Bioanalytical analysis of site-specific antibody drug conjugates

Abstract

Methods to rapidly and accurately detect, characterize, measure, and quantify site-specific antibody drug conjugates, that may be present in pre-clinical animal biological samples, or human biological samples, including plasma/serum and tissue samples.

What is claimed is: 
     
         1 . A method of evaluating an antibody drug conjugate (ADC) comprising:
 a. digesting an ADC comprising at least one drug moiety linked to an antibody at a recombinantly-engineered site selected from:
 a cysteine amino acid residue, 
 a selenocysteine amino acid residue, 
 a glutamine amino acid residue, 
 a non-naturally occurring amino acid residue, and 
 a sugar-modified glycan residue, 
   with a protease that cleaves the ADC, to form a digested ADC composition comprising at least one peptide fragment that is not linked to the at least one drug moiety, and at least one peptide fragment that is linked to the at least one drug moiety; and,   b. analyzing the digested ADC composition by high performance liquid chromatography (HPLC) and/or mass spectrometry (MS) to detect at least one peptide fragment that is not linked to the at least one drug moiety.   
     
     
         2 . The method of  claim 1 , wherein the antibody is selected from an IgG antibody, an antibody fragment, a human or humanized antibody, a glycosylated or phosphorylated antibody, and a cysteine-engineered antibody. 
     
     
         3 . The method of  claim 1 , 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, CD79β, 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) IL20Rα;   (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; FLJ3 5226);   (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.   
     
     
         4 . The method of  claim 1 , wherein the drug moiety is linked to the antibody portion of the ADC through a linker and is selected from a peptide, a polyamide, a maytansinoid, dolastatin, auristatin, calicheamicin, pyrrolobenzodiazepine (PBD), PNU-159682, anthracyclines, duocarmycins, vinca alkaloids, taxanes, trichothecene, CC1065, duocarmycin, camptothecin, elinafide, an antibiotic, a fluorophore, a radioisotope, and stereoisomers, isosteres, metabolites, analogs or derivatives thereof. 
     
     
         5 . The method of  claim 1 , wherein the protease is selected from: an IdeS protease, an IdeZ protease, an IgdE protease, a SpeB protease, a gingipain protease, an endoglycosidase, and combinations thereof. 
     
     
         6 . The method of  claim 1 , wherein the digesting comprises incubating the ADC with the protease at a temperature between about 20° C. and about 45° C.; at a pH between about pH 5 and about pH 9; and for a time period between about 0.1 hour and about 48 hours. 
     
     
         7 . The method of  claim 1 , wherein the digesting comprises incubating the ADC with the protease for a time period of about 1 hour, at a pH of about 7, at a temperature of about 37° C. 
     
     
         8 . The method of  claim 1 , wherein the analyzing comprises at least one of RP-LC, RP-LC/MS and LC-MS/MS. 
     
     
         9 . The method of  claim 1 , 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 digesting step. 
     
     
         10 . The method of  claim 1 , wherein the ADC is suspended in whole blood, serum, plasma, or tissue of a mammal selected from a human, a cynomolgus monkey, a rat, and a mouse. 
     
     
         11 . The method of  claim 1 , 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, SpinTrap columns including protein A and protein G, NHS and streptavidin iron or phosphorus or immobilized antibodies or lectin, paramagnetic beads, immuno-depletion, fractionation, solid phase extraction, phosphopeptide enrichment, polyacrylamide gel electrophoresis, and desalting, prior to the digesting step. 
     
     
         12 . The method of  claim 1 , wherein the ADC 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. 
     
     
         13 . The method of  claim 12 , further comprising washing ADC bound to the affinity capture media to reduce non-antibody proteins in contact with the ADC. 
     
     
         14 . The method of  claim 12 , further comprising dephosphorylating ADC bound to the affinity capture media. 
     
     
         15 . The method of  claim 12 , wherein the step of digesting occurs while ADC is bound to the affinity capture media. 
     
     
         16 . The method of  claim 12 , further comprising eluting ADC from the affinity capture media prior to the step of digesting the ADC. 
     
     
         17 . The method of  claim 1 , wherein the total antibody concentration of the ADC is calculated from the analysis of the digested ADC composition. 
     
     
         18 . The method of  claim 1 , wherein an antibody-conjugated drug concentration of the ADC is calculated from the analysis of the digested ADC composition. 
     
     
         19 . The method of  claim 1 , wherein the average drug-to-antibody ratio (DAR) of the ADC is calculated from the analysis of the digested ADC composition. 
     
     
         20 . The method of  claim 1 , wherein a metabolite or catabolite structure is determined from the analysis of the digested ADC composition. 
     
     
         21 . The method of  claim 1 , wherein the protein concentration of the ADC is calculated from the analysis of the digested ADC composition. 
     
     
         22 . The method of  claim 21 , wherein the protein concentration is correlated with a peak area from an RP-LC and/or MS analysis of at least one Fc fragment from the digested ADC. 
     
     
         23 . The method of  claim 1 , wherein the extinction coefficient of the ADC is calculated from the analysis of the digested ADC composition. 
     
     
         24 . The method of  claim 1 , wherein the average drug-to-antibody ratio (DAR) of the ADC, a metabolite or catabolite structure is determined from the analysis of the digested ADC, and the protein concentration is correlated with a peak area from an RP-LC and/or MS analysis of at least one Fc fragment from the digested ADC.