WO2010085542A2

WO2010085542A2 - Biomarkers related to age-related macular degeneration (amd)

Info

Publication number: WO2010085542A2
Application number: PCT/US2010/021615
Authority: WO
Inventors: Martin Neuner-Jehle; Baldo Scassellati-Sforzolini; Thaddeus Dryja; Charles Paulding; Steven Lewitzky; Yunsheng He; Joanne Meyer; Lindsay Farrer
Original assignee: Novartis Ag
Priority date: 2009-01-23
Filing date: 2010-01-21
Publication date: 2010-07-29
Also published as: WO2010085542A3

Abstract

A method of determining the therapeutic outcome of treating an AMD patient with an AMD therapeutic agent is provided. The method includes obtaining a sample from the patient, and analyzing the sample to determine the existence of one or more biomarkers associated with an improved response to treatment with the AMD therapeutic agent.

Description

BIOMARKERS RELATED TO AGE-RELATED MACULAR DEGENERATION (AMD)

[0001 ] FIELD OF INVENTION

[0002] The present inventions relate to biomarkers to predict the outcome of anti-vascular endothelial growth factor (anti-VEGF) treatment in patients with age-related macular degeneration.

[0003] BACKGROUND

[0004] Age-related macular degeneration (AMD) is a degenerative disease of the macula and a common cause of irreversible vision loss. Conditions associated with AMD include small yellow deposits called drusen external to the outer retina and retinal pigment epithelium (RPE). Large numbers of drusen and damage to the RPE markedly increase the risk of complications (atrophy of the RPE and abnormal neovascularization of the outer retina), which can lead to severe vision loss. Cellular remnants and debris derived from degenerate RPE cells become sequestered between the RPE basal lamina and Bruch's membrane. It has been suggested that this cellular debris creates chronic inflammatory stimuli, and a potential "nucleation" site for drusen formation. The entrapped cellular debris then become the targets of encapsulation by a variety of inflammatory mediators, some of which are contributed by the RPE or other local cell types, and some of which are extravasated from the choroidal circulation. It is believed that key elements that contribute to AMD include drusen deposition, the combined events of cellular dysfunction and death, and the activation of immunomodulatory processes at the level of the RPE-choroid-retina complex. One set of immunomodulatory molecules which have been proposed to have a role in the development of AMD are the proteins of the complement system.

[0005] A locus for AMD (ARMDl) was reported in a single extended family linked to chromosome 1 q25.3- 31.3. Variants of the human gene encoding complement factor H (CFH) have been found to be highly associated with age-related macular degeneration (AMD). [0006] Diagnosis of AMD includes detection of symptoms such as center of vision blurring where the blurred region grows larger as the disease progresses. Symptoms also include straight lines appearing wavy and rapid central vision loss. However, a patient may not report symptoms if only one eye is affected. [0007] AMD treatments include prophylaxis with agents which slow or prevent the progression of the disease. Such therapies include laser based therapies to destroy blood vessels, such as photodynamic laser therapy (PDT or Visudyne®) or pegaptanib sodium (Macugen®). One method of treatment includes administration of an angiogenesis inhibitor. One class of angiogenesis inhibitors utilized in such a treatment includes VEGF inhibitors. Three particular VEGF inhibitors that have been used to treat AMD are pegaptanib sodium, bevacizumab (AVASTIN®) and ranibizumab (LUCENTIS®). Ranibizumab is a humanized version of muMAb VEGF A4.6.1. Neovascularization (sprouting of blood vessels from the choroid and growth into the subretinal space) occurs in the late stage of AMD (wet AMD) and leads to RPE detachment, vascular leakage, RPE and retinal atrophy, and irreversible loss of vision. These neovascular forms of AMD are treated with anti-VEGF therapies (e.g. ranibizumab or pegabtanib sodium and PDT using photosensitive agents (e.g. verteporfϊn (Visudyne®).

[0008] Several therapeutic agents are known for treatment or prophylaxis of AMD.

However, there remains a need for methods of predicting the effectiveness of particular therapies or therapeutic agents suited for a specific patient with AMD.

[0009] SUMMARY

[0010] In one aspect, the invention relates to a method of treating an age-related macular degeneration (AMD) patient. The method includes obtaining a sample from the patient, analyzing the sample to determine the existence of one or more variants associated with an improved response to treatment with an AMD therapeutic agent, and administering the AMD therapeutic agent to the patient upon a positive determination that the patient has at least one of the one or more variants associated with an improved response to treatment with the AMD therapeutic agent.

[0011] In another aspect, the invention relates to a method of determining the therapeutic outcome of treating an AMD patient with an AMD therapeutic agent. The method includes obtaining a sample from the patient, and analyzing the sample to determine the existence of one or more biomarkers associated with an improved response to treatment with the AMD therapeutic agent.

[0012] In another aspect, the invention relates to a kit for predicting the therapeutic outcome of treating an AMD patient with an AMD therapeutic agent comprising an oligonucleotide having fifteen contiguous nucleotides with at least 90% identity to fifteen contiguous nucleotides of a gene including a mutation associated with an improved response to treatment with the AMD therapeutic agent, wherein the oligonucleotide includes one or more nucleotides complementary to the mutation.

[0013] In another aspect, the invention relates to a method of treating an AMD patient. The method includes obtaining a sample from the patient, analyzing the sample to determine the existence of a Tyr402His variant of Human Complement Factor H (CFH), and administering an

AMD therapeutic agent including an anti-VEGF agent to the patient upon a positive determination that the patient has the Tyr402His variant of Human CFH.

[0014] In another aspect, the invention relates to a method of determining the therapeutic outcome of treating an AMD patient with an AMD therapeutic agent including an anti-VEGF agent.

The method includes obtaining a sample from the patient, and analyzing the sample to determine the existence of a Tyr402His variant of Human CFH.

[0015] In another aspect, the invention relates to a kit for predicting the therapeutic outcome of treating an AMD patient with an anti-VEGF agent. The kit includes an oligonucleotide having fifteen contiguous nucleotides that hybridize under conditions of high stringency to fifteen contiguous nucleotides of a nucleic acid having the sequence of SEQ ID NO: 2. The fifteen contiguous nucleotides of the nucleic acid include the sequence of SEQ ID NO: 2 at a position including nucleotide 1277 of SEQ ID NO: 2.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The following detailed description of the preferred embodiments will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It is understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings:

[0018] Figure 1 illustrates a sequence of a CFH nucleic acid (SEQ IDNO: 1) encoding a wild type CFH protein.

[0019] Figure 2 illustrates a sequence of a CFH nucleic acid (SEQ ID NO: 2) encoding a

Tyr402His variant of the CFH protein.

[0020] Figure 3 illustrates a sequence of a wild type CFH protein (SEQ ID NO: 3). [0021] Figure 4 illustrates a sequence of a Tyr402His variant of the CFH protein (SEQ ID

NO: 4).

[0022] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The methods of the embodiments herein may be substituted or combined with other screening and treatment methods known to those of skill in the art.

[0024] "Biomarker," as used herein includes genes or gene expression products (e.g. proteins, polypeptides, and mRNA) which are present in a sample taken from a patient and associated with a differential or improved response to an AMD therapeutic agent.

[0025] "Sample" as used herein includes a sample from a patient obtained for the purposes, including but not limited to, identification, diagnosis, prediction, or monitoring. Preferred samples include blood, serum, plasma, lymph, urine, tear, saliva, cerebrospinal fluid, leukocyte or tissue samples. In addition, samples can be subjected to further processing including fractionation or purification, for example, by isolation of nucleic acid.

