity with any one of the expression products of SEQ ID N°1, or fragments thereof.
- the polypeptides of the invention have at least 90% sequence identity with any one of the peptides encoded by SEQ ID N°s 35 to 38, or fragments thereof, as shown in figure 21.
- the nucleic acids of the invention can be used, in prognostic and/or diagnostic methods.
- the nucleic acids of the invention can be used as probes or primers to determine whether a subject has or is at risk of developing a disease or disorder associated with a specific allelic variant of a PD-1 polymorphism, for example, a disease or disorder associated with an aberrant PD-1 activity.
- nucleic acids of the invention can be used in the treatment of diseases associated with aberrant PD-1 function.
- the expression products of nucleic acids of the invention can be used, in prognostic and/or diagnostic methods.
- the expression products of the invention can be used as probes to determine whether a subject has or is at risk of developing a disease or disorder associated with a specific allelic variant of a PD-1 polymorphism, for example, a disease or disorder associated with an aberrant PD-1 activity.
- the expression products of nucleic acids of the invention can be used in the treatment of diseases associated with aberrant PD-1 function.
- Antibody probes that specifically bind to polymorphs of PD-1 peptide or fragments thereof are also part of the invention.
- Preferred polymorphs of PD-1 to which antibody probes are raised have at least 90% sequence identity any one of the peptides encoded in SEQ ID N°s35 to 38, or fragments thereof, as shown in figure 21.
- the invention further describes vectors which encode the claimed nucleic acids; host cells transfected with said vectors whether prokaryotic or eukaryotic; and transgenic non-human animals that contain a heterologous form of a functional or non-functional PD-1 allele described herein.
- a transgenic animal can serve as an animal model for studying, for example, the effect of specific allelic variations, including mutations of a PD-1 gene, especially a human PD-1 gene or for use in drug screening or recombinant protein production.
- the invention further provides methods for determining the molecular structure of at least a portion of a PD-1 gene.
- the method comprises contacting a sample nucleic acid comprising a PD-1 gene sequence with a probe or primer having a sequence which is complementary to a PD-1 gene sequence and comparing the molecular structure of the sample nucleic acid with the molecular structure of a control (known) PD-1 gene (for example, a PD-1 gene from a human not afflicted with a condition or a disease associated with an aberrant PD-1 activity).
- a control (known) PD-1 gene for example, a PD-1 gene from a human not afflicted with a condition or a disease associated with an aberrant PD-1 activity.
- the method of the invention can be used for example in determining the molecular structure of at least a portion of an exon, an intron, a promoter, or a 3'UTR.
- the method comprises determining the identity of at least one nucleotide.
- the nucleotide is guanine or adenine at nucleotide position 126 of the PD-1 gene, as shown in figure 1, cytosine or thymine at position 6371, as shown in figure 1, guanine or adenine at position 7101, as shown in figure 1, guanine or adenine at position 7809, as shown in figure 1, cytosine of thymine at position 7872 as shown in figure 1 , guanine or adenine at position 8162 as shown in figure 1, cytosine or thymine at position 8288, as shown in figure 1 and SEQ ID N°1, cytosine or thymine at position 8448, as shown in figure 1 or guanine or adenine at position 9400, as shown in figure 1.
- the method comprises determining the nucleotide content of at least a portion of a PD-1 gene, such as by sequence analysis. In yet another embodiment, determining the molecular structure of at least a portion of a PD-1 gene is carried out by single-stranded conformation polymorphism.
- Non-limiting examples of methods within the scope of the invention for determining the molecular structure of at least a portion of a PD-1 gene include hybridisation of allele-specific oligonucleotides, sequence specific amplification, and primer specific extension.
- the probe or primer is allele specific. Preferred probes or primers are single stranded nucleic acids, which optionally are labelled.
- the methods of the invention can be used for determining the identity of the allelic variant of a polymorphic region of a human PD-1 gene present in a subject.
- the method of the invention can be useful for determining whether a subject has, or is at risk of developing, a disease or condition associated with a specific allelic variant of a polymorphic region in the human PD-1 gene.
- the disease or condition is characterized by an aberrant PD-1 activity, such as an aberrant PD-1 protein level, which can result from an aberrant expression of a PD-1 gene.
- the disease or condition can be autoimmune disorders such as myasthenia gravis multiple sclerosis, Type 1 diabetes, rheumatoid arthritis, Sjogrens syndrome, atopy, allergy, systemic lupus erythematosus and diseases associated with SLE which include fatigue, fever, loss of appetite, nausea, weight loss, hives, loss of scalp hair, red "butterfly rash” and raised rash, sensitivity to sun, ulcers in mouth, nose, or vagina, arthritis, joint pain, loss of blood supply to bone, pain, infections within joints, decrease in kidney function including, blood, aberrant amounts of protein or white blood cells in urine, intracerebral haemorrhage, headaches, loss of coordination, memory loss, seizures, strokes, anaemia, low white blood cell or low platelet count, pericardial effusion, heart attack, inflammation in the heart, infection in the heart, inflammation of the lining of the heart, infection of the lining of the heart, heart valve problems, shortness of breath, cough, inflammation of the
- the invention provides methods for predicting or diagnosing autoimmune disorders, conditions and diseases, specifically disorders conditions and diseases associated with myasthenia gravis multiple sclerosis, Type 1 diabetes, rheumatoid arthritis, Sjogrens syndrome, atopy, allergy and most specifically disorders conditions and diseases associated with SLE.
- the methods of the invention can also be used in selecting the appropriate drug to administer to a subject to treat a disease or condition, such as autoimmune disorders.
- a disease or condition such as autoimmune disorders.
- specific allelic variants of PD-1 polymorphic regions may be associated with a specific response to a specific drug by an individual having such an allele.
- a specific PD-1 allele may encode a PD-1 protein having a modified affinity for certain types of molecules. Accordingly, the action of a drug necessitating interaction with a PD-1 protein will be different in individuals carrying such a PD-1 allele.
- a specific PD-1 allele may encode a variant which modifies the level of protein expression.
- the invention provides a method for treating a subject having a disease or condition associated with a specific allelic variant of a polymorphic region of a PD-1 gene.
- the method comprises (a) determining the identity of the allelic variant; and (b) administering to the subject a compound that compensates for the effect of the specific allelic variant.
- the specific, allelic variant is a mutation. The mutation can be located, for example, in a promoter region, an intron, or an exon of the gene, or in the 3'UTR of the gene.
- the compound has an antagonistic or agonistic effect or other modulatory effect on PD-1 protein levels which prevents or alleviates autoimmune disorders such as myasthenia gravis multiple sclerosis, Type 1 diabetes, rheumatoid arthritis, Sjogrens syndrome, atopy, allergy, systemic lupus erythematosus and diseases associated with SLE which include fatigue, fever, loss of appetite, nausea, weight loss, hives, loss of scalp hair, red "butterfly rash” and raised rash, sensitivity to sun, ulcers in mouth, nose, or vagina, arthritis, joint pain, loss of blood supply to bone, pain, infections within joints, decrease in kidney function including, blood, aberrant amounts of protein or white blood cells in urine, intracerebral haemorrhage, headaches, loss of coordination, memory loss, seizures, strokes, anaemia, low white blood cell or low platelet count, pericardial effusion, heart attack, inflammation in the heart, infection in the heart, inflammation of the lining of autoimmune disorders
- the compound is selected from the group consisting of a nucleic acid, a protein, a peptidomimetic, or a small molecule.
- the disorders may be prevented from occurring or may be reduced, by administering to the subject a pharmaceutically effective amount of a compound which provides compensation for the dysfunction caused by aberrant PD-1 function, especially where caused by an allelic variant.
- the invention also provides substantially purified nucleic acids and oligonucleotides which can be used as a therapy to treat diseases and conditions associated with PD-1.
- Preferred nucleic acids are any one of SEQ ID N°s1 to 34.
- the invention also provides probes and primers comprising substantially purified oligonucleotides, which correspond to a region of nucleotide sequence which hybridises to at least 10 consecutive nucleotides of the sequence set forth in any one of SEQ ID N°s1 , 2, 3, 4, 5, 7, 8, 9, 10 11 or 12 or to any one of the complementary sequences set forth as SEQ ID N°s1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.
- the probe/primer further includes a label group attached thereto, which is capable of being detected.
- the invention provides a kit for amplifying and/or for determining the molecular structure of at least a portion of a PD-1 gene, comprising a probe or primer capable of hybridising to a PD-1 gene and instructions for use.
- the probe or primer is capable of hybridising to or amplifying a PD-1.
- the probe or primer is capable of hybridising to or amplifying an allelic variant of a PD-1.
- the polymorphic regions encode guanine or adenine at nucleotide position 126 of the PD-1 gene, cytosine or thymine at position 6371 , guanine or adenine at position 7101, guanine or adenine at position 7809 cytosine of thymine at position 7872, guanine or adenine at position 8162, cytosine or thymine at position 8288, cytosine or thymine at position 8448, or guanine or adenine at position 9400.
- determining the molecular structure of a region of a PD-1 gene comprises determining the identity of the allelic variant of the polymorphic region.
- kits of the invention can be used, for example, for determining whether a subject has or is at risk of developing a disease associated with a specific allelic variant of a polymorphic region of a PD-1 gene.
- the invention provides a kit for determining whether a subject has or is at risk of developing a disease or condition associated with autoimmune disorders such as multiple sclerosis, myasthenia gravis Type 1 diabetes, rheumatoid arthritis, Sjogrens syndrome, atopy, allergy, systemic lupus erythematosus and diseases associated with SLE.
- the disease or condition can be associated with an aberrant PD-1 activity, which can result, for example, from a mutation in the PD-1 gene.
- the kit of the invention can also be used in selecting the appropriate drug to administer to a subject to treat a disease or condition, such as a disease or condition set forth above.
- a disease or condition such as a disease or condition set forth above.
- pharmacogenetic studies have shown that the genetic background of individuals plays a role in determining the response of an individual to a specific drug.
- determining the allelic variants of PD-1 polymorphic regions of an individual can be useful in predicting how an individual will respond to a specific drug, for example, a drug for treating a disease or disorder associated with an aberrant PD-1 activity and/or a autoimmune disorders such as multiple sclerosis, Type 1 diabetes, rheumatoid arthritis, Sjogrens syndrome, atopy, allergy, systemic lupus erythematosus associated aberrant PD-1.
- the inventors have identified nucleic acid sequences associated with autoimmune diseases in humans, most notably SLE (NCBI REF 986034, Banklt Ref 392218 and GeneBank AF363458, Unpublished). Furthermore, the inventors have identified polymorphisms in the gene that make a subject more susceptible or prone to such conditions.
- the present invention is based at least in part on the discovery of the genomic structure of the human PD-1 gene and on the identification of polymorphic regions within the gene which correlate with specific diseases or conditions, including autoimmune disorders such as myasthenia gravis multiple sclerosis, Type 1 diabetes, rheumatoid arthritis, Sjogrens syndrome, atopy, allergy, systemic lupus erythematosus and diseases associated with SLE which include fatigue, fever, loss of appetite, nausea, weight loss, hives, loss of scalp hair, red "butterfly rash” and raised rash, sensitivity to sun, ulcers in mouth, nose, or vagina, arthritis, joint pain, loss of blood supply to bone, pain, infections within joints, decrease in kidney function including, blood, aberrant amounts of protein or white blood cells in urine, intracerebral haemorrhage, headaches, loss of coordination, memory loss, seizures, strokes, anaemia, low white blood cell or low platelet count, pericardial effusion, heart attack, inflammation in the
- the human PD-1 gene is at least 9625 base pairs long and has 5 coding exons and 4 introns.
- the exons are numbered 1 to 5 from 5' to 3' and the introns are numbered 1 through 4 from 5' to 3'.
- nucleic acids 1 to 663 encode a promoter region, as shown in figure 1 and SEQ ID N° 1 or 6.
- nucleic acids 8512 to 9625 is the 3' UTR, as shown on figure 1 and SEQ ID N° 1 or 7.
- Exon 1 corresponds to the first Exon, nucleic acids 664 to 807, as shown in figure 1 and SEQ ID N°1 or SEQ ID N°8 is situated 3' of the promoter and 5' to intron 1 on the sense coding strand of the gene, and contains the initiation codon.
- nucleic acids 808 to 6588 is situated immediately downstream of exon 1, as shown in figure 1, SEQ ID N°1 or SEQ ID N°2 on the sense coding strand of the gene.
- nucleic acids 6589 to 6948, as shown in figure 1, SEQ ID N°1 or SEQ ID N°9 is situated 3' to intron 1 and 5' to intron 2 on the sense coding strand of the gene.
- nucleic acids 6949 to 7215 as shown in figure 1 , SEQ ID N°1 or SEQ ID N°3 is situated 3' with respect to exon 2 and 5' with respect to exon 3 on the sense coding strand of the gene.
- Exon 3 nucleic acids 7216 to 7371 as shown in figure 1 SEQ ID N°1 or SEQ ID N°10 is situated 3' with respect to intron 2 and 5' with respect to intron 3 on the sense coding strand of the gene.
- This polymorphism is located in the 5' promotor region of the PD-1 gene and comprises an Ikaros transcription factor binding site 126 to 130 bp GGGAA - binding site, (ref: Molnar A., Georg ⁇ pulos, K., Mol. Cel. Biol., 14: 8292 - 8303, 1994) as shown in SEQ ID N°1. Replacement of guanine for adenine disrupts the binding of the transcription factor Ikaros for the binding site.
- a second polymorphism, located in intron 1 is a change from cytosine to thymine at position 6371, as shown in figure 1 and SEQ ID N°1 or SEQ ID N°2.
- a third polymorphism located in intron 2 is a change of guanine to adenine at position 7101 , as shown in figure 1 and SEQ ID N°s1 , 3, 19 or 20.
- a fourth, fifth and six polymorphism is located in intron 4 at nucleotide positions 7809, 7872 and 8162 respectively.
- the substitution at position 7809 is of guanine with adenine, as shown in figure 1 and SEQ ID N°1, 5 or SEQ ID N° 26 to 29.
- the substitution at position 7872 is of cytosine with thymine, as shown in figure 1 and SEQ ID N°1, 5 or SEQ ID N°26 to 29.
- the substitution at position 8162 is of guanine with adenine, as shown in figure 1 and SEQ ID N°1 or 5.
