Note: Descriptions are shown in the official language in which they were submitted. The technology in part relates to prenatal diagnostics and enrichment methods. Non-invasive prenatal testing is becoming a field of rapidly growing interest.
of crucial importance, as it allows early medical intervention necessary for
Prenatal diagnosis has been conducted using cells isolated from procedures such as chorionic villus sampling (CVS) or amniocentesis.
methods are invasive and present an appreciable risk to both the mother and Health Service currently cites a miscarriage rate of between 1 and 2 per cent
amniocentesis and chorionic villus sampling (CVS) tests. An alternative to these invasive approaches has been developed for prenatal maternal plasma and serum (Lo et al., Lancet 350:485-487, 1997; and U.S. cell free fetal nucleic acid (cffNA) has several advantages making it more
For example, cell free nucleic acid is present at higher hours after delivery, preventing contamination from previous pregnancies. Examples of prenatal tests performed by detecting fetal DNA in maternal plasma
In addition, quantitative abnormalities of fetal DNA in maternal
The invention provides inter alia human epigenetic biomarkers that are useful detection of fetal genetic traits, including, but not limited to, the presence
genomic DNA that display differential CpG methylation patterns between the add in a maternal sample based on the methylation status of the nucleic acid
specifically, the amount of fetal nucleic acid from a maternal sample can be total amount of nucleic acid present, thereby providing the percentage of
specific) manner and with sufficient sensitivity to allow for accurate example, to detect the presence or absence of a fetal aneuploidy).
In the first aspect of the invention, a method is provided for enriching fetal maternal biological sample, based on differential methylation between fetal
comprising the steps of: (a) binding a target nucleic acid, from a sample, and
includes the sequences of SEQ ID NOs: 1-261, and variations thereto. In a related embodiment, a method is provided for enriching fetal nucleic acid which comprises the following steps: (a) obtaining a biological sample from a fetal and maternal nucleic acid based on the methylation status of a CpG- (1) a genomic locus selected from Tables 1A-1C; and (2) a DNA sequence of no
a biological sample from a woman is not meant to limit the scope of the In a related embodiment, a method is provided for enriching fetal nucleic acid
which comprises the following steps: (a) obtaining a biological sample from removing maternal nucleic acid based on the methylation status of a CpG-
Maternal nucleic acid may be digested using one or more methylation enzymes that selectively digest or cleave maternal nucleic acid based on its cytosine, further wherein the region consists of (1) a genomic locus selected sequence of a fetal nucleic acid, which comprises the following steps: (a) pregnant female according to a different methylation state between the fetal maternal nucleic acid counterpart, wherein the nucleotide sequence of the
polynucleotide sequence from a gene or locus that contains one of the ID NOs: 1-261; and (c) preparing nucleic acid comprising a nucleotide sequence
by an amplification process in which fetal nucleic acid separated in part (b)
an embodiment, a method is provided for preparing nucleic acid having a nucleic acid, which comprises the following steps: (a) providing a sample from digesting or removing maternal nucleic acid from the sample of the pregnant
different methylation state between the fetal nucleic acid and the maternal wherein the nucleotide sequence of the fetal nucleic acid comprises one or
gene that contains one of the polynucleotide sequences of SEQ ID NOs: 1-261; restriction enzymes that selectively digest or cleave maternal nucleic acid
In either embodiment, the polynucleotide sequences of SEQ ID NOs: 1- yet an embodiment, the method further comprises quantifying the fetal nucleic
In a third aspect of the invention, a method Is provided for enriching fetal from a pregnant female with respect to maternal nucleic acid, which comprises
providing a sample from a pregnant female; and (b) separating or capturing maternal nucleic acid from the sample of the pregnant female according to a
fetal nucleic acid comprises one or more CpG sites from one or more of the SEQ ID NOs: 1-261 within a polynucleotide sequence from a gene that contains
SEQ ID NOs: 1-261 may be within a polynucleotide sequence from a CpG island In a fourth aspect of the invention, a composition is provided comprising an
fetus of a pregnant female, wherein the nucleotide sequence of the nucleic consists essentially of a nucleotide sequence of a gene, or portion thereof. The polynucleotide sequences of SEQ ID NOs: 1-261 are further characterized in fetus of a pregnant female, wherein the nucleotide sequence of the nucleic CpG sites from one or more of the polynucleotide sequences of SEQ ID NOs: 1- polynucleotide sequence from a gene, or portion thereof, that contains one of 261 within a polynucleotide sequence from a CpG island, or portion thereof, embodiment, the nucleic acid from the fetus is enriched relative to maternal
hypomethylated nucleic acid sequences of the invention are identified in example, the agent may be a methyl-CpG binding protein (MBD) or fragment
In some embodiments, a nucleotide sequence of the invention includes three or the nucleotide sequence is from a gene region that comprises a PRC2 domain embodiment, the nucleotide sequence is from a gene region involved with
(SRY-related HMG-box) family of transcription factors involved in the
woman is unmethylated and the genomic sequence from the fetus is methylated. Fetal genomic sequences found to be hypermethylated relative to
hypomethylated relative to the genomic sequence from the mother. be hypomethylated relative to maternal genomic sequence are provided in SEQ ID
restriction enzymes of the invention may be sensitive to hypo- or hyper-
or separated from or relative to the digested maternal nucleic acid based on
primers may be designed to amplify nucleic acid greater than about 75, 80, 85, In some embodiments, the present invention provides a method in which the
chorionic villus sample, biopsy material from a pre-implantation embryo, fetal reproductive tract and a sample obtained by celocentesis or lung lavage. In some embodiments, all nucleated and anucleated cell populations are removed transported in a manner known to the person of ordinary skill in the art to
quality of fetal nucleic acid present in the sample. The sample can be from any animal, including but not limited, human, non-
cattle, cat, dog, goat, swine, pig, monkey, ape, gorilla, bull, cow, bear, fish, dolphin, whale, and shark, or any animal or organism that may have a
or more enzymes that preferentially cleave methylated DNA; or the reagent may enzymes include, but are not limited to, Hhal and Hpall. In one embodiment, the fetal nucleic acid is separated from the maternal specifically binds to methylated nucleotides in the fetal nucleic acid.
methylated nucleotides in the maternal nucleic acid counterpart. In a sixth aspect of the invention, a method is provided for determining the fetal DNA in a maternal sample that comprises differentially methylated method is performed by a) distinguishing between the maternal and fetal DNA
methylation status; and b) quantifying the fetal DNA of step a). restriction enzymes thereby enriching the fetal DNA; and b) determining the
The amount of fetal DNA can be used inter alio to confirm the in conjunction with other fetal diagnostic methods to improve sensitivity or
embodiment, the method for determining the amount of fetal DNA does not
an embodiment, the method for determining the amount of fetal DNA does not of DNA with bisulfite to convert cytosine residues to uracil. further reducing the already limited fetal nucleic acid present in maternal
determining the amount of fetal DNA in step b) is done by introducing one or is determined using BEAMing technology as described in US Patent Publication another related embodiment, the amount of nucleic acid is determined using the used to further determine the amount of fetal DNA.