[0026] "Age-related macular degeneration patient" refers to an individual that has age-related macular degeneration or has a predisposition to developing age-related macular degeneration.

[0027] "Antibody," as used herein, includes but is not limited to an immunoglobulin protein that is capable of binding an antigen or fragments thereof, including but not limited to F(ab')2, Fab',

Fab, and Fv fragments, which are capable of binding the antigen or antigenic fragment of interest.

[0028] "Isolated nucleic acid," "isolated polynucleotide," "isolated DNA," or "isolated

RNA," as used herein includes but is not limited to, the referenced nucleic acid separated from the organism from which it originates or from the naturally occurring genome, location, or molecules with which it is normally associated.

[0029] "Isolated protein," "isolated polypeptide," or "isolated peptide," as used herein includes, but is not limited to, a protein, polypeptide, or peptide separated from the organism from which it originates or from the naturally occurring location, or molecules with which it is normally associated. [0030] "Differential response," as used herein, means that the patient has a variant of a gene or gene product such that the patient's response to an AMD therapeutic agent would be different than the response achieved with a patient that did not have the particular variant. [0031 ] "Improved response," as used herein, means that the patient has a variant of a gene or gene product such that the patient's response to an AMD therapeutic agent results in a greater improvement in AMD symptoms than the response achieved with a patient that did not have the particular variant.

[0032] "AMD therapeutic agent," as used herein, includes any agent or combination of agents delivered to treat AMD. An AMD therapeutic agent can include but is not limited to verteporfin in combination with PDT, an anti-VEGF agent(s), pegaptanib sodium, bevacizumab, ranibizumab, zinc, or an antioxidant(s), alone or in any combination. An AMD therapeutic agent can also include pharmaceutically acceptable carriers.

[0033] "Increased response rate" with respect to a biomarker means that more patients having a particular variant in a gene or gene product experience an improvement in AMD conditions after treatment with a particular AMD therapeutic agent relative to patients that have a different variant at the same position that received the same AMD therapeutic agent.

[0034] "Variant," as used herein, refers to a particular wild type or mutant disposition in a gene(s) or gene product(s).

[0035] In an embodiment, methods of improving the treatment response in an age-related macular degeneration patient are provided. In a preferred embodiment, the patient has the neovascular form of AMD called wet AMD. The methods include obtaining a sample from the patient, analyzing the sample to determine whether the patient has one or more variants associated with an improved response to an AMD therapeutic agent, and administering the AMD therapeutic agent to the patient if the patient has at least one of the one or more variants associated with an improved response to the AMD therapeutic agent. In a preferred embodiment, the AMD therapeutic agent includes an anti-VEGF agent. In a more preferred embodiment, the AMD therapeutic agent is ranibizumab. The presence of the T1277C polymorphism in the human complement factor gene, which corresponds to position 1277 in SEQ ID NO: 1 and SEQ ID NO: 2, is predictive of an improved response when ranibizumab is selected as the AMD treatment. It was found that patients having a CC genotype at this position showed an increased response rate to treatment with ranibizumab in comparison to patients with the CT genotype. In an even more preferred embodiment, the genotype CC at this position of CFH is predictive of a higher level increase in the response rate to ranibizumab compared to the CT genotype. However, both the CC and CT genotypes are predictive of an increase in response rate compared to the TT wild type. The sequence of a wild type CFH gene is listed in SEQ ID NO: 1 and the sequence of a CFH gene including a variant C at position 1277 is listed in SEQ ID NO: 2.

[0036] In an embodiment, methods to predict the therapeutic outcome of treating a patient with an AMD therapeutic agent are provided. In a preferred embodiment, the patient has the neovascular form of AMD called wet AMD. The methods include obtaining a sample from the patient and analyzing the sample to determine whether the patient has one or more variants associated with a differential response to an AMD therapeutic agent. In a preferred embodiment the AMD therapeutic agent includes an anti-VEGF agent. In a more preferred embodiment, the AMD therapeutic agent includes ranibizumab. In a still more preferred embodiment, the presence of a T 1277C polymorphism in the CFH gene, which corresponds to position 1277 in SEQ IDNO: 1 and SEQ ID NO: 2, is predictive of an increased response rate when the selected AMD therapeutic agent includes administration of ranibizumab. It was found that patients having a CC genotype at this position showed an increased response rate to treatment with ranibizumab in comparison to patients with the CT genotype. In an even more preferred embodiment, the genotype CC at this position of CFH is predictive of a higher level increase in the response rate to ranibizumab compared to the CT genotype. However, both the CC and CT genotypes are predictive of an increase in response rate as compared to the TT wild type. The sequence of a wild type CFH gene is listed in SEQ ID NO: 1 and the sequence of a CFH gene including a variant C at position 1277 is listed in SEQ IDNO: 2. [0037] It has been determined that the (Tyr402His) variant CFH gene is a prognostic marker predicting an improved treatment response when ranibizumab is administered to patients with AMD. The sequence of a CFH including Tyr at position 402 is listed in SEQ ID NO: 3 and sequence of a CFH Tyr402His variant is listed in SEQ ID NO: 4.

[0038] Improved treatment outcome can be measured by monitoring patient vision with a visual acuity test, examples of which have been described in the literature. See Ricci et al. Opthalmic Epidemiology 5(1): 41 - 43 (1998), which is incorporated by reference as if fully set forth. In a preferred embodiment, the visual acuity test is measured by the ETDRS or Snellen method. Other clinical parameters can also be used to assess the treatment outcome. The following list provides examples of such clinical parameters (methods): central retinal thickness (measured by optical coherence tomography); visual field, contrast, or speed sensitivity (measured by perimetry); reading speed; vascular leakage (assessed by fluorescein or indocyanine green angiography); or neuronal activity (measured by electroretinography).

[0039] In an embodiment, the methods involve using a probe to determine whether a patient has a variant genotype or phenotype (e.g. mutations, polymorphisms, single nucleotide polymorphisms (SNPs), alternate splicing, or post-translational modifications) associated with differential or improved responses to AMD therapeutic agents. The probe can be any moiety that preferentially binds to a gene or gene product associated with a differential or improved response to an AMD therapeutic agent. The probe can also be a moiety that is processed by the gene or gene product associated with a differential or improved response to an AMD therapeutic agent. In preferred embodiments, the probe can be one or more of a nucleic acid, a DNA, an RNA, an aptamer, a ribozyme, an antibody, an enzymatic substrate, or an enzyme. In a preferred embodiment, the AMD therapeutic agent is an anti-VEGF agent, more preferably an anti-VEGF antibody, and still more preferably ranibizumab.

[0040] In an embodiment, the methods involve sequencing a gene or gene product to determine whether a patient has a variant genotype or phenotype (e.g. mutations, polymorphisms, single nucleotide polymorphisms (SNPs), alternate splicing, or post-translational modifications) associated with differential or improved responses to AMD therapeutic agents. In a preferred embodiment, the AMD therapeutic agent is an anti-VEGF agent, more preferably and anti-VEGF antibody, and still more preferably ranibizumab.

