Attorney Docket No. AGS-013C2
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`In an embodiment, the cell includes a heterologous copy of a mutant IDH gene,
`
`e.g., a mutant IDHl or IDH2 gene. (Heterologous copy refers to a copy introduced or
`
`formed by a genetic engineering manipulation.)
`
`In an embodiment, the cell is transfected (e.g., transiently or stably transfected)
`
`or transduced (e.g., transiently or stably transduced) with a nucleic acid sequence
`
`encoding an IDH, e.g., IDHl or IDH2, described herein, e.g., an IDHl having other
`
`than an Arg at residue 132. In an embodiment, the IDH, e.g., IDHl or IDH2, is
`
`epitope-tagged, e.g., myc-tagged.
`
`In an embodiment, the cell, e.g., a cancer cell, is non-mutant or wild type for
`
`the IDH, e.g., IDHl or IDH2, allele. The cell can include a heterologous IDHl or
`
`IDH2 mutant.
`
`In an embodiment, the cell is a cultured cell, e.g., a primary cell, a secondary
`
`cell, or a cell line. In an embodiment, the cell is a cancer cell, e.g., a glioma cell (e.g.,
`
`a glioblastoma cell), a prostate cancer cell, a leukemia cell (e.g., an ALL, e.g., B-ALL
`
`or T-ALL, cell or AML cell) or a cell characterized by myelodysplasia or
`
`myelodysplastic syndrome. In embodiment, the cell is a 293T cell, a U87MG cell, or
`
`an LN-18 cell (e.g., ATCC HTB-14 or CRL-2610).
`
`In an embodiment, the cell is from a subject, e.g., a subject having cancer, e.g.,
`
`a cancer characterized by an IDH, e.g., IDHl or IDH2, allele described herein, e.g., an
`
`IDHl allele having His, Ser, Cys, Gly, Val, Pro or Leu at residue 132 (SEQ ID NO:8);
`
`specifically His or Cys; or an IDH2 allele having Lys, Gly, Met, Trp, Thr, or Ser at
`
`residue 172 (SEQ ID NO: 10), specifically Lys, Gly, Met, Trp, or Ser.
`
`In an embodiment, the evaluating step comprises evaluating the presence
`
`and/or amount of an alpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG, e.g.,
`
`in the cell lysate or culture medium, e.g., by LC-MS.
`
`In an embodiment, the evaluating step comprises evaluating the presence
`
`and/or amount of an alpha hydroxy neoactivity, e.g., 2HG neoactivity, in the cell
`
`lysate or culture medium.
`
`In an embodiment, the method further comprises evaluating the
`
`presence/amount one or more of TCA metabolite(s), e.g., citrate, a-KG, succinate,
`
`fumarate, and/or malate, e.g., by LC-MS, e.g., as a control.
`
`In an embodiment, the method further comprises evaluating the oxidation state
`
`of NADPH, e.g., the absorbance at 340 nm, e.g., by spectrophotometer.
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`In an embodiment, the method further comprises evaluating the ability of the
`
`candidate compound to inhibit a second enzymatic activity, e.g., the forward reaction
`
`of non-mutant or wild type enzyme activity, e.g., in the case of IDHl or IDH2 (e.g.,
`
`IDHl), the conversion of isocitrate to a-ketoglutarate ( or an intermediate thereof,
`
`including the reduced hydroxyl intermediate).
`
`In an embodiment, the candidate compound is a small molecule, a polypeptide,
`
`peptide, a carbohydrate based molecule, or an aptamer (e.g., a nucleic acid aptamer,
`
`or a peptide aptamer). The method can be used broadly and can, e.g., be used as one
`
`or more of a primary screen, to confirm candidates produced by this or other methods
`
`or screens, or generally to guide drug discovery or drug candidate optimization.
`
`In an embodiment, the method comprises evaluating, e.g., confirming, the
`
`ability of a candidate compound (e.g., a candidate compound which meets a
`
`predetermined level of inhibition in the evaluating step) to inhibit the neoactivity or
`
`proxy activity in a second assay.
`
`In an embodiment, the second assay comprises repeating one or more of the
`
`contacting and/or evaluating step(s) of the basic method.