In a seventh aspect of the invention, a method is provided for determining the restriction enzymes thereby enriching the fetal DNA; c) determining the amount
thereby determining the concentration of fetal DNA in the maternal sample. DNA can be used inter alia in conjunction with other fetal diagnostic methods In an embodiment, the method for determining the amount of fetal DNA treatment of DNA with bisulfite to convert cytosine residues to uracil.
In an embodiment, determining the amount of fetal DNA in step In an eighth aspect of the invention, a method is provided for determining the
differentially methylated maternal and fetal DNA, comprising a) selectively in a maternal sample using one or more methylation sensitive restriction the method for determining the amount of fetal DNA does not require the use of
For example, an allelic ratio is not used to quantify the fetal DNA
embodiment, the method for determining the amount of fetal DNA does not DNA with bisulfite to convert cytosine residues to uracil.
In an embodiment, determining the amount of fetal DNA in steps b) and c) is determined in step b) is compared to a standard control, for example, the
In a ninth aspect of the invention, a method is provided for detecting the chromosomal abnormality by analyzing the amount or copy number of target
methylation state; (b) performing a copy number analysis of the enriched one of the fractions; (c) performing a copy number analysis of the enriched
absence of a chromosomal abnormality by analyzing the amount or copy number of agent; (b) eluting the bound nucleic acid based on methylation status, wherein
nucleic acids elute at least partly into separate fractions; (c) performing a
eluted target nucleic acid in at least one of the fractions; (d) performing a
eluted control nucleic acid in at least one of the fractions; (e) comparing the comparison in step (e), wherein the target nucleic acid and control
In a tenth aspect of the invention, a method is provided for detecting the chromosomal abnormality by analyzing the allelic ratio of target nucleic acid from a sample of differentially methylated nucleic acids comprising the steps
the bound nucleic acid based on methylation status, wherein differentially elute at least partly into separate fractions; (c) performing an allelic ratio nucleic acid in at least one of the fractions; (d) performing an allelic ratio
nucleic acid in at least one of the fractions; (e) comparing the allelic ratio step d; and (f) determining if a chromosomal abnormality exists based on the
within the differentially methylated nucleic acids are provided in Table 2. In an eleventh aspect of the invention, the amount of maternal nucleic acid is
example, digested using a methylation-sensitive enzyme) from the maternal and the maternal nucleic acid can be quantified using the methods of the
of maternal nucleic acid is determined, that amount can subtracted from the For all aspects and embodiments of the invention described herein, the methods
Nucleic acid species with a different methylation status digestion of maternal nucleic acid by a methylation sensitive restriction the fetal nucleic acid is enriched by the selective digestion of maternal least a portion of a chromosome which may be abnormal and the control nucleic
Also, the enriched or eluted nucleic acid is amplified not require the treatment of DNA with bisulfite to convert cytosine residues In some embodiments, the methods of the invention include the additional step amount of fetal nucleic acid in a sample as determined by using the
the invention is compared to the amount of Y-chromosome nucleic acid present. Methods for differentiating nucleic acid based on methylation status include,
methylation sensitive capture, for example using, MBD2-Fc fragment; bisuifite extension (Ms-SNuPE) or Sequenom MassCLEAVETM technology; and the use of on methylation status can be used with the compositions and methods of the the amplification reaction is performed on single molecules, for example, by by counting the fetal DNA (or sequence tags attached thereto) with a flow determined by an amplification reaction that generates amplicons larger than
In some embodiments, the fetal nucleic acid (alone or in combination with the one or more of a compomer, sugar, peptide, protein, antibody, chemical distinguishable product (MDP) or part of an MOP detected by mass spectrometry.
embodiment, the detection moiety is a fluorescent tag or label that is
oligonucleotide, or the detection moiety is at the 5′ terminus of a non- more non-target chromosomes are labeled with a different detection moiety,
target chromsome can be compared to the amount of non-target chromosome. may be used: a primer extension method (e.g., iPLEX ; Sequenom, Inc.), direct restriction fragment length polymorphism (RFLP analysis), real-time PCR, for (Scalable Transcription Analysis Routine) technology (see US Patent No. thereof, allele specific oligonucleotide (ASO) analysis, methylation-specific allele-specific hybridization (DASH), Peptide nucleic acid (PNA) and locked
TaqManrm, Molecular Beacons, Intercalating dye, FRET primers, fluorescence AlphaScreen, SNPstream, genetic bit analysis (GSA), Multiplex nninisequencing,
Coded microspheres, Template-directed incorporation (TDI), fluorescence one fluorescently labeled probe, electrophoresis, cloning and sequencing, for
Analyzer (or Solexa platform) or SOLID System (Applied Biosystems) or the Molecule DNA sequencing technology (Harris T D et al. 2008 Science, 320, 106- that measures an electrical charge associated with each individual base of DNA through a tiny pore at the bottom of a sample well, or Oxford Nanopore device
to measure the electrical charge associated with each individual unit of DNA, Nanopore-based methods may include sequencing nucleic acid using a counting nucleic acid molecules using a nanopore, for example, based on size The absolute copy number of one or more nucleic acids can be determined, for
spectrometry, a system that uses a competitive PCR approach for absolute copy See for example, Ding C, Cantor CR (2003) A high-throughput gene
analysis technique using competitive PCR and matrix-assisted laser desorption flight MS. Proc Natl Acad Sci U S A 100:3059-3064, and US Patent Application
aneuploidy, the amount of fetal nucleic acid from target chromosome may be amount of fetal nucleic acid from a reference chromosome.
reference fetal nucleic acid may be compared to the same ratio from a normal, For example, a control ratio may be determined from a DNA sample obtained from chromosomal aneuploidies in a maternal plasma of a pregnant female, one control DNAs that have been isolated from mothers who are known to carry a
In some embodiments, the present invention provides a method in which the an embodiment, the fetal nucleic acid should comprise at least one high
ratio of the nucleic acid in order to assess the presence or absence of the polymorphic alleles that can be used as part of the invention.