[0041 ] In an embodiment, CFH, Complement Component 3 (C3), Complement Component 2

(C2), Factor B (BF), and Age-Related Maculopathy Susceptibility 2 (ARMS2) gene or gene products are examined to determine if a patient has one or more variants associated with a differential or improved response to an AMD therapeutic agent. In a preferred embodiment, one or more of the following variants are considered in the examination: CFH Tyr402His, C3 ArglO2Gly, C2 Asp318Glu, C2 Intron 10, BF Lys565Glu, BF Arg32Gln, BF His9Leu, BF Arg31Trp, BF b_rail7, BF Argl 50Arg, and ARMS2 Ala69Ser. The patient can also be examined to determine the genotype of the variant. Particular genotypes that may be screened for include one or more of the following: CFH Tyr402His (CC/CT/TT), C3 Argl 02GIy (CC/CG/GG), C2 Asp318GIu (CG/GG), C2 Intron 10 (AC/CC), BF Lys565Glu (AA/AG), BF Arg32Gln (CC/CT), BF His9Leu (AT/TT), BF Arg3 lTφ (CC/CT/TT), BF Intron 17 (CC/CT/TT), BF Argl50Arg (AA/AG/GG), and ARMS2 Ala69Ser (GG/GT/TT). In a preferred embodiment, the AMD therapeutic agent is an anti-VEGF agent, more preferably an anti-VEGF antibody, and still more preferably ranibizumab. Sequences coding for a C3 and ARMS2 wild type can be found at Accession Nos. NM_000064 and NM_001099667, respectively. Sequences coding for a CFH Tyr402His, C2 Asp318Glu, and BF His9Leu can be found at Accession Nos. CR616715, NM 000063, and NP 000054, respectively. Protein sequences of a C3 and ARMS2 wild type can be found at Accession Nos. NP_OOOO55 and NP_001093137, respectively. Protein sequences of a CFH Tyr402His, C2 Asp318GIu, and BF His9Leu can be found at Accession Nos. NP_000177 and NP_000054, and NP OO 1701 , respectively. The C2 Intron 10 and BF Intron 17 polymorphisms can be found at Accession Nos. RS547154 and RS2072633, respectively. Corresponding wild type or mutant nucleic acid or protein sequences can be readily determined based on any of these references sequences.

[0042] A biomarker can be a variant indicating a differential or improved response to an

AMD therapeutic agent and can be a polymorphism or mutation within a coding portion of a gene, preferably the CFH gene, hi a preferred embodiment, the biomarker is a polymorphism or mutation which encodes an amino acid residue located within a SCR of CFH; more preferably, the SCR is SCR7; and still more preferably, the polymorphism of the CFH gene causes a change from tyrosine at amino acid 402 of CFH; and still more preferably, the polymorphism changes a tyrosine at position 402 to a histidine.

[0043] A biomarker can be a variant indicating a differential or improved response to an

AMD therapeutic agent and can be a polymorphism or mutation within a non-coding region of a gene. Non-coding regions include, for example, intron sequences as well as 5' and 3' untranslated sequences.

[0044] Detection of Biomarkers

[0045] In an embodiment, determining whether a patient has one or more variants associated with an improved response to an AMD therapeutic agent includes determining the content of a gene product. This can be readily done by standard methods such as, but not limited to, binding of antibodies that recognize specific epitopes associated with a differential or improved response to an AMD therapeutic agent. Preferably, the antibody discriminatively binds to either the wildtype CFH or the Tyr402His CFH.

[0046] In an embodiment, a biomarker is detected using a nucleic acid hybridization assay.

In a nucleic acid hybridization assay, the presence or absence of the biomarker is determined based on the ability of the nucleic acid from the sample to hybridize to a complementary nucleic acid molecule (e.g., an oligonucleotide or polynucleotide probe). A variety of hybridization assays are available. Other assays for analyzing gene products include, but are not limited to, protein sequencing, protein fragmentation profiles, and nucleic acid sequencing of, for example, RNA. [0047] Oligonucleotides or polynucleotides used as hybridization probes or primers are not limited to but may have a length in the range from 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20 or 10 to 15 nucleotides, preferably from 20 to 30 nucleotide residues, and more preferably having a length of 25 nucleotide residues. In an embodiment, the hybridization probes or primers are 85 to 100%, 90 to 100%, 93.3 to 100%, or 95 to 100% complementary to a nucleic acid having the sequence of a biomarker gene or transcript along the length of the hybridization probe. In an embodiment, the hybridization probes or primers are 85 to 100%, 90 to 100%, 93.3 to 100%, or 95 to 100% complementary to a nucleic acid having the sequence of either SEQ ID NO: 1 or SEQ ID NO: 2 along the length of the hybridization probe or primer. Further, a probe or primer with a length selected from the lengths listed above may be complementary to this degree to any corresponding length of a nucleic acid having the sequence of either SEQ ID NO: 1 or SEQ ID NO: 2. In an embodiment, the hybridization probes or primers hybridize to a nucleic acid having the sequence of a biomarker gene or transcript under conditions of low, moderate or high stringency. In an embodiment, the hybridization probes or primers hybridize to a nucleic acid having the sequence of either SEQ ID NO: 1 or SEQ ID NO: 2 under conditions of low, moderate or high stringency. Further, a probe or primer with a length selected from the lengths listed above may be hybridized to this degree to any corresponding length of a nucleic acid having either SEQ ID NO: 1 or SEQ ID NO: 2.

[0048] In an embodiment, one or more nucleic acids having a sequence with at least 75, 80,

85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a nucleic acid having the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or the complement thereof is provided as a biomarker, detection reagent, or a control. In another embodiment, one or more nucleic acids having a sequence with at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a nucleic acid having the sequence of SEQ IDNO: 1 or SEQ IDNO: 2 along 10 to 50, 10 to 100, 10 to 150, 10 to 300, 10 to 600, 10 to 1200, 10 to 1300, 10 to 1400, 10 to 1500, 10 to 1600, or 10 to 1642 nucleotides of a nucleic acid having the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or the complement thereof is provided as a biomarker, detection reagent, or a control.

[0049] In an embodiment, one or more proteins having a sequence with at least 75, 80, 85,

90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a protein having the sequence of SEQ ID NO: 3 or SEQ ID NO: 4 is provided as a biomarker, detection reagent, or a control. In another embodiment, one or more proteins having a sequence with at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a protein having the sequence of SEQ ID NO: 3 or SEQ ID NO: 4 along 10 to 50, 10 to 100, 10 to 150, 10 to 300, 10 to 600, or 10 to 1201 amino acids of one of a protein having the sequence of SEQ ID NO: 3 or SEQ ID NO: 4 is provided as a biomarker, detection reagent, or a control.

[0050] By way of example, but not limitation, procedures using conditions of low stringency are as follows: filters containing DNA are pretreated for 6 h at 4O⁰C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 10⁶ cpm ³²P-labeled or fluorescently labeled probes can be used. Filters are incubated in hybridization mixture for 18-20 h at 40⁰C and then washed for 1.5 h at 55⁰C in a solution containing 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60⁰C. Filters are blotted dry and exposed for development in an imager or by autoradiography. If necessary, filters are washed for a third time at 65-68⁰C and re-exposed for development. [0051] By way of example, but not limitation, high stringency refers to hybridizing conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1 % SDS at 5O⁰C; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1 % FicolI/0.1 % polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42⁰C; or (3) employ 50% formamide, 5X SSC (0.75 M NaCl, 0.075 M sodium pyrophosphate), 5X Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42⁰C, with washes at 42⁰C in 0.2X SSC and 0.1% SDS. [0052] One of ordinary skill in the art will recognize that moderate stringency lies between low and high stringency. For example, moderate stringency includes hybridizing in 3X SSC at 42⁰C. [0053] Oligonucleotides or polynucleotides used as sequencing primers are not limited to but may have a length in the range from 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20 or 10 to 15 nucleotides, preferably from 20 to 30 nucleotide residues, and more preferably having a length of 25 nucleotide residues.

