Patent application title: METHODS AND COMPOSITIONS TO EVALUATE ANTIBODY TREATMENT RESPONSE
Inventors:
Hervé Watier (Ballan-Mire, FR)
Guillaume Cartron (Savonnieres, FR)
Philippe Colombat (Larcay, FR)
IPC8 Class: AC12Q168FI
USPC Class:
435 611
Class name: Measuring or testing process involving enzymes or micro-organisms; composition or test strip therefore; processes of forming such composition or test strip involving nucleic acid nucleic acid based assay involving a hybridization step with a nucleic acid probe, involving a single nucleotide polymorphism (snp), involving pharmacogenetics, involving genotyping, involving haplotyping, or involving detection of dna methylation gene expression
Publication date: 2011-05-26
Patent application number: 20110123996
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.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 claim 1, comprising determining amino acid residue at position 158 of FcγRIIIa 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 claim 1, wherein determining amino acid residue at position 158 of FcγRIIIa receptor comprises a step of sequencing the FcγRIIIa receptor gene or RNA or a portion thereof comprising the nucleotides encoding amino acid residue 158.
6. The method of claim 1, wherein determining amino acid residue at position 158 of FcγRIIIa receptor comprises a step of amplifying the FcγRIIIa 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 claim 1, wherein determining amino acid residue at position 158 of FcγRIIIa receptor comprises a step of allele-specific restriction enzyme digestion.
9. The method of claim 1, wherein determining amino acid residue at position 158 of FcγRIIIa receptor comprises a step of hybridization of the FcγRIIIa 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 claim 1, wherein determining amino acid residue at position 158 of FcγRIIIa receptor comprises: Obtaining genomic DNA from a biological sample, Amplifying the FcγRIIIa receptor gene or a portion thereof comprising the nucleotides encoding amino acid residue 158, and determining amino acid residue at position 158 of said FcγRIIIa receptor gene.
11. The method of claim 1, wherein determining amino acid residue at position 158 of FcγRIIIa receptor comprises: Obtaining genomic DNA from a biological sample, Amplifying the FcγRIIIa 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 claim 1, wherein determining amino acid residue at position 158 of FcγRIIIa 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 claim 1, wherein determining amino acid residue at position 158 of FcγRIIIa receptor comprises a step of sequencing the FcγRIIIa receptor polypeptide or a portion thereof comprising amino acid residue 158.
14. The method. of claim 1, 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 claim 1, 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.
Description:
[0001] 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
[0002] 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®, Rituxan®), which is a chimeric anti-CD20 IgG1 monoclonal antibody made with human γ1 and κ constant regions linked to murine variable domains1. 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®), which is used in the treatment of B cell malignancies or trastuzumab (Herceptin®, which is used in the treatment of breast cancer. Additional examples of therapeutic antibodies under development are disclosed in the art.
[0003] 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-intermediate2-8 and high-grade NHL6, 9, 30% to 50% of patients with low grade NHL have no clinical response to rituximab4, 5. It has been suggested that the level of CD20 expression on lymphoma cells2, the presence of high tumor burden at the time of treatment6 or low serum rituximab concentrations2 may explain the lack of efficacy of rituximab in some patients. Nevertheless, the actual causes of treatment failure remain largely unknown.
[0004] 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)10, 11, complement-dependant cytotoxicity10, 12, 13, direct signalling leading to apoptosis14, 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 controversial12, 13.
[0005] 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.
[0006] 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 FcγRIIIa receptor directly correlates with the subject's response to therapeutic antibody treatment.
[0007] Three classes of FcγR (FcγRJ, FcγRII and FcγRIII) 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 diseases19-21. One of these genetic factors is a gene dimorphism in FCGR3A, which encodes FcγRIIIa with either a phenylalanine (F) or a valine (V) at amino-acid position 15822,23. This residue directly interacts with the lower hinge region of IgG1 as recently shown by IgG1-FcγRIII co-cristallization24. It has been clearly demonstrated that human IgG1 binds more strongly to homozygous FcγRIIIa-158V natural killer cells (NK) than to homozygous FcγRIIIa-158F or heterozygous NK cells22, 23.