may also be considered: (a) the SNP has a heterozygosity frequency greater In other embodiments, the sequence variation is a short tandem repeat (STR)
some embodiments, the sequence variation falls in a restriction site, whereby In some embodiments, performing an allelic ratio analysis comprises an nucleic acid-containing biological sample from the pregnant woman, wherein maternal nucleic acid based on differential methylation, discriminating the
detecting the presence or absence of a chromosomal disorder in the fetus based In some embodiments, the present invention is combined with other fetal presence or absence of multiple chromosomal abnormalities, wherein the example, the amount of fetal nucleic acid may be determined at multiple loci
when detecting the presence or absence of fetal aneuploidy, the amount of determined at multiple loci on one or more target chromosomes (e.g., intermediate levels, polynucleotide sequences of the invention are enriched,
processes of the invention may be used to assay samples that have been divided including single molecule analyses, are provided in US Application No, In a further embodiment, the present invention provides a method wherein a
an increased risk of a fetus having a chromosomal disorder if the ratio of the number of the target nucleic acid is higher or lower by 1 standard deviation
having a chromosomal disorder if the ratio of the alleles or absolute copy
other embodiments, the comparison step shows an increased risk of a fetus disorder if the ratio of the alleles or absolute copy number of the target comparison step shows an increased risk of a fetus having a chromosomal
alleles or absolute copy number of the target nucleic acid is higher or lower maternal reference, and in an embodiment the standard control is a fetal
confirmation of the presence or absence of fetal nucleic acid, such as a sex acid in a maternal sample in order to enable another diagnostic method that When determining an allelic ratio to diagnose a fetal aneuploidy sample, the amount or concentration of fetal nucleic acid may be required to
mathematical models have suggested that a combined first-trimester screening maternal age (MA), nuchal translucency (NT) thickness, serum-free beta-hCG, detect more than 80% of fetuses with Down’s syndrome for a 5% invasive testing
aneuploidy detection methods combined with the non-invasive free fetal nucleic described herein may offer improved accuracy with a lower false positive rate. combined diagnostic methods are provided in PCT Publication Number
based methods, wherein fetal cells are procured invasively or non-invasively. on the outcome or result(s) produced from the compositions or methods provided
An example of an outcome is a deviation from the euploid absolute copy number or allelic ratio, which indicates the presence of chromosomal
This increase or decrease in the absolute copy number or ratio from the control indicates an increased risk of having a fetus with a chromosomal
outcome, result, or risk of trisomy or aneuploidy, for example, may be outcome may be transfixed in a medium to save, store, share, communicate or
(e.g., electronic medium), and examples of media include, but are not limited from one location to another using a physical medium (e.g., paper) or a In practicing the present invention within all aspects mentioned above, a CpG other cases the CpG-containing genomic sequence may not be a CpG island.
In some embodiments, the present invention provides a kit for performing the The invention as claimed relates to a method for determining the presence or
sample using a methylation sensitive restriction enzyme thereby enriching the
chromosome using a non-polymorphic-based and non-bisulfite-based quantitative
method in a multiplex reaction by determining the amount of fetal DNA at selected from the differentially methylated nucleic acids as provided in SEQ 164-261, and wherein the fetal nucleic acid comprises one or more CpG sites determining the amount of fetal nucleic acid from a reference chromosome using reaction by determining the amount of fetal DNA at multiple loci selected from differentially methylated nucleic acids as provided in SEQ ID NOs: 90-163, and wherein the fetal nucleic acid comprises one or more CpG sites from one or the polynucleotide sequences of SEQ ID NOs: 90-163; d) comparing the amount of
between the amount of target and reference fetal nucleic acid is indicative of FIGURE 1: Shows the design of the recombinant MBD-Fc protein used to separate
FIGURE 2: Shows the methyl-CpG-binding, antibody-like protein has a high and high avidity to its “antigen”, which is preferably DNA that is methylated
solutions of increasing salt concentrations can be used to fractionate non- FIGURE 4: Shows the experiment used to identify differentially methylated DNA a fetus and mother using the recombinant MBD-Fc protein and a microarray. FIGURE 5: Shows typical results generated by Sequenom EpiTYPERTm method, which was used to validate the results generated from the experiment FIGURE 6: Shows the correlation between the log ratios derived from microarray Each data point represents the average for one region across all measured The microarray analysis is comparative in nature because the highly methylated
fraction of the maternal DNA is hybridized together with the highly methylated Positive values indicate higher methylation of the placenta
results with the microarray data we calculated the average of the differences FIGURE 8: Shown is the correlation between the number of gDNA molecules that
number of molecules measured by competitive PCR in combination with mass this experiment we used DNA derived from whole blood (black plus signs) and
fully methylated DNA(red crosses) in a 90 to 10 ratio. copy number variations and is commercially available for gene expression
Using an input of 300 total gDNA copies we expect to reaction and shows that this initial proof of concept experiment needs to be
the approach for capturing and quantifying of a few copies of methylated DNA FIGURE 9A-9C: Shown are bar graph plots of the methylation differences
axis depicts the log ration (in case of the microarrays) and the methylation Bars showing values greater than zero indicate higher DNA methylation in the FIGURE 10: Shows one embodiment of the Fetal Quantifier Method. digested and the remaining fetal nucleic acid is quantified using a competitor
In this schema, the analyte is separated and quantified by a mass spectromter. FIGURE 11: Shows one embodiment of the Methylation-Based Fetal Diagnostic greater than 100 base pair amplicons) such that they favor the longer, non- over the digested maternal nucleic acid, thereby further enriching the fetal the bottom of the Figure show an increased amount of chromosome 21 fetal FIGURE 12: Shows the total number of amplifiable genomic copies from four DNA/competitor ratio obtained from two total copy number assays (ALB and Figure 12 shows, the total copy number is accurate and stable across the
FIGURES 13A and B: A model system was created that contained a constant number spiked with male placental amounts ranging from approximately 0 to 25% The fraction of placental DNA was calculated using the ratios FIGURES 14 A and B: Show the results of the total copy number assay from
FIGURES 15A and B: The amount (or copy numbers) of fetal nucleic acid from 33 samples taken from pregnant women with male fetuses are plotted.
FIGURE 16: Shows a paired correlation between the results obtained using the FIGURE 17: Shows the digestion efficiency of the restriction enzymes using the
control versus the competitor and comparing this value to the mean total copy
FIGURE 18: Provides a specific method for calculating fetal DNA fraction (or
using the Y-chromosome-specific markers for male pregnancies and the mean of FIGURE 19: Provides a specific method for calculating fetal DNA fraction (or
were used to determine the concentration of fetal DNA. FIGURE 20: Shows a power calculation t-test for a simulated trisomy 21 and the power to discriminate the assay populations using a simple t-test (y- The term “pregnancy-associated disorder,” as used in this application, refers disease may manifest its symptoms during a limited time period, e.g., during may last the entire life span of the fetus following its birth.
