WO2009051672A2 - Dosage des récepteurs de la famille des immunoglobulines de tueurs naturels (kir) - Google Patents

Dosage des récepteurs de la famille des immunoglobulines de tueurs naturels (kir) Download PDF

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WO2009051672A2
WO2009051672A2 PCT/US2008/011671 US2008011671W WO2009051672A2 WO 2009051672 A2 WO2009051672 A2 WO 2009051672A2 US 2008011671 W US2008011671 W US 2008011671W WO 2009051672 A2 WO2009051672 A2 WO 2009051672A2
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kir
primer
cells
expression
sample
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WO2009051672A3 (fr
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Xiaohua Chen
Rupert Handgretinger
Gregory A. Hale
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St. Jude Children's Research Hospital
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention provides methods and assays and enabling compositions for genotyping natural killer (NK) cells according to the type of immunoglobulin-like receptors (KIR) they express.
  • KIR immunoglobulin-like receptors
  • the present invention allows for KIR expression profiling in, for example, samples from recipients and donors of allogeneic bone marrow and hematopoietic stem cell transplants, wherein NK cells are expected to be therapeutic for recipients if the matching of donor NK cells to recipient is optimized for that purpose.
  • ABO blood group system ABO blood group system
  • HLA human leukocyte antigen
  • T-cell receptors T-cell receptors
  • B-lymphocyte antigen receptors B-lymphocyte antigen receptors
  • transplanting a population of cells from a donor who is immunologically non-identical to the recipient (or “host") exposes the host's immune system to an array of foreign antigens, with the result that fragments (generally, small peptides) from at least some of the donor cells find their way to so-called major histocompatibility complex molecules, or "MHCs" (referred to, for historical reasons, as human leukocyte antigens or "HLAs”), arrayed on the surfaces of host cells.
  • MHCs major histocompatibility complex molecules
  • HLAs human leukocyte antigens
  • the interaction sets into motion a process whereby the body's immune system acquires the ability to recognize any future encounter with the antigen as a signal that more of the donor cells have invaded.
  • an elaborate defense is mounted against the invading cells until the host has destroyed (“rejected") them.
  • T-cells As endogenous T-cells mature, they interact with a multitude of (autologous) MHC molecules (whether or not complexed to a foreign fragment). Immature T-cells bearing receptors that react with an autologous MHC molecule, will not mature. An individual's mature T-cells therefore tend to fit the individual's repertoire of MHC molecules in a "self-self manner.
  • the T-cell receptor repertoire of the T-cell population that descends from the colony assuming that the mismatch doesn't lead to outright rejection, also comes to conform to the host's HLA genotype, leaving behind only a residuum of chimeras.
  • NK cells are polymorphonuclear leukocytes (their nuclei take many forms). Interferons, other cytokines, and the Fc portion of antibodies activate NK cells, which thereupon release enzymes that induce apoptosis in nearby cells, especially virus- infected cells, cancer cells and allogeneic ("foreign") cells. Like T-cells, NK cells are lymphocytes sensitive to HLAs, but their tolerance of "self ' antigens arises from a quite different mechanism. Two broad classes of receptors on NK cells modulate the activation of NK cells. One class is lectin-like.
  • KIRs immunoglobulin-like receptors
  • a re-constituted population of NK cells carries a KIR repertoire that remains highly reflective of the donor's repertoire (Vilches et al. Annu. Rev. Immunol. 20: 217-251, 2002). "Mismatched" donor KIRs therefore interact with host HLAs at a high frequency. Nevertheless, a self-non-self reaction is unlikely: Each NK cell displays its own "mini- repertoire" of iKIRs.
  • NK cells In general, if even one of those iKIRs interacts with an HLA on a host cell, the killer cell will remain quiescent (Borrego et al. MoI. Immunol. 38: 637-660, 2001), leaving the host cell undisturbed. An NK cell mounts a destructive response when it encounters a cell that is broadly deficient in expressed HLAs. Thus, NK cells often tolerate non-self, but not "missing self.”
  • Therapeutic obliteration of bone marrow and of hematopoietic stem cells, followed by transplantation of "new" marrow or stem cells, are established methods for treating hematologic diseases such as leukemias. Since T cell receptors and the HLA molecules with which they interact are both highly polymorphic, clinical hematopoietic cell transplantation predictably involves "mismatches" in T cell receptor type and in HLA type between allogeneic donors and recipients. If grossly mismatched (unrelated donor- recipient pair), graft rejection or graft-vs-host disease (GVHD) can be expected. Less severe mismatches (siblings), on the other hand, can be therapeutic for the leukemic patient.
  • GVHD graft rejection
  • siblings graft-vs-host disease
  • cancer cells can grow, multiply, and spread. Thus, cancer cells tend not to alert the body's immune defenses. Transplanted, unmatched T-cells, on the other hand, do not accept the host's cancer cells as self. A destructive interaction, called the graft versus tumor effect, results. The effect appears to be most powerful in diseases that progress slowly, like chronic leukemia, low-grade lymphoma, and in some cases multiple myeloma, but is weaker in the rapidly growing acute leukemias.