- this nucleotide 7809 TGCGGT comprise an AML1 transcription factor binding site in the PD-1 gene, as shown in figure 1, SEQ ID N°1 , SEQ ID N°5 (7806 to 7811 (tgcg/agt). Substitution of guanine with adenine results in an inhibition of binding of the AML-1 transcription factor to bind to its binding site (ref: Meyers S., Downing J. R., Hiebert S. W., Mol. Cell Biol. 13: 6336-6345, 1993).
- a seventh polymorphism is located in exon 5 at position 8288 as shown in figure 1, SEQ ID N°1 or SEQ ID N°12. This polymorphism is a change from cytosine to thymine, which changes the codon for alanine at amino acid 215 to a codon encoding valine. This modification is close to the PD-1 cytoplasmic domain ITIM motif.
- An eighth polymorphism is located in exon 5 at position 8448 as shown in figure 1, SEQ ID .N°1 or SEQ ID N°12. This polymorphism is a change from cytosine to thymine.
- An ninth polymorphism is to be found 3' to the PD-1 stop codon at position 9400, as shown in figure 1 and SEQ ID N°s1 , 7, 33 or 34. This polymorphism is a change from guanine to adenine.
- the inventors have identified at least one NF B transcription factor binding site in intron 4, as shown in SEQ ID N°1 or SEQ ID N°5 (7817- 7825 GGGGTGCCC) and that binding of the transcription factor NFKB to this site is not perturbed by the indicated polymorphisms at positions 7809, 7872 or 8162.
- the inventors have identified at least one E-box transcription factor binding site in intron 4, as shown in SEQ ID N°1 or SEQ ID N°5 and figure 6 and that binding of the transcription factor NFKB to this site is not perturbed by the indicated polymorphisms at positions 7809, 7872 or 8162.
- the invention provides nucleic acids, for example, intronic sequences, useful as probes or primers for determining the identity of an allelic variant of a PD-1 polymorphic region.
- the invention also provides methods for determining the identity of the alleles of a specific polymorphic region of a PD-1 gene. Such methods can be used, for example, to determine whether a subject has or is at risk of developing a disease or condition associated with one or more specific alleles of polymorphic regions of a PD-1 gene.
- the disease or condition is caused or contributed to by an aberrant PD-1 bioactivity.
- alleles refers to alternative forms of a gene or portions thereof. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for the gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene. Alleles of a specific gene can differ from each other in a single nucleotide, or several nucleotides, and can include substitutions, deletions, and insertions of nucleotides. An allele of a gene can also be a form of a gene containing a mutation.
- allelic variant of a polymorphic region of an PD-1 gene refers to a region of a PD-1 gene having one of several nucleotide sequences found in that region of the gene in other individuals.
- Antigenic functions include possession of an epitope or antigenic site that is capable of cross-reacting with antibodies raised against a naturally occurring or denatured PD-1 polypeptide or fragment thereof.
- Biologically active PD-1 polypeptides include polypeptides having both an effector and antigenic function, or only one of such functions.
- PD-1 polypeptides include antagonist polypeptides and native PD-1 polypeptides, provided that such antagonists include an epitope of a native PD-1 polypeptide.
- An effector function of PD-1 polypeptide can be the ability to bind to a Iigand.
- bioactive fragment of a PD-1 protein refers to a fragment of a full-length PD-1 protein, wherein the fragment specifically mimics or antagonizes the activity of a wild ype PD-1 protein.
- the bioactive fragment preferably is a fragment capable of binding to a second molecule, such as a Iigand.
- exon refers to the nucleotide sequence of an exon or portion thereof.
- an aberrant activity or "abnormal activity", as applied to an activity of a protein such as PD-1 , refers to an activity which differs from the activity of the wild-type or native protein or which differs from the activity of the protein in a healthy subject, for example, a subject not afflicted with a disease associated with a specific allelic variant of an PD-1 polymorphism.
- An activity of a protein can be aberrant because it is stronger than the activity of its native counterpart.
- an activity can be aberrant because it is weaker or absent related to the activity of its native counterpart.
- An aberrant activity can also be a change in an activity.
- an aberrant protein can interact with a different protein relative to its native counterpart.
- a cell can have an aberrant PD-1 activity due to over expression or under expression of the gene encoding PD-1.
- An aberrant PD-1 activity can a protein which has greater or less activity that its wild type counterpart.
- An aberrant PD-1 activity can also result from a lower or higher level of PD-1 on cells, which can result, for example, from a mutation in the 5' flanking region of the PD-1 gene or any other regulatory, element of the PD-1 gene, such as a regulatory element located in an intron. Accordingly, an aberrant PD-1 activity can result from an abnormal PD-1 promoter activity, or abnormal enhancer activity.
- Cells “host cells” or “recombinant host cells” are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
- the term “gene” or “recombinant gene” refers to a nucleic acid molecule comprising an open reading frame and including at least one exon and (optionally) an intron sequence.
- the term “intron” refers to a DNA sequence present in a given gene which is spliced out during mRNA maturation.
- Homology refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
- a homolog of a nucleic acid refers to a nucleic acid having a nucleotide sequence having a certain degree of homology with the nucleotide sequence of the nucleic acid or complement thereof.
- a homolog of a double stranded nucleic acid having SEQ ID N°: x is intended to include nucleic acids having a nucleotide sequence which has a certain degree of homology with SEQ ID N°: x or with the complement thereof.
- Preferred homologs of nucleic acids are capable of hybridising to the nucleic acid or complement thereof.
- interact as used herein is meant to include detectable interactions between molecules, such as can be detected using, for example, a hybridisation assay.
- interact is also meant to include "binding" interactions between molecules. Interactions may be, for example, protein-protein, protein-nucleic acid, protein-small molecule or small molecule-nucleic acid in nature.
- intron refers to the nucleotide sequence of an intron or portion thereof.
- isolated refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the macromolecule.
- isolated as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
- isolated nucleic acid is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
- locus refers to a specific position in a chromosome.
- locus of a PD-1 gene refers to the chromosomal position of the PD-1 gene.
- modulation refers to both upregulation, (i.e., activation or stimulation), for example by agonizing; and downregulation (i.e. inhibition or suppression), for example by antagonizing of a bioactivity (for example expression of a gene).
- molecular structure of a gene or a portion thereof refers to the structure as defined by the nucleotide content (including deletions, substitutions, additions of one or more nucleotides), the nucleotide sequence, the state of methylation, and/or any other modification of the gene or portion thereof.
- nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
- DNA deoxyribonucleic acid
- RNA ribonucleic acid
- Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine.
- nucleotide of a nucleic acid which can be DNA or an RNA
- adenosine cytidine
- guanosine guanosine
- thymidine a nucleotide having a uracil base
- complement of a nucleic acid strand can be the complement of a coding strand or the complement of a non-coding strand.
- complement of a nucleic acid having SEQ ID N°: x refers to the complementary strand of the strand having SEQ ID N°: x or to any nucleic acid having the nucleotide sequence of the complementary strand of SEQ ID N°: x.
- the complement of this nucleic acid is a nucleic acid having a nucleotide sequence which is complementary to that of SEQ ID N°: x.
- the nucleotide sequences and complementary sequences thereof are always given in the 5' to 3' direction.
- the term “complement” and “reverse complement” are used interchangeably herein.
- a "non-human animal” of the invention can include mammals such as rodents, non-human primates, sheep, goats, horses, dogs, cows, chickens, amphibians, reptiles, etc.
- Preferred non-human animals are selected from the rodent family including rat and mouse, most preferably mouse, though transgenic amphibians, such as members of the Xenopus genus, and transgenic chickens can also provide important tools for understanding and identifying agents which can affect, for example, embryogenesis and tissue formation.
- transgenic amphibians such as members of the Xenopus genus
- transgenic chickens can also provide important tools for understanding and identifying agents which can affect, for example, embryogenesis and tissue formation.
- chimeric animal is used herein to refer to animals in which an exogenous sequence is found, or in which an exogenous sequence is expressed in some but not all cells of the animal.
- tissue-specific chimeric animal indicates that an exogenous sequence is present and/or expressed or disrupted in some tissues, but not others
- operably linked is intended to mean that the promoter is associated with the nucleic acid in such a manner as to facilitate transcription of the nucleic acid from the promoter.
- polymorphism refers to the coexistence of more than one form of a gene or portion thereof.
- a portion of a gene of which there are at least two different forms, i.e., two different nucleotide sequences, is referred to as a "polymorphic region of a gene".
- a polymorphic region can be a single nucleotide, the identity of which differs in different alleles.
- a polymorphic region can also be several nucleotides long.
- a “polymorphic gene” refers to a gene having at least one polymorphic region.
- protein protein
- polypeptide peptide
- recombinant protein refers to a polypeptide which is produced by recombinant DNA techniques, wherein generally, DNA encoding the polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein.
- probe refers to a molecule which can be used to identify the presence of a PD-1 nucleic acid sequence or protein. Such probes may themselves be nucleic acid sequences of PD-1, expression products of PD-1, or binding molecules to PD-1 such as a Iigand or antibody.
- regulatory element also termed herein "regulatory sequence is intended to include elements which are capable of modulating transcription from a basic promoter and include elements such as enhancers and silencers.
- enhancer also referred to herein as “enhancer element” is intended to include regulatory elements capable of increasing, stimulating, or enhancing transcription from a basic promoter.
- the term “silencer”, also referred to herein as “silencer element” is intended to include regulatory elements capable of decreasing, inhibiting, or repressing transcription from a basic promoter. Regulatory elements are typically present in 5' flanking regions of genes. However, regulatory elements have also been shown to be present in other regions of a gene, in particular introns. Thus, it is possible that PD-1 genes have regulatory elements located in introns, exons, coding regions, and 3' flanking sequences.
- regulatory elements are also intended to be encompassed by the present invention and can be identified by any of the assays that can be used to identify regulatory elements in 5' flanking regions of genes.
- regulatory element further encompasses "tissue specific” regulatory elements, i.e., regulatory elements which effect expression of the selected DNA sequence preferentially in specific cells (for example, cells of a specific tissue). Gene expression occurs preferentially in a specific cell if expression in this cell type is significantly higher than expression in other cell types.
- regulatory element also encompasses non-tissue specific regulatory elements, i.e., regulatory elements which are active in most cell types.
- a regulatory element can be a constitutive regulatory element, i.e., a regulatory element which constitutively regulates transcription, as opposed to a regulatory element which is inducible, i.e., a regulatory element which is active primarily in response to a stimulus.
- a stimulus can be, for example, a molecule, such as a hormone, cytokine, heavy metal, phorbol ester, cyclic AMP (cAMP), or retinoie acid.
- transcription factor is intended to include proteins or modified forms thereof, which interact preferentially with specific nucleic acid sequences, i.e., regulatory elements, and which in appropriate conditions stimulate or repress transcription. Some transcription factors are active when they are in the form of a monomer. Alternatively, other transcription factors are active in the form of a dimer consisting of two identical proteins or different proteins (heterodimer). Modified forms of transcription factors are intended to refer to transcription factors having a postranslational modification, such as the attachment of a phosphate group. The activity of a transcription factor is frequently modulated by a postranslational modification.
- transcription factors are active only if they are phosphorylated on specific residues.
- transcription factors can be active in the absence of phosphorylated residues and become inactivated by phosphorylation.
- a list of known transcription factors and their DNA binding site can be found, for example, in public databases, for example, TFMATRIX Transcription Factor Binding Site
- the term “specifically hybridises” or “specifically detects” refers to the ability of a nucleic acid molecule of the invention to hybridise to at least approximately 6, 12, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 or 140 consecutive nucleotides of either strand of a PD-1 gene.
- substantially pure or “purified” does not require absolute purity; rather it is intended as a relative definition of purification of starting materials or natural materials to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
- PD-1 refers to a programmed cell death protein-1.
- PD-1 therapeutic refers to various forms of PD-1 polypeptides, as well as peptidomimetics, nucleic acids, or small molecules, which can modulate at least one activity of a PD-1 for the treatment of autoimmune disorders such as multiple sclerosis, Type 1 diabetes, rheumatoid arthritis, Sjogrens syndrome, atopy, allergy, systemic lupus erythematosus and diseases associated with SLE which include fatigue, fever, loss of appetite, nausea, weight loss, hives, loss of scalp hair, red "butterfly rash” and raised rash, sensitivity to sun, ulcers in mouth, nose, or vagina, arthritis, joint pain, loss of blood supply to bone, pain, infections within joints, decrease in kidney function including, blood, aberrant amounts of protein or white blood cells in urine, intracerebral haemorrhage, headaches, loss of coordination, memory loss, seizures stokes, anaemia, low white or platelet count, pericardial effusion, heart attack,
- transfection means the introduction of a nucleic acid, for example, an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.
- transformation is generally used herein when the transfection with a nucleic acid is by viral delivery of the nucleic acid.
- Transformation refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of a polypeptide or, in the case of anti-sense expression from the transferred gene, the expression of a naturally-occurring form of the recombinant protein is disrupted.
- transgene refers to a nucleic acid sequence which has been introduced into a cell.
- Daughter cells deriving from a cell in which a transgene has been introduced are also said to contain the transgene (unless it has been deleted).
- a transgene can encode, for example, a polypeptide, or an antisense transcript, partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (for example, it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).
- a transgene can also be present in an episome.
- a transgene can include one or more transcriptional regulatory sequence and any other nucleic acid, (for example intron), that may be necessary for optimal expression of a selected nucleic acid.
- a “transgenic animal” refers to any animal, preferably a non-human animal, for example a mammal, bird or an amphibian, in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
- the nucleic acid is introduced into the cell,- directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
- the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
- transgenic animal also includes those recombinant animals in which gene disruption of one or more genes is caused by human intervention, including both recombination and antisense techniques.
- transgenic may also refer to a nucleic acid sequence which has been introduced into a plant cell.
- treating is intended to encompass curing as well as ameliorating at least one symptom of the condition or disease.
- vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
- One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication.
- Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.
- Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors”.
- expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome.
- plasmid and "vector” are used interchangeably as the plasmid is the most commonly used form of vector.
- vector is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
- wild-type allele refers to an allele of a gene which, when present in two copies in a subject results in a wild-type phenotype. There can be several different wild-type alleles of a specific gene, since certain nucleotide changes in a gene may not affect the phenotype of a subject having two copies of the gene with the nucleotide changes.