[0054] Oligonucleotides or polynucleotide probes or sequencing primers may be deoxyribonucleic acids, ribonucleic acids, peptide nucleic acids or hybrids thereof. The oligonucleotides or polynucleotides may include synthetic nucleotides in whole or in part. [0055] Although non-labeled oligonucleotide probes can be used in the embodiments herein, the probes are preferably detectably labeled. Primers can also be labeled. Exemplary labels include, but are not limited to, radionuclides, light-absorbing chemical moieties, dyes, and fluorescent moieties. Preferably, the label is a fluorescent moiety, such as 6-carboxyfluorescein (FAM), 6- carboxy-4,7,2',7'-tetrachlorofluoroscein (TET), rhodamine, JOE (2,7-dimethoxy-4,5-dichloro-6- carboxyfluorescein), HEX (hexachIoro-6-carboxyfluorescein), or VIC.

[0056] In an embodiment, solid supports containing oligonucleotide probes for identifying the biomarkers are provided. The solid supports can be filters, polyvinyl chloride dishes, silicon or glass based chips, etc. Any solid surface to which oligonucleotides can be bound, either directly or indirectly, either covalently or noncovalently, can be used.

[0057] A preferred solid support includes an array of oligonucleotide probes where different oligonucleotides in the array are capable of hybridizing to different biomarkers. In one embodiment, the array is a matrix in which each position represents a discrete binding site for a biomarker, preferably a biomarker binding site is capable of binding a biomarker corresponding to a CFH, C3, C2, BF, or ARMS2 gene or transcripts. More preferably, the biomarker binding site is capable of binding a biomarker corresponding to a gene or transcripts encoding CFH Tyr402His, C3 ArglO2G)y, C2 Asp318Glu, C2 Intron 10, BF Lys565Glu, BF Arg32Gln, BF His9Leu, BF Arg3 ITrp, BF Intron 17, BF Argl 50Arg, or ARMS2 Ala69Ser mutations. Still more preferably, the array includes binding sites that in combination can be used to distinguish between the alleles of CFH Tyr402His (CC/CT/TT), C3 Argl 02GIy (CC/CG/GG), C2 Asp318GIu (CG/GG), C2 Intron 10 (AC/CC), BF (AA/AG), BF Arg32Gln (CC/CT), BF His9Leυ (AT/TT), BF Arg3 ITrp (CC/CT/TT), BF Intron 17 (CC/CT/TT), BF Argl50Arg (AA/AG/GG), and ARMS2 Ala69Ser (GG/GT/TT). [0058] In an embodiment, a biomarker is detected by analysis of proteins, polypeptides, or peptides. Immunoassay devices and methods are often used to detect proteins, polypeptides, or peptides. These devices and methods can utilize labeled molecules in various sandwich, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of the biomarker of interest. Additionally, certain methods and devices, such as biosensors and optical immunoassays, may be employed to determine the presence or amount of analytes without the need for a labeled molecule.

[0059] The presence or amount of a protein, polypeptide, or peptides can be determined using specific antibodies and detecting specific binding. Any suitable immunoassay may be utilized, for example, enzyme-linked immunoassays (ELISA), radioimmunoassays (RIAs), competitive binding assays, and the like. Specific immunological binding of the antibody to the protein or polypeptide can be detected directly or indirectly. Direct labels include, but are not limited to, fluorescent or luminescent tags, metals, dyes, and radionuclides attached to the antibody. Indirect labels include various enzymes well known in the art, including but not limited to alkaline phosphatase, and horseradish peroxidase.

[0060] The use of immobilized antibodies specific for a protein, polypeptide, or peptide is also contemplated in the present embodiments. The antibodies can be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay place (such as microtiter wells), and pieces of a solid substrate material (such as plastic, nylon, paper). An assay strip can be prepared by coating the antibody or a plurality of antibodies in an array on a solid support. This strip can then be dipped into the test sample and then processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot. [0061] Analysis of biomakers through detection of proteins, polypeptides, or peptides can also take place on an array of detection moieties. In one embodiment, the array is a matrix in which each position represents a discrete binding site for a biomarker. Preferably, a biomarker binding site is capable of binding a biomarker corresponding to a CFH, C3, C2, BF, or ARMS2 protein, polypeptide, or peptide. More preferably, the biomarker binding site is capable of binding a biomarker corresponding to a protein, polypeptide, or peptide including the CFH Tyr402His, C3 ArglO2Gly, C2 Asp318Glu, C2 Intron 10, BF Lys565GIu, BF Arg32Gln, BF His9Leu, BF Arg31Trp, BF Intron 17, BF ArglSOArg, or ARMS2 Ala69Ser mutations. [0062] The analysis of a plurality of genes, proteins, polypeptides, or peptides may be carried out separately or simultaneously with one test sample. The analysis of biomarkers can be carried out in a variety of physical formats as well. For example, the use of microtiter plates or automation could be used to facilitate the processing of large numbers of test samples in a high through-put manner.

[0063] In an embodiment, the step of determining includes a method for determining whether a patient is homozygous for a polymorphism, heterozygous for a polymorphism, or lacking the polymorphism altogether (i.e., homozygous wildtype). For example, for the Tyr402His polymorphism, a method for determining whether the patient is homozygous CC, heterozygous CT, or homozygous wild type TT with respect to this polymorphism in the human CFH gene is provided. [0064] Numerous additional methods and devices are well known to the skilled artisan for the detection and analysis of biomarkers. Any approach that detects mutations or polymorphisms in a gene or gene product can be used in the embodiments herein. The additional approaches include, but are not limited to, single-strand conformational polymorphism (SSCP) analysis (Orita, M. et al. Proc. Natl. Acad. Sci. USA 86: 2766-2770 (1989)), heteroduplex analysis (Prior, T.W. et al. Hum. Mutat. 5: 263-268 (1995)), oligonucleotide ligation (Nickerson D.A. et al. Proc. Natl. Acad. Sci. USA 87: 8923-8927 (1990)), artificial mismatch hybridization (U.S. Patent No. 5,780,233) and hybridization assays (Conner BJ. et al. Proc. Natl. Acad. Sci. USA 80: 278-282 (1983)). Traditional Taq polymerase PCR-based strategies, such as PCR-RFLP, allele-specific amplification (ASA) (Ruano G. et al. Nucleic Acids Res. 17: 8392 (1989)), single-molecule dilution (SMD) (Ruano G. et al. Proc. Natl. Acad. Sci. USA 87: 6296-6300 (1990)), and coupled amplification and sequencing (CAS) (Ruano G. et al. Nucleic Acids Res. 19: 6877-6882 (1991)) are also contemplated. These references are incorporated by reference as if fully set forth.