  • the therapeutic aspect of transplanting incompletely matched blood products is most apparent when leukemia returns ("relapse") in a treated patient and a follow-up dose of the original donor's white blood cells is administered:
  • the donated cells sharply combat the leukemic cells immunologically, which keeps the leukemic cell population from expanding.
  • NK cells transplanted into a non-identical leukemic host can also be curative. Because any given NK cell displays a plurality of iKIR types, and healthy host cells display a plurality of HLAs, at least one NK cell-inhibiting iKIR:HLA interaction is likely. Leukemic host cells, however, are HLA-deficient. The probability that a host's leukemic cells will inhibit the host's NK cells is therefore reduced. Thus, transplanted NK cells would appear to be well-suited to counter leukemias, particularly if their aKERs do not match the aKIRs of their host (Ruggeri et al. Transpl. Immunol. 14: 203-206, 2005).
  • KIR mismatch decreases relapse rate (Leung et al. J. Immunol. 172: 644-650, 2004; Hsu et al. Blood 105: 4878-4884, 2005) but no such benefit is seen in patients with SCID (Keller et al. J. Clin. Immunol. 27: 109-116, 2007). It is likely that full KIR genotyping could reveal the basis for these divergent results and lead, ultimately, to a thorough understanding of how to conduct therapeutic mismatching in an optimal manner. Accordingly, what are needed are methods and systems that allow practitioners and investigators to more accurately and efficiently determine the KIR genotype of a sample. Such methods and systems would provide tools for use in transplant therapeutics and in KIR-related research.
  • the present invention provides compositions, methods and assays for genotyping natural killer (NK) cells according to their expression of immunoglobulin-like receptors (KIRs).
  • KIRs immunoglobulin-like receptors
  • embodiments of the present invention allow for KTR expression profiling in, for example, samples from donors and patients, results of which are useful in optimizing transplantation therapies involving NK cells.
  • the invention comprises a set of oligonucleotide-primer pairs, the set configured to amplify amplicons of KIRs such that each amplicon is specific to a KIR family, wherein the length of each specific amplicon is different.
  • the present invention provides methods and assays for the specific detection of KIRs in donor and recipient samples ⁇ e.g., blood samples) using mRNA expression typing.
  • the present invention provides amplification primers in such combination as to result in short amplicons ⁇ e.g., shorter than 500bp), each of a length specific for a particular KIR family and, at the same time, amenable to visualization ⁇ e.g., using fluorescently labeled primers) by means of instruments ⁇ e.g., genetic sequence analyzers), especially instruments wherein the amplifications can be performed and the amplicons displayed in a multiplex fashion.
  • the incorporation of a means of measuring CD56 mRNA or other marker that is specific to NK cells provides for normalization of the samples for NK cell numbers.
  • the assembled set of primers are provided in a kit, which kit preferably includes a means for identifying a specific NK-cell marker, and reagents, enzymes and buffers that enable the assays to be conducted.
  • the present invention provides methods and assays for performing individual RT-PCR or other amplification reactions, preferably using primers selected according to criteria provided by the present invention, on mRNA from NK cells for determining KIR expression. In some embodiments, the present invention provides methods and assays for conducting the amplification of a plurality of mRNA species ⁇ e.g. multiple amplification reactions in one tube) using primers of the present invention.
  • the present invention provides for the visualization of the products of the amplification reactions
  • an aliquot of one RT- PCR or other amplification reaction is loaded into one well (or other single point of origin) in a separation medium ⁇ e.g., an agarose or polyacrylamide gel), thereby allowing for the visualization of one amplification reaction per well, wherein the separation medium may be configured as a slab or column and may be disposed in an electric, magnetic or gravitational field
  • the products of separate amplification reactions are combined into a mixture or complex ⁇ i.e., "multiplexed" and loaded into one well or point of origin, thereby allowing for the visualization of multiple amplification reaction products emanating from a single point of origin into the separation medium.
  • the amplification reactions themselves are conducted simultaneously on a plurality of mRNA species as a complex in a single reaction vessel and an aliquot of the products of the multiplex amplification reaction is loaded into one well for separation and visualization as a spectrum of amplification products.
  • the invention is embodied as a method of assembling or constructing a set of oligonucleotide-primer pairs wherein at least one forward KIR oligonucleotide primer is provided, and a plurality of reverse KIR oligonucleotide primers is provided, wherein each reverse KIR primer is specific for one and only one KIR family, and each forward KIR primer and reverse KIR primer defines an amplicon having a nucleotide length less than about 500 nucleotides and each such nucleotide length is different.
  • the invention is embodied in a method for determining the expression of a KTR family in a sample.
  • a nucleic acid sample from a population of polymorphonuclear cells is provided.
  • At least one pair of oligonucleotide primers is applied to the sample, wherein one member of the pair is selected from the group consisting of SEQ ID NO. 15 and SEQ ID NO. 16 and one member is selected from the group consisting of SEQ ID NO. 1 through SEQ ID NO. 14.
  • the nucleic acid sample is amplified with the oligonucleotide primers and, based on the amplification, the presence or absence of expression of the KIR family in the sample is determined.
  • the invention is also embodied in a method for determining the expression of a plurality of KIR families in a sample.
