Multiplexed total antibody and antibody-conjugated drug quantification assay

Abstract

Methods are disclosed to detect, characterize, measure, and quantify human and humanized antibodies, and their conjugates, that may be present in pre-clinical animal biological samples, or human biological samples, including plasma/serum and tissue samples.

1 - 35 . (canceled) 
     
     
         36 . A method of quantifying both an antibody-conjugated drug concentration and a total antibody concentration of an antibody-drug conjugate (ADC), the method comprising:
 (a) contacting the ADC which is bound to an affinity capture media comprising at least one of bead- or resin-supported Protein A/G, target antigen-paramagnetic bead capture media, anti-idiotypic antibodies, anti-Hu antibodies, and anti-drug antibodies with (i) a reductant that reduces the antibody portion of the ADC to form a denatured antibody portion of the ADC, wherein the reductant is selected from dithiolthreitol (DTT), 2-mercaptoethanol, and tris(2-carboxyethyl)phosphine (TCEP); (ii) at least one denaturant selected from formamide, dimethylformamide, acetonitrile, SDS, urea, an acid labile surfactant, a decyl furanyl sulfonic acid salt, and guanidine HCl and; (iii) at least one chemical selected from methanol, ethanol, HCl, ammonium bicarbonate, Tris buffer, HEPES, ammonium acetate, and acetonitrile; whereby the antibody portion of the ADC is denatured;   (b) digesting the denatured antibody portion of the ADC to form a digested ADC peptide mixture in a single analysis sample with a proteolytic enzyme selected from the group consisting of trypsin, chymotrypsin, papain, pepsin, LysN, LysC, AspN, GluC, ArgC, and PNGaseF, wherein a drug moiety is separated from the denatured antibody to form a digested ADC peptide mixture; and   (c) analyzing the digested ADC peptide mixture by LC-MS/MS to detect a peptide-linker-drug complex comprising at least one antibody signature peptide and the drug moiety;   with the proviso that the ADC is not a disulfide-linked ADC.   
     
     
         37 . The method of  claim 36 , wherein the ADC is suspended in a matrix selected from a buffer, whole blood, serum, plasma, cerebrospinal fluid, saliva, urine, lymph, bile, feces, sweat, vitreous, tears, and tissue, prior to the contacting step. 
     
     
         38 . The method of  claim 36 , wherein the ADC is enriched by a technique selected from size exclusion chromatography, dialysis, selective precipitation, differential centrifugation, filtration, gel electrophoresis, liquid chromatography, reversed-phase chromatography, immunoprecipitation, spin columns including protein A and protein G, and desalting, prior to the contacting step. 
     
     
         39 . The method of  claim 36 , further comprising washing ADC bound to the affinity capture media to reduce non-antibody proteins in contact with the ADC. 
     
     
         40 . The method of  claim 36 , further comprising dephosphorylating the ADC bound to the affinity capture media. 
     
     
         41 . The method of  claim 36 , wherein the step of digesting the denatured antibody to form a digested antibody-drug conjugate peptide mixture occurs while the ADC is bound to the affinity capture media. 
     
     
         42 . The method of  claim 36 , further comprising eluting the denatured ADC from the affinity capture media prior to the step of digesting the denatured ADC. 
     
     
         43 . The method of  claim 36 , further comprising eluting the digested ADC peptide mixture from the affinity capture media prior to the step of analyzing the digested ADC peptide mixture. 
     
     
         44 . The method of  claim 36 , wherein the total antibody concentration of the ADC is calculated from the analysis of the digested ADC peptide mixture. 
     
     
         45 . The method of  claim 36 , wherein the antibody-conjugated drug concentration of the ADC is calculated from the analysis of the digested ADC peptide mixture. 
     
     
         46 . The method of  claim 36 , wherein the average drug-to-antibody ratio (DAR) of the ADC is calculated from the analysis of the digested ADC peptide mixture. 
     
     
         47 . The method of  claim 36 , wherein the drug moiety is selected from the group consisting of a maytansinoid, dolastatin, auristatin, calicheamicin, pyrrolobenzodiazepine (PBD), PNU-159682, anthracycline, duocarmycin, vinca alkaloid, taxane, trichothecene, CC1065, duocarrnycin, camptothecin, and elinafide. 
     
     
         48 . The method of  claim 36 , wherein the antibody portion of the ADC is an antibody fragment. 
     
     
         49 . The method of  claim 36 , wherein the antibody portion of the ADC is an antibody variant in which one or more residues of the antibody are substituted with cysteine residues. 
     
     
         50 . The method of  claim 36 , wherein the antibody portion of the ADC is a human or humanized antibody. 
     
     
         51 . The method of  claim 36 , wherein the antibody portion of the ADC is an antibody which binds to one or more tumor-associated antigens or cell-surface receptors selected from (1)-(53):
 (1) BMPR1B (bone morphogenetic protein receptor-type IB);   (2) E16 (LAT1, SLC7A5);   (3) STEAP1 (six transmembrane epithelial antigen of prostate);   (4) MUC16 (0772P, CA125);   (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin);   (6) Napi2b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b);   (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B);   (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene);   (9) ETBR (Endothelin type B receptor);   (10) MSG783 (RNF124, hypothetical protein FLJ20315);   (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein);   (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4);   (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor);   (14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs 73792);   (15) CD79b (CD79B, CD79(3, IGb (immunoglobulin-associated beta), B29);   (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C);   (17) HER2;   (18) NCA;   (19) MDP;   (20) IL2ORα;   (21) Brevican;   (22) EphB2R;   (23) ASLG659;   (24) PSCA;   (25) GEDA;   (26) BAFF-R (B cell -activating factor receptor, BLyS receptor 3, BR3);   (27) CD22 (B-cell receptor CD22-B isoform);   (28) CD79a (CD79A, CD79α, immunoglobulin-associated alpha);   (29) CXCR5 (Burkitt's lymphoma receptor 1);   (30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen));   (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5);   (32) CD72 (B-cell differentiation antigen CD72, Lyb-2);   (33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family);   (34) FcRH1 (Fc receptor-like protein 1);   (35) FcRH5 (IRTA2, Immunoglobulin superfamily receptor translocation associated 2);   (36) TENB2 (putative transmembrane proteoglycan);   (37) PMEL17 (silver homolog; SILV; D12S53E; PMEL17; SI; SIL);   (38) TMEFF1 (transmembrane protein with EGF-like and two follistatin-like domains 1; Tomoregulin-1);   (39) GDNF-Ra1 (GDNF family receptor alpha 1; GFRA1; GDNFR; GDNFRA; RETL1; TRNR1; RET1L; GDNFR-alpha1; GFR-ALPHA-1);   (40) Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E,SCA-2,TSA-1);   (41) TMEM46 (shisa homolog 2 (Xenopus laevis); SHISA2);   (42) Ly6G6D (lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1);   (43) LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5; GPR49, GPR67);   (44) RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC; CDHF12; Hs.168114; RET51; RET-ELE1);   (45) LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ35226);   (46) GPR19 (G protein-coupled receptor 19; Mm.4787);   (47) GPR54 (KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR12);   (48) ASPHD1 (aspartate beta-hydroxylase domain containing 1; LOC253982);   (49) Tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP3);   (50) TMEM118 (ring finger protein, transmembrane 2; RNFT2; FLJ14627);   (51) GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856; D15Ertd747e);   (52) CD33; and   (53) CLL-1.