[0065] Kits for predicting the response to an AMD therapeutic agent or designing a method of treating a patient with AMD

[0066] In an embodiment, a kit is provided for predicting the therapeutic outcome of treating a patient with an AMD therapeutic agent, preferably ranibizumab. Such a kit preferably comprises devices and reagents for the analysis of at least one test sample and instructions for performing the assay. Optionally the kits may contain one or more means for converting detection of a biomarker to a prediction of the therapeutic outcome. Control samples may be included in the kit so that patient sample assay results can be compared to control sample assay results. The controls can be positive or negative controls. In an embodiment, both positive and negative controls are included in the kit. [0067] In an embodiment, the kits contain one or more antibodies specific for binding to one or more biomarkers corresponding to CFH, C3, C2, BF, or ARMS2 proteins, polypeptides, or peptides. More preferably, the kit includes one or more antibodies specific for binding to biomarkers corresponding to proteins, polypeptides, or peptides including one or more of the CFH Tyr402His, C3 ArglO2Gly, C2 Asp318Glu, C2 Intron 10, BF Lys565Glu, BF Arg32Gln, BF His9Leu, BF Arg31Trp, BF Intron 17, BF Argl50Arg, or ARMS2 Ala69Ser mutations. [0068] In an embodiment, the kits contain one or more oligonucleotides or polynucleotides specific for biomarkers corresponding to one or more of CFH, C3, C2, BF₇ or ARMS2 genes or transcripts. More preferably, the kit includes one or more oligonucleotides or polynucleotides specific for genes or transcripts encoding biomarkers corresponding to one or more of the CFH Tyr402His, C3 ArglO2Gly, C2 Asp318Glu, C2 Intron 10, BF Lys565Glu, BF Arg32Gln, BF His9Leu, BF Arg31Trp, BF Intron 17,BF Argl50Arg, or ARMS2 Ala69Ser mutations. Preferably, the oligonucleotides are can be used to discriminate between the CC, CT, and TT genotypes in a CFH gene. In an embodiment the one or more oligonucleotide or polynucleotide can be probes. In another embodiment, the one or more oligonucleotide or polynucleotide can be primers. In another embodiment, the oligonucleotides or polynucleotides include probes and primers. [0069] In preferred embodiments, the kits contain all of the components necessary to perform a detection assay(s), including all controls and instructions for performing assays and for analysis of results. In one embodiment, a kit contains instructions including a statement of intended use as required by the Environmental Protection Agency or U.S. Food and Drug Administration (FDA) for the labeling of in vitro diagnostic assays and/or of pharmaceutical or food products. [0070] Example

[0071] A clinical trial including a 1-year study of three regimens of ranibizumab for the treatment of subfoveal choroidal neovascularization (CNV) secondary to AMD was conducted. The primary objective was to demonstrate the non-inferiority of particular dosing regimens of ranibizumab versus continuous monthly dosing of 0.3 mg ranibizumab. The particular dosing regimens were defined as A, B, C, A + B, and A + B + C, where A = 0.3 mg ranibizumab quarterly dosing, B — 0.5 mg ranibizumab quarterly dosing, and C = 0.3 mg ranibizumab monthly dosing. The primary clinical endpoint was a change from baseline of a best-corrected visual acuity (BCVA) to 12 months. The clinical variables assessed were the mean value of BCVA at baseline for the study eye, mean change in BCVA from baseline to 12 months for the study eye, and an extreme response of gain or loss of at least 15 letters from baseline to 12 months for the study eye. [0072] The 0.3 mg and 0.5 mg quarterly dosing regimens of ranibizumab showed a similar efficacy in the study. The continuous monthly dosing of 0.3 mg ranibizumab was significantly more efficacious than the 0.3 mg and 0.5 mg quarterly dosing regimens. The two arms of the 0.3 mg and 0.5 mg quarterly dosing were, therefore, combined in the pharmacogenetic analysis. The 0.3 mg monthly dosing was analyzed independently. In addition, a combined analysis of all treatment arms was performed in an effort to maximize the statistical power. All statistical tests were two-tailed. [0073] Primary analysis

[0074] The primary analysis was to identify an association between genetic factors and the primary clinical endpoint, the mean change in the best-corrected visual acuity (BCVA) from baseline to 12 months. A linear regression model was used, and an additive genetic model was evaluated. Age, gender, treatment and baseline value were included in the model as covariates. [0075] To determine whether the genotyped population is representative of the overall clinical study, demographic characteristics and the primary efficacy variable BCVA were compared between the two populations. The results are summarized in TABLE I, below. There were a total of 334 Caucasian patients with the primary endpoint data in the overall clinical study. 114 patients were genotyped, which represents approximately 34% of the overall study. The demographic characteristics and baseline BCVA appeared similar between the two populations. For the change from baseline BCVA, relatively more positive changes were seen in the genotyped population compared to the overall study (7.0 vs 5.1). In addition, the monthly dosing regimen showed higher efficacy than the quarterly dosing regimens in both populations. TABLE I, below, shows a comparison of the genotyped population versus the overall clinical study. TABLE I

[0076] Patients participating in the study were asked to provide a separate consent for participation in pharmacogenetic analysis. A total of 116 patients consented for the pharmacogenetic assessment and were genotyped for 11 genetic variants in 5 candidate genes. Among the 116 patients genotyped, 2 were non-Caucasians and they were excluded in the association analysis. The genomic DNA of each patient was extracted from the blood by BARC (Lake Success, NY) using the PUREGENE™ DNA Isolation Kit (D-50K) (Gentra, Minneapolis, MN) and subsequently genotyped. A total of 116 DNA samples were genotyped for the genetic variants selected. Genotyping was performed using TaqMan™ Assays-by-Design and Assays-on-Demand™ (Applied Biosystems, Foster City, CA) on an ABI 7900 sequencer. Genotyping used 1 ng of genomic DNA according to the manufacturer's instructions.

[0077] The genes and genetic variants selected for this analysis include those that have been shown to be strongly associated with AMD and are listed in TABLE II, below. The allele frequencies of each variant are also summarized in TABLE II, below.

TABLE II Genotype counts and allele frequencies

[0078] Because none of the genetic variants tested deviated from Hardy- Weinberg equilibrium with p < 0.05 in a chi-square test, no evidence of genotyping failure or population admixture was seen.

[0079] The primary analysis testing for presence of an association between genetic factors and the primary clinical endpoint using linear regression was performed. As shown in TABLE III below, no significant association was found except the Arg31Trp variant in the BF gene, which showed an association with the primary endpoint in the monthly dosing arm (p=0.02). However, this association disappeared in the quarterly dosing arms (p=0.36). An opposite numerical trend was noted between the monthly dosing arm and the quarterly dosing arms. It is likely that this association was due to random chance. In addition, the Arg31Trp variant did not show any effect on the drug response in the secondary analysis, as shown in TABLE IV below. Moreover, the variants Lys565Glu and Arg32Gln in the BF gene and the intronic polymorphism in the C2 gene showed a trend of association with the primary endpoint. However, this trend was not seen in the secondary analyses. (See TABLE IV, below).

TABLE III

Influence of genetic variants on primary clinical endpoint and baseline BCVA levels

Gene Variant BCVA Treatment Mean value by genotype P value*

CC CT TT

Baseline A+B+C n=33 56.85 (12.94) n=55 57.36 (12.67) N=26 59.46(10.32) 0.66 A+B n=20 3.95(11.47) n=38 6.74(10.35) N=20 2.45 (12.22) 0.35

CFH Tyr402His Change from baseline

14.08(13.50) n=17 10.35(8.15) N=6 8.50(14.92) at month 12 C n=13 0.31 A+B+C n=33 7.94(13.10) n=55 7.85 (9.79) N=26 3.85 (12.84) 0.54

CC CG GG

Baseline A+B+C n=71 59.20(11.27) n=34 54.24 (14.22) N=8 60.50(8.16) 0.17

C3 A+B n=48 5.56(11.68) n=25 3.76 (9.45) N=4 2.50(17.02) 0.71

ArglO2Gly Change from baseline at month 12 C n=23 9.87(12.83) n=9 14.00 (9.38) N=4 14.25 (4.35) 0.45 A+B+C n=71 6.96(12.14) n=34 6.47(10.36) N=8 8.38(13.10) 0.95

CG GG

Baseline A+B+C n=3 55.33 (8.96) n=109 57.84(12.35) 0.97

C2 Asp318Glu A+B n=2 12.50 (3.54) n=74 4.81(11.19) 0.17 VO

Change from baseline at month 12 C n=l 11.00(0.00) n=35 11.40(11.54) 0.83 A+B+C n=3 12.00 (2.65) n=109 6.93(11.67) 0.22

