Functional cell-based potency assay for measuring biological activity of interleukin 2 (il-2) analogs
AssigneeMerck Sharp & Dohme Corp.
InventorsDANILA G. GIACONE, DAVID P. SANDEN, AARRON WILLINGHAM, JUNMING YIE
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Abstract
A functional cell-based potency assay for measuring the biological activity of IL-2 mutants with biased activity for the IL-2 receptor beta-gamma complex is described. In particular, the present invention relates to a Kit225 cell line that lacks expression of the IL-2 receptor alpha and its use in said functional cell-based potency assay.
Claims27
A method for determining the potency of an interleukin 2 (IL-2) analog biased for the IL-2 receptor beta-gamma (IL-2Rβγ) complex over the IL-2 receptor alpha-beta-gamma (IL-2Rαβγ) complex, comprising:
- (a) providing (i) a cell line that expresses an IL-2Rβγ complex without expression of an IL-2Rαβγ complex and a signal transducer and activator of transcription 5 (STAT5) signaling transduction pathway reporter comprising a STAT5 response element and a promoter linked to an open reading frame encoding a detectable polypeptide, and (ii) serial dilutions of an IL-2 analog biased for the IL-2Rβγ complex;
- (b) contacting each serial dilution of the IL-2 analog biased for the IL-2Rβγ complex with an aliquot of the cell line to provide a plurality of cultures;
- (c) incubating the cultures for a time sufficient to enable expression of the detectable polypeptide over time;
- and
- (d) measuring expression of the detectable polypeptide to determine the potency of the IL-2 analog biased for the IL-2Rβγ complex.
5-10. (canceled)
A method for producing an interleukin 2 (IL-2) analog biased for the IL-2 receptor beta-gamma (IL-2Rβγ) complex over the IL-2 receptor alpha-beta-gamma (IL-2Rαβγ) complex, comprising:
- (a) providing an IL-2 analog capable of binding the IL-2Rαβγ complex;
- (b) substituting one or more amino acids of the IL-2 analog that are at the interface between the IL-2 and the IL-2Rα of the IL-2Rαβγ complex with a natural amino acid or non-natural amino acid to provide an IL-2 analog biased for the IL-2Rβγ complex;
- (c) making a serial dilution of the IL-2 analog biased for the IL-2Rβγ complex;
- (d) contacting each serial dilution of the IL-2 analog biased for the IL-2Rβγ complex with an aliquot of a cell line that expresses (i) the IL-2Rβγ complex without expression of the IL-2Rαβγ complex and (ii) a signal transducer and activator of transcription 5 (STAT5) signaling transduction pathway reporter comprising a STAT5 response element and promoter linked to an open reading frame encoding a detectable polypeptide, to provide a plurality of cultures;
- (e) incubating the cultures for a time sufficient to enable expression of the detectable polypeptide over time;
- (f) measuring expression of the detectable polypeptide to determine the potency of the IL-2 analog biased for the IL-2Rβγ complex;
- and
- (g) isolating the IL-2 analog biased for the IL-2Rβγ complex if the potency of the IL-2 analog biased for the IL-2Rβγ complex has a potency within a predetermined range.
14-18. (canceled)
A manufacturing process for producing a production batch of an interleukin 2 (IL-2) analog biased for the IL-2 receptor beta-gamma (IL-2Rβγ) complex comprising the steps of:
- (a) synthesizing the IL-2 analog biased for the IL-2Rβγ complex;
- (b) purifying the IL-2 analog biased for the IL-2Rβγ complex;
- (c) formulating the IL-2 analog biased for the IL-2Rβγ complex into a production batch;
- (d) obtaining a sample of the IL-2 analog biased for the IL-2Rβγ from the production batch;
- (e) making a serial dilution of the IL-2 analog biased for the IL-2Rβγ complex;
- (f) contacting each serial dilution of the IL-2 analog biased for the IL-2Rβγ complex with an aliquot of a cell line that expresses (i) the IL-2Rβγ complex without expression of the IL-2Rαβγ complex and (ii) a signal transducer and activator of transcription 5 (STAT5) signaling transduction pathway reporter, which comprises a STAT5 response element and promoter linked to an open reading frame encoding a detectable polypeptide, to provide a plurality of cultures;
- (g) incubating the cultures for a time sufficient for expression of the detectable polypeptide over time;
- and
- (h) measuring expression of the detectable polypeptide to determine the potency of the IL-2 analog biased for the IL-2Rβγ complex.
21-22. (canceled)
A Kit225 cell modified to lack expression of the CD25 gene and comprising a nucleic acid molecule comprising a signal transducer and activator of transcription 5 (STAT5) response element and promoter operably linked to an open reading frame encoding a detectable polypeptide.
A cell line comprising Kit225 cells modified to lack expression of the CD25 gene and comprising a nucleic acid molecule comprising a signal transducer and activator of transcription 5 (STAT5) response element and promoter operably linked to an open reading frame encoding a detectable polypeptide.
27-28. (canceled)
A method for determining the potency of an interleukin 2 (IL-2) analog, comprising:
- (a) providing (i) a cell line that expresses an IL-2Rαβγ complex and a signal transducer and activator of transcription 5 (STAT5) signaling transduction pathway reporter comprising a STAT5 response element and a promoter linked to an open reading frame encoding a detectable polypeptide, and (ii) serial dilutions of an IL-2 analog;
- (b) contacting each serial dilution of the IL-2 analog with an aliquot of the cell line to provide a plurality of cultures;
- (c) incubating the cultures for a time sufficient to enable expression of the detectable polypeptide over time;
- and
- (d) measuring expression of the detectable polypeptide to determine the potency of the IL-2 analog.
33-35. (canceled)
A manufacturing process for producing a production batch of an interleukin 2 (IL-2) analog comprising the steps of:
- (a) synthesizing the IL-2 analog;
- (b) purifying the IL-2 analog;
- (c) formulating the IL-2 analog into a production batch;
- (d) obtaining a sample of the IL-2 analog from the production batch;
- (e) making a serial dilution of the IL-2 analog;
- (f) contacting each serial dilution of the IL-2 analog with an aliquot of a cell line that expresses (i) the IL-2Rαβγ complex and (ii) a signal transducer and activator of transcription 5 (STAT5) signaling transduction pathway reporter, which comprises a STAT5 response element and promoter linked to an open reading frame encoding a detectable polypeptide, to provide a plurality of cultures;
- (g) incubating the cultures for a time sufficient for expression of the detectable polypeptide over time;
- and
- (h) measuring expression of the detectable polypeptide to determine the potency of the IL-2 analog biased.
38-42. (canceled)
A cell line comprising Kit225 cells comprising a nucleic acid molecule comprising a signal transducer and activator of transcription 5 (STAT5) response element and promoter operably linked to an open reading frame encoding a detectable polypeptide.
44-45. (canceled)
Dependencies are inferred from the claim text.