  • a nucleic acid sample from a population of polymorphonuclear cells is provided, to which sample is applied a set of oligonucleotide primers.
  • the set is assembled with at least one forward KIR oligonucleotide primer, together with a plurality of reverse KIR oligonucleotide primers, wherein each reverse KIR primer is specific for one and only one KIR family.
  • Each forward KER. primer and reverse KTR primer defines an amplicon having a nucleotide length less than about 500 nucleotides and each such nucleotide length is different.
  • Amplicons of the nucleic acid sample are generated and used to determine the KIR families expressed in the sample.
  • the amplicons are generated in a single reaction vessel.
  • the amplicons are separated from one another in a single separation step.
  • the invention provides an assay for tissue typing blood products for KIR genotype.
  • the assay can be part of a procedure for determining tissue compatibility for use in transplantation therapy. DETAILED DESCRIPTION OF THE DRAWINGS
  • Figure 1 shows an exemplary electropherogram of KJR spectratypes in a mixed PBMNC sample from healthy donors. The sample was tested by using a single PCR for each KIR gene. Each peak represents a unique KIR gene fragment. The size value is shown on the x-axis and the peak height on the y-axis.
  • FIG. 2 shows an exemplary experiment demonstrating KIR expression in donors after stem cell mobilization and selection.
  • Each KIR gene family is shown on the x-axis and its expression level on the y-axis.
  • KJR expression was quantified before mobilization (grey) after mobilization (open) and after stem cell selection (hatched).
  • Figure 3 shows an exemplary electropherogram of KIR spectratypes in two mixed PCR populations. The size value is shown on the x-axis and the peak high on the y-axis. Each peak represents one KIR gene fragment.
  • PCR polymerase chain reaction
  • oligonucleotides With the appropriate reagents and enzymes, PCR makes copies (or the complements thereof) of the region of the longer polymer that is defined between the binding sites, resulting in an exponentially expanded population of so-called “amplicons.”
  • the oligonucleotides "prime” the reaction.
  • a primer pair consists of a primer having a sequence that complements a sequence running "forward” in the larger polymer and a primer that complements a sequence running in the "reverse” direction.
  • the allowable length of the region between the primers can be varied widely, but lengths of less than 500 consecutive nucleotides (the “nucleotide length") are preferred in embodiments of the invention.
  • the term “nucleotide sequence of interest” refers to any nucleotide sequence (e.g.,
  • RNA or DNA the manipulation of which may be deemed desirable for any reason (e.g., treat disease, confer improved qualities, expression of a protein of interest in a host cell, expression of a ribozyme, etc.), by one of ordinary skill in the art.
  • nucleotide sequences include, but are not limited to, coding sequences of structural genes (e.g., reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.), and non-coding regulatory sequences which do not encode an mRNA or protein product (e.g., promoter sequence, polyadenylation sequence, termination sequence, enhancer sequence, etc.).
  • protein of interest refers to a protein encoded by a nucleic acid of interest.
  • exogenous gene refers to a gene that is not naturally present in a host organism or cell, or is artificially introduced into a host organism or cell.
  • the term "gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of a polypeptide or precursor (e.g., proinsulin).
  • the polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the full-length or fragment are retained.
  • the term also encompasses the coding region of a structural gene and includes sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb or more on either end such that the gene corresponds to the length of the full-length mRNA.
  • sequences that are located 5' of the coding region and which are present on the mRNA are referred to as 5' untranslated sequences.
  • sequences that are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' untranslated sequences.
  • the term "gene” encompasses both cDNA and genomic forms of a gene.
  • a genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns" or "intervening regions” or “intervening sequences.”
  • Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers.
  • RNA messenger RNA
  • mRNA messenger RNA
  • the mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.
  • gene expression refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of the gene (i.e., via the enzymatic action of an RNA polymerase), and for protein encoding genes, into protein through “translation” of mRNA. Gene expression can be regulated at many stages in the process.
  • Up- regulation or “activation” refers to regulation that increases the production of gene expression products (i.e., RNA or protein), while “down-regulation” or “repression” refers to regulation that decrease production.
  • Molecules e.g. , transcription factors
  • activators and “repressors,” respectively.
  • amino acid sequence is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule
  • amino acid sequence and like terms, such as “polypeptide” or “protein” are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.
  • nucleic acid molecule encoding refers to the order or sequence of deoxyribonucleotides or ribonucleotides along a strand of deoxyribonucleic acid or ribonucleic acid. The order of these deoxyribonucleotides or ribonucleotides determines the order of amino acids along the polypeptide (protein) chain.
  • the DNA or RNA sequence thus codes for the amino acid sequence.
  • complementarity are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules.
  • sequence “5'-A-G-T-S','' is complementary to the sequence "3'-T- C-A-5'.”
  • Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
  • a partially complementary sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid sequence and is referred to using the functional term "substantially homologous.”
  • the inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency.
  • a substantially homologous sequence or probe i.e., an oligonucleotide which is capable of hybridizing to another oligonucleotide of interest
  • conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction.
  • the absence of non-specific binding may be tested by the use of a second target which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.
  • a partial degree of complementarity e.g., less than about 30% identity
  • low stringency conditions factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions.