`
`In another embodiment, the second assay is different from the first. E.g.,
`
`where the first assay can use a cell or cell lysate or other non-whole animal model the
`
`second assay can use an animal model, e.g., a tumor transplant model, e.g., a mouse
`
`having an IDH, e.g., IDHl or IDH2, mutant cell or tumor transplanted in it. E.g., a
`
`U87 cell, or glioma, e.g., glioblastoma, cell, harboring a transfected IDH, e.g., IDHl
`
`or IDH2, neoactive mutant can be implanted as a xenograft and used in an assay.
`
`Primary human glioma or AML tumor cells can be grafted into mice to allow
`
`propagation of the tumor and used in an assay. A genetically engineered mouse
`
`model (GEMM) harboring an IDHl or IDH2 mutation and/or other mutation, e.g., a
`
`p53 null mutation, can also be used in an assay.
`
`In an embodiment the method comprises:
`
`optionally supplying the candidate compound;
`
`contacting the candidate compound with a cell comprising a nucleic acid
`
`sequence, e.g., a heterologous sequence, encoding an IDHl having other than an Arg
`
`at residue 132 (e.g., IDH1R132H) or an IDH2 having other than an Arg at residue 172
`
`(specifically an IDHl having other than an Arg at residue 132); and
`
`evaluating the presence and/or amount of an alpha hydroxy neoactivity
`
`product, e.g., 2HG, e.g., R-2HG, in the cell lysate or culture medium, by LC-MS,
`
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`thereby evaluating the compound.
`
`In an embodiment the result of the evaluation is compared with a reference,
`
`e.g., the level of product, e.g., an alpha hydroxy neoactivity product, e.g., 2HG. e.g.,
`
`R-2HG, in a control cell, e.g., a cell having inserted therein a wild type or non-mutant
`
`copy of IDHl or IDH2 (e.g., IDHl).
`
`In another aspect, the invention features, a method of evaluating a candidate
`
`compound, e.g., for the ability to inhibit an RNA encoding a mutant enzyme having a
`
`neoactivity, e.g., for use as an anti-proliferative or anti-cancer agent. In an
`
`embodiment the mutant enzyme is an IDH, e.g., an IDHl or IDH2 mutant, e.g., a
`
`mutant described herein. In an embodiment the neaoctivity is alpha hydroxy
`
`neoactivity, e.g., 2HG neoactivity. The method comprises:
`
`optionally supplying the candidate compound, e.g., a nucleic acid based
`
`inhibitor (e.g., a dsRNA (e.g., siRNA or shRNA), an antisense, or a microRNA);
`
`contacting the candidate compound with an RNA, e.g., an mRNA, which
`
`encodes IDH, e.g., an IDHl or IDH2, e.g., an RNA that encode mutant enzyme
`
`having a neoactivity (or with a cell or cell lysate comprising the same); and
`
`evaluating the ability of the candidate compound to inhibit the RNA,
`
`thereby evaluating the candidate compound. By inhibit the RNA means, e.g., to
`
`cleave or otherwise inactivate the RNA.
`
`In an embodiment the RNA encodes a fusion of all or part of the IDH, e.g.,
`
`IDHl or IDH2, wildtype or mutant protein to a second protein, e.g., a reporter protein,
`
`e.g., a fluorescent protein, e.g., a green or red fluorescent protein.
`
`In an embodiment the mutant enzyme is a mutant IDHl, e.g., an IDHl mutant
`
`described herein, and the neoactivity is an alpha hydroxy neoactivity, e.g., 2HG
`
`neoactivity.
`
`In an embodiment the mutant enzyme is a mutant IDH2, e.g., an IDH2 mutant
`
`described herein, and the neoactivity is an alpha hydroxy neoactivity, e.g., 2HG
`
`neoactivity.
`
`In an embodiment, the contacting step comprises contacting the candidate
`
`compound with a cell, or a cell lysate thereof, wherein the cell comprises RNA
`
`encoding IDH, e.g., IDHl or IDH2, e.g., a mutant IDH, e.g., IDHl or IDH2, enzyme
`
`having the neoactivity.