[0008] 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 high5. The FCGR2A-131H/R was also determined as control since this gene co-localizes with FCGR3A on chromosome 1q22 and encodes the macrophage FcγRIIa receptor.
[0009] 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).
[0010] 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.
[0011] 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 FcγRIIIa receptor of said subject. More specifically, the method comprises determining in vitro the FCGR3A158 genotype of said subject.
[0012] 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 FcγRIIIa receptor of said subject. More specifically, the method comprises determining in vitro the FCGR3A 158 genotype of said subject.
[0013] 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 FcγRIIIa receptor of said subject. More specifically, the method comprises determining in vitro the FCGR3A 158 genotype of said subject.
[0014] More specifically, determining in vitro the FCGR3A 158 genotype of a subject comprises determining amino acid residue at position 158 of FcγRIIIa 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.
[0015] 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 (FcγR) whose function is to link the IgG-sensitized antigens to FcγR-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, αVβ3, etc., particularly lymphoma antigens (e.g., CD20). The therapeutic antibodies are preferably IgG1 or IgG3, more preferably IgG1.
[0016] 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 ®, LNH B Rituxan ® Anti-CD52 alemtuzumab CAMPATH-1H ® LLC, allograft Anti-CD33 Zamyl ® Acute myeloid Leukemia Anti-HLA- Remitogen ® LNH B DR Anti-CD22 epratuzumab LymphoCide ® LNH B Anti-erbB2 trastuzumab Herceptin ®, Metastatic breast cancer (HER-2/neu) Anti-EGFR cetuximab ORL and colorectal Cancers (HER-1, erbB1) Anti-MUC-1 Therex ® Breast and epithelial cancers Anti-CEA labetuzumab CEA-Cide ® Anti-αVβ3 Vitaxin Cancers (anti-angiogenic) Anti-KDR Cancers (anti-angiogenic) (VEGFR2) anti-VRS palivizumab Synagis ® Viral diseases fusion protein anti-VRS Numax ® '' fusion protein CMV sevirumab Protovir CMV Infection HBs tuvirumab Ostavir ® Hepatitis B Anti-CD25 basiliximab Simulect ® Prevention/treatment allograft rejection Anti-CD25 daclizumab Zenapax ® Prevention/treatment allograft rejection anti-TNF-α infliximab Remicade ® Crohn disease, polyarthrite rhumatoid anti-IgE omalizumab Xolair ® Asthma anti-integrin efalizumab Xanelim ® psoriasis αL (CD11a, LFA-1) anti-CD4 keliximab anti-CD2 siplizumab Anti-CD64 anemia anti-CD147 GvH anti-integrin natalizumab Antegren ® Sclerosis, Crohn α4 (α4β1-α4β7) Anti-integrin Crohn, RCH β7 anti-CD4* clenoliximab
[0017] Within the context of the present invention, a subject or patient includes any mammalian subject or patient, more preferably a human subject or patient.
[0018] According to the invention the term FCGR3A gene refers to any nucleic acid molecule encoding a FcγRIIIa 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 FcγRIIIa polypeptide having the sequence of human FcγRIIIa 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 FcγRIIIa 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--000569 for partial sequence).
[0019] 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 4980 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
[0020] 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 FcγRIIIa polypeptide.
[0021] 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.
[0022] In a particular embodiment, determining amino acid residue at position 158 of FcγRIIIa receptor comprises a step of sequencing the FCGR3A receptor gene or RNA or a portion thereof comprising the nucleotides encoding amino acid residue 158.
[0023] In an other particular embodiment, determining amino acid residue at position 158 of FcγRIIIa 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 polymerase chain reaction (PCR), such as simple PCR, RT-PCR or nested PCR, for instance, using conventional methods and primers.
[0024] 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.
[0025] Specific examples of such amplification primers are, for instance, SEQ ID NO: 1-4.
[0026] 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.