disorder include ectopic pregnancy, preeclampsia, preterm labor, RhD chromosomal abnormalities such as trisomy 21, and genetically inherited fetal
associated with quantitative abnormalities of fetal DNA in maternal For example, an elevated level of fetal nucleic acid in maternal
to enrich fetal nucleic from a maternal sample may prove particularly useful diagnosis of autosomal recessive diseases such as the case when a mother and
disease causing mutation, an occurrence previously perceived as a challenge The term “chromosomal abnormality” or “aneuploidy” as used herein refers to a refers to the predominate karyotype or banding pattern found in healthy chromosomal abnormality may be detected by quantitative analysis of nucleic The terms refer to nucleic acids of any composition from, such as backbone and the like), RNA/DNA hybrids and polyamide nucleic acids (PNAs),
nucleotides that can function in a similar manner as naturally occurring nucleic acids provided in SEQ ID NOs: 1-261 (see Tables 4A-4C) can be in any processes herein (e.g., linear, circular, supercoiled, single-stranded, double- may include variations (e.g., insertions, deletions or substitutions) that do Unless specifically limited, the term encompasses nucleic acids containing nucleotides that have similar binding properties as the reference nucleic acid
sequence also implicitly encompasses conservatively modified variants thereof substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), The term nucleic acid is used interchangeably with locus, gene,
synthesized from nucleotide analogs, single-stranded (“sense” or “antisense”,
RNA, the base cytosine is replaced with uracil. A “nucleic acid comprising one or more CpG sites” or a “CpG-containing genomic
herein refers to a segment of DNA sequence at a defined location in the genome given genetic locus (see Tables 1A-1C), nucleotide sequence variations may
boundary of the genetic locus, respectively) can be as long as 10 kb, in other
methylcytosine contains a methyl moiety at position 5 of its pyrimidine ring. contains a methyl moiety at position 5 of its pyrimidine ring, however, for not considered a methylated nucleotide when present in DNA since thymine is a
Typical nucleoside bases for DNA are thymine, adenine, cytosine and RNA are uracil, adenine, cytosine and guanine. the target gene nucleic acid region where methylation has, or has the example a location containing CpG is a methylation site wherein the cytosine
A “CpG island” as used herein describes a segment of DNA sequence that have described a set of standards for determining a CpG island: it must be at
molecular level of a nucleic acid (e.g., DNA or RNA) other than the primary instance, the epigenetic state of a genomic DNA may include its secondary or
determined or influenced by, e.g., its methylation pattern or its association the state of methylation of a genomic sequence, refers to the characteristics profile” or “methylation status” also refers to the relative or absolute sequence (e.g., fetal nucleic acid) are methylated as compared to another
region or from a different individual (e.g., relative to maternal nucleic residue(s) within a DNA sequence are not methylated as compared to another the sequences are considered “differentially methylated maternal and fetal
The term “agent that binds to methylated nucleotides” as used herein refers to
different nucleic acid species according to their respective methylation agent that binds to methylated nucleotides is described in PCT Patent
polypeptide comprising the DNA-binding domain of a protein belonging to the binding proteins (MBDs) and an Fc portion of an antibody (see Figure 1). CpG-binding, antibody-like protein can preferably bind CpG methylated DNA in
avidity to its “antigen”, which is preferably DNA that is methylated at CpG The term “polymorphism” as used herein refers to a sequence variation within to, single nucleotide polymorphisms (SNPs), restriction fragment length
according to the present invention can be used to specifically differentiate paternal allele in the enriched fetal nucleic acid sample.
The terms “single nucleotide polymorphism” or “SNP” as used herein refer to sequence variation present at a single nucleotide residue within different This variation may occur within the coding region or non-coding The term “allele” as used herein is one of several alternate forms of a gene from any organism including but not limited to bacteria, viruses, fungi, The term “non-polymorphism-based quantitative method” as used herein refers to
determining the amount of an analyte (e.g., total nucleic acid, Y-chromosome nucleic acid) that does not require the use of a polymorphic marker or The terms “absolute amount” or “copy number” as used herein refers to the processes for determining the absolute amount of fetal nucleic acid in a mixed
proportion of a substance in a mixture or solution (e.g., the amount of fetal sample that comprises a mixture of maternal and fetal nucleic acid). target nucleic acid) include, but are not limited to, mass spectrometery, synthesis platforms (e.g., pyrosequencing), fluorescence spectroscopy and flow The term “sample” as used herein refers to a specimen containing nucleic acid. include, but are not limited to, tissue, bodily fluid (for example, blood,
breast milk, lymph fluid, cerebrospinal fluid or mucosa secretion), umbilical cord blood, chorionic villi, amniotic
matter, an individual cell or extract of the such sources that contain the
subcellular structures such as mitochondria, using protocols well established Fetal DNA can be obtained from sources including but not limited to maternal maternal plasma, fetal cells, umbilical cord blood, chorionic villi, amniotic
capable of chemically converting a cytosine (C) to a uracil (U) without cytosine and therefore can be used to differentially modify a DNA sequence As used herein, a reagent that “differentially modifies” methylated or non-
encompasses any reagent that modifies methylated and/or unmethylated DNA in a
which distinguishable products result from methylated and non-methylated DNA, efficiency when the DNA is methylated, whereas an enzyme that preferentially refer to any method for quantifying methylated or non-methylated nucleic acid The terms also refer to methods for preparing a nucleic
to, methods for digesting nucleic acid using one or more methylation sensitive
The terms “methyl-sensitive enzymes” and “methylation sensitive restriction disclosed herein to determine if the nucleic acid is part of a pregnancy- according to the methods disclosed herein to determine if the nucleic acid is
The term “sequence-specific” or “locus-specific method” as used herein refers interrogates (for example, quantifies) nucleic acid at a specific location (or The term “gene” means the segment of DNA involved in producing a polypeptide as intervening sequences (introns) between individual coding segments (exons).
amino acid residue is an artificial chemical mimetic of a corresponding used herein, the terms encompass amino acid chains of any length, including
antigens), wherein the amino acid residues are linked by covalent peptide analogs and amino acid mimetics that function in a manner similar to the
Naturally occurring amino acids are those encoded by the genetic code, one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Nucleotides, likewise, may be referred to by their commonly accepted single- a polymerase chain reaction (PCR), to amplify a nucleotide sequence based on
The term “template” refers to any nucleic acid molecule that can be used for RNA or DNA that is not naturally double stranded can be made into The term “amplification reaction” as used herein refers to a process for amplification, or splice overlap extension polymerase chain reaction.