  • conditions that promote hybridization under conditions of high stringency e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.).
  • substantially homologous refers to any probe that can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.
  • substantially homologous refers to any probe that can hybridize ⁇ i.e., it is the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above.
  • hybridization is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the T m of the formed hybrid, and the G:C ratio within the nucleic acids. A single molecule that contains pairing of complementary nucleic acids within its structure is said to be “self-hybridized.”
  • T m is used in reference to the "melting temperature" of a nucleic acid.
  • the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
  • stringency is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. With “high stringency” conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences. Thus, conditions of "weak” or “low” stringency are often required with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually less.
  • High stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42 0 C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PCvH 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt's reagent and 100 ⁇ g/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1X SSPE, 1.0% SDS at 42°C when a probe of about 500 nucleotides in length is employed.
  • 5X SSPE 43.8 g/1 NaCl, 6.9 g/1 NaH 2 PCvH 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH
  • SDS 5X Denhardt's reagent
  • 100 ⁇ g/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1X SSPE, 1.0% SDS at 42°
  • “Medium stringency conditions” when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 -H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt's reagent and 100 ⁇ g/ml denatured salmon sperm DNA followed by washing in a solution comprising 1.0X SSPE, 1.0% SDS at 42°C when a probe of about 500 nucleotides in length is employed.
  • Low stringency conditions comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 ⁇ H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5X Denhardt's reagent [5OX Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and 100 ⁇ g/ml denatured salmon sperm DNA followed by washing in a solution comprising 5X SSPE, 0.1% SDS at 42°C when a probe of about 500 nucleotides in length is employed.
  • 5X SSPE 43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 ⁇ H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH
  • 5X Denhardt's reagent [5OX Denhard
  • in operable combination refers to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced.
  • the term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced. It is advantageous for some embodiments of the invention and the various applications thereof to identify the expression products of the genes that encode KIRs rather than the genes themselves as they are encoded in the DNA (desoxyribonucleic acid) of chromosome 19.
  • RNA messenger RNA
  • mRNA messenger RNA
  • mRNA messenger RNA
  • each NK cell displays several KIR family members, but each cell does not necessarily display the same set.
  • the full spectrum, across all NK cells, comprises an individual's KIR "repertoire.”
  • "repertoire” may be used interchangeably with "genotype,” although practitioners will understand that the former term generally refers to phenotype (i.e., a trait or set of traits) while the latter refers to the combination of alleles located on homologous chromosomes that determines phenotype.
  • KIR KIR
  • the term “expression profiling” refers generally to a method of assessing the genotype (in this case the KIR genotype) of an individual by detecting the presence or absence of the family- specific mRNAs in a sample under inspection. Expression profiling yields data useful in "expression typing.”
  • the term relates to procedures for tissue typing, that is, determining the compatibility or incompatibility of a donor tissue with a host in connection with tissue transplantation.
  • the polymerase chain reaction preferred for use herein is the so-called reverse transcriptase PCR or RT-PCR, wherein mRNA transcripts are enzymatically converted to DNA and then the DNA is subjected to PCR.
  • the method is not limited to these means for determining the KIR repertoire of an individual or a sample suspected of containing KIRs.
  • a sample as used herein relates to a sample of blood obtained from a donor or prospective donor of an organ or tissue, including but not limited to blood or elements thereof (e.g., lymphocytes), collectively referred to as "blood products.”
  • the recipient of a donor's organ or tissue typically a patient (or subject), may also be referred to herein as a "host.”
  • multiplex as used herein relates to two or more events that can take place at separate times or in separate spaces being made to take place at the same time and space.
  • a plurality of amplification reactions could be conducted, each in its own reaction vessel. If they are conducted at once in a single vessel, the reactions are said to be multiplexed.
  • the analysis, visualization or measurement of the products of two or more amplification reactions could be analyzed, visualized or measured separately in time or space (e.g, electrophoresed in separate lanes of a gel). If such analyses, visualizations or measurements are conducted at once on a single gel lane, the analysis is said to be multiplexed.
  • multiplexing is a means of displaying or visualizing amplification products as a spectrum.
  • a sample of blood from a patient for example, is subjected to a multiplex amplification reaction using a set of primers assembled according to the invention and the mixed products of the reaction are separated from one another by electrophoresis, the amplification product of each KIR family in the patient's KIR repertoire travels to its own site in the separation medium, so that the repertoire is visualized as a spectrum.
  • Each patient has a patient-specific spectrum, which is referred to herein as a "spectratype.”
  • KIRs Killer cell immunoglobulin-like receptors
  • LRC 1 Mb leukocyte receptor complex
  • the gene content of the KIR gene cluster varies among haplotypes, although several 'framework' genes are found in all haplotypes (KIR3DL3, KTR3DL1, KIR3DL4, KIR3DL2).
  • KIR proteins are classified by the number of extracellular immunoglobulin domains (2D or 3D) and by whether they have a long (L) or short (S) cytoplasmic domain.
  • ITIM immune tyrosine-based inhibitory motif
  • KIR proteins with the short cytoplasmic domain lack the ITIM motif and instead associate with the tyrosine kinase binding protein TYRO to transduce activating signals (aKIR).
