USOO8865417B2
(12) United States Patent (10) Patent No.: US 8,865,417 B2 Singh et al. (45) Date of Patent: Oct. 21, 2014
(54) ASSAYS FOR THE DETECTION OF (52) U.S. Cl. ANT-TNF DRUGS AND AUTOANTIBODES CPC ...... G0IN33/6854 (2013.01); G0IN33/5091 (2013.01); G0IN33/564 (2013.01) (71) Applicant: Nestec S.A., Vevey (CH) USPC ...... 435/7.1; 435/7.2 (58) Field of Classification Search (72) Inventors: Sharat Singh, Rancho Santa Fe, CA None (US); Shui Long Wang, San Diego, CA See application file for complete search history. S. Linda Ohrmund, San Diego, CA (56) References Cited U.S. PATENT DOCUMENTS (73) Assignee: Nestec S.A., Vevey (CH) 4.459,359 A 7/1984 Neurath (*) Notice: Subject to any disclaimer, the term of this 5,094,740 A 3/1992 Brandley et al. patent is extended or adjusted under 35 (Continued) U.S.C. 154(b) by 0 days. FOREIGN PATENT DOCUMENTS (21) Appl. No.: 14/046,863 9 EP O440044 8, 1991 (22) Filed: Oct. 4, 2013 EP O492448 A1 7, 1992 (Continued) (65) Prior Publication Data OTHER PUBLICATIONS US 2014/OO57367 A1 Feb. 27, 2014 Aarden, L. et al., “Immunogenicity of anti-tumor necrosis factor O O antibodies—toward improved methods of anti antibody measure Related U.S. Application Data ment.” Current Opinion in Immunology, 2008, 20(4): 431-435. (63) Continuation of application No. 13/441.727, filed on Arcangelo & Peterson, Pharmacotherapeutics for Advanced Prac Apr. 6, 2012, now Pat. No. 8,574,855, which is a tice: a Practical Approach, Philadelphia, PA 2006, vol. 536, p. 18. continuation of application No. PCT/US2010/054125 s Aybay,infliximab C. inducedet al., “Demonstration during treatment of of specific a patient antibodies with ankylosing against filed on Oct. 26, 2010. spondylitis. Rheumatology International, Clin. and Exper. Invest. (60) Provisional application No. 61/255,048, filed on Oct. 2006, 26(5):473-480. 26, 2009, provisional application No. 61/262,877, (Continued) filed on Nov. 19, 2009, provisional application No. 61/324,635, filed on Apr. 15, 2010, provisional Primary Examiner Jacob Cheu application No. 61/345,567, filed on May 17, 2010, (74) Attorney, Agent, or Firm — Kilpatrick Townsend & provisional application No. 61/351.269, filed on Jun. Stockton LLP 3, 2010, provisional application No. 61/389,672, filed (57) ABSTRACT on Oct. 4, 2010, provisional application No. Th id ford d 61/393,581 filed on Oct. 15, 2010 epresent 1nvention provides assays Ior etecting an mea sa- Y - us • u a-s suring the presence or level of anti-TNFC. drug therapeutics (51) Int. Cl and autoantibodies in a sample. The present invention is use coiN s3/53 (2006.01) ful for optimizing therapy and monitoring patients receiving GOIN33/68 (200 6. 01) anti-TNFC. drug therapeutics to detect the presence or level of GOIN33/50 (200 6,015 autoantibodies (e.g., HACA and/or HAHA) against the drug. GOIN33/564 (2006.01) 20 Claims, 30 Drawing Sheets
HACA F-Remicade ACA ImmunoComplex 2 . — A Free F1-Remicade F1-Renicade
HACA
HACA Immuno-Complex 3
Free F2-F-ct,
F1 Remicade US 8,865,417 B2 Page 2
(56) References Cited Flood, J., “Tumor necrSosis factor inhibitors in the treatment of chronic inflammatory diseases: A review of immunogenicity and U.S. PATENT DOCUMENTS potential implications,” Suppl. to Managed Care, 2009, 18(4): 1-5. Gisbert, Javier et al., “Loss of Response and Requirement of 5,223,395 A 6, 1993 Gero 5,582,998 A 12/1996 Adolfetal. Infliximab Dose Intensification in Chrohns Disease: A Review.” Jour 5,698,419 A 12/1997 Wolpe et al. nal of Gastroenterology, Mar. 2009, vol. 104, pp. 760-767. 5,795,967 A 8/1998 Aggarwal et al. Hagg, D. et al., “Measurement and biological correlates of antibody 5,837,242 A 1 1/1998 Holliger et al. bioactivity during antibody immunotherapies. J. Immunol. Meth. 6,284.471 B1 9, 2001 Le et al. 219(1-2): 7-21, 1998. 6,444,461 Bl 9/2002 Knapp et al. Harris, Quantitative Chemical Analysis, Sixth Ed., New York, W.H. 7,189,515 B2 3/2007 Buechler et al. Freeman Co., 2003, p. 85-86. 7,276.477 B2 10/2007 Osslund et al. 7,524,502 B2 4/2009 Hellendoorn et al. Harris, Quantitative Chemical Analysis, Sixth Ed., New York, W.H. 7,601,335 B2 10/2009 McCutcheon et al. Freeman Co., 2003, p. 91. 7,662,569 B2 2/2010 Targan et al. Hosono, M. et al., “Human-mouse chimeric antibodies show low 8,574,855 B2 11/2013 Singh et al. reactivity with human anti-murine antibodies HAMA.” British J. 2003/004.0027 A1 2/2003 Ritter et al. Cancer, 1992, 65(2): 197-200. 2003/0077246 A1 4/2003 Welcher et al. Invitrogen, “Looking on the bright side with AlexaFluor R secondary 2006,0003384 A1 1/2006 Wagner et al. antibodies.” 2008, URL http://www.jimmunol.org/content/181/3/lo 2006,0078944 A1 4/2006 Kuai et al. 2006, O110407 A1 5/2006 Stopera et al. cal/advertising.pdf, retrieved on Oct. 11, 2013. 2006/0240480 A1 10, 2006 Curdt et al. Kawate, T. et al., “Fluorescence-detection size-exclusion chromatog 2007/0077249 A1 4/2007 Silence et al. raphy for precrystallization screening of integral membrane pro 2008/028.0311 A1 11/2008 Strohner teins. Structure, 2006, 14:673-681. 2009 OO35216 A1 2/2009 Svenson et al. Lofgren, J. et al., “Detection of neutralizing anti-therapeutic protein 2009, 02342O2 A1 9, 2009 Goix et al. antibodies in serum or plasma samples containing high levels of the 2009/0275496 A1 11, 2009 Baldwin et al. 2010, O130367 A1 5/2010 Murthy et al. therapeutic protein.” J. Immunol. Meth., 2006, 308(1-2): 101-108. 2012/0329172 A1 12/2012 Singh O'Keefe, Michael, Ed., Residue Analysis in Food Principles and 2013/0266963 A1 10, 2013 Hauenstein et al. Applications, Amsterdam, Hardwood Academic Publishers, 2000, p. 2013/0295685 A1 1 1/2013 Singh 20. 2013/0344621 A1 12/2013 Wang et al. Panchuk-Voloshina, N. et al., “Alexa dyes, a series of new fluorescent 2014/0045276 A1 2/2014 Singh et al. dyes that yield exceptionally bright, photostable conjugates. J. 2014/0051184 A1 2/2014 Singh et al. Histochem & Cytochem., 1999, 47(9): 1179-1188. Patton et al., “An acid dissociation bridging ELISA for detection of FOREIGN PATENT DOCUMENTS antibodies directed against therapeutic proteins in the presence of EP O642021 A2 3, 1995 antigen.” J. Immunol. Meth., 2005, 304(1-2): 189-195. EP O882984 A1 12, 1998 Reynolds, J.C. et al., “Anti-murine antibody response to mouse EP 1237.926 B1 9, 2002 monoclonal antibodies: Clinical findings and implications.” Intl. J. EP 1769.244 4/2007 Radiation Applications and Instrumentation, Part B. Nuclear Medi EP 1902320 3, 2008 cine and Biology, 1989, 16(2): 121-125. JP H5-96 A 1, 1993 Scallon, B. et al., “Binding and functional comparisons of two types JP H7-110331 A 4f1995 of tumor necrosis factor antagonists. J. Pharmacol. Exper. Ther. JP H11-5006O7T A 1, 1999 2002, 301(2):418-426. JP 2001-24.9127 A 9, 2001 Sickert, D. et al., “Improvement of drug tolerance in immunogenicity WO 96.20219 A2 T 1996 WO 2005/O19271 A1 3, 2005 testing by acid treatment on Biacore.” J. Immunol. Meth., 2008, WO 2007/OO9469 A2 1, 2007 334(1-2):29-36. WO 2009/091240 A1 T 2009 Smith et al., “Detection of antibodies against therapeutic proteins in WO 2011/056590 A1 5, 2011 the presence of residual therapeutic protein using a Solid-phase WO 2012/054532 A1 4/2012 extraction with acid dissociation (SPEAD) sample treatment prior to WO 2012/154253 A1 11, 2012 ELISA.” Regulatory Toxicology and Pharmacology, 2007, 49(3): WO 2013,00681.0 A1 1, 2013 230-237. WO 2014/083520 A1 6, 2014 Svenson, M. et al., “Monitoring patients treated with anti-TNFO. OTHER PUBLICATIONS biopharmaceuticals: assessing serum infliximab and anti-infliximab Bendtzen, K. et al., “Individualized monitoring of drug bioavail antibodies.” Rheumatology, 2007, 46:1828-34. ability and immunogenicity in rheumatoid arthritis patients treated Tayyab, S. et al., “Size exclusion chromatography and size exclusion with the tumor necrosis factor alpha inhibitor infliximab.” Arthritis & HPLC of proteins.” Biochemical Education, 1991, 19(3):149-152. Rheumatism, 2006, 54(12):3782-3789. Van Der Laken, C. et al., “Imaging and serum analysis of immune Bourdage et al., “An affinity capture elution (ACE) assay for detec complex formation of radiolabeled infliximab and anti-infliximab in tion of anti-drug antibody to monoclonal antibody therapeutics in the responders and non-responders to therapy for rheumatoid arthritis.” presence of high levels of drug.” J. Immunol. Methods, 2007, 327(1- Ann. Rheum. Dis., 2007, 66(2):253-256. 2):10-17. Van Schouwenburg, P. et al., “A novel method for the detection of Cheifetz, A. et al., “Monoclonal antibodies: immunogenicity, and antibodies to adalimumab in the presence of drug reveals “hidden' associated infusion reactions.” Mount Sinai J. Medicine, 2005, immunogenicity in rheumatoid arthritis patients.” J. Immunol. Meth. 72(4):250-256. 2010, 362(1-2):82-88. Cisbio Bioassays, “HTRF human kappa and lambda MAbassay: A Wang, S. et al., “Analysis of anti-drug antibodies (ADA) to new solution for human IgG characterisation.” 2009, URL: http:// adalimumab in patient serum using a novel homogeneous mobility www.biolab.cn/plus/view-241835-1.html. Accessed on Feb. 20, shift assay.” Am. J. Gastro., 2010, 105(Suppl. 1): S444-S445. 2014. Hagg, D. et al., “Measurement and biological correlates of antibody Deventer, S. et al., “Anti-tumour necrosis factor therapy in Crohn's bioactivity during antibody immunotherapies. J. Immunol. Meth. disease: Where are we now?” Gut, 2002, 51(3):362-63. 219(1-2): 7-21, Year 1998. Elliott, M. et al., “Repeated therapy with monoclonal antibody to Rojas, J.R. et al., “Formation, distribution, and elimination of tumour necrosis factor alpha (cA2) in patients with rheumatoid infliximab and anti-infliximab immune complexes in cynomolgus arthritis.” Lancet, 1994, 334(8930): 1125-1127. monkeys.” JPET, May 1, 2005, 313(2):578-585. U.S. Patent Oct. 21, 2014 Sheet 1 of 30 US 8,865,417 B2
U.S. Patent Oct. 21, 2014 Sheet 2 of 30 US 8,865,417 B2
U.S. Patent Oct. 21, 2014 Sheet 3 of 30 US 8,865,417 B2
Bridging A
Biotinylated x8& infixirab Biotin
Murine
HACA Human
infiximab
FIG. 3
U.S. Patent US 8,865,417 B2
U.S. Patent Oct. 21, 2014 Sheet 6 of 30 US 8,865,417 B2
(p,quo3)G'91-J U.S. Patent US 8,865,417 B2
U.S. Patent US 8,865,417 B2
U.S. Patent US 8,865,417 B2
U.S. Patent Oct. 21, 2014 Sheet 11 of 30 US 8,865,417 B2
Biotin and DIG-Based HACA Assay of the Bridge HACA Assay Homogeneous Present invention Bridging ELISA F-Remicade HACA HRP-AWii HRP-anti-DIG
Bio Y
& Remicade DIG-Remicade
Assay Format HACA HACA
Bio-Remicade Remicade Avidin
Non-specific Background Interference
Sensitivity Possibility of False Positive and False Negative IgG4 HACA Detection g isotype identification No
Tolerance of Poor Drug in the Sample
FIG. 1 O U.S. Patent Oct. 21 2014 Sheet 12 of 30 US 8,865,417 B2
U.S. Patent Oct. 21, 2014 Sheet 13 of 30 US 8,865,417 B2
s h 62. 2, (2.
9. e 2. e -
- - s 62. O 2. 1. V.d O. O SCO. SN. SO. S D Y of CN wond d5 2. von que (%) --X-puno Jo effeueoued CN vs
9l v, (2)
2. 62.
9. ca 62
O O. O. O. O. O. O. O. o O O O O OO. N. CO D N C CN val va (%) --X eel-JO effeueoued U.S. Patent 865,417 B2
H-OHN13uOOT+SHN%7
SHN%7 U.S. Patent Oct. 21, 2014 Sheet 15 Of 30 US 8,865,417 B2
Co o to o o o o O do o co o s v O or oo N. co to r of on ten v. v. (%) NL pun Oyo e.eue) led
2 e
s
O o O O o O o O o o O O s: vo o or oo N. co r of CN ve to in %) - NLee Joe feueoued U.S. Patent US 8,865,417 B2
-X eel pue puno JO Oe U.S. Patent Oct. 21, 2014 Sheet 17 Of 30 US 8,865,417 B2
is a S is to e o . . S. Y st 5 8 9 s cus O E SESB Es 3 S e 8 9C >S as we E E 88: X 9. E O. LS en o n N se wn re
S s sa cid up t E > SSS s as LL EEEEEEEEEEEEEEE| r O 222 w 9 w SS ------...- . C. g.
years is 8 ()s is C l as wasS S s. As As me . SSSSSSSSSS k H O Cld as %22 S. c g O o o to ed 1. c. c. (N (N V. V. c. c. o o o O o o o d
E San E on S r we we s .gs SB ce we wo as:vs Y. ... O ced - se cS ce
a. d 59 s 2 g ce d co cred Cc Co N o N. N C N ed eco o to o ced co e ced -X eel pue puno Jo Oe U.S. Patent Oct. 21, 2014 Sheet 18 of 30 US 8,865,417 B2
A. Non-neutralizing HACA Assay
F1-Remicade F2-TNF-o. HACA . Y . F1-Remicade
immuno-Complex
Immuno-Complex 1 2
Free F1-Remicade Free F2TNFO,
B. Neutralizing HACA Assay
HACA
F1-Remicade F2-TNF-cy.