[0027] 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).
[0028] In an other particular embodiment, determining amino acid residue at position 158 of FcγRIIIa 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.
[0029] 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.
[0030] In a further particular embodiment, determining amino acid residue at position 158 of FcγRIIIa 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.
[0031] 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.
[0032] In a preferred embodiment of this invention, determining amino acid residue at position 158 of FcγRIIIa receptor comprises: [0033] Obtaining genomic DNA from a biological sample, [0034] Amplifying the FcγRIIIa receptor gene or a portion thereof comprising the nucleotides encoding amino acid residue 158, and [0035] determining amino acid residue at position 158 of said FcγRIIIa receptor gene.
[0036] 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: [0037] Obtaining genomic DNA from a biological sample, [0038] Amplifying the FcγRIIIa receptor gene or a portion thereof comprising the nucleotides encoding amino acid residue 158, [0039] Introducing an allele-specific restriction site, [0040] Digesting the nucleic acids with the enzyme specific for said restriction site and, [0041] Analysing the digestion products, i.e., by electrophoresis, the presence of digestion products being indicative of the presence of the allele.
[0042] 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.
[0043] The method of this invention may also comprise determining amino acid residue at position 158 of FcγRIIIa receptor directly by sequencing the FcγRIIIa receptor polypeptide or a portion thereof comprising amino acid residue 158 or by using reagents specific for each allele of the FcγRIIIa 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 FcγRIIIa158 polypeptide, more preferably any antibody or fragment or derivative thereof. In a particular embodiment, the FcγRIIIa158 polypeptide is detected with an anti-FcγRIIIa158 antibody (or a fragment thereof) that discriminates between FcγRIIIa158V and FcγRIIIa158F, more preferably a monoclonal antibody. The antibody (or affinity reagent) may be labelled by any suitable method (radioactivity, fluorescence, enzymatic, chemical, etc.). Alternatively, FcγRIIIa158 antibody immune complexes may be revealed (and/or quantified) using a second reagent (e.g., antibody), labelled, that binds to the anti-FcγRIIIa158 antibody, for instance.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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
[0048] FIG. 1: Adjusted KAPLAN-METER estimates of progression-free survival after rituximab treatment according to FcγR3a-158V/F genotype (p=0.05).
[0049] FIG. 2: Genetic organization of the human FCGR3A gene
[0050] FIG. 3: Amino acid sequences of human FcγRIIIa158F (SEQ ID NO:7)
[0051] FIG. 4: Nucleic acid sequence of human FCGR3A158F (SEQ ID NO:8)
MATERIALS AND METHODS
Patients and Treatment
[0052] Clinical trial design, eligibility criteria and end-point assessment have been previously reported.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.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.27 A total of four 375 mg/m2 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.5 The study protocol was approved by an ethics committee, and all patients gave their informed consent.
Monitoring and Endpoints
[0053] 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.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.
[0054] Molecular analysis of BCL2-JH gene rearrangement was performed by PCR, as previously described,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
[0055] 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 al22 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 μg of genomic DNA, 200 ng of each primer, 200 μmol/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° C., then 35 cycles (each consisting in 3 steps at 95° C. for 1 min, 57° C. for 1.5 min, 72° C. for 1.5 min) and 8 min at 72° 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 μL of the amplified DNA, 150 ng of each primer, 200 μmol/L of each dNTP and 1 U of Taq DNA polymerase. The first cycle consisted in 5 min at 95° C. then 35 cycles (each consisting in 3 steps at 95° C. for 1 min, 64° C. for 1 min, 72° C. for 1 min) and 9.5 min at 72° C. to complete extension. The amplified DNA (10 μL) was then digested with 10 U of NlaIII (New England Biolabs, Hitchin, England) for 12 h at 37° 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
[0056] Genotyping of FCGR2A-1311-1/R was done by PCR followed by an allele-specific restriction enzyme digestion according to Liang et al28. 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 μL reaction with 1.25 μg genomic DNA, 170 ng of each primer, 200 μmol/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° C. followed by 35 cycles (each consisting in 3 steps at 94° C. for 15 seconds, 55° C. for 30 seconds, 72° C. for 40 seconds) and 7 min at 72° C. to complete extension. The amplified DNA (7 μL) were then digested with 20 U of BstUI (New England Biolabs) for 12 h at 60° 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
[0057] 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 ≦60 years), number of extra-nodal sites involved (≧ 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 Meier29 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
[0058] 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
[0059] 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; P=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
[0060] 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.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.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 insufficient to significantly improve the progression-free survival in homozygous FCGR3A-158V patients.
