BRPI0914368B1 - METHOD FOR PREDICTING WHETHER A PATIENT WITH WET AGE-RELATED MACULAR DEGENERATION (AMD) HAS A GREATER LIKELIHOOD OF BENEFITING FROM TREATMENT WITH AN ANTI-VEGF ANTIBODY AND METHOD FOR PREDICTING WHETHER AN INDIVIDUAL HAS A GREATER LIKELIHOOD OF DEVELOPING WET AMD OR DRY AMD WITH GEOGRAPHIC ATROPHY (GA) - Google Patents

Abstract

METHOD AND KIT FOR PREDICTING WHETHER A PATIENT WITH WET AGE-RELATED MACULAR DEGENERATION (AMD) HAS AN INCREASED LIKELIHOOD OF BENEFITING FROM TREATMENT WITH AN ANTI-VEGF ANTIBODY AND METHOD FOR PREDICTING WHETHER AN INDIVIDUAL HAS AN INCREASED LIKELIHOOD OF DEVELOPING WET AMD OR AMD DROUGHT WITH GEOGRAPHIC ATROPHY (GA). The present invention relates to methods for determining whether a patient is at increased risk for developing wet AMD or whether a patient is more likely to benefit from treatment with a high affinity anti-VEGF antibody.

Description

RELATED PATENT APPLICATIONS

[001] This application claims the benefit of priority, under USC title 35 § 119(e), of United States provisional patent application number US 61/111,667, filed November 5, 2011, and patent application United States provisional number US 61/174,856, filed May 1, 2009, the contents of which are fully incorporated herein by reference.

FIELD OF INVENTION

[002] The present invention is generally related to the treatment of a human disease. More specifically, the invention relates to the wet form of age-related macular degeneration (AMD).

BACKGROUND OF THE INVENTION

[003] AMD is one of the main causes of irreversible and serious vision loss among the elderly. Bressler (2004) JAMA 291:1900-01. It is characterized by a broad spectrum of clinical and pathological findings, including pale yellow patches known as drusen, disruption of the retinal pigment epithelium (RPE), choroidal neovascularization (CNV), and disciform macular degeneration. The manifestations of the disease are classified into two forms: non-exudative (dry) and exudative (wet or neovascular). Recently, several therapies for the treatment of wet AMD have been approved - photodynamic therapy with verteporfin (Visudyne®), a VEGF-binding aptamer, pegaptantib (Macugen®); and an anti-VEGF antibody fragment, ranibizumab (Lucentis®).

[004] Genetic polymorphisms may occur in a population when different alleles in specific genes result in different phenotypes, including the development or progression of the disease and the ability to respond to therapeutic drugs. Several polymorphisms have been identified that are associated with the development or progression of AMD (e.g., Despriet et al. (2007) Ophthalmology 125:1270-71; Seddon et al. (2007) 297:1793-99 JAMA, 2585; Boon et al (2008) Am. J. Genet Human. 82:516-23). Previous work has demonstrated that specific polymorphisms at amino acid position 402 of the complement factor H (CFH) gene are associated with response to photodynamic therapy (PDT) with verteporfin or off-label therapy with bevacizumab for the treatment of AMD. . The identification of polymorphisms predictive of efficacy and safety in specific therapies can be used to more effectively tailor therapies to patients who would best benefit from them.

BRIEF DESCRIPTION OF THE INVENTION

[005] The present invention is based in part on the identification of genetic polymorphisms that are predictive of the risk of AMD or a greater probability that treatment with high affinity anti-VEGF antibodies will benefit patients with AMD.

[006] In one aspect, the present invention provides a method for predicting whether a patient with wet AMD is more likely to benefit from treatment with a high-affinity anti-VEGF antibody, comprising screening for a genomic polymorphism in the Y402H allele of the complement factor H (CFH) gene corresponding to rs1061170 in an isolated sample from said patient, so that the patient is more likely to benefit from said treatment if the corresponding genotype comprises CC or CT. In another aspect, the present invention provides a method for predicting whether a patient with wet AMD is more likely to benefit from treatment with an anti-VEGF antibody, which comprises screening for a genomic polymorphism in the (C5) I802V allele of the gene of complement component C5 corresponding to rs17611 in a sample isolated from said patient, wherein the patient has a greater probability of benefiting from said treatment if the corresponding genotype comprises AA or AG. In yet another aspect, the present invention provides a method for predicting whether a patient with wet AMD is more likely to benefit from treatment with an anti-VEGF antibody, comprising screening for a genomic polymorphism in the HrtA serine protease 1 gene ( HTRA1) A69S allele corresponding to rs10490924 in a sample isolated from said patient, so that the patient is more likely to benefit from said treatment if the corresponding genotype comprises GT. In some exemplary embodiments, the method further comprises treating the patient with an anti-VEGF antibody.