- one aspect of the invention pertains to isolated nucleic acids comprising a polymorphic sequence of a PD-1 gene.
- This gene being characterised by containing intron and exon nucleic acid sequences encoding PD-1 as well as 5' and 3' nucleic acid sequence which flanks the intron and exon nucleic acid sequences.
- the invention provides an intronic sequence of the genomic DNA sequence encoding a PD-1 protein, comprising an intronic sequence shown in figure 1 or set forth in any of SEQ ID N°s1 to 5 (and associated sequences) or complements thereof or homologs thereof.
- Nucleic acids of the invention can function as probes or primers, for example, in methods for determining the identity of an allelic variant of a PD-1 polymorphic region.
- the nucleic acids of the invention can also be used to determine whether a subject is at risk of developing a disease associated with a specific allelic variant of a PD-1 polymorphic region, for example, a disease or disorder associated with an aberrant PD-1 activity.
- the nucleic acids of the invention can further be used to prepare PD-1 polypeptides encoded by specific alleles, such as mutant alleles. Such nucleic acids and polypeptides can be used in gene therapy.
- Polypeptides encoded by specific PD-1 alleles can also be used for preparing reagents, for example, antibodies, for detecting PD-1 proteins encoded by these alleles. Accordingly, such reagents can be used to detect mutant PD-1 proteins, for the diagnosis and treatment of autoimmune diseases disorders and conditions.
- Preferred polymorphs of PD-1 have at least 90% sequence identity any one of the peptides encoded in SEQ ID N°s35 to 38, or fragments thereof, as shown in figure 21.
- nucleic acids of the invention comprise an intronic sequence of a PD- 1 gene.
- PD-1 intronic sequence refers to a nucleotide sequence of an intron of a PD-1 gene.
- An intronic sequence can be directly adjacent to an exon or located further away from the exons.
- Preferred nucleic acids of the invention include an intronic sequence of a PD-1 gene which is adjacent to an exon and comprises at least about 3 consecutive nucleotides, at least about 6 consecutive nucleotides, at least about 9 consecutive nucleotides, at least about 12 consecutive nucleotides, at least about 15 consecutive nucleotides, at least about 18 consecutive nucleotides, or at least about 20 consecutive nucleotides.
- Isolated nucleic acids which comprise a PD-1 intronic sequence which is immediately adjacent to an exon and comprises at least about 25 consecutive nucleotides, at least about 30 consecutive nucleotides, at least about 35 consecutive nucleotides, at least about 40 consecutive nucleotides, at least about 50 consecutive nucleotides, or at least about 100 consecutive nucleotides are also within the scope of the invention.
- Preferred isolated nucleic acids of the invention also include those having a PD-1 intronic sequence having a nucleotide sequence of at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides or at least about 100 nucleotides.
- Other preferred nucleic acids of the invention can comprise a PD-1 intronic sequence having less than about 10 nucleotides, provided that the nucleotide sequence is novel.
- Yet other preferred isolated nucleic acids of the invention include PD-1 intronic nucleic acid sequences of a PD-1 intron, having at least about 150 consecutive nucleotides, at least about 200 consecutive nucleotides, at least about 250 consecutive nucleotides, at least about 300 consecutive nucleotides, at least about 350 consecutive nucleotides, at least about 400 consecutive nucleotides, at least about 500 consecutive nucleotides or at least about 1000 consecutive nucleotides.
- Preferred nucleic acids of the invention comprise a PD-1 intronic or non codon encoding nucleic acid sequence having a nucleotide sequence shown in figure 1, and/or in any of SEQ ID N°s 1, 2, 3, 4, 5, 6 or 7, complement thereof, reverse complement thereof or homolog thereof.
- the invention provides an isolated nucleic acid comprising an PD-1 intronic or non codon encoding nucleic acid sequence which is at least about 80% or preferably at least about 98%, and most preferably at least about 99% identical to an intronic nucleotide sequence shown in figure 1 or set forth in any of SEQ ID N°s 1, 2, 3, 4, 5, 6 or 7 or a complement thereof.
- nucleic acids of the invention comprise an exon coding sequence of a PD-1 gene.
- PD-1 exon of exonic sequence refers to a nucleotide sequence of an exon of a PD-1 gene.
- An exonic sequence can be directly adjacent to an intron or located further away from the intron.
- Preferred nucleic acids of the invention include an exonic sequence of a PD-1 gene which is adjacent to an intron and comprises at least about 3 consecutive nucleotides, at least about 6 consecutive nucleotides, at least about 9 consecutive nucleotides, at least about 12 consecutive nucleotides, at least about 15 consecutive nucleotides, at least about 18 consecutive nucleotides, or at least about 20 consecutive nucleotides.
- Isolated nucleic acids which comprise a PD-1 exonic sequence which is immediately adjacent to an intron and comprises at least about 25 consecutive nucleotides, at least about 30 consecutive nucleotides, at least about 35 consecutive nucleotides, at least about 40 consecutive nucleotides, at least about 50 consecutive nucleotides, or at least about 100 consecutive nucleotides are also within the scope of the invention.
- Preferred isolated nucleic acids of the invention also include those having a PD-1 exonic sequence having a nucleotide sequence of at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides or at least about 100 nucleotides.
- Other preferred nucleic acids of the invention can comprise a PD-1 exonic sequence having less than about 10 nucleotides, provided that the nucleotide sequence is novel.
- nucleic acids of the invention include PD-1 exonic nucleic acid sequences of a PD-1 exon, having at least about 150 consecutive nucleotides, at least about 200 consecutive nucleotides, at least about 250 consecutive nucleotides, at least about 300 consecutive nucleotides, at least about 350 consecutive nucleotides, at least about 400 consecutive nucleotides, at least about 500 consecutive nucleotides or at least about 1000 consecutive nucleotides.
- Preferred nucleic acids of the invention comprise a PD-1 exonic or codon encoding nucleic acid sequence having a nucleotide sequence shown in figure 1, and/or in any of SEQ ID N°s 1 , 8, 9, 10, 11 or 12, or complement thereof, reverse complement thereof or homolog thereof.
- the invention provides an isolated nucleic acid comprising an PD-1 exonic or codon encoding nucleic acid sequence which is at least about 80% or preferably at least about 98%, and most preferably at least about 99% identical to an intronic nucleotide sequence shown in figure 1 or set forth in any of SEQ ID N°s 1, 8,9, 10, 11 or 12, or a complement thereof.
- several alleles of human PD-1 genes have been identified. The invention is intended to encompass all of these alleles and PD-1 alleles not yet identified, which can be identified, for example, according to the methods described herein.
- Preferred nucleic acids of the invention are from vertebrate genes encoding PD-1 proteins. Particularly preferred vertebrate nucleic acids are mammalian nucleic acids.
- a particularly preferred nucleic acid of the invention is a human nucleic acid, such as a nucleic acid comprising an PD-1 intronic or non codon encoding nucleic acid sequence shown in figure 1 or set forth in any of SEQ ID N°s1 , 2, 3, 4, 5, 6 or 7, Other preferred nucleic acid sequences are those which encode exonic sequences in humans shown in figure 1 or set forth in any SEQ ID N°s1, 8, 9, 10, 11 or 12.
- Another aspect of the invention provides a nucleic acid which hybridises under appropriate stringency to an PD-1 intronic or non codon coding nucleic acid sequences having a nucleotide sequence shown in introns shown in figure 1 or in intronic sequences set forth in any of SEQ ID N°s1 , 2, 3, 4, 5, 6 or 7 or complement thereof.
- a further aspect of the invention provides a nucleic acid which hybridises under appropriate stringency to an PD-1 exonic or codo ⁇ coding nucleic acid sequences having a nucleotide sequence shown in figure 1 or in exonic sequences set forth in any of SEQ ID N°s 1, 8, 9, 10, 11 or 12, or complement thereof.
- Appropriate stringency conditions which promote DNA hybridisation for example, ⁇ .O.times. sodium chloride/sodium citrate (SSC) at about 45.degree. C, followed by a wash of 2.0.times.SSC at ⁇ O.degree. C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
- the salt concentration in the wash step can be selected from a low stringency of about 2.0.times.SSC at 50.degree. C. to a high stringency of about 0.2.times.SSC at 50.degree. C.
- a nucleic acid of the present invention will bind to at least about 20, preferably at least about 25, more preferably at least about 30 and most preferably at least about 50 consecutive nucleotides of a sequence shown in figure 1 or set forth in any of SEQ ID N°1, 2, 3, 4, 5, 6 or 7 under moderately stringent conditions, for example at about 2.0.times.SSC and about 40.degree. C.
- nucleic acids of the invention are capable of hybridising under stringent conditions to an intronic sequence of at least about 20, 30, 40, or at least about 50 nucleotides as shown in figure 1 or as set forth in an intronic or non codon encoding nucleic acid sequence of any of SEQ ID N°s1 , 2, 3, 4, 5, 6 or 7.
- Hybridisation as described above, can be used to isolate nucleic acids comprising an PD-1 intron or portion thereof from various animal species. A comparison of these nucleic acids should be indicative of intronic sequences which may have a regulatory or other function, since these regions are expected to be conserved among various species. Hybridisation can also be used to isolate PD-1 alleles.
- the nucleic acid of the invention can be single stranded DNA (for example, an oligonucleotide), double stranded DNA (for example, double stranded oligonucleotide) or RNA.
- Preferred nucleic acids of the invention can be used as probes or primers.
- Primers of the invention refer to nucleic acids which hybridise to a nucleic acid sequence which is adjacent to the region of interest or which covers the region of interest and is extended.
- a primer can be used alone in a detection method, or a primer can be used together with at least one other primer or probe in a detection method.
- Primers can also be used to amplify at least a portion of a nucleic acid.
- Probes of the invention refer to nucleic acids which hybridise to the region of interest and which are not further extended.
- a probe is a nucleic acid which hybridises to a polymorphic region of a PD-1 gene, and which by hybridisation or absence of hybridisation to the DNA of a subject will be indicative of the identity of the allelic variant of the polymorphic region of the PD-1 gene.
- nucleic acid amplification step which can be carried out by, for example, polymerase chain reaction (PCR).
- the invention provides primers for amplifying portions of a PD-1 gene, such as portions of exons and/or portions of introns.
- the exons and/or sequences adjacent to the exons of the human PD-1 gene will be amplified to, for example, detect which allelic variant of a polymorphic region is present in the PD- 1 gene of a subject.
- Preferred primers comprise a nucleotide sequence complementary to an PD-1 intronic sequence or a specific allelic variant of an PD-1 polymorphic region and of sufficient length to selectively hybridise with an PD-1 gene.
- the primer for example, a substantially purified oligonucleotide, comprises a region having a nucleotide sequence which hybridises under stringent conditions to consecutive nucleotides of an PD-1 gene.
- the primer is capable of hybridising to a PD-1 intron or non codon encoding sequence and has a nucleotide sequence of an intronic or non codon encoding sequence shown in figure 1 or set forth in any of SEQ ID N°s1 to 7 complements thereof, allelic . variants thereof, or complements of allelic variants thereof.
- primers comprising a nucleotide sequence of at least about 10 consecutive nucleotides, at least about 20 nucleotides or having from about 15 to about 25 nucleotides or set forth in any of SEQ ID N°s 13 to 34 or complement thereof are provided by the invention. Primers having a sequence of more than about 25 nucleotides are also within the scope of the invention.
- Preferred primers of the invention are primers that can be used in PCR for amplifying each of the exons of a PD-1 gene. Even more preferred primers of the invention have the nucleotide sequence set forth in any of SEQ ID N°s13 to 34.
- Primers can be complementary to nucleotide sequences located close to each other or further apart, depending on the use of the amplified DNA.
- primers may be chosen such that they amplify DNA fragments of at least about 10 nucleotides or as much as several kilobases.
- the primers of the invention will hybridise selectively to nucleotide sequences located about 50 to about 9000 nucleotides apart.
- a forward primer i.e., 5' primer
- a reverse primer i.e., 3' primer
- Forward and reverse primers hybridise to complementary strands of a double stranded nucleic acid, such that upon extension from each primer, a double stranded nucleic acid is amplified.
- a forward primer can be a primer having a nucleotide sequence or a portion of the nucleotide sequence shown in figure 1 or in any one of SEQ ID N°s 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.
- a reverse primer can be a primer having a nucleotide sequence or a portion of the nucleotide sequence that is complementary to a nucleotide sequence shown in figure 1 or in any one of SEQ ID N°s 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.
- Preferred forward primers comprise a nucleotide sequence set forth in SEQ ID N°s. 13 to 34.
- Preferred reverse primers comprise a nucleotide sequence set forth in SEQ ID N°s13 to 34.
- primers of the invention are nucleic acids which are capable of selectively hybridising to an allelic variant of a polymorphic region of a PD-1 gene.
- such primers can be specific for a PD-1 gene sequence, so long as they have a nucleotide sequence which is capable of hybridising to a PD- 1 gene.
- Preferred primers are capable of specifically hybridising to an allelic variant indicated in figure 1 and positions 126, 6371, 7101, 7809, 7872, 8162, 8288, 8448 or 9400 respectively.
- Such primers can be used, for example, in sequence specific oligonucleotide priming as described further herein.
- the PD-1 nucleic acids of the invention may also be used as probes, for example, in therapeutic and diagnostic assays.
- the present invention provides a probe comprising a substantially purified oligonucleotide, which oligonucleotide comprises a region having a nucleotide sequence that hybridises under stringent conditions to at least approximately 6, 8, 10 or 12, preferably about 25, 30, 40, 50 or 75 consecutive nucleotides of an PD-1 gene.
- the probes preferably hybridise to an intron of an PD-1 gene, having an intronic or non codon encoding nucleotide sequence shown in figure 1 or set forth in any of SEQ ID N°s1, 2, 3, 4, 5, 6 or 7 allelic variants thereof, complements thereof or complements of allelic variants thereof.
- the probes are capable of hybridising to a nucleotide sequence encompassing an intron/exon border of a PD-1 gene, or complements thereof.
- the probes are capable of hybridising to a nucleic acid sequence encoding a PD-1 amino acid sequence, such as those shown for example in figure 1 , and SEQ ID N°s1, 8, 9, 10, 11 or 12.
- probes of the invention are capable of hybridising specifically to a region of a PD-1 gene which is polymorphic.