molecule of nucleic acid is amplified, for example, by digital PCR. The term “sensitivity” as used herein refers to the number of true positives
true positives plus the number of false negatives, where sensitivity (sens) sens 5 1. ideally, method embodiments herein have the number of false
to classify negatives correctly, a complementary measurement to sensitivity. number of false positives, where sensitivity (spec) may be within the range of methods embodiments herein have the number of false positives equaling zero or One or more prediction algorithms may be used to determine significance or
detection data collected under variable conditions that may be weighed chromosome that results in a viable being are variables that are dependent One of skill in the art may use any type of method or prediction algorithm to algorithms such as Chi-squared test, z-test, t-test, ANOVA (analysis of
data having different independent and/or dependent variables of the present having different independent and/or dependent variables of the present
or change parameters of the different variables of methods described herein or more prediction algorithms (e.g., number of sets analyzed, types of
For example, applying the Chi-squared test to detection data may suggest that
maternal age are correlated to a higher likelihood of having an offspring with abnormality, hence the variable of maternal age may be weighed differently samples, the trained algorithms’ performance may be assessed based on
The presence of fetal nucleic acid in maternal plasma was first reported in possibility for non-invasive prenatal diagnosis simply through the analysis of associated disorders, including preeclampsia, preterm labor, antepartum acid analysis in maternal plasma represents a powerful mechanism for the reported applications have relied on the detection of Y-chromosome sequences fetal nucleic acid from a maternal sample. fetal nucleic acid may be susceptible to varying heterozygasity rates across It was previously demonstrated that fetal and maternal DNA can be
refers to processes that alter a phenotype without involving changes in the
mother and fetus, one can successfully detect and analyze fetal nucleic acid fetal and maternal genomic DNA and new methods for accurately quantifying Practicing the invention utilizes routine techniques in the field of molecular
disclosing the general methods of use in the invention include Sambrook and For nucleic acids, sizes are given in either kilobases (kb) or base pairs from agarose or acryiamide gel electrophoresis, from sequenced nucleic acids,
For proteins, sizes are given in kilodaltons (kDa) or amino acid are estimated from gel electrophoresis, from sequenced proteins, from derived the solid phase phosphoramidite triester method first described by Beaucage &
strategy, e.g., native acrylamide gel electrophoresis or anion-exchange high chromatography (HPLC) as described in Pearson & Reanier, J. Chrom.
Acquisition of Blood Samples and Extraction of DNA The present invention relates to separating, enriching and analyzing fetal DNA as a non-invasive means to detect the presence and/or to monitor the progress A blood sample is obtained from a pregnant woman at a gestational age suitable
standard protocol hospitals or clinics generally follow. e.g., typically between 5-50 ml, is collected and may be stored according to
the person of ordinary skill in the art to minimize degradation or the quality The analysis of fetal DNA found in maternal blood according to the present performed using, e.g., the whole blood, serum, or plasma. woman’s blood can be placed in a tube containing EDTA or a specialized Vacutainerm SST (Becton Dickinson, Franklin Lakes, to prevent blood Plasma or serum may be subjected to additional centrifugation steps before
fraction, enriched in the buffy coat portion, which can be obtained following There are numerous known methods for extracting DNA from a biological sample
The general methods of DNA preparation (e.g., described by Sambrook and Madison, Wis.), and GFXTM Genomic Blood DNA Purification Kit (Amersham,
also be used to obtain DNA from a blood sample from a pregnant woman.
AU2009299791A1 – Cross-species-specific PSCAxCD3, CD19xCD3, C-METxCD3, EndosialinxCD3, EpCAMxC D3, IGF-1RxCD3 or FAPalpha xCD3 bispecific single chain antibody – Google Patents
The invention also provides nucleic acids encoding said bispecific single chain antibody molecule as well as vectors and host cells and a process for its production. The mechanism by which TcR ligation is directly communicated to the signalling apparatus remains a fundamental question in T cell biology (Alarcon, loc. It seems clear that sustained T cell responses involve coreceptor engagement, TcR oligomerization, and a higher order arrangement of TcR-pMHC complexes in the immunological synapse (Davis & van der Merwe, Curr.
However very early TcR signalling occurs in the absence of these events and may involve a ligand-induced conformational change in CD3 epsilon (Alarcon, loc. Although the cysteine-rich stalk appears to play an important role in driving CD3 dimerization (Su, loc. 273 (1998), 12807-12816), interaction by means of the extracellular domains of CD3 epsilon and CD3 gamma is sufficient for assembly of these proteins with TcR beta (Manolios, Eur.
Given the central role of the human CD3 epsilon gamma heterodimer in the immune response, the crystal structure of this complex bound to the therapeutic antibody OKT3 has recently been elucidated (Kjer-Nielsen, PNAS 101, (2004), 7675-7680). A number of therapeutic strategies modulate T cell immunity by targeting TcR signalling, particularly the anti-human CD3 monoclonal antibodies (mAbs) that are widely used clinically in immunosuppressive regimes. It is due to this later blocking of T cell function that OKT3 has found such wide application as an immunosuppressant in therapy regimens for reduction or even abolition of allograft tissue rejection. As indicated above, such CD3 specific antibodies are able to induce various T cell responses such as lymphokine production (Von Wussow, J. Immunol. That is, depending on the experimental conditions, CD3 specific monoclonal antibody can either inhibit or induce cytotoxicity (Leewenberg, J. Immunol. Conformational epitopes are characterized by the presence of two or 3 WO 2010/037835 PCT/EP2009/062792 more discrete amino acid residues which are separated in the primary sequence, but come together on the surface of the molecule when the polypeptide folds into the native protein/antigen (Sela, (1969) Science 166, 1365 and Laver, (1990) Cell 61, 553-6).
For example, it has been found in several studies that the most widely used CD3 epsilon monoclonal antibodies OKT3, WT31, UCHT1, 7D6 and Leu 4 did not bind to cells singly transfected with the CD3-epsilon chain. However, these antibodies stained cells doubly transfected with a combination of CD3 epsilon plus either CD3 gamma or CD3 delta (Tunnacliffe, loc. A member of this group is for instance mAb APA 1/1 which has been raised against denatured CD3 epsilon (Risueno, Blood 106 (2005), 601-8). The aim of pre-clinical testing is to prove that the drug candidate has the desired activity and most importantly is safe.
Between rodents and man there are significant differences in the physiology and the safety results cannot be easily extrapolated to humans. Therefore, preclinical data generated in rodents are of limited 5 WO 2010/037835 PCT/EP2009/062792 predictive power with respect to the drug candidate.
The limitation now of monoclonal antibodies suitable for therapeutic intervention in man described in the art is that the relevant species are higher primates, in particular chimpanzees. Chimpanzees are considered as endangered species and due to their human-like nature, the use of such animals for drug safety testing has been banned in Europe and is highly restricted elsewhere.
Doses as low as 0.005 milligrams per square meter per day in non-Hodgkin’s lymphoma patients led to an elimination of target cells in blood. In the late 1980s, the widespread adoption of the prostate-specific antigen (PSA) test represented a major improvement in the management of prostate cancer.
This test measures the amount of PSA protein in the blood, which is often elevated in patients with prostate cancer. Due to the widespread implementation of PSA testing in the United States, approximately 90 percent of all prostate cancers are currently diagnosed at an early stage, and, consequently, men are surviving longer after 6 WO 2010/037835 PCT/EP2009/062792 diagnosis.