  • the ligands for several KIR proteins are subsets of HLA molecules; thus, KIR proteins are thought to play an important role in regulation of the immune response.
  • NK cells When it comes to transplantation therapies involving NK cells, such as transplantation therapies for leukemias ⁇ e.g., bone marrow transfusions and hematopoietic stem cell transplantation), the matching (or, perhaps more to the point, the strategic "mismatching" of NK cells from donor to recipient may be advantageous in ameliorating relapse and graft vs host disease (GvH).
  • transplantation therapies for leukemias ⁇ e.g., bone marrow transfusions and hematopoietic stem cell transplantation
  • the matching or, perhaps more to the point, the strategic "mismatching"
  • GvH graft vs host disease
  • the explicit use of NK cells for therapeutic purposes will preferably employ methods that provide accurate, convenient and reliable determinations of the KIR repertoire of both donor and recipient.
  • NK cells are called "natural” because of the role they play in the innate immune response (Carrington and Martin, 2006, Curr Topics Micro Immuno 298:225-257). KIRs are also expressed on a minor subset of memory/effector T-cells that participate in acquired immunity.
  • the KIR repertoire of NK cells is shaped in subtle ways by the HLA environment to which the cells are exposed (Shilling et al., Blood 101:3730-3740, 2003), but is by no means determined by an individual's HLA repertoire as T-cell receptors are (Vilches and Parham, Ann Rev Immunol 20:217-251, 2002).
  • the KIR gene repertoire is diverse, being determined not only by gene locus but also by allelic polymorphism.
  • iKIRs regulate natural killer (NK) cell function by recognizing self-HLA ligands that belong to Class I of the major histocompatibility complex. Recognition leads to NK cell inhibition, which prevents NK cells from killing healthy autologous cells.
  • aKTRs recognize non-self HLAs to initiate NK-cell activity against infected or transformed cells (Carrington et al. Curr. Top. Microbiol. Immunol. 298: 225-257, 2006; Borrego et al, MoI Immunol 38:637-660, 2001).
  • the "missing self hypothesis assumes that NK activation depends upon a balance between inhibitory and activating signals such that a cell that cannot present sufficient self-signals to an NK cell will tip the balance toward NK activation, especially if that NK cell is picking up non-self signals at the same time.
  • allogeneic hematopoietic stem cell transplantation In recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT),
  • NK cells do not cause GvH disease even when donor and recipient are mismatched, because the graft NK cells attack only mismatched host cells that express little or no HLA class-I, leukemic cells being a prominent example (Ruggeri et al., Transplant Immunol 14:203-206, 2005). These advantages have made NK-cell treatment attractive as an adjunct in allo-HSCT. Available information about KIR genotypes derives largely from investigations directed at identifying KIR genotypes that correlate with disease or untoward post- transplant effects. It is known that the genotype is not fully expressed transcriptionally or phenotypically even in healthy individuals (Leung et al., J Immunol 174:6540-6545, 2005).
  • KIR genes may be down-regulated during hematologic reconstitution after HSCT, particularly when myeloablative conditioning is used (Shilling et al, Blood 101:3730-3740, 2003). And, when the appearance of KIR transcripts is delayed post-transplant, the risk of acute GvH increases (Denis et al., Hum Immunol 66:447-459, 2005). Taken together, these results suggest that KIR expression undergoes kinetic changes that may affect the development of disease and the outcome of treatment. However, KIR expression in the healthy population, in donors during stem cell recruitment and selection, and in patients during the peri-transplant period all remain to be investigated.
  • KIR typing such as the QIAGEN® Olerup SSP Kits for KIR, DYNAL® KIR Genotyping SSP Kit, and the Miltenyi Biotec, Inc. KIR Typing Kit. All these products utilize amplification methods for typing KIRs present in a sample followed by agarose gel electrophoresis of the single amplifications. Other systems for KIR typing are reported (Lin et al., Am. J. Pathol. 159:1671-1679, 2001; Uhrberg, Immunity 7:753-763, 1997; Leung et al, J. Immunol.
  • Embodiments of the present invention provide such methods.
  • One embodiment comprises a set of KIR polymerase chain reaction (PCR) primers designed to quantitatively determine repertoire expression.
  • the invention comprises the criteria by which the set is designed. These criteria can be used to admit or deny entry of primer pairs to the repertoire-determining set for purposes of expanding the set to accommodate any newly discovered KIRs.
  • the criteria include, (1) the specificity of each primer pair must be unambiguous; (2) each primer pair of the set must define an amplicon of a nucleotide length amenable to amplification in an automated system; (3) the length of the amplicon each primer pair defines must be sufficiently different for each primer pair that each amplicon can be resolved from the others in a suitable separation step such as gel electrophoresis, and (4) a means of amplifying an expression product unique to NK cells must be provided.
  • a set of KIR primers that includes only two fluorescently-labeled common forward primers and 14 reverse primers specific to each KIR family was generated.
  • each specific primer not only hybridizes to a specific KIR transcript, but also generates fluorescently-labeled PCR fragments, each having its own distinct length, that are easily recognized and quantified by a DNA genetic analyzer.
  • the PCR fragments were designed to be approximately 148-329 bp to conform to a preferred size range less than 500 bp.