- the probes are capable of hybridising specifically to one allelic variant of an PD-1 gene.
- Such probes can then be used to specifically detect which allelic variant of a polymorphic region of a PD-1 gene is present in a subject.
- the polymorphic region can be located in the promoter, exon, intron or 3'UTR sequences of a PD-1 gene.
- the probe or primer further comprises a label attached thereto, which, for example, is capable of being detected, for example the label group is selected from amongst radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.
- the isolated nucleic acid which is used, for example, as a probe or a primer, is modified, such as to become more stable.
- exemplary nucleic acid molecules which are modified include phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775).
- the nucleic acids of the invention can also be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule.
- the nucleic acids for example, probes or primers, may include other appended groups such as peptides (for example, for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, for example, Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. WO88/09810, published Dec.
- nucleic acid of the invention may be conjugated to another molecule, for example, a peptide, hybridisation triggered cross-linking agent, transport agent, triggered-triggered cleavage agent, and the like.
- the isolated nucleic acid comprising an PD-1 intronic sequence may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouraciI, 5- iodouracil, hypoxanthine, xantine, 4-acetylcytidine, 5-(carboxyhydroxymethyI) uracil, 5-carboxymethyIaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2- dimethylguanine, 2-methyIadenine, 2-methylguanine, 3-methylcytidine, 5- methylcytidine, N6-adenine, 7-methyIguanine, 5-methyIaminomethyluraciI, 5- methoxyamin
- the isolated nucleic acid may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2- fluoroarabinose, xylulose, and hexose.
- the nucleic acid comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
- the nucleic acid is an ⁇ -anomeric oligonucleotide.
- An ⁇ -anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual P-units, the strands run parallel to each other (Gautier et al., 1987, Nucl Acids Res. 15:6625- 6641).
- the oligonucleotide is a 2'-0-methyl . ribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).
- nucleic acid fragment of the invention can be prepared according to methods well known in the art and described, for example, in Sambrook, J. Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
- discrete fragments of the DNA can be prepared and cloned using restriction enzymes.
- discrete fragments may be prepared using the Polymerase Chain Reaction (PCR) using primers having an appropriate sequence.
- Oligonucleotides of the invention may be synthesized by standard methods known in the art, for example by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
- an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
- phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209)
- methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.
- the invention also provides vectors and plasmids containing the nucleic acids of the invention.
- the invention provides a vector comprising at least a portion of a PD-1 gene comprising a polymorphic region and/or intronic sequence.
- the invention provides vectors for expressing at least a portion of the newly identified allelic variants of the human PD-1 gene, as well as other allelic variants.
- the allelic variants can be expressed in eukaryotic cells, for example, cells of a subject, or in prokaryotic cells.
- the vector comprising at least a portion of a PD-1 allele is introduced into a host cell, such that a protein encoded by the allele is synthesized.
- the PD-1 protein produced can be used, for example, for the production of antibodies, which can be used, for example, in methods for detecting mutant forms of PD-1.
- the vector can be used for gene therapy, and be, for example, introduced into a subject to produce PD-1 protein.
- Host cells comprising a vector having at least a portion of a PD-1 gene are also within the scope of the invention.
- the present invention makes available isolated PD-1 polypeptides, such as PD-1 polypeptides which are encoded by specific allelic variants of PD-1 , such as those identified herein.
- the PD-1 polypeptides are isolated from, or otherwise substantially free of other cellular proteins.
- substantially free of other cellular proteins also referred to herein as "contaminating proteins”
- substantially pure or purified preparations are defined as encompassing preparations of PD-1 polypeptides having less than about 20% (by dry weight) contaminating protein, and preferably having less than about 5% contaminating protein.
- Functional forms of the subject polypeptides can be prepared, for the first time, as purified preparations by using a cloned gene as described herein.
- Preferred PD-1 proteins of the invention have an amino acid sequence which is at least about 90%, or 95% identical or homologous to an expression product of the nucleic acid of SEQ ID N°1 or fragments thereof. Even more preferred PD- 1 proteins comprise an amino acid sequence which is at least about 97, 98, or 99% homologous or identical to an amino acid sequence of SEQ ID N°1 or fragments thereof.
- Preferred polymorphs of PD-1 have at least 90% sequence identity any one of the peptides encoded in SEQ ID N°s35 to 38, or fragments thereof, as shown in figure 21.
- Such proteins can be recombinant proteins, and can be, for example, produced in vitro from nucleic acids comprising a specific allele of a PD-1 polymorphic region.
- recombinant polypeptides preferred by the present invention can be encoded by a nucleic acid, which is at least 80% homologous and more preferably 90% homologous and most preferably 95% homologous with a nucleotide sequence set forth in SEQ ID N°1 or fragment thereof.
- Polypeptides which are encoded by a nucleic acid that is at least about 98-99% homologous with the sequence of SEQ ID N° 1 and comprises an allele of a polymorphic region that differs from that set forth in SEQ ID N°1 are also within the scope of the invention.
- a PD-1 protein of the present invention is a mammalian PD-1 protein.
- the PD-1 protein is a human protein, such as an PD-1 polypeptide comprising any of the polymorphic amino acid sequence encoded by SEQ ID N°1.
- the polymorphs of PD-1 have at least 90% sequence identity any one of the peptides encoded in SEQ ID N°s35 to 38, or fragments thereof, as shown in figure 21 , or an expression product o SEQ ID N°1.
- Full length proteins or fragments corresponding to one or more particular motifs and/or domains or to arbitrary sizes, for example, at least 5, 10, 25, 50, 75 and 100, amino acids in length are within the scope of the present invention.
- Isolated peptidyl portions of PD-1 proteins can be obtained by screening peptides recombinantly produced from the corresponding fragment of the nucleic acid encoding such peptides.
- fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.
- an PD-1 polypeptide of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length. The fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments which can function as either agonists or antagonists of a wild-type (for example, "authentic") PD-1 protein.
- polypeptides referred to herein as having an activity (for example, are "bioactive") of an PD-1 protein are defined as polypeptides which mimic or antagonize all or a portion of the biological/biochemical activities of an PD-1 protein having a nucleic acid sequence of SEQ ID N°1.
- Other biological activities of the subject PD-1 proteins are described herein or will be reasonably apparent to those skilled in the art.
- a polypeptide has biological activity if it is a specific agonist or antagonist of a naturally-occurring form of an PD-1 protein.
- fusion proteins for example, PD-1- immunoglobulin fusion proteins.
- fusion proteins can provide, for example, enhanced stability and solubility of PD-1 proteins and may thus be useful in therapy. Fusion proteins can also be used to produce an irnmunogenic fragment of an PD-1 protein.
- the VP6 capsid protein of rotavirus can be used as an immunologic carrier protein for portions of the PD-1 polypeptide, either in the monomeric form or in the form of a viral particle.
- nucleic acid sequences corresponding to the portion of a subject PD-1 protein to which antibodies are to be raised can be incorporated into a fusion gene construct which includes coding sequences for a late vaccinia virus structural protein to produce a set of recombinant viruses expressing fusion proteins comprising PD-1 epitopes as part of the virion. It has been demonstrated with the use of irnmunogenic fusion proteins utilizing the Hepatitis B surface antigen fusion proteins that recombinant Hepatitis B virions can be utilized in this role as well.
- chimeric constructs coding for fusion proteins containing a portion of a PD-1 protein and the poliovirus capsid protein can be created to enhance immunogenicity of the set of polypeptide antigens (see, for example, EP Publication No: 0259149; and Evans et al. (1989) Nature 339:385; Huang et al. (1988) J. Virol. 62:3855; and Schlienger et al. (1992) J. Virol. 66:2).
- the Multiple antigen peptide system for peptide-based immunization can also be utilized to generate an immunogen, wherein a desired portion of a PD-1 polypeptide is obtained directly from organo-chemical synthesis of the peptide onto an oligomeric branching lysine core (see, for example, Posnett et al. (1988) JBC 263:1719 and Nardelli et al. (1992) J. Immunol. 148:914).
- Antigenic determinants of PD-1 proteins can also be expressed and presented by bacterial cells.
- fusion proteins can also facilitate the expression of proteins, and accordingly, can be used in the expression of the PD-1 polypeptides of the present invention.
- PD-1 polypeptides can be generated as glutathione— S--transferase (GST-fusion) proteins.
- GST-fusion proteins can enable easy purification of the PD-1 polypeptide, as for example by the use of glutathione-derivatized matrices (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. (N.Y.: John Wiley & Sons, 1991).
- the present invention further pertains to methods of producing the subject PD-1 polypeptides.
- a host cell transfected with a nucleic acid vector directing expression of a nucleotide sequence encoding the subject polypeptides can be cultured under appropriate conditions to allow expression of the peptide to occur. Suitable media for cell culture are well known in the art.
- the recombinant PD-1 polypeptide can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for such peptide.
- the recombinant PD-1 polypeptide is a fusion protein containing a domain which facilitates its purification, such as GST fusion protein.
- homologs of one of the subject PD-1 polypeptides which function in a limited capacity as one of either a PD-1 agonist (mimetic) or a PD-1 antagonist, in order to promote or inhibit only a subset of the biological activities of the naturally-occurring form of the protein.
- a PD-1 agonist mimetic
- a PD-1 antagonist a PD-1 antagonist
- Homologs of each of the subject PD-1 proteins can be generated by mutagenesis, such as by discrete point mutation(s), or by truncation. For instance, mutation can give rise to homologs which retain substantially the same, or merely a subset, of the biological activity of the PD-1 polypeptide from which it was derived.
- antagonistic forms of the protein can be generated which are able to inhibit the function of the naturally occurring form of the protein, such as by competitively binding to an PD-1 receptor.
- the recombinant PD-1 polypeptides of the present invention also include homologs of PD-1 polypeptides which differ from the PD-1 proteins encoded by the nucleic acid of SEQ ID N°1, such as versions of those protein which are resistant to proteolytic cleavage, as for example, due to mutations which alter ubiquitination or other enzymatic targeting associated with the protein.
- PD-1 polypeptides may also be chemically modified to create PD-1 derivatives by forming covalent or aggregate conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like.
- Covalent derivatives of PD-1 proteins can be prepared by linking the chemical moieties to functional groups on amino acid side chains of the protein or at the N- terminus or at the C-terminus of the polypeptide.
- Modification of the structure of the subject PD-1 polypeptides can be for such purposes as enhancing therapeutic or prophylactic efficacy, stability (for example, ex vivo shelf life and resistance to proteolytic degradation), or post- translational modifications (for example, to alter phosphorylation pattern of protein).
- modified peptides when designed to retain at least one activity of the naturally-occurring form of the protein, or to produce specific antagonists thereof, are considered functional equivalents of the PD-1 polypeptides described in more detail herein.
- Such modified peptides can be produced, for instance, by amino acid substitution, deletion, or addition.
- the substitutional variant may be a substituted conserved amino acid or a substituted non- conserved amino acid.
- aliphatic glycine, alanine, valine, leucine, isoleucine, serine, threonine, with serine and threonine optionally be grouped separately as aliphatic-hydroxyl;
- aromatic phenyIalanine, tyrosine, tryptophan;
- amide asparagine, glutamine;
- su!fur-containing cysteine and methionine.
- PD-1 homolog (for example, functional in the sense that the resulting polypeptide mimics or antagonizes the wild-type form) can be readily determined by assessing the ability of the variant peptide to produce a response in cells in a fashion similar to the wild-type protein, or competitively inhibit such a response.
- Polypeptides in which more than one replacement has taken place can readily be tested in the same manner.
- Kits as set forth herein the invention provides methods, for example, diagnostic and therapeutic methods, for example, for determining the type of allelic variant of a polymorphic region present in a PD-1 gene, such as a human PD-1 gene.
- the methods use probes or primers comprising nucleotide sequences which are complementary to a PD-1 intronic sequence or to a polymorphic region of a PD-1 gene. Accordingly, the invention provides kits for performing these methods.
- the invention provides a kit for determining whether a subject has or is at risk of developing a disease or condition associated with a specific allelic variant of a PD-1 polymorphic region.
- the disease or disorder is characterized by an abnormal PD-1 activity.
- the invention provides a kit for determining whether a subject has or is at risk of developing a cardiovascular disease, for example, autoimmune disorders such as multiple sclerosis, Type 1 diabetes, rheumatoid arthritis, Sjogrens syndrome, atopy, allergy, systemic lupus erythematosus and diseases associated with SLE.
- a preferred kit provides reagents for determining whether a male or female subject is likely to develop SLE or is suffering from SLE.
- kits comprise at least one probe or primer which is capable of specifically hybridising to a PD-1 sequence or polymorphic region and instructions for use.
- the kits preferably comprise at least one of the above described nucleic acids, for example, including nucleic acids hybridising to an exon/intron border.
- Even more preferred kits comprise a pair of primers selected from the group consisting of SEQ ID N°s13 to 34, or complement thereof.
- kits of the invention can also comprise one or more control nucleic acids or reference nucleic acids, such as nucleic acids comprising a PD-1 intronic sequence.
- a kit can comprise primers for amplifying a polymorphic region of a PD-1 gene and a control DNA corresponding to such an amplified DNA and having the nucleotide sequence of a specific allelic variant.
- the control nucleic acid comprises at least a portion of a PD-1 gene of an individual, who does not have an autoimmune disease, disorder or condition, associated with an aberrant PD-1 activity.
- kits of the invention comprise at least one reagent necessary to perform the assay.
- the kit can comprise an enzyme.
- the kit can comprise a buffer or any other necessary reagent.
- the invention further features predictive medicines, which are based, at least in part, on determination of the identity of PD-1 polymorphic regions which are associated with specific diseases, conditions or disorders.
- information obtained using the diagnostic assays described herein is useful for diagnosing or confirming that a symptomatic subject has an allele of a polymorphic region which is associated with a particular disease or disorder.
- the information (alone or in conjunction with information on another genetic defect, which contributes to the same disease) can be used prognostically for predicting whether a non- symptomatic subject is likely to develop a disease or condition, which is associated with one or more specific alleles of PD-1 polymorphic regions in a subject.
- a doctor can recommend a regimen (for example diet or exercise) or therapeutic protocol, useful for preventing or prolonging onset of the particular disease or condition in the individual.