F1-Remicade
immuno-Complex Free F2-TNFO, 2
Free F1-Remicade
FIG. 17 U.S. Patent US 8,865,417 B2
VOVH
VOVH }—Y),
VOVH U.S. Patent Oct. 21, 2014 Sheet 20 of 30 US 8,865,417 B2
9|
to Lo N. Loco Lo Ca Lo N- La C - Yi cd cN we do U.S. Patent Oct. 21, 2014 Sheet 21 of 30 US 8,865,417 B2
rrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrry'erry'rry wers: **** *****r-se olor Loco Lo Cn LON - Loo its ord on - co rt ord on were co
U.S. Patent Oct. 21, 2014 Sheet 22 of 30 US 8,865,417 B2
1.OO
O. 7 5 O.O 25 5O
O.OO O.OO 0.01 O. 10 100 Anti-Human lgg giml)
FIG. 20
U.S. Patent 865,417 B2
(p,quo3)LZ'91-'
U.S. Patent Oct. 21, 2014 Sheet 25 of 30 US 8,865,417 B2
0.75
O. 5 O
0.25
0.00 0.001 0.01 0.1 10 100 Adalimumab giml)
HILLSLOPE -1813 EC50 1213
FIG. 22
U.S. Patent US 8,865,417 B2
(p,quo3)CZ'91-'
U.S. Patent Oct. 21, 2014 Sheet 28 of 30 US 8,865,417 B2
0043 S844, 289 | | res
... On
------o
U.S. Patent US 8,865,417 B2
US 8,865,417 B2 1. 2 ASSAYS FOR THE DETECTION OF Tumor necrosis factor alpha (TNF-C.) is a cytokine pro ANT-TNF DRUGS AND AUTOANTIBODES duced by numerous cell types, including monocytes and mac rophages, that was originally identified based on its ability to CROSS-REFERENCE TO RELATED induce the necrosis of certain mouse tumors. Subsequently, a APPLICATIONS factor termed cachectin, associated with cachexia, was shown to be identical to TNF-C. TNF-C. has been implicated in the This application is a continuation U.S. application Ser. No. pathophysiology of a variety of other human diseases and 13/441.727, filed Apr. 6, 2012, which is a continuation of disorders, including shock, sepsis, infections, autoimmune PCT/US2010/054125, filed Oct. 26, 2010, which application diseases, RA, Crohn's disease, transplant rejection and graft claims priority to U.S. Provisional Application No. 61/255, 10 Versus-host disease. 048, filed Oct. 26, 2009, U.S. Provisional Application No. Because of the harmful role of human TNF-C. (hTNF-C.) in 61/262,877, filed Nov. 19, 2009, U.S. Provisional Application a variety of human disorders, therapeutic strategies have been No. 61/324,635, filed Apr. 15, 2010, U.S. Provisional Appli designed to inhibit or counteract hTNF-C. activity. In particu cation No. 61/345,567, filed May 17, 2010, U.S. Provisional 15 lar, antibodies that bind to, and neutralize, hTNF-C., have Application No. 61/351,269, filed Jun. 3, 2010, U.S. Provi been sought as a means to inhibit hTNF-C. activity. Some of sional Application No. 61/389,672, filed Oct. 4, 2010, and the earliest of Such antibodies were mouse monoclonal anti U.S. Provisional Application No. 61/393,581, filed Oct. 15, bodies (mAbs), secreted by hybridomas prepared from lym 2010, the disclosures of which are hereby incorporated by phocytes of mice immunized with hTNF-C. (see e.g., U.S. Pat. reference in their entirety for all purposes. No. 5,231,024 to Moeller et al.). While these mouse anti hTNF-C. antibodies often displayed high affinity for hTNF-C. REFERENCE TO A “SEQUENCE LISTING. A and were able to neutralize hTNF-C. activity, their use in vivo TABLE, ORACOMPUTER PROGRAM LISTING has been limited by problems associated with the administra APPENDIX SUBMITTED AS ANASCII TEXT tion of mouse antibodies to humans, such as a short serum FILE 25 half-life, an inability to trigger certain human effector func tions, and elicitation of an unwanted immune response The Sequence Listing written in file-182-7.TXT, created against the mouse antibody in a human (the “human anti on Jul. 16, 2012, 4,096 bytes, machine format IBM-PC, MS mouse antibody” (HAMA) reaction). Windows operating system, is hereby incorporated by refer More recently, biological therapies have been applied to ence in its entirety for all purposes. 30 the treatment of autoimmune disorders such as rheumatoid arthritis. For example, four TNFC. inhibitors, REMICADETM BACKGROUND OF THE INVENTION (infliximab), a chimericanti-TNFC. mAb, ENBRELTM (etan Autoimmune disorders are a significant and widespread ercept), a TNFR-Ig Fc fusion protein, HUMIRATM (adali medical problem. For example, rheumatoid arthritis (RA) is 35 mumab), a human anti-TNFC. mab, and CIMZIAR (certoli an autoimmune disease affecting more than two million Zumab pegol), a PEGylated Fab fragment, have been people in the United States. RA causes chronic inflammation approved by the FDA for treatment of rheumatoid arthritis. of the joints and typically is a progressive illness that has the CIMZIAR) is also used for the treatment of moderate to severe potential to cause joint destruction and functional disability. Crohn's disease (CD). While such biologic therapies have The cause of rheumatoid arthritis is unknown, although 40 demonstrated Success in the treatment of rheumatoid arthritis genetic predisposition, infectious agents and environmental and other autoimmune disorders such as CD, not all Subjects factors have all been implicated in the etiology of the disease. treated respond, or respond well, to Such therapy. Moreover, In active RA, symptoms can include fatigue, lack of appetite, administration of TNFC. inhibitors can induce an immune low grade fever, muscle and joint aches and stiffness. Also response to the drug and lead to the production of autoanti during disease flare ups, joints frequently become red, Swol 45 bodies such as human anti-chimeric antibodies (HACA), len, painful and tender, due to inflammation of the synovium. human anti-humanized antibodies (HAHA), and human anti Furthermore, since RA is a systemic disease, inflammation mouse antibodies (HAMA). Such HACA, HAHA, or HAMA can affect organs and areas of the body other than the joints, immune responses can be associated with hypersensitive including glands of the eyes and mouth, the lung lining, the reactions and dramatic changes in pharmacokinetics and bio pericardium, and blood vessels. 50 distribution of the immunotherapeutic TNFC. inhibitor that Traditional treatments for the management of RA and other preclude further treatment with the drug. Thus, there is a need autoimmune disorders include fast acting “first line drugs' in the art for assays to detect the presence of anti-TNFC. and slower acting 'second line drugs.” The first line drugs biologics and/or their autoantibodies in a patient sample to reduce pain and inflammation. Example of Such first line monitor TNFC. inhibitor therapy and to guide treatment deci drugs include aspirin, naproxen, ibuprofen, etodolac and 55 sions. The present invention satisfies this need and provides other non-steroidal anti-inflammatory drugs (NSAIDs), as related advantages as well. well as corticosteroids, given orally or injected directly into tissues and joints. The second line drugs promote disease BRIEF SUMMARY OF THE INVENTION remission and prevent progressive joint destruction and are also referred to as disease-modifying anti-rheumatic drugs or 60 TNFC. has been implicated in inflammatory diseases, DMARDs. Examples of second line drugs include gold, autoimmune diseases, viral, bacterial and parasitic infections, hydrochloroquine, aZulfidine and immunosuppressive malignancies, and/or neurodegenerative diseases and is a use agents, such as methotrexate, azathioprine, cyclophospha ful target for specific biological therapy in diseases, such as mide, chlorambucil and cyclosporine. Many of these drugs, rheumatoid arthritis and Crohn's disease. TNFO inhibitors however, can have detrimental side-effects. Thus, additional 65 such as anti-TNFC. antibodies are an important class of there therapies for rheumatoid arthritis and other autoimmune dis apuetics. Assay methods are needed to detect the presence of orders have been sought. anti-TNFC. biologics and/or their autoantibodies. US 8,865,417 B2 3 4 As such, in one embodiment, the present invention pro of a neutralizing form of the autoantibody (i.e., wherein vides a method for detecting the presence or level of an the autoantibody interferes with the binding between the anti-TNFC. drug in a sample, comprising: anti-TNFC. drug and TNFC.) when only the second (a) contacting labeled TNFC. with a sample having or Sus labeled complex is present. pected of having an anti-TNFC. drug to form a labeled In certain instances, the methods are useful for measuring complex (i.e., immuno-complex or conjugate) with the the levels of autoantibodies including, but not limited to, anti-TNFC. drug (i.e., wherein the labeled TNFC. and human anti-chimeric antibodies (HACA), human anti-hu anti-TNFC. drug are not covalently attached to each manized antibodies (HAHA), and human anti-mouse anti other); bodies (HAMA) in a sample, e.g., from a subject receiving (b) Subjecting the labeled complex to size exclusion chro 10 matography to separate the labeled complex (e.g., from anti-TNFC. drug therapy. free labeled TNFC); and In a related embodiment, the present invention provides a (c) detecting the labeled complex, thereby detecting the method for detecting the presence or level of an autoantibody presence or level of an anti-TNFC. drug. to an anti-TNFC. drug in a sample, comprising: In certain instances, the methods are useful for measuring 15 (a) contacting a labeled anti-TNFC. drug with a sample the levels of REMICADETM (infliximab), ENBRELTM (etan having or Suspected of having an autoantibody to the ercept), HUMIRATTM (adalimumab), and CIMZIAR (cer anti-TNFC. drug to form a first labeled complex (i.e., tolizumab pegol) in a sample, e.g., from a subject receiving immuno-complex or conjugate) between the labeled such anti-TNFC. drug therapy. anti-TNFC. drug and the autoantibody (i.e., wherein the In another embodiment, the present invention provides a labeled anti-TNFC. drug and autoantibody are not method for detecting the presence or level of an autoantibody covalently attached to each other); to an anti-TNFC. drug in a sample, comprising: (b) subjecting the first labeled complex to a first size exclu (a) contacting a labeled anti-TNFC. drug with a sample sion chromatography to separate the first labeled com having or Suspected of having an autoantibody to the plex (e.g., from free labeled anti-TNFC. drug); anti-TNFC. drug to form a labeled complex (i.e., 25 (c) detecting the first labeled complex, thereby detecting immuno-complex or conjugate) with the autoantibody the presence or level of the autoantibody; (i.e., wherein the labeled anti-TNFC. drug and autoanti (d) contacting labeled TNFC. with the first labeled complex body are not covalently attached to each other); to form a second labeled complex (i.e., immuno-com (b) Subjecting the labeled complex to size exclusion chro plex or conjugate) between the labeled anti-TNFC. drug matography to separate the labeled complex (e.g., from 30 free labeled anti-TNFC. drug); and and the labeled TNFC. (i.e., wherein the labeled anti (c) detecting the labeled complex, thereby detecting the TNFC. drug and the labeled TNFC. are not covalently presence or level of the autoantibody. attached to each other), wherein the labeled anti-TNFC. In certain instances, the methods are useful for measuring drug and the labeled TNFC. comprise different labels; the levels of autoantibodies including, but not limited to, 35 (e) Subjecting the second labeled complex to a second size human anti-chimeric antibodies (HACA), human anti-hu exclusion chromatography to separate the second manized antibodies (HAHA), and human anti-mouse anti labeled complex (e.g., from free labeled TNFC.); and bodies (HAMA) in a sample, e.g., from a subject receiving (f) detecting the second labeled complex, thereby detecting anti-TNFC. drug therapy. the presence or level of a neutralizing form of the In yet another embodiment, the present invention provides 40 autoantibody (i.e., wherein the autoantibody interferes a method for detecting the presence or level of an autoanti with the binding between the anti-TNFC. drug and body to an anti-TNFC. drug in a sample, comprising: TNFo). (a) contacting a labeled anti-TNFC. drug and labeled TNFC. In certain instances, the methods are useful for measuring with a sample having or Suspected of having an autoan the levels of autoantibodies including, but not limited to, tibody to the anti-TNFC. drug to form a first labeled 45 human anti-chimeric antibodies (HACA), human anti-hu complex (i.e., immuno-complex or conjugate) between manized antibodies (HAHA), and human anti-mouse anti the labeled anti-TNFC. drug, the labeled TNFC., and the bodies (HAMA) in a sample, e.g., from a subject receiving autoantibody (i.e., wherein the components of the first anti-TNFC. drug therapy. labeled complex are not covalently attached to each In another embodiment, the present invention provides a other) and a second labeled complex (i.e., immuno-com 50 method for determining an effective amount of an anti-TNFC. plex or conjugate) between the labeled anti-TNFC. drug drug for a subject receiving therapy with the anti-TNFC. drug, and the autoantibody (i.e., wherein the components of the method comprising: the second labeled complex are not covalently attached (a) measuring the level of the anti-TNFC. drug in a first to each other), wherein the labeled anti-TNFC. drug and sample from the Subject, comprising: the labeled TNFC. comprise different labels; 55 (i) contacting the first sample with an amount of a (b) subjecting the first labeled complex and the second labeled TNFC. to form a first complex comprising the labeled complex to size exclusion chromatography to labeled TNFC. with the anti-TNFC. drug; and separate the first labeled complex and the second labeled (ii) detecting the first complex by size exclusion chro complex (e.g., from each other and from free labeled matography, thereby measuring the level of the anti TNFC. and free labeled anti-TNFC. drug); and 60 TNFC. drug; (c) detecting the first labeled complex and the second (b) measuring the level of an autoantibody to the anti labeled complex, thereby detecting the presence or level TNFC. drug in a second sample from the Subject, com of a non-neutralizing form of the autoantibody (i.e., prising: wherein the autoantibody does not interfere with the (i) contacting the second sample with an amount of a binding between the anti-TNFC. drug and TNFC) when 65 labeled anti-TNFC. drug to form a second complex both the first labeled complex and the second labeled comprising the labeled anti-TNFC. drug with the complex are present, and detecting the presence or level autoantibody; and US 8,865,417 B2 5 6 (ii) detecting the second complex by size exclusion chro the labeled TNFC. to the labeled internal control of step (f) in matography, thereby measuring the level of the the previous paragraph, thereby optimizing the therapeutic autoantibody; and amount of the anti-TNFC. drug. (c) subtracting the level of the autoantibody measured in In some embodiments, the present invention provide a step (b) from the level of the anti-TNFC. drug measured method for determining the presence or level of an autoanti in step (a), body to an anti-TNFC. drug with reference to an internal thereby determining the effective amount of the anti-TNFC. control in a sample having or Suspected of having the autoan drug. tibody, the method comprising: In another embodiment, the present invention provides a (a) contacting an amount of labeled anti-TNFC. drug and an method for optimizing the therapeutic amount of an anti 10 TNFC. drug in a subject receiving therapy with the anti-TNFC. amount of labeled internal control with the sample to drug, the method comprising: form a complex of the labeled anti-TNFC. drug and the (a) determining an effective amount of the anti-TNFC. drug autoantibody; in accordance with a method of the present invention; (b) detecting the labeled anti-TNFC. and the labeled inter (b) comparing the effective amount of the anti-TNFC. drug 15 nal control by size exclusion chromatography; with the level of the anti-TNFC. drug; and (c) integrating the area-under-the curve for a peak of the (c) determining a Subsequent dose of the anti-TNFC. drug labeled anti-TNFC. drug from a plot of signal intensity as for the Subject based upon the comparison of step (b). a function of elution time from the size exclusion chro thereby optimizing the therapeutic amount of the anti-TNFC. matography; drug. (d) integrating the area-under-the curve for a peak of the In another embodiment, the present invention provides a labeled internal control from the plot of signal intensity method for optimizing therapy and/or reducing toxicity to an as a function of elution time from the size exclusion anti-TNFC. drug in a subject receiving therapy with the anti chromatography; TNFC. drug, the method comprising: (e) determining a first ratio by dividing the amount of (a) measuring the level of the anti-TNFC. drug in a first 25 labeled anti-TNFC. drug by the amount of labeled inter sample from the Subject; nal control; and (b) measuring the level of an autoantibody to the anti (f) determining a second ratio by dividing the resultant TNFC. drug in a second sample from the subject; and integration from step (c) and (d); and (c) determining a Subsequent course of therapy for the (g) comparing the first ratio determined in step (e) with the subject based upon the levels of the anti-TNFC. drug and 30 second ratio determined in step (f). the autoantibody, thereby determining the presence or level of the autoantibody thereby optimizing therapy and/or reducing toxicity to the with reference to an internal control. anti-TNFC. drug. In another embodiment, the present invention provides a In another embodiment, the present invention provides a kit for measuring the presence or level of an anti-TNFC. drug method for determining the presence or level of an anti-TNFC. 35 and the presence or level of an autoantibody to an anti-TNFC. drug with reference to an internal control in a sample having drug in a sample, the kit comprising: or suspected of having the anti-TNFC. drug, the method com (a) a first measuring Substrate comprising an amount of a prising: labeled TNFO: (a) contacting an amount of a labeled TNFC. and an amount (b) a second measuring Substrate comprising an amount of of a labeled internal control with the sample to form a 40 a labeled anti-TNFO: complex of the labeled TNFC. and the anti-TNFC. drug; (c) optionally a third measuring Substrate comprising an (b) detecting the labeled TNFC. and the labeled internal amount of a labeled TNFC. and an amount of a labeled control by size exclusion chromatography; internal control; (c) integrating the area-under-the curve for a peak of the (d) optionally a fourth measuring Substrate comprising an labeled TNFC. from a plot of signal intensity as a func 45 amount of a labeled anti-TNFC. drug and an amount of a tion of elution time from the size exclusion chromatog labeled internal control; raphy; (e) optionally a means for extracting a sample from a (d) integrating the area-under-the curve for a peak of the Subject; and labeled internal control from the plot of signal intensity (f) optionally a pamphlet of instructions for using the kit. as a function of elution time from the size exclusion 50 Other objects, features, and advantages of the present chromatography; invention will be apparent to one of skill in the art from the (e) determining a first ratio by dividing the amount of following detailed description and figures. labeled TNFC. by the amount of labeled internal control; (f) determining a second ratio by dividing the resultant BRIEF DESCRIPTION OF THE DRAWINGS integration from step (c) by the resultant integration 55 from step (d); and FIG. 1 shows an exemplary embodiment of the assays of (g) comparing the first ratio determined in step (e) with the the present invention wherein size exclusion HPLC is used to second ratio determined in step (f). detect the binding between TNFC.-Alexa, and thereby determining the presence or level of the anti-TNFC. HUMIRATTM. drug with reference to an internal control. 60 FIG.2 shows dose responsecurves of HUMIRATM binding In another embodiment, the present invention provides a to TNFC.-Alexa. method for optimizing the therapeutic amount of an anti FIG. 3 shows a current ELISA-based method for measur TNFC. drug in a subject receiving therapy with the anti-TNFC. ing HACA levels, known as the bridging assay. drug, the method comprising: determining a Subsequent dose FIG. 4 illustrates an exemplary outline of the autoantibody of the anti-TNFC. drug for the subject based upon a compari 65 detection assays of the present invention for measuring the son of the ratio of the labeled TNFC. to the labeled internal concentrations of HACA/HAHA generated against REM control in step (e) of the previous paragraph with the ratio of ICADETTM. US 8,865,417 B2 7 8 FIG. 5 shows a dose response analysis of anti-human IgG FIG. 21 shows mobility shift profiles of Fl-labeled TNF-C. antibody binding to REMICADETTM-Alexa. incubated with normal human serum (NHS) in the presence FIG. 6 shows a second dose response analysis of anti of different amounts of ADL. Ex=494 nm; Em=519 mm. The human IgG antibody binding to REMICADETTM-Alexa7. addition of increasing amounts of ADL to the incubation FIG. 7 shows dose response curves of anti-human IgG mixture dose-dependently shifted the free TNF-Fl peak (FT) antibody binding to REMICADETTM-Alexa. to the higher molecular mass eluting positions, while the FIG.8 shows REMICADETTM-Alexa, immunocomplex internal control (IC) peak did not change. formation in normal human serum and HACA positive serum. FIG.22 shows a dose-responsecurve of ADL on the shift of FIG. 9 provides a summary of HACA measurements from free TNF-C.