`
`In an embodiment, the cell comprises a mutation, or preselected allele, of a
`
`mutant IDHl gene. E.g., in an embodiment, the IDHl allele encodes an IDHl having
`
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`other than an Arg at residue 132. E.g., the allele can encode His, Ser, Cys, Gly, Val,
`
`Pro or Leu, or any other residue described in Yan et al., at residue 132, according to
`
`the sequence of SEQ ID NO:8 (see also FIG. 21), specifically His, Ser, Cys, Gly, Val,
`
`or Leu.
`
`In an embodiment the allele encodes an IDHl having His at residue 132.
`
`In an embodiment the allele encodes an IDHl having Ser at residue 132.
`
`In an embodiment the allele is an Arg132His mutation, or an Arg132Ser
`
`mutation, according to the sequence of SEQ ID NO:8 (see FIGs. 2 and 21).
`
`In an embodiment, the cell comprises a mutation, or preselected allele, of a
`
`mutant IDH2 gene. E.g., in an embodiment, the IDH2 allele encodes an IDH2 having
`
`other than an Arg at residue 172. E.g., the allele encodes Lys, Gly, Met, Trp, Thr, Ser,
`
`or any residue described in described in Yan et al., at residue 172, according to the
`
`sequence of SEQ ID NO:lO(see also Fig. 22), specifically Lys, Gly, Met, Trp or Ser.
`
`In an embodiment the allele encodes an IDH2 having Lys at residue 172. In an
`
`embodiment the allele encodes an IDH2 having Met at residue 172.
`
`In an embodiment, the cell includes a heterologous copy of a wildtype or
`
`mutant IDH gene, e.g., a wildtype or mutant IDHl or IDH2 gene. (Heterologous copy
`
`refers to a copy introduced or formed by a genetic engineering manipulation.) In an
`
`embodiment the heterologous gene comprises a fusion to a reporter protein, e.g., a
`
`fluorescent protein, e.g., a green or red fluorescent protein.
`
`In an embodiment, the cell is transfected (e.g., transiently or stably transfected)
`
`or transduced (e.g., transiently or stably transduced) with a nucleic acid sequence
`
`encoding an IDH, e.g., IDHl or IDH2, described herein, e.g., an IDHl having other
`
`than an Arg at residue 132 or an IDH2 having other than an Arg at residue 172 (e.g.,
`
`an IDHl having other than an Arg at residue 132). In an embodiment, the IDH, e.g.,
`
`IDHl or IDH2, is epitope-tagged, e.g., myc-tagged.
`
`In an embodiment, the cell, e.g., a cancer cell, is non-mutant or wild type for
`
`the IDH, e.g., IDHl or IDH2, allele. The cell can include a heterologous IDHl or
`
`IDH2 mutant.
`
`In an embodiment, the cell is a cultured cell, e.g., a primary cell, a secondary
`
`cell, or a cell line. In an embodiment, the cell is a cancer cell, e.g., a glioma cell (e.g.,
`
`a glioblastoma cell), a prostate cancer cell, a leukemia cell (e.g., an ALL, e.g., B-ALL
`
`or T-ALL cell or AML cell) or a cell characterized by myelodysplasia or
`
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`myelodysplastic syndrome. In embodiment, the cell is a 293T cell, a U87MG cell, or
`
`an LN-18 cell (e.g., ATCC HTB-14 or CRL-2610).
`
`In an embodiment, the cell is from a subject, e.g., a subject having cancer, e.g.,
`
`a cancer characterized by an IDH, e.g., IDHl or IDH2, allele described herein, e.g., an
`
`IDHl allele having His, Ser, Cys, Gly, Val, Pro or Leu at residue 132 (SEQ ID NO:8);
`
`specifically His or Cys. In an embodiment, the cancer is characterized by an IDH2
`
`allele having Lys, Gly, Met, Trp, Tor, or Ser at residue 172 (SEQ ID NO: 10),
`
`specifically Lys, Gly, Met, Trp, or Ser.
`
`In an embodiment, the method comprises a second assay and the second assay
`
`comprises repeating one or more of the contacting and/or evaluating step(s) of the
`
`basic method.
`
`In another embodiment, the second assay is different from the first. E.g.,
`
`where the first assay can use a cell or cell lysate or other non-whole animal model the
`
`second assay can use an animal model
`
`In an embodiment the efficacy of the candidate is evaluated by its effect on
`
`reporter protein activity.