- an individual's PD-1 genetic profile or the genetic profile of a disease or condition associated with a specific allele of a PD-1 polymorphic region can enable a doctor: 1) to more effectively prescribe a drug that will address the molecular basis of the disease or condition; and 2) to better determine the appropriate dosage of a particular drug.
- the expression level of PD-1 proteins can be measured in many patients at various stages of the disease to generate a transcriptional or expression profile of the disease. Expression patterns of individual patients can then be compared to the expression profile of the disease to determine the appropriate drug and dose to administer to the patient.
- the ability to target populations expected to show the highest clinical benefit, based on the PD-1 or disease genetic profile, can enable: 1) the repositioning of marketed drugs with disappointing market results; 2) the rescue of drug candidates whose clinical development has been discontinued as a result of safety or efficacy limitations, which are patient subgroup-specific; and 3) an accelerated and less costly development for drug candidates and more optimal drug labelling (for example since the use of PD-1 as a marker is useful for optimising effective dose).
- the present methods provide means for determining if a subject has (diagnostic) or is at risk of developing (prognostic) a disease, condition or disorder that is associated a specific PD-1 allele, for example, autoimmune disorders such as multiple sclerosis, Type 1 diabetes, rheumatoid arthritis, Sjogrens syndrome, atopy, allergy, systemic lupus erythematosus and diseases associated with SLE.
- autoimmune disorders such as multiple sclerosis, Type 1 diabetes, rheumatoid arthritis, Sjogrens syndrome, atopy, allergy, systemic lupus erythematosus and diseases associated with SLE.
- the present invention provides methods for determining the molecular structure of a PD-1 gene, such as a human PD-1 gene, or a portion thereof.
- determining the molecular structure of at least a portion of a PD-1 gene comprises determining the identity of the allelic variant of at least one polymorphic region of an PD-1 gene.
- a polymorphic region of a PD-1 gene can be located in an exon, an intron, at an intron/exon border, or in the promoter of the PD-1 gene, or the 3'UTR.
- the invention provides methods for determining whether a subject has, or is at risk of developing, a disease or condition associated with a specific allelic variant of a polymorphic region of a PD-1 gene.
- diseases can be associated with an aberrant PD-1 activity, for example, aberrant PD-1 protein level.
- An aberrant PD-1 protein level can result from an aberrant transcription or post-transcriptional regulation.
- allelic differences in specific regions of a PD-1 gene can result in differences of PD-1 protein due to differences in regulation of expression.
- some of the identified polymorphisms in the human PD-1 gene may be associated with differences in the level of transcription, RNA maturation, splicing, or translation of the PD-1 gene or transcription product.
- Analysis of one or more PD-1 polymorphic region in a subject can be useful for predicting whether a subject has or is likely to develop a autoimmune disorders such as multiple sclerosis, Type 1 diabetes, rheumatoid arthritis, Sjogrens syndrome, atopy, allergy, systemic lupus erythematosus and diseases associated with SLE.
- a autoimmune disorders such as multiple sclerosis, Type 1 diabetes, rheumatoid arthritis, Sjogrens syndrome, atopy, allergy, systemic lupus erythematosus and diseases associated with SLE.
- the methods of the invention can be characterized as comprising detecting, in a sample of cells from the subject, the presence or absence of a specific allelic variant of one or more polymorphic regions of an PD-1 gene.
- the allelic differences can be: (i) a difference in the identity of at least one nucleotide or (ii) a difference in the number of nucleotides, which difference can be a single nucleotide or several nucleotides.
- the invention also provides methods for detecting differences in PD-1 genes such as chromosomal rearrangements, for example, chromosomal dislocation.
- the invention can also be used in prenatal diagnostics.
- a preferred detection method is allele specific hybridisation using probes overlapping the polymorphic site and having about 5, 10, 20, 25, or 30 nucleotides around the polymorphic region.
- probes for detecting specific allelic variants of the polymorphic regions are probes comprising a nucleotide sequence set forth in any of SEQ ID N°s13 to 34.
- several probes capable of hybridising specifically to allelic variants are attached to a solid phase support, for example, a "chip".
- Oligonucleotides can be bound to a solid support by a variety of processes, including lithography. For example a chip can hold up to 250,000 oligonucleotides or more (GeneChip, Affymetrix).
- a chip comprises all the allelic variants of at least one polymorphic region of a gene.
- the solid phase support is then contacted with a test nucleic acid and hybridisation to the specific probes is detected. Accordingly, the identity of numerous allelic variants of one or more genes can be identified in a simple hybridisation experiment.
- genomic DNA of a cell is exposed to two PCR primers and amplification for a number of cycles sufficient to produce the required amount of amplified DNA.
- the primers are located between about 50 and 9000 base pairs apart.
- Alternative amplification methods include: self sustained sequence replication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. et al., 1988, Bio/Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
- any of a variety of sequencing reactions known in the art can be used to directly sequence at least a portion of an PD-1 gene and detect allelic variants, for example, mutations, by comparing the sequence of the sample sequence with the corresponding wild-type (control) sequence.
- Exemplary sequencing reactions include those based on techniques developed by Maxam and Gilbert (Proc. Natl Acad Sci USA (1977) 74:560) or Sanger (Sanger et al (1977) Proc. Nat. Acad. Sci 74:5463). It is also contemplated that any of a variety of automated sequencing procedures may be utilized when performing the subject assays (Biotechniques (1995) 19:448), including sequencing by mass spectrometry (see, for example, U.S. Pat. No. 5,547,835 and international patent application Publication Number WO 94/16101, entitled
- PCT/US96/03651 entitled DNA Diagnostics Based on Mass Spectrometry by H.
- A-track or the like, for example, where only one nucleotide is detected, can be carried out.
- protection from cleavage agents can be used to detect mismatched bases in RNA/RNA DNA/DNA, or RNA DNA heteroduplexes (Myers, et al. (1985) Science 230:1242).
- cleavage agents such as a nuclease, hydroxylamine or osmium tetroxide and with piperidine
- cleavage agents such as a nuclease, hydroxylamine or osmium tetroxide and with piperidine
- RNA DNA heteroduplexes Myers, et al. (1985) Science 230:1242).
- the technique of "mismatch cleavage” starts by providing heteroduplexes formed by hybridising a control nucleic acid, which is optionally labelled, for example, RNA or DNA, comprising a nucleotide sequence of a PD-1 allelic variant with a sample nucleic acid, for example, RNA or DNA, obtained from a tissue sample.
- RNA/DNA duplexes can be treated with RNase and DNA DNA hybrids treated with S1 nuclease to enzymatically digest the mismatched regions.
- DNA/DNA can be treated with RNase and DNA DNA hybrids treated with S1 nuclease to enzymatically digest the mismatched regions.
- RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine whether the control and sample nucleic acids have an identical nucleotide sequence or in which nucleotides they are different. See, for example, Cotton et al (1988) Proc. Natl Acad Sci USA
- control or sample nucleic acid is labelled for detection.
- alterations in electrophoretic mobility is used to identify the type of PD-1 allelic variant.
- SSCP single strand conformation polymorphism
- SSCP single strand conformation polymorphism
- RNA rather than DNA
- the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
- the identity of an allelic variant of a polymorphic region is obtained by analysing the movement of a nucleic acid comprising the polymorphic region in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al (1985) Nature 313:495).
- DGGE denaturing gradient gel electrophoresis
- DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
- a temperature gradient is used in place of a denaturing agent gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:1275).
- oligonucleotide probes may be prepared in which the known polymorphic nucleotide is placed centrally (allele-specific probes) and then hybridised to target DNA under conditions which permit hybridisation only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al (1989) Proc. Natl Acad. Sea USA 86:6230; and Wallace et al. (1979) Nucl. Acids Res. 6:3543).
- Such allele specific oligonucleotide hybridisation techniques may be used for the simultaneous detection of several nucleotide changes in different polylmorphic regions of PD-1.
- oligonucleotides having nucleotide sequences of specific allelic variants are attached to a hybridising membrane and this membrane is then hybridised with labelled sample nucleic acid. Analysis of the hybridisation signal will then reveal the identity of the nucleotides of the sample nucleic acid.
- Oligonucleotides used as primers for specific amplification may carry the allelic variant of interest in the centre of the molecule (so that amplification depends on differential hybridisation) (Gibbs et al (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238; Newton et al. (1989) Nuc). Acids Res. 17:2503). This technique is also termed "PROBE” for Probe Oligo Base Extension.
- identification of the allelic variant is carried out using an oligonucleotide ligation assay (OLA), as described, for example, in U.S. Pat. No. 4,998,617 and in Landegren, U. et al., Science 241 :1077-1080 (1988).
- OLA oligonucleotide ligation assay
- the OLA protocol uses two oligonucleotides which are designed to be capable of hybridising to abutting sequences of a single strand of a target.
- One of the oligonucleotides is linked to a separation marker, for example, biotinylated, and the other is detectably labelled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridise such that their termini abut, and create a ligation substrate. Ligation then permits the labelled oligonucleotide to be recovered using avidin, or another biotin Iigand.
- Nickerson, D. A. et al. have described a nucleic acid detection assay that combines attributes of PCR and OLA (Nickerson, D. A. et al., Proc. Natl. Acad. Sci. (U.S.A.) 87:8923-8927 (1990). In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.
- U.S. Pat. No. 5593826 discloses an OLA using an oligonucleotide having 3'-amino group and a 5'-phosphorylated oligonucleotide to form a conjugate having a phosphoramidate linkage.
- OLA OLA combined with PCR permits typing of two alleles in a single microtiter well. By marking each of the allele-specific primers with a unique hapten, i.e.
- each OLA reaction can be detected by using hapten specific antibodies that are labelled with different enzyme reporters, alkaline phosphatase or horseradish peroxidase.
- This system permits the detection of the two alleles using a high throughput format that leads to the production of two different colours.
- the invention further provides methods for detecting single nucleotide polymorphisms in a PD-1 gene. Because single nucleotide polymorphisms constitute sites of variation flanked by regions of invariant sequence, their analysis requires no more than the determination of the identity of the single nucleotide present at the site of variation and it is unnecessary to determine a complete gene sequence for each patient. Several methods have been developed to facilitate the analysis of such single nucleotide polymorphisms.
- the single base polymorphism can be detected by using a specialized exonuclease-resistant nucleotide, as disclosed, for example, in
- a primer complementary to the allelic sequence immediately 3' to the polymorphic site is permitted to hybridise to a target molecule obtained from a particular animal or human. If the polymorphic site on the target molecule contains a nucleotide that is complementary to the particular exonuclease-resistant nucleotide derivative present, then that derivative will be incorporated onto the end of the hybridised primer. Such incorporation renders the primer resistant to exonuclease, and thereby permits its detection.
- a primer is employed that is complementary to allelic sequences immediately 3' to a polymorphic site.
- the method determines the identity of the nucleotide of that site using labelled dideoxynucleotide derivatives, which, if complementary to the nucleotide of the polymorphic site will become incorporated onto the terminus of the primer.
- GBA.TM Genetic Bit Analysis
- Goelet, P. et al. PCT Appln. No. 92/157112.
- the method of Goelet, P. et al. use ⁇ mixtures of labelled terminators and a primer that is complementary to the sequence 3' to a polymorphic site.
- the labelled terminator that is incorporated is thus determined by, and complementary to, the nucleotide present in the polymorphic site of the target molecule being evaluated.
- the method of Goelet, P. et al. is preferably a heterogeneous phase assay, in which the primer or the target molecule is immobilized to a solid phase.
- allelic variants of a polymorphic region located in the coding region of a PD-1 gene yet other methods than those described above can be used. For example, identification of an allelic variant which encodes a mutated PD-1 protein can be performed by using an antibody specifically recognizing the mutant protein in, for example, immunohistochemistry or immunoprecipitation.
- Antibodies to wild-type PD-1 protein are described, for example, in Acton et al. (1999) Science 271:518 (anti-mouse PD-1 antibody cross-reactive with human PD-1).
- Other antibodies to wild-type PD-1 or mutated forms of PD-1 proteins can be prepared according to methods known in the art.
- Binding assays are known in the art and involve, for example, obtaining cells from a subject, and performing binding experiments with a labelled Iigand, to determine whether binding to the mutated form of PD-1 differs from binding to the wild-type of the PD-1.
- Antibodies directed against wild type or mutant PD-1 polypeptides or allelic variant thereof, which are discussed above, may also be used in disease diagnostics and prognostics. Such diagnostic methods, may be used to detect abnormalities in the level of PD-1 polypeptide expression, or abnormalities in the structure and/or tissue, cellular, or subceliular location of an PD-1 polypeptide. Structural differences may include, for example, differences in the size, electronegativity, or antigenicity of the mutant PD-1 polypeptide relative to the normal PD-1 polypeptide. Protein from the tissue or cell type to be analysed may easily be detected or isolated using techniques which are well known to one of skill in the art, including but not limited to western blot analysis.
- the antibodies (or fragments thereof) useful in the present invention may, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of PD-1 polypeptides.
- In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labelled antibody of the present invention.
- the antibody (or fragment) is preferably applied by overlaying the labelled antibody (or fragment) onto a biological sample.
- a solid phase support or carrier is used as a support capable of binding an antigen or an antibody.
- supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
- the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
- the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
- the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
- the surface may be flat such as a sheet, test strip, etc.
- Preferred supports include polystyrene beads.
- EIA enzyme immunoassay
- the enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means.
- Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, aipha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6- phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
- the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
- Detection may also be accomplished using any of a variety of other immunoassays.
- a radioimmunoassay RIA
- the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography. It is also possible to label the antibody with a fluorescent compound.
- fluorescent labelling compounds fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
- the antibody can also be detectably labelled using fluorescence emitting metals such as 152 Eu or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
- DTPA diethylenetriaminepentacetic acid
- EDTA ethylenediaminetetraacetic acid
- the antibody also can be detectably labelled by coupling it to a chemiluminescent compound.
- the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
- chemiluminescent labelling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
- Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labelling are luciferin, luciferase and aequorin.
- any of the above methods for detecting alterations in a gene or gene product or polymorphic variants can be used to monitor the course of treatment or therapy.
- the identity of the allelic variant can be determined by determining the molecular structure of the MRNA, pre-mRNA, or cDNA.
- the molecular structure can be determined using any of the above described methods for determining the molecular structure of the genomic DNA, for example, sequencing and SSCP.