[0061] 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 FcγRIIIa. 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 polymorphism31 or in other FcγR-coding genes, since FCGR3A is located on the long arm of chromosome 1, which includes the three FCGR2 genes and FCGR3B.32 A linkage disequilibrium has been reported between FCGR2A and FCGR3B.33 However, the fact that FCGR2A-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.
[0062] Several in vitro studies argue in favor of direct involvement of FCGR3A-158V/F polymorphism. First, Koene et a/23 have shown that the previously reported differences in IgG binding among the three FcγRIIIa-48L/H/R isoforms31 are a consequence of the linked FcγRIIIa-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 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 FcγRIIIa. Secondly, NK cell- and macrophage-mediated ADCC is one of the mechanisms triggered by anti-CD20 antibodies in vitro8,11,12 as well as in murine models in vivo, 17-19 and rituximab-mediated apoptosis is amplified by FcγR-expressing cells.15,16 Out of all FcγR, FcγRIIIa 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 cytotoxicity11,13,14 and/or apoptosis.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.
[0063] 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 homozygotes23. 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.
[0064] Since FcγRIIIa 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 al have recently obtained IgG1 mutants which bind more strongly to FcγRIIIa-158F than native IgG134.
[0065] 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.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®). 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 [0101] 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 ≧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 FCGRA-158F patients against FCGR3A/158V carriers.
TABLE-US-00004 TABLE 2 CLINICAL RESPONSE TO RITUXIMAB BY FCGR3A-158V/F POLYMORPHISM. FCGR3A-158F FCGR3A-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-158F FCGR3A-158VV carriers p Molecular response at M2 ns Cleaning of BCL2-JH 3 5 rearrangement Persistent BCL2-JH 3 14 rearrangement Molecular response at M12 0.03 Cleaning of BCL2-JH 5 5 rearrangement Persistent BCL2-JH 1 12 rearrangement
Sequence CWU
1
10121DNAArtificial 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 antisens primer. 6caacagcctg
actacctatt acgcggg
277254PRTArtificial SequenceDescription of Artificial Sequence Amino acid
sequence of human FCGR3A158F. 7Met Trp Gln Leu Leu Leu Pro Thr Ala
Leu Leu Leu Leu Val Ser Ala1 5 10
15Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu
Pro 20 25 30Gln Trp Tyr Arg
Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35
40 45Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp
Phe His Asn Glu 50 55 60Ser Leu Ile
Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr65 70
75 80Val Asp Asp Ser Gly Glu Tyr Arg
Cys Gln Thr Asn Leu Ser Thr Leu 85 90
95Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu
Leu Gln 100 105 110Ala Pro Arg
Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115
120 125His Ser Trp Lys Asn Thr Ala Leu His Lys Val
Thr Tyr Leu Gln Asn 130 135 140Gly Lys
Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro145
150 155 160Lys Ala Thr Leu Lys Asp Ser
Gly Ser Tyr Phe Cys Arg Gly Leu Phe 165
170 175Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile
Thr Ile Thr Gln 180 185 190Gly
Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln 195
200 205Val Ser Phe Cys Leu Val Met Val Leu
Leu Phe Ala Val Asp Thr Gly 210 215
220Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp225
230 235 240Lys Asp His Lys
Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 245
250822685DNAArtificial SequenceDescription of Artificial Sequence
Nucleic 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 Ser1 5 10
151045DNAArtificial 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 Ser1 5
10 15
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