`
`In another aspect, the invention features, a method of evaluating a candidate
`
`compound, e.g., for the ability to inhibit transcription of an RNA encoding a mutant
`
`enzyme having a neoactivity, e.g., for use as an anti-proliferative or anti-cancer agent.
`
`In an embodiment the mutant enzyme is an IDH, e.g., an IDHl or IDH2 mutant, e.g.,
`
`a mutant described herein. In an embodiment the neaoctivity is alpha hydroxy
`
`neoactivity, e.g., 2HG neoactivity. The method comprises:
`
`optionally supplying the candidate compound, e.g., a small molecule,
`
`polypeptide, peptide, aptomer, a carbohydrate-based molecule or nucleic acid based
`
`molecule;
`
`contacting the candidate compound with a system comprising a cell or cell
`
`lysate; and
`
`evaluating the ability of the candidate compound to inhibit the translation of
`
`IDH, e.g., IDHl or IDH2, RNA, e.g,
`
`thereby evaluating the candidate compound.
`
`In an embodiment the the system comprises a fusion gene encoding of all or
`
`part of the IDH, e.g., IDHl or IDH2, wildtype or mutant protein to a second protein,
`
`e.g., a reporter protein, e.g., a fluorescent protein, e.g., a green or red fluorescent
`
`protein.
`
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`In an embodiment the mutant enzyme is a mutant IDHl, e.g., an IDHl mutant
`
`described herein, and the neoactivity is alpha hydroxy neoactivity, e.g., 2HG
`
`neoactivity.
`
`In an embodiment the mutant enzyme is a mutant IDH2, e.g., an IDH2 mutant
`
`described herein, and the neoactivity is alpha hydroxy neoactivity, e.g., 2HG
`
`neoactivity.
`
`In an embodiment, the system includes a heterologous copy of a wildtype or
`
`mutant IDH gene, e.g., a wildtype or mutant IDHl or IDH2 gene. (Heterologous copy
`
`refers to a copy introduced or formed by a genetic engineering manipulation.) In an
`
`embodiment the heterologous gene comprises a fusion to a reporter protein, e.g., a
`
`fluorescent protein, e.g., a green or red fluorescent protein.
`
`In an embodiment the cell, e.g., a cancer cell, is non-mutant or wild type for
`
`the IDH, e.g., IDHl or IDH2, allele. The cell can include a heterologous IDHl or
`
`IDH2 mutant.
`
`In an embodiment, the cell is a cultured cell, e.g., a primary cell, a secondary
`
`cell, or a cell line. In an embodiment, the cell is a cancer cell, e.g., a glioma cell (e.g.,
`
`a glioblastoma cell), a prostate cancer cell, a leukemia cell (e.g., an ALL, e.g., B-ALL
`
`or T-ALL, cell or AML cell) or a cell characterized by myelodysplasia or
`
`myelodysplastic syndrome. In embodiment, the cell is a 293T cell, a U87MG cell, or
`
`an LN-18 cell (e.g., ATCC HTB-14 or CRL-2610).
`
`In an embodiment, the cell is from a subject, e.g., a subject having cancer,
`
`e.g., a cancer characterized by an IDH, e.g., IDHl or IDH2, allele described herein,
`
`e.g., an IDHl allele having His, Ser, Cys, Gly, Val, Pro or Leu at residue 132 (SEQ
`
`ID NO:8); specifically His, Ser, Cys, Gly, Val, or Leu. In an embodiment, the cancer
`
`is characterized an IDH2 allele having Lys, Gly, Met, Trp, Thr, or Ser at residue 172
`
`(SEQ ID NO: 10).
`
`In an embodiment, the method comprises a second assay and the second assay
`
`comprises comprises repeating the method.
`
`In another embodiment, the second assay is different from the first. E.g.,
`
`where the first assay can use a cell or cell lysate or other non-whole animal model the
`
`second assay can use an animal model.
`
`In an embodiment the efficacy of the candidate is evaluated by its effect on
`
`reporter protein activity.
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`In another aspect, the invention features, a method of evaluating a candidate
`
`compound, e.g., a therapeutic agent, or inhibitor, described herein in an animal model.
`
`The candidate compound can be, e.g., a small molecule, polypeptide, peptide, aptomer,
`
`a carbohydrate-based molecule or nucleic acid based molecule. The method
`
`comprises, contacting the candidate with the animal model and evaluating the animal
`
`model.