Ongoing clinical trials over the past 25 years have investigated the effectiveness of natural and synthetic compounds in the prevention of prostate cancer. Other prostate cancer prevention trials are currently evaluating the protective potential of multivitamins, vitamins C and D, soy, green tea, and lycopene, which is a natural compound found in tomatoes. Other studies have shown that genetic variations in a specific region of chromosome 8 can increase a man’s risk of developing prostate cancer. There is also ongoing research that examines how proteins circulating in a patient’s blood can be used to improve the diagnosis of prostate and other cancers. In 2005, scientists identified a group of specific proteins that are produced by a patient’s immune system in response to prostate tumors. These proteins, a type of autoantibody, were able to detect the presence of prostate cancer cells in blood specimens with greater than 90 percent accuracy.
Stem cell antigen type 2 has been shown to prevent apoptosis in immature thymocytes (Classon and Coverdale, PNAS 91 (1994), 5296-5300). High levels of PSCA protein expression were also detected in nine out of nine prostate cancer bone metastases examined.
Deregulated activation of MET is critical not only for the acquisition of tumorigenic properties but also for the achievement of the invasive phenotype (Trusolino, L. & Comoglio, P. M. (2002) Nat.
The role of MET in human tumors emerged from several experimental approaches and was unequivocally proven by the discovery of MET-activating mutations in inherited forms of carcinomas (Schmidt et al., Nat.
9 WO 2010/037835 PCT/EP2009/062792 The Scatter Factor (SF) secreted in culture by fibroblasts, that has the ability to induce intercellular dissociation of epithelial cells, and the Hepatocyte Growth Factor (HGF), a potent mitogen for hepatocytes in culture derived from platelets or from blood of patients with acute liver failure, independently identified as Met ligands turned out to be the same molecule. The precursor is proteolytically cleaved at a furin site to produce a highly glycosylated and entirely extracellular a-subunit of 50 kd and a p-subunit of 145 kd with a large extracellular region (involved in binding the ligand), a membrane spanning segment, and an intracellular region (containing the catalytic activity) (Giordano (1989) 339: 155-156).
269 (1994), 1815-20); and (b) a tyrosine (Tyr 1003) that binds the ubiquitin ligase Cbl responsible for Met polyubiquitination, endocytosis and degradation (Peschard et al., Mol. Inhibition of tumor angiogenesis is one of the anticancer strategies which has generated much excitement among clinicians and cancer research scientists in the last few years. Despite the fact that their functions have not been characterized in detail so far, it is well established that they are strongly expressed on vascular endothelial cells in developing embryos and tumors studies (Carson-Walter et al., Cancer Res.
The Endosialin core protein carries abundantly sialylated, 0-linked oligosaccharides and is sensitive to 0-sialoglycoprotein endopeptidase, placing it in the group of sialomucin-like molecules.
In addition, Endosialin is expressed at a low level on a subset of tumor stroma fibroblasts (Brady et al., J. Neuropathol. EpCAM has already been employed as a target antigen in several antibody-based therapeutic approaches, including a human antibody (Oberneder et al., Eur.
This antibody is active against a variety of human carcinoma lines expressing the target antigen and is currently being tested in a phase I study for safety and early signs of efficacy. Altered gene expression in these non-transformed stromal cells has been discussed to provide potential targets for therapy.
The predicted 760-amino acid human FAP alpha protein is a type II integral membrane protein with a large C-terminal extracellular domain, which contains 6 potential N-glycosylation sites, 13 cysteine residues, and 3 segments that correspond to highly conserved catalytic domains of serine proteases; a hydrophobic transmembrane segment; and a short cytoplasmic tail. Seprase is a 170-kD integral membrane gelatinase whose expression correlates with the invasiveness of human melanoma and carcinoma cells. Due to its degrading activity of gelatine and heat-denatured type-I and type-IV collagen, a role for seprase/FAP alpha in extracellular matrix remodeling, tumor growth, and metastasis of cancers has been suggested. It is a hetero-tetrameric receptor of which each half-linked by disulfide bridges-is composed of an extracellular a-subunit and of a transmembrane [beta]-subunit.
The sequence differences observed in the a-subunit are situated in the binding zone of the ligands and are therefore at the origin of the relative affinities of IGF-1 R and of 1 R for the IGFs and insulin respectively. The differences in the C-terminal part of the [beta]-subunit result in a divergence in the signalling pathways of the two receptors; 14 WO 2010/037835 PCT/EP2009/062792 IGF-1R mediating mitogenic, differentiation and antiapoptosis effects, while the activation of the IR principally involves effects at the level of the metabolic pathways (Baserga et al., Biochim.
The activation of the kinases in its turn involves the stimulation of different intra-cellular substrates, including IRS-1, IRS-2, Shc and Grb 10 (Peruzzi F. et al., J. This interest followed the discovery of the fact that in addition to its mitogenic and antiapoptosis properties, IGF-1R seems to be required for the establishment and the maintenance of a transformed phenotype. This in itself is not a unique property since a great variety of products of overexpressed genes can transform cells, including a good number of receptors of growth factors. It has likewise been shown in the works of Jiang et al. (Oncogene, 18:6071-6077, 1999) that a negative dominant of IGF-1R is capable of inhibiting tumor proliferation.
The present invention provides for a bispecific single chain antibody molecule comprising a first binding domain capable of binding to an epitope of human and non chimpanzee primate CD38 (epsilon) chain, wherein the epitope is part of an amino acid sequence comprised in the group consisting of SEQ ID NOs. Though T cell-engaging bispecific single chain antibodies described in the art have great therapeutic potential for the treatment of malignant diseases, most of these bispecific molecules are limited in that they are species specific and recognize only human antigen, and – due to genetic similarity – likely the chimpanzee counterpart. In the present invention, an N-terminal 1-27 amino acid residue polypeptide fragment of the extracellular domain of CD3 epsilon was surprisingly identified which – in contrast to all other known epitopes of CD3 epsilon described in the art – maintains its three-dimensional structural integrity when taken out of its native environment in the CD3 complex (and optionally fused to a heterologous amino acid sequence such as EpCAM or an immunoglobulin Fc part). The present invention, therefore, provides for a bispecific single chain antibody molecule comprising a first binding domain capable of binding to an epitope of an N terminal 1-27 amino acid residue polypeptide fragment of the extracellular domain of CD3 epsilon (which CD3 epsilon is, for example, taken out of its native environment and/or comprised by (presented on the surface of) a T-cell) of human and at least one non-chimpanzee primate CD3 epsilon chain, wherein the epitope is part of an amino acid sequence comprised in the group consisting of SEQ ID NOs. It is thus envisaged that antibodies of the invention bind to (are capable of binding to) the context independent epitope of an N-terminal 1-27 amino acid residue polypeptide fragment of the extracellular domain of CD3 epsilon of human and Callithrix jacchus, Saguinus oedipus, Saimiri sciureus, and Macaca fascicularis (either SEQ ID 2225 or 2226 or both), and optionally also to Macaca mulatta. To this end, it is envisaged to (a) immunize mice with an N-terminal 1-27 amino acid residue polypeptide fragment of the extracellular domain of CD3 epsilon of human and/or Saimiri sciureus; (b) generation of an immune murine antibody scFv library; (c) identification of CD3 epsilon specific binders by testing the capability to bind to at least SEQ ID NOs.