-Fl. Increasing amounts of ADL were incubated 20 patient serum samples that were performed using the 10 with 100 ng of TNF-C-Fland internal control. The more the bridging assay or the mobility shift assay of the present inven antibody ADL was added to the reaction mixture the lower the tion. ratio of free TNF-C-F to internal control. FIG. 10 provides a summary and comparison of current FIG. 23 shows the mobility shift profiles of Fl-labeled Remicade (IFX) Incubated with Normal (NHS) or Pooled methods for measuring serum concentrations of HACA to the 15 HACA Positive Patient Serum. novel HACA assay of the present invention. FIG. 24 shows the mobility shift profiles of Fl-Labeled FIG. 11 shows SE-HPLC profiles of fluorophore (Fl)-la HUMIRA (ADL) incubated with normal (NHS) or Mouse beled IFX incubated with normal (NHS) or HACA-positive Anti-Human IgG 1 Antibody. (HPS) serum. The addition of increasing amounts of HACA FIG. 25 shows the mobility shift profiles of Fl-Labeled positive serum to the incubation mixture dose-dependently HUMIRA (ADL) incubated with normal (NHS) or pooled shifted the IFX-F1 peak to the higher molecular mass eluting HAHA positive patient serum. positions, C1 and C2. FIG. 12 shows dose-response curves of the bound and free DETAILED DESCRIPTION OF THE INVENTION IFX-F1 generated with increasing dilutions of HACA-posi tive serum as determined by the mobility shift assay. (A) 25 I. Introduction Increasing dilutions of HACA-positive serum were incubated with 37.5ng of IFX-Fl. The higher the dilution (less HACA) The present invention is based in part on the discovery that the more free IFX-F1 was found in the SE-HPLCanalysis. (B) a homogeneous mobility shift assay using size exclusion Increasing dilutions of HACA-positive serum were incubated chromatography is particularly advantageous for measuring with 37.5ng of IFX-Fl. The higher the dilution (less HACA) 30 the presence or level of TNFC. inhibitors as well as autoanti the less HACA bound IFX-F1 was found in the SE-HPLC bodies (e.g., HACA, HAHA, etc.) that are generated against analysis. them. In particular, the present invention provides “mix and FIG. 13 shows SE-HPLC profiles of TNFC-F1 incubated read' assays that do not require any wash steps. As a result, with normal (NHS) or IFX-spiked serum. The addition of complexed and uncomplexed protein therapeutics are easily increasing amounts of IFX-spiked serum to the incubation 35 separated from each other. In addition, any potential interfer mixture dose-dependently shifted the fluorescent TNFC. peak ence from the free drug is minimized using the assays of the to the higher molecular mass eluting positions. present invention. In contrast, a typical ELISA for measuring FIG. 14 shows dose-response curves of the bound and free HACA or HAHA levels cannot be performed until the TNFC. TNFC. generated with increasing dilutions of IFX-spiked inhibitor is eliminated from the body, which can take up to 3 serum as determined by the mobility shift assay. Increasing 40 months. Moreover, the present invention is generally appli concentrations of IFX added to the incubation mixture cable to a wide variety of protein therapeutics in addition to decreases the percentage of free TNFC. while increasing the anti-TNFC. antibodies. The assays of the present invention are percentage of bound TNFC. also advantageous because they avoid the attachment of anti FIG. 15 shows the measurement of relative HACA level gens to Solid Surfaces, eliminate the non-specific binding of and IFX concentration in IBD patients treated with IFX at 45 irrelevant IgGs, detect antibodies with weak affinities, and different time points by the mobility shift assay. exhibit increased sensitivity and specificity over currently FIG. 16 shows patient management-measurement of available detection methods such as enzyme immunoassays. HACA leveland IFX concentration in the sera of IBD patients The importance of measuring serum concentrations of treated with IFX at different time points. anti-TNFC. biologics as well as other immunotherapeutics is FIG. 17 shows exemplary embodiments of the assays of the 50 illustrated by the fact that the FDA requires pharmacokinetic present invention to detect the presence of (A) non-neutral and tolerability (e.g., immune response) studies to be per izing or (B) neutralizing autoantibodies such as HACA. formed during clinical trials. The present invention also finds FIG. 18 shows an alternative embodiment of the assays of utility in monitoring patients receiving these drugs to make the present invention to detect the presence of neutralizing Sure they are getting the right dose, that the drug isn't being autoantibodies such as HACA. 55 cleared from the body too quickly, and that they are not FIG. 19 shows mobility shift profiles of Fl-labeled ADL developing an immune response against the drug. Further incubated with normal human serum (NHS) in the presence more, the present invention is useful in guiding the Switch of different amounts of anti-human IgG. The addition of between different drugs due to failure with the initial drug. increasing amounts of anti-human IgG to the incubation mix ture dose-dependently shifted the free Fl-ADL peak (FA) to 60 II. DEFINITIONS the higher molecular mass eluting positions, C1 and C2, while the internal control (IC) did not change. As used herein, the following terms have the meanings FIG. 20 shows a dose-responsecurve of anti-human IgG on ascribed to them unless specified otherwise. the shift of free Fl-ADL. Increasing amounts of anti-human The terms “anti-TNFC drug or “TNF C inhibitor” as used IgG were incubated with 37.5 ng of Fl-ADL and internal 65 herein is intended to encompass agents including proteins, control. The more the antibody was added to the reaction antibodies, antibody fragments, fusion proteins (e.g., Ig mixture the lower the ratio of free Fl-ADL to internal control. fusion proteins or Fc fusion proteins), multivalent binding US 8,865,417 B2 9 10 proteins (e.g., DVD Ig). Small molecule TNF C. antagonists in the pathophysiology, e.g., but not limited to, shock, sepsis, and similar naturally- or normaturally-occurring molecules, infections, autoimmune diseases, RA, Crohn's disease, trans and/or recombinant and/or engineered forms thereof, that, plant rejection and graft-Versus-host disease, can be the directly or indirectly, inhibit TNF C activity, such as by inhib amount that is capable of preventing or relieving one or more iting interaction of TNFC. with a cell surface receptor for TNF symptoms associated therewith. C., inhibiting TNF C protein production, inhibiting TNF C. The phrase “area-under-the-curve' is a mathematical term gene expression, inhibiting TNF C. Secretion from cells, of art used to describe an integration of a portion of a two inhibiting TNF C. receptor signaling or any other means dimensional plot. For example, in a plot of signal intensity as resulting in decreased TNF C. activity in a subject. The term a function of elution time from a size exclusion chromatog “anti-TNFC drug” or “TNF C inhibitor” preferably includes 10 raphy, various peaks in the plot indicate the detection of agents which interfere with TNF C activity. Examples of TNF particular molecules. The integration of these peaks includes C. inhibitors include etanercept (ENBRELTM, Amgen), inflix the area circumscribed by a minimum y-axis value, e.g. the imab (REMICADETM, Johnson and Johnson), human anti baseline of the two dimensional plot, and circumscribed by TNF monoclonal antibody adalimumab (D2E7/HUMIRATM, the two dimensional plot itself. The integration of a peak is Abbott Laboratories), certolizumab pegol (CIMZIAR, UCB, 15 also equally described by the calculus formula, Area-under Inc.), CDP 571 (Celltech), and CDP870 (Celltech), as well as the-curve-?,f(x) dx, where f(x) is a function describing the other compounds which inhibit TNF C activity, such that two dimensional plot and variables “a” and “b’ indicate the when administered to a subject suffering from or at risk of X-axis limits of the integrated peak. suffering from a disorder in which TNF C activity is detri The phrase “fluorescence label detection' includes a mental (e.g., R.A), the disorder is treated. means for detecting a fluorescent label. Means for detection The term “predicting responsiveness to a TNF C inhibitor, include, but are not limited to, a spectrometer, a fluorimeter, a as used herein, is intended to refer to an ability to assess the photometer, a detection device commonly incorporated with likelihood that treatment of a subject with a TNF C. inhibitor a chromatography instrument Such as, but not limited to, a will or will not be effective in (e.g., provide a measurable size exclusion-high performance liquid chromatography, benefit to) the Subject. In particular, Such an ability to assess 25 such as, but not limited to, an Agilent-1200 HPLC System. the likelihood that treatment will or will not be effective Brackets, “I” indicate that the species within the brackets typically is exercised after treatment has begun, and an indi are referred to by their concentration. cator of effectiveness (e.g., an indicator of measurable ben The phrase “optimize therapy” includes optimizing the efit) has been observed in the subject. Particularly preferred dose (e.g., the effective amount or level) and/or the type of a TNFC. inhibitors are biologic agents that have been approved 30 particular therapy. For example, optimizing the dose of an by the FDA for use in humans in the treatment of TNFC.- anti-TNFC. drug includes increasing or decreasing the mediated diseases or disorders such as, e.g., rheumatoid amount of the anti-TNFC. drug subsequently administered to arthritis, or inflammatory bowel disease (IBD), which agents a Subject. In certain instances, optimizing the type of an include adalimumab (HUMIRATTM), infliximab (REMI anti-TNFC. drug includes changing the administered anti CADETM) etanercept (ENBRELTM), and certolizumab pegol 35 TNFC. drug from one drug to a different drug (e.g., a different (CIMZIAR, UCB, Inc.). anti-TNFC. drug). In certain other instances, optimizing The term “size exclusion chromatography” (SEC) is therapy include co-administering a dose of an anti-TNFC. intended to include a chromatographic method in which mol drug (e.g., at an increased, decreased, or same dose as the ecules in Solution are separated based on their size and/or previous dose) in combination with an immunosuppressive hydrodynamic Volume. It is applied to large molecules or 40 drug. macromolecular complexes such as proteins and their conju The term “co-administer' includes to administer more than gates. Typically, when an aqueous solution is used to trans one active agent, such that the duration of physiological effect port the sample through the column, the technique is known of one active agent overlaps with the physiological effect of a as gel filtration chromatography. second active agent. The terms “complex,” “immuno-complex,” “conjugate.” 45 The term “subject,” “patient or “individual typically and “immunoconjugate' include, but are not limited to, refers to humans, but also to other animals including, e.g., TNFC. bound (e.g., by non-covalent means) to an anti-TNFC. other primates, rodents, canines, felines, equines, Ovines, por drug, an anti-TNFC. drug bound (e.g., by non-covalent cines, and the like. means) to an autoantibody against the anti-TNFC. drug, and The term “course of therapy” includes any therapeutic an anti-TNFC. drug bound (e.g., by non-covalent means) to 50 approach taken to relieve or prevent one or more symptoms both TNFC. and an autoantibody against the anti-TNFC. drug. associated with a TNFC.-mediated disease or disorder. The As used herein, an entity that is modified by the term term encompasses administering any compound, drug, pro "labeled includes any entity, molecule, protein, enzyme, cedure, and/or regimen useful for improving the health of an antibody, antibody fragment, cytokine, or related specie that individual with a TNFC.-mediated disease or disorder and is conjugated with another molecule or chemical entity that is 55 includes any of the therapeutic agents described herein. One empirically detectable. Chemical species suitable as labels skilled in the art will appreciate that either the course of for labeled-entities include, but are not limited to, fluorescent therapy or the dose of the current course of therapy can be dyes, e.g. Alexa Fluor R. dyes such as Alexa Fluor R 647, changed (e.g., increased or decreased) based upon the pres quantum dots, optical dyes, luminescent dyes, and radionu ence or concentration level of an anti-TNFC. drug and/or an clides, e.g. 'I. 60 autoantibody to the anti-TNFC. drug. The term “effective amount includes a dose of a drug that The term “immunosuppressive drug or “immunosuppres is capable of achieving atherapeutic effectina Subject in need sive agent” includes any Substance capable of producing an thereofas well as the bioavailable amount of a substance. The immunosuppressive effect, e.g., the prevention or diminution term “bioavailable' includes the fraction of an administered of the immune response, as by irradiation or by administra dose of a drug that is available for therapeutic activity. For 65 tion of drugs such as anti-metabolites, anti-lymphocyte Sera, example, an effective amount of a drug useful for treating antibodies, etc. Examples of Suitable immunosuppressive diseases and disorders in which TNF-C. has been implicated drugs include, without limitation, thiopurine drugs such as US 8,865,417 B2 11 12 azathioprine (AZA) and metabolites thereof anti-metabo of TNFC. is in the regulation of immune cells. TNFC. is also lites such as methotrexate (MTX); sirolimus (rapamycin); able to induce apoptotic cell death, to induce inflammation, temsirolimus; everolimus; tacrolimus (FK-506); FK-778: and to inhibit tumorigenesis and viral replication. TNF is anti-lymphocyte globulin antibodies, anti-thymocyte globu primarily produced as a 212-amino acid-long type II trans lin antibodies, anti-CD3 antibodies, anti-CD4 antibodies, and membrane protein arranged in stable homotrimers. antibody-toxin conjugates; cyclosporine, mycophenolate; mizoribine monophosphate; scoparone; glatiramer acetate; The term “TNF-C. as used herein, is intended to include a metabolites thereof: pharmaceutically acceptable salts human cytokine that exists as a 17 kDa secreted form and a 26 thereof; derivatives thereof; prodrugs thereof; and combina kDa membrane associated form, the biologically active form tions thereof. 10 of which is composed of a trimer of noncovalently bound 17 The term “thiopurine drug includes azathioprine (AZA). kDa molecules. The structure of TNF-C. is described further 6-mercaptopurine (6-MP), or any metabolite thereof that has in, for example, Jones, et al. (1989) Nature, 338:225-228. The therapeutic efficacy and includes, without limitation, 6-thioguanine (6-TG), 6-methylmercaptopurine riboside, term TNF-C. is intended to include human, a recombinant 6-thioinosine nucleotides (e.g., 6-thioinosine monophos 15 human TNF-C. (rhTNF-C.), or at least about 80% identity to phate, 6-thioinosine diphosphate, 6-thioinosine triphos the human TNFC. protein. Human TNFC. consists of a 35 phate), 6-thioguanine nucleotides (e.g., 6-thioguanosine amino acid (aa) cytoplasmic domain, a 21 aa transmembrane monophosphate, 6-thioguanosine diphosphate, 6-thiogua segment, and a 177 aa extracellular domain (ECD) (Pennica, nosine triphosphate), 6-thioxanthosine nucleotides (e.g., D. et al. (1984) Nature 312:724). Within the ECD, human 6-thioxanthosine monophosphate, 6-thioxanthosine diphos TNFC. shares 97% aa sequence identity with rhesus and 71% phate, 6-thioxanthosine triphosphate), derivatives thereof, analogues thereof, and combinations thereof. 92% with bovine, canine, cotton rat, equine, feline, mouse, The term "sample' includes any biological specimen porcine, and rat TNFC. TNFC. can be prepared by standard obtained from an individual. Suitable samples for use in the recombinant expression methods or purchased commercially present invention include, without limitation, whole blood, 25 (R& D Systems, Catalog No. 210-TA, Minneapolis, Minn.). plasma, serum, saliva, urine, stool, tears, any other bodily In certain embodiments, “TNF-C is an “antigen.” which is fluid, tissue samples (e.g., biopsy), and cellular extracts a molecule or a portion of the molecule capable of being thereof (e.g., red blood cellular extract). In a preferred embodiment, the sample is a serum sample. One skilled in the bound by an anti-TNF-C. antibody. TNF-C. can have one or art will appreciate that samples Such as serum samples can be 30 more than one epitope. In certain instances, TNF-C. will react, diluted prior to the analysis. In certain instances, the term in a highly selective manner, with an anti-TNF-C. antibody. “sample” includes, but is not limited to blood, body tissue, Preferred antigens that bind antibodies, fragments and blood serum, lymph fluid, lymph node tissue, spleen tissue, regions of anti-TNF C. antibodies of the present invention bone marrow, or an immunoglobulin enriched fraction include at least 5 amino acids of SEQ ID NO:1. In certain derived from one or more of these tissues. In certain other 35 instances, TNF-C. is a sufficient length having an epitope of instances, the term "sample includes blood serum or is an TNF C. that is capable of binding anti-TNF-C. antibodies, immunoglobulin enriched fraction derived from blood serum fragments and regions thereof. or blood. In certain instances, the term “sample' includes a In certain embodiments, the length of TNF-C. having an bodily fluid. epitope of sufficient length to bind an anti-TNF-C. antibody, 40 fragments and regions thereof is at least 5, 10, 15, 20, 25, 30. 35, 40, 45,50,55, 60, 65,70, 75,80, 85,90, 95, 100,105,110, III. DESCRIPTION OF THE EMBODIMENTS 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165,170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220 amino acids in length. In one embodiment, the TNF-C., used includes The steps of the methods of the present invention do not 45 necessarily have to be performed in the particular order in residues 77-233 of SEQ ID NO:1. which they are presented. A person of ordinary skill in the art In certain instances, at least one amino acid in the soluble would understand that other orderings of the steps of the portion of TNF-C. is labeled i.e., residues 77-233 of SEQ ID methods of the present invention are encompassed within the NO:1. In certain instances, an amine reactive fluorophore Scope of the present invention. 50 derivative is used. In most instances, the amine reactive group In one embodiment, the present invention provides a is an acylating reagent that forms carboxamides, Sulfona method for detecting the presence or level of an anti-TNFC. mides or thioureas upon reaction with amines. Virtually all drug in a sample, comprising: proteins have lysine residues, and most have a free amine at (a) contacting labeled TNFC. with a sample having or Sus the N-terminus and thus can be used as a point of attachment pected of having an anti-TNFC. drug to form a labeled com 55 to label. In a preferred embodiment, residue 77 is labeled, and plex with the anti-TNFC. drug; the TNF-C. used includes residues 77-233 of SEQIDNO:1. In (b) Subjecting the labeled complex to size exclusion chro other embodiments, many primary amines are labeled and the matography to separate the labeled complex; and TNF-C. is multiply labeled. (c) detecting the labeled complex, thereby detecting the In certain instances, a portion of TNF-C. is not labeled, i.e., anti-TNFC. drug. 60 portions that are recognized by antibodies, and fragments and In certain instances, the methods are especially useful for regions thereof. In other words, certain portions of TNF-C. the following anti-TNFC. antibodies: REMICADETM (inflix antigens provide a topographical or three dimensional imab), ENBRELTM (etanercept), HUMIRATTM (adali epitope of TNF which is recognized by, and/or binds with mumab), and CIMZIAR (certolizumab pegol). anti-TNF C activity, an antibody, and fragments, and variable Tumor necrosis factor C. (TNFC.) is a cytokine involved in 65 regions thereof. These portions are preferably free to bind, systemic inflammation and is a member of a group of cytok and thus are not labeled. They include residues 136-157 and ines that stimulate the acute phase reaction. The primary role 164-185 of SEQID NO:1: US 8,865,417 B2 13 14
(SEQ ID NO: 2) Tyr-Ser-Gln-Wal-Leu-Phe-Lys-Gly-Gln-Gly-Cys-Pro-Ser-Thr-His-Val-Leu-Leu-Thr-His
Thr-Ile; and
(SEQ ID NO : 3) Tyr-Gln-Thr-Lys-Val-Asn-Leu-Leu-Ser-Ala-Ile-Lys-Ser-Pro-Cys-Gln-Arg-Glu-Thr-Pro Glu-Gly.
TNFC. or an anti-TNFC. drug can be labeled with a variety 650 to about 900 nm. Use of near infrared fluorescence tech of detectable group(s). Preferably, TNFC. or an anti-TNFC. nology is advantageous in biological assays as it substantially drug is labeled with a fluorophore or a fluorescent dye. Exem eliminates or reduces background from auto fluorescence of plary fluorophores suitable for use in the present invention 15 biosubstrates. Another benefit to the near-IR fluorescent tech include those listed in the Molecular Probes Catalogue, which nology is that the scattered light from the excitation source is is herein incorporated by reference (see, R. Haugland, The greatly reduced since the scattering intensity is proportional Handbook A Guide to Fluorescent Probes and Labeling to the inverse fourth power of the wavelength. Low back Technologies, 10" Edition, Molecular probes, Inc. (2005)). ground fluorescence and low scattering result in a high signal Such exemplary fluorophores include, but are not limited to, to noise ratio, which is essential for highly sensitive detection. Alexa Fluor R, dyes such as Alexa Fluor R350, Alexa Fluor R Furthermore, the optically transparent window in the near-IR 405, Alexa Fluor R 430, Alexa Fluor R. 488, Alexa Fluor R region (650 nm to 900 nm) in biological tissue makes NIR 514, Alexa Fluor R. 532, Alexa Fluor R. 546, Alexa Fluor R fluorescence a valuable technology for in vivo imaging and 555, Alexa Fluor R 568, Alexa Fluor R 594, Alexa Fluor R Subcellular detection applications that require the transmis 610, Alexa Fluor R 633, Alexa Fluor R 635, Alexa Fluor R 25 sion of light through biological components. Within aspects 647, Alexa Fluor R 660, Alexa Fluor R 680, Alexa Fluor R of this embodiment, the fluorescent group is preferably 700, Alexa Fluor R 750, and/or Alexa Fluor R 790, as well as selected form the group consisting of IRDyeR 700DX, other fluorophores such as, for example, Dansyl Chloride IRDye R 700, IRDyeR 800RS, IRDyeR 800CW, IRDyeR (DNS C1), 5-(iodoacetamida) fluoroscein (5-IAF), fluoros 800, Alexa Fluorr 660, Alexa Fluor R 680, Alexa Fluor R cein 5-isothiocyanate (FITC), tetramethylrhodamine 5-(and 30 700, Alexa Fluor R 750, Alexa Fluor R 790, Cy5, Cy5.5, Cy7, 6-)isothiocyanate (TRITC), 6-acryloyl-2-dimethylaminon DY 676, DY680, DY682 and DY780. In certain embodi aphthalene (acrylodan), 7-nitrobenzo-2-oxa-1,3-diazol-4-yl ments, the near infrared group is JRDyeR 800CW, IRDye(R) chloride (NBD-C1), ethidium bromide, Lucifer Yellow, 800, IRDye R 700DX, IRDye R 700, or Dynomic DY676. 5-carboxyrhodamine 6G hydrochloride, Lissamine Fluorescent labeling is accomplished using a chemically rhodamine B sulfonyl chloride, Texas RedTM sulfonyl chlo 35 reactive derivative of a fluorophore. Common reactive groups ride, BODIPYTM, naphthalamine sulfonic acids (e.g., 1-anili include amine reactive isothiocyanate derivatives such as nonaphthalene-8-sulfonic acid (ANS), 6-(p-toluidinyl)maph FITC and TRITC (derivatives of fluorescein and rhodamine), thalene-2-sulfonic acid (TNS), and the like), Anthroyl fatty amine reactive succinimidyl esters such as NHS-fluorescein, acid, DPH, Parinaric acid, TMA-DPH, Fluorenyl fatty acid, and sulfhydryl reactive maleimide activated fluors such as fluorescein-phosphatidylethanolamine, Texas Red-phos 40 fluorescein-5-maleimide, many of which are commercially phatidylethanolamine, Pyrenyl-phophatidylcholine, Fluore available. Reaction of any of these reactive dyes with TNFC. nyl-phosphotidylcholine, Merocyanine 540, 1-(3-sulfonato oran anti-TNF(R) drug results in a stable covalent bond formed propyl)-4-3-2(di-n-butylamino)-6naphthylvinyl between a fluorophore and TNFR) or an anti-TNFC. drug. pyridinium betaine (Naphtyl Styryl), 3,3' In certain instances, following a fluorescent labeling reac dipropylthiadicarbocyanine (dis-C-(5)), 4-(p-dipentyl ami 45 tion, it is often necessary to remove any nonreacted fluoro nostyryl)-1-methylpyridinium (di-5-ASP), Cy-3 Iodo Aceta phore from the labeled target molecule. This is often accom mide, Cy-5-N-Hydroxysuccinimide, Cy-7-Isothiocyanate, plished by size exclusion chromatography, taking advantage rhodamine 800, IR-125. Thiazole Orange, Azure B, Nile of the size difference between fluorophore and labeled pro Blue, Al Phthalocyanine, Oxaxine 1,4,6-diamidino-2-phe tein. nylindole (DAPI), Hoechst 33342, TOTO, Acridine Orange, 50 Reactive fluorescent dyes are available from many sources. Ethidium Homodimer, N(ethoxycarbonylmethyl)-6-methox They can be obtained with different reactive groups for ycuinolinium (MQAE). Fura-2, Calcium Green, Carboxy attachment to various functional groups within the target SNARF-6, BAPTA, coumarin, phytofluors, Coronene, metal molecule. They are also available in labeling kits that contain ligand complexes, IRDyeR 700DX, IRDyeR 700, IRDye(R) all the components to carry out a labeling reaction. In one 800RS, IRDyeR 800CW, IRDyeR 800, Cy5, Cy5.5, Cy7, 55 preferred aspect, Alexa Fluor R 647 C2 maleimide is used DY676, DY680, DY682, DY780, and mixtures thereof. Addi from Invitrogen (Cat. No. A-20347). tional Suitable fluorophores include enzyme-cofactors; lan Specific immunological binding of an anti-TNFC. antibody thanide, green fluorescent protein, yellow fluorescent protein, to TNFC. or of an anti-drug antibody (ADA) to an anti-TNFC. red fluorescent protein, or mutants and derivates thereof. In antibody can be detected directly or indirectly. Direct labels one embodiment of the invention, the second member of the 60 include fluorescent or luminescent tags, metals, dyes, radio specific binding pair has a detectable group attached thereto. nuclides, and the like, attached to the antibody. In certain Typically, the fluorescent group is a fluorophore selected instances, TNFC. or an anti-TNFC. antibody labeled with from the category of dyes comprising polymethines, pthalo iodine-125 ('I) can be used for determining the concentra cyanines, cyanines, Xanthenes, fluorenes, rhodamines, cou tion levels of anti-TNFC. antibody or ADA in a sample, marins, fluoresceins and BODIPYTM. 