[007] In some embodiments, the anti-VEGF antibody binds to the same epitope as the anti-VEGF monoclonal antibody A4.6.1 produced by the hybridoma ATCC® HB 10709. In some embodiments, the anti-VEGF antibody has a domain heavy chain variable, comprising the following heavy chain complementarity determining region (CDR) amino acid sequences: CDRH1 (GYDFTHYGMN; SEQ ID NO: 1), CDRH2 (WINTYTGEPTYAADFKR; SEQ ID NO: 2) and CDRH3 (YPYYYGTSHWYFDV; SEQ ID NO: 3) and a light chain variable domain comprising the following light chain CDR amino acid sequences: CDRL1 (SASQDISNYLN; SEQ ID NO: 4), CDRL2 (FTSSLHS; SEQ ID NO: 5) and CDRL3 (QQYSTVPWT; SEQ ID NO: 6). In some exemplary embodiments, the anti-VEGF antibody has the heavy chain variable domain and the light chain variable domain of Y0317. In some exemplary embodiments, the anti-VEGF antibody is ranibizumab.

[008] In another aspect, the invention provides a kit for predicting whether a patient with wet AMD is more likely to benefit from treatment with ranibizumab comprising a first oligonucleotide and a second oligonucleotide specific for the C/T polymorphism in the CFH allele. Y402H corresponding to rs1061170. In some exemplary embodiments, oligonucleotides can be used to amplify a portion of the CFH gene comprising a C/T polymorphism in the CFH allele Y402H.

[009] In another aspect, the invention provides a kit for predicting whether a patient with wet AMD is more likely to benefit from treatment with an anti-VEGF antibody, which comprises a first oligonucleotide and a second oligonucleotide specific for the A polymorphism. /G on the C5 I802V allele corresponding to rs17216529. In some exemplary embodiments, oligonucleotides can be used to amplify a portion of the CFH gene comprising an A/G polymorphism in the C5 I802V allele.

[010] In another aspect, the invention provides a kit for predicting whether a patient with wet AMD is more likely to benefit from treatment with an anti-VEGF antibody comprising a first oligonucleotide and a second oligonucleotide specific for the G/T polymorphism. in HTRA1 A69S allele corresponding to rs10490924. In some exemplary embodiments, oligonucleotides can be used to amplify a portion of the CFH gene comprising a G/T polymorphism in the HTRA1 A69S allele.

[011] In another aspect, the invention provides a method for predicting whether an individual has a greater probability of developing AMD, comprising screening for a genomic polymorphism in the I145V allele of C5 corresponding to rs17216529 in a sample isolated from said patient, where the patient is more likely to develop AMD if the corresponding genotype comprises GG or AG.

[012] In another aspect, the invention provides a method for predicting whether an individual has a greater probability of developing wet AMD or dry AMD with geographic atrophy (GA), which comprises screening for a genomic polymorphism in the C5 I802V allele corresponding to rs17661 in a sample isolated from said patient, wherein the patient is more likely to develop AMD if the corresponding genotype comprises the T (ile) allele.

DETAILED DESCRIPTION OF THE INVENTION

[013] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the state of the art. Such techniques are fully elucidated in the literature, such as in "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984); "Animal Cell Culture" (RI Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., Eds., 1987, and periodic updates); "PCR: The Polymerase Chain Reaction", (Mullis et al., Ed., 1994).

[014] Unless otherwise defined, the technical and scientific terms used in the present specification have the same meaning as is commonly understood by a person skilled in the art to which this invention belongs. Singleton et al. “Dictionary of Microbiology and Molecular Biology” 2nd Ed., J. Wiley & Sons (New York, NY, 1994), and March, “Advanced Organic Chemistry Reactions, Mechanisms and Structure” 4th Ed. John Wiley & Sons (New York, NY, 1992), provide one skilled in the art with a general guide to many of the terms used in the present application.

[015] All references cited herein, including patents and publications, are incorporated herein by reference in their entirety.

DEFINITIONS

[016] As used in the present invention, the singular forms “a/a” and “the/a” include the plural unless the context clearly indicates otherwise. For example, “a” cell will also include “cells”.

[017] The term “comprises/understanding” is intended to say that the compositions and methods include the recited elements, but do not exclude others.

[018] The terms “VEGF” and “VEGF-A” are used interchangeably in the present invention to refer to the 165 amino acids of endothelial cell growth factor and/or the 121-, 189-, and 206-amino acids related to endothelial cell growth factors, as described by Leung et al. Science, 246:1306 (1989), and Houck et al. Mol. Endocrin., 5:1806 (1991), together with naturally occurring and transformed allelic forms thereof. An “anti-VEGF antibody” is an antibody that binds to VEGF with sufficient affinity and specificity. Preferably, the anti-VEGF antibody of the present invention can be used as a therapeutic agent in targeting and interfering with diseases or conditions in which VEGF activity is involved. An anti-VEGF antibody does not normally bind to other VEGF homologues, such as VEGF-B or VEGF-C, nor to other growth factors, such as PlGF, PDGF, or bFGF. A preferred anti-VEGF antibody is a monoclonal antibody that binds to the same epitope as the anti-VEGF A4.6.1 monoclonal antibody produced by the ATCC® HB 10709 hybridoma and is a high affinity anti-VEGF antibody. A “high-affinity anti-VEGF antibody” has at least 10 times better affinity for VEGF than the anti-VEGF monoclonal antibody A4.6.1. Preferably, the anti-VEGF antibody is a recombinant humanized anti-VEGF monoclonal antibody fragment generated according to WO 98/45331, including an antibody comprising the CDRs or variable regions of Y0317. More preferably, the anti-VEGF antibody is the antibody fragment known as ranibizumab (Lucentis).