  • the two common forward primers simplify the PCR procedure and provide the opportunity to further reduce the number of PCR reactions by generating mixed PCR products (e.g., multiplex PCR) in which the distinct length of PCR fragments is retained (Figure 3).
  • a transcript of CD56 is amplified as an endogenous control (or “amplification control") to effectively reduce error by providing an index of the number of NK cells in the sample.
  • KIR primers were designed according to the sequence profile described by Garcia et al., Immunogenetics 55:227-239, 2003. To confirm the specificity of the primers, PCR products generated by each of the reverse (RV) primers in combination with a forward (FW) primer were sequenced. The specific sequence was found in the PCR products of all primer pairs except 3DL3, for which no positive signal in any of the tested healthy donors and patients was found. Each pair of KIR primers generated a specific PCR fragment with distinct length as demonstrated on an electropherogram ( Figure 1). In some embodiments, the present invention provides methods and systems for
  • the methods and systems comprise fluorescently labeled forward primers in operable combination with unlabeled reverse primers, wherein each pair of primers amplifies one KTR family.
  • the methods and systems of the present invention further comprise reagents, buffers, enzymes, and other solutions, proteins and the like for performing RT-PCR and/or PCR reactions.
  • the methods and systems comprise an internal amplification control for each amplification reaction.
  • the amplification control is a CD56 encoding nucleic acid.
  • methods and systems further comprise reagents, buffers, enzymes, and the like for performing polyacrylamide gel electrophoresis.
  • the methods and systems of the present invention are used in conjunction with a genetic sequence analyzer, such as an ABI PRISM®
  • the present invention is not limited by the instrumentation used in performing the amplification and/or visualization reaction and it is contemplated that any instrument capable of performing one or both of amplification and/or visualization of an amplified product is amenable for use with the methods of the present invention.
  • the label affixed to a forward primer is fluorescent.
  • fluorescent labels include, but are not limited to, 6-FAM, fluorescein isothiocyanate, phycoerythrin, allophycocyanin, Cy 3 and Cy 5.
  • Dyes useful in labeling primers are known to those skilled in the art, and can be found in, for example, the Handbook of Fluorescent Probes and Research Chemicals,
  • the combinations of forward and reverse primers provide for amplification products less than 500bp, less than 400bp, less than 350bp. In some embodiments, the combinations of primers provides for amplification products preferably from about lOObp to about 400bp, from about 120bp to about 380bp, from about 140bp to about 350bp, or from about 148bp to about 329bp.
  • a KER amplification reaction is performed as a single amplification reaction, wherein only one set of KIR primers is present in one tube or one well of a 96 well plate or other plate format used for amplification purposes. In some embodiments, several sets of KIR primers are present in one tube or one well of a plate, such that multiple KER amplification are performed at one time (e.g., a multiplex reaction). In some embodiments, an aliquot of a KIR amplification reaction is visualized on a polyacrylamide gel, such that one KIR reaction is run in one well of a polyacrylamide gel.
  • aliquots of separate KIR amplification reactions are combined and visualized on a polyacrylamide gel, such that multiple reaction products are run in one well of a polyacrylamide gel for visualization.
  • an aliquot of a KIR multiplex reaction is visualized on a polyacrylamide gel, such that all products of a multiplex reaction are run in one well of a polyacrylamide gel for visualization.
  • Multiplexing of amplification reactions and of the separation process required to visualize a complete spectrum is enabled with the primer set of Table 4.
  • the present invention provides methods and systems for typing the KIR genotype of a donor and/or a recipient blood sample contemplated for use in transplantation therapy.
  • the present invention is used in combination with other diagnostic assays for tissue typing blood products, such as methods and assays found in US Patents 5,256,543, 5,420,013, 5,702,885, 6,670,124, and 5,972,604 (all incorporated herein by reference in their entireties).
  • embodiments of the present invention include determining the compatibility of a donor sample for use by a recipient for transplantation therapy, for example, for leukemic diseases and other blood related disorders where NK cells and or stem cells are used for transplantation.
  • KIR expression has not previously been described in leukemia patients.
  • reduced expression of the majority of KIR families in leukemia patients before transplantation, especially in patients with ALL and NHL was found.
  • the present invention provides a diagnostic in determining relapse of a patient following HSCT.
  • KIR gene expression was determined in healthy donors (in two groups; 1-20 years and 21-51 years of age). Ranges of KIR gene expression in the healthy donors tested are shown in Table 1.
  • the group of 21-51 years of age showed significantly lower mean expression of six KJR families (2DLl, 2DlA, 2DSl, 2DS3, 3DSl, 3DL2) than the younger group.
  • KJR families Four of those KIR families were aKIR.
  • the overall KIR expression decreased as age increased.
  • a statistically significant inverse correlation between age and expression was seen in 2DLl, 2DL4, 2DS3 and 3DSl.
  • KIR spectratyping profiles of healthy donors were compared with their KIR phenotypes as determined by flow cytometry. There was no qualitative difference between these phenotypes and the KLR gene expression profiles.
  • KJR repertoire in pediatric leukemic patients (acute lymphoblastic leukemia (ALL), 18; acute myelogenous leukemia (AML), 9; chronic myelogenous leukemia (CML), 8; non-Hodgkin lymphoma (NHL), 2) before allo-HSCT was examined.
  • ALL acute lymphoblastic leukemia
  • AML acute myelogenous leukemia
  • CML chronic myelogenous leukemia
  • NHL non-Hodgkin lymphoma
  • KIR genotypes in the healthy population has been broadly studied and 37 KIR haplotypes have been described (Car ⁇ ngton and Martin, 2006) However, the KTR expression pattern in the healthy population has not been reported In developing embodiments of the present invention, 32 catego ⁇ es of KIR expression patterns in healthy donors were found, suggesting a wide va ⁇ ation in KIR expression in healthy individuals. Similarly, is it was found that of the KIR genotypes, 2DLl, 2DL2, 2DL3, 2DL4, 2DS2, 2DS4, 3DLl, and 3DL2 were expressed frequently. However, three aKIRs (2DS3, 2DS5 and 3DS 1) were expressed less frequently in adult donors. An overall inverse correlation was found between KIR gene expression and age that affected mainly the activating KIR families.
  • aKTR expression declines quantitatively as well as qualitatively with age.
  • the present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nonetheless, it is contemplated that that this decline may delay activation of the NK killing function.
  • the overall quantity of iKIR is higher than that of aKIR in the healthy population, suggesting the dominance of the self-protective iKIR function. Subjects and transplant regimens
  • KIR gene expression was examined in 50 healthy sibling donors whose ages ranged from 1 to 20 years matching the patient age range. Thirty-seven healthy adult donors 21 to 51 years of age were also studied. A total of 126 samples were obtained from 37 patients pre- transplantation and 12 donor-recipient pairs during stem cell processing and post-transplant. All 12 patients received CD3 depleted haplo-HSCT. Six of these patients were conditioned with a reduced-intensity regimen comprising fludarabine, thiotepa, melphalan and OKT3 without total-body irradiation (TBI) and antithymocyte globulin (ATG). Mycophenolate mofetil was used for GvHD prophylaxis.
  • RNA (l ⁇ g) was purified from IO 6 Ficoll-enriched peripheral blood mononuclear cells (PBMNCs) by using the RNeasy Mini Kit (QIAgen Inc, Valencia, CA).
  • cDNA Complementary DNA
  • cDNA Complementary DNA
  • Superscript II reverse transcriptase and random hexamer primers (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.
  • PCR was performed in a volume of 25 ⁇ l containing 1/200 of the cDNA (corresponding to approximately 5ng original RNA), IX AmpliTaq Gold Master Mix (Applied Biosystems, Branchburg, New Jersey), and 25OnM of 1 of 14 specific reverse (RV) primers combined with 1 of 2 forward (FW) primers conjugated to the fluorescent dye 6-FAM (Table 4).
  • the 2DFW-FAM primer (SEQ ID NO. 15) was used with the RV primers 2DLl RV (SEQ ID NO. 1), 2DL2RV (SEQ ID NO.
  • 2DL3RV SEQ ID NO. 3
  • 2DS IRV SEQ ID NO. 6
  • 2DS2RV SEQ ID NO. 7
  • 2DS3RV SEQ ID NO. 8
  • 2DS4RV SEQ ID NO. 9
  • 2DS5RV SEQ ID NO.10
  • the 2DL4-3DFW-FAM primer SEQ ID NO. 16 was used with RV primers 2DL4RV (SEQ ID NO. 4), 2DL5RV (SEQ ID NO. 5), 3DL1RV (SEQ ID NO. 11), 3DL2RV (SEQ ID NO. 12), 3DL3RV (SEQ ID NO. 13) and 3DS1RV (SEQ ID NO. 14).
  • the CD56-mRNA-encoding marker was used as an endogenous control to normalize for input RNA and to calculate the percentile of each KIR family in the total CD56 transcripts (using forward amplification primerCD56W-FAM (SEQ ID NO. 17) and reverse amplification primer CD56RV (SEQ ID NO. 18)).
  • the PCR conditions were 95°C for 6 min followed by 35 cycles of 95 0 C for 15 sec, 58 °C for 60 sec. and a final extension step of 72 0 C for 5 min.
  • PCR products (4 ⁇ l) were diluted in 1 O ⁇ l of 1 X TE buffer and mixed with 20 ⁇ l of formamide/size standard mixture (20 ⁇ l 500LIZ size standard per ml of formamide).
  • the final mixture was denatured at 95 0 C for 7 min and chilled to 4 0 C for more than 2 min before analysis on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). Data were collected and analyzed by Genemapper Software version 3.7. All samples were measured in duplicate PCR reactions.
  • the percent expression of each KIR gene was calculated as follows:
  • Peripheral blood was drawn in VACUTAINERTM tubes containing trisodium citrate, citric acid and dextrose (ACD).
  • the KIR phenotype was determined by using a 6- COLOR BDTM LSRII Flow Cytometer (Becton Dickinson, San Jose, CA).
  • Whole blood was incubated with the monoclonal antibodies and red cells were lysed with an ammonium chloride-based reagent.