`
`In an embodiment evaluating comprises;
`
`determining an effect of the compound on the general health of the animal;
`
`determining an effect of the compound on the weight of the animal;
`
`determining an effect of the compound on liver function, e.g, on a liver
`
`enzyme;
`
`determining an effect of the compound on the cardiovascular system of the
`
`animal;
`
`determining an effect of the compound on neurofunction, e.g., on
`
`neuromuscular control or response;
`
`determining an effect tof the compound on eating or drinking;
`
`determining the distribution of the compound in the animal;
`
`determining the persistence of the compound in the animal or in a tissue or
`
`oragn of the animal, e.g., determining plasma half-life; or
`
`determining an effect of the compound on a selected cell in the animal;
`
`determining an effect of the compound on the growth, size, weight,
`
`invasiveness or other phenotype of a tumor, e.g., an endogenous tumor or a tumor
`
`arising from introduction of cells from the same or a different species.
`
`In an embodiment the animal is a non-human primate, e.g., a cynomolgus
`
`monkey or chimpanzee.
`
`In an embodiment the animal is a rodent, e.g., a rat or mouse.
`
`In an embodiment the animal is a large animal, e.g., a dog or pig, other than a
`
`non-human primate.
`
`In an embodiment the evaluation is memorialized and optionally transmetted
`
`to another party.
`
`In one aspect, the invention provides, a method of evaluating or processing a
`
`therapeutic agent, e.g., a therapeutic agent referred to herein, e.g., a therapeutic agent
`
`that results in a lowering of the level of a product of an IDH, e.g., IDHl or IDH2,
`
`mutant having a neoactivity. In an embodiment the neoactivity is an alpha hydroxy
`
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`neoactivity, e.g., 2HG neoactivity, and the level of an alpha hydroxy neoactivity
`
`product, e.g., 2HG, e.g., R-2HG, is lowered.
`
`The method includes:
`
`providing, e.g., by testing a sample, a value (e.g., a test value) for a parameter
`
`related to a property of the therapeutic agent, e.g., the ability to inhibit the conversion
`
`of alpha ketoglutarate to 2 hydroxyglutarate (i.e., 2HG), e.g., R-2 hydroxyglutarate
`
`(i.e., R-2HG), and,
`
`optionally, providing a determination of whether the value determined for the
`
`parameter meets a preselected criterion, e.g., is present, or is present within a
`
`preselected range,
`
`thereby evaluating or processing the therapeutic agent.
`
`In an embodiment the therapeutic agent is approved for use in humans by a
`
`government agency, e.g., the FDA.
`
`In an embodiment the parameter is correlated to the ability to inhibit 2HG
`
`neoactivity, and, e.g., the therapeutic agent is an inhibitor which binds to IDHl or
`
`IDH2 protein and reduces an alpha hydroxy neoactivity, e.g., 2HG neoactivity.
`
`In an embodiment the parameter is correlated to the level of mutant IDH, e.g.,
`
`IDHl or IDH2, protein, and, e.g., the therapeutic agent is an inhibitor which reduces
`
`the level of IDHl or IDH2 mutant protein.
`
`In an embodiment the parameter is correlated to the level of an RNA that
`
`encodes a mutant IDH, e.g., IDHl or IDH2, protein, and, e.g., the therapeutic agent
`
`reduces the level of RNA, e.g., mRNA, that encodes IDHl or IDH2 mutant protein.
`
`In an embodiment the method includes contacting the therapeutic agent with a
`
`mutant IDH, e.g., IDHl or IDH2, protein (or corresponding RNA).
`
`In an embodiment, the method includes providing a comparison of the value
`
`determined for a parameter with a reference value or values, to thereby evaluate the
`
`therapeutic agent. In an embodiment, the comparison includes determining if a test
`
`value determined for the therapeutic agent has a preselected relationship with the
`
`reference value, e.g., determining if it meets the reference value. The value need not
`
`be a numerical value but, e.g., can be merely an indication of whether an activity is
`
`present.