[019] The term “antibody” is used herein in the broadest sense and includes monoclonal antibodies (including full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments , as long as they exhibit the desired biological activity.

[020] The term "treatment" refers to both therapeutic treatment and prophylactic or preventive measures. Those who require such treatments include those who already have the disease as well as those in whom the disease should be avoided.

[021] The term “polymorphism” refers to a position in the sequence of a gene that varies within a population. A polymorphism is made up of different “alleles”. The location of such a polymorphism can be identified by its position in the gene and the different amino acids or bases that are found in this position. For example, Y402H of CFH indicates that there is variation between tyrosine (Y) and histidine (H) at amino acid position 402 of the CFH gene. This amino acid change is the result of two possible base variants, C and T, which are two different alleles. Because the genotype is made up of two distinct alleles, any one of several possible variants can be observed in an individual (e.g., for this example, CC, CT, or TT). Individual polymorphisms are also assigned with unique identifiers (“Reference SNP”, “refSNP” or “rs#”) known to those skilled in the art and used, for example, in the single nucleotide polymorphism database “Single Nucleotide Polymorphism Database ” (dbSNP) from the “Nucleotide Sequence Variation” available on the NCBI web page.

[022] The term “genotype” refers to the specific alleles of a given gene in a sample of cells or tissues. In the example above, CC, CT or TT are possible genotypes of the Y402H polymorphism in CHF.

[023] The term “sample” encompasses a sample of cells or tissues taken from a patient. For example, a sample may include a skin sample, a cheek cell sample, or blood cells.

[024] Identification of the genotype in a sample can be carried out by any of a number of methods well known to a person skilled in the art. For example, identification of the polymorphism can be done by cloning the allele and sequencing using techniques well known in the art. Alternatively, gene sequences can be amplified from genomic DNA, for example using PCR, and the product sequenced. Several non-limiting methods for analyzing mutations at a given genetic locus in a patient's DNA are described below.

[025] DNA microarray technology, for example, DNA chip devices and high-density microarrays for high-throughput selection applications and low-density microarrays, can be used. Methods for microarray fabrication are known in the art and include various spotting or microjet and inkjet deposition technologies and processes, in situ or on-chip photolithographic oligonucleotide synthesis processes, and electronic addressing processes. of the DNA probe. DNA microarray hybridization applications have been successfully used in the areas of gene expression analysis and genotyping of point mutations, single nucleotide polymorphisms (SNPs), and short tandem repeats (STRs). Additional methods include RNA interference microarrays and combinations of microarrays and other methods such as laser capture and microdissection (LCM), comparative genomic hybridization (CGH), and chromatin immunoprecipitation (ChiP). See, for example, He et al. (2007) Adv. Exp. Med. Biol. 593:117-133 and Heller (2002) Annu. Rev. Biomed. Eng 4:129-153. Other methods include PCR, xMAP, invasive assay, mass spectrometry, and pyrosequencing (Wang et al. (2007). Microarray Technology and Cancer Gene Profiling Vol. 593 of the book series “Advances in Experimental Medicine and Biology”, pub. Springer, New York).

[026] Another method of detection is allele-specific hybridization using probes that overlap the polymorphic site and have about 5 or, alternatively 10, or alternatively, 20, or alternatively 25, or alternatively 30 nucleotides throughout the polymorphic region. For example, several probes capable of specifically hybridizing the allelic variant are attached to a solid phase support, e.g., a "chip". Oligonucleotides can attach to a solid support by a variety of processes, including lithography. Mutation detection analysis using such chips comprising oligonucleotides, also called “DNA probe arrays” is described, for example, in Cronin et al. (1996 ) Human Mutation 7:244.

[027] In other detection methods, it is first necessary to amplify at least a portion of the gene before identifying the allelic variant. Amplification can be carried out, for example, by PCR and/or LCR or by other methods known in the art.

[028] In some cases, the presence of the specific allele in an individual's DNA can be demonstrated by analysis with restriction enzymes. For example, the specific nucleotide polymorphism may result in a nucleotide sequence comprising a restriction site that is absent from the nucleotide sequence of another allelic variant.

[029] In an additional exemplary embodiment, protection against cleavage agents (such as a nuclease, hydroxylamine or osmium tetroxide and with piperidine) can be used to detect unpaired bases in RNA/RNA, DNA/DNA, or heteroduplexes RNA/DNA (see, for example, Myers et al. (1985) Science 230:1242). In general, the “mismatch cleavage” technique begins with the provision of heteroduplexes formed by the hybridization of a control nucleic acid, which is optionally labeled, for example, RNA or DNA, comprising a nucleotide sequence of the allelic variant of the gene with a nucleic acid sample, for example, RNA or DNA obtained from a tissue sample. Double-stranded duplexes are treated with an agent that cleaves the single-stranded (single) regions of the duplex, such as duplexes formed based on base pair mismatches between the control strand and the sample strand. For example, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically digest the unpaired regions. Alternatively, both DNA/DNA duplexes and RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide with piperidine in order to digest the unpaired regions. After digestion of the incompatible regions, the resulting material is then separated by size in polyacrylamide gels to determine whether the sample and control nucleic acids have an identical nucleotide sequence or whether the nucleotides in these samples are different. See, for example, US Patent 6,455,249, Cotton et al. (1988) Proc. Natl. Academic. Sci USA 85:4397; Saleeba et al. (1992) Method. Enzymol. 217:286-295.

[030] Changes in electrophoretic mobility can also be used to identify the allelic variant. For example, a single-strand conformation polymorphism (SSCP) can be used to detect differences in electrophoretic mobility between mutant and wild-type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA 86:2766; Cotton (1993) Mutat. Res. 285:125-144 and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments from sample and control nucleic acids are denatured and renatured. The secondary structure of single-stranded nucleic acids varies depending on the sequence, the resulting change in electrophoretic mobility allows detection of the change of even a single base. DNA fragments can be labeled or detected with labeled probes. The sensitivity of the assay can be improved by using RNA (rather than DNA), where the secondary structure is more sensitive to a change in sequence. In another preferred embodiment, the method in question utilizes heteroduplex analysis to separate double-stranded heteroduplex molecules based on changes in electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

[031] The identity of the allelic variant can also be obtained by analyzing the mobility of a nucleic acid comprising the polymorphic region in the polyacrylamide gel containing a denaturing gradient, which is analyzed using denaturing gradient gel electrophoresis (DGGE). (Myers et al. (1985) Nature 313:495). When DGGE is used as an analysis method, the DNA will be modified to ensure that it does not completely denature, for example, by the addition by PCR of a “GC-clamp” of about 40 bp of DNA rich in guanine and cytosine with a high melting point. In a further exemplary embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in mobility between the control and the DNA sample (Rosenbaum and Reissner (1987) Biophys. Chem. 265:1275).

[032] Examples of techniques for detecting differences of at least one nucleotide between the two nucleic acids include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide probes can be prepared so that the known polymorphic nucleotide is placed centrally (allele-specific probes) and hybridized to the target DNA under conditions that allow hybridization to occur only if a perfect match is found (Saiki et al. al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Academic. Sci. USA 86:6230). Such allele-specific oligonucleotide hybridization techniques can be used for the detection of nucleotide changes in the polymorphic region of the gene. For example, oligonucleotides having the nucleotide sequence of the allele-specific variant are linked to a hybridizing membrane and this membrane is then hybridized with the labeled nucleic acid sample. Analysis of the hybridization signal will then reveal the identity of the nucleotides in the nucleic acid sample.

[033] Alternatively, allele-specific amplification technology that depends on selective amplification can be used in conjunction with the present invention. Oligonucleotides used as primers for specific amplification can carry the allelic variant of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Acids Nucl. Res. 17:2437-2448) or at the 3' end of a primer where, under suitable conditions, mismatch can prevent or reduce polymerase extension (Prossner (1993) Tibtech 11:238 and Newton et al. (1989) Acids Nucl. Res. 17:2503). . This technique is also called “PROBE” for Oligo Base Probe Extension. Furthermore, it may be desirable to introduce a new restriction site into the mutation region to create cleavage-based detection systems (Gasparini et al. (1992) Mol. Cell. Probes 6:1).

[034] In another exemplary embodiment, identification of the allelic variant was performed using an oligonucleotide binding assay (OLA), as described, for example, in US Patent 4,998,617 and in Laridegren, U. et al. Science 241:1077-1080 (1988). The OLA protocol uses two oligonucleotides that are designed to be capable of hybridizing to adjacent sequences of a single-stranded target. One of the oligonucleotides is linked to a separation marker, for example biotin, and the other is labeled for detection. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize to their terminal ends, and create a binding substrate. Ligation then allows the labeled oligonucleotide to be recovered using avidin, or another biotin ligand. Nickerson, D. A. et al. described a nucleic acid detection test that combines the attributes of PCR and OLA (Nickerson, D.A. et al. (1990) Proc. Natl. Acad. Sci. USA 87:8923-8927). In this method, PCR is used to obtain exponential amplification of target DNA, which is then detected using the OLA method.

[035] The invention provides methods for detecting a single nucleotide polymorphism (SNP) in CFH and C5. Because single nucleotide polymorphisms are flanked by regions of non-variable sequence, their analysis requires no more than determining the identity of the single variant nucleotide and it is not necessary to determine the complete genetic sequence of each patient. Several methods have been developed to facilitate SNP analysis.

[036] The single base polymorphism can be detected using a specialized exonuclease resistant nucleotide, as disclosed, for example, in US Patent 4,656,127. According to the method, a primer complementary to the allelic sequence immediately 3' of the polymorphic site is allowed to hybridize to a target molecule obtained from a particular animal or human. If the polymorphic site of the target molecule contains a nucleotide that is complementary to the specific exonuclease-resistant nucleotide derived from the present invention, then that derivative will be incorporated into the end of the hybridized primer. This incorporation makes the primer resistant to exonuclease, and thus allows its detection. Since the identity of the sample's exonuclease-resistant derivative is known, the conclusion that the primer has become exonuclease-resistant reveals that the current nucleotide in the polymorphic site of the target molecule was complementary to that of the derivative nucleotide used in the reaction. This method has the advantage of not requiring the determination of large amounts of extraneous sequence data.

[037] A solution-based method can also be used to determine the identity of the nucleotides of the polymorphic site (document WO 91/02087). As stated above, a primer is used that is complementary to the allelic sequences immediately 3' to a polymorphic site. The method allows determining the identity of the site's nucleotides using labeled dideoxynucleotide derivatives, which, if complementary to the polymorphic site's nucleotide, will be incorporated into the end of the primer.

[038] An alternative method is described in document WO 92/15712. This method uses mixtures of labeled terminators and a primer that is complementary to the 3' sequence of a polymorphic site. The labeled terminator that is incorporated is thus determined by, and complementary to, the nucleotide of the present invention in the polymorphic site of the target molecule being evaluated. The method is generally a heterogeneous phase assay, in which the primer or target molecule is immobilized on a solid phase.

[039] Many other primer-guided nucleotide incorporation procedures for evaluating polymorphic sites in DNA have been described (Komher, J. S. et al. (1989) Nucl. Acids. Res. 17:7779-7784; Sokolov, B. P. (1990 ) Nucl. Acids Res. 18:3671; Syvanen, A.-C., et al. (1990) Genomics 8:684-692; Kuppuswamy, M. N. et al. (1991) Proc. Natl. Acad. Sci. USA 88 :1143-1147; Prezant, T. R. et al. (1992) Hum. Mutat. 1: 159-164; Ugozzoli, L. et al. (1992) GATA 9:107-112; Nyren, P. et al. (1993 ) Anal. Biochem. 208:171-175). All of these methods rely on the incorporation of labeled deoxynucleotides to discriminate between the bases of a polymorphic site.

[040] Furthermore, it will be understood that any of the above methods for detecting changes in a gene or gene product or polymorphic variants can be used to monitor the course of treatment or therapy.

[041] The methods described herein can be carried out, for example, through the use of pre-packaged diagnostic kits, such as those described below, including at least one nucleic acid probe or primer, which can be conveniently used, for example, to determine whether an individual has an increased likelihood of developing AMD or whether a patient with exudative (wet type) AMD has a greater likelihood of benefiting from treatment with an anti-VEGF antibody.

[042] The nucleic acid sample for use in the diagnostic and prognostic methods described above can be obtained from any type of cell or tissue from a subject. For example, a subject's bodily fluid (e.g., blood) can be obtained using known techniques. Alternatively, nucleic acid tests can be performed on dry samples (e.g. hair or skin).

[043] The invention described herein refers to methods and compositions for the determination and identification of alleles present in various alleles, including the alleles Y402H in CFH, I145V in C5, and I802V in C5. Probes can be used to directly determine the genotype of the sample or can be used simultaneously or after amplification. The term “probes” includes naturally occurring or recombinant single-stranded (single) or double-stranded nucleic acids or chemically synthesized nucleic acids. They can be marked by translation by nick translation, Klenow reaction, PCR or other methods known in the art. The probes of the present invention, their preparation and/or labeling, are described in Sambrook et al. (1989) above. A probe may be a polynucleotide of any length suitable for selective hybridization to a nucleic acid containing a polymorphic region of the invention. The length of the probe used will depend, in part, on the nature of the assay used and the hybridization conditions employed.

[044] Labeled probes can also be used in conjunction with the amplification of a polymorphism. (Holland et al. (1991) Proc. Natl. Acad. Sci. USA 88:7276-7280). US Patent 5,210,015 describes fluorescence-based approaches to provide real-time measurements of amplification products during PCR. These approaches have been employed either by intercalating dyes (such as ethidium bromide) to indicate the amount of double-stranded DNA present, or by using probes containing pairs of fluorescence quenchers (also known as the “TaqMan®” approach) where the probe is cleaved during amplification to release a fluorescent molecule whose concentration is proportional to the amount of double-stranded DNA present. During amplification, the probe is digested by the nuclease activity of a polymerase when hybridized with the target sequence to cause the separation of the fluorescent molecule from the quencher molecule, causing the appearance of fluorescence from the reporter molecule. The TaqMan® approach uses a probe that contains a molecule-reporter molecule-fluorescence quencher pair that specifically anneals to a region of a target polynucleotide containing the polymorphism.

[045] The probes can be glued to surfaces for use as "gene chips". Such gene chips can be used to detect genetic variations by a variety of techniques known to those skilled in the art. In one technique, oligonucleotides are arranged on a gene chip to determine the DNA sequence by sequencing a hybridization approach, such as that mentioned in US Patents 6,025,136 and US 6,018,041. The probes of the present invention can also be used for fluorescent detection of a genetic sequence. Such techniques have been described, for example, in US Patents 5,968,740 and US 5,858,659. The probe may also be affixed to an electrode surface for the electrochemical detection of nucleic acid sequences, as described in US Patent 5,952,172 and by Kelley, S. O. et al. (1999) Nucl. Acids Res 27:4830-4837.

[046] Furthermore, isolated nucleic acids used as probes or primers can be modified to become more stable. Exemplary nucleic acid molecules that are modified include phosphoramidate, phosphothioate and methylphosphonate analogues of DNA (see also US Patents 5,176,996; US 5,264,564 and US 5,256,775).

[047] As set forth herein, the invention also provides diagnostic methods for determining the type of allelic variants of the polymorphic regions present in CFH or C5. In some exemplary embodiments, the methods use probes or primers comprising nucleotide sequences that are complementary to a polymorphic region of CFH or C5. Thus, the invention provides kits for carrying out these methods.

[048] In some exemplary embodiments, the invention provides a kit for determining whether a patient with wet type AMD is more likely to benefit from treatment with an anti-VEGF antibody, including a high affinity anti-VEGF antibody. These kits contain one or more of the compositions and instructions for use described below. As just one example, the invention provides a kit for predicting whether a patient with wet AMD is more likely to benefit from treatment with ranibizumab comprising a first oligonucleotide and a second oligonucleotide specific for the C/T polymorphism in the Y402H allele of CFH. Oligonucleotides “specific for” a genetic locus bind both the polymorphic region of the locus and the region adjacent to the polymorphic region of the locus. For oligonucleotides a are used as primers for amplification, the primers are adjacent if they are sufficiently close together to be used to produce a polynucleotide comprising the polymorphic region. In an exemplary embodiment, the oligonucleotides are adjacent if they bind within about 1-2 kb, e.g., less than 1 kb of the polymorphism. Specific oligonucleotides are capable of hybridizing to a sequence, and, under appropriate conditions, do not bind to a sequence that differs by a single nucleotide.

[049] The kit may comprise at least one probe or primer that is capable of specifically hybridizing to the CFH or C5 polymorphic region and instructions for use. Kits typically include at least one of the nucleic acids described above. Kits for amplification of at least a portion of CFH or C5 generally comprise at least two primers, one of which is capable of hybridizing to the allelic variant sequence. These kits are suitable for genotype detection, for example, fluorescence detection, electrochemical detection, or other types of detection.

[050] Oligonucleotides, when used as probes or primers, contained in a kit can be labeled for detection. Markers can be detected directly, for example fluorescent markers, or indirectly. Indirect detection may include any detection method known to those of skill in the art, including avidin-biotin interactions, antibody binding, and the like. Fluorescently labeled oligonucleotides may also contain a fluorescence quenching molecule. Oligonucleotides can be attached to a surface. In some embodiments, the surface is silica or glass. In some embodiments, the surface is a metal electrode.

[051] However, other kits of the invention comprise at least one reagent necessary to carry out the assay. For example, the kit may comprise an enzyme. Alternatively, the kit may comprise a buffer or any other necessary reagent.

[052] Kits may include all or some of the positive controls, negative controls, reagents, primers, sequencing markers, probes and antibodies described herein to determine the individual's genotype in the CFH or C5 polymorphic region.

[053] The following example is intended only to illustrate the practice of the present invention and is not provided for the purpose of limiting the invention.

EXAMPLE EXAMPLE 1 GENETIC POLYMORPHISMS IN CFH, HTRA1 AND C5 AND THESE ASSOCIATION WITH THE OCCURRENCE OF AMD AND TREATMENT RESULTS POLYMORPHISM

[054] We tested a variety of polymorphisms for variation associated with a favorable outcome during anti-VEGF therapy using ranibizumab. These polymorphisms were examined and were chosen because they are eight 5-locus alleles previously reported to be associated with susceptibility to the development of AMD (Table 1). Specifically, two alleles of factor H (CFH), HTRa serine peptidase/age-related maculopathy susceptibility 2 (HTRA1/ARMS2), Complement Factor 2/Complement Factor B preprotein (C2/BF), and a single Complement Factor 3 (C3) allele and chemokine (C-X3-C motif) receptor 1 (CX3CR1) were genotyped in 352 AMD samples from the DAWN study using quantitative PCR, via the TaqMan® system. Additionally, we tested two alleles of complement component 5 (C5) (Table 2). The standard experimental protocol provided by ABI was used for genotyping of all assays in Table 1. Briefly, assays were run on an ABI 7500 machine, using the following cycling conditions: 2 min at 50 °C, 10 min at 95 °C, followed by 40 cycles of 15 seconds at 92 °C and 1 min at 60 °C. TABLE 1 SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS) TESTED CX3CR1 rs3732378 3 39,282,166 M280T Chan et al. (2005) Histol. Histopath. 20:857-63 * Position in base pairs from the May 2004 human genome assembly. TABLE 2 SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS) TESTED FOR C5 * Position in base pairs as of the May 2004 human genome assembly.

SAMPLES

[055] Peripheral blood samples from 352 anonymous individuals from the main Lucentis® studies (MARINA, ANCHOR and FOCUS) who participated in the DAWN genetic sub-study, an extension of the HORIZONTE study, were collected and genomic DNA was isolated. All samples had a confirmed diagnosis of neovascular AMD, 60% were female, and the mean age at the beginning of the study was 75.0 years of age for sham(placebo)/PDT and 75.6 years of age for those treated with ranibizumab. Written informed consent was obtained from all study subjects and study protocols were approved by institutional review boards (research ethics committee). DNA was extracted using the “Dneasy® Tissue kit” Qiagen, Valencia, CA). The SNPs were genotyped with TaqMan® based on Real-Time PCR. For two, custom primers for SNPs were used (Table 4) and for the others, six proprietary primers from ABI were used (Table 3). The two C5 SNPs (oligonucleotides shown in Table 5) were typed as part of a custom SNP 96 assay panel using the Illumina® GoldenGate platform. TABLE 3 ASSAYS USED FOR SNPS GENOTYPE *Pre-designed TaqMan SNP genotyping assays (Applied Biosystems, Foster City, CA) were available for six of the alleles. The ABI Assay ID (ABI Assay ID) is shown for the pre-designed assays as the primer sequences are proprietary to this. Custom TaqMan® assays were designed for the remaining two alleles, and primers are in Table 4. TABLE 4 PRIMERS USED FOR GENOTYPING RS1061170 AND RS11200638 TABLE 5 PRIMERS USED FOR GENOTYPING OF SNPS C5 RS17216529 AND RS17611

CLINICAL INFORMATION

[056] Clinical information from the Lucentis MARINA, ANCHOR and FOCUS studies were examined. Specifically, information regarding self-identified race, sex, age at study entry, initial study treatment group, Lucentis Dose, treatment arm crossover at year 2, Presence of dosing error, Study Best Acuity score baseline corrected visual (VA) (BL) in letters, VA score of the contralateral eye in BL (letters), Study of the VA score in the eye at the 12th month (letters), VA score of the contralateral eye at the 12th month (letters ), presence of neovascular AMD in the contralateral eye at BL time, and CNV classification.

ASSOCIATION FOR SENSITIVITY ANALYSIS

[057] The eight alleles were examined for an association with disease susceptibility by comparing the allele frequency in DAWN study cases with control samples obtained from publicly available sources. For rs10490924, rs1410996, rs9332739 and rs3732378, information on the frequency of the control allele was obtained from freely available summary statistics from the Wellcome Trust Case Control Consortium (WTCCC (2007) Nature 447:661-78). Controlling allele frequencies and genotype counts of the remaining SNPs were taken from a paper reporting the association of rs11200638 3, rs2230199 6 and rs547154 1. Statistical associations were calculated using standard 2x2 results tables.

ASSOCIATION OF GENOTYPE TO BASEMENTAL CLINICAL CHARACTERISTICS

[058] The association of alleles 8 to baseline visual acuity (VA) was performed using VA (letters) in the study eye at baseline as a quantitative characteristic, with or without baseline age added as a covariate. The three genotypic classes were tested for significant differences in median baseline VA. The analysis was performed using the PLINK software (Purcell et al. (2007). Am. J. Hum. Genet. 81:559-75). The association of the eight alleles with the presence of neovascular AMD in the contralateral eye, and classification of neovascular disease (minimally classic, predominantly classic and occult without classic) of the study eye at baseline was evaluated. The frequency of the clinical condition was stratified by genotype and significance determined by the t test.

ENRICHMENT OF AMD RISK ALLELES IN THE DAWN STUDY

[059] Confirming previously published observations, alleles from all eight loci were associated with the risk of neovascular AMD with P < 0.05. However, only the CX3CR1 locus allele did not show an association consistent with the original report. In the DAWN study samples the CX3CR1 locus had an odds ratio of 1.12, in contrast to an odds ratio > 3 in the original report. A significant association was observed between rs17216529 (I145V in C5) and the occurrence of choroidal neovascularization (CNV) in the contralateral eyes of individuals with AMD (Table 6). Furthermore, an association was observed between rs17611 (I802V in C5) and the occurrence of exudative AMD (wet type), dry type with AG AMD, or both (Table 7). This association was stronger in cases of AMD (p = 0.0014) than in cases of dry-type AMD with AG. TABLE 6 ASSOCIATION OF THE GENOTYPE IN RS17216529 (I145V IN C5) WITH CNV. TABLE 7 ASSOCIATION OF THE GENOTYPE IN RS17611 (I802V IN GENE C5) WITH WET TYPE AMD, DRY TYPE AMD WITH AG AND BOTH WET TYPE AMD AND DRY TYPE AMD WITH AG, OR BOTH

ASSOCIATION OF ALLELES TO BASEMENTAL CLINICAL FEATURES

[060] No significant association of the 8 alleles on baseline visual acuity was observed. The number of risk alleles of the Y402H allele in CFH and the A69S allele in HTRA1 were associated with the prevalence of neovascular AMD in the contralateral eye in the samples, suggesting a link between the genotype at these loci and disease severity. The CFH Y402H allele was associated with the “predominantly classic” subtype of neovascular AMD in the study eye at baseline. The CFH intronic rs1410996 allele was associated with the lesion area in CNV and classic CNV and the I802V allele of C5 was associated with the lesion area in classic CNV.

ASSOCIATION OF GENOTYPE WITH RESPONSE TO RANIBIZUMAB THERAPY

[061] The DAWN study samples were separated into 3 groups based on treatment status during the MARINA ANCHOR and FOCUS studies. The ranibizumab-treated group included subjects who received doses of 0.3 mg, 0.5 mg, or 0.5 mg + PDT. The Sham/PDT group consisted of individuals who received a sham injection (SHAM) or photodynamic therapy (PDT) alone. The association of change in visual acuity (VA, measured in letters) after 12 months of treatment was assessed for an association with the genotype of each of the eight alleles. Significant differences in response to treatment were associated with the genotype in the Y402H alleles of the CFH gene, I802V of the C5 gene, and A69S of the HTRA1 gene (Tables 8, 9 and 10). These variations are associated with changes in VA in patients treated monthly with ranibizumab. Average VA variation in 12 months was +14.5, +10.8 and +7.0 letters for the Y402H CC, CT and TT genotypes, respectively, +15.6, +12.2 and +8, 8 letters for genotypes I802V AA, AG and GG, respectively; and +9.3, +14.1 and +10.5 for the A69S GG, GT and TT genotypes, respectively. For the CFH Y402H alleles, a corresponding inverse trend was observed in the control group (SHAM/PDT), with mean changes in visual acuity at 12 months of -4.8, -10.2 and -11.5 for the Y402H genotypes. CC, CT and TT, respectively. The difference in mean 12-month BCVA results between the control and ranibizumab treatment groups was the same across all Y402H risk genotypes. TABLE 8 ASSOCIATION OF GENOTYPE IN Y402H CFH WITH RESPONSE TO RANIBIZUMAB THERAPY TABLE 9 ASSOCIATION OF THE GENOTYPE IN C5I802V WITH RESPONSE TO RANIBIZUMAB THERAPY TABLE 10 ASSOCIATION OF THE GENOTYPE AT A69S OF THE HTRA1 GENE WITH RESPONSE TO RANIBIZUMAB THERAPY

Claims (7)

1. METHOD TO PREDICT WHETHER A PATIENT WITH WET AGE-RELATED MACULAR DEGENERATION (AMD) HAS A GREATER LIKELIHOOD OF BENEFITING FROM TREATMENT WITH AN ANTI-VEGF ANTIBODY, characterized by comprising screening for a genomic polymorphism in the (C5) I802V allele of complement component C5 gene corresponding to rs17611 in a sample isolated from said patient, wherein the patient is more likely to benefit from said treatment if the corresponding genotype comprises AA or AG. 2. METHOD, according to claim 1, characterized in that said anti-VEGF antibody binds to the same epitope as the anti-VEGF monoclonal antibody A4.6.1 produced by the ATCC® HB 10709 hybridoma. 3. METHOD according to claim 2, characterized in that said anti-VEGF antibody has a heavy chain variable domain comprising the following amino acid sequences of the heavy chain complementarity determining region (CDR): CDRH1 (GYDFTHYGMN; SEQ ID NO : 1), CDRH2 (WINTYTGEPTYAADFKR; SEQ ID NO: 2) and CDRH3 (YPYYYGTSHWYFDV; SEQ ID NO: 3) and a light chain variable domain comprising the following light chain CDR amino acid sequences: CDRL1 (SASQDISNYLN; SEQ ID NO: 4), CDRL2 (FTSSLHS; SEQ ID NO: 5) and CDRL3 (QQYSTVPWT; SEQ ID NO: 6). 4. METHOD, according to claim 1, characterized in that said anti-VEGF antibody is ranibizumab. 5. METHOD, according to any one of claims 1 to 4, characterized in that the corresponding genotype comprises AA. 6. METHOD, according to any one of claims 1 to 4, characterized in that the corresponding genotype comprises AG. 7. METHOD FOR PREDICTING WHETHER AN INDIVIDUAL HAS A GREATER LIKELIHOOD OF DEVELOPING WET AMD OR DRY AMD WITH GEOGRAPHIC ATROPHY (GA), characterized by comprising screening for a genomic polymorphism in the C5 I802V allele corresponding to rs17661 in a sample isolated from said patient, wherein the patient is more likely to develop AMD if the corresponding genotype comprises the allele for ile.