- the methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits, such as those described above, comprising at least one probe or primer nucleic acid described herein, which may be conveniently used, for example, to determine whether a subject has or is at risk of developing a disease associated with a specific PD-1 allelic variant.
- Sample nucleic acid for using in the above-described diagnostic and prognostic methods can be obtained from any cell type or tissue of a subject.
- a subject's bodily fluid for example blood
- nucleic acid tests can be performed on dry samples (for example hair or skin).
- Fetal nucleic acid samples can be obtained from maternal blood as described in International Patent Application No. WO91/07660 to Bianchi.
- amniocytes or chorionic villi may be obtained for performing prenatal testing.
- Diagnostic procedures may also be performed in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary.
- Nucleic acid reagents may be used as probes and/or primers for such in situ procedures (see, for example, Nuovo, G. J., 1992, PCR in situ hybridisation: protocols and applications, Raven Press, NY).
- Fingerprint profiles may be generated, for example, by utilizing a differential display procedure, Northern analysis and/or RT-PCR.
- Pharmacogenomics Knowledge of the identity of the allele of one or more PD-1 gene polymorphic regions in an individual (the PD-1 genetic profile), alone or in conjunction with information on other genetic defects contributing to the same disease (the genetic profile of the particular disease) allows a customisation of the therapy for a particular disease to the individual's genetic profile, the goal of "pharmacogenomics". For example, subjects having a specific allele of a PD-1 gene may or may not exhibit symptoms of a particular disease or be predisposed to developing symptoms of a particular disease. Further, if those subjects are symptomatic, they may or may not respond to a certain drug, for example, a specific PD-1 therapeutic, but may respond to another. Thus, generation of a PD-1 genetic profile, (for example, categorization of alterations in
- PD-1 genes which are associated with the development of a particular disease from a population of subjects, who are symptomatic for a disease or condition that is caused by or contributed to by a defective and/or deficient PD-1 gene and/or protein (a PD-1 genetic population profile) and comparison of an individual's PD-1 profile to the population profile, permits the selection or design of drugs that are expected to be safe and efficacious for a particular patient or patient population (i.e., a group of patients having the same genetic alteration).
- an PD-1 population profile can be performed by determining the PD-1 profile, for example, the identity of PD-1 alleles, in a patient population having a disease, which is associated with one or more specific alleles of PD-1 polymorphic regions.
- the PD-1 population profile can further include information relating to the response of the population to an PD-1 therapeutic, using any of a variety of methods, including, monitoring: 1) the severity of symptoms associated with the PD-1 related disease, 2) PD-1 gene expression level, 3) PD-1 mRNA level, and/or 4) PD-1 protein level, and (iii) dividing or categorizing the population based on particular PD-1 alleles.
- the PD-1 genetic population profile can also, optionally, indicate those particular PD-1 alleles which are present in patients that are either responsive or non-responsive to a particular therapeutic. This information or population profile, is then useful for predicting which individuals should respond to particular drugs, based on their individual PD-1 profile.
- the PD-1 profile is a transcriptional or expression level profile and step (i) is comprised of determining the expression level of PD-1 proteins, alone or in conjunction with the expression level of other genes known to contribute to the same disease at various stages of the disease.
- transgenic animals For example, one can produce transgenic mice, for example, as described herein, which contain a specific allelic variant of a PD-1 gene. These mice can be created, for example, by replacing their wild-type PD-1 gene with an allele of the human PD-1 gene. The response of these mice to specific PD-1 therapeutics can then be determined.
- the treatment of an individual with a PD-1 therapeutic can be monitored by determining PD-1 characteristics, such as PD-1 protein level or activity, PD-1 mRNA level, and/or PD-1 transcriptional level. This measurement will indicate whether the treatment is effective or whether it should be adjusted or optimised.
- PD-1 can be used as a marker for the efficacy of a drug during clinical trials.
- the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (for example, an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a preadministration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a PD-1 protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the PD-1 protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the PD-1 protein, mRNA, or genomic DNA in the preadministration sample with the PD-1 protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly.
- an agent for example, an agonist,
- increased administration of the agent may be desirable to increase the expression or activity of PD-1 to higher levels than detected, i.e., to increase the effectiveness of the agent.
- decreased administration of the agent may be desirable to decrease expression or activity of PD-1 to lower levels than detected, i.e., to decrease the effectiveness of the agent.
- Cells of a subject may also be obtained before and after administration of a PD-1 therapeutic to detect the level of expression of genes other than PD-1, to verify that the PD-1 therapeutic does not increase or decrease the expression of genes which could be deleterious. This can be done, for example, by using the method of transcriptional profiling.
- mRNA from cells exposed in vivo to a PD-1 therapeutic and mRNA from the same type of cells that were not exposed to the PD-1 therapeutic could be reverse transcribed and hybridised to a chip containing DNA from numerous genes, to thereby compare the expression of genes in cells treated and not treated with a PD-1 therapeutic. If, for example a PD-1 therapeutic turns on the expression of a proto-oncogene in an individual, use of this particular PD-1 therapeutic may be undesirable.
- the present invention provides for both prophylactic and therapeutic methods of treating a subject having or likely to develop a disorder associated with specific PD-1 alleles and/or aberrant PD-1 expression or activity.
- the invention provides a method for preventing in a subject, a disease or condition associated with a specific PD-1 allele and/or an aberrant PD-1 expression or activity, by administering to the subject an agent which counteracts the unfavourable biological effect of the specific PD-1 allele.
- Subjects at risk for such a disease can be identified by a diagnostic or prognostic assay, for example, as described herein.
- Administration of a prophylactic agent can occur prior to the manifestation of symptoms associated with specific PD-1 alleles, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
- a compound that counteracts the effect of this allele is administered.
- the compound can be a compound modulating the level. of PD-1 in a patient.
- the invention further provides methods of treating subjects having a disease or disorder associated with a specific allelic variant of a polymorphic region of a
- the method comprises (a) determining the identity of the allelic variant; and (b) administering to the subject a compound that compensates for the effect of the specific allelic variant.
- the polymorphic region can be localized at any location of the gene, for example, in the promoter (for example, in a regulatory element of the promoter), in an exon,
- a subject having a specific variant of the polymorphic region which is associated with a specific disease or condition can be treated with compounds which specifically compensate for the allelic variant.
- the identity of one or more of the following nucleotides of a PD-1 gene of a subject is determined: nucleotide 126, 6371, 7101, 7809, 7872, 8162, 8288, 8448 or 9400.
- the allelic variant can be a mutant allele, i.e., an allele which when present in one, or preferably two copies, in a subject results in a change in the phenotype of the subject.
- a mutation can be a substitution, deletion, and/or addition of at least one nucleotide relative to the wild-type allele.
- the subject can be treated to specifically compensate for the mutation.
- the subject can be treated, for example, by administration to the subject of a nucleic acid encoding a wild- type PD-1 protein, such that the expression of the wild-type PD-1 protein compensates for the endogenous mutated form of the PD-1 protein.
- Nucleic acids encoding wild-type and variant human PD-1 protein are set forth in the expression product of SEQ ID N°1.
- the PD-1 protein is a membrane type protein consisting of 288 amino acids. It contains two hydrophobic regions, one at the N- terminus and the other in the middle, which are likely to serve as a signal peptide and transmembrane segment respectively (US 5629204).
- a treatment can be designed which up regulates the expression of PD-1 or improves signal transduction.
- a compound or molecule which promotes expression or signal transduction of PD-1 is administered to the subject.
- a mutant PD-1 protein can also be an PD-1 protein having a mutation in the cytoplasmic domain of the protein which results in an aberrant signal transduction from the PD-1.
- Subjects having such a mutation can be treated, for example, by administration of compounds which induce the same or similar signal transduction or compounds which act downstream of PD-1.
- the effect of a mutation in a PD-1 protein can be determined according to methods known in the art. For example, if the mutation is located in the extracellular portion of the protein, one can perform binding assays using an appropriate Iigand, and determine whether the binding affinity of such a Iigand with the mutated PD-1 protein is different from the binding affinity of the Iigand with the wild-type protein. Such assays can be performed using a soluble form of a PD-1 protein or a membrane bound form of the protein. If the mutation in the PD-1 protein is located in the cytoplasmic domain of the protein, signal transduction experiments can be performed to determine whether the signal transduced from the mutated PD-1 differs from the signal transduced from the wild-type PD-1. Alternatively, one can also investigate whether binding to a protein which interacts with the cytoplasmic domain of the receptor is affected by the mutation. Such determination can be made by, for example, by immunoprecipitation.
- the invention provides methods for treating a subject having a mutated PD-1 gene, in which the mutation is located in a regulatory region of the gene.
- a regulatory region can be localized in the promoter of the gene, in the 5' or 3' untranslated region of an exon, or in an intron or in the 3'UTR.
- a mutation in a regulatory region can result in increased production of PD-1 protein, decreased production of PD-1 protein, or production of PD-1 having an aberrant tissue distribution.
- the effect of a mutation in a regulatory region upon the PD-1 protein can be determined, for example, by measuring the PD-1 protein level or mRNA level in cells having a PD-1 gene having this mutation and which, normally (i.e., in the absence of the mutation) produce PD-1 protein.
- the effect of a mutation can also be determined in vitro.
- a reporter construct can be constructed which comprises the mutated promoter linked to a reporter gene, the construct transfected into cells, and comparison of the level of expression of the reporter gene under the control of the mutated promoter and under the control of a wild-type promoter.
- Such experiments can also be carried out in mice transgenic for the mutated promoter.
- the effect of the mutation can be determined, for example, by producing transgenic animals in which the mutated PD-1 gene has been introduced and in which the wild-type gene may have been knocked out. Comparison of the level of expression of PD-1 in the mice transgenic for the mutant human PD-1 gene with mice transgenic for a wild-type human PD-1 gene will reveal whether the mutation results in increased, decreased synthesis of the PD-1 protein and/or aberrant tissue distribution of PD-1 protein. Such analysis could also be performed in cultured cells, in which the human mutant PD-1 gene is introduced and, for example, replaces the endogenous wild-type PD-1 gene in the cell.
- a specific treatment can be administered to a subject having such a mutation.
- the subject can be treated by administration of a compound which increases synthesis, such as by increasing PD-1 gene expression, and wherein the compound acts at a regulatory element different from the one which is mutated.
- the subject can be treated by administration of a compound which reduces PD-1 protein production, for example, by reducing PD-1 gene expression or a compound which inhibits or reduces the activity of PD-1.
- a correlation between drug responses and specific alleles of PD-1 can be shown, for example, by clinical studies wherein the response to specific drugs of subjects having different allelic variants of a polymorphic region of a PD-1 gene is compared. Such studies can also be performed using animal models, such as mice having various alleles of human PD-1 genes and in which, for example, the endogenous PD-1 has been inactivated such as by a knock-out mutation. Test drugs are then administered to the mice having different human PD-1 alleles and the response of the different mice to a specific compound is compared. Accordingly, the invention provides assays for identifying the drug which will be best suited for treating a specific disease or condition in a subject. For example, it will be possible to select drugs which will be devoid of toxicity, or have the lowest level of toxicity possible for treating a subject having a disease or condition.
- the identification of different alleles of PD-1 can also be useful for identifying an individual among other individuals from the same species.
- DNA sequences can be used as a fingerprint for detection of different individuals within the same species (Thompson, J. S. and Thompson, eds., Genetics in Medicine, WB Saunders Co., Philadelphia, Pa. (1991)). This is useful, for example, in forensic studies.
- Figure 1 Shows the nucleic acid sequence of the human PD-1 gene
- Figure 2 Shows the physical map of the 2q37.3 region
- Figure 3 Shows expression of PD-1 mRNA in patients
- Figure 4 Shows the major haplotype for the SLEB2 locus and subhaplotype groups identified with the inclusion of the PD-1 polymorphisms;
- Figure 5 Shows the geneology of one of the Icelandic families with the group IV disease sub-haplotype containing the PD1.3 A allele and with two affected recombinants (IV-10 and IV-11);
- Figure 6a Is a map of the 172 bp region of intron 4 of the PD-1 gene containing predicted transcription factor binding sites;
- Figure 6b Shows the results of an electrophoretic mobility shift assay
- Figure 7 Shows results of the reporter gene assay for the intron 4 of PD-1 ;
- FIG. 8 Shows sequence number 1
- Figure 9 Shows sequence number 2 which is the nucleic acid sequence encoding intron 1 , polymorphism is shown in bold;
- Figure 10 Shows sequence number 3 which is the nucleic acid sequence encoding intron 2, polymorphism is shown in bold;
- Figure 11 Shows sequence number 4 which is the nucleic acid sequence encoding intron 3;
- Figure 12 Shows sequence number 5 which is the nucleic acid sequence encoding intron 4, polymorphism is shown in bold;
- Figure 13 Shows sequence number 6 which is the nucleic acid sequence encoding Promoter region, polymorphism is shown in bold;
- Figure 14 Shows sequence number 7 which is the nucleic acid sequence encoding 3'UTR, polymorphism us shown in bold;
- Figure 15 Shows sequence number 8 which is the nucleic acid sequence encoding exon 1 ;
- Figure 16 Shows sequence number 9 which is the nucleic acid sequence encoding exon 2;
- Figure 17 Shows sequence number 10 which is the nucleic acid sequence encoding exon 3;
- Figure 18 Shows sequence number 11 which is the nucleic acid sequence encoding the exon 4;
- Figure 19 Shows sequence number 12 which is the nucleic acid sequence encoding exon 5, polymorphism is shown in bold;
- Figure 20 Shows sequence numbers 13 to 34 which are oligonucleotides
- Figure 21 Shows sequence numbers 35 to 38 which are polypeptides encoding PD-1.
- Figure 22 Shows the structure of the predicted binding sites in the intronic enhancer within the PD-1 gene.
- Figure 23 Shows an electrophoretic mobility shift, supershift and competition assays with Jurkat nuclear cell extract and allelic variants of SNP PD-1.3.
- Figure 24 Shows enhancer activity of intron 4 of the PD-1 gene in a Luciferase reporter assay
- Figure 25 Shows expression of the PD-1 mRNA in SLE patients and controls.
- the top line shows the distance in bases
- the second line shows contiguous sequences as found in the Ensemble database.
- the clones used for FISH are shown as black bars.
- the position of PD-1 and other genes and ESTs relative to the contiguous information available through the public databases is also shown, as well as other genes according to Ensemble and the physical map published for NIDDM1.