65 respectively. In other instances, a chemiluminescence assay In one embodiment, the fluorescent group is a near-infrared using a chemiluminescent TNFC. or anti-TNFC. antibody spe (NIR) fluorophore that emits in the range of between about cific for anti-TNFC. antibody or ADA in a sample, respec US 8,865,417 B2 15 16 tively, is suitable for sensitive, non-radioactive detection of detection techniques useful in the present invention include, anti-TNFC. antibody or ADA concentration levels. In particu but are not limited to, fluorometry, refractive index (RI), and lar instances, TNFC. or an anti-TNFC. antibody labeled with a ultraviolet (UV). In certain instances, the elution volume fluorochrome is also Suitable for determining the concentra decreases roughly linearly with the logarithm of the molecu tion levels of anti-TNFC. antibody or ADA in a sample, lar hydrodynamic Volume (i.e., heavier moieties come off respectively. Examples of fluorochromes include, without first). limitation, Alexa Fluor R. dyes, DAPI, fluorescein, Hoechst In certain aspects, the methods are useful in detecting the 33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, amount of anti-TNFC. drugs such as, e.g., antibodies includ rhodamine, Texas red, and lissamine. Secondary antibodies ing REMICADETM (infliximab), a chimericanti-TNFC. mAb, linked to fluorochromes can be obtained commercially, e.g., 10 goat F(ab') anti-human IgG-FITC is available from Tago ENBRELTM (etanercept), a TNFR-Ig Fc fusion protein, Immunologicals (Burlingame, Calif.). HUMIRATM (adalimumab), a human anti-TNFC. mAb, and Indirect labels include various enzymes well-known in the CIMZIAR (certolizumab pegol), a PEGylated Fab fragment. art, such as horseradish peroxidase (HRP), alkaline phos In certain instances, after the anti-TNFC. drug (e.g., anti phatase (AP), B-galactosidase, urease, and the like. A horse 15 TNFC. antibody) is detected, the anti-TNF C drug is measured radish-peroxidase detection system can be used, for example, using a standard curve. with the chromogenic substrate tetramethylbenzidine In another embodiment, the present invention provides a (TMB), which yields a soluble product in the presence of method for detecting the presence or level of an autoantibody hydrogen peroxide that is detectable at 450 nm. An alkaline to an anti-TNFC. drug in a sample, the method comprising: phosphatase detection system can be used with the chro (a) contacting labeled anti-TNFC. drug with the sample to mogenic Substrate p-nitrophenyl phosphate, for example, form a labeled complex with the autoantibody; which yields a soluble product readily detectable at 405 nm. (b) Subjecting the labeled complex to size exclusion chro Similarly, a B-galactosidase detection system can be used matography to separate the labeled complex; and with the chromogenic Substrate o-nitrophenyl-B-D-galacto (c) detecting the presence or level labeled complex, thereby pyranoside (ONPG), which yields a soluble product detect 25 detecting the autoantibody. able at 410 nm. An urease detection system can be used with In certain instances, the autoantibodies include human a Substrate such as urea-bromocresol purple (Sigma Immu anti-chimeric antibodies (HACA), human anti-humanized nochemicals; St. Louis, Mo.). A useful secondary antibody antibodies (HAHA), and human anti-mouse antibodies linked to an enzyme can be obtained from a number of com (HAMA). mercial sources, e.g., goat F(ab') anti-human IgG-alkaline 30 In yet another embodiment, the present invention provides phosphatase can be purchased from Jackson ImmunoRe a method for detecting the presence or level of an autoanti search (West Grove, Pa.). body to an anti-TNFC. drug in a sample, comprising: A signal from the direct or indirect label can be analyzed, (a) contacting a labeled anti-TNFC. drug and labeled TNFC. for example, using a spectrophotometer to detect color from with a sample having or Suspected of having an autoantibody a chromogenic Substrate; a radiation counter to detect radia 35 to the anti-TNFC. drug to form a first labeled complex tion such as a gamma counter for detection of ''I; or a between the labeled anti-TNFC. drug, the labeled TNFC, and fluorometer to detect fluorescence in the presence of light of the autoantibody and a second labeled complex between the a certain wavelength. For detection of enzyme-linked anti labeled anti-TNFC. drug and the autoantibody, wherein the bodies, a quantitative analysis of the amount of anti-TNFC. labeled anti-TNFC. drug and the labeled TNFC. comprise dif antibody or ADA levels can be made using a spectrophotom 40 ferent labels; eter such as an EMAX Microplate Reader (Molecular (b) subjecting the first labeled complex and the second Devices: Menlo Park, Calif.) in accordance with the manu labeled complex to size exclusion chromatography to sepa facturers instructions. If desired, the assays of the present rate the first labeled complex and the second labeled complex: invention can be automated or performed robotically, and the and signal from multiple samples can be detected simultaneously. 45 (c) detecting the first labeled complex and the second In certain embodiments, size exclusion chromatography is labeled complex, thereby detecting a non-neutralizing form used. The underlying principle of SEC is that particles of of the autoantibody when both the first labeled complex and different sizes will elute (filter) through a stationary phase at the second labeled complex are present, and detecting a neu different rates. This results in the separation of a solution of tralizing form of the autoantibody when only the second particles based on size. Provided that all the particles are 50 labeled complex is present. loaded simultaneously or near simultaneously, particles of In certain instances, the autoantibodies include human the same size elute together. Each size exclusion column has anti-chimeric antibodies (HACA), human anti-humanized a range of molecular weights that can be separated. The antibodies (HAHA), and human anti-mouse antibodies exclusion limit defines the molecular weight at the upper end (HAMA). of this range and is where molecules are too large to be 55 In a related embodiment, the present invention provides a trapped in the stationary phase. The permeation limit defines method for detecting the presence or level of an autoantibody the molecular weight at the lower end of the range of separa to an anti-TNFC. drug in a sample, comprising: tion and is where molecules of a small enough size can pen (a) contacting a labeled anti-TNFC. drug with a sample etrate into the pores of the stationary phase completely and all having or Suspected of having an autoantibody to the anti molecules below this molecular mass are so Small that they 60 TNFC. drug to form a first labeled complex between the elute as a single band. labeled anti-TNFC. drug and the autoantibody; In certain aspects, the eluent is collected in constant Vol (b) subjecting the first labeled complex to a first size exclu umes, or fractions. The more similar the particles are in size, sion chromatography to separate the first labeled complex; the more likely they will be in the same fraction and not (c) detecting the first labeled complex, thereby detecting detected separately. Preferably, the collected fractions are 65 the presence or level of the autoantibody; examined by spectroscopic techniques to determine the con (d) contacting labeled TNFC. with the first labeled complex centration of the particles eluted. Typically, the spectroscopy to form a second labeled complex between the labeled anti US 8,865,417 B2 17 18 TNFC. drug and the labeled TNFC, wherein the labeled anti (4) multiplying the amount of the labeled TNFC. by the TNFC. drug and the labeled TNFC. comprise different labels; ratio of step (3). (e) Subjecting the second labeled complex to a second size In a related embodiment, the present invention further pro exclusion chromatography to separate the second labeled vides that the detection in step (b)(ii) comprises: complex; and 5 (1) integrating the area-under-the curve for a peak of the (f) detecting the second labeled complex, thereby detecting labeled anti-TNFC. drug from a second plot of signal the presence or level of a neutralizing form of the autoanti intensity as a function of elution time from the size body. exclusion chromatography; In certain instances, the autoantibodies include human (2) integrating the area-under-the curve for a peak of the anti-chimeric antibodies (HACA), human anti-humanized 10 second complex from the second plot; antibodies (HAHA), and human anti-mouse antibodies (3) determining a ratio by dividing the resultant integration (HAMA). from step (2) by the resultant integration from step (1): In other embodiments, the assay methods described herein and can be used to predict responsiveness to a TNF C. inhibitor, 15 (4) multiplying the amount of the labeled anti-TNFC. drug especially to an anti-TNF C. antibody in a subject having an by the ratio of step (3). autoimmune disorder (e.g., rheumatoid arthritis, Crohn's In a related embodiment, the present invention further pro Disease, and the like). In this method, by assaying the Subject vides that both the first and second samples are serum for the correct or therapeutic dose of anti-TNF C antibody, samples. In another related embodiment, the present inven i.e., the therapeutic concentration level, it is possible to pre tion further provides that both the first and second samples are dict whether the individual will be responsive to the therapy. obtained from the subject during therapy with the anti-TNFC. In another embodiment, the present invention provides drug. In yet another related embodiment, the present inven methods for monitoring an autoimmune disorder in a subject tion further provides that the detection in step (a)(ii) and/or having the autoimmune disorder, wherein the method com step (b)(ii) comprises fluorescence label detection. prises assaying the Subject for the correct or therapeutic dose 25 In another embodiment, the present invention provides a of anti-TNF C. antibody, i.e., the therapeutic concentration method for optimizing the therapeutic amount of an anti level, over time. In this manner, it is possible to predict TNFC. drug in a subject receiving therapy with the anti-TNFC. whether the individual will be responsive to the therapy over drug, the method comprising: the given time period. (a) determining an effective amount of the anti-TNFC. drug In another embodiment, the present invention provides a 30 method for determining an effective amount of an anti-TNFC. in accordance with the methods of the present invention; drug for a subject receiving therapy with the anti-TNFC. drug, (b) comparing the effective amount of the anti-TNFC. drug the method comprising: with the level of the anti-TNFC. drug; and (a) measuring the level of the anti-TNFC. drug in a first (c) determining a Subsequent dose of the anti-TNFC. drug sample from the Subject, comprising: 35 for the Subject based upon the comparison of step (c), (i) contacting the first sample with an amount of a thereby optimizing the therapeutic amount of the anti-TNFC. labeled TNFC. to form a first complex comprising the drug. labeled TNFC. with the anti-TNFC. drug; and In a related embodiment, the present invention further pro (ii) detecting the first complex by size exclusion chro vides increasing the subsequent dose of the anti-TNFC. drug matography, 40 when the effective amount of the anti-TNFC. drug is less than thereby measuring the level of the anti-TNFC. drug; the level of the anti-TNFC. drug. In a related embodiment, the (b) measuring the level of an autoantibody to the anti present invention provides that the Subsequent dose of the TNFC. drug in a second sample from the Subject, com anti-TNFC. drug is increased such that the effective amount of prising: the anti-TNFC. drug is about equal to the level of the anti (i) contacting the second sample with an amount of a 45 TNFC. drug. labeled anti-TNFC. drug to form a second complex In some other embodiments, the present invention further comprising the labeled anti-TNFC. drug with the provides that the anti-TNFC. drug is a member selected from autoantibody; and the group consisting of REMICADETM (infliximab), (ii) detecting the second complex by size exclusion chro ENBRELTM (etanercept), HUMIRATM (adalimumab), CIM matography, 50 ZIAR (certolizumab pegol), and combinations thereof. thereby measuring the level of the autoantibody; and In some embodiments, the anti-TNFC. drug is infliximab (c) subtracting the level of the autoantibody measured in (REMICADETM). In some other embodiments, the anti step (b) from the level of the anti-TNFC. drug measured TNFC. drug is adalimumab (HUMIRATM). In other embodi in step (a), ments, the anti-TNFC drug is etanercept (ENBRELTM). In thereby determining the effective amount of the anti-TNFC. 55 some other embodiments, the anti-TNFC. drug is CIMZIAR drug. (certolizumab pegol). In other embodiments, the labeled In a related embodiment, the present invention further pro TNFC. is a fluorophore labeled TNFC. In some other embodi vides that the detection in step (a)(ii) comprises: ments, the measured anti-TNFC. drug is quantitated. In some (1) integrating the area-under-the curve for a peak of the embodiments, the measured TNFC. is quantitated. In other labeled TNFC. from a first plot of signal intensity as a 60 embodiments, the labeled complex is eluted first, followed by function of elution time from the size exclusion chroma free labeled TNFC. In some other embodiments, the labeled tography: complex is eluted first, followed by free labeled anti-TNFC. (2) integrating the area-under-the curve for a peak of the antibody. In some embodiments, the size exclusion chroma first complex from the first plot; tography is size exclusion-high performance liquid chroma (3) determining a ratio by dividing the resultant integration 65 tography (SE-HPLC). In some other embodiments, the from step (2) by the resultant integration from step (1): autoantibody is a member selected from human anti-mouse and antibody (HAMA), human anti-chimeric antibody (HACA), US 8,865,417 B2 19 20 human anti-humanized antibody (HAHA), or combinations about 2.0 to about 20 ng/10 ul, about 2.0 to about 10 ng/10 ul, thereof. In some embodiments, the measured autoantibody is about 2.0 to about 5.0 ng/10 ul, or about 2.0 to about 5.0 ng/10 quantitated. ul. In another embodiment, the present invention provides a In some embodiments, the phrase “low level of an anti method for optimizing therapy and/or reducing toxicity to an drug antibody' includes anti-drug antibody levels of about anti-TNFC. drug in a subject receiving therapy with the anti 0.0 to about 5.0 ng/10 ul, about 0.1 to about 5.0 ng/10 ul, TNFC. drug, the method comprising: about 0.0 to about 2.0 ng/10 ul, about 0.1 to about 2.0 ng/10 (a) measuring the level of the anti-TNFC. drug in a first ul, or about 0.5 to about 2.0 ng/10 ul. In other embodiments, sample from the Subject; the phrase “low level of anti-drug antibody' includes anti (b) measuring the level of an autoantibody to the anti 10 drug antibody levels of about less than about 5.0, 4.0.3.0, 2.0, TNFC. drug in a second sample from the subject; and 1.0, or 0.5 ng/10 ul. (c) determining a Subsequent course of therapy for the In some embodiments, the methods of the present inven subject based upon the levels of the anti-TNFC. drug and tion further provide that anti-TNFC. drug is measured with an the autoantibody, assay comprising: thereby optimizing therapy and/or reducing toxicity to the 15 (i) contacting the first sample with an amount of a labeled anti-TNFC. drug. TNFC. to form a first complex comprising the labeled In a related embodiment, the Subsequent course of therapy TNFC. with the anti-TNFC. drug; and comprises co-administering an immunosuppressive drug (ii) detecting the first complex by size exclusion chroma with the anti-TNFC. drug when the level of the anti-TNFC. tography, thereby measuring the level of the anti-TNFC. drug is a high level and the level of the autoantibody is a low drug. level. In another related embodiment, the subsequent course In some embodiments, the methods of the present inven of therapy comprises increasing the level of the anti-TNFC. tion further provide that the autoantibody is measured with an drug and co-administering an immunosuppressive drug when assay comprising: the level of the anti-TNFC. drug is a medium level and the (i) contacting the second sample with an amount of a level of the autoantibody is a low level. In another related 25 labeled anti-TNFC. drug to form a second complex com embodiment, the Subsequent course of therapy comprises prising the labeled anti-TNFC. drug with the autoanti administering a different anti-TNFC. drug when the level of body; and the anti-TNFC. drug is a medium level and the level of the (ii) detecting the second complex by size exclusion chro autoantibody is a medium level. In yet another related matography, embodiment, the Subsequent course of therapy comprises 30 thereby measuring the level of the autoantibody. administering a different anti-TNFC. drug when the level of In a related embodiment, the methods of the present inven the anti-TNFC. drug is a low level and the level of the autoan tion further provide that the anti-TNFC. drug is a member tibody is a high level. In another related embodiment, adali selected from the group consisting of REMICADETM (inflix mumab (HUMIRATM) is administered instead of infliximab imab), ENBRELTM (etanercept), HUMIRATM (adalimumab), (REMICADETM). 35 CIMZIAR (certolizumab pegol), and combinations thereof. In some embodiments, the phrase “high level of an anti In a related embodiment, the methods of the present invention TNFC. drug includes drug levels of about 10 to about 100 further provide that the anti-TNFC. drug is infliximab (REMI ng/10 uL, about 10 to about 70 ng/10 uL, or about 10 to about CADETM). In some other embodiments, the anti-TNFC. drug 50 ng/10 uL. In other embodiments, the phrase “high level of is adalimumab (HUMIRATM). In some other embodiments, anti-TNFC. drug includes drug levels greater than about 10, 40 the anti-TNFC drug is etanercept (ENBRELTM). In another 20, 30, 40, 50, 60, 70, 80,90, or 100 ng/10 uL. related embodiment, the methods of the present invention In some embodiments, the phrase “medium level of an further provide that the anti-TNFC. drug is CIMZIAR (cer anti-TNFC. drug” includes drug levels of about 5.0 to about 50 tolizumab pegol). In another related embodiment, the meth ng/10 uL, about 5.0 to about 30 ng/10 uL, about 5.0 to about ods of the present invention further provide that the measured 20 ng/10 uL, or about 5.0 to about 10 ng/10 uL. In other 45 anti-TNFC. drug is quantitated. In another related embodi embodiments, the phrase “medium level of anti-TNFC. drug’ ment, the methods of the present invention further provide includes drug levels of about 10,9,8,7,6, 5, 4, 3, 2, or 1 ng/10 that both the first and second samples are serum. In another LL. related embodiment, the methods of the present invention In some embodiments, the phrase “low level of an anti further provide that both the first and second samples are TNFC. drug includes drug levels of about 0 to about 10 ng/10 50 obtained from the subject during therapy with the anti-TNFC. ul, about 0 to about 8 ng/10 ul, or about 0 to about 5 ng/10 ul. drug. In another related embodiment, the methods of the In other embodiments, the phrase “low level of an anti-TNFC. present invention further provide that the autoantibody is a drug includes drug levels of about less than about 10, 9.0, member selected from human anti-mouse antibody (HAMA), 8.0, 7.0, 6.0, 5.0, 4.0, 3.0, 2.0, 1.0, or 0.5 ng/10 ul human anti-chimeric antibody (HACA), human anti-human The acronym 'ADA’ includes the phrase “anti-drug anti 55 ized antibody (HAHA), or combinations thereof. In another body.” related embodiment, the methods of the present invention In some embodiments, the phrase “high level of an anti further provide that the measured autoantibody is quantitated. drug antibody' includes anti-drug antibody levels of about In some embodiments, the present invention provides a 3.0 to about 100 ng/10 uL, about 3.0 to about 50 ng/10 uL. method for determining the presence or level of an anti-TNFC. about 10 to about 100 ng/104, about 10 to about 50 ng/10 uL. 60 drug with reference to an internal control in a sample having or about 20 to about 50 ng/10 uL. In some other embodiments, or suspected of having the anti-TNFC. drug, the method com the phrase “high level of anti-drug antibody' includes anti prising: drug antibody levels of about greater than about 10, 20, 30. (a) contacting an amount of a labeled TNFC. and an amount 40, 50, 60, 70, 80,90, or 100 ng/10 ul. of a labeled internal control with the sample to form a In some embodiments, the phrase “medium level of an 65 complex of the labeled TNFC. and the anti-TNFC. drug; anti-drug antibody includes anti-drug antibody levels of (b) detecting the labeled TNFC. and the labeled internal about 0.5 to about 20 ng/10 about 0.5 to about 10 ng/10 ul, control by size exclusion chromatography; US 8,865,417 B2 21 22 (c) integrating the area-under-the curve for a peak of the TNFC. In a related embodiment, the present invention further labeled TNFC. from a plot of signal intensity as a func provides that the sample is serum. In a related embodiment, tion of elution time from the size exclusion chromatog the present invention further provides that the sample is raphy; obtained from a subject receiving therapy with the anti-TNFC. (d) integrating the area-under-the curve for a peak of the drug. In a related embodiment, the present invention further labeled internal control from the plot of signal intensity provides that the size exclusion chromatography is size exclu as a function of elution time from the size exclusion sion-high performance liquid chromatography (SE-HPLC). chromatography; In some embodiments, the present invention provides a (e) determining a first ratio by dividing the amount of method for determining the presence or level of an autoanti labeled TNFC. by the amount of labeled internal control; 10 body to an anti-TNFC. drug with reference to an internal (f) determining a second ratio by dividing the resultant control in a sample having or Suspected of having the autoan integration from step (c) by the resultant integration tibody, the method comprising: from step (d); and (a) contacting an amount of labeled anti-TNFC. drug and an (g) comparing the first ratio determined in step (e) with the amount of labeled internal control with the sample to second ratio determined in step (0. 