`
`In an embodiment the method includes determining if a test value is equal to
`
`or greater than a reference value, if it is less than or equal to a reference value, or if it
`
`falls within a range (either inclusive or exclusive of one or both endpoints). In an
`
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`embodiment, the test value, or an indication of whether the preselected criterion is
`
`met, can be memorialized, e.g., in a computer readable record.
`
`In an embodiment, a decision or step is taken, e.g., a sample containing the
`
`therapeutic agent, or a batch of the therapeutic agent, is classified, selected, accepted
`
`or discarded, released or withheld, processed into a drug product, shipped, moved to a
`
`different location, formulated, labeled, packaged, contacted with, or put into, a
`
`container, e.g., a gas or liquid tight container, released into commerce, or sold or
`
`offered for sale, or a record made or altered to reflect the determination, depending on
`
`whether the preselected criterion is met. E.g., based on the result of the determination
`
`or whether an activity is present, or upon comparison to a reference standard, the
`
`batch from which the sample is taken can be processed, e.g., as just described.
`
`The evaluation of the presence or level of activity can show if the therapeutic
`
`agent meets a reference standard.
`
`In an embodiment, methods and compositions disclosed herein are useful from
`
`a process standpoint, e.g., to monitor or ensure batch-to-batch consistency or quality,
`
`or to evaluate a sample with regard to a reference, e.g., a preselected value.
`
`In an embodiment, the method can be used to determine if a test batch of a
`
`therapeutic agent can be expected to have one or more of the properties. Such
`
`properties can include a property listed on the product insert of a therapeutic agent, a
`
`property appearing in a compendium, e.g., the US Pharmacopea, or a property
`
`required by a regulatory agency, e.g., the FDA, for commercial use.
`
`In an embodiment the method includes testing the therapeutic agent for its
`
`effect on the wildtype activity of an IDH, e.g., IDHl or IDH2, protein, and providing
`
`a determination of whether the value determined meets a preselected criterion, e.g., is
`
`present, or is present within a preselected range.
`
`In an embodiment the method includes:
`
`contacting a therapeutic agent that is an inhibitor of IDHl an alpha hydroxy
`
`neoactivity, e.g., 2HG neoactivity, with an IDHl mutant having an alpha hydroxy
`
`neoactivity, e.g., 2HG neoactivity,
`
`determining a value related to the inhibition of an alpha hydroxy neoactivity,
`
`e.g., 2HG neoactivity, and
`
`comparing the value determined with a reference value, e.g., a range of values,
`
`for the inhibition of an alpha hydroxy neoactivity, e.g., 2HG neoactivity. In an
`
`embodiment the reference value is an FDA required value, e.g., a release criteria.
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`In an embodiment the method includes:
`
`contacting a therapeutic agent that is an inhibitor of mRNA which encodes a
`
`mutant IDHl having an alpha hydroxy neoactivity, e.g., 2HG neoactivity, with an
`
`mRNA that encodes an IDHl mutant having an alpha hydroxy neoactivity, e.g., 2HG
`
`neoactivity,
`
`determining a value related to the inhibition of the mRNA, and,
`
`comparing the value determined with a reference value, e.g., a range of values
`
`for inhibition of the mRNA. In an embodiment the reference value is an FDA
`
`required value, e.g., a release criteria.
`
`In one aspect, the invention features a method of evaluating a sample of a
`
`therapeutic agent, e.g., a therapeutic agent referred to herein, that includes receiving
`
`data with regard to an activity of the therapeutic agent; providing a record which
`
`includes said data and optionally includes an identifier for a batch of therapeutic agent;
`
`submitting said record to a decision-maker, e.g., a government agency, e.g., the FDA;
`
`optionally, receiving a communication from said decision maker; optionally, deciding
`
`whether to release market the batch of therapeutic agent based on the communication
`
`from the decision maker. In one embodiment, the method further includes releasing,
`
`or other wise processing, e.g., as described herein, the sample.