- the extension of the SLEB2 locus is shown as a grey square. The recombinations found in affected individuals and the families to which they belonged are also depicted.
- the haplotype blocks are shadowed in grey to distinguish the recombinations.
- the relevant PD-1 allele for the sub-haplotype I and IV is underlined.
- sub-haplotype I the numbers of the affected individuals with the haplotype are shown.
- the other sub-haplotypes the number of the family is given.
- this figure shows the recombinational history of the group IV disease sub-haplotype in the multicase families and trios (haplotypes transmitted to the SLE individual) where PD-1.3 A is found.
- This figure shows how only the polymorphisms of the PD-1 gene are conserved among all individuals having the group IV sub-haplotype.
- the disease haplotype is shown as a black bar.
- the alleles of the markers for each haplotype are shown with PD1.3A underlined (shown as a number 1).
- Individual IV-2 had clinical manifestations but negative serology and is inmarried.
- this individual has the sub-haplotype of the group I (including PD1.1) and is related to a second SLE patient not studied here but known to us by genealogy information (not shown).
- the bars of Figure 7 show the Luciferase activity after normalization with the ⁇ -gal control.
- Jurkat cells were activated with PMA + Ionomycin 8 hours after transfection and Luciferase activity was measured after 10 hours of induction.
- PD-1 is located within SLEB2
- PD-1 was considered a strong candidate for the SLEB2 locus.
- PD-1 is expressed in lymphocytes and it is known to regulate T and B cell activation and mice made deficient for PD-1 develop a syndrome characterized by high levels of autoantibodies and immune-complex- mediated glomerulonephritis, similar to human SLE 17 .
- This gene had previously been mapped to 2q37.3, but its precise position was not known 11,12 .
- PD-1 was not found among the clones described in the physical map for NIDDM1 (ref. 18) nor in a second unpublished map of the region (provided by Dr. Patrick Concannon as a personal communication) or in any commercially available BAC and PAC libraries. Instead, we used fluorescent in situ hybridisation (FISH) on metaphase and interphase chromosomes to define the position of PD-1 and some other ESTs. This identified the position of PD-1 centromeric of AC025684.00001 (represented by the clone RP11-463B12) at the very end of the 2q37.3 (247.50-247.70Mb, chr2) (Fig. 2). PD-1 was therefore included within SLEB2.
- FISH fluorescent in situ hybridisation
- PD-1 is differentially expressed in SLE, nephritis and controls
- PBMC peripheral blood mononuclear cells
- Polymorphisms of PD-1 reveal genetic/allelic heterogeneity and the presence of founder disease haplotypes within SLEB2
- the complete PD-1 gene was sequenced in 10 unrelated individuals from multicase families (4 healthy and 6 with SLE) and 6 single nucleotide polymorphisms were identified in 9,6kb.
- PD1.1 located in the promoter, SNP PD1.2 in intron 2, SNP PD1.3 and SNP PD1.4 in intron 4, SNP PD1.5 in exon 5 (synonymous substitution), and SNP PD1.6 in the 3' UTR.
- the multicase families were genotyped for the SNPs PD1.3, PD1.5 and PD1.6 and analysed for linkage.
- PD1.1 and PD1.2 were in complete linkage disequilibirum, as well as PD1.4 and PD1.5.
- Table 1A Linkage and Association Analysis of PD-1 Polymorphisms in Scandinavian Multicase Families (n-31).
- PD1.6 (A/G) RFLP 0.22 0.49
- the SNPs were assayed by restriction enzyme analysis (RFLP) and/or dynamic allele-specific hybridization (DASH). * he nucleotide change is shown within parentheses.
- RFLP restriction enzyme analysis
- DASH dynamic allele-specific hybridization
- N I A refers to the non-transmitted alleles ot parents to SLL
- ⁇ refers to the number of chromosomes.
- the present inventors found that the major haplotype was partitioned and new disease haplotypes were observed supporting the presence of allelic and/or genetic heterogeneity.
- the present inventors discovered 4 sub-haplotypes segregating with SLE in multicase families (Fig. 4) probably suggesting the presence of at least 4 different underlying mutations.
- Each sub-haplotype was found in four groups of families I,
- the sub-haplotype from group IV was distinct due to the presence of allele A of PD1.3 that was observed only in one, possibly founder disease haplotype (Fig. 4). This haplotype segregated in 10 out of the 31 multicase families (7 Icelandic, 2 Norwegian and 1 Swedish). All four recombination events delimiting the centromeric end of SLEB2 (Fig. 2 and 5) were found in individuals carrying this haplotype. So we decided to analyse this haplotype and the PD1.3 A variant further. The present inventors studied the association of PD-1 polymorphisms in our independent set of 190 trios. Only PD1.3A was associated with SLE (TDT, p ⁇ 0.02, transmitted in 11%, non-transmitted in 4%) (Table 1).
- PD1.3 A was present in a unique disease haplotype, the present inventors analysed all affected individuals with PD1.3 allele A from the multicase families and the trios for the past history of recombinational events. The present inventors reasoned that if PD1.3 A was a mutation occurring only once in a founder haplotype, we could obtain information as to whether PD-1 , and in particular PD-1.3A was the susceptibility variant for SLE by studying the decay of the haplotype in past generations (Fig. 4).
- PD1.3A was present in a "complete" haplotype covering markers M64098 to AB023160.
- the telomeric marker AB012360 was excluded in 42% of the haplotypes and markers M64098 and D63878a could be excluded in 13% of the cases.
- the excluded markers have been recombined out of the disease haplotype sometime in the past with frequencies depending on the degree of linkage disequilibrium in the region and on the degree of polymorphism of each marker.
- This analysis allows us to exclude other genes beyond PD-1 from consideration in our mutation search (Fig. 4), and give support for PD-1 as the susceptibility gene for SLE in this group of families and for PD1.3A as the susceptibility variant.
- Glomerulonephritis is an important clinical feature of SLE that usually represents a very defined immune complex induced manifestation and has been observed in mouse models with deficiency of PD-1 17 .
- Our own results showed differences in PD-1 expression between patients with and without nephritis and controls, so the present inventors studied this subgroup of patients for association with the SNPs.
- 30% were diagnosed with nephritis (see Methods).
- AML-1 in the first repeat (Fig. 6a).
- the present inventors performed an electrophoretic mobility shift assay (EMSA) (Fig. 6b). Nuclear extracts from ESA and Fig. 6b.
- Jurkat cells expressing AML-1 (Ref. 19) formed a complex with a probe covering the first AML-1 site (PD1.3G in Fig. 6b). In contrast, the probe for PD1.3 A
- PD1.3G oligonucleotide was specifically competed by unlabelled self- oligonucleotide. Upon addition of AML-1 polyclonal antisera, a supershifted band was detected suggesting that AML-1 indeed is part of a complex that binds to
- the present inventors cloned the complete intron 4 (560 bp) into the pGL3-promoter vector and transfected the construct into the human T cell line Jurkat.
- the diminished activation-induced gene transcription by the nephritis-associated allele A is in line with the results obtained for PD-1 expression in the patients with nephritis. These results are consistent with the 40 bp repeat of intron 4 being a regulatory element and that PD1.3 allele A influences its activity.
- allelic and genetic heterogeneity was found at the SLEB2 locus. It is therefore not possible to assume that a general mutation for the disease could be found. Instead, the present inventors discovered the presence of several (at least 4) independent mutations through haplotype analysis. With such analysis the present inventors could identify conserved fragments of linkage disequilibrium that segregated non-randomly with the disease.
- rare polymorphisms like PD1.1 and PD1.3 with frequencies between 1-15% could be themselves disease mutations, or alternatively, belong to rare and distinct haplotypes useful for fine mapping. Due to the very rare allelic frequency of PD1.1A (less than 0,5% in controls and about 1 % in sporadic SLE and its presence in 7 individuals from the multicase families) no association analysis was applicable at this point. This SNP is, nevertheless, functionally interesting (data not shown). If indeed regulatory, this polymorphism could be relevant for a small proportion of SLE patients.
- PD1.3 was more polymorphic than PD1.1 and this allowed us to make statistical evaluations, which showed that out of all PD-1 SNPs found, PD1.3 was the strongest candidate as a mutation for PD-1, due to the association of allele A with SLE and lupus nephritis.
- PD1.3 A was a mutation that had occurred once in a founder disease haplotype
- the present inventors analysed its recombinational history.
- the analysis of past recombinational events allowed us to exclude other markers that, although tightly linked in the major disease haplotype, had undergone recombination from the disease sub-haplotype in past generations.
- Only PD-1 was conserved. Therefore, PD-1 is the susceptibility gene for the group of families and patients that have PD1.3A, but the present inventors cannot exclude other polymorphisms and/or genes within SLEB2 to be involved in susceptibility to SLE in the other families and patients.
- the present inventors observed that the "affected" recombinants delimiting SLEB2 from the centromeric end were found in families with individuals carrying the PD1.3 A associated haplotype, supporting the exclusion of the region upstream UCSNP-19 from further mutational searching.
- a mouse model made deficient for PD-1 develops, at late age, a lupus-like syndrome with high levels of autoantibodies and glomerulonephritis 17 .
- the present inventors observed that the PD1.3 A disrupts binding of an important transcription factor, de-represses basal transcription and is unable to enhance gene transcription in response to cell activation, suggesting a defect on the on-off switch affecting PD-1 expression.
- This result is in line with the function of PD-1 as an immunoreceptor tyrosine-based inhibitory motif-containing molecule 11"17 . It should be expected that a decrease in the expression of PD-1 would prevent its inhibitory function upon cellular activation.
- our results support the possibility that the reduced expression of PD-1 after cellular activation or antigenic stimulation in lupus nephritis can be partly explained by the presence of the PD-1.3 allele A.
- SLE is a disease characterized by a state of chronic lymphocyte hyperactivity 22"24 .
- Persistent T cell activation is thought to allow autoreactive T cells to induce autoantibody production and increase the formation of immune complexes that could eventually deposit in the kidney glomerulus.
- Genes coding for molecules similar to PD-1 , carrying inhibitory motifs (ITIM) have been described as involved in autoimmune-like syndromes when deleted by homologous recombination 25 or found as mutations in lupus-prone mice 26 . These models show T or B cell hyperactivity and autoantibody production. These receptors and many of the pathways in which they act, are involved in the fine regulation of T and B cell activation and tolerance, possibly at different developmental stages.
- PD-1 contributes to the risk for the expression of the disease possibly together with other factors known to affect immune complex deposition such as partial deficiency of C4A (ref. 4) or reduced handling of immune complexes by the FcG receptors IIA and IIIA 27, 2 ⁇ .
- the present inventors are at present analysing the genetic effect of PD-1 together with other genes associated with SLE.
- the polymorphism PD1.3 is located within an intronic direct repeat with transcription factor-binding sites for molecules exclusively involved in hematopoietic differentiation and inflammation 29"31 .
- Previous studies have identified other intronic regulatory sequences 18 ' 32,33 .
- Some examples are the silencers for the genes CD4 and CD21, both of which are members of the immunogiobulin superfamily 32,33 and the recently described polymorphism in intron 3 of calpain 10 (CAPN10) for NIDDM1 (Ref. 18).
- the present inventors expect to identify other important intronic or non-coding regulatory sequences to play a role in complex disease pathogenesis in the future.
- the present inventors show here for the first time the possible role of the transcription factor AML-1 in peripheral tolerance and inflammation. Support for the role of AML-1 in T cell regulation is found in the co-activation by AML-1 on the T cell receptor, alpha chain enhancer 34 .
- the present work gives evidence for a regulatory role of AML-1 in T-lymphocyte-expressed co-receptors.
- the transcription interactions taking place at the regulatory element of the intron 4 of PD-1 appear to be very complex. Within the region, transcription factor binding sites for NFkB and E boxes were also found, but their role in the regulatory element has to be established.
- the present inventors analyzed here 2510 individuals from 5 independent sets for SNPs found in the PD-1 gene.
- This SNP alters a binding site for the transcription factor AML-1 in an intronic enhancer. This change causes alterations in the enhancer activity upon cellular activation and in PD-1 mRNA expression in SLE patients.
- the PD-1 gene considered a strong candidate for SLEB2 because it is an immunoreceptor, member of the immunogiobulin family, has a tyrosine-based inhibitory motif (ITIM), is known to regulate T and B cell activation and mice knockout for pc develop an SLE-like disease 11, 2, 14, 17 .
- ITIM tyrosine-based inhibitory motif
- AFBAC - affected family-based controls RR - relative risk
- a) one allele of each SNP is shown to simplify the table
- b) n/N number of alleles out of total number of chromosomes
- PD-1.3.A was less frequent in Mexicans than in Europeans and almost not present in African- Americans, suggesting that this mutation is of recent origin affecting mostly Europeans and to a lesser extent populations admixed with them.
- Alleles of SNPs PD-1.5 and PD-1.6 demonstrated residual association with SLE due to linage disequilibrium with SNP PD-1.3, but no increase in the relative risk (Table 4).
- PD-1.3 is located in an enhancer-like structure in intron 4 of the PD-1 gene, where four imperfect tandem repeats contain binding sites for transcription factors exclusively involved in hematopoietic differentiation and inflammation: AML-1, E box-binding factors and NFKB (p50) 29,31 .
- the SNP PD-1/3A disrupts the predicted DNA-binding site for AML-1 in the first repeat (Fig. 22).
- FIG. 22 the predicted binding sites for the transcription factors AML- 1 , NFKB (p50) and E-box-binding factors are shown.
- the first AML-1 binding site is disrupted by SNP PD1.3A, hence, the predicted binding sites in the intronic enhancer are within the PD-1 gene.
- AML-1 also known as CBF ⁇ 2
- CBF ⁇ 2 is a transcription factor inactivated by translocations found in acute myeloid leukaemia, and is shown to either repress or activate transcription 29 .
- the inventors show here the specific binding of nuclear extract from a human T cell- line, Jurkat, to the wild-type AML-1 binding site (PD-1.3, allele G), confirmed by supershifting upon addition of antibodies against AML-1. No binding was found to the mutated site (PD-1.3, allele A) at any concentration of the nuclear extract (Fig.23).
- Figure 23 shows the 18 bp oligonucleotides containing both allelic variants of SNP PD-1.3 which represent native and mutated AML-1 binding sites.