  • the monoclonal antibodies used were anti-CD45- FITC (fluorescein isothiocyanate), anti-CD3-ECD (Phycoerythrin-Texas Red), anti-CD5- APC (allophycocyanin), anti-CD 14- APC-Cy7-A, anti-CD 158a,h-(EB6)-PE
  • the CD14-negative population (equivalent to the lymphocyte population) was selected and gated for the CD56 positive/CD3 negative population.
  • This cell population was analyzed on the basis of KIR 2DLl (Clone EB6) positive vs. KIR 2DL2/3 (Clone CH-L) negative; KIR3DL1 (Clone DX9) negative; KIR 3DLl (Clone DX9) positive vs. KJR 2DL2/3 (Clone GL 183) negative; KIR 2DLl (Clone EB6) negative; KJR 2DL2/3 (CH-L) positive vs. KIR 2DLl (Clone EB6) negative; IQR 3DLl (Clone DX9) negative. Results were calculated as absolute cell numbers. Data were analyzed by using FACS DIVA software or FLOWJO software. Quantitative analysis of chimerism
  • PCR products generated by each pair of FW and RV primers were sequenced.
  • PCR product 50 ⁇ l was purified by using a QIAquick PCR purification kit (QIAGEN, Maryland USA) and was sequenced by using BIG DYE® Terminator (v3.1 ) Chemistry on an ABI Prism 3730XL DNA analyzer.
  • KIR expression levels were compared by using the Mann Whitney U test. Fisher's exact test was used to compare the frequency of KIR expression. Univariate regression models were used to assess the relationship between KER expression levels and age. In the model, each category of KTR expression level was used as a dependent variable and age was used as the independent variable.

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La présente invention porte sur des procédés, des dosages et des compositions pour établir le génotype des cellules tueuses naturelles (NK) par l'expression des récepteurs de la famille des immunoglobulines (KIR). En particulier, la présente invention porte sur le profilage de l'expression de KIR, par exemple, dans des échantillons provenant de donneurs et de patients, dont les résultats sont utiles dans des thérapies de transplantation mettant en jeu les cellules NK.
PCT/US2008/011671 2007-10-12 2008-10-10 Dosage des récepteurs de la famille des immunoglobulines de tueurs naturels (kir) WO2009051672A2 (fr)

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CN102575292A (zh) * 2009-09-22 2012-07-11 霍夫曼-拉罗奇有限公司 与疾病相关的kir单元型的测定
EP3385395A1 (fr) 2015-08-17 2018-10-10 Kura Oncology, Inc. Méthodes de traitement de patients cancéreux à l'aide d'inhibiteurs de farnésyltransférases
EP3519581A4 (fr) * 2016-10-01 2020-09-09 Memorial Sloan-Kettering Cancer Center Procédés et kits de typage d'allèles kir2dl
CN112442525A (zh) * 2020-11-20 2021-03-05 江苏伟禾生物科技有限公司 用于检测人类自然杀伤细胞免疫球蛋白样受体kir基因分型的试剂盒
CN112725428A (zh) * 2021-02-22 2021-04-30 深圳荻硕贝肯精准医学有限公司 Kir2dl4基因分型试剂盒和分型方法

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102575292A (zh) * 2009-09-22 2012-07-11 霍夫曼-拉罗奇有限公司 与疾病相关的kir单元型的测定
US9005894B2 (en) 2009-09-22 2015-04-14 Roche Molecular Systems, Inc. Determination of KIR haplotypes associated with disease
CN102575292B (zh) * 2009-09-22 2015-07-29 霍夫曼-拉罗奇有限公司 与疾病相关的kir单元型的测定
US9914970B2 (en) 2009-09-22 2018-03-13 Roche Molecular Systems, Inc. Determination of KIR haplotypes associated with disease
EP3385395A1 (fr) 2015-08-17 2018-10-10 Kura Oncology, Inc. Méthodes de traitement de patients cancéreux à l'aide d'inhibiteurs de farnésyltransférases
EP3640345A1 (fr) 2015-08-17 2020-04-22 Kura Oncology, Inc. Méthodes de traitement de patients cancéreux à l'aide d'inhibiteurs de farnésyltransférases
EP3995589A1 (fr) 2015-08-17 2022-05-11 Kura Oncology, Inc. Méthodes de traitement de patients cancéreux à l'aide d'inhibiteurs de farnésyltransférases
EP3519581A4 (fr) * 2016-10-01 2020-09-09 Memorial Sloan-Kettering Cancer Center Procédés et kits de typage d'allèles kir2dl
US11453908B2 (en) 2016-10-01 2022-09-27 Memorial Sloan Kettering Cancer Center Methods and kits for typing KIR2DL alleles
CN112442525A (zh) * 2020-11-20 2021-03-05 江苏伟禾生物科技有限公司 用于检测人类自然杀伤细胞免疫球蛋白样受体kir基因分型的试剂盒
CN112442525B (zh) * 2020-11-20 2022-11-04 江苏伟禾生物科技有限公司 用于检测人类自然杀伤细胞免疫球蛋白样受体kir基因分型的试剂盒
CN112725428A (zh) * 2021-02-22 2021-04-30 深圳荻硕贝肯精准医学有限公司 Kir2dl4基因分型试剂盒和分型方法

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