15 form a complex of the labeled anti-TNFC. drug and the thereby determining the presence or level of the anti-TNFC. autoantibody; drug with reference to an internal control. (b) detecting the labeled anti-TNFC. and the labeled inter In a related embodiment, the present invention further pro nal control by size exclusion chromatography; vides that the first ratio determined in step (e) is from about (c) integrating the area-under-the curve for a peak of the 80:1 to about 100:1. In another related embodiment, the labeled anti-TNFC. drug from a plot of signal intensity as present invention further provides that the first ratio deter a function of elution time from the size exclusion chro mined in step (e) is about 100:1. In a related embodiment, the matography; present invention further provides that the labeled internal (d) integrating the area-under-the curve for a peak of the control is Biocytin-Alexa 488. In certain embodiments, the labeled internal control from the plot of signal intensity amount of the labeled internal control (e.g., Biocytin-Alexa 25 as a function of elution time from the size exclusion 488) is from about 1 to about 25 ng per 100 uL of sample chromatography; analyzed. In certain other embodiments, the amount of the (e) determining a first ratio by dividing the amount of labeled internal control (e.g., Biocytin-Alexa 488) is from labeled anti-TNFC. drug by the amount of labeled inter about 5 to about 25 ng per 100 uL, from about 5 to about 20 nal control; and ng per 100 uL, from about 1 to about 20 ng per 100 uL, from 30 (f) determining a second ratio by dividing the resultant about 1 to about 10 ng per 100 uL, or from about 1 to about 5 integration from step (c) and (d); and ng per 100 uL of sample analyzed. In further embodiments, (g) comparing the first ratio determined in step (e) with the the amount of the labeled internal control (e.g., Biocytin second ratio determined in step (f). Alexa 488) is about 1, 5, 10, 15, 20, or 25 ng per 100 uL of thereby determining the presence or level of the autoantibody sample analyzed. 35 with reference to an internal control. In some embodiments, the present invention provides a In a related embodiment, the first ratio determined in step method for optimizing the therapeutic amount of an anti (e) is from about 8.0:1 to about 100:1. In another related TNFC. drug in a subject receiving therapy with the anti-TNFC. embodiment, the first ratio determined in step (e) is about drug, the method comprising: determining a Subsequent dose 100:1. In a related embodiment, the labeled internal control is of the anti-TNFC. drug for the subject based upon the com 40 Biocytin-Alexa 488. In certain embodiments, the amount of parison of the first and second ratios in accordance with the the labeled internal control (e.g., Biocytin-Alexa 488) is from methods of the present invention, thereby optimizing the about 50 to about 200 pg per 100 uL of sample analyzed. In therapeutic amount of the anti-TNFC. drug. In a related certain other embodiments, the amount of the labeled internal embodiment, the present invention further provides that the control (e.g., Biocytin-Alexa 488) is from about 100 to about method further comprises: increasing the Subsequent dose of 45 200 pg per 100 uL, from about 150 to about 200 pg per 100 the anti-TNFC. drug when the first ratio is about 100:1 and the uL, from about 50 to about 150pg per 100 uL, or from about second ratio is less than about 95:1, thereby optimizing the 50 to about 100pg per 100 uL of sample analyzed. In further therapeutic amount of the anti-TNFC. drug. In another related embodiments, the amount of the labeled internal control (e.g., embodiment, the present invention further provides that the Biocytin-Alexa 488) is about 50, 75, 100, 125, 150, 175, or anti-TNFC. drug is a member selected from the group con 50 200pg per 100 uL of sample analyzed. sisting of REMICADETM (infliximab), ENBRELTM (etaner In a related embodiment, the anti-TNFC. drug is a member cept), HUMIRATM (adalimumab), CIMZIAR (certolizumab selected from the group consisting of REMICADETM (inflix pegol), and combinations thereof. In a related embodiment, imab), ENBRELTM (etanercept), HUMIRATM (adalimumab), the present invention further provides that the anti-TNFC. CIMZIAR (certolizumab pegol), and combinations thereof. drug is infliximab (REMICADETM). In a related embodi 55 In a related embodiment, the anti-TNFC. drug is infliximab ment, the present invention further provides that the anti (REMICADETM). In a related embodiment, the anti-TNFC. TNFC. drug is adalimumab (HUMIRATM). In a related drug is adalimumab (HUMIRATM). In a related embodiment, embodiment, the present invention further provides that the the anti-TNFC. drug is etanercept (ENBRELTM). In a related anti-TNFC drug is etanercept (ENBRELTM). In a related embodiment, the anti-TNFC. drug is CIMZIAR (certoli embodiment, the present invention further provides that the 60 Zumab pegol). In a related embodiment, the detected autoan anti-TNFC. drug is CIMZIAR (certolizumab pegol). In a tibody is quantitated. In a related embodiment, the labeled related embodiment, the present invention further provides complex is eluted first, followed by free labeled anti-TNFC. that the labeled TNFC. is a fluorophore labeled TNFC. In a antibody. In a related embodiment, the sample is serum. In a related embodiment, the present invention further provides related embodiment, the sample is obtained from a subject that the detected anti-TNFC. drug is quantitated. In a related 65 receiving therapy with the anti-TNFC. drug. In a related embodiment, the present invention further provides that the embodiment, the size exclusion chromatography is size labeled complex is eluted first, followed by free labeled exclusion-high performance liquid chromatography (SE US 8,865,417 B2 23 24 HPLC). In a related embodiment, the autoantibody is a mem related embodiment, the labeled TNFC. is a fluorophore ber selected from the group consisting of human anti-mouse labeled TNFC. In a related embodiment, the sample is serum. antibody (HAMA), human anti-chimeric antibody (HACA), In a related embodiment, the sample is obtained from a sub human anti-humanized antibody (HAHA), and combinations ject receiving therapy with the anti-TNFC. drug. In a related thereof. In a related embodiment, the detected autoantibody is 5 embodiment, the autoantibody is a member selected from the quantitated. group consisting of human anti-mouse antibody (HAMA), In a related embodiment, the methods of the present inven human anti-chimeric antibody (HACA), human anti-human tion further provide determining a Subsequent course of ized antibody (HAHA), and combinations thereof. therapy. The Subsequent course of therapy comprises co administering an immunosuppressive drug with the anti 10 IV. PHYSIOLOGICAL RANGES AND LEVELS TNFC. drug when the level of the anti-TNFC. drug is a high OF SPECIES RELEVANT TO THE PRESENT level and the level of the autoantibody is a low level. In INVENTION another related embodiment, the Subsequent course of therapy comprises increasing the level of the anti-TNFC. drug Therapeutically effective levels of REMICADETM in a and co-administering an immunosuppressive drug when the 15 patient treated with REMICADETM are in the range of about level of the anti-TNFC. drug is a medium level and the level of 1 to 10 mg REMICADETM per kg patient body weight. In the autoantibody is a low level. In another related embodi some embodiments, the therapeutically effective levels of ment, the Subsequent course of therapy comprises adminis REMICADETM are in the range of 3 to 8 mg REMICADETM tering a different anti-TNFC. drug when the level of the anti per kg patient body weight. In some other embodiments, the TNFC. drug is a medium level and the level of the therapeutic effective levels of REMICADETM are about 5 mg autoantibody is a medium level. In yet another related REMICADETM per kg of patient body weight. embodiment, the Subsequent course of therapy comprises A typical dose of REMICADETM (infliximab) is in the administering a different anti-TNFC. drug when the level of range of about 0.05ug to about 80g per ml. In some embodi the anti-TNFC. drug is a low level and the level of the autoan ments, a dose of REMICADETM is in the range of about 0.05 tibody is a high level. In another related embodiment, the 25 ug to about 50 ug per ml. In some other embodiments, a dose adalimumab (HUMIRATM) is administered instead of inflix of REMICADETM is about 0.05ug to about 30 ug per ml. In imab (REMICADETM). some embodiments, a dose of REMICADETM is about 30 ug In some embodiments, the present invention provides a kit per ml. In some other embodiments, a dose of REMICADETM for measuring the presence or level of an anti-TNFC. drug and is about 50 ug per ml. the presence or level of an autoantibody to an anti-TNFC. drug 30 Therapeutically effective levels of HUMIRATM in a patient in a sample, the kit comprising: treated with HUMIRATM are in the range of about 0.1 to 10 (a) a first measuring substrate comprising an amount of a mg HUMIRATM per kg patient body weight. In some embodi labeled TNFO: ments, the therapeutically effective levels of HUMIRATM are (b) a second measuring Substrate comprising an amount of in the range of 0.1 to 8 mg HUMIRATM per kg patient body a labeled anti-TNFC. drug; 35 weight. In some other embodiments, the therapeutic effective (c) optionally a third measuring Substrate comprising an levels of HUMIRATM are about 1 mg HUMIRATM per kg of amount of a labeled TNFC. and an amount of a labeled patient body weight. In some embodiments, the therapeutic internal control; effective levels of HUMIRATM are about 0.8 mg HUMIRATM (d) optionally a fourth measuring Substrate comprising an per kg of patient body weight. amount of a labeled anti-TNFC. drug and an amount of a 40 A typical dose of HUMIRATM is in the range of about 0.05 labeled internal control; ug is to about 150g per ml. In some embodiments, a dose of (e) optionally a means for extracting a sample from a HUMIRATM is in the range of about 0.05ug to about 100 ug Subject; and per ml. In some other embodiments, a dose of HUMIRATM is (f) optionally a pamphlet of instructions for using the kit. about 0.05ug to about 50 ug per ml. In some embodiments, a In a related embodiment, the Substrate comprises any mate 45 dose of HUMIRATM is about 30 g per ml. In some embodi rial that can be deposited with the chemicals of the present ments, a dose of HUMIRATM is about 32 ug per ml. In some invention and include, but are not limited to, nitrocellulose, other embodiments, a dose of HUMIRATM is about 50 lug per silica gel, thin-layer chromatography Substrates, wooden ml. Sticks, cellulose, cotton, polyethylene, combinations thereof, and the like. In another related embodiment, the chemicals of 50 V. EXEMPLARY DISEASES AND the present invention can be deposited on the aforementioned THERAPEUTIC ANTIEBODIES FOR THE materials in an ordered array, matrix, or matrix array. TREATMENT THEREOF In a related embodiment, the kit further comprises a means for detecting the labeled TNFC, the labeled anti-TNFC. drug, In certain embodiments, the present invention may employ and/or the labeled internal control. In a related embodiment, 55 therapeutic monoclonal antibodies. Table 1 provides an the kit further comprises means for detection including, but exemplary list of therapeutic monoclonal antibodies which not limited to, fluorescence label detection, UV-radiation have either been approved or are currently in development. detection, or iodine exposure. In a related embodiment, the kit An extensive list of monoclonal antibody therapeutics in further comprises a size exclusion-high performance liquid clinical development and approved products are disclosed in chromatography (SE-HPLC) instrument. In a related 60 the 2006 PhRMA Reportentitled “418 Biotechnology Medi embodiment, the first, second, third, and fourth measuring cines in Testing Promise to Bolster the Arsenal Against Dis Substrates are selected from nitrocellulose, silica gel, and a ease. size-exclusion chromatographic medium. In a related Particularly preferred therapeutic antibodies include, but embodiment, the anti-TNFC. drug is a member selected from are not limited to, anti-TNFC. monoclonal antibodies such as, the group consisting of REMICADETM (infliximab), 65 e.g., (1) RemicadeTM (infliximab), a mouse-human IgG ENBRELTM (etanercept), HUMIRATM (adalimumab), CIM I-kappa antiTNFC. monoclonal antibody, (2) EnbrelTM (etan ZIAR (certolizumab pegol), and combinations thereof. In a ercept), a fusion protein of human TNF receptor 2 and human US 8,865,417 B2 25 26 IgG I, and (3) HumiraTM (adalimumab), a fully human IgG1 Blood disorders, such as sepsis, septic shock, paroxysmal kappa antiTNFC. monoclonal antibody. Two other anti-TNFC. nocturnal hemoglobinuria, and hemolytic uremic syndrome. antibody constructs have shown promise in pivotal phase III Cancer, such as colorectal cancer, non-Hodgkin’s lym trials inpatients with some of the same diseases: (4) CimziaTM phoma, B-cell chronic lymphocytic leukemia, anaplastic 5 large-cell-lymphoma, squamous cell cancer of the head and CDP870 (certolizlunab pegol), a PEGylated Fab fragment of neck, treatment of HER2-overexpressing metastatic breast a humanized anti-TNFC. monoclonal antibody, and (5) CNTO cancer, acute myeloid leukemia, prostate cancer (e.g., adeno 148 (golimlunab), a fully human IgGI-kappa antiTNFC. carcinoma), Small-cell lung cancer, thyroid cancer, malignant monoclonal antibody. melanoma, Solid tumors, breast cancer, early stage HER2 A preferred class of therapeutic antibodies are anti-TNFC. positive breast cancer, first-line non-squamous NSCLC can single chain monoclonal antibodies used in treatment of 10 cers, AML, hairy cell leukemia, neuroblastoma, renal cancer, numerous autoimmune diseases, such as rheumatoid arthritis, brain cancer, myeloma, multiple myeloma, bone metastases, juvenile idiopathic arthritis, ankylosing spondylitis (Bech SCLC, head/neck cancer, first-line pancreatic, SCLC, terew's disease), inflammatory bowel diseases (Crohn's dis NSCLC, head and neck cancer, hematologic and solid eases and ulcerative colitis), severe psoriasis, chronic uveitis, tumors, advanced solid tumors, gastrointestinal cancer, pan severe sarcoidosis, and Wegener's granulomatosis. 15 creatic cancers, cutaneous T-cell lymphoma, non-cutaneous In addition to being useful in determining the bioavailabil T-cell lymphoma, CLL, ovarian, prostate, renal cell cancers, ity/concentration of anti-TNFC. antibodies, the present inven mesothelin-expressing tumors, glioblastoma, metastatic pan tion is also suitable for use in the determination of the bio creatic, hematologic malignancies, cutaneous anaplastic availability/concentration of any therapeutic antibody used large-cell MAb lymphoma, AML, myelodysplastic Syn within the body, Such as for therapeutic or diagnostic pur dromes. poses. Table 1 below also provides a list of medical indica Cardiovascular disease. Such as atherosclerosis acute myo tions which correlate with various therapeutic monoclonal cardial infarction, cardiopulmonary bypass, angina. antibodies used in vivo. Metabilic disorders such as diabetes, such as type-I diabe In further embodiments, the methods of the present inven tes mellitus. tion are suitable for use in the determination of the presence or 25 Digestive disorders, such as Crohn's disease, C. difficile concentration levels of autoantibodies to the therapeutic anti disease, ulcerative colitis. bodies. Eye disorders such as uveitis. The present invention can therefore be used in methods of Genetic disorders such as paroxysmal nocturnal hemoglo optimizing therapy where the treatment (or diagnosis) com binuria (PNH). prises administering a therapeutic antibody to the Subject. 30 Neurological disorders such as osteoarthritis pain and The methods can be for optimizing the treatment (or diagno Alzheimer's disease. sis) of one or more of the diseases or disorders referred to Respiratory disorders such as respiratory diseases, asthma, herein, including one or more of the following: chronic obstructive pulmonary disorders (COPD, nasal poly Infectious diseases, such as respiratory syncytial virus posis, pediatric asthma. (RSV), HIV, anthrax, candidiasis, staphylococcal infections, 35 Skin diseases, such as psoriasis, including chronic moder hepatitis C. ate to severe plaque psoriasis. Autoimmune diseases, such as rheumatoid arthritis, Transplant rejection, such as acute kidney transplant rejec Crohn's disease, B-cell nonhodgkin’s lymphoma, Multiple tion, reversal of heart and liver transplant rejection, preven Scleorisis, SLE, ankylosing spondylitis, lupus, psoriatic tion of renal transplant rejection, prophylaxis of acute kidney arthritis, erythematosus. 40 transplant rejection, renal transplant rejection. Inflammatory disorders such as rheumatoid arthritis (RA), Other disorders. Such as diagnosis of appendicitis, kidney juvenile idiopatmc arthritis, ankylosing spondylitis (Bech inflammation, postmenopausal osteoporosis (bone disor terew's disease), inflammatory bowel diseases (Crohn's dis ders), hypereosinophilic syndrome, eosinophilic esophagitis eases and ulcerative colitis), severe psoriasis, chronic uveitis, and peanut allergy. sarcoidosis, Wegener's granulomatosis, and other diseases 45 In one embodiment, the disease is selected from one or with inflammation as a central feature. more of the above groups of specific diseases and disorders. TABLE 1. Therapeutic and diagnostic monoclonal antibodies Product Name Sponsor Indication Infectious diseases Synagis (R) palivizumab MedImmune prevention of respiratory syncytial virus (RSV) anti-HIV-1 MAb Polymun Scientific HIV infection treatment Vienna, Austria CCRS MAb Hunan Genome Sciences HIV infection Rockville, MD Cytolin (R) anti-CD8 MAb CytoDyn HIV infection Santa Fe, NM NMO1 SRD Pharmaceuticals HIV infection Los Angeles, CA PRO 140 Progenies HIV infection Pharmaceuticals Tarrytown, NY 355 Tanox MAb HIV 355 Tainox MAb HIV infection Please II infection Phase II US 8,865,417 B2 27 28 TABLE 1-continued Therapeutic and diagnostic monoclonal antibodies Product Name Sponsor Indication ABthraxTM raxibacumab Human Genome anthrax Sciences Anthim. TM (ETI-204) Elusys Therapeutics anthrax (Orphan Drug) anti-hsp90 MAb NeuTec Pharma candidiasis anti-staph MAb MedImmune Prevention of staphylococcal infections Aurexistefibazumab Inhibitex prevention and treatment of S. aureus bacteremia Bavituximab Peregrine hepatitis C treatment Pharmaceuticals MDX-1303 Medarex anthrax PharmAthene NumaxTM motavizumab MedImmune RSV Tarwacin TM Peregrine hepatitis C bavituximab Pharmaceuticals XTL 686S XTL Biopharmaceuticals hepatitis C Autoimmune disorders
Humira (Radalimumab Abbott Laboratories rheumatoid arthritis Remicade TM infiximab Centocor Crohn's disease, rheumatoid arthritis Rituxan (R) ritiximab Genentech B-cell nonhodgkin's Biogen Idec lymphoma, relapse in patients following rituxan treatment. Rheumatoid arthritis Tysarbi (R) natalizumab Biogen Idec Multiple scleorisis ART 874 Abbott Laboratories multiple Sclerosis Actemra Roche rheumatoid arthritis AME S27 Applied Molecular rheumatoid arthritis AMG 108 Amgen rheumatoid arthritis AMG 714 Amgen rheumatoid arthritis anti-CD16 MAb MacroGenics immune thrombocytopenic CNTO 1275 Centocor Horham, PA multiple Sclerosis daclizumab PDL BioPharma multiple Sclerosis (anti-CD25 MAb) Fremont, CA (see also respiratory) Biogen Idec Cambridge, MA denosumah Amgen rheumatoid arthritis (AMG 162) Thousand Oaks, CA ETI-2O1 Elusys Therapeutics SLE Pine Brook, NJ golimumab Centocor rheumatoid arthritis Horsham, PA HuMax-CD2O Genmab rheumatoid arthritis (ofatumumab) Princeton, NJ Humira (R) Abbott Laboratories ankylosing spondylitis adalimumab juvenile rheumatoid arthritis HuZAFTM fontolizumab PDL BioPharma rheumatoid arthritis Fremont, CA Biogen Idec Cambridge, MA MMU-106 mmunomedics autoimmune disease (hCD20) Morris Plains, NJ LymphoStat-BTM belimumab Human Genome rheumatoid arthritis, Sciences SLE Rockville, MD MEDI-545 Medarex lupus (MDX-1103) Princeton, NJ MedImmune Gaithersburg, MD MLN 1202 Millennium multiple Sclerosis Pharmaceuticals Cambridge, MA ocrelizumab Genentech rheumatoid arthritis (2nd anti-CD20) South San (R1594) Francisco, CA Biogen Idec Cambridge, MA Roche Nutley, NJ US 8,865,417 B2 29 30 TABLE 1-continued Therapeutic and diagnostic monoclonal antibodies Product Name Sponsor Indication OKT3-gamma-1 Johnson & Johnson psoriatic arthritis Pharmaceutical Research & Development Raritan, NJ Rituxan (R) rituximab Genentech rheumatoid arthritis South San (DMARD inadequate Francisco, CA responders), lupus, Biogen Idec primary progressive multiple Cambridge, MA sclerosis, SLE (see also cancer) relapsing remitting multiple Sclerosis TRX1 TolerRX cutaneous lupus (anti CD4) Cambridge, MA erythetinatosus Blood disorders
ReoPro (R) Centocor anti-platelet prevention of blood clots abciximab Eli Lilly (PTCA), angina (PTCA) urtoxazumab Teijin Pharma hemolytic uremic Afelimomab Abbot Laboratories Sepsis, septic shock Ecullizumab Alexion Paroxysmall nocturnal Pharmaceuticals hemoglobinurea Cancer