AC CC

Baseline A+B+C n=9 59.78 (7.87) n=105 57.51 (12.51) 0.52 A+B n=9 9.44 (14.23) n=69 4.33 (10.67) 0.07

C2 Intron 10 Change from baseline at month 12 C n=0 n=36 11.39(11.37) A+B+C n=9 9.44 (14.23) n=105 6.75(11.37) 0.09

AA AG

Baseline A+B+C n=lll 57.83 (12.00) n=3 52.67(21.03) 0.50

BF A+B n=77 5.03(11.18) n=l -3.00 (0.00) 0.44

Lys565Glu Change from baseline at month 12 C n=34 11.67(11.59) n=2 6.50 (6.36) 0.06 A+B+C n=lll 7.06(11.67) n=3 3.33 (7.09) 0.21

CC CT

BF Arg32Gln Baseline A+B+C n=103 57.44(12.43) n=9 59.78 (7.87) 0.51

Change from baseline A+B n=67 4.24(10.77) n==9 9.44(14.23) 0.06

at month 12 C n=36 11.39 (11.37) n=0

A+B+C n=103 6.74 (11.45) n=9 9.44 (14.23) 0.09

AT TT

Baseline A+B+C n=4 53.00 (8.68) n=109 57.85 (12.36) 0.66

F A+B n=3 12.67 (2.52) n=74 4.59 (11.32) 0.12

His9Leu Change from baseline at tnonth 12 C n=l 11.00 (0.00) n=35 11.40 (11.54) 0.83

A+B+C n=4 12.25 (2.22) n=109 6.78 (11.78) 0.18

CC CT TT

Baseline A+B+C n=95 57.64 (12.42) n=13 58.54 (9,43) N=2 67.50 (2.12) 0.56

N=2

F A+B n=64 4.94 (11.18) n=9 1.11 (11.54) 11.50 (10.61) 0.36

Arg31Trp Change from baseline at month 12 C n=31 9.48 (9.93) n=4 23.75 (15.84) N=O 0.02

A+B+C n=95 6.42 (10.94) n=13 8.08 (16.42) N=2 1 1.50 (10.61) 0.49

CC CT TT

Baseline A+B+C n=46 55.52 (13.24) n=56 58.68 (12.03) N=12 61.42 (6.84) 0.29

F A+B n=32 5.53 (9.31) n=36 5.28 (10.99) N=IO 1.70 (16.76) 0.61

Intron 17 Change from baseline at month 12 C n=14 10.14 (9.98) n=20 12.05 (12.88) N=2 13.50 (6.36) 0.53

A+B+C n=46 6.93 (9.64) n=56 7.70 (12.04) N=12 3.67 (15.96) 0.85 κ>

AA AG GG O

Baseline A+B+C n=l 63.00 (0.00) n=34 59.26 (11.98) N=73 57.40 (12,13) 0,75

F A+B 2.00 (0.00) n=23 2.26 (11.79) N=49 5.47 (10.11) 0.49

ArglSOArg n=l

Change from baseline at month 12 C n=0 n=l l 12.27 (15.72) N=24 10.50 (9.07) 0.74

A+B+C n=l 2.00 (0.00) n=34 5.50 (13.79) N=73 7.12 (10.00) 0.75

GG GT TT

Baseline A+B+C n=34 56.91 (13.65) n=51 56.76 (12.01) N=29 60.24 (10.67) 0.62

KMS A+B n=23 7.61 (12.34) n=35 3.83 (10.23) N=20 3.75 (11.28) 0.39

Ala69Ser Change from baseline at month 12 C n=l l 8.27 (9.32) n=16 11.00 (14.21) N=9 15.89 (6.49) 0.50

A+B+C n=34 7.82 (11.31) n=51 6.08 (11.96) N=29 7.52 (11.45) 0.67

T population. Caucasians only. alues are mean (SD) for the study eye.

^■eatment: A = 0.3 mg quarterly dosing, B = 0.5 mg quarterly dosing, C = 0.3 mg monthly dosing

Jnear regression model. In the analysis for change from baseline, age, gender, treatment and baseline value were adjusted for. In the alysis for baseline, adjustments were made for age and gender. An additive genetic model was evaluated.

[0080] Secondary analysis

[0081] The secondary analysis tested for an association between genetic factors and drug response measured by gain or loss of at least 15 letters from baseline to 12 months. In addition, a potential association between genetic factors and baseline disease severity was also explored. A similar linear regression model was used in the analysis for baseline disease severity. Age and gender were included in the model as covariates. Due to the sample size, only descriptive statistics are provided in TABLE IV below.

TABLE IV

Influence of genetic variants on drug response measured by gain or loss of at least 15 letters from baseline to 12 months.

I *Responder = gain of at least 15 letters, Non-responder = loss of at least of 15 letters. (

[0082] It was determined that the CFH Tyr402His variant was the only genetic variant that showed an interesting numerical trend towards the association with the drug response rate (TABLE V, below). The patients with CC and CT genotypes showed greater response to ranibizumab (100% and 94%, respectively) compared to those with TT genotype (63%) in the combined treatment arms. A similar trend was seen in the monthly dosing regimen (CC/CT/TT, 100%/100%/67%) and the quarterly dosing regimens (CC/CT/TT, 100%/90%/60%) as shown in TABLE V below.

TABLE V

Influence of Tyr402His on drug response measured by gain or loss of at least 15 letters from baseline to 12 months

Genotype

Gene Variant Treatment Responder* CC CT TT

No 0 1 (10.00) 2 (40.00)

A+B Yes 3 (100.00) 9 (90.00) 3 (60.00)

No 0 0 1 (33.33)

CFH Tyr402His C Yes 7 (100.00) 6 (100.00) 2 (66.67)

No 0 1 (6.25) 3 (37.50)

A+B+C Yes 10 (100.00) 15 (93.75) 5 (62.50)

ITT population. Caucasians only

Values are number of patients (%).

*Responder = gain of at least 15 letters, Non-responder = loss of at least of 15 letters.

[0083] Furthermore, patients with CC and CT genotypes also exhibited greater mean improvement measured by the primary clinical endpoint (7.94 and 7.85, respectively) compared to those with TT genotype (3.85) (TABLE III, above). The demographic characteristics and baseline BCVA according to Tyr402His genotype are summarized in Table VI, below.

TABLE VI Demographic characteristics and baseline BCVA by Tyr402His genotype

Genotype

Variable CC (n=33) CT (n=55) TT (n=26) P value

Age 75.36 (8.03) 75.18 (7.96) 75.65 (10.18) 0.9732*

Gender, female

% 72.73 58.18 61.54 0.3959**

Baseline BCVA 56.85 (12.94) 57.36 (12.67) 59.46 (10.32) 0.6592***

ITT population. Caucasians only. Values are mean (SD). BCVA is for the study eye.

*Linear regression model

**Fisher's exact test

***Linear regression, age and gender were adjusted for.

[0084] Discussion

[0085] The Tyr402His CHF genetic variant showed a numerical trend towards an association with drug response in both primary and secondary analyses. This coding variant in the CFH gene on chromosome Iq32 is the result of a T-to-C transition at nucleotide position 1277 in exon-9 and causes tyrosine-to-histidine substitution at amino acid position 402.

[0086] Patients with CC and CT genotypes tended to have greater response rate to ranibizumab (100% and 94%, respectively) compared to those with TT genotype (63%).

Furthermore, the patients with CC and CT genotypes also tended to have greater improvement measured by change from baseline of BCVA (7.94 and 7.85, respectively) compared to those with

TT genotype (3.85).

[0087] ARMS2 Ala69Ser

[0088] In an additional study, BPD952A2309 (the "BPD" study), the influence of the

ARJVIS2 gene variant Ala69Ser on drug response was determined, as measured by clinical variables

(1) mean change in BCVA from baseline to 12 months; and (2) gain of at least 15 letters from baseline to 12 months.

[0089] BPD952A2309 had two treatment arms. For the first treatment arm (data shown in row 1 of Tables VII and VIII), all patients received 3 loading doses of Lucentis (at baseline, month 1 , and month T). Thereafter, i.e, after month 3, Lucentis was administered as needed ("as needed" refers to a determination by the treating physician).

[0090] For the second treatment arm (data shown in row 2 of Tables VII and VIII), Lucentis was again administered at baseline, month 1, and month 2, and either Verteporfin or a placebo was co-administered at baseline only. Thereafter, i.e, after month 3, Lucentis alone was administered as needed (as above, "as needed" refers to a determination by the treating physician). The results are shown in Table VII for the change in BCVA from baseline to 12 months, and in Table VIII for the gain of at least 15 letters from baseline to 12 months. TABLE VII

Influence of ARMS2 variant Ala69Ser on drug measured by mean change in BCVA from baseline to 12 months (study 2)

ITT population

Values are mean (SD) * ANCOVA, age, gender and baseline value are adjusted for

TABLE VIII

Influence of ARMS2 variant Ala69Ser on drug response measured by gain of at least 15 letters from baseline to 12 months (study 2)

[0091 ] As one can see in Tables VIl and VIH, the ARMS2 Ala69Ser polymorphism showed a clear association with Lucentis efficacy, both when Lucentis was administered alone and when Lucentis was co-administered with Verteporfin. The patients with the normal, or major, allele, G, tended to show greater improvement than those with the risk, or minor, allele, T. [0092] The inventors then re-reviewed data obtained in an earlier-performed study,

RFB002A2302 (the "RFB" study), to determine if the association between the ARMS2 Ala69Ser polymorphism and Lucentis efficacy was evident in that study as well In the RFB study, Lucentis was administered either monthly or quarterly for 12 months. The results are shown in Table IX for the change in BCVA from baseline to 12 months, and in Table X for the gain of at least 15 letters from baseline to 12 months.

TABLE IX

Influence of ARMS2 variant Ala69Ser on drug measured by mean change in BCVA from baseline to 12 months (study 1)

ITT population Values are mean (SD).

TABLE X

Influence of ARMS2 variant Ala69Ser on drug response measured by gain of at least 15 letters from baseline to 12 months (study 1)

ITT population Values are number of patients (%).

[0093] As one can see in Tables IX and X, the ARMS2 Ala69Ser polymorphism showed a similar trend toward association with Lucentis efficacy when Lucentis was administered quarterly (see Tables IX and X, row 1). The patients with the normal, or major, allele, G, tended to show greater improvement than those with the risk, or minor, allele, T. But, this trend was not evident when Lucentis was administered monthly (see Tables IX and X, row 2). However, the inventors note that the small size of the sample population who received monthly Lucentis rendered the data for this treatment arm of the "RFB" study statistically insignificant, at least when compared to the data for the other treatment arm.

[0094] Overall, as shown in Tables VII, VIH, IX and X, the ARMS2 Ala69Ser polymorphism is likely associated with Lucentis efficacy. Patients with the normal, or major, allele, G, tended to show greater improvement than those with the risk, or minor, allele, T.

[0095] It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but is intended to cover all modifications which are within the spirit and scope of the invention as defined by the appended claims and/or the above description.

Claims

CLAIMS What is claimed is:

1. A method of treating an age-related macular degeneration (AMD) patient comprising: a) obtaining a sample from the patient; b) analyzing the sample to determine the existence of one or more variants associated with an improved response to treatment with an AMD therapeutic agent; and c) administering the AMD therapeutic agent to the patient upon a positive determination that the patient has at least one of the one or more variants associated with an improved response to treatment with the AMD therapeutic agent.

2. The method of claim 1 wherein the one or more variants are in a gene encoding one or more of the group consisting of Complement Factor H (CFH), Complement Component 3 (C3), Complement Component 2 (C2), Factor B (BF), and Age-Related Maculopathy Susceptibility 2 (ARMS2).

3. The method of claim 2 wherein the one or more variants are selected from the group consisting of CFH Tyr402His, C3 ArglO2Gly, C2 Asp318Glu, C2 Intron 10, BF Lys565Glu, BF Arg32Gln, BF His9Leu, BF Arg31Trp, BF Intron 17, BF Argl50Arg, and ARMS2 Ala69Ser.

4. The method of claim 1 wherein the one or more variants are in one or more proteins selected from the group consisting of Complement Factor H (CFH), Complement Component 3 (C3), Complement Component 2 (C2), Factor B (BF), and Age-Related Maculopathy Susceptibility 2 (ARMS2).

5. The method of claim 4 wherein the one or more variants are selected from the group consisting of CFH Tyr402His, C3 ArglO2Gly, C2 Asp318Glu, C2 Intron 10, BF Lys565Glu, BF Arg32Gln, BF His9Leu, BF Arg31Trp, BF Intron 17, BF Argl50Arg, and ARMS2 Ala69Ser.

6. The method of claim 1 wherein the AMD therapeutic agent is an anti-VEGF agent.

7. The method of claim 6 wherein the anti-VEGF agent is ranibizumab.

8. The method of claim 1 wherein the one or more variants associated with an improved response to treatment with the therapeutic agent include a polymorphism in the human Complement Factor H (CFH) gene.

9. The method of claim 8 wherein the polymorphism is a change in the human CFH gene corresponding to a change in a thymine at position 1277 of SEQ ID NO: 1 to a cytosine.

10. The method of claim 9 wherein the patient has a human Complement Factor H genotype at the position corresponding to nucleotide 1277 of SEQIDNO: 1 selected from the group consisting of CC and CT.

11. The method of claim 9 wherein and the AMD therapeutic agent is ranibizumab.

12. The method of claim 8 wherein the step of analyzing the sample includes analyzing the nucleic acid content of the sample.

13. The method of claim 12 wherein the step of analyzing the nucleic acid content includes sequencing a portion of the human CFH gene or an mRNA encoded by the human CFH gene including a nucleotide corresponding to the nucleotide at position 1277 of SEQ ID NO: 2.

14. The method of claim 12 wherein the step of analyzing the nucleic acid content includes exposing the sample to a first nucleic acid probe that hybridizes under conditions of high stringency to a first nucleic acid including a nucleotide that is complementary to the nucleotide at position 1277 of SEQ ID NO: 1 and exposing the sample to a second nucleic acid probe that hybridizes under conditions of high stringency to a second nucleic including a nucleotide that is complementary to the nucleotide at position 1277 of SEQ ID NO: 2.

15. The method of claim 14 wherein the first nucleic acid includes the sequence of nucleotides 1277 - 1291 of SEQ ID NO: 1 and the first nucleic acid probe is complementary to at least 14 nucleotides of the first nucleic acid; and wherein the second nucleic acid includes the sequence of nucleotides 1277 - 1291 of SEQ ID NO: 2 and the second nucleic acid probe is complementary to at least fourteen nucleotides of the second nucleic acid.

16. The method of claim 14 wherein the first nucleic acid probe is fifteen nucleotides in length and complementary to fourteen nucleotides of the first nucleic acid; and wherein the second nucleic acid probe is fifteen nucleotides in length and complementary to fourteen nucleotides of the second nucleic acid.