Without being bound by theory, since the CD3 epitope is context-independent, forming an autonomous selfsufficient subdomain without much influence on the rest of the CD3 complex, the CD3 binding molecules/domains provided herein induce less allosteric changes in CD3 conformation than the conventional CD3 binding molecules, which recognize context-dependent CD3 epitopes (e.g. as disclosed in WO 99/54440 or WO 04/106380). The context-independence of the CD3 epitope which is recognized by the CD3 binding domain of the bispecific single chain antibody of the invention (PSCAxCD3, CD19xCD3, C-METxCD3. This results in a better safety profile of the bispecific single chain antibodies of the invention compared to conventional CD3 binding molecules known in the art, which recognize context-dependent CD3 epitopes. Particularly, because T cell redistribution during the starting phase of treatment with CD3 binding molecules is a major risk factor for adverse events, like CNS adverse events, the bispecific single chain antibodies of the invention has a substantial safety 18 WO 2010/037835 PCT/EP2009/062792 advantage over the CD3 binding molecules known in the art by recognizing a context-independent rather than a context-dependent CD3 epitope. Patients with such CNS adverse events related to T cell redistribution during the starting phase of treatment with conventional CD3 binding molecules usually suffer from confusion and disorientation, in some cases also from urinary incontinence. Patients with CNS adverse events related to T cell redistribution during the starting phase of treatment with conventional CD3 binding molecules may also suffer from loss of memory. Frequently, the confusion leads to the loss of ability to recognize people, places, time or dates. CNS adverse events related to T cell redistribution during the starting phase of treatment with conventional CD3 binding molecules may further comprise blurred speech and/or word finding difficulties.
Besides urinary incontinence, vertigo and dizziness may also accompany CNS adverse events related to T cell redistribution during the starting phase of treatment with conventional CD3 binding molecules in some patients. These data strongly indicate that the N-terminal fragment as described herein forms a tertiary conformation, which is similar to its structure normally existing in vivo.
A very sensitive test for the importance of the structural integrity of the amino acids 1-27 of the N-terminal polypeptide fragment of CD3 epsilon was performed. While, for at least some of the, preferably human bispecific single chain antibodies of the invention, two amino acid residues at the C-terminus of the mentioned fragment T (Threonine at position 23) and I (Isoleucine at position 25) reduced the binding energy to the, preferably human, bispecific single chain antibodies of the invention.
The amino acid sequence of the aformentioned N-terminal fragments of CD3 epsilon are depicted in SEQ ID No. Said amino acid residues 26 to 61 of the EGF-like domain 1 of human EpCAM are shown in SEQ ID NO.
It will be understood that in a preferred embodiment, the cross-species specificity of the first and second binding domain of the antibodies of the invention is identical. The proportion of older persons is projected to more than double worldwide over the next half century, which means that a further increase in incidence of diagnosed prostate cancer has to be expected. In a preferred embodiment, the bispecific single chain antibody thus comprises a second binding domain exhibiting cross-species specificity to the human and a non-chimpanzee primate Prostate stem cell antigen (PSCA). In this case, the identical bispecific single chain antibody molecule can be used both for preclinical 22 WO 2010/037835 PCT/EP2009/062792 evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and as drugs in humans.
Since both the CD3 and the PSCA binding domain of the PSCAxCD3 bispecific single chain antibody of the invention are cross-species specific, i.e. reactive with the human and non chimpanzee primates, it can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and – in the identical form – as drugs in humans. In a preferred embodiment, the bispecific single chain antibody of the invention comprises a second binding domain exhibiting cross-species specificity to the human and a non-chimpanzee primate CD19.
In this case, the identical bispecific single chain antibody molecule can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and as drug in humans.
Since in this embodiment both the CD3 and the CD19 binding domain of the CD19xCD3 bispecific single chain antibody of the invention are cross-species specific, i.e. reactive with the human and non-chimpanzee primates, it can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and – in the identical form – as drug in humans.
Since preferably both the CD3 and the C-MET binding domain of the C METxCD3 bispecific single chain antibody of the invention are cross-species specific, i.e. reactive with the human and non-chimpanzee primates’ antigens, the C METxCD3 bispecific single chain antibody of the invention can be used for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and – in the identical form – as drugs in humans. In a preferred embodiment, the bispecific single chain antibody thus comprises a second binding domain exhibiting cross species specificity to the human and a non-chimpanzee primate C-MET.
In this case, the identical bispecific single chain antibody molecule can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and as drugs in humans. Since both the CD3 and the C-MET binding domain of the C-METxCD3 bispecific single chain antibody of the invention are cross-species specific, i.e. reactive with the human and non chimpanzee primates’ antigens, it can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and – in the identical form – as drugs in humans.
Angiogenesis, i.e. the formation of new capillaries, is essential to a number of important physiological events, both normal and pathological. In a preferred embodiment, the bispecific single chain antibody thus comprises a second binding domain exhibiting cross-species specificity to the human and a non-chimpanzee primate Endosialin.
In this case, the identical bispecific single chain antibody molecule can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and as drug in humans. Since both the CD3 and the 25 WO 2010/037835 PCT/EP2009/062792 Endosialin binding domain of the EndosialinxCD3 bispecific single chain antibody of the invention are cross-species specific, i.e. reactive with the human and non chimpanzee primates, it can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and – in the identical form – as drug in humans. In a preferred embodiment, the bispecific single chain antibody of the invention comprises a second binding domain exhibiting cross-species specificity to the human and a non-chimpanzee primate EpCAM. In this case, the identical bispecific single chain antibody molecule can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and as drug in humans.
Since in this embodiment both the CD3 and the EpCAM binding domain of the EpCAMxCD3 bispecific single chain antibody of the invention are cross-species specific, i.e. reactive with the human and non-chimpanzee primates, it can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and – in the identical form – as drug in humans. Previous study have shown that 26 WO 2010/037835 PCT/EP2009/062792 most of the common types of epithelial cancers, including more than 90% of primary and malignant breast, lung and colorectal carcinomas, contain abundant FAP alpha reactive stromal fibroblasts (Scanlan et al., PNAS 91 (1994), 5657-5661 and references cited therein). In contrast, normal tissues and benign and premalignant epithelial lesions only rarely contain FAP alpha-positive stromal cells. In a preferred embodiment, the bispecific single chain antibody thus comprises a second binding domain exhibiting cross-species specificity to the human and a non-chimpanzee primate FAP alpha.
In this case, the identical bispecific single chain antibody molecule can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and as drugs in humans. Since both the CD3 and the FAP alpha binding domain of the FAPalphaxCD3 bispecific single chain antibody of the invention are cross-species specific, i.e. reactive with the human and non chimpanzee primates, it can be used both for preclinical evaluation of safety, activity 27 WO 2010/037835 PCT/EP2009/062792 and/or pharmacokinetic profile of these binding domains in primates and – in the identical form – as drugs in humans. In this case, the identical bispecific single chain antibody molecule can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and as drug in humans. Since in this embodiment both the CD3 and the IGF-1 R binding domain of the IGF-1 RxCD3 bispecific single chain antibody of the invention are cross-species specific, i.e. reactive with the human and non-chimpanzee primates, it can be used both for preclinical evaluation of safety, activity and/or pharmacokinetic profile of these binding domains in primates and – in the identical form – as drug in humans.
As shown in the following Examples, said preferably human, PSCAxCD3 (respectively CD19xCD3, C-METxCD3, EndosialinxCD3, EpCAMxCD3, IGF-1 RxCD3 or FAPaxCD3) bispecific single chain antibody of the invention can be used as therapeutic agent or drug against various diseases, including, but not limited, to cancer. Said preferably human c-METxCD3 bispecific single chain antibody of the invention can be used as therapeutic agent against various diseases, including but not limited to cancer, preferably carcinoma, sarcoma, glioblastoma/astrocytoma, melanoma, mesothelioma, Wilms tumor or a hematopoietic malignancy such as leukemia, lymphoma or multiple myeloma.
Said preferably human EndosialinxCD3 bispecific single chain antibody of the invention provides a novel and inventive approach for tumor endothelium targeting and killing for including (but not limited to) carcinomas (breast, kidney, lung, colorectal, colon, pancreas mesothelioma), sarcomas, and neuroectodermal tumors (melanoma, glioma, neuroblastoma). The EndosialinxCD3 bispecific single chain antibody of the invention can deprive solid tumors of their supporting blood vessels, thereby inhibiting angiogenesis and consequently the growth of said neoplasms.
IGF-1 RxCD3 bispecific single chain antibody of the invention can be used as therapeutic agent against autoimmune diseases, preferably psoriasis. 29 WO 2010/037835 PCT/EP2009/062792 In view of the above, the need to construct a surrogate PSCAxCD3 (respectively CD19xCD3, C-METxCD3, EndosialinxCD3, EpCAMxCD3, IGF-1RxCD3 or FAPaxCD3) bispecific single chain antibody for testing in a phylogenetic distant (from humans) species disappears. Thus, the molecule to be used in human therapy in fact differs in sequence and also likely in structure from the surrogate molecule used in preclinical testing in pharmacokinetic parameters and/or biological activity, with the consequence that data obtained in preclinical animal testing have limited applicability / transferability to the human case. Another major advantage of the, preferably human, PSCAxCD3 (respectively CD19xCD3, C-METxCD3, EndosialinxCD3, EpCAMxCD3, IGF-1RxCD3 or FAPaxCD3) bispecific single chain antibody of the invention is its applicability for preclinical testing in various primates.
As a result, the data obtained from such preclinical testing should therefore generally have a highly predictive power for the human case. The results of these dramatic, non-desired negative events suggest that it may not be sufficient to limit preclinical testing to only one (non-chimpanzee primate) species. The fact that the PSCAxCD3 (respectively CD1 9xCD3, C-METxCD3, EndosialinxCD3, EpCAMxCD3, IGF-1 RxCD3 or FAPaxCD3) bispecific single chain antibody of the invention binds to a series of New-World and Old-World Monkeys may help to overcome the problems faced in the case mentioned above. Accordingly, the present invention provides means and methods for minimizing species differences in effects when drugs for human therapy are being developed and tested. A further advantage of the uses of the preferably human PSCAxCD3 (respectively CD19xCD3, C-METxCD3, EndosialinxCD3, EpCAMxCD3, IGF-1RxCD3 or FAPaxCD3) bispecific single chain antibody of the invention exhibiting cross-species specificity is the fact that chimpanzees as an endangered species are avoided for animal testing. Chimpanzees are the closest relatives to humans and were recently grouped into the family of hominids based on the genome sequencing data (Wildman et al., PNAS 100 (2003), 7181).
In light of this, the uses of the, preferably human, PSCAxCD3 (respectively CD1 9xCD3, C-METxCD3, EndosialinxCD3, EpCAMxCD3, IGF-1 RxCD3 or FAPaxCD3) bispecific single chain antibody of the invention provide for a reasonable alternative for studies in chimpanzees. Multiple blood extractions can be much more readily obtained with a non-chimpanzee primate than with lower animals, e.g. a mouse. Furthermore, the extraction of multiple blood samples enables the researcher to evaluate the pharmacokinetic profile of the, preferably human, PSCAxCD3 (respectively CD1 9xCD3, C-METxCD3, EndosialinxCD3, EpCAMxCD3, IGF-1 RxCD3 or FAPaxCD3) bispecific single chain antibody of the invention as defined herein. This allows the determination of the potential toxicity profile of the, preferably human, PSCAxCD3 (respectively CD1 9xCD3, C-METxCD3, EndosialinxCD3, EpCAMxCD3, IGF-1RxCD3 or FAPaxCD3) bispecific single chain antibody of the invention as defined herein. This is highly advantageous in that e.g. the pharmacokinetic results are more directly transferable and applicable to the human setting than e.g. in conventional surrogate approaches. * the therapeutic use of the C-METxCD3 bispecific single chain antibody of the invention provides a novel and inventive therapeutic approach for cancer, 34 WO 2010/037835 PCT/EP2009/062792 preferably carcinoma, sarcoma, glioblastoma/astrocytoma, melanoma, mesothelioma, Wilms tumor or a hematopoietic malignancy such as leukemia, lymphoma or multiple myeloma.
e the therapeutic use of the EndosialinxCD3 bispecific single chain antibody of the invention provides a novel and inventive approach for tumor endothelium targeting and killing for including (but not limited to) carcinomas (breast, kidney, lung, colorectal, colon, pancreas mesothelioma), sarcomas, and neuroectodermal tumors (melanoma, glioma, neuroblastoma). The EndosialinxCD3 bispecific single chain antibody of the invention can deprive solid tumors of their supporting blood vessels, thereby inhibiting angiogenesis and consequently the growth of said neoplasms.
The advantage of bispecific single chain antibody molecules as drug candidates fulfilling the requirements of the preferred bispecific single chain antibody of the invention is the use of such molecules in preclinical animal testing as well as in clinical studies and even for therapy in human.
The term “antibody” comprises derivatives or functional fragments thereof which still retain the binding specificity. The term “antibody” also comprises immunoglobulins (Ig’s) of different classes (i.e. IgA, IgG, IgM, IgD and IgE) and subclasses (such as IgG1, IgG2 etc.). Also, transgenic animals may be used to express humanized antibodies specific for polypeptides and fusion proteins of this invention.
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