Avastin TM bevacizumab Genentech metastatic colorectal cancer Bexxar (R) to situmomab, GlaxoSmithKline non-Hodgkin's lymphoma iodine 1131 to situmomab Campath (R) alemtuzumab Berlex Laboratories B-cell chronic lymphocytic Genzyme leukemia ErbituxTM cetuximab Bristol-Myers colorectal cancer Squibb squamous cell cancer of the Medarex head and neck Herceptin (R) trastuzumab Genentech treatment of HER2 overexpressing metastatic breast cancer Mylotarg TM gemtuzumab Wyeth Acute myeloid leukemia OZogamicin OncoScint (R) CROW CYTOGEN detection, staging and follow Satumomab up of colorectal cancers pendetide ProstaScint (R) capiromab CYTOGEN detection, staging and follow pentetate up of prostate adenocarcinoma Rituxan (R) Genentech B-cell nonhodgkin's lymphoma, relapse ritiximab Biogen Idec in patients following rituxan treatment. Verluma (R) moletumomab DuPont Pharmaceuticals detection of Small-cell lung C8Ce Zevalin TM ibritumomab IDFC Pharmaceuticals Non-hodgkin's lymphoma tiuxetan 1311 huA33 Life Science colorectal cancer Pharmaceuticals Greenwich, CT GPC Biotech relapsed/refractory B-cell Waltham, MA lymphomas AGS PSCA MAb Agensys prostate cancer Santa Monica, CA Merck Whitehouse Station, NJ AMG 1 O2 Amgen Thousand Oaks, CA AMG 479 Amgen Thousand Oaks, CA C8Ce AMG 623 Amgen Thousand Oaks B-cell chronic lymphocytic leukemia (CLL) (see also autoimmune) AMG 6SS Amgen Thousand Oaks C8Ce AMG 706 Amgen Thousand Oaks imatinib-resistant GIST, advanced thyroid cancer AMG 706 Arngen imatinib resistant GIST, Thousand Oaks, CA advanced thyroid cancer anti-CD23 MAb Biogen Idec Cambridge, MA CLL anti-CD80 MAb Biogen Idec Cambridge, MA non-Hodgkin's B anti-idiotype Viventia Biotech malignant melanoma US 8,865,417 B2 31 32 TABLE 1-continued Therapeutic and diagnostic monoclonal antibodies Product Name Sponsor Indication cancer vaccine Toronto, Ontario anti-lymphotoxin Biogen Idec Cambridge, MA. Solid tumors beta receptor MAb anti-PEMMAb Somanta Pharmaceuticals C8Ce rvine, CA anti-Tac(Fv)- National Cancer Institute leukemia, lymphoma. PE38 immunotoxin Bethesda, MD Avastin (R) bevacizumab Genentech relapsed metastatic South San colorectal cancer Francisco, CA first line metastatic breast, first-line non-squamous NSCLC cancers AVE 9633 Sanofi aventis AML maytansin Bridgewater, NJ loaded anti-CD33 MAb bavituximab Peregrine Pharmaceuticals Solid cancers (see also Tustin, CA infectious) CAT 3888 Cambridge Antibody hairy cell leukemia Technology chimeric MAb National Cancer Institute neuroblastoma CNTO 328 Centocor renal cancer Cotara TM Peregrine Pharmaceuticals brain cancer bivatuzumab Boehringer Ingelheim C8Ce Pharmaceuticals Ridgefield, CT CP-751,871 Pfizer Daiichi multiple myeloma cancer CS 1008 Sankyo Sankyo Sankyo Pharma Development Parsippany, NJ BrewaRexTM WiRexX breast cancer, multiple antibody based Edmonton, Alberta myeloma immunotherapy denosumab Amgen bone loss induced by hormone ablation therapy for breast or prostate cancer, prolonging bonemetastases free survival (see also autoimmune, other) bone metastases in breast cancer ecromeximab Kyowa Hakko USA malignant melanoma EMD 273063 EMD Lexigen Solid tumors malignant melanoma, neuroblastoma, SCLC ErbitxTM Bristol Myers Squibb head/neck cancer, first-line pallicreatic, first-line NSCLC, -second-line NSCLC, first line colorectal, second-line colorectal cancers GMK Progenies Pharmaceuticals prevention of recurrence following Surgery to remove primacy melanoma in high risk patients Campath (R) alemtuzumab National Cancer Institute leukemia, lymphoma Bethesda, MD Berlex Laboratories Montville, NJ Herceptin (R) trastuzumab Genentech South San early stage HER2-positive Francisco, CA breast cancer first-line metastatic HER2-positive breast cancer in combination with Taxotere (R) HGS-ETR1 Human Genome Sciences hematologic and solid tumors Rockville, MD HGSETR2 Human Genome Sciences hematologic and solid tumors (mapatumumab) Rockville, MD HGS-TR2J Human Genome Sciences advanced solid tumors Rockville, MD HuC242-DM4 mmunoGen colorectal, gastrointestinal, Cambridge, MA NSCLC, pancreatic cancers HuMax-CD4 Genmab cutaneous T-cell lymphoma (Zanolimumab) Princeton, NJ non-cutaneous T-cell Serono lymphoma Rockland, MA HuMax CD2O Geminab CLL, non-Hodgkin's US 8,865,417 B2 33 34 TABLE 1-continued Therapeutic and diagnostic monoclonal antibodies Product Name Sponsor Indication (ofatumumab) Princeton, NJ lymphoma (see also autoimmune) HuMax-EGFr Genmab head and neck cancer Princeton, NJ huN901-DM1 ImmunoGen SCLC multiple myeloma Cambridge, MA ipilimumab (MDX Bristol-Myers Squibb melanoma monotherapy Medarex, Princeton leukemia, lymphoma, ovarian, prostate, renal cell cancers melanoma (MCX 010 +/- DTIC) second-line metastatic melanoma (MDX 010 disomotide? overmotide MDX-1379) M195-bismuth Actinium AML 213 conjugate Pharmaceuticals Florham Park, NJ M200 (volocixirnab) PDL BioPharma Fremont, advanced solid tumors CA Biogen Idec Cambridge, MA MAb HeFi-1 National Cancer Institute lymphoma, non-Hodgkin's Bethesda, MD lymphoma MDX-060 Medarex Hodgkin's disease, anaplastic (iratumumab) Princeton, NJ large-cell-lymphoma MDX-070 Medarex Princeton, NJ prostate cancer MDX-214 Medarex Princeton, NJ ECFR-expressing cancers MEDI-SO7 MedImmune T-cell lymphoma infections Siplizumab Gaithersburg, MD melanoma, prostate cancer MEDI-522 MedImmune Solid tumors Gaithersburg, MD National Cancer Institute Bethesda, MD MedImmune Gaithersburg, MD MORAb OO3 Morphotek ovarian cancer Exton, PA MORAb O09 Morphotek mesothelin-expressing Exton, PA tumors neuradiab Bradmer glioblastoma Pharmaceuticals Louisville, KY nimotuzumab YM Biosciences metastatic pancreatic, (Orphan Drug) Mississauga, Ontario NSCLC hematologic ocrelizumab Genentech malignancies (2nd anti-CD20) South San Francisco, CA (see also autoimmune) (R1594) Biogen Idec Cambridge, MA Roche Nutley, NJ OmnitargTM pertuzumab Genentech ovarian cancer South San Francisco, CA OvaRex (R) oregovomab ViRexX MAb Edmonton, ovarian cancer Alberta PAM4 Merck pancreatic cancer Whitehouse Station, NJ panitumumab Abgenix colorectal cancer (rIIuMAb EGFr) Proleukin (R) Chiron Emeryville, CA Non-hodgkin's lymphoma PSMA Progenies Pharmaceuticals prostate cancer Tarrytown, NY R1550 Roche Nutley, NJ YMYM metastatic breast cancer RadioTheraCIM BioSciences Mississauga, glioma Ontario RAV12 Raven Biotechnologies South San Francisco, CA Renearex (R) G250 Wilex Miulich, Germany renal cancer Rituxan (R) rituximab Genentech South San indolent non-Hodgkin's Francisco, CA Biogen Idec lymphoma induction therapy Cambridge, MA (see also autoimmune) relapsed or refractory CLL SGN3O Seattle Genetics cutaneous anaplastic large (Orphan Drug) Bothell, WA cell MAb lyrphoma, systemic anaplastic large-cell lymphoma, Hodgkin's disease SGN-33 Seattle Genetics Bothell, WA AML, myelodysplastic (lintuzumab) Seattle Genetics Bothell, WA syndromes CLL, multiple US 8,865,417 B2 35 36 TABLE 1-continued Therapeutic and diagnostic monoclonal antibodies Product Name Sponsor Indication SGN-40 myeloma, non Hodgkin's lymphoma sibroturtunab Life Science Pharmaceuticals colorectal, head and neck, Greenwich, CT lung cancers Tarwacin TMbawituximab Peregrine Pharmaceuticals Solid tumors (see also Tustin, CA infectious) ticilimumab Pfizer metastatic melanoma prostate New York, NY cancerticilimumab TNX-6SO Zewain TM Tanox Houston, TX Hodgkin's disease leukemia, ibritumomab National Cancer Institute lymphoma non-Hodgkin's tiuxetan Bethesda lymphoma Biogen Cardiovascular disease
MLN 1202 Millennium Pharmaceuticals atherosclerosis Cambridge, MA (see also autoimmune) pexelizumab Alexion Pharmaceuticals acute myocardial infarction, Cheshire, CT cardiopulmonary bypass Procter & Gamble Pharmaceuticals Mason, OH Diabetes and Related Conditions anti-CD3 MAb MacroGenics Rockville, MD type-1 diabetes mellitus OKT3-gamma-1 Johnson & Johnson type-1 diabetes mellitus Pharmaceutical Research & Development TRX4 TolerRX type-1 diabetes mellitus (anti-CD3) Cambridge, MA Digestive Disorders
Remicade TM infiximab Centocor Crohn's disease ABT 874 Abbott Laboratories Crohn's disease Abbott Park, IL, (see also autoimmmune) CNTO 1275 Centocor Crolin's disease Phase II (see Horsham, PA also autoinunurle, skin) (610) 651-6000 Humira (Radalimumab Abbott Laboratories Crohn's disease Phase III MDX-066 (CDA-1) Abbott Park, IL (see also autoimmune, skin) Medarex (847) 936,1189 Princeton, NJ C. difficile disease MDX-1100 Millennium Pharmaceuticals ulcerative colitis Cambridge, MA Nuvion (R) visilizumab PDT, BioPharma Fremont, I.V. steroid-refractory CA ulcerative colitis Crohn's disease Tysarbi (R) natalizumab Biogen Idec Crohn's disease Cambridge, MA Eye Conditions golimumab Centocor Horsham, PA uveitis (see also autoimmune) Genetic Disorders
SolirisTM eculizumab Alexion Pharmaceuticals paroxysmal nocturnal (Orphan Drug) Cheshire, CT hemoglobinuria (PNH) Neurological Disorders RN624 Rinat Neuroscience Osteoarthritis pain South San Francisco, CA RN1219 Rinat Neuroscience Alzheimer's disease South San Francisco, CA Respiratory Disorders ABN 912 Novartis Pharmaceuticals asthma, chronic East Hanover, NJ obstructive pulmonary disorders (COPD) Amgen COPD Thousand Oaks, CA AMG 317 Amgen asthma Thousand Oaks, CA daclizumab Protein Design Labs asthma (see also (anti-CD25 MAb) Fremont, CA Roche autoimmune) Nutley, NJ MEDI-528 MedImmune asthma anti-TL-9 MAb Gaithersburg, MD US 8,865,417 B2 37 38 TABLE 1-continued Therapeutic and diagnostic monoclonal antibodies Product Name Sponsor Indication mepolizumab GlaxoSmithKline asthma and nasal polyposis (anti-TL5 MAb) Philadelphia, PA (see also other) Rsch. Triangle Park, NC Tanox respiratory diseases Houston, TX Xolair (Romalizumab Genentech pediatric asthma South San Francisco, CA (see also other) Novartis Pharmaceuticals Skin Disorders Raptiva (R) efalizumab Genentech chronic moderate to severe CNTO1275 XOMA plaque psoriasis Centocor psoriasis See also autoimmune, digestive) Humira (Radalimumab Abbott Laboratories psoriasis See also autoimmune, digestive) TRX4 TolerRX psoriasis (see also diabetes) Transplatation
ORTHOCLONEOKT (R 3 Ortho Biotech acute kidney transplant muromonab-CD3 rejection, reversal of heart and liver transplant rejection Simulect (R) basiliximab Novartis prevention of renal transplant Pharmaceuticals rejection Zenapax (R) daclizumab Roche prophylaxis of acute kidney OKT3-gamma-1 Protein Design Labs transplant rejection Johnson & Johnson renal transplant rejection (see also autoimmune, diabetes) Other NeutroSpec TM Palatin Technologies diagnosis of appendicitis technetium 99m Tc fanolesomab CROOO2 CuraGen kidney inflammation denosumab Amgen Postmenopausal (AMG 162) Osteoporosis, see also autoimmune and cancer mepolizumab GlaxoSmithKline hypereosinophilic syndrome, (anti-IL5 MAb) eosinophlic esophagitis (see also respiratory) Xolair (Romalizumab Genentech peanut allergy (see also Tanox respiratory)
45 VI. THERAPY AND THERAPEUTIC anti-TNFC. drug (e.g., an immunosuppressive agent). MONITORING Examples of anti-TNFC. drugs are described herein include, but are not limited to, REMICADETM (infliximab), Once the diagnosis or prognosis of a subject receiving ENBRELTM (etanercept), HUMIRATM (adalimumab), CIM anti-TNFC. drug therapy has been determined or the likeli 50 ZIAR (certolizumab pegol), biologic agents, conventional hood of response to anti-TNFC. drug has been predicted in an drugs, and combinations thereof. As such, the present inven individual diagnosed with a disease and disorder in which tion advantageously enables a clinician to practice “person TNFC. has been implicated in the pathophysiology, e.g., but alized medicine by guiding treatment decisions and inform not limited to, shock, sepsis, infections, autoimmune dis ing therapy selection and optimization for anti-TNFC. drugs eases, RA, Crohn's disease, transplant rejection and graft 55 Versus-host disease, according to the methods described Such that the right drug is given to the right patient at the right herein, the present invention may further comprise recom time. mending a course of therapy based upon the diagnosis, prog Anti-TNFC. drug can be administered with a suitable phar nosis, or prediction. In certain instances, the present invention maceutical excipient as necessary and can be carried out via may further comprise administering to the individual athera 60 any of the accepted modes of administration. Thus, adminis peutically effective amount of an anti-TNFC. drug useful for tration can be, for example, intravenous, topical. Subcutane treating one or more symptoms associated with disease and ous, transcutaneous, transdermal, intramuscular, oral, buccal, disorder in which TNF-C. has been implicated in the patho Sublingual, gingival, palatal, intra joint, parenteral, intra-ar physiology. For therapeutic applications, the anti-TNFC. drug teriole, intradermal, intraventricular, intracranial, intraperito can be administered alone or co-administered in combination 65 neal, intralesional, intranasal, rectal, vaginal, or by inhala with one or more additional anti-TNFC. drugs and/or one or tion. By “co-administer it is meant that an anti-TNFC. drug is more drugs that reduce the side-effects associated with the administered at the same time, just prior to, or just after the US 8,865,417 B2 39 40 administration of a second drug (e.g., another anti-TNFC. gelatin, cellulose and derivatives thereof. An anti-TNFC. drug drug, a drug useful for reducing the side-effects of the anti can also be formulated into a Suppository disposed, for TNFC. drug, etc.). example, in a polyethylene glycol (PEG) carrier. A therapeutically effective amount of an anti-TNFC. drug Liquid dosage forms can be prepared by dissolving or may be administered repeatedly, e.g., at least 2, 3, 4, 5, 6, 7, 8, dispersing an anti-TNFC. drug and optionally one or more or more times, or the dose may be administered by continuous pharmaceutically acceptable adjuvants in a carrier Such as, infusion. The dose may take the form of solid, semi-solid, for example, aqueous Saline (e.g., 0.9% w/v Sodium chlo lyophilized powder, or liquid dosage forms, such as, for ride), aqueous dextrose, glycerol, ethanol, and the like, to example, tablets, pills, pellets, capsules, powders, Solutions, form a solution or Suspension, e.g., for oral, topical, or intra Suspensions, emulsions, Suppositories, retention enemas, 10 venous administration. An anti-TNFC. drug can also be for creams, ointments, lotions, gels, aerosols, foams, or the like, mulated into a retention enema. preferably in unit dosage forms suitable for simple adminis For topical administration, the therapeutically effective tration of precise dosages. dose can be in the form of emulsions, lotions, gels, foams, As used herein, the term “unit dosage form' includes creams, jellies, solutions, Suspensions, ointments, and trans physically discrete units Suitable as unitary dosages for 15 dermal patches. For administration by inhalation, an anti human Subjects and other mammals, each unit containing a TNFC. drug can be delivered as a dry powder or in liquid form predetermined quantity of an anti-TNFC. drug calculated to via a nebulizer. For parenteral administration, the therapeuti produce the desired onset, tolerability, and/or therapeutic cally effective dose can be in the form of sterile injectable effects, in association with a suitable pharmaceutical excipi Solutions and sterile packaged powders. Preferably, inject ent (e.g., an ampoule). In addition, more concentrated dosage able solutions are formulated at a pH of from about 4.5 to forms may be prepared, from which the more dilute unit about 7.5. dosage forms may then be produced. The more concentrated The therapeutically effective dose can also be provided in dosage forms thus will contain Substantially more than, e.g., a lyophilized form. Such dosage forms may include a buffer, at least 1, 2,3,4,5,6,7,8,9, 10, or more times the amount of e.g., bicarbonate, for reconstitution prior to administration, or the anti-TNFC. drug. 25 the buffer may be included in the lyophilized dosage form for Methods for preparing Such dosage forms are known to reconstitution with, e.g., water. The lyophilized dosage form those skilled in the art (see, e.g., REMINGTON'S PHARMA may further comprise a Suitable vasoconstrictor, e.g., epi CEUTICAL SCIENCES, 18TH ED., Mack Publishing Co., nephrine. The lyophilized dosage form can be provided in a Easton, Pa. (1990)). The dosage forms typically include a Syringe, optionally packaged in combination with the buffer conventional pharmaceutical carrier or excipient and may 30 for reconstitution, Such that the reconstituted dosage form can additionally include other medicinal agents, carriers, adju be immediately administered to an individual. vants, diluents, tissue permeation enhancers, solubilizers, and In therapeutic use for the treatment of diseases and disor the like. Appropriate excipients can be tailored to the particu ders in which TNFC. has been implicated in the pathophysi lar dosage form and route of administration by methods well ology, an anti-TNFC. drug can be administered at the initial known in the art (see, e.g., REMINGTON'S PHARMACEU 35 dosage of from about 0.001 mg/kg to about 1000 mg/kg daily. TICAL SCIENCES, supra). A daily dose range of from about 0.01 mg/kg to about 500 Examples of suitable excipients include, but are not limited mg/kg, from about 0.1 mg/kg to about 200 mg/kg, from about to, lactose, dextrose. Sucrose, Sorbitol, mannitol, starches, 1 mg/kg to about 100 mg/kg, or from about 10 mg/kg to about gum acacia, calcium phosphate, alginates, tragacanth, gela 50 mg/kg, can be used. The dosages, however, may be varied tin, calcium silicate, microcrystalline cellulose, polyvi 40 depending upon the requirements of the individual, the sever nylpyrrolidone, cellulose, water, Saline, syrup, methylcellu ity of disease, disorder, or symptoms, and the anti-TNFC. drug lose, ethylcellulose, hydroxypropylmethylcellulose, and being employed. For example, dosages can be empirically polyacrylic acids such as Carbopols, e.g., Carbopol 941, Car determined considering the type and severity of the disease, bopol 980, Carbopol 981, etc. The dosage forms can addi disorder, and/or symptoms according to the methods tionally include lubricating agents such as talc, magnesium 45 described herein. The dose administered to an individual, in Stearate, and mineral oil; wetting agents; emulsifying agents; the context of the present invention, should be sufficient to Suspending agents; preserving agents such as methyl-, ethyl-, affect a beneficial therapeutic response in the individual over and propyl-hydroxy-benzoates (i.e., the parabens); pH time. The size of the dose can also be determined by the adjusting agents such as inorganic and organic acids and existence, nature, and extent of any adverse side-effects that bases; Sweetening agents; and flavoring agents. The dosage 50 accompany the administration of a particular anti-TNFC. drug forms may also comprise biodegradable polymer beads, dex in an individual. Determination of the proper dosage for a tran, and cyclodextrin inclusion complexes. particular situation is within the skill of the practitioner. Gen For oral administration, the therapeutically effective dose erally, treatment is initiated with Smaller dosages which are can be in the form of tablets, capsules, emulsions, Suspen less than the optimum dose of the anti-TNFC. drug. Thereaf sions, Solutions, syrups, sprays, lozenges, powders, and Sus 55 ter, the dosage is increased by Small increments until the tained-release formulations. Suitable excipients for oral optimum effect under circumstances is reached. For conve administration include pharmaceutical grades of mannitol, nience, the total daily dosage may be divided and adminis lactose, starch, magnesium Stearate, sodium saccharine, tal tered in portions during the day, if desired. cum, cellulose, glucose, gelatin, Sucrose, magnesium carbon As used herein, the term “anti-TNFC. drug includes all ate, and the like. 60 pharmaceutically acceptable forms of a drug that is useful for In some embodiments, the therapeutically effective dose treating one or more symptoms associated with the diseases takes the form of a pill, tablet, or capsule, and thus, the dosage and disorders in which TNF-C. has been implicated in the form can contain, along with an anti-TNFC. drug, any of the pathophysiology. For example, the anti-TNFC. drugs can be in following: a diluent such as lactose. Sucrose, dicalcium phos a racemic or isomeric mixture, a solid complex bound to an phate, and the like; a disintegrant such as starch orderivatives 65 ion exchange resin, or the like. In addition, the anti-TNFC. thereof a lubricant Such as magnesium Stearate and the like; drugs can be in a Solvated form. The term is also intended to and a binder Such a starch, gum acacia, polyvinylpyrrolidone, include all pharmaceutically acceptable salts, derivatives, and US 8,865,417 B2 41 42 analogs of the anti-TNFC. drugs being described, as well as bodies (e.g., autoantibodies to anti-TNFC. antibodies) in an combinations thereof. For example, the pharmaceutically individual over a threshold amount indicates the individual acceptable salts of an anti-TNFC. drugs include, without limi has a significantly higher risk of developing complications or tation, the tartrate. Succinate, tartarate, bitartarate, dihydro risk of side-effects. Information obtained from serial testing chloride, Salicylate, hemisuccinate, citrate, maleate, hydro in the form of a marker Velocity (i.e., the change in antibody chloride, carbamate, Sulfate, nitrate, and benzoate salt forms levels over a time period) may be associated with the severity thereof, as well as combinations thereof and the like. Any of the disease, the risk of complications of disease, and/or the form of an anti-TNFC. drugs is suitable for use in the methods risk of side-effects. of the present invention, e.g., a pharmaceutically acceptable The methods of the present invention also provide for salt of an anti-TNFC. drugs, a free base of an anti-TNFC. 10 identifying primary non- or low-responders, e.g., for treat drugs, or a mixture thereof. Examples of suitable anti-TNFC. drugs include, but are not limited to, biologic agents, conven ment with a therapeutic monoclonal antibody. These primary tional drugs, and combinations thereof. non- or low-responders may, for example, be patients that Therapeutic agents include, e.g., anti-cytokine and happen to have an innate or a pre-developed immunoglobulin chemokine antibodies Such as anti-tumor necrosis factor C. 15 response to the therapeutic agent. Where the therapeutic alpha (TNFC) antibodies. Non-limiting examples of anti agent is a diagnostic antibody, the identification of primary TNFC. antibodies include: chimeric monoclonal antibodies non-or-low responders can ensure the selection of a Suitable such as infliximab (Remicade(R) (Centocor, Inc.; Horsham, therapeutic agent for each individual patient. Pa.), which is a chimeric IgG1 anti-TNFC. monoclonal anti The method according to the invention may, for example, body; humanized monoclonal antibodies such as CDP571 be used for identifying patients with secondary response fail and the PEGylated CDP870; fully human monoclonal anti ure. Secondary response failures can be asymptomatic, i.e. bodies such as adalimumab (HumiraR) (Abbott Laborato the only symptoms are that the treatment has become less ries; Abbott Park, Ill.); p75 fusion proteins such as etanercept effective or even non-effective. In certain instances, the meth (Enbrel(R) (Amgen; Thousand Oaks, Calif.; Wyeth Pharma ods of the present invention are useful for identifying the ceuticals Inc.; Collegeville, Pa.), small molecules (e.g., MAP 25 development of secondary response failure before the patient kinase inhibitors); and combinations thereof. See, Ghosh, or medical practitioner has noticed that the treatment is less Novartis Found Symp., 263:193-205 (2004). effective. A higher dosage of treatment may be applied to Other biologic agents include, e.g., anti-cell adhesion anti ensure the correct in vivo concentration is achieved, or an bodies such as natalizumab (Tysabri R) (Elan Pharmaceuti alternative treatment can be selected, or a combination cals, Inc.; Dublin, Ireland; Biogen Idec; Cambridge, Mass.), 30 thereof. Where the therapeutic agent is a diagnostic agent, the which is a humanized monoclonal antibody against the cel development of secondary response failure can be particu lular adhesion molecule C4-integrin, and MLN-02 (Millen larly catastrophic. Radio-labeled monoclonal antibodies are nium Pharmaceuticals; Cambridge, Mass.), which is a routinely used in the monitoring of diseases such as cancers, humanized IgG1 anti-C437-integrin monoclonal antibody; and some infectious diseases, where it is important to deter anti-T cell agents; anti-CD3 antibodies such as visilizumab 35 mine the size and/or location of the disease/agent, e.g., in (Nuvion(R) (PDL BioPharma: Incline Village, Nev.), which is identifying the presence and/or location of any secondary a humanized IgG2M3 anti-CD3 onoclonal antibody; anti metastases. When the development of response failure (either CD4 antibodies such as priliximab (cM-T412) (Centocor, primary or secondary) occurs unnoticed, the patient may be Inc.; Horsham, Pa.), which is a chimeric anti-CD4 mono given the all clear, i.e., a false negative result, which can lead clonal antibody; anti-IL-2 receptor alpha (CD25) antibodies 40 to the cessation of treatment and the latter reappearance of the such as daclizumab Zenapax(R) (PDL BioPharma; Incline disease, often in a far more developed and possibly untreat Village, Nev.; Roche: Nutley, N.J.), which is a humanized able condition. IgG1 anti-CD25 monoclonal antibody, and basiliximab A further category of response failure is the development of (Simulect(R) (Novartis; Basel, Switzerland), which is a chi (e.g., secondary) response failure associated with adverse meric IgG1 anti-CD25 monoclonal antibody; and combina 45 side-effects. The development of a host-immune response in tions thereof. a Subject can be accompanied by deleterious or unpleasant An individual can also be monitored at periodic time inter side-effects. These may be caused by the development of vals to assess the efficacy of a certain therapeutic regimen antibodies which recognize the human or humanized thera once diagnostic, prognostic and/or predictive information has peutic agents but which fail to distinguish between other host been obtained from the individuals sample. For example, the 50 immunoglobulins. The methods of the present invention are presence or concentration level of a therapeutic antibody therefore suitable to identify subjects who have either an and/or an autoantibody directed to the therapeutic antibody innate or previously-developed immune response to the may change based on the therapeutic effect of treatment with therapeutic agent and who may, for example, be vulnerable to the therapeutic antibody. In certain embodiments, the patient adverse side-effects associated with response failure. can be monitored to assess response and understand the 55 The methods of the present invention are suitable for the effects of certain drugs or treatments in an individualized selection and management of treatment regimes which approach. Additionally, patients may not respond to a drug, involve the administration of monoclonal therapeutic agents but the antibody levels may change, suggesting that these to patients. Accordingly, the methods for determining the patients belong to a special population (not responsive) that concentration or bioavailability of a therapeutic antibody as can be identified by their antibody levels. These patients can 60 described herein can be incorporated into a method of treat be discontinued on their current therapy and alternative treat ment of a disease or a disorder. By monitoring the immuno ments prescribed. logical status of a patient using the methods of the present An individual can also be monitored at periodic time inter invention during the course of therapeutic treatment, the vals to assess the concentrations or levels of various antibod selection and/or administration of the therapeutic agent can ies. The antibody levels at various time points, as well as the 65 be tailored to ensure maximum therapeutic benefit to the rate of change of the antibody levels over time is significant. patient, while ensuring the cost-effective use of expensive In certain instances, the rate of increase of one or more anti therapeutic agents. US 8,865,417 B2 43 44 The methods of the present invention may be used to deter FIG. 1 shows an example of the assay of the present inven mine whether a patient requires either an altered dosage tion wherein size exclusion HPLC is used to detect the bind regime of the therapeutic agent (e.g., therapeutic antibody) or ing between TNFC.