US 12,270,081 B2
Method for quantification of PD-L1 expression
Evrykleia Lianidou, Athens (GR); and Areti Strati, Athens (GR)
Assigned to PHARMASSIST LTD, Athens (GR)
Filed by PHARMASSIST LTD, Athens (GR)
Filed on Aug. 11, 2021, as Appl. No. 17/399,339.
Application 17/399,339 is a division of application No. 15/771,165, abandoned, previously published as PCT/GR2015/000054, filed on Oct. 27, 2015.
Prior Publication US 2022/0073995 A1, Mar. 10, 2022
Int. Cl. C12P 19/34 (2006.01); C12Q 1/6853 (2018.01); C12Q 1/6886 (2018.01)
CPC C12Q 1/6886 (2013.01) [C12Q 1/6853 (2013.01); C12Q 2600/106 (2013.01); C12Q 2600/112 (2013.01); C12Q 2600/118 (2013.01); C12Q 2600/156 (2013.01); C12Q 2600/16 (2013.01); C12Q 2600/166 (2013.01)] 21 Claims
 
1. An in vitro method for quantitative determination of the expression of Programmed Death Ligand 1 (PD-L1) mRNA in a sample, said method comprising:
subjecting a sample to reverse transcription using RNA present in the sample as a template in order to synthesize a corresponding cDNA sequence;
forming a reaction mixture comprising the sample, nucleic acid amplification reagents, a target primer pair, and a target hydrolysis probe, said target primer pair and target hydrolysis probe being capable of hybridizing to PD-L1 mRNA;
subjecting the reaction mixture to amplification conditions optimized to generate at least one copy of a nucleic acid sequence complementary to a target sequence, said target sequence being a mRNA transcript of the PD-L1 mRNA sequence (SEQ ID NO: 1);
determining the amount of PD-L1 mRNA in the sample;
normalizing the expression of PD-L1 with respect to an expression of a reference gene; and
comparing the amount of PD-L1 mRNA expressed in the sample to a positive and negative control in order to estimate an overexpression of the PD-L1 mRNA sequence;
wherein the forward PD-L1 target primer has a forward sequence of 5′-GCTGAATTGGTCATCCCAGAA-3′ (SEO ID NO: 4); and
wherein the reverse target primer of PD-L1 is designed to hybridize between exon 5 and 6 of the PD-L1 mRNA sequence and has a reverse sequence of 5′-TTTCACATCCATCATTCTCCCTT-3′ (SEO ID NO: 6).