17. The method of claim 14 wherein the first nucleic acid probe is fifteen nucleotides in length and complementary to fifteen nucleotides of the first nucleic acid; and wherein the second nucleic acid probe is fifteen nucleotides in length and complementary to fifteen nucleotides of the second nucleic acid.

18. The method of claim 14 wherein the first nucleic acid probe includes a first fluorescent dye and the second nucleic acid probe includes a second fluorescent dye.

19. The method of claim 8, wherein the step of analyzing the sample includes analyzing the human Complement Factor H protein.

20. The method of claim 19 wherein the polymorphism is a Tyr402His mutation.

21. The method of claim 20 wherein the step of analyzing the sample includes exposing the sample to an antibody the binds a protein having the sequence of SEQ ID NO : 4 preferentially to a protein having the sequence of SEQ ID NO: 3.

22. A method of determining the therapeutic outcome of treating an age-related macular degeneration (AMD) patient with an AMD therapeutic agent comprising: a) obtaining a sample from the patient; and b) analyzing the sample to determine the existence of one or more biomarkers associated with an improved response to treatment with the AMD therapeutic agent.

23. The method of claim 22 wherein the one or more biomarkers are in a gene encoding one or more of the group consisting of Complement Factor H (CFH), Complement Component 3 (C3), Complement Component 2 (C2), Factor B (BF), and Age-Related Maculopathy Susceptibility 2 (ARMS2).

24. The method of claim 23 wherein the one or more biomarkers are selected from the group consisting of CFH Tyr402His, C3 ArglO2Gly, C2 Asp318GIu, C2 Intron 10, BF Lys565Glu, BF Arg32Gln, BF His9Leu, BF Arg31Trp, BF Intron 17, BF ArglSOArg, and ARMS2 Ala69Ser.

25. The method of claim 22 wherein the one or more biomarkers are in one or more proteins selected from the group consisting of Complement Factor H (CFH), Complement Component 3 (C3), Complement Component 2 (C2), Factor B (BF), and Age-Related Maculopathy Susceptibility 2 (ARMS2).

26. The method of claim 25 wherein the one or more biomarkers are selected from the group consisting of CFH Tyr402His, C3 ArglO2Gly, C2 Asp318Glu, C2 Intron 10, BF Lys565GIu, BF Arg32Gln, BF His9Leu, BF Arg31Trp, BF Intron 17, BF Argl50Arg, and ARMS2 Ala69Ser.

27. The method of claim 22 wherein the AMD therapeutic agent is an anti-VEGF agent.

28. The method of claim 27 wherein the anti-VEGF agent is ranibizumab.

29. The method of claim 22 wherein the one or more biomarkers include a polymorphism in the human Complement Factor H (CFH) gene.

30. The method of claim 29 wherein the polymorphism is a change in the human CFH gene corresponding to a change in a thymine at position 1277 of SEQ ID NO: 1 to a cytosine.

31. The method of claim 30 wherein the patient has a human Complement Factor H genotype at the position corresponding to nucleotide 1277 of SEQ ID NO: I selected from the group consisting of CC and CT.

32. The method of claim 31 wherein and the AMD therapeutic agent is ranibizumab.

33. The method of claim 30 wherein the step of analyzing the sample includes analyzing the nucleic acid content of the sample.

34. The method of claim 33 wherein the step of analyzing the nucleic acid content includes sequencing a portion of the human complement factor H (CFH) gene DNA or an mRNA encoded by the human CFH gene including a nucleotide corresponding to the nucleotide at position 1277 of SEQ ID NO: 1 or SEQ ID NO: 2.

35. The method of claim 33 wherein the step of analyzing the nucleic acid content includes exposing the sample to a first nucleic acid probe that hybridizes under conditions of high stringency to a first nucleic acid including a nucleotide that is complementary to the nucleotide at position 1277 of SEQ ID NO: 1 and exposing the sample to a second nucleic acid probe that hybridizes under conditions of high stringency to a second nucleic including a nucleotide that is complementary to the nucleotide at position 1277 of SEQ ID NO: 2.

36. The method of claim 35 wherein the first nucleic acid includes the sequence of nucleotides 1277 - 1291 of SEQ ID NO: 1 and the first nucleic acid probe is complementary to at least fourteen nucleotides of the first nucleic acid; and wherein the second nucleic acid includes the sequence of nucleotides 1277 - 1291 of SEQ ID NO: 2 and the second nucleic acid probe is complementary to at least fourteen nucleotides of the second nucleic acid.

37. The method of claim 36 wherein the first nucleic acid probe is fifteen nucleotides in length and complementary to fourteen nucleotides of the first nucleic acid; and wherein the second nucleic acid probe is fifteen nucleotides in length and complementary to fourteen nucleotides of the second nucleic acid.

38. The method of claim 36 wherein the first nucleic acid probe is fifteen nucleotides in length and complementary to fifteen nucleotides of the first nucleic acid; and wherein the second nucleic acid probe is fifteen nucleotides in length and complementary to fifteen nucleotides of the second nucleic acid.

39. The method of claim 35 wherein the first nucleic acid probe includes a first fluorescent dye and the second nucleic acid probe includes a second fluorescent dye.

40. The method of claim 29 wherein the step of analyzing the sample includes analyzing the human Complement Factor H protein.

41. The method of claim 40 wherein the polymorphism is a Tyr402His mutation.

42. The method of claim 41 wherein the step of determining includes exposing the sample to an antibody that binds a protein having the sequence of SEQ ID NO: 4 preferentially to a protein having the sequence of SEQ ID NO: 3.

43. A kit for pred i cting the therapeutic outcome of treating an AMD patient with an AMD therapeutic agent comprising an oligonucleotide having fifteen contiguous nucleotides with at least 90% identity to fifteen contiguous nucleotides of a gene including a mutation associated with an improved response to treatment with the AMD therapeutic agent, wherein the oligonucleotide includes one or more nucleotides complementary to the mutation.

44. The kit of claim 43 wherein the gene is a human Complement Factor H (CFH) gene.

45. The kit of claim 44 wherein the oligonucleotide hybridizes to a nucleic acid having the sequence of SEQ ID NO: 2 under high stringency conditions.

46. The kit of claim 45 further comprising instructions for predicting the therapeutic outcome of treating a subject with an AMD therapeutic agent.

47. A method of treating an age-related macular degeneration (AMD) patient compri sing : a) obtaining a sample from the patient; b) analyzing the sample to determine the existence of a Tyr402His variant of Human Complement Factor H (CFH); and c) administering an AMD therapeutic agent including an anti-VEGF agent to the patient upon a positive determination that the patient has the Tyr402His variant of Human CFH.

48. The method of claim 47 wherein the anti-VEGF agent is ranibizumab.

49. A method of determining the therapeutic outcome of treating an AMD patient with an AMD therapeutic agent including an anti-VEGF agent, the method comprising: a) obtaining a sample from the patient; and b) analyzing the sample to determine the existence of a Tyr402His variant of Human Complement Factor H (CFH).

50. The method of claim 49 wherein the anti-VEGF agent is ranibizumab.

51. A kit for pred icting the therapeutic outcome of treating an AMD patient with an anti- VEGF agent comprising an oligonucleotide having fifteen contiguous nucleotides that hybridize under conditions of high stringency to fifteen contiguous nucleotides of a nucleic acid having the sequence of SEQ ID NO: 2; wherein the fifteen contiguous nucleotides of the nucleic acid include the sequence of SEQ ID NO: 2 at a position including nucleotide 1277 of SEQ ID NO: 2.