-Alexa, and HUMIRATM (adali an alternative pharmaceutical therapy. mumab). As shown in FIG. 1, the binding of HUMIRATM to The methods of the present invention may further involve a TNFC-Alexa, caused a shift of the TNFC.-Alexa, peak to periodic assessment of the serum concentration or bioavail the left. ability of the therapeutic agent (e.g., therapeutic antibody) in FIG.2 shows dose responsecurves of HUMIRATM binding the patient. to TNFC.-Alexaaz. In particular, FIG. 2A shows that The methods of the present invention also provide for HUMIRATM dose-dependently increased the shift of TNFC.- determining whether a subject has developed an immune 10 Alexa, in the size exclusion chromatography assay. FIG. response to a therapeutic agent Such as atherapeutic antibody. 2B shows that the presence of 1% human serum did not have The present invention also provides for a method of deter a significant effect on the shift of TNFC-Alexa, in the size mining whether the lack of treatment response in a patient is exclusion chromatography assay. FIG. 2C shows that the due to the formation of patient-derived immunoglobulins presence of pooled RF-positive serum did not have a signifi against a therapeutic antibody Such as, e.g., an anti-TNFC. 15 cant effect on the shift of TNFC.-Alexa, in the size exclusion antibody. chromatography assay. The present invention further provides for a method of As such, this example demonstrates the utility of the selecting the appropriate drug treatment for a patient Suffer present invention in monitoring patients receiving an anti ing from a disease which is treatable with a therapeutic anti TNFC. drug such as HUMIRATM: (1) to guide in the determi body. nation of the appropriate drug dosage; (2) to evaluate drug The present invention also provides for a prognostic pharmacokinetics, e.g., to determine whether the drug is method for the determination of the likelihood of whether a being cleared from the body too quickly; and (3) to guide patient will develop secondary response failure to a therapeu treatment decisions, e.g., whether to Switch from the current tic antibody. anti-TNFC. drug to a different TNFC. inhibitor or to another 25 type of therapy. VII. EXAMPLES Example 2 The present invention will be described in greater detail by way of specific examples. The following examples are Novel Mobility Shift Assay for Measuring HACA offered for illustrative purposes, and are not intended to limit 30 and HAHA Levels the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which This example illustrates a novel homogeneous assay for can be changed or modified to yield essentially the same measuring autoantibody (e.g., HACA and/or HAHA) con results. centrations in a patient sample (e.g., serum) using size exclu 35 sion chromatography to detect the binding of these autoanti Example 1 bodies to fluorescently labeled anti-TNFC. drug. The assay is advantageous because it obviates the need for wash steps Novel Mobility Shift Assay for Measuring Levels of which remove low affinity HACA and HAHA, uses fluoro Anti-TNFC. Biologics phores that allow for detection on the visible and/or fluores 40 cent spectra which decreases background and serum interfer This example illustrates a novel homogeneous assay for ence issues, increases the ability to detect HACA and HAHA measuring anti-TNFC. drug concentration in a patient sample in patients with a low titer due to the high sensitivity of (e.g., serum) using size exclusion chromatography to detect fluorescent label detection, and occurs as a liquid phase reac the binding of the anti-TNFC. drug to fluorescently labeled tion, thereby reducing the chance of any changes in the TNFC. The assay is advantageous because it obviates the 45 epitope by attachment to a solid Surface Such as an ELISA need for wash steps, uses fluorophores that allow for detection plate. on the visible and/or fluorescent spectra which decreases The clinical utility of measuring autoantibodies (e.g., background and serum interference issues, increases the abil HACA, HAHA, etc.) that are generated against TNFC. inhibi ity to detect anti-TNFC. drugs in patients with a low titer due tors is illustrated by the fact that HACAs were detected in to the high sensitivity of fluorescent label detection, and 50 53%. 21%, and 7% of rheumatoid arthritis patients treated occurs as a liquid phase reaction, thereby reducing the chance with 1 mg/kg, 3 mg/kg, and 10 mg/kg infliximab. When of any changes in the epitope by attachment to a Solid Surface infliximab was combined with methotrexate, the incidence of such as an ELISA plate. antibodies was lower 15%, 7%, and 0%, which indicates that In one exemplary embodiment, TNFC. is labeled with a concurrent immunosuppressive therapy is effective in lower fluorophore (e.g., Alexaaz), wherein the fluorophore can be 55 ing anti-drug responses, but also indicates that a high dose of detected on either or both the visible and fluorescent spectra. anti-TNFC. drug might lead to tolerance. In Crohn's disease, The labeled TNFC. is incubated with human serum in a liquid a much higher incidence was reported; after the fifth infusion, phase reaction to allow the anti-TNFC. drug present in the 61% of patients had HACA. The clinical response was short serum to bind. The labeled TNFO. can also be incubated with ened when HACAS were present. See, Rutgeerts, N. Engl. J. known amounts of the anti-TNFC. drug in a liquid phase 60 Med., 348:601-608 (2003). A retrospective study of inflix reaction to create a standard curve. Following incubation, the imab and HACA levels measured over a 3 year period from samples are loaded directly onto a size exclusion column. 2005 to 2008 in 155 patients demonstrated that HACAS were Binding of the anti-TNFC. drug to the labeled TNFC. results in detected in 22.6% (N-35) of patients with inflammatory a leftward shift of the peak compared to labeled TNFC. alone. bowel disease. See, Afif et al., “Clinical Utility of Measuring The concentration of the anti-TNFC. drug present in the serum 65 Infliximab and Human Anti-Chimeric Antibody Levels in sample can then be compared to the standard curve and con Patients with Inflammatory Bowel Disease'; paper presented trols. at Digestive Disease Week; May 30-Jun. 3, 2009; Chicago, US 8,865,417 B2 45 46 Ill. The authors concluded that changing treatment based on using the size exclusion chromatography assay of the present clinical symptoms alone may lead to inappropriate manage invention. The binding of anti-IgG antibody to REMI ment. CADETM-Alexa, caused a shift of the REMICADETM-Al The homogeneous mobility shift assay is advantageous exa, peak to the left. FIG. 6 shows a second dose response over current methods such as the bridging assay shown in 5 analysis of anti-human IgG antibody binding to REMI FIG. 3 for measuring autoantibody (e.g., HACA and/or CADETM-Alexa, as detected using the size exclusion chro HAHA) concentrations in a patient sample because the inven matography assay of the present invention. Higher amounts tive method is capable of measuring the concentration of of anti-IgG antibody resulted in a dose-dependent increase in autoantibodies such as HACA without non-specific binding the formation of anti-IgG/REMICADETM-Alexa, com and solid phase interference from the ELISA plate, without 10 plexes, as indicated by a shift of the REMICADETM-Alexa, interference from the anti-TNFC. drug (e.g., with the bridging peak to the left. FIG. 7 shows dose response curves of anti assay, HACA measurements must be taken at anti-TNFC. IgG antibody binding to REMICADETM-Alexa7. drug trough levels), and without any dependency on the mul FIG. 8 shows REMICADETM-Alexa, immunocomplex tivalency of the antibody (e.g., IgG4 antibodies are not formation in normal human serum and HACA positive serum detected using the bridging assay because IgG4 antibodies 15 as detected using the size exclusion chromatography assay of are bispecific and cannot cross-link the same antigen). As the present invention with 100 ul of injected sample. As Such, the present invention has at least the following advan shown in FIG. 8, the binding of HACA present in patient tages over current methods: avoids attachment of antigens to samples to REMICADETM-Alexa, caused a shift of the Solid Surfaces (denaturation avoided); eliminates the IgG4 REMICADETM-Alexa, peak to the left. As such, the size effect; overcomes therapeutic antibody trough issues; detects exclusion chromatography assay of the invention is particu antibodies with weak affinities; eliminates non-specific bind larly advantageous because it measures HACA in the pres ing of irrelevant IgGs; and increases the sensitivity and speci ence of REMICADETM, can be utilized while the patient is on ficity of detection. therapy, measures both weak and strong HACA binding, is a In one exemplary embodiment, an anti-TNFC. drug (e.g., mix and read mobility shift assay, and can be extended to REMICADETM) is labeled with a fluorophore (e.g., 25 other approaches which use labeled REMICADETM to equili Alexaaz), wherein the fluorophore can be detected on either brate with HACA and REMICADETM. or both the visible and fluorescent spectra. The labeled anti FIG. 9 provides a summary of HACA measurements from TNFC. drug is incubated with human serum in a liquid phase 20 patient serum samples that were performed using the reaction to allow HACA and HAHA present in the serum to bridging assay or the mobility shift assay of the present inven bind. The labeled anti-TNFC. drug can also be incubated with 30 tion. This comparative study demonstrates that the present known amounts of an anti-IgG antibody in a liquid phase methods have increased sensitivity over current methods reaction to create a standard curve. Following incubation, the because 3 samples that were negative for HACA as measured samples are loaded directly onto a size exclusion column. using the bridging assay were actually HACA positive when Binding of the autoantibodies to the labeled anti-TNFC. drug measured using the mobility shift assay of the present inven results in a leftward shift of the peak compared to labeled drug 35 tion (see, Patient # SK07070305, SK07070595, and alone. The concentration of HACA and HAHA present in the SK07110035). serum sample can then be compared to the standard curve and As such, this example demonstrates the utility of the controls. FIG. 4 illustrates an exemplary outline of the present invention in monitoring patients receiving an anti autoantibody detection assays of the present invention for TNFC. drug (e.g., REMICADETM) to detect the presence or measuring the concentrations of HACA/HAHA generated 40 level of autoantibodies (e.g., HACA and/or HAHA) against against REMICADETM. In certain instances, high HACA/ the drug, because Such immune responses can be associated HAHA levels indicate that the current therapy with REMI with hypersensitive reactions and dramatic changes in phar CADETM should be switched to anotheranti-TNFC. drug such macokinetics and biodistribution of the anti-TNFC. drug that as HUMIRATTM. preclude further treatment with the drug. The principle of this assay is based on the mobility shift of 45 In conclusion, Examples 1 and 2 demonstrate that TNFC. the antibody bound Alexa-labeled REMICADETM com and anti-TNFC. antibodies can be efficiently labeled with plex versus free Alexa-labeled REMICADETM on size Alexa. When labeled TNFC.-Alexa, was incubated with exclusion-high performance liquid chromatography (SE anti-TNFC. antibodies, the retention time of the labeled HPLC) due to the increase in molecular weight of the com TNFO/anti-TNFC. drug complex was shifted, and the amount plex. 50 of anti-TNFC. drug that caused the shift could be quantitated The chromatography in this example was performed on an with HPLC. Furthermore, when labeled anti-TNFO. drug was Agilent- 1200 HPLC System, using a Bio-Sep 300x7.8 mm incubated with anti-human IgG antibody, the retention time SEC-3000 column (Phenomenex) with a molecular weight of the labeled anti-TNFC. drug/anti-IgG antibody complex fractionating range of 5,000-700,000 and a mobile phase of was shifted, and the amount of anti-IgG antibody that caused 1xRBS, pH 7.4, at a flow-rate of 0.5 mL/min with UV detec 55 the shift could be quantitated with HPLC. Moreover, low tion at 650 nm. In front of the Agilent-1200 HPLC System serum content in the assay system was shown to have little with a Bio-Sep300x7.8 mm SEC-3000 column is a analytical effect on HPLC analysis. Finally, a standard curve could be pre-column which is a BioSep 75x7.8 mm SEC-3000. A 100 generated for the anti-TNFC. drug and HACA/HAHA assays LL sample Volume is loaded onto the column for each analy and could be used to quantitate patient serum anti-TNFC. drug S1S. 60 or HACA/HAHA levels. Advantageously, the present inven The antibody bound Alexa-labeled REMICADETM tion provides in certain aspects a mobility shift assay, Such as complex is formed by incubating a known amount of the a homogeneous mix and read assay developed to measure antibody and Alexa-labeled REMICADETM in the 1xPBS, both drug and antibodies against the drug. A standard curve pH 7.3, elution buffer at room temperature for 1 hour before was generated for the anti-TNFC. biologic REMICADETM SE-HPLC analysis. 65 and HUMIRA and also for the HACA antibodies against FIG. 5 shows a dose response analysis of anti-human IgG REMICADETM. The mobility shift assay format, unlike antibody binding to REMICADETM-Alexa, as detected ELISA, eliminates coating of antigens to solid Surface and is US 8,865,417 B2 47 48 not affected by non-specific binding of irrelevant IgGs. The Introduction assay format is simple, but very sensitive and can be used to Tumor necrosis factor-alpha (TNFO) plays a pivotal role in detect all anti-TNFC. biologic drugs (e.g., REMICADTM, the pathogenesis of autoimmune diseases Such as Crohn's HUMIRA, Enbrel and Cimzia) as well as the neutralizing disease (CD) and rheumatoid arthritis (RA). It is well docu antibody (anti-REMICADETM) in patient serum. mented that blocking TNFC. with therapeutic antibodies such as Infliximab (human-murine chimeric monoclonal IgG1K) Example 3 or adalimumab (fully human monoclonal antibody) reduces disease activity in CD and RA. However, about 30-40% of the Measurement of Human Anti-Chimeric Antibodies patients do not respond to anti-TNFC. therapy and some (HACA) and Infliximab (IFX) Levels in Patient 10 patients need higher doses or dosing frequency adjustments Serum Using a Novel Mobility Shift Assay due to lack of sufficient response. Differences of drug bio availability and pharmacokinetics in individual patients may Abstract contribute to the failure of the treatment. Immunogenicity of Background: the drugs, which causes patients to develop HACA/HAHA. Infliximab (IFX) is a chimeric monoclonal antibody thera 15 could result in a range of adverse reactions from mild allergic peutic against TNFC. that has been shown to be effective in response to anaphylactic shock. These problems are now treating autoimmune diseases such as rheumatoid arthritis recognized by many investigators, drug-controlling agencies, (RA) and inflammatory bowel disease (IBD). However, anti health insurance companies, and drug manufacturers. Fur bodies against IFX were found in some IFX-treated patients thermore, many patients with secondary response failure to through the detection of human anti-chimeric antibodies one anti-TNFC. drug benefit from a switch to other anti-TNFC. (HACA), which may reduce the drug's efficacy or induce drugs, suggesting a role of neutralizing antibodies directed adverse effects. Monitoring of HACA and IFX levels in indi specifically against the protein used for treatment (Radstake vidual patients may help to optimize the dosing and treatment et al., Ann. Rheum. Dis., 68(11):1739-45 (2009)). Monitoring with IFX. Current methods for detecting HACA are based on of patients for drug and HACA/HAHA levels is therefore solid-phase assays, which are limited by the fact that the 25 warranted so that drug administration can be tailored to the presence of IFX in the circulation may mask the presence of individual patient and prolonged therapies can be given effec HACA and, therefore, measurement can only be done at least tively and economically with little or no risk to patients 8 weeks following a dose of IFX. Moreover, this time-lapse (Bendtzen et al., Scand. J. Gastroenterol., 44(7):774-81 further confounds the assays because of the rapid clearance of (2009)). the high molecular weight immune complexes in the blood 30 Several enzyme-linked immunoassays have been used to circulation. To overcome these drawbacks, we have devel assess the circulating levels of drugs and HACA/HAHA. FIG. oped and evaluated a new method to measure serum IFX and 10 provides a summary of the current assays available for the HACA levels in patients treated with IFX. measurement of HACA in comparison to the novel HACA Methods: assay of the present invention. One of the limitations of cur A novel non-radiolabeled, liquid-phase, size-exclusion 35 rent methodologies is that antibody levels are difficult to (SE)-HPLC mobility shift assay was developed to measure measure when there is a measurable amount of drug in the the HACA and IFX levels in serum from patients treated with circulation. In contrast to current Solid-phase methods for IFX. The immuno-complex (e.g., TNFO/IFXor IFX/HACA), detecting HACA in which measurements can only be per free TNFC. or IFX, and the ratio of bound/free can be resolved formed at least 8 weeks following a dose of IFX, the novel and calculated with high sensitivity. Serum concentrations of 40 assay of the present invention is a non-radiolabeled, liquid IFX or HACA were determined with standard curves gener phase, size-exclusion (SE)-HPLC assay that is capable of ated by incubating with different concentrations of IFX or measuring HACA and IFX levels in serum from patients pooled HACA-positive serum. Using this novel assay, we while being treated with IFX. have measured IFX and HACA levels in sera collected from The following are rationales for measuring the serum con IBD patients treated with IFX who had relapsed and com 45 centrations of anti-TNFC. biologic drugs and antibodies pared the results with those obtained by the traditional Bridge against TNFC. biologic drugs in patients: (1) for PK studies in ELISA assay. clinical trials; (2) it may be required by the FDA during Results: clinical trials to monitor a patient’s immune response to the Dose-response curves were generated from the novel assay biologic drug; (3) to monitor a patient's response to the bio with high sensitivity. Detection of HACA was demonstrated 50 logic drug by measuring HACA or HAHA to guide the drug in the presence of excess IFX. In the 117 serum samples from dosage for each patient; and (4) for use as a guide for Switch patients treated with IFX, 65 samples were found to have IFX ing to a different biologic drug when the initial drug fails. levels above the detection limit and the average was 11.0+6.9 Methods mg/mL. For HACA levels, 33 (28.2%) samples were found to SE-HPLC Analysis of Infliximab (IFX) Levels in Patient be positive while the Bridge ELISA assay detected only 24 55 Serum. positive samples. We also identified 9 false negatives and 9 Human recombinant TNFC. was labeled with a fluorophore false positives from the samples determined by the Bridge (“Fl' such as, e.g., Alexa Fluor R. 488) according to the manu assay. HACA levels were found to be increased in 11 patients facture's instructions. Labeled TNFC. was incubated with during the course of IFX treatment while the IFX levels were different amounts of IFX or patient serum for one hour at found to be significantly decreased. 60 room temperature. Samples of 100 u, volume were analyzed Conclusions: by size-exclusion chromatography on an HPLC system. FLD A novel non-radiolabeled, liquid-phase, mobility shift was used to monitor the free TNFC.-Fland the bound TNFC.- assay has been developed to measure the IFX and HACA F1 immuno-complex based on their retention times. Serum levels in serum from patients treated with IFX. The assay has IFX levels were calculated from the standard curve. high sensitivity and accuracy, and the obtained results were 65 SE-HPLC Analysis of HACA Levels in Patient Serum. reproducible. This novel assay can advantageously be used to Purified IFX was labeled with Fl. Labeled IFX was incu measure HACA and IFX levels while patients are on therapy. bated with different dilutions of pooled HACA-positive US 8,865,417 B2 49 50 serum or diluted patient serum for one hour at room tempera TABLE 2 ture. Samples of 100 uL volume were analyzed by size exclusion chromatography on an HPLC system. FLD was Correlation of Relative HACA Levels in Patient Serum from Strong used to monitor the free IFX-F1 and the bound IFX-Fl Positive and Negative on Bridge Assay to SE-HPLC Assay. immuno-complex based on their retention times. The ratio of 5 HPLC shift bound and free IFX-F1 was used to determine the HACA Bridge assay assay Correlation level. Positive 82 81 99% Mobility Shift Assay Procedure to Measure HACA in Negative 12 12 100% Serum. 10 The principle of this assay is based on the mobility shift of the HACA bound Fl-labeled Infliximab (IFX) complex ver TABLE 3 sus free Fl-labeled IFX on size exclusion-high performance Patient Sample Analysis on Serum Levels of HACA with liquid chromatography (SE-HPLC) due to the increase in Bridge Assay (Cut Off 1.69 g/ml) and HPLC Shift molecular weight of the complex. The chromatography is 15 Assay (Cut Off 0.19. Ratio of Bound and Free IFX). performed in an Agilent- 1200 HPLC System, using a Bio-Sep HACA Positive Bridge Assay 300x7.8 mm SEC-3000 column (Phenomenex) with a Subjects Bridge HPLC False False molecular weight fractionating range of 5,000-700,000 and a (n) Assay Assay Negative Positive mobile phase of 1xPBS, pH 7.3, at a flow-rate of 0.5 mL/min Healthy 50 NA O NA NA with FLD detection. In front of the Agilent-1200 HPLC Sys Control Patient 117 24 (20.5%) 33 (28.2%) 9 9 tem with a Bio-Sep 300x7.8 mm SEC-3000 column is a treated (High IFX) analytical pre-column which is a BioSep 75x7.8 mm SEC with IFX 3000. A 100 uL sample volume is loaded onto the column for 25 False negative results are caused by patient serum containing high levels of DFX which interferes with the Bridge assay on HACA determination while the SE-HPLC assay is not each analysis. The HACA bound Fl-labeled IFX complex is affected. False positive results are caused by patient serum containing high levels of non formed by incubating serum from IFX treated patient and specific interference substance which may interfere with the Bridge assay, Fl-labeled IFX in the 1xPBS, pH 7.3, elution buffer at room FIG. 15 shows the relationship of the HACA level and IFX temperature for 1 hour before SE-HPLC analysis. 30 concentration in IBD patients during the course of IFX treat Results ment. HACA could be detected as early as V10 (30 Weeks) FIG. 11 shows the separation of the HACA bound IFX-Fl and continued to increase in some patients during IFX treat complex from the free IFX-F1 due to the mobility shift of the ment. FIG. 16 shows that HACA can be detected in the high molecular weight complex. As seen in panels candid, the presence of IFX using the assay of the present invention. A 35 higher level of HACA in the serum was associated with a retention time of the fluorescent peak shifted from 21.8 minto lower level of IFX that could be detected (e.g., reduced the 15.5-19.0 min. The more the HACA is present in the reaction bioavailability). As such, early detection of HACA while on mixture, the less the free IFX-F1 remains in the chromatogram treatment with IFX can guide the physician and/or patient to and the more the immuno-complex is formed. FIG. 12 shows switch to other anti-TNF drugs or increase the dose of IFX. the dose-response curves of the fluorescent peak shift caused 40 by the addition of HACA. Using the HACA positive sample, CONCLUSION we could detect the peak shift with 1:1000 dilutions of the S. Anti-TNFC. biologic drugs can be readily labeled with a FIG. 13 shows the separation of the IFX bound TNFC-Fl fluorophore (“Fl') and the mobility shift assay format used complex from the free TNFO-F1 due to the mobility shift of 45 for measuring HACA/HAHA is a homogeneous assay with the high molecular weight complex. As seen in panels candid, out the coating of antigens to a solid Surface and multiple the retention time of the fluorescent peak shifted from 24 min washing and incubation steps like a typical ELISA. Incuba to 13-19.5 min. The more the IFX is present in the reaction tion of Fl-labeled IFX with HACA-positive serum results in mixture, the less the free TNFC.-Fl remains in the chromato the formation of an immune complex which elutes at a dif gram and the more the immuno-complex is formed. FIG. 14 50 ferent position compared to free Fl-labeled IFX in SE-HPLC and thus the amount of HACA can be quantitated. The pres shows the dose-response curves of the TNFC.-Fl peak shift ence of other serum components has little effect on the mobil caused by the addition of IFX. Based on the added IFX, the ity shift assay. The mobility shift assay format, unlike ELISA, detection limit is 10 ng/mL of IFX in serum. is not affected by non-specific binding of irrelevant IgGs and The novel mobility shift assay of the present invention was 55 detects the IgG4 isotype. Healthy serum samples do not cause validated by testing serum samples from HACA positive and mobility shift of the Fl-labeled IFX and 28.2% of the patients negative patients measured by the Bridge assay (Table 2). treated with IFX were found to have HACA by the assay of Using this assay, we have analyzed serum samples from 50 the present invention. As such, the assay format described healthy subjects and 117 IBD patients treated with IFX. All herein is very sensitive and can be applied to detect all bio 50 healthy subject samples have an IFX level below the limit 60 logic drugs (e.g., REMICADETM, HUMIRA, Enbrel and of detection, whereas 65 of the patient samples have an aver Cimzia) as well as their antibodies (e.g., anti-REMICADETM, age IFX concentration of 11.0 lug/ml. Table 3 shows the anti-HUMIRA, anti-Enbrel and anti-Cimzia) in patient HACA levels in the serum of healthy controls and IBD serum. Notably, since HACA can be detected in the presence patients treated with IFX measured by the Bridge assay and of IFX using the mobility shift assay of the invention, early the mobility shift assay. The Bridge assay detected less 65 detection of HACA while on treatment with IFX can guide HACA-positive patients than the mobility shift assay and the physician and/or patient to switch to other anti-TNF drugs more false negatives as well as more false positives. or increase the subsequent dose of IFX. US 8,865,417 B2 51 52 We have developed a novel non-radiolabeled, liquid-phase, CADETM and F2-TNFO, resulting in an increase in both the SE-HPLC assay to measure the IFX and HACA levels in Immuno-Complex 1 and Immuno-Complex 2 peaks and a serum samples obtained from patients treated with IFX. The decrease in the free F1-REMICADETM and free F2-TNFC. novel assay has high sensitivity, accuracy, and precision, and peaks. However, the presence of the binary complex between the results are highly reproducible, which makes this assay the autoantibody (e.g., HACA) and the labeled anti-TNFC. Suitable for routine testing of a large number of human serum drug (e.g., “Immuno-Complex 2 in FIG. 17B) in the absence samples. The new assay format, unlike ELISA, eliminates of the ternary complex of autoantibody (e.g., HACA), labeled coating of antigens to Solid Surfaces and is not affected by TNFC, and labeled anti-TNFC. drug indicates that the autoan non-specific binding of irrelevant IgGs. These advantages of tibody present in the serum sample is a neutralizing form of the assay format described herein reduce the false negative 10 the autoantibody (e.g., HACA), such that the autoantibody and false positive results of the test. Advantageously, the interferes with the binding between the anti-TNFC. drug and assay format of the present invention is very sensitive and can TNFC. In one particular embodiment, as shown in FIG. 17B. be used to detect all biologic drugs as well as their antibodies if neutralizing HACA is present in the serum, a shift will be present in the serum while the patient is on therapy. observed for F1-REMICADETM, resulting in an increase in 15 the Immuno-Complex 2 peak, a decrease in the free Example 4 F1-REMICADETM peak, and no change in the free F2-TNFC. peak. In certain instances, the presence of neutralizing HACA Differentiation Between Neutralizing and indicates that the current therapy with REMICADETM should Non-Neutralizing Human Anti-Chimeric Antibodies be switched to another anti-TNFC. drug such as (HACA) in Patient Serum Using Novel Mobility HUMIRATTM. Shift Assays In an alternative embodiment, the labeled anti-TNFC. drug is first incubated with human serum in a liquid phase reaction This example illustrates novel homogeneous assays for to allow the formation of complexes (i.e., immuno-com measuring autoantibody (e.g., HACA) concentrations in a plexes) between the labeled anti-TNFC. drug and HACA patient sample (e.g., serum) and for determining whether 25 present in the serum. Following incubation, the samples are Such autoantibodies are neutralizing or non-neutralizing loaded directly onto a first size exclusion column. Binding of autoantibodies using size exclusion chromatography to detect the autoantibody (e.g., HACA) to the labeled anti-TNFC. drug the binding of these autoantibodies to fluorescently labeled results in a leftward shift of the peak (e.g., “Immuno-Com anti-TNFC. drug in the presence of fluorescently labeled plex 2 in FIG. 18) compared to the labeled drug alone. The TNFC. These assays are advantageous because they obviate 30 labeled TNFC. is then added to the reaction to determine the need for wash steps which remove low affinity HACA, use whether it is capable of displacing (e.g., competing with) the distinct fluorophores that allow for detection on the visible autoantibody (e.g., HACA) for binding to the labeled anti and/or fluorescent spectra which decreases background and TNFC. drug, to thereby allow the formation of complexes (i.e., serum interference issues, increase the ability to detect neu immuno-complexes) between the labeled anti-TNFC. drug tralizing or non-neutralizing HACA in patients with a low 35 and the labeled TNFC. Following incubation, the samples are titer due to the high sensitivity of fluorescent label detection, loaded directly onto a second size exclusion column. Binding and occur as a liquid phase reaction, thereby reducing the of the labeled anti-TNFC. drug to the labeled TNFC. results in chance of any changes in the epitope by attachment to a solid a leftward shift of the peak (e.g., “Immuno-Complex 3” in surface such as an ELISA plate. FIG. 18) compared to the labeled TNFC. alone. Disruption of In one exemplary embodiment, an anti-TNFC. drug (e.g., 40 the binding between the autoantibody (e.g., HACA) and the REMICADETM) is labeled with a fluorophore “F1 (see, e.g., labeled anti-TNFC. drug by the addition of the labeled TNFC. FIG. 17A), wherein the fluorophore can be detected on either indicates that the autoantibody present in the serum sample is or both the visible and fluorescent spectra. Similarly, TNFC. is a neutralizing form of the autoantibody (e.g., HACA). Such labeled with a fluorophore “F2 (see, e.g., FIG. 17A), that the autoantibody interferes with the binding between the wherein the fluorophore can also be detected on either or both 45 anti-TNFC. drug and TNFC. In certain instances, the presence the visible and fluorescent spectra, and wherein “F1 and of neutralizing HACA indicates that the current therapy with “F2 are different fluorophores. The labeled anti-TNFC drug REMICADETM should be switched to another anti-TNFC. is incubated with human serum in a liquid phase reaction and drug such as HUMIRATTM. the labeled TNFC. is added to the reaction to allow the forma tion of complexes (i.e., immuno-complexes) between the 50 Example 5 labeled anti-TNFC drug, labeled TNFC, and/or HACA present in the serum. Following incubation, the samples are Analysis of Human Anti-Drug Antibodies (ADA) to loaded directly onto a size exclusion column. Binding of both Adalimumab in Patient Serum Using a Novel the autoantibody (e.g., HACA) and the labeled TNFC. to the Homogeneous Mobility Shift Assay labeled anti-TNFC. drug results in a leftward shift of the peak 55 (e.g., “Immuno-Complex 1 in FIG. 17A) compared to a Background and Aim: binary complex between the autoantibody and the labeled Monoclonal antibodies against TNF-C. such as infliximab anti-TNFC. drug (e.g., “Immuno-Complex 2 in FIG. 17A), (IFX), adalimumab (HUMIRATM), and certolizumab have the labeled drug alone, or the labeled TNFO, alone. The pres been shown to be effective in treating inflammatory bowel ence of this ternary complex of autoantibody (e.g., HACA), 60 disease (IBD) and other inflammatory disorders. Anti-drug labeled TNFC, and labeled anti-TNFC. drug indicates that the antibodies (ADA) may reduce the drug’s efficacy and/or autoantibody present in the serum sample is a non-neutraliz induce adverse effects. However, ADAs have been found not ing form of the autoantibody (e.g., HACA), such that the only inpatients treated with the chimericantibody infliximab, autoantibody does not interfere with the binding between the but also inpatients treated with the humanized antibody adali anti-TNFC. drug and TNFC. In one particular embodiment, as 65 mumab. Monitoring of ADA and drug levels in individual shown in FIG. 17A, if non-neutralizing HACA is present in patients may help optimize treatment and dosing of the the serum, a shift will be observed for both F1-REMI patient. We have developed a non-radio labeled liquid-phase US 8,865,417 B2 53 54 homogeneous mobility shift assay to accurately measure in inability to accurately detect HACA in the presence of IFX in the serum both HACA (Human Anti-Chimeric Antibody) and circulation. In the present study, we have evaluated this new IFX from patients. This assay method overcomes a major assay to measure serum ADA and drug levels in patients limitation of the current Solid-phase assays for detecting treated with the fully human antibody drug, ADL. HACA, namely the inability to accurately detect HACA in the Methods: presence of IFX in circulation. In the present study, we have The mobility shift assay was based on the shift in retention evaluated this new method for measuring serum ADA and time of the antigen-antibody immunocomplex versus free drug levels in patients treated with the humanized antibody antigen on size-exclusion chromatography. Fluorophore-la drug, adalimumab. beled ADL or TNF-C. and internal control were mixed with Methods: 10 serum samples to measure the mobility shift of labeled ADL The mobility shift assay was based on the shift in retention and TNF-C. in the presence of ADA or drug. The changes in time of a free antigen versus antigen-antibody immunocom the ratio of free ADL or TNF-C. to internal control are the plex on size-exclusion separation. Fluorophore-labeledadali indicators of the immunocomplex formation. Serum concen mumab or TNF-C. and internal control were mixed with serum trations of ADA or ADL were determined with standard samples to measure the mobility shift of free adalimumab and 15 curves generated by incubating with different concentrations INF-C. in the presence of ADA or drug. The changes in the of anti-human IgG antibody or purified ADL. Using this ratio of free adalimumab or TNF-C. to internal control are assay, we measured ADL and ADA levels in sera collected indicators of immunocomplex formation. Serum concentra from IBD patients treated with ADL. tions of ADA or adalimumab were determined with standard Results: curves generated by incubating with different concentrations Dose-response curves were generated with anti-human of anti-human IgG antibody or purified adalimumab. Using IgG antibody for the measurement of mobility shift of labeled the mobility shift assay, we measured adalimumab and ADA ADL. The detection limit of the assay was 10 ng of anti levels in sera collected from IBD patients treated with adali human IgG. Sera from 100 healthy controls were tested for mumab who had lost response. the ADA and all of the samples had an ADA level below Results: 25 detection limit (no shift of free labeled ADL). Detection of Dose-response curves were generated with anti-human ADA was demonstrated in five out of 114 IBD patient IgG antibody for the measurement of mobility shift of labeled samples treated with ADL. To measure the drug concentra adalimumab. The detection limit of the assay was 1 ng of tion in patients treated with ADL, we generated a standard anti-human IgG. Sera from fifty healthy controls were tested curve with different amounts of ADL on the shift of labeled for ADA and all of the samples had ADA levels below the 30 TNF-C. with the detection limit of 10 ng. detection limit (i.e., no shift of the free labeled-adalimumab). Conclusions: Detection of ADA was also demonstrated in the presence of We have applied our proprietary non-radio labeled liquid exogenously added adalimumab. To measure the drug con phase homogeneous mobility shift assay to measure the ADA centration in patients treated with adalimumab, we generated and ADL levels in serum from patients treated with ADL. The a standard curve with different amounts of adalimumab on the 35 assays are reproducible with high sensitivity and accuracy, mobility shift of labeled TNF-C., and the detection limit of and are useful for evaluating ADA levels in serum samples adalimumab was 10 ng. from patients treated with ADL. Conclusions: Introduction The non-radio labeled liquid-phase homogeneous mobility Anti-tumor necrosis factor-alpha (TNF-C.) biologics such shift assay of the present invention has been applied to mea 40 as infliximab (IFX), etanercept, adalimumab (ADL) and cer Sure ADA and adalimumab levels in serum samples from tolizumab pegol have been shown to reduce disease activity in patients treated with adalimumab. The assay is found to be a number of autoimmune diseases, including Crohn's Dis reproducible with high sensitivity and accuracy, and can be ease (CD) and rheumatoid arthritis (RA). However, some used to evaluate ADA levels in serum samples from patients patients do not respond to anti-TNF-C. therapy, while others treated with adalimumab. 45 need higher or more frequent dosage due to lack of Sufficient response, or develop infusion reactions. Example 6 Immunogenicity of therapeutic antibodies which causes the patients to develop antibodies against the drugs may con Analysis of Anti-Drug Antibodies (ADA) to tribute to the failure of the treatments and infusion reactions. Adalimumab in Patient Serum Using a Novel 50 Chimeric antibodies like IFX have a higher potential of Proprietary Mobility Shift Assay inducing antibody generation compared to fully humanized antibodies such as ADL. The prevalence of antibodies to IFX Abstract (HACA) in RA patients varies from 12% to 44% and seems to Background: be inversely proportional to the level of IFX in patient serum Anti-TNF-C. drugs such as infliximab (IFX) and adali 55 and therapeutic response. While the fully humanized ADL is mumab (ADL) have been shown to be effective in treating Supposed to be less immunogenic than murine or chimeric inflammatory bowel disease (IBD). However, induction of antibodies, several studies have reported the formation of ADA in the treated patients may reduce the drug’s efficacy human anti-humanized antibodies (HAHA) and showed the and/or induce adverse effects. Indeed, ADAs have been found prevalence of antibody generation from 1% to 87% in RA and not only in patients treated with IFX, but also in patients 60 CD patients (Aikawa et al., Immunogenicity of Anti-TNF treated with ADL. Monitoring of ADA and drug levels in Clpha agents in autoimmune diseases. Clin. Rev. Allergy individual patients may help to optimize treatment and dosing Immunol., 38(2-3):82-9 (2010)). of the patient. We have developed a proprietary mobility shift Many patients with secondary response failure to one anti assay to accurately measure in the serum both HACA (Hu TNF-C. drug may benefit from switching to another anti man Anti-Chimeric Antibody) and IFX from IFX-treated 65 TNF-C. drug or increasing dosage and/or dosing frequency. patients. This assay overcomes the major limitation of the Monitoring of patients for drug and anti-drug antibody current Solid-phase assays for detecting HACA, namely the (ADA) levels is therefore warranted so that drug administra US 8,865,417 B2 55 56 tion can be tailored to the individual patient. This approach ADL), whereas 5 out of the 114 patient samples had an ADA allows dose adjustment when warranted or cessation of medi concentration of 0.012 to >20 ug/ml. The mean of ADL levels cation when ADA levels are present. (Bendtzen et al., Indi in 100 healthy subject samples was 0.76+ 1.0 ug/ml (range 0 vidual medicine in inflammatory bowel disease: monitoring to 9.4 ug/ml). The mean of ADL levels in 114 serum samples bioavailability, pharmacokinetics and immunogenicity of from patients treated with ADL was 10.8+17.8 ug/ml (range anti-tumour necrosis factor-alpha antibodies. Scand. J. Gas 0-139 g/ml). Four out of five of the ADA positive samples troenterol., 44(7):774-81 (2009): Afifet al., Clinical utility of had undetectable levels of ADL. measuring infliximab and human anti-chimericantibody con centrations in patients with inflammatory bowel disease. Am. TABLE 4 J. Gastroenterol., 105(5):1133-9 (2010)). 10 A number of assays have been developed to measure Patient Serum Levels of ADA and ADL HACA and HAHA. One of the limitations of the current Measured by the Mobility Shift ASSay methodologies is that ADA levels cannot be reliably mea Subjects Sex Age (Years) ADA ADL level sured when there is a high level of drugs in the circulation. (n) (M/F) (Mean) Positive (ig/ml) We have developed a proprietary non-radiolabeled, liquid 15 Healthy 100 38/62 18-62 (37.1) O O.76 1.OO phase, mobility shift assay to measure the ADA and ADL Control levels in serum from patients treated with ADL which is not IBD Patient 114 51/63 20-69 (39.9) 5 (4.3%) 10.80 + 17.80 affected by the presence of the drug in the serum. Treated with Methods ADL Fluorophore (FI)-labeled ADL was incubated with patient serum to form the immunocomplex. A Fl-labeled small pep Table 5 provides a further analysis of the serum samples tide was included as an internal control in each reaction. from 100 healthy subjects and 114 IBD patients treated with Different amounts of anti-human IgG were used to generate a ADL. In particular, 4 out of the 42 patient samples with 0-4 standard curve to determine the serum ADA level. Free Fl ug/ml ADL had an average ADA concentration of 0.012 to labeled ADL was separated from the antibody bound complex 25 based on its molecular weight by size-exclusion chromatog >20 ug/ml. Four out of four of the ADA positive samples had raphy. The ratio of free F1-labeled ADL to internal control undetectable levels of ADL. For the detection of ADA, the from each sample was used to extrapolate the HAHA con 114 IBD patients treated with ADL were divided into two centration from the standard curve. A similar methodology categories, 0-4 ug/ml of ADL and >4 ug/ml of ADL. Patients was used to measure ADL levels in patient serