Methods And Compositions To Evaluate Antibody Treatment Response

Abstract

The present invention relates to methods and compositions to evaluate or assess the response of a subject to particular therapeutic treatment. More particularly, the invention provides methods to determine the response of subjects, or to adapt the treatment protocol of subjects treated with therapeutic antibodies. The invention is based on a determination of the FCGR3A genotype of a subject. The invention can be used for patients with malignancies, particularly lymphoma, and is suited to select best responders and/or adjust treatment condition or protocol for low responders.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No. 12/858,343, filed Aug. 17, 2010, which in turn, is a continuation of U.S. application Ser. No. 10/492,183, filed Apr. 9, 2004, now U.S. Pat. No. 7,858,300 issued on Dec. 28, 2010, which, in turn, is the U.S. National Stage application of International Patent Application No. PCT/EP02/11397, filed Oct. 11, 2002, which claims the benefit of European Patent Application No. EP 01402718.9, filed Oct. 19, 2001. The entire contents of each of these applications are expressly incorporated herein by reference.

[0002] The present invention relates to methods and compositions to evaluate or assess the response of a subject to particular therapeutic treatment. More particularly, the invention provides methods to determine the response of subjects, or to adapt the treatment protocol of subjects treated with therapeutic antibodies. The invention can be used for patients with malignancies, particularly lymphoma, and is suited to select best responders and/or adjust treatment condition or protocol for low responders.

INTRODUCTION

[0003] Various therapeutic strategies in human beings are based on the use of therapeutic antibodies. This includes, for instance, the use of therapeutic antibodies developed to deplete target cells, particularly diseased cells such as virally-infected cells, tumor cells or other pathogenic cells, including allogenic immunocompetent cells. Such antibodies are typically monoclonal antibodies, of IgG species, typically IgG1 and IgG3. These antibodies can be recombinant antibodies and humanized antibodies, comprising functional domains from various species or origin or specificity. A particular example of such therapeutic antibodies is rituximab (Mabthera.RTM., Rituxan.RTM.), which is a chimeric anti-CD20 IgG1 monoclonal antibody made with human .gamma.l and .kappa. constant regions linked to murine variable domains.sup.1. For a few years, rituximab has been considerably modifying the therapeutical strategy against B lymphoproliferative malignancies, particularly non-Hodgkin's lymphomas (NHL). Other examples of intact humanized IgG1 antibodies include alemtuzumab (Campath-1H.RTM.), which is used in the treatment of B cell malignancies or trastuzumab (Herceptin.RTM.), which is used in the treatment of breast cancer. Additional examples of therapeutic antibodies under development are disclosed in the art.

[0004] While these antibodies represent a novel efficient approach to human therapy, particularly for treatment of tumors, they do not always exhibit a strong efficacy and their use could be improved by evaluating the response of subjects thereto. For instance, while rituximab, alone or in combination with chemotherapy was shown to be effective in the treatment of both low-intermediate.sup.2-8 and high-grade NHL.sup.6,9, 30% to 50% of patients with low grade NHL have no clinical response to rituximab.sup.4,5. It has been suggested that the level of CD20 expression on lymphoma cells.sup.2, the presence of high tumor burden at the time of treatment.sup.6 or low serum rituximab concentrations may explain the lack of efficacy of rituximab in some patients. Nevertheless, the actual causes of treatment failure remain largely unknown.

[0005] The availability of methods allowing the evaluation of patient response to antibody treatment would greatly enhance the therapeutic efficacy of these products. However, the precise mode of action in vivo of such therapeutic antibodies is not clearly documented. Indeed, while in vitro studies suggest various possible modes of action of rituximab (antibody-dependant cell-mediated cytotoxicity (ADCC).sup.10,11, complement-dependant cytotoxicity.sup.10, 12, 13, direct signalling leading to apoptosis.sup.14, 15, etc.), the clear action of these target cell-depleting antibodies in vivo is not documented in humans. Furthermore, while ADCC is an important effector mechanism in the eradication of intracellular pathogens and tumor cells, the role of an ADCC is still controversial.sup.12,13.

[0006] The present invention now proposes novel methods and compositions to assess the therapeutic response of a subject to a therapeutic antibody. The invention also proposes methods to select patients having best responding profile to therapeutic antibody treatment. The invention also relates to methods of treating patients with therapeutic antibodies, comprising a prior step of evaluating the patient's response. The invention also relates to compositions and kits suitable to perform the invention. The invention may as well be used in clinical trials or experimental settings, to assess or monitor a subject's response, or to verify the mode of action of an antibody.

[0007] The invention is based, in part, on the demonstration of a correlation between the genotype of a subject and its ability to respond to therapeutic antibody treatment. More specifically, the invention shows that the genotype of the Fc.gamma.RIIIa receptor directly correlates with the subject's response to therapeutic antibody treatment.

[0008] Three classes of Fc.gamma.R (Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII) and their subclasses are encoded by eight genes in humans, all located on the long arm of chromosome 1. Some of these genes display a functional allelic polymorphism generating allotypes with different receptor properties. These polymorphisms have been identified as genetic factors increasing the susceptibility to autoimmune or infectious diseases.sup.19, 21. One of these genetic factors is a gene dimorphism in FCGR3A, which encodes Fc.gamma.RIIIa with either a phenylalanine (F) or a valine (V) at amino-acid position 158.sup.22,23. This residue directly interacts with the lower hinge region of IgG1 as recently shown by IgG1-Fc.gamma.RIII co-cristallization.sup.24. It has been clearly demonstrated that human IgG1 binds more strongly to homozygous Fc.gamma.RIIIa-158V natural killer cells (NK) than to homozygous Fc.gamma.RIIIa-158F or heterozygous NK cells.sup.22,23.

[0009] We undertook to evaluate a possible correlation between the FCGR3A genotype and a patient response to therapeutic antibody treatment in vivo. Our invention stems in part from the unexpected discovery that a very strong correlation exists between said genotype and said response profile, the presence of a valine residue at position 158 being indicative of a high response rate. More specifically, the genotyping of FCGR3A was performed in patients with previously untreated follicular NHL who had received rituximab alone, a particular situation in which the response rate is very high.sup.5. The FCGR2A-131H/R was also determined as control since this gene co-localizes with FCGR3A on chromosome lq22 and encodes the macrophage Fc.gamma.RIIa receptor.

[0010] The FCGR3A-158V/F genotype was determined in 47 patients having received rituximab for a previously untreated follicular non-Hodgkin's lymphoma. Clinical and molecular response were evaluated at two months (M2) and at one year (M12). Positive molecular response was defined as a disappearance of the BCL2-JH gene rearrangement in both peripheral blood and bone marrow. FCGR3A-158V homozygous patients were 21% whereas FCGR3A-158F homozygous and heterozygous patients (FCGR3A-158F carriers) were 34% and 45%, respectively. The objective response rates at M2 and M12 were 100% and 90% in FCGR3A-158V homozygous patients compared with 65% (p=0.02) and 51% (p=0.03) in FCGR3A-158F carriers. A positive molecular response was observed at M12 in 5/6 of homozygous FCGR3A-158V patients compared with 5/16 of FCGR3A-158F carriers (p=0.04). Furthermore, the homozygous FCGR3A-158V genotype was confirmed to be the single parameters associated with clinical and molecular responses in multivariate analysis and was also associated with a lower rate of disease progression (p=0.05).

[0011] Accordingly, the present invention establishes, for the first time, an association between the FCGR3A genotype and clinical and molecular responses to therapeutic antibodies. The invention thus provides a first unique marker that can be used to monitor, evaluate or select a patient's response. This invention thus introduces new pharmacogenetical approaches in the management of patients with malignancies, viral infections or other diseases related to the presence of pathological cells in a subject, particularly non-Hodgkin's lymphoma.

[0012] An object of this invention resides in a method of assessing the response of a subject to a therapeutic antibody treatment, comprising determining in vitro the FCGR3A genotype and/or the presence of a polymorphism in the Fc.gamma.RIIIa receptor of said subject. More specifically, the method comprises determining in vitro the FCGR3A158 genotype of said subject.

[0013] A further object of this invention is a method of selecting patients for therapeutic antibody treatment, the method comprising determining in vitro the FCGR3A genotype and/or the presence of a polymorphism in the Fc.gamma.RIIIa receptor of said subject. More specifically, the method comprises determining in vitro the FCGR3A158 genotype of said subject.

[0014] An other object of this invention is a method of improving the efficacy or treatment condition or protocol of a therapeutic antibody treatment in a subject, comprising determining in vitro the FCGR3A genotype and/or the presence of a polymorphism in the Fc.gamma.RIIIa receptor of said subject. More specifically, the method comprises determining in vitro the FCGR3A158 genotype of said subject.

[0015] More specifically, determining in vitro the FCGR3A158 genotype of a subject comprises determining amino acid residue at position 158 of Fc.gamma.RIIIa receptor (or corresponding codon in the FCGR3A gene), a valine at position 158 being indicative of a better response to said treatment and a phenylalanine at position 158 being indicative of a lower response to said treatment.

[0016] Within the context of this invention, the term "therapeutic antibody or antibodies" designates more specifically any antibody that functions to deplete target cells in a patient. Specific examples of such target cells include tumor cells, virus-infected cells, allogenic cells, pathological immunocompetent cells (e.g., B lymphocytes, T lymphocytes, antigen-presenting cells, etc.) involved in allergies, autoimmune diseases, allogenic reactions, etc., or even healthy cells (e.g., endothelial cells in an anti-angiogenic therapeutic strategy). Most preferred target cells within the context of this invention are tumor cells and virus-infected cells. The therapeutic antibodies may, for instance, mediate a cytotoxic effect or a cell lysis, particularly by antibody-dependant cell-mediated cytotoxicity (ADCC). ADCC requires leukocyte receptors for the Fc portion of IgG (Fc.gamma.R) whose function is to link the IgG-sensitized antigens to Fc.gamma.R-bearing cytotoxic cells and to trigger the cell activation machinery. While this mechanism of action has not been evidenced in vivo in humans, it may account for the efficacy of such target cell-depleting therapeutic antibodies. The therapeutic antibodies may by polyclonal or, preferably, monoclonal. They may be produced by hybridomas or by recombinant cells engineered to express the desired variable and constant domains. The antibodies may by single chain antibodies or other antibody derivatives retaining the antigen specificity and the lower hinge region or a variant thereof. These may be polyfunctional antibodies, recombinant antibodies, ScFv, humanized antibodies, or variants thereof. Therapeutic antibodies are specific for surface antigens, e.g., membrane antigens. Most preferred therapeutic antibodies are specific for tumor antigens (e.g., molecules specifically expressed by tumor cells), such as CD20, CD52, ErbB2 (or HER2/Neu), CD33, CD22, CD25, MUC-1, CEA, KDR, .alpha.V.beta.3, etc., particularly lymphoma antigens (e.g., CD20). The therapeutic antibodies are preferably IgG1 or IgG3, more preferably IgG1.

[0017] Typical examples of therapeutic antibodies of this invention are rituximab, alemtuzumab and trastuzumab. Such antibodies may be used according to clinical protocols that have been authorized for use in human subjects. Additional specific examples of therapeutic antibodies include, for instance, epratuzumab, basiliximab, daclizumab, cetuximab, labetuzumab, sevirumab, tuvurimab, palivizumab, infliximab, omalizumab, efalizumab, natalizumab, clenoliximab, etc., as listed in the following table:

TABLE-US-00001 Ab specificity DCI Commercial name Typical Indications Anti-CD20 rituximab MabThera .RTM., LNH B Rituxan .RTM. Anti-CD52 alemtuzumab CAMPATH-1H .RTM. LLC, allograft Anti-CD33 Zamyl .TM. Acute myeloid Leukemia Anti-HLA-DR Remitogen .TM. LNH B Anti-CD22 epratuzumab LymphoCide .TM. LNH B Anti-erbB2 trastuzumab Herceptin .RTM., Metastatic breast cancer (HER-2/neu) Anti-FGFR cetuximab ORL and colorectal Cancers (HER-1, erbBl) Anti-MUC-1 Therex .RTM. Breast and epithelial cancers Anti-CEA labetuzumab CEA-Cide .TM. Anti-.alpha.V.beta.3 Vitaxin Cancers (anti-angiogenic) Anti-KDR Cancers (anti-angiogenic) (VEGFR2) anti-VRS palivizumab Synagis .RTM. Viral diseases fusion protein anti-VRS Numax .TM. '' fusion protein CMV sevirumab Protovir CMV Infection HBs tuvirumab Ostavir .TM. Hepatitis B Anti-CD25 basiliximab Simulect .RTM. Prevention/treatment allograft rejection Anti-CD25 daclizumab Zenapax .RTM. Prevention/treatment allograft rejection anti-TNF-.alpha. infliximab Remicade .TM. Crohn disease, polyarthrite rhumatoid anti-IgE omalizumab Xolair .TM. Asthma anti-integrin .alpha.L efalizumab Xanelim .TM. psoriasis (CD11a, LFA-1) anti-CD4 keliximab anti-CD2 siplizumab Anti-CD64 anemia anti-CD 147 GvH anti-integrin .alpha.4 natalizumab Antegren .RTM. Sclerosis, Crohn (.alpha.4B.beta.1-.alpha.4.beta.7) Anti-integrin .beta.7 Crohn, RCH anti-CD4* clenoliximab

[0018] Within the context of the present invention, a subject or patient includes any mammalian subject or patient, more preferably a human subject or patient.

[0019] According to the invention the term FCGR3A gene refers to any nucleic acid molecule encoding a Fc.gamma.RIIIa polypeptide in a subject. This term includes, in particular, genomic DNA, cDNA, RNA (pre-rRNA, messenger RNA, etc.), etc. or any synthetic nucleic acid comprising all or part of the sequence thereof. Synthetic nucleic acid includes cDNA, prepared from RNAs, and containing at least a portion of a sequence of the FCGR3A genomic DNA as for example one or more introns or a portion containing one or more mutations. Most preferably, the term FCGR3A gene refers to genomic DNA, cDNA or mRNA, typically genomic DNA or mRNA. The FCGR3A gene is preferably a human FCGRIIIa gene or nucleic acid, i.e., comprises the sequence of a nucleic acid encoding all or part of a Fc.gamma.RIIIa polypeptide having the sequence of human Fc.gamma.RIIIa polypeptide. Such nucleic acids can be isolated or prepared according to known techniques. For instance, they may be isolated from gene libraries or banks, by hybridization techniques. They can also be genetically or chemically synthesized. The genetic organization of a human FCGRIIIa gene is depicted on FIG. 2. The amino acid sequence of human Fc.gamma.RIIIa is represented FIG. 3. Amino acid position 158 is numbered from residue 1 of the mature protein. It corresponds to residue 176 of the pre-protein having a signal peptide. The sequence of a wild type FCGR3A gene is represented on FIG. 4 (see also Genbank accession Number AL590385 or NM.sub.--000569 for partial sequence).

[0020] Within the context of this invention, a portion or part means at least 3 nucleotides (e.g., a codon), preferably at least 9 nucleotides, even more preferably at least 15 nucleotides, and can contain as much as 1000 nucleotides. Such a portion can be obtained by any technique well known in the art, e.g., enzymatic and/or chemical cleavage, chemical synthesis or a combination thereof. The sequence of a portion of a FCGR3A gene encoding amino acid position 158 is represented below, for sake of clarity:

TABLE-US-00002 cDNA 540 550 560 570 580 genomic DNA 4970 4900 4990 5000 158F allele tcctacttctgcagggggctttttgggagtaaaaatgtgtcttca S Y F C R G L F G S K N V S S 158V allele tcctacttctgcagggggcttgttgggagtaaaaatgtgtcttca S Y F C R G L V G S K N V S S

[0021] As indicated above, the invention comprises a method of determining in vitro the FCGR3A158 genotype of said subject. This more particularly comprises determining the nature of amino acid residue present (or encoded) at position 158 of the Fc.gamma.RIIIa polypeptide.

[0022] Genotyping the FCGR3A gene or corresponding polypeptide in said subject may be achieved by various techniques, comprising analysing the coding nucleic acid molecules or the encoded polypeptide. Analysis may comprise sequencing, migration, electrophoresis, immuno-techniques, amplifications, specific digestions or hybridisations, etc.

[0023] In a particular embodiment, determining amino acid residue at position 158 of Fc.gamma.RIIIa receptor comprises a step of sequencing the FCGR3A receptor gene or RNA or a portion thereof comprising the nucleotides encoding amino acid residue 158.

[0024] In an other particular embodiment, determining amino acid residue at position 158 of Fc.gamma.RIIIa receptor comprises a step of amplifying the FCGR3A receptor gene or RNA or a portion thereof comprising the nucleotides encoding amino acid residue 158. Amplification may be performed by polmerase chain reaction (PCR), such as simple PCR, RT-PCR or nested PCR, for instance, using conventional methods and primers.

[0025] In this regard, amplification primers for use in this invention more preferably contain less than about 50 nucleotides even more preferably less than 30 nucleotides, typically less than about 25 or 20 nucleotides. Also, preferred primers usually contain at least 5, preferably at least 8 nucleotides, to ensure specificity. The sequence of the primer can be prepared based on the sequence of the FCGR3A gene, to allow full complementarity therewith, preferably. The probe may be labelled using any known techniques such as radioactivity, fluorescence, enzymatic, chemical, etc. This labeling can use for example Phosphor 32, biotin (16-dUTP), digoxygenin (11-dUTP). It should be understood that the present invention shall not be bound or limited by particular detection or labelling techniques. The primers may further comprise restriction sites to introduce allele-specific restriction sites in the amplified nucleic acids, as disclosed below.

[0026] Specific examples of such amplification primers are, for instance, SEQ ID NO: 1-4.

[0027] It should be understood that other primers can be designed by the skilled artisan, such as any fragment of the FCGR3A gene, for use in the amplification step and especially a pair of primers comprising a forward sequence and a reverse sequence wherein said primers of said pair hybridize with a region of a FCGR3A gene and allow amplification of at least a portion of the FCGR3A gene containing codon 158. In a preferred embodiment, each pair of primers comprises at least one primer that is complementary, and overlaps with codon 158, and allows to discriminate between 158V (gtt) and 158F (ttt). The amplification conditions may also be adjusted by the skilled person, based on common general knowledge and the guidance contained in the specification.

[0028] In a particular embodiment, the method of the present invention thus comprises a PCR amplification of a portion of the FCGR3a mRNA or gDNA with specific oligonucleotide primers, in the cell or in the biological sample, said portion comprising codon 158, and a direct or indirect analysis of PCR products, e.g., by electrophoresis, particularly Denaturing Gel Gradient Electrophoresis (DGGE).

[0029] In an other particular embodiment, determining amino acid residue at position 158 of Fc.gamma.RIIIa receptor comprises a step of allele-specific restriction enzyme digestion. This can be done by using restriction enzymes that cleave the coding sequence of a particular allele (e.g., the 158V allele) and that do not cleave the other allele (e.g., the 158F allele, or vice versa). Where such allele-specific restriction enzyme sites are not present naturally in the sequence, they may be introduced therein artificially, by amplifying the nucleic acid with allele-specific amplification primers containing such a site in their sequence. Upon amplification, determining the presence of an allele may be carried out by analyzing the digestion products, for instance by electrophoresis. This technique also allows to discriminate subjects that are homozygous or heterozygous for the selected allele.

[0030] Examples of allele-specific amplification primers include for instance SEQ ID NO:3. SEQ ID NO:3 introduces the first 3 nucleotides of the NlaIII site (5'-CATG-3'). Cleavage occurs after G. This primer comprises 11 bases that do not hybridise with FCGR3A, that extend the primer in order to facilitate electrophoretic analysis of the amplification products) and 21 bases that hybridise to FCGR3A, except for nucleotide 31 (A) which creates the restriction site.

[0031] In a further particular embodiment, determining amino acid residue at position 158 of Fc.gamma.RIIIa receptor comprises a step of hybridization of the FCGR3A receptor gene or RNA or a portion thereof comprising the nucleotides encoding amino acid residue 158, with a nucleic acid probe specific for the genotype Valine or Phenylalanine, and determining the presence or absence of hybrids.

[0032] It should be understood that the above methods can be used either alone or in various combinations. Furthermore, other techniques known to the skilled person may be used as well to determine the FCGR3A158 genotype, such as any method employing amplification (e.g. PCR), specific primers, specific probes, migration, etc., typically quantitative RT-PCR, LCR (Ligase Chain Reaction), TMA (Transcription Mediated Amplification), PCE (an enzyme amplified immunoassay) and bDNA (branched DNA signal amplification) assays.

[0033] In a preferred embodiment of this invention, determining amino acid residue at position 158 of Fc.gamma.RIIIa receptor comprises: [0034] Obtaining genomic DNA from a biological sample, [0035] Amplifying the Fc.gamma.RIIIa receptor gene or a portion thereof comprising the nucleotides encoding amino acid residue 158, and [0036] determining amino acid residue at position 158 of said Fc.gamma.RIIIa receptor gene.

[0037] Amplification can be accomplished with any specific technique such as PCR, including nested PCR, using specific primers as described above. In a most preferred embodiment, determining amino acid residue at position 158 is performed by allele-specific restriction enzyme digestion. In that case, the method comprises: [0038] Obtaining genomic DNA from a biological sample, [0039] Amplifying the Fc.gamma.RIIIa receptor gene or a portion thereof comprising the nucleotides encoding amino acid residue 158, [0040] Introducing an allele-specific restriction site, [0041] Digesting the nucleic acids with the enzyme specific for said restriction site and, [0042] Analysing the digestion products, i.e., by electrophoresis, the presence of digestion products being indicative of the presence of the allele.

[0043] In an other particular embodiment, the genotype is determined by a method comprising: total (or messenger) RNA extraction from cell or biological sample or biological fluid in vitro or ex vivo, optionally cDNA synthesis, (PCR) amplification with FCGR3A-specific oligonucleotide primers, and analysis of PCR products.

[0044] The method of this invention may also comprise determining amino acid residue at position 158 of Fc.gamma.RIIIa receptor directly by sequencing the Fc.gamma.RIIIa receptor polypeptide or a portion thereof comprising amino acid residue 158 or by using reagents specific for each allele of the Fc.gamma.RIIIa polypeptide. This can be determined by any suitable technique known to the skilled artisan, including by immuno-assay (ELISA, EIA, RIA, etc.). This can be made using any affinity reagent specific for a Fc.gamma.RIIIa158 polypeptide, more preferably any antibody or fragment or derivative thereof. In a particular embodiment, the Fc.gamma.RIIIa158 polypeptide is detected with an anti-Fc.gamma.RIIIa158 antibody (or a fragment thereof) that discriminates between Fc.gamma.RIIIa158V and Fc.gamma.RIIIa158F, more preferably a monoclonal antibody. The antibody (or affinity reagent) may be labelled by any suitable method (radioactivity, fluorescence, enzymatic, chemical, etc.). Alternatively, Fc.gamma.RIII158 antibody immune complexes may be revealed (and/or quantified) using a second reagent (e.g., antibody), labelled, that binds to the anti-Fc.gamma.RIIIa158 antibody, for instance.

[0045] The above methods are based on the genotyping of FCGR3A158 in a biological sample of the subject. The biological sample may be any sample containing a FCGR3A gene or corresponding polypeptide, particularly blood, bone marrow, lymph node or a fluid, particularly blood or urine, that contains a FCGR3A158 gene or polypeptide. Furthermore, because the FCGR3A 158 gene is generally present within the cells, tissues or fluids mentioned above, the method of this invention usually uses a sample treated to render the gene or polypeptide available for detection or analysis. Treatment may comprise any conventional fixation techniques, cell lysis (mechanical or chemical or physical), or any other conventional method used in immunohistology or biology, for instance.

[0046] The method is particularly suited to determine the response of a subject to an anti-tumor therapeutic antibody treatment. In this regard, in a particular embodiment, the subject has a tumor and the therapeutic antibody treatment aims at reducing the tumor burden, particularly at depleting the tumor cells. More preferably, the tumor is a lymphoma, such as more preferably a B lymphoma, particularly a NHL. As indicated above, the antibody is preferably an IgG1 or an IgG3, particularly an anti-CD20 IgG1 or IgG3, further preferably a humanized antibody, for instance rituximab.

[0047] The invention also relates to a bispecific antibody, wherein said bispecific antibody specifically binds CD16 and a tumor antigen, for instance a CD20 antigen. The invention also encompasses pharmaceutical compositions comprising such a bispecific antibody and a pharmaceutically acceptable excipient or adjuvant.

[0048] Further aspects and advantages of this invention will be disclosed in the following examples, which should be regarded as illustrative and not limiting the scope of this application.

FIGURE LEGENDS

[0049] FIG. 1: Adjusted KAPLAN-MEIER estimates of progression-free survival after rituximab treatment according to Fc.gamma.R3a-158V/F genotype (p=0.05). Patients carrying two "V" alleles (top line) had a markedly improved and statistically significant progression free survival relative to those carrying either "V/F" or "F/F" genotypes (bottom line). For example, at the 20 month time point, .about.90% of "V/V" patients were alive whereas only .about.55% of patients with either the "V/F" or "F/F" genotypes survived. These levels remained stable through the 30 month time point.

[0050] FIG. 2: Genetic organization of the human FCGR3A gene. The FCGR3A 158V/F polymorphism is within Exon 4 at nucleotide position 4987.

[0051] FIG. 3: Amino acid sequences of human Fc.gamma.RIIIa158F (SEQ ID NO:7)

[0052] FIGS. 4A-H: Nucleic acid sequence of human FCGR3A158F (SEQ ID NO:8).

MATERIALS AND METHODS

Patients and Treatment

[0053] Clinical trial design, eligibility criteria and end-point assessment have been previously reported..sup.5 In brief, patients were eligible for inclusion in this study if they had previously untreated follicular CD20 positive NHL according to the REAL classification..sup.26 Patients were required to present with stage II to IV disease according to Ann-Arbor classification and at least one measurable disease site. All patients were required to have low tumor burden according to the GELF criteria..sup.27 A total of four 375 mg/m.sup.2 doses of rituximab (Roche, Neuilly, France) were administered by intravenous infusion (days 1, 8, 15, 22). The management of infusion and adverse events has already been reported..sup.5 The study protocol was approved by an ethics committee, and all patients gave their informed consent.

Monitoring and Endpoints

[0054] Baseline evaluation included clinical examination, chest X-ray, computed tomography (CT) of the chest, abdomen and pelvis, and unilateral bone marrow biopsy. Response was assessed by an independent panel of radiologists who reviewed all the CT scans of the included patients. The primary efficacy endpoint was the objective response rate, i.e the proportion of patients achieving either complete remission (CR), unconfirmed CR (CRu) or partial response (PR) according to the criteria recently proposed by an international expert committee..sup.28 Clinical response was evaluated at days 50 and 78. Only the maximum response was taken into account and that assessment time point named M2. All patients were evaluated for progression at one year (M12). Patients in CR or CRu with disappearance of bone marrow infiltration at M2 and reappearance of lymphoma cells in bone marrow at M12 were considered "progressive"; patients in PR with negative bone marrow biopsy at M2 and positive biopsy at M12 were considered in PR.

[0055] Molecular analysis of BCL2-JH gene rearrangement was performed by PCR, as previously described,.sup.5 on a lymph node obtained at diagnosis and on both peripheral blood and bone marrow at diagnosis, M2 and M12.

FCGR3A-158V/F Genotyping

[0056] Out of the 50 patients included in the clinical trial, one patient was excluded after histological review and DNA was not available for two other patients. Forty seven patients were therefore available for FCGR3A genotype analysis. All samples were analysed in the same laboratory and DNA was extracted using standard procedures including precautions to avoid cross-contamination. DNA was isolated from peripheral blood (n=43), bone marrow (n=3) or lymph node (n=1). Genotyping of FCGR3A-158V/F polymorphism was performed as described by Koene et al.sup.22 with a nested PCR followed by an allele-specific restriction enzyme digestion. Briefly, two FCGR3A specific primers (5'-ATATTTACAGAATGGCACAGG-3', SEQ ID NO: 1; 5'-GACTTGGTACCCAGGTTGAA-3', SEQ ID NO: 2) (Eurobio, Les Ulis, France) were used to amplify a 1.2 kb fragment containing the polymorphic site. The PCR assay was performed with 1.25 .mu.g of genomic DNA, 200 ng of each primer, 200 .mu.mol/L of each dNTP (MBI Fermentas, Vilnius, Lithuania) and 1 U of Taq DNA polymerase (Promega, Charbonniere, France) as recommended by the manufacturer. This first PCR consisted in 10 min at 95.degree. C., then 35 cycles (each consisting in 3 steps at 95.degree. C. for 1 min, 57.degree. C. for 1.5 min, 72.degree. C. for 1.5 min) and 8 min at 72.degree. C. to achieve complete extension. The second PCR used primers (5'-ATCAGATTCGATCCTACTTCTGCAGGGGGCAT-3' SEQ ID NO: 3; 5'-ACGTGCTGAGCTTGAGTGATGGTGATGTTCAC-3' SEQ ID NO: 4) (Eurobio) amplifying a 94 bp fragment and creating a NlaIII restriction site only in the FCGR3A-158V allele. This nested PCR was performed with 1 .mu.L of the amplified DNA, 150 ng of each primer, 200 .mu.mol/L of each dNTP and 1 U of Taq DNA polymerase. The first cycle consisted in 5 min at 95.degree. C. then 35 cycles (each consisting in 3 steps at 95.degree. C. for 1 min, 64.degree. C. for 1 min, 72.degree. C. for 1 min) and 9.5 min at 72.degree. C. to complete extension. The amplified DNA (10 .mu.L) was then digested with 10 U of NlaIII (New England Biolabs, Hitchin, England) for 12 h at 37.degree. C. and separated by electrophoresis on a 8% polyacrylamide gel. After staining with ethidium bromide, DNA bands were visualized with UV light. For homozygous FCGR3A-158F patients, only one undigested band (94 bp) was visible. Three bands (94 bp, 61 bp and 33 bp) were seen in heterozygous individuals whereas for homozygous FCGR3A-158V patients, only two digested bands (61 bp and 33 bp) were obtained.

FCGR2A-131H/R Genotyping

[0057] Genotyping of FCGR2A-131H/R was done by PCR followed by an allele-specific restriction enzyme digestion according to Liang et al.sup.28. The sense primer (5'-GGAAAATCCCAGAAATTCTCGC-3' SEQ ID NO: 5) (Eurobio) has been modified to create a BstUI restriction site in case of R allele whereas the antisense primer (5'-CAACAGCCTGACTACCTATTACGCGGG-3' SEQ ID NO: 6) (Eurobio) has been modified to carry a second BstUI restriction site that served as an internal control. PCR amplification was performed in a 50 .mu.L reaction with 1.25 .mu.g genomic DNA, 170 ng of each primer, 200 .mu.mol/L of each dNTP, 0.5 U of Taq DNA polymerase, and the manufacturer's buffer. The first cycle consisted of 3 minutes at 94.degree. C. followed by 35 cycles (each consisting in 3 steps at 94.degree. C. for 15 seconds, 55.degree. C. for 30 seconds, 72.degree. C. for 40 seconds) and 7 min at 72.degree. C. to complete extension. The amplified DNA (7 .mu.L) were then digested with 20 U of BstUI (New England Biolabs) for 12 h at 60.degree. C. Further analysis was performed as described for FCGR3A genotyping. The FCGR2A-131H and -131R alleles were visualized as a 337 bp and 316 bp DNA fragments, respectively.

Statistical Analysis

[0058] Clinical and biological characteristics as well as clinical and molecular responses of the patients in the different genotypic groups were compared using a Chi-squared test or by Fisher's exact test when appropriated. A logistic regression analysis including: sex, age (> or .ltoreq.60 years), number of extra-nodal sites involved (.gtoreq. or <2), bone marrow involvement, BCL2-JH rearrangement status at diagnosis and FCGR3A genotype was used to identify independent prognostic variables influencing clinical and molecular responses. Progression-free survival was calculated according to the method of Kaplan and Meier.sup.29 and was measured from the start of treatment until progression/relapse or death. Comparison of the progression-free survival by FCGR3A genotype was performed using the log-rank test. P<0.05 was considered as statistically significant.

RESULTS

Clinical Response

[0059] Out of the 49 patients tested for the FCGR3A-158V/F polymorphism, 10 (20%) and 17 (35%) were homozygous for FCGR3A-158V and FCGR3A-158F, respectively, and 22 (45%) were heterozygous. The three groups were not different in terms of sex, disease stage, bone marrow involvement, number of extra-nodal sites involved or presence of BCL2-JH rearrangement in peripheral blood and bone marrow at diagnosis (Table 1). No difference was found when homozygous FCGR3A-158V patients were compared with FCGR3A-158F carriers (FCGR3A-158F homozygous and heterozygous patients) or when homozygous FCGR3A-158F patients were compared with FCGR3A-158V carriers (FCGR3A-158V homozygous and heterozygous patients). The objective response rate at M2 was 100% (CR+CRu=40%), 70% (CR+CRu=29%) and 64% (CR+CRu=18%) in FCGR3A-158V homozygous, FCGR3A-158F homozygous and heterozygous patients respectively (P=0.09). A significant difference in objective response rate was observed between FCGR3A-158V homozygous patients and FCGR3A-158F carriers with 67% (CR+CRu=23%) objective response rate for this latter group (relative risk=1.5; 95% CI, 1.2-1.9; P=0.03) (Table 2). No difference was observed between FCGR3A-158F homozygous patients and FCGR3A-158V carriers. At M12, the objective response rate was 90% (CR+CRu=70%), 59% (CR+CRu=35%) and 45% (CR+CRu=32%) in FCGR3A-158V homozygous, FCGR3A-158F homozygous and heterozygous patients respectively (P=0.06). The difference in objective response rate was still present one year after treatment between FCGR3A-158V homozygous group and FCGR3A-158F carriers with 51% (CR+CRu=33%) objective response rate for this latter group (relative risk=1.7; 95% CI, 1.2-2.5; P=0.03). The logistic regression analysis showed that the homozygous FCGR3A-158V genotype was the only predictive factor for clinical response both at M2 (P=0.02) and at M12 (P=0.01). The progression-free survival at 3 years (median follow-up: 35 months; 31-41) (FIG. 1) was 56% in FCGR3A-158V homozygous patients and 35% in FCGR3A-158F carriers (ns). Out of the 45 patients analyzed for FCGR2A-131H/R polymorphism, 9 (20%) and 13 (29%) were homozygous for FCGR2A-131R and FCGR2A-131H, respectively, while 23 (51%) were heterozygous. There was no difference in the characteristics at inclusion or clinical response to rituximab treatment for these three groups or for homozygous FCGR2A-131H patients and FCGR2A-131R carriers, or for homozygous FCGR2A-131R patients and FCGR2A-131H carriers (data not shown).

Molecular Response

[0060] At diagnosis, BCL2-JH rearrangement was detected in both peripheral blood and in bone marrow in 30 (64%) patients, enabling further follow-up. Twenty-five patients (six FCGR3A-158V homozygous patients and 19 FCGR3A-158F carriers) and 23 patients (six FCGR3A-158V homozygous patients and 17 FCGR3A-158F carriers) were analysed for BCL2-JH rearrangement in both peripheral blood and bone marrow at M2 and at M12 (Table 3). At M2, a cleaning of BCL2-JH rearrangement was observed in 3/6 of the FCGR3A-158V homozygous patients and in 5/19 of the FCGR3A-158F carriers (ns). In contrast, the rate of BCL2-JH rearrangement cleaning at M12 was higher (5/6) in the FCGR3A-158V homozygous patients than in the FCGR3A-158F carriers (5/17) (relative risk=2.8; 95% CI, 1.2-6.4; E=0.03). The logistic regression analysis showed that the FCGR3A-158V homozygous genotype was the only factor associated with a greater probability of exhibiting BCL2-JH rearrangement cleaning at M12 (P=0.04). The single homozygous FCGR3A-158V patient still presenting with BCL2-JH rearrangement in peripheral blood and bone marrow at M12 was in CR 23 months after rituximab treatment. In contrast, the molecular responses at M2 and M12 were not influenced by the FCGR2A-131H/R polymorphism (data not shown).

DISCUSSION

[0061] Because of the increasing use of rituximab in B cell lymphoproliferative malignancies, enhanced understanding of treatment failures and of the mode of action of rituximab is required. In this regard, we genotyped FCGR3A in follicular NHL patients with well-defined clinical and laboratory characteristics and treated with rituximab alone..sup.5 In particular, all the patients included in this study had a low tumor burden NHL and a molecular analysis of BCL2-JH at diagnosis and during follow-up. The FCGR3A allele frequencies in this population were similar to those of a general Caucasian population..sup.23,24 Our results show an association between the FCGR3A genotype and the response to rituximab. Indeed, homozygous FCGR3A-158V patients, who account for one fifth of the population, had a greater probability of experiencing clinical response, with 100% and 90% objective response rates at M2 and M12, respectively. Moreover, five of the six FCGR3A-158V homozygous patients analysed for BCL2-JH rearrangement showed molecular response at M12, compared to 5 of the 17 FCGR3A-158F carriers. FCGR3A-158V homozygosity was the only factor associated with the clinical and molecular responses. However, these higher clinical and molecular responses were still unsufficient to significantly improve the progression-free survival in homozygous FCGR3A-158V patients.

[0062] This is the first report of an easily assessable genetic predictive factor for both clinical and molecular responses to rituximab. However, the genetic association does not demonstrate the mode of action of rituximab involves Fc.gamma.RIIIa. The association observed between FCGR3A genotype and response to rituximab might be due to another genetic polymorphism in linkage disequilibrium. Those polymorphisms could be located in FCGR3A itself like the triallelic FCGR3A-48L/H/R polymorphism.sup.31 or in other Fc.gamma.R-coding genes, since FCGR3A is located on the long arm of chromosome 1, which includes the three FCGR2 genes and FCGR3B..sup.32 A linkage disequilibrium has been reported between FCGR2A and FCGR3B..sup.33 However, the fact that FCGR3A-131H/R polymorphism was not associated with a better response to rituximab strongly supports the fact that a gene very close to FCGR3A or FCGR3A itself is directly involved.

[0063] Several in vitro studies argue in favor of direct involvement of FCGR3A-158V/F polymorphism. First, Koene et al.sup.23 have shown that the previously reported differences in IgG binding among the three Fc.gamma.RIIIa-48L/H/R isoforms.sup.31 are a consequence of the linked Fc.gamma.RIIIa-158V/F polymorphism and several teams have demonstrated that NK cells from individuals homozygous for the FCGR3A-158V allotype have a higher affinity for human complexed IgG1 and are more cytotoxic towards IgG1-sensitized targets..sup.23,24,34 Our present results establish that FCGR3A-158V homozygous patients have a better response to rituximab, which is probably due to a better in vivo binding of that chimeric human IgG1 to Fc.gamma.RIIIa. Secondly, NK cell- and macrophage-mediated ADCC is one of the mechanisms triggered by anti-CD20 antibodies in vitro.sup.8,11,12 as well as in murine models in vivo,.sup.17-19 and rituximab-mediated apoptosis is amplified by Fc.gamma.R-expressing cells..sup.15,16 Out of all Fc.gamma.R, Fc.gamma.RIIIa is the only receptor shared by NK cells and macrophages. We thus postulate that FCGR3A-158V patients show a better response to rituximab because they have better ADCC activity against lymphoma cells. The fact that more than 50% of the FCGR3A-158F carriers nonetheless present a clinical response to rituximab could be explained by lower, but still sufficient, ADCC activity or, more likely, by other mechanisms operating in vivo such as complement-dependent cytotoxicity, complement-dependent cell-mediated cytotoxicity.sup.11,13,14 and/or apoptosis..sup.15,16 ADCC could then be viewed as an additional mechanism in the response to rituximab that is particularly effective in FCGR3A-158V homozygous patients.

[0064] The in vitro studies suggest a "gene-dose" effect with a level of IgG1 binding to NK cells from FCGR3A heterozygous donors intermediate between that observed with NK cells from FCGR3A-158V and FCGR3A-158F homozygotes.sup.23. However, the clinical response of heterozygous patients appears similar to that of FCGR3A-158F homozygous patients. Further studies with larger groups of patients will be required to conclude against a "gene-dose" effect in vivo.

[0065] Since Fc.gamma.RIIIa is strongly associated with a better response to rituximab, it needs to be taken into account in the development of new drugs targetting the CD20 antigen. For example, it may be possible to use engineered rituximab to treat FCGR3A-158F-carrier patients with B cell lymphomas. Indeed, by modifying various residues in the IgG1 lower hinge region, Shields et at have recently obtained IgG1 mutants which bind more strongly to Fc.gamma.RIIIa-158F than native IgG1.sup.34.

[0066] Taken together, these results allow to set up new therapeutic strategies against B lymphoproliferative disorders based upon prior determination of the patients FCGR3A genotype. Since this polymorphism has the same distribution in various ethnic population, including blacks and Japanese, such a strategy may be applied worldwide..sup.23,35,36 Furthermore, such a pharmacogenetic approach may also be applied to other intact humanized IgG1 antibodies used in the treatment of B cell malignancies, such as Campath-1H, or those used in the treatment of other malignancies, such as trastuzumab (Herceptin.RTM.). Even more generally, this approach may apply to other intact (humanized) therapeutic (IgG1) antibodies developed to deplete target cells.

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TABLE-US-00003 [0102] TABLE 1 CHARACTERISTICS OF PATIENTS ACCORDING TO THE FCGR3A-158V/F POLYMORPHISM FCGR3A- FCGR3A- FCGR3A- 158VV 158VF 158FF p* n (%) 10 (20%) 22 (45%) 17 (35%) Sex M 3 12 10 ns F 7 10 7 Disease stage II-III 3 6 6 ns IV 7 16 11 Bone marrow involvement yes 7 16 9 ns no 3 6 8 Extra-nodal sites involved <2 8 20 13 ns .gtoreq.2 2 2 4 BCL2-JH rearrangement 8 12 11 ns in peripheral blood BCL2-JH rearrangement 7 12 11 ns in bone marrow *Satistical comparisons of the three groups of homozygous FCGR3A-158V patients vs FCGR3A-158F carriers and of homozygous FCGR3A-158F patients against FCGR3A-158V carriers.

TABLE-US-00004 TABLE 2 CLINICAL RESPONSE TO RITUXIMAB BY FCGR3A-158V/F POLYMORPHISM. FCGR3A- FCGR3A-158F 158VV carriers p* Clinical response at M2 Objective response 10 (100%) 26 (67%) 0.03 complete remission 3 7 complete remission 1 2 unconfirmed partial response 6 17 No response 0 (0%) 13 (33%) no change 0 10 progressive disease 0 3 Clinical response at M12 Objective response 9 (90%) 20 (51%) 0.03 complete remission 6 11 complete remission 1 2 unconfirmed partial response 2 7 No response 1 (10%) 19 (49%) no change 0 2 progressive disease 1 17 *Satistical comparison of homozygous FCGR3A-158V patients against FCGR3A-158F carriers. Data concerning the three genotype subgroups are given in the text.

TABLE-US-00005 TABLE 3 MOLECULAR RESPONSE TO RITUXIMAB AT M2 AND AT M12 BY THE FCGR3A-158V/F POLYMORPHISM. FCGR3A- FCGR3A-158F 158VV carriers p Molecular response at M2 ns Cleaning of BCL2-JH rearrangement 3 5 Persistent BCL2-JH rearrangement 3 14 Molecular response at M12 0.03 Cleaning of BCL2-JH rearrangement 5 5 Persistent BCL2-JH rearrangement 1 12

Sequence CWU 1

1

13121DNAArtificial SequenceDescription of Artificial Sequence FCGR3A specific primer. 1atatttacag aatggcacag g 21220DNAArtificial SequenceDescription of Artificial Sequence FCGR3A specific primer. 2gacttggtac ccaggttgaa 20332DNAArtificial SequenceDescription of Artificial Sequence Amplification primer. 3atcagattcg atcctacttc tgcagggggc at 32432DNAArtificial SequenceDescription of Artificial Sequence Amplification primer. 4acgtgctgag cttgagtgat ggtgatgttc ac 32522DNAArtificial SequenceDescription of Artificial Sequence Amplification sense primer. 5ggaaaatccc agaaattctc gc 22627DNAArtificial SequenceDescription of Artificial Sequence Amplification antisense primer. 6caacagcctg actacctatt acgcggg 277254PRTHomo sapiensmisc_featureAmino acid sequence of human FCGR3A158F. 7Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala 1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30 Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60 Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr 65 70 75 80 Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140 Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190 Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln 195 200 205 Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220 Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp 225 230 235 240 Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 245 250 822685DNAHomo sapiensmisc_featureNucleic acid sequence of human FCGR3A158F. 8cagcctggct gacacagtga gacctcatct ctaaaaaaaa aagcaagcag aatattttct 60taaaaggcaa ttatattcct tcttggccag gcccagtggc tcacacctgt aatcccagca 120ctttgggagg ccgagatggg tggatcacct gaggtcagga gttcgagacc agcctggcca 180acatggcgaa aacccgtgtc tactaaaaat acaaaaatta gctgggcatg ggggcatatg 240cctgtaatcc cagctacttg ggaggctgag acaggagaat cgcttgtacc cgggaggcag 300agattgcagt gagccgagat catgccactg cactccagcc ttggcgacag agtgaggctt 360tgtctaaaaa aaaaaaggta ttttttgcct ctctgttggt accaattgtt aaattctttg 420tggaccactg atgcttacca aaaaaaaaaa aaaaaaaagt gggggcatca tatttcctct 480agttgacatt aagacacagt aatttagcca gaggagatct tagcaaacat acagtccaca 540ctccactttc tcatttcatg attgtagaga ctgagatcta gacaatttaa tcggtggtca 600ccctgggtga catagctagg tctagagctc ctggtctcca ggtcagcatt tctttcttct 660tcattaaatg tcaagtttcc tcccctgttc attattagct ccttccagaa agagagtttc 720ttatcttttt agtaggtact cagtaaatac caaggtattc actaggatgc ctttggatga 780aggtaacaag ccctgactta aattggctta aacagcaggg aaatttactt gaaattgtaa 840gaaatctggg ttggttgcgt tagaagctca gtgatgtcac caagaccata ttctatccct 900ccactctgtc ctccttggct atttggcatt gacctcagac tggctgcctt caaaatctta 960ggttttgcca gcagaaccta ggacaaaatg agcccttgtt catgtacagt gggagagaga 1020gatcatctct cccaaacatg gcactcccct ctaccagatt ggccctattt aggacaaagt 1080tccgtcttct cccacttaac caataaaagc caggggaatg ctacaccctg agtggcttag 1140atcagtcaag atccacctct gcatatgagg gtgattcctg aatagaatca aggttatatt 1200agaagggagg gagagggatg gatatcaggc tagtacatca tattctattt gttgagttaa 1260ctgagtcata gcaattgttg agttggaaaa aactcagaac ctactgtgga ttcaagttca 1320agaaatcatt ctttcctaca tacaacagca ttgctctgta gccctgagct aagagagcat 1380cacgaaatac agtcttcttg ctgtttataa tcgtaagcaa actcttggac ctgggagggg 1440atgaatggat aatgtctgtc tgacttgctt ctttctagtt agtaccaact acctcccttc 1500ttcctgtgat tgttcttaga ataggataaa aaatcttccc ttccctagat cttacagtct 1560ccccttcccc caggcctttc tatttttcag gattttactc taatcacacc accgaagaat 1620caagaaatct ttaaagtgta ttagagtagc tagttgtggc agcactaaaa cacggctgca 1680aattctttga cactctctcc atcaagaaat gaggcctaca tcctctaccc ttgaatctgg 1740gtgggcttat aacttctggt gattagacta cagcagaaag agaagctgta tagcttccta 1800aatgttataa agtcttaagg atagctgcca gcaagacctt aggacaaaat gagcccttgt 1860tcacgtccag tgggagagag agagagagac cctctctccc aaatatggca atcctctcta 1920cctataacat ttacataaat gttataggtt aaacgttcca caacaaacta agtactattt 1980aacatcaaga ggaaaaagag acaggagaaa gggttaataa gcctgttgat gaggatctaa 2040gaagaacaaa ggaggcctgg tttgggcctg gctacccgtt ggtcttgcaa agaagagtct 2100gaggtggcag agccttcagt ggcagatgcc aaattatcat catgagtgac tgcaagacag 2160tgtcagctaa gatagccatt tcaagctgct gaaggccttc tcttttagtc gtggagtcct 2220gtgataagaa ctgaaagttg gaagagtgtg cttgtctgtg gccttatttg gtcggatgca 2280gtctttatca tttttaattt gtttcttaga acattttatc ttgttggcca aatgccctac 2340gaaatataaa atggagtctt tttctaagat ggagttagtt atgtcaaggg tcctttatac 2400agtcttcatc ctttttcctg gcatacaact cctaaaatcc ttagaatctc caaagtgatg 2460tcttttggtg tgctaatgag gtaactgatg gctggcagct cttaggtagc ttcataacag 2520gggctgggca caagaaagat catggcaagg tcagaggatt ggggctttca gctccaccct 2580ccaaactccc tctgggaagt ggagaggggc tgaaggttga attgatcacc aatagccaat 2640gacttaatta atcattccta agtaataaag ctcccataaa aacccaaaag gacagggttt 2700ggagatcctc cagagagccg aacacagaga ggttcttgga gggtagtgca ccagagggca 2760tggaagctcc aagccccttc ccacaggtct tgccctatgt actctttact tgtgtccttt 2820gtaatattct ttatcacaaa ctgataaatg taaatgtttc cctgagtact gagagccact 2880ctagcaaatt aattgaaccc aagatgcagg tggtgggaac ccccatttat aactggttgg 2940tcaaaagcac aggtaaaaca acctggggct tcatcctgga gtatcagaag tgtcttgtga 3000gactgagccc ttcacttgtg tcacctgatg ctatttccag ttagatagtg ttggaattca 3060attgaatttg agcagaagtc ccaatcccca gacctgtagt tgtcagtgac ctcttaggaa 3120ctgggctgca cagcaggaag tgaggggcag gtggggagca aagctttatc tgtatttaca 3180gtagatcccc atggctcaca tcaccgcctg agctcctcct cctatcagat cagctgtggc 3240attaaattat cacaggagca tgaaccctat tgtgaagtac gcatgcaagg gatctaggtt 3300gcattctcct tatgagaatc taatgcctga tgacctggca ttgtctccca tcaccctaga 3360tgggactgtc tagttgcaag aaaacaagct cggggctccc cctgattcta cattatggta 3420agttgtataa ttacttcatt aaatattaca ctgtaataat aatagaaatg aagtgcacaa 3480taaatgtaat gcacttgaat catcctgaaa ccatccccca cccccctacc cctgtccgag 3540gaaaaattgt cttccatgaa gccaatccct ggtgccaaaa attttggaga ccactggatt 3600agaagacacc cagttggtgc ccactgctga attgcttgct tgcttgcttg cttgcttgcc 3660agtggagaga aatccccaca tatctgttgt cagaaatgtg ttgtgagagc atagtgggag 3720gaactgagtt tgttttttct acagttacag caataggtaa ctggaattca actgctggac 3780tataccaaag actgccaggc cagcctacct ttctcacagc cttcttgact acctgtcttg 3840gatgagctca ctgaaagccc acataccttc attctagcat ttcctcagtc tggttgagct 3900gctttggagg taatacaggt tgtaggactt ccctccactc ctgctcagga ccgttttcag 3960caggctaatc agacagcagt tggcactgag tacaactgga gaaatgttat cagcactgaa 4020gactgctcca gctacaaatg cacagcgacc cacctcagct ggacctttgg cattgcttgg 4080caatccttac aactgtttgg aattcttggt aattcttgta catacaaaca gtcggctcct 4140tctcccatgc accacaatgg ccattctcaa ctctggtgtt ctcaaacccc caccacaact 4200gttacccact ctttctcgac aagtgtcttt gactcctcct taaccaagaa aatcaggacc 4260agcagatgtg gatggtcact caacatctga aaatggattt gcatatgtac cccctcagct 4320cctgccttca gctcagagcg agaggtaatc cgtatccagt tcacagccaa ctccctgtcc 4380atgtcccatt cccatctcct caggacccac acttgcttct ctaggtgttc cgtccctgtt 4440aggcatccaa cttctcccac cctgctggct tcttcccaaa gacctataac caagctcgta 4500tctttcactt aaaagaaaac aaaaactccc attctcttgt aactaccttt gcagaattgt 4560gtctgaaaca gtgagagaga tctaacttaa ttgactccat cttgcttcta acctccaagc 4620tgtctttcct cattcttggg cataggctga actaactttg ggagaaactt agtttatagt 4680ttgtggttta aagcaaagat gataacagcc ctttcccagg gcagacctcc tttttttctg 4740aagactagat tgtctttgta ggactaacat tagccacaag attggaaatt atggtttagg 4800aatcatgcag gtggaggcta caagattctg acccttccta agcactgatc ctaagatcgg 4860tgcttgagat attttgcaga ccctgcactt gatggatcac ctggcaacac ccagatcaat 4920aaactggctc atctgatctt gtggtgccca cccaggaact gactcagaac aagaagacag 4980cttcaacttc ctgtgatttc atccctgacc aatcaacact cctggctcac tggcttcccc 5040tccaccaacc aagttgtcct taaaaactct gctccccgaa tgctctggaa gactgatttg 5100agtaataata aaactccagt ctctggctca gtcagctctg catgaattac tctttctcta 5160ttgcaattcc cctgtcttga tgaatcagct ctgtctagtt accatcctcc acttctcctt 5220tcttattgtg tcacttaggg ctctgggtta taaacaactt tatcagaatc cagatctttt 5280aagtagagga aaaagattta ttggatggaa actagaggag gtgagcccat cccactgctg 5340tgatgactgg gacccaacca tctccctctc ctagaagtga atctccctta tgagtaaaca 5400agtgtcactt attcaggatt catctcagaa gagactctaa tgggccaaac ctcagttata 5460tgcctgtcct ctgtctgcct gtatcagtta gctagcactt ttataacaaa gtaccacaga 5520ctgggtggct taaacaacaa aaatgtattt tcttacagtt cttgaggctg caagtccaag 5580atcaaggtgt tgacagggtt ggtttctttt aatttttttt ttaaaaattt tattttagat 5640ttaggggtat gcgtgcaggt ttgctacata ggtaaactcc tatcacgggg gtttgttgca 5700cacattattt catcacccat gtactaagcc tagttactca atagttattt tttccgatcc 5760tctccctctt cctaccctcc accctcaagt aggccccagt gtgtctgttg ttcttccttt 5820gagtccatag ggttggtttc ttctgaggcc tctctccttg gcttgtaggt gtccatcttc 5880tccctgtatg ggtctgtgtc ccaactaaca aggacatcag tcatattgga ctagagccca 5940cctaatgatt ttattttaac ttaattacct ctttaaaggc tctatatcca catatagtca 6000catcctgagg tatagggggt tccacatgtg gacttcaaca tatgaactgg gagagacaaa 6060atttagtcct taatagtgcc ccaaagtggg gaaaaggaag atctggaccc tcgggtttcc 6120atagtagaaa gcaatcactg ctttctatta agtactcaca gtggggcttc tccagaaaga 6180atgatatgct aataagaagg ggaggaggaa gtgatcctgg acagccagat gatatgtgca 6240ctattccttc ataatggaga ttctgaagag gagaagcact tgactaaaca ctttttcatt 6300cctactccgt cttcaaccaa aagctgtcaa acttctgttt ctcagcccca gcccctgaaa 6360ttgctcagga aaaggtcatt aatagttcct tgattgccat atttcaatca aactcttgtt 6420tgaattcttt tctacaacat taatactgtt actgttgact actccttcct tgaagatctg 6480ttcccactaa acttccttgt tccctcctct tcagcccctc ctatacaaac tcctttgtca 6540gctatttttc ctgtgcacgc ttcaaaaatg tttgcatgtc aagtttctgt cattgactct 6600ctcctcttct cctctccctc tcaatccctc cttcctttcc ctcactgttt ccctttaatt 6660ctctctcaat actcttacag tttcagagat cttatcctta ctttatctta acctaggatc 6720tctggatgga ttcaaataga gcttcttaaa ttaaaggaaa cataatgtgt atatttgcat 6780cctttcttgg gagaaggccc aaaggttttt atcagaggtt tgaaacctca accgtgttgg 6840tgcctcctaa attgtgtctt ttgtcaagac ctgtcttctg agttccaggg ccatgtgtct 6900cactgcctac tggaaatctt cacctgaaac cttcacagct acctcaaact caataacatc 6960aaaagctgaa atcattgtct ctccctccca aagcctgctc atcttcccat ttttcttttg 7020tccatgaaag ctactgccat cctccttatc acccaaatta gaaatccgag catcacccag 7080acctctcccc cttcatcacc cctcagccaa tcactcacca agtcttgtcc atccttcctt 7140cctaacttct ctcctggatg cttccattgc atatccactt tttaaacaga gtggctcttg 7200tctcaactag actgttgaaa taatcttcca acttttccct ccaccttcca tctctctccc 7260ctctaactca ttccttggac tgctgtcaga gtacttttca taaaatataa aacagatctt 7320gtgattcccc agtctaaagc ctttttatta gttcccatta ccttttagaa taaaatatgt 7380actgttcatc ctgacacaca aaactcttcg tgataaatac taattgagtg cctagtatgt 7440gcctgccctt gtgctaaatg ttgagggtac aggggtaaac aaggtgaaca gcttccctgc 7500tctccaagac ctttcagtcc acaaatgcaa tgagtttaca gaggagaagc acaagctcct 7560aaaggagttg gggtggggtt gggggtcaga acctaattta gaaaattgag gaaggtctca 7620acctcccatc ttgcatttac aatagtaatc agcaggtgtg gtaccaaata tggaaccaac 7680aattttatct gcattatctc atttaagcca tgagtgccat tattgttagc ctcactttac 7740agataaggaa actgagggct agaaggttaa ataagtggca gagttgggat ttcctccaga 7800ttcctgtgag acccagacat cttaatcctt ttggaacctg tgcttctcct ttgtagtact 7860cactacactt gtggaactac atccaactac acttgtggaa ctacagccag ctctgcaaac 7920atgacagtct acttcactcc aagtctttgc tcatgctgct cctcttgcct ggaatgccta 7980tttctctcaa aaatcttcct gctgaatatt ttgcgatcta attaaagtgt tctctcttcc 8040atgtacactc ctccctcaga tagaattagc cactgtcttc tttgtgcata cacagcattt 8100cataaatact gtcacagtcc ctctagcact tcaaatactt atctgatgtt ctccccctaa 8160gaaactgtaa gtcctagagg atgacaatca actgaattcc atagtcagaa acttctgctg 8220tgcctggcct tccaatgaga aaaggagaga agaggagggg aaggaagaaa aagggaagga 8280gaagaaagaa aagcaaacat gaagataaac acttcaatat atgatatccc aagaccatct 8340acccttttgt aaaaattttg cttttttttt ttccccccca agagtcaggg tctcactctg 8400tcgcccaggc tagagtgcag tgccatgaac ataactcact gtagcctcta actccggggc 8460tcaagcaatc ctcctgcctc agcctcctgg gtagctggga ctacaggcat gcaccaccac 8520atctggctat tattattatt actatattag tagagatggg gtctttctat gttgcctagg 8580ctggtctcaa attcctggcc tcaagcaatt cttccacctc acattggcct tccaaagtgc 8640tgggattaca ataagccacc ataggccaaa attttgcatt ttatccatta ctgtaaaatt 8700aacccttaga aatccaacaa cactcaattt gagaattgtt caacaaccac ttaatgaaaa 8760ccccctgaaa gcttcccatc ctgttgcagt ccctttctct cctcctgtgc tctctcctct 8820tcttcctatc tagcccaccc ttttggcagc taagaattcc tccctccatt ggagagccac 8880agaccaaaga ggagtcaaat aagaaaataa gacctcaaag aaggaaaaca aagtgaaggc 8940cttgcatcag aagtcacgtg gcagaaagcc acctggatat ctgaaaagaa gaaagaattg 9000agggatatcc gctttttgcc tcagagacca tccttagccc tgaaggcttt gtttctgctt 9060taggtttccc agataagcat ccgaagtgct acagcaagga actttaagtt tccagatact 9120tgtctggatt ttgcaaggcg tagatgagtc acttgagaag gagaactgga atggctgcct 9180gggttcattt ccattgtcca atccaagggc ctgtggagaa ggggctgctg caagactctg 9240tgtgtggcgg ggggaggggt gggtacgtgg atggcaatgg gaggatcaat taactccacc 9300caggagccaa atgaaacaca caaataaaaa acaaaacctg agtagtggtt tttaggtcat 9360tctggagtag aaagagcatt catttatagc aaaggttggc gggcacctgt gtcagcccct 9420gcctccactc cacccctaac aagtatcagg tgcccacacg ggcctgctgc tcgcctcctg 9480ggcttttcta agccaggtga gacctgtccc agatgtccac gaatccactg ggggagtggc 9540actatcaagc agagtcatct gattttctgc ctgggacctg gaccattgtg agagtaacca 9600acgtggggtt acgggggaga atctggagag aagagaagag gttaacaacc ctcccacttc 9660ctggccaccc ccctccacct tttctggtaa ggagccctgg agccccggct cctaggctga 9720cagaccagcc cagatccagt ggcccggagg ggcctgagct aaatccgcag gacctgggta 9780acacgaggaa ggtaaagagt tcctgtcctc gcccctcccc acccccacct tttctgtgat 9840cttttcagcc tttcgctggt gacttgttct tccagggccc atttctctac cctacctggg 9900tttcttctaa cctggaaatc taatgatcaa atcacactaa aaagtcagta gctcctgtgg 9960attacatatc ccaggagcat atagattttg aattttgaat tttgaaagaa attctgcgtg 10020gagataatat tgaggcagag acactgctag tggtctgaag atttgaaagg accactttct 10080gtgtgcaggc agggcctcag ctggagatag atgggtctgg gcgaggcagg agagtgacaa 10140gttctgaggt gaaatgaagg aagccctcag agaatgctcc tcccaccttg aatctcatcc 10200ccagggtctc actgtcccat tcttggtgct gggtggatcc aaatccagga gatggggcaa 10260gcatcctggg atggctgagg gcacactctg gcagattctg tgtgtgtcct cagatgctca 10320gccacagacc tttgagggag taaagggggc agacccaccc accttgcctc caggctcttt 10380ccttcctggt cctgttctat ggtggggctc ccttgccaga cttcagactg agaagtcaga 10440tgaagtttca agaaaaggaa attggtgggt gacagagatg ggtggagggg ctggggaaag 10500gctgtttact tcctcctgtc tagtcggttt ggtcccttta gggctccgga tatctttggt 10560gacttgtcca ctccagtgtg gcatcagggg ctggggaaag gctgtttact tcctcctgtc 10620tagtcggttt ggtcccttta gggctccgga tatctttggt gacttgtcca ctccagtgtg 10680gcatcatgtg gcagctgctc ctcccaactg ctctgctact tctaggtaag tcagggtctc 10740cctggttgag ggagaagttt gagatgcctt gggttcagca gagacccctt ttcaggctac 10800gaatgagact cccacgaagg gatgggaccc ctcaccacat ctatagctgt ggattgagct 10860cctaggacaa gccaagatgg ggctagaaat gaggagaatg ctggttccaa ttggggcata 10920ctcatgagtg aggccagtca cttcacccct ctgggtccca gaatcactct gtggaaccaa 10980agagcttcga ctagatggtc cctagggtct gtctctttca gtttgacatt ccagggttct 11040cctctatgat tttcaatttc taccctttct tgtggggata tgggttgagg ctctttctgt 11100agcttggttc agggaaattc aacctgtacc cttaatttgt gagtttgcac agggagcaag 11160gggtaaggga gcagtgttga aaatagggat ttgtgttgac agtggcgcaa gaggcatgaa 11220cagtggagac cagagagcag gtagcaaggt ttccaccaga aacatcctga ttcttgggaa 11280aattgggctc ctggggcaga ggagggcagg ggagttttaa actcactcta tgttctaatc 11340actctgatct ctgcccctac tcaatatttg atttatcttt tttcttgcag tttcagctgg 11400catgcggact ggtgagtcag cttcatggtc ttggattgac ccagtggggc acatatgggg 11460acaaaggcca taagatattg ggaaatgctt gttgaatggg aaaatgctga tgtggggtta 11520gcagggatag ttcctccaac acagcagaac ttggccctgt gcttctctgg ccagctttcc 11580ttaagatact gaacaggcca aaaatggggc caagatgctc taagactgag ccaccaagca 11640tgggtttgca atgagctcat tctggctttg aggctccctg ggaatggcag tgtagagcct 11700gctcctctcc ctgtcctcac cccacattat cttggctcct cagaagatct cccaaaggct 11760gtggtgttcc tggagcctca atggtacagg gtgctcgaga aggacagtgt gactctgaag 11820tgccagggag cctactcccc tgaggacaat tccacacagt ggtttcacaa tgagagcctc 11880atctcaagcc aggcctcgag ctacttcatt gacgctgcca cagtcgacga cagtggagag 11940tacaggtgcc agacaaacct ctccaccctc agtgacccgg tgcagctaga agtccatatc 12000ggtgagttga tgaaggggaa gaggaaaatc accaataaag ggtgaaacaa agggtcctga 12060aatacttggt aagagccaga gatgatattc ttagagataa aagctaagat gagatgatgt 12120gtggtcccac tgaatggtat cagagttgta gtcctagctc taagtaggtc ttgggcaaaa 12180tgtcaaagcc tgtcagacag tagatatagg actgctgcat tgcacaattc caagaatccc 12240catatggagt gcatacaatg tgaatgtgtc atgtgaaggt taggccatgg catagatgct 12300caataatagt tatttatata tttattttca ttttttttaa ttttattttt tgagacagag 12360tatcactctg tcacccaggc tggagtgcaa tgcggcaatc tcagctcact gcaacttctg 12420cccccttggg

ttgtagtgat tctcctgcct cagcctcccg agtagctgag attacaggca 12480cccgccacca cgcccagcta atttttgtat ttttagtaga gacagggttt caccatgttg 12540gtcagtctgg tctcaaactc ctgacctcag gtgattcacc agccttggct tcccaaagtg 12600ctgggactac aggcgtgagc caccacacct ggccaataat atttattgaa taaattaatg 12660aatttggtgt taggacctca atctccttct cgctctcaga catgtaatgc cctaagccac 12720ctcccaaagc aatcctagtg gcctagcatc atatctttct gtctcctcat caatgctata 12780ctcaaaccta taattaagca taaatttggt aatgtgatag ctcttccaat agaggcagat 12840acatgttcag cctgcacatt aatcatgaca tgaaagttct tgtgtactat taacagaata 12900tagacgtcag acacaggtag gagaaatatt ttgaaggcag aggtctttcc tggtgtccct 12960acaatcttac cacataggct ggtccctgca gtgtcgccct gcaaacctaa ctctacttcc 13020acggctgttc cattcataca atgtttatgg gtggaacaag ctttggggga agaagggcat 13080aaggaggtgg atctgcaaga gagctccatg gaattgggcc tctgaaactg atttttgtgg 13140ctctttggcc tctgacagta ccactcaact gacatggtct tcactctcca gagctacaag 13200aagatatgtc catttctagc taggtaagag atgtccacct acaaccaaat aaaatggggg 13260aattaccaag agaaagcaat agaaaaatca agtctaagag ttactagttt gccttgaact 13320tggctctaga aactggcttt agaagtctag ccaatcaagg ctatattaaa ctgtgaccat 13380gagaattagc ttcaccaggt aaacttctga gcatccttta atcctttagg acccatttca 13440cttatgtcct cctctgagaa gcatttttta cttctttttt tgtttgtttg tttgtgtttg 13500tttttgtttt tgtttttgag acagagtctc tctctgtcac ccaagctgga gtgcagtggc 13560gcaatcttgg ctcactgcaa cctccacctc ccgggttcaa gcaattctcc tgcctcagcc 13620tcccaagtag ctgggactac aggtgcatgc caccacgccc ggctaatttt ttgtattttt 13680agtagagaca gggtttcgca gcgttagcca ggatggtctt gatctcctga cttcatgatc 13740tgcccacctc ggcctcccaa agtactagga ttacagatgt gagccaccgc gcccagcctg 13800cattttttac ttctttcagg cagaatttct ttattccaat ctagtcagcc ccgcagtcct 13860ttattcttag cctgttgtag cacttgtcat attgtattgt gattatttct gaatatttat 13920gtttctatgt ctagactgta gattctttga ggctgagaac tatatgtccc atcatctggg 13980tatctccagt ccacagtgtg tcatacatag tgagtgcttg atgaaatatc acttgaagga 14040atatacatat ggacattcac tgggtccatg acaggataga ttcgaacaag aatgttcctc 14100caaaggccac cagactatat actaaccatg actttatgct aataatgatt catctctctg 14160ctgaaaaagt aagtggatag ataggcacat ggcttctttt gataaatgat atctcttaat 14220aggtaatgaa gattactttc tgtttggcaa atctttgtgg tagagaatca tgaccaacac 14280acgtcctacc aattttgttt agcatcaggt agtagatttt ttaaattata gtaattcaag 14340ctgagaatgt agatttaaaa aataaaatta ttgtaaattt tgttttgttc ttattacaaa 14400agtcatttgg ggtcaatttc aaaaatatat aaaagtaaac aggagaaatt taaaatgtcc 14460ttcagtccca ctccttcaga gaaaacccct gttaatatgt aagtgcatat ccttcttttt 14520tctgtgcata atacttttta aaatatttga agtattatgc ttttttaact taaaattgtc 14580tcatgaatat tttcttatgc cattataata cttacctata acatcattat tttttaatta 14640ttcaggccct ttcccgacca tgacctcatg ttctctcttt gtgaagtctg attacttggt 14700gacatgatcg tgagaataag ctctggcgat ataagaattt cctctcttga aggccatgct 14760cagtaaatta cttggtgaca tgatcgtgag aataagctct ggcgatacaa gaatttcctc 14820tcttgaaggc catgctcagt aataaagttg gtctcaccga ggccctgtga caccttagaa 14880accacgaatt gccaggctga gcaataccag tcccgccctt cccctccctg gtgtttacat 14940tgagttctcc ttcacaattt ctgcagccac tccgtggcca ccgtcacctt attcctgact 15000gccacaagag tctttcaata ttcctttgat tgcctattcc ttctgaaatc taccttttcc 15060tctaataggg caattcatca ttttcaaatg caatttttac tctgatctag aacttactgt 15120gaatccttgt cacctgccac agcaaatcta agtctagcac ttaaggatcc tgcagatatg 15180ctcatcgttg cttctcactt acctcattgc ttagtccctc tgctctaacc ctgtgtgttg 15240atcacatgtg tgtgtgtccc tcttccccat tagacaaagg tcttggtatg acttcagttc 15300tcttgcaggg ccccatcagc tcttccccaa agggagctat gcagggttga ctcccaatct 15360ggctttccct tatgtctcag gatctgggtg gtacgtggcc ccttcacaaa gctctgcact 15420gagagctgag gcctcccggg cctggggtgt ctgtgtcttt caggctggct gttgctccag 15480gcccctcggt gggtgttcaa ggaggaagac cctattcacc tgaggtgtca cagctggaag 15540aacactgctc tgcataaggt cacatattta cagaatggca aaggcaggaa gtattttcat 15600cataattctg acttctacat tccaaaagcc acactcaaag acagcggctc ctacttctgc 15660agggggcttt ttgggagtaa aaatgtgtct tcagagactg tgaacatcac catcactcaa 15720ggtgagacat gtgccaccct ggaatgccca gggacgcctg tgtgtggaac ctgcaatcac 15780actgggaagt tgagttggga ggagattcct gattcttaca cgcacttctt catatgtggt 15840tccctcctgg tgatcaccag gaggtcccca aaagtccctg attgcagggt aggtttgcag 15900ctctgtttca gtccattctt ttggggtagc taggaggtgt cattcactct gcagcatgat 15960ggcaggagca gaagccacat ctcctcccca ataaatacct ctgtctttcc ttacgctaat 16020cacacccacg gtgtcatatg ttcctatcgt gctggcctcc ttcttatcca agccttttag 16080ccacgatcca aactggcagg agcccctcat cccctcacag aaagagccca gaacctgggt 16140tctggccctg cagctaatta accatctgac cagaggtgag ccacttagtc tctctgaacc 16200ccaatttctt cttccgtaac aaaaataagc tgacatttat tgggcacctt tcagtgtgct 16260agactctgtg ctaaacaatt ctttacatgc acctggtttg actatcacag tagaccttca 16320caacatgaga taggtaatat tccattttac agatgaagta accgaggtgc aaaaataaat 16380aaataagttt ccctaaggtc acatcaaaga cttcaaagcc tgtatattta accagtaagt 16440aaaagatttg aacaagcact aatatcctat gatcccatta agtcatccac aaaacatctc 16500taggttctgt agcaccagcc tccagaatca gagctctaga gtggtgtgcc tggactttcc 16560agtttcacag aacttctatc tgtaactagc ccaagacata aattgtaaac aatttgcatg 16620tagaaaggca gcaaaacacc ttttgagatt ttgacactac aatgccataa tttgtacaaa 16680aataatttca tgacacttta aactgaaagt aaatactccc aagtggttag ggaaagagag 16740caaataaagc aaatggggta acatgtaaac aatgagtgga tctgggtaaa ggatatacga 16800gattaaacta ttctggtcat ttttttttta agtttggaaa tatatcaaaa tcaagagttt 16860aaaaaattga aatgcaaaat caacaaattt gtcccagttt ctagaccata gcattgtctg 16920acaatttctt aactgtcaca caaaacccag cttacaacct aacttgttaa cgctccctgt 16980cacatctctg tcaaacaagc aggagccttt gctcagtgtt tggtgagctg tcctctgctc 17040agatagcact aagatcagga accaatggga ggaagcaata ctttccccca gacttcccca 17100ccattcctac cacttgcctg ttggctgttg tcaaagactt tctactggtg acctcactgt 17160ttgttccaaa tatctgcctt agtgactgtc attttttttc atctctccac ttctcctaat 17220aggtttggca gtgtcaacca tctcatcatt ctttccacct gggtaccaag tctctttctg 17280cttggtgatg gtactccttt ttgcagtgga cacaggacta tatttctctg tgaagacaaa 17340cattcgaagc tcaacaagag actggaagga ccataaattt aaatggagaa aggaccctca 17400agacaaatga cccccatccc atgggggtaa taagagcagt agcagcagca tctctgaaca 17460tttctctgga tttgcaaccc catcatcctc aggcctctct acaagcagca ggaaacatag 17520aactcagagc cagatccctt atccaactct cgacttttcc ttggtctcca gtggaaggga 17580aaagcccatg atcttcaagc agggaagccc cagtgagtag ctgcattcct agaaattgaa 17640gtttcagagc tacacaaaca ctttttctgt cccaaccgtt ccctcacagc aaagcaacaa 17700tacaggctag ggatggtaat cctttaaaca tacaaaaatt gctcgtgtta taaattaccc 17760agtttagagg ggaaaaaaaa acaattattc ctaaataaat ggataagtag aattaatggt 17820tgaggcagga ccatacagag tgtgggaact gctggggatc tagggaattc agtgggacca 17880atgaaagcat ggctgagaaa tagcaggtag tccaggatag tctaagggag gtgttcccat 17940ctgagcccag agataagggt gtcttcctag aacattagcc gtagtggaat taacaggaaa 18000tcatgagggt gacgtagaat tgagtcttcc aggggactct atcagaactg gaccatctcc 18060aagtatataa cgatgagtcc tcttaatgct aggagtagaa aatggtccta ggaaggggac 18120tgaggattgc ggtggggggt ggggtggaaa agaaagtaca gaacaaaccc tgtgtcactg 18180tcccaagttg ctaagtgaac agaactatct cagcatcaga atgagaaagc ctgagaagaa 18240agaaccaacc acaagcacac aggaaggaaa gcgcaggagg tgaaaatgct ttcttggcca 18300gggtagtaag aattagaggt taatgcaggg actgtaaaac caccttttct gcttcaatat 18360ctaattcctg tgtagctttg ttcattgcat ttattaaaca aatgttgtat aaccaatact 18420aaatgtacta ctgagcttcg ctgagttaag ttatgaaact ttcaaatcct tcatcatgtc 18480agttccaatg aggtggggat ggagaagaca attgttgctt atgaaagaaa gctttagctg 18540tctctgtttt gtaagcttta agcgcaacat ttcttggttc caataaagca ttttacaaga 18600tcttgcatgc tactcttaga tagaagatgg gaaaaccatg gtaataaaat atgaatgata 18660aaattctttc ttcttccctt tgtccaacat tgtaacagag attggtttgg attggtaaga 18720aacaccccct cctcccagca accatctcac cacaactcat ataaattagc cagcttgctt 18780tccaaatctt gctgagacaa ttgggctaag gaggattctt atgggaagta tgggatagga 18840gggtgaataa gcattagaga tcgttttaga gcattggggc agataggaga aggcacagct 18900acacaggagg tagaggcctg ggcagaggta gagggtcagc ctgattgtat gaattatgag 18960ctatatacca agacgattca agctagattg catacataaa tattacataa gattccgaca 19020cgacacaggt gcatttggaa accttggaca ttcaactcac atttatttac tacctacaat 19080gtgcaagctt gagttcaggt gctgaagata ccagatgaac aacacagggt cattccctgg 19140agaagcttta tttctagtga gaaaaacagt taaataggaa gagaatgaag aaagggctgc 19200agaaaagagg cttgatttgg ggggtgtggt catgaaggat gagtaggagt tcgccaggca 19260aagaagagaa gaaaagccca aggttcatag gcaaagattc aaaaaccaga gtgtgagttc 19320aagaaagcag tttggttctg tgtcggtgag ggagaggaaa gagtttcagg gccagatcat 19380gaagggcatt accttccaaa ctaaggagat cgtatcagac cctgcaatac attgagagag 19440tttaagcaga ccaggtttgt accgtatagt attttagaag gattctctcg caactacttg 19500atggatggac gggacaggag agttgaagac cagaagccaa atagggcagc aaggcaggat 19560gcagtaaccc aaagggagca atgaggaagt aactggcggt gaggctggag aggaaggtgc 19620ttaatcaaca aggtatttag gaggccgact ctccaagaat tggcagccag cagtacacgg 19680cgtgactaag gaccaggttc cacacatagt gcccgttttc tgagttagga aatagaaagg 19740caaggcaggt acaggtttgg tggaaagaca aacaattcgt tttggtatta ttagtactta 19800cttcctttgg tcagtaaatt ttcttaaagt gtcagtttcc ataacgtaat tgccgtggtt 19860aagcagctaa gagttatcac tacaacccta gtcggaaaaa ccaaatacct caaaattacc 19920cgtacagcac taaggcagaa gaggacattg ggaaccacac aacgcggagg tctgctacca 19980gagctccctg cggttagcac cgcggctggt tttgagcgcc aaggccccag cgctcccagc 20040ggatagcatc gcacgcagtt ttttcagtca aagtttcaaa aacccagggt tcacaaaatg 20100cgacttccgt ccctgggtgg gatcgaacca ccaacctttc ggttaacagc cgaacgcgct 20160aaccgattgc gccacagaga cgggcgttgg cgattttggc tgccaagtca cttcactgaa 20220gaaaaaatgc tcagcactca cgtctccaaa aaaattgagg ttgatttgaa accagtgaca 20280caattagctt tccgtgcttc agggcgcggc tcatagccct gagcgaggca ggtctttttt 20340ctgcgctagc acttgcctag atctggagca ggactcagct tccagcagaa gaggttgaga 20400aaaggagagc agaagagaat gcaggaacga agggtcttcg gggaatccaa aatggatgct 20460ctctgtgggt tcgggggttc cgttgatttt ggtcagagaa gtacgacgat aagctttttt 20520tgctgatgta gacaacttat gtatgcatgt gcacacgttt agtgctgact cataataagc 20580ttattatcgt gagcattaaa aatattttct ttcaggtcca atcacgtcca gcaaaatgtg 20640atgtctaagt aagtgagttt tgtgttacaa aattagtctt caacccacgc tgttttgaaa 20700ggtttctacc ggcatattag acatgcagac agaacacgga gcttaaaaag cctgtaacat 20760tccaattaat ggtattcagc ttggaaataa aaaatatttt ttaaaaaatg cgtgcaactt 20820aaggactttc atgctgacat atccagatcc aaatatctga ggacagagac ccctaattcc 20880accaccatcg acctagggaa cctcgtcagt gctgggtcta aaaaggcttt ttttttttct 20940ttaattcata tgtatatata ctttattcat atatatatat actttaagtt ctaggctaca 21000tgtgcacaag gtgcaggttc gttacatata catgtgccat gttggtgtgc tgcacccatt 21060aactcgtcat ctacattagg tgtttctcct aatgttatcc ctccctcctt cccccaccca 21120cgacaggtcc cggtgtgtga tgttccctac catgcacacg tatgtttatt gtggaactat 21180tcacaatagc aaagacttgg aaccaaccca aatgtccatc aatgatagac tggattaaga 21240aaatgtggca catatacact atggaatact atgcagccat aaaaaaggat gagttcatgt 21300cctttgtagg gacatagatg aagctggaaa ccatcagtct gaacaaacta tcacaaggac 21360agaaaatcag acaccgcatg ttctcactca taggtgggaa ttgaacaata agaacacttg 21420gacgaaagcc attttctata ttgcccaaaa accagggtct ctccatagcc tccacacaga 21480atctcctttc tttctgccct gccatcctct gtcatcagtg ggctccagtt taggagcagg 21540tggaagtttt caatgatgtt cagtgaaatg agaagacatg caaacataga tatgtatatg 21600cagaaattat atatgcatat atgtttatat gtacacagta tcatatgtat aataaataag 21660taaataaata aataaatttg ccaaatgatc tttaaactag agtcatttat tttttttatt 21720aatttttttt tttgagatgg agtcttgctc tgtcgcccag gctggagtgc agtggcgcaa 21780tcttggctca ctgcaacctc cacctcccac attcaagcaa ttctcctgtc tcagcctcct 21840gagtagctga gattacagtc atgggccacc atgcccggct aatttttgta tttttttttt 21900tttttttttg agacagagtc tcgctgtcgc ccaggttgga gtgcagtggc gcgatctcgg 21960ctcactgcag gctccgcccc ccgggattca cgccattctc ctgcctcagc ctcctgagta 22020gctgggacta caggcacctg ccacctcgcc cggctaattt ttttgtattt ttggtagaga 22080tgggctttcg ctatgttggc caggctggtc tcaaactcct gacctcaggt gatcctcctg 22140cctcagcctc ccaaactgct gggattacag gtgagagcca ctgtaccagg cctagagtca 22200tttcttttat actttaaatt tttgtctctg ttcttttgct cagacctgtg gagctggcaa 22260tatgggcaag tgtcatggac tgtctactgc caggaagctc cattgtcacc gacaggatca 22320gaagtggcat ggtaaatggt acaagaaagc ccattcgggc acagtcctga agaccagcct 22380ttttggaggt gcttctcatg caaagggaat tgggctggaa aaagtaggga ttggagccaa 22440atagcccagc tctgccactg agaagtgtgc cagggccaag ctgatcatcc agcataagct 22500agatgctgtg gtctccactg gcacagctga tcctcttgtt acaggatgga ggctgtgagg 22560cagatgagag aacagcaaga aaatcacagc ctttgtacct gatgatgatt gcttgaattt 22620tattgaaaaa aatgatgaag ttctgtatca ggggaaccag cacccaatat ttcaatgtag 22680gttct 22685945DNAArtificial sequenceFCGR3A gene 158F allele 9tcc tac ttc tgc agg ggg ctt ttt ggg agt aaa aat gtg tct tca 45Ser Tyr Phe Cys Arg Gly Leu Phe Gly Ser Lys Asn Val Ser Ser 1 5 10 15 1045DNAArtificial sequenceFCGR3A gene 158V allele 10tcc tac ttc tgc agg ggg ctt gtt ggg agt aaa aat gtg tct tca 45Ser Tyr Phe Cys Arg Gly Leu Val Gly Ser Lys Asn Val Ser Ser 1 5 10 15 11887DNAHomo sapiensmisc_featurecDNA sequence of human FCGR3A158F 11tctttggtga cttgtccact ccagtgtggc atcatgtggc agctgctcct cccaactgct 60ctgctacttc tagtttcagc tggcatgcgg actgaagatc tcccaaaggc tgtggtgttc 120ctggagcctc aatggtacag ggtgctcgag aaggacagtg tgactctgaa gtgccaggga 180gcctactccc ctgaggacaa ttccacacag tggtttcaca atgagagcct catctcaagc 240caggcctcga gctacttcat tgacgctgcc acagtcgacg acagtggaga gtacaggtgc 300cagacaaacc tctccaccct cagtgacccg gtgcagctag aagtccatat cggctggctg 360ttgctccagg cccctcggtg ggtgttcaag gaggaagacc ctattcacct gaggtgtcac 420agctggaaga acactgctct gcataaggtc acatatttac agaatggcaa aggcaggaag 480tattttcatc ataattctga cttctacatt ccaaaagcca cactcaaaga cagcggctcc 540tacttctgca gggggctttt tgggagtaaa aatgtgtctt cagagactgt gaacatcacc 600atcactcaag gtttggcagt gtcaaccatc tcatcattct ttccacctgg gtaccaagtc 660tctttctgct tggtgatggt actccttttt gcagtggaca caggactata tttctctgtg 720aagacaaaca ttcgaagctc aacaagagac tggaaggacc ataaatttaa atggagaaag 780gaccctcaag acaaatgacc cccatcccat gggggtaata agagcagtag cagcagcatc 840tctgaacatt tctctggatt tgcaacccca tcatcctcag gcctctc 8871215PRTArtificial SequenceAmino acid sequence of FCGR3A gene 158F allele 12Ser Tyr Phe Cys Arg Gly Leu Phe Gly Ser Lys Asn Val Ser Ser 1 5 10 15 1315PRTArtificial SequenceAmino acid sequence of FCGR3A gene 158V allele 13Ser Tyr Phe Cys Arg Gly Leu Val Gly Ser Lys Asn Val Ser Ser 1 5 10 15

Claims

1. A method of assessing the response of a subject to a therapeutic antibody treatment, comprising determining in vitro the FCGR3A158 genotype of said subject.

2. A method of selecting patients for therapeutic antibody treatment, the method comprising determining in vitro the FCGR3A158 genotype of said subject.

3. A method of improving the efficacy or treatment condition or protocol of a therapeutic antibody treatment in a subject, comprising determining in vitro the FCGR3A158 genotype of said subject.

4. The method of any one of claims 1 to 3, comprising determining amino acid residue at position 158 of Fc.gamma.RIIIa receptor, a Valine at position 158 being indicative of a better response to said treatment and a phenylalanine at position 158 being indicative of a lower response to said treatment.

5. The method of any one of claims 1 to 4, wherein determining amino acid residue at position 158 of Fc.gamma.RIIIa receptor comprises a step of sequencing the Fc.gamma.RIIIa receptor gene or RNA or a portion thereof comprising the nucleotides encoding amino acid residue 158.

6. The method of any one of claims 1 to 5, wherein determining amino acid residue at position 158 of Fc.gamma.RIIIa receptor comprises a step of amplifying the Fc.gamma.RIIIa receptor gene or RNA or a portion thereof comprising the nucleotides encoding amino acid residue 158.

7. The method of claim 6, wherein amplification is performed by polymerase chain reaction (PCR), such as PCR, RT-PCR and nested PCR.

8. The method of any one of claims 1 to 7, wherein determining amino acid residue at position 158 of Fc.gamma.RIIIa receptor comprises a step of allele-specific restriction enzyme digestion.

9. The method of any one of claims 1 to 8, wherein determining amino acid residue at position 158 of Fc.gamma.RIIIa receptor comprises a step of hybridization of the Fc.gamma.RIIIa receptor gene or RNA or a portion thereof comprising the nucleotides encoding amino acid residue 158, with a nucleic acid probe specific for the genotype Valine or Phenylalanine.

10. The method of any one of claims 1 to 9, wherein determining amino acid residue at position 158 of Fc.gamma.RIIIa receptor comprises: Obtaining genomic DNA from a biological sample, Amplifying the Fc.gamma.RIIIa receptor gene or a portion thereof comprising the nucleotides encoding amino acid residue 158, and determining amino acid residue at position 158 of said Fc.gamma.RIIIa receptor gene.

11. The method of any one of claims 1 to 9, wherein determining amino acid residue at position 158 of Fc.gamma.RIIIa receptor comprises: Obtaining genomic DNA from a biological sample, Amplifying the Fc.gamma.RIIIa receptor gene or a portion thereof comprising the nucleotides encoding amino acid residue 158, Introducing an allele-specific restriction site, Digesting the nucleic acids with the enzyme specific for said restriction site and, Analysing the digestion products, i.e., by electrophoresis, the presence of digestion products being indicative of the presence of the allele.

12. The method of any one of claims 1 to 9, wherein determining amino acid residue at position 158 of Fc.gamma.RIIIa receptor comprises: total (or messenger) RNA extraction from cell or biological sample or biological fluid in vitro or ex vivo, optionally cDNA synthesis, (PCR) amplification with specific FCGRIIIa oligonucleotide primers, and analysis of PCR products.

13. The method of any one of claims 1 to 4, wherein determining amino acid residue at position 158 of Fc.gamma.RIIIa receptor comprises a step of sequencing the Fc.gamma.RIIIa receptor polypeptide or a portion thereof comprising amino acid residue 158.

14. The method of any one of the preceding claims, wherein the subject is a human subject.

15. The method of claim 14, wherein the subject has a tumor, a viral infection, or a disease condition associated with allogenic or pathological immunocompetent cells.

16. The method of claim 15, wherein the subject has a tumor and the therapeutic antibody treatment aims at reducing the tumor burden.

17. The method of claim 16, wherein the tumor is a lymphoma, particularly a NHL.

18. The method of any one of the preceding claims, wherein the antibody is an IgG1 or an IgG3.

19. The method of claim 18, wherein the antibody is an anti-CD20 antibody, particularly rituximab.