`
`In another aspect, the invention features, a method of selecting a payment
`
`class for treatment with a therapeutic agent described herein, e.g., an inhibitor of IDH,
`
`e.g., IDHl or IDH2, neoactivity, for a subject having a cell proliferation-related
`
`disorder. The method includes:
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`providing (e.g., receiving) an evaluation of whether the subject is positive
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`for increased levels of an alpha hydroxy neoactivity product, e.g., 2HG, e.g., R-2HG,
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`or neoactivity, e.g., an alpha hydroxy neoactivity, e.g., 2HG neoactivity, a mutant
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`IDHl or IDH2 having neoactivity, e.g., an alpha hydroxy neoactivity, e.g., 2HG
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`neoactivity, (or a corresponding RNA), or a mutant IDH, e.g., IDHl or IDH2, somatic
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`gene, e.g., a mutant described herein, and
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`performing at least one of (1) if the subject is positive selecting a first
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`payment class, and (2) if the subject is a not positive selecting a second payment
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`class.
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`system.
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`In an embodiment the selection is memorialized, e.g., in a medical records
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`In an embodiment the method includes evaluation of whether the subject is
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`positive for increased levels of an alpha hydroxy neoactivity product, e.g., 2HG, e.g.,
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`R-2HG, or neoactivity, e.g., an alpha hydroxy neoactivity, e.g., 2HG neoactivity.
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`In an embodiment the method includes requesting the evaluation.
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`In an embodiment the evaluation is performed on the subject by a method
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`described herein.
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`In an embodiment, the method comprises communicating the selection to
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`another party, e.g., by computer, compact disc, telephone, facsimile, email, or letter.
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`In an embodiment, the method comprises making or authorizing payment for
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`said treatment.
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`In an embodiment, payment is by a first party to a second party. In some
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`embodiments, the first party is other than the subject. In some embodiments, the first
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`party is selected from a third party payor, an insurance company, employer, employer
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`sponsored health plan, HMO, or governmental entity. In some embodiments, the
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`second party is selected from the subject, a healthcare provider, a treating physician,
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`an HMO, a hospital, a governmental entity, or an entity which sells or supplies the
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`drug. In some embodiments, the first party is an insurance company and the second
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`party is selected from the subject, a healthcare provider, a treating physician, an
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`HMO, a hospital, a governmental entity, or an entity which sells or supplies the drug.
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`In some embodiments, the first party is a governmental entity and the second party is
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`selected from the subject, a healthcare provider, a treating physician, an HMO, a
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`hospital, an insurance company, or an entity which sells or supplies the drug.
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`As used herein, a cell proliferation-related disorder is a disorder characterized
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`by unwanted cell proliferation or by a predisposition to lead to unwanted cell
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`proliferation (sometimes referred to as a precancerous disorder). Examples of
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`disorders characterized by unwanted cell proliferation include cancers, e.g., tumors of
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`the CNS, e.g., a glioma. Gliomas include astrocytic tumors, oligodendroglial tumors,
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`oligoastrocytic tumors, anaplastic astrocytomas, and glioblastomas. Other examples
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`include hematological cancers, e.g., a leukemia, e.g., AML (e.g., an adult or pediatric
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`form) or ALL, e.g., B-ALL or T-ALL (e.g., an adult or pediatric form), localized or
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`metastatic prostate cancer, e.g., prostate adenocarcinoma, fibrosarcoma, and
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`paraganglioma; specificallya leukemia, e.g., AML (e.g., an adult or pediatric form) or
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`ALL, e.g., B-ALL or T-ALL (e.g., an adult or pediatric form), localized or metastatic
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`prostate cancer, e.g., prostate adenocarcinoma. Examples of disorders characterized
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`by a predisposition to lead to unwanted cell proliferation include myelodysplasia or
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`myelodysplastic syndrome, which are a diverse collection of hematological conditions
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`marked by ineffective production (or dysplasia) of myeloid blood cells and risk of
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`transformation to AML.
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`As used herein, specifically inhibits a neoactivity (and similar language),
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`means the neoactivity of the mutant enzyme is inhibted to a significantly greater
`
`degree than is the wildtype enzyme activity. By way of example, "specifically
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`inhibits the 2HG neoactivity of mutant IDHl (or IDH2)" means the 2HG neoactivity
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`is inhibited to a significantly greater degree than is the forward reaction (the
`
`conversion of isocitrate to alpha ketoglutarate) of wildtype IDHl ( or IDH2) activity.
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`In embodiments the neactivity is inhibited at least 2, 5, 10, or 100 fold more than the
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`wildtype activity. In embodiments an inhibitor that is specfic for the 2HG neaoctivity
`
`of IDH, e.g., IDHl or IDH2, will also inhibit another dehydrogenase, e.g., malate
`
`dehydrogenase. In other embodiments the specific inhibitor does inhibit other
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`dehydrogenases, e.g., malate dehydrogenase.