- Binding sites were assayed with increasing (0-4 ⁇ g) amounts of Jurkat cells nuclear extract. Lanes 1-4 show that there is a lack of binding to oligonucleotide containing PD-1.3A; lanes 5 -12 show that there is binding to native PD-1.3G containing oligonucleotide. Binding to wild-type G allele results in a complex (C) that is specifically competed by 100x excess of unlabelled PD-1.3 G oligonucleotide (lane 11) but not by the same amount of unrelated oligonucleotide (lane 12). Antiserum against AML-1 reveals a supershifted band (S) as indicated in lane 9, while unrelated serum does not produce the supershift as indicated in lane 10.
- C complex
- S supershifted band
- FIG. 25 shows the results of PBMC of 17 controls with SNP PD-1.3G/G genotypes, 8 patients with SNP PD-1.3G/G genotypes, 4 patients with SNP PD-1.3A/G genotypes and one patient with SNP PD-1.3AA.
- Genotype were activated with MPA & ionomycin for 2 and 4 hours and compared with untreated samples. Untreated samples are marked as 1, activated for 2 hours as 2 and activated for 4 hours as 3.
- Level of PD-1 mRNA normalized by ⁇ 2-microglobuIin was measured in triplicate using TaqMan technology. The ⁇ Ct value is derived as a difference between Ctpo-i and Ct p2 . microgiobuiin values. High ⁇ Ct value means low PD-1 expression and vice versa. Results of PD-1 mRNA expression are presented as box-and-whiskers plots. Black bars represent 50% of sample distribution and whiskers show high and low extremes of distribution.
- SLE patients particularly those positive for allele A of SNP PD-1.3 show higher degree of variation.
- PD-1 expression in SLE patients had much higher degree of inter-individual variation than in controls.
- Level of PD-1 expression in patients homozygous for SNP PD-1.3G (8 individuals) was also higher at 2 hours after activation, (p 0.05).
- Dr. Graeme Bell kindly provided a physical map of 2q37.3, prior to publication 18 .
- Dr. Patrick Concannon also provided a preliminary physical map (unpublished). The described clones in those maps were obtained from Research Genetics and tested by PCR for the PD-1 gene. Further search for PD- 1 in BAC and PAC libraries was attempted through services provided commercially covering all available libraries (Research Genetics and Incyte Genomics).
- FISH Fluorescent in situ hybridisation
- the BAC clone RP11-463B12 representing contig AC025684.00001 at the location described in www.ensemble.org, was used as a FISH probe.
- the Scaffold X54KRCE619J from Celeras' February frozen dataset (www.celera.com) was found to contain fragments of the ESTs L16991 (gene human thymidylate kinase, HTHYK) and AB023160 (mRNA for the KIAA0943 gene) from the public database at NCBI (www.ncbi.nlm.nih.gov).
- the present inventors aligned these sequences to generate a joint FISH probe covering both genes as they were very close to each other (about 2,5kb apart).
- the joint FISH probe was made of 6 PCR fragments (free from repetitive elements) of 9.0kb of total length, covering about 20kb of genomic sequence.
- the FISH probe for PD-1 was generated by PCR from the genomic sequence obtained by us and included two PCR fragments of 1.2kb and 6.7kb covering almost the entire gene.
- FISH with metaphase and interphase chromosomes was performed essentially as described elsewhere 37 .
- DNA from the BAC clones was labelled either with biotin or digoxigenin using nick translation.
- the probes were detected by the application of a single layer of FITC-avidin (Vector labs) and rhodamine labelled anti-digoxigenin antibodies (Boehringer-Mannheim). Images were merged using a Zeiss Axioscope microscope with a cooled CCD camera (Photometries) and the IPlab software (Vysis).
- the complete PD-1 gene was sequenced using the dye-terminator kit following the manufacturer's instructions (PE Biosystems).
- the SNPs for M64098 (HDLBP), D63878a (NEDD5), AA15760 (Cda0fd11) and AB023160 (KIAA0943) were discovered by in silico search and sequencing and have been previously described 11 . Sequences for the polymorphisms UCSNP-6, -11, -12, -15 and -19 were kindly provided by Prof. Graeme Bell.
- the SNPs were genotyped by restriction enzyme analysis (RFLP) or dynamic allele-specific hybridisation (DASH) 38 or PCR followed by agarose gel analysis (for the UCSNP-19 minisatellite).
- RFLP restriction enzyme analysis
- DASH dynamic allele-specific hybridisation
- PD1.2 The following primers and restriction enzymes were used for PD-1: PD1.2, DASH assay: PD1.2f: 5'CTG CAT CTG GGG GAA TGG TGA C 3', PD1.2r: 5' GAT TCC AGA GCT AGA GGA CAG A 3'-biotin, PD1.2probe1: 5' GGT GAC CGG CAT CTC 3', PD1.2probe2: 5' GGT GAC CAG CAT CTC 3'. PD1.3f. 5' CCC CAG GCA GCA ACC TCA AT 3', PD1.3r. 5' GAC CGC AGG CAG GCA CAT AT 3', 180bp (130+50) Pstl.
- DPD1.3f 5' TGG TGC CCC AGC CCA CCT G 3'
- DPD1.3r 5' CAT GGG ACT GGC ACC CCC GGA 3'-biotin and as PD1.3 probe: 5'CAC CTG CGG TCT CCG 3'.
- PD1.5f 5'CTC AAA GAA GGA GGA CCC CTC A 3'
- PD1.5r 5' GCC AAG AGC AGT GTC CAT CCT 3', 240bp (180+60), Pvull
- PD1-6 PD1.6f: 5' CAT CCT ACG GTC CCA AGG TCA 3'
- PD1.6r 5' TGT GTG GAT GTG AGG AGT GGA TAG 3', 267bp (153+114), Ndel. All primers and probes were synthesized by Interactiva (Interactiva Division, ThermoHybaid).
- TFSEARCH database was used to predict potential transcription factor binding sites in the different promoter and intronic sequences of PD-1.
- Nuclear extracts from Jurkat T cells was prepared according to established methodology 39 .
- EMSA was performed using P -labelled ds-oligonucleotides (10 fmole) with the same specific activity for PD1.3G: 5' gat ctC CCA CCT GCG GTC TCC GG 3' and PD1.3A: 5' gat ctC CCA CCT GCA.GTC TCC GG 3', in an DNA-binding reaction [2 mM HEPES (pH 7.9), 10 mM Tris-HCl (pH 7.5), 25 mM NaCl, 10 mM KCI, 1.5 mM EDTA, 0.1 mM ZnSO4, 15% glycerol, 0.25 mg/ml BSA, 0.6 mM DTT, 2 ⁇ g poly(dl-dC)] for 20 min on ice, separated on 6% PAGE and visualized by autoradiography.
- the complete intron 4 (560bp) sequence from the PD1.3A or PD1.3G alleles were amplified by PCR and the fragments were cloned into the BamHl site of a pGL3-promoter vector containing the SV40 viral promoter (Promega).
- a pGL3-promoter vector containing the SV40 viral promoter Promega.
- PBMC peripheral blood mononuclear cells
- PDITaqManR 5' AAT CCA GCT CCC CAT AGT CCA 3' and the PD1 probe was: 5'FAM-AGA GAA CAC AGG CAC GGC TGA GGG- TAMRA 3'. 2nM MgCI 2 , 40nM of the probe and of each primer together with the TaqMan PCR Core Reagents kit (PE Biosystems) were used for the PD-1 assay. Human ⁇ 2-microglobulin (PE Biosystems) was used as an endogenous control as recommended. Samples were run and analysed in the Sequence Detection system, ABI Prism 7700 (PE Biosystems). Samples were run in triplicates from which the mean was calculated. Ct values were used to determine differences in the expression of PD-1 and ⁇ 2-microglobuIin, and then converted into x-folds. Statistical analysis
- Linkage analysis was performed using the MLINK routine of the ANALYZE software as described previously 9,10 and developed by Joseph Terwilliger 40 . All linkage analyses were performed using an "affected-only" analysis, with a dominant mode of inheritance and a disease gene frequency of 0.002. Association analysis of the major haplotype with SLE in the multicase and single case families (trios) was performed using the TDT routine of ANALYZE. Otherwise association was tested with 2x2 contingency tables and X 2 analysis. P values were calculated using the Fisher's exact test. Multiple testing was conservatively corrected with the Bonferroni method.
- the complete sequence of the PD-1 gene has been deposited in GeneBank with the accession number AF363458.
- the databases used were www.ensemble.org and ncbi.nlm.nih.gov, www.celera.com and (http://molsun1.cbrc.aist.go.ip/research/db/TFSEARCH.html.
- FISH for the PD-1 gene (10 kb) and for a joint probe for the ESTs L16991 and AB023160 (20 kb) on metaphase and interphase chromosomes was performed as described 37 .
- the complete PD-1 gene (9.6 kb) was sequenced and 7 SNPs were detected and genotyped by RFLP, DASH adapted for FRET signal generation 38,44 or sequencing (primers and probes available upon request).
- Nuclear extracts from Jurkat T cells was prepared as described 39 .
- EMSA was performed using 32 P-labelled ds-oligonucleotides (10 fmole) with the same specific activity for PD1.3G:5' gat ctC CCA CCT GCG GTC TCC GG3 ' and PD1.3A: 5' gat ctC CCA CCT GCA GTC TCC GG3 ' , in a DNA-binding reaction [2 mM HEPES (pH 7.9), 10 mM Tris-HCl (pH 7.5), 25 mM NaCl, 10 mM KCI, 1.5 mM EDTA, 0.1 mM ZnSO , 15% glycerol, 0.25 mg/ml BSA, 0.6 mM DTT, 2 ⁇ g poly(dl.dC)] for 20 min on ice, separated on 6% PAGE and visualized by autoradiography.
- the complete intron 4 (560 bp) containing allele PD1.3A or PD1.3G was cloned into a BamHl site of the pGL3 promoter vector (Promega).
- Jurkat cells (3x10 6 ) were transiently co-transfected with 0.4 ⁇ g of the pGL3 constructs and 0.1 ⁇ g of ⁇ -actin-LacZ reporter using Effectene (Qiagen, Valencia, CA). Cells were activate by a combination of PMA (phorbo!-12-myristate-13-acetate, 20ng/ml) and Ionomycin (0.5 ⁇ M) (Sigma) for 10 hours
- Primers and probes were designed to cover exon-exon borders in cDNA and therefore amplification cannot be achieved from genomic DNA.
- the primers and probes were: PD1 aqManF:5' CCA GCC CTG AAG GAG 3 ' .
- PDITaqManR 5 ' AAT CCA GCT CCC CAT AGT CCA 3' and the PD1 probe was: 5 AM-AGA GAA CAC AGG CAC GGC TGA GGG-TAMRA 3 ' .
- PCR was run as recommended (PE Biosystems) with 2nM MgCI 2 , and 40nM of each primers and probe.
- AFBAC analysis was performed as described 41 . Alleles of all SNPs were confirmed to be in Hardy-Weinberg equilibrium in non-affected sibs in all families (no transmission distortion was observed). Association was tested with 2x2 contingency tables and X 2 analysis. Relative risk (RR) was calculated with 95% confidence intervals. Expression and transfection assays were analysed with an unpaired Student's t-test.
- Finger LR et al. The human PD-1 gene: complete cDNA, genomic organization, and developmentally regulated expression in B cell progenitors. Get7e 197,177-187(1997).
- Ghozi MC Bernstein Y, Negreanu V, Levanon D and Groner Y. Expression of the human acute myeloid leukemia gene AML1 is regulated by two promoter regions. Proc Natl Acad Sci (USA) 93, 1935.1940 (1996).
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Abstract
Cette invention est fondée au moins partiellement sur l'identification de la structure génomique du gène PD-1 humain et sur l'identification des zones polymorphes à l'intérieur de ce gène. Cette invention concerne par conséquent des acides nucléiques comportant une séquence nucléotidique codant des variants du gène PD-1 et concerne également des acides nucléiques contenant un promoteur de PD-1, un intron, un exon et des séquences UTR 3', ainsi que des produits d'expression. Cette invention se rapporte également à des procédés servant à identifier des allèles spécifiques des zones polymorphes d'un gène PD-1, à des procédés permettant de déterminer si un sujet est atteint ou risque de développer une maladie qui est associée à un allèle spécifique d'une zone polymorphe d'un gène PD-1, ainsi qu'à des kits de réalisation de ces procédés.
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GB0121674A GB0121674D0 (en) | 2001-09-07 | 2001-09-07 | Nucleic acids encoding PD-1, polymorphs and uses thereof |
GB0121674 | 2001-09-07 | ||
US219446 | 2002-08-13 | ||
US10/219,446 US20040033497A1 (en) | 2002-08-13 | 2002-08-13 | Polymorphisms of PD-1 |
PCT/EP2002/010011 WO2003022875A2 (fr) | 2001-09-07 | 2002-09-06 | Polymorphismes de pd-1 |
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WO2005089080A2 (fr) * | 2004-03-22 | 2005-09-29 | Everygene Ab | Procede et trousse de detection, compositions pharmaceutiques et methode de traitement de troubles auto-immuns |
US8563314B2 (en) | 2007-09-27 | 2013-10-22 | Sangamo Biosciences, Inc. | Methods and compositions for modulating PD1 |
WO2009042163A2 (fr) | 2007-09-27 | 2009-04-02 | Sangamo Biosciences, Inc. | Identification in vivo rapide de nucléases biologiquement actives |
US11235026B2 (en) | 2007-09-27 | 2022-02-01 | Sangamo Therapeutics, Inc. | Methods and compositions for modulating PD1 |
EP3763810A3 (fr) | 2012-10-10 | 2021-07-14 | Sangamo Therapeutics, Inc. | Composés de modification de cellules t et leurs utilisations |
SG11201706947TA (en) | 2015-03-02 | 2017-09-28 | Innovative Cellular Therapeutics Co Ltd | Reducing immune tolerance induced by pd-l1 |
WO2018084706A1 (fr) * | 2016-11-04 | 2018-05-11 | Erasmus University Medical Center Rotterdam | Marqueurs d'identification de classes de patients et leur utilisation |
WO2020068500A1 (fr) * | 2018-09-26 | 2020-04-02 | Albert Einstein College Of Medicine | Variants mutants du récepteur pd-1 avec liaison sélective à pd-l1 et leurs utilisations |
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