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`As used herein, a cell proliferation-related disorder, e.g., a cancer,
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`characterized by a mutation or allele, means a cell proliferation-related disorder
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`having a substantial number of cells which carry that mutation or allele. In an
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`embodiment at least 10, 25, 50, 75, 90, 95 or 99% of the cell proliferation-related
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`disorder cells, e.g., the cells of a cancer, or a representative, average or typical sample
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`of cancer cells, e.g., from a tumor or from affected blood cells, carry at least one copy
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`of the mutation or allele. A cell proliferation-related disorder, characterized by a
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`mutant IDH, e.g., a mutant IDHl or mutant IDH2, having 2HG neoactivity is
`
`exemplary. In an embodiment the mutation or allele is present as a heterozygote at
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`the indicated frequencies.
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`As used herein, a "SNP" is a DNA sequence variation occurring when a single
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`nucleotide (A, T, C, or G) in the genome (or other shared sequence) differs between
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`members of a species (or between paired chromosomes in an individual).
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`As used herein, a subject can be a human or non-human subject. Non-human
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`subjects include non-human primates, rodents, e.g., mice or rats, or other non-human
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`animals.
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`The details of one or more embodiments of the invention are set forth in the
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`description below. Other features, objects, and advantages of the invention will be
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`apparent from the description and the drawings, and from the claims.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`FIG. 1 depicts DNA sequence verification of pET41a-IDH1 and alignment against
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`published IDHl CDS. The sequence of IDHl (CDS) corresponds to SEQ ID NO:5.
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`The sequence of pET41a-IDH1 corresponds to SEQ ID NO:6, and the "consensus"
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`sequence corresponds to SEQ ID NO:7.
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`FIG. 2 depicts DNA sequence verification of R132S and R132H mutants according to
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`the SEQ ID NO:8. The amino acid sequence ofIDHl (SEQ ID NO:8) is provided in
`
`FIG. 21.
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`FIG. 3 depicts separation of wild type IDHl protein on Ni-Sepharose column.
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`FIG. 4 depicts protein analysis of wild type IDHl on SDS gel pre and post Ni column
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`fractionation. T: total protein; I: insoluble fractions; S: soluble fraction; L: sample for
`
`loading on Ni-column. The numbers in the figure indicates the fraction numbers.
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`Fractions #17 - #27 were collected for further purification.
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`FIG. SA depicts separation of wild type IDHl protein through SEC column S-200.
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`FIG. 5B depicts protein analysis of wild type IDHl on SDS gel pre and post S-200
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`column fractionation. M: molecular weight marker; Ni: nickel column fraction prior
`
`to S-200; S200: fraction from SEC column.
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`FIG. 6 depicts separation of mutant R132S protein on Ni-Sepharose column.
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`FIG. 7 depicts protein analysis of mutant R132S on SDS gel pre and post Ni column
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`fractionation. M: protein marker (KDa): 116, 66.2, 45, 35, 25, 18.4, 14.4; T: total cell
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`protein; So: soluble fraction; In: insoluble fraction; Ft: flow through. #3-#7 indicate
`
`the corresponding eluted fraction numbers.
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`FIG. SA depicts separation of mutant R132S protein through SEC column S-200.
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`FIG. 8B depicts protein analysis of mutant R132S on SDS gel post S-200 column
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`fractionation. M: molecular weight marker; R132S: fraction from SEC column.
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`FIG. 9 depicts separation of mutant R132H protein on Ni-Sepharose column.
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`FIG. 10 depicts protein analysis of mutant R132H on SDS gel pre and post Ni column
`
`fractionation. M: protein marker (KDa): 116, 66.2, 45, 35, 25, 18.4, 14.4; T: total cell
`
`protein; So: soluble fraction; In: insoluble fraction; Ft: flow through; #5-#10 indicate
`
`the corresponding eluted fraction numbers; Ni: sample from Ni-Sepharose column,
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`pool #5-#10 together.
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`FIG. llA depicts separation of mutant R132H protein through SEC column S-200.
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`FIG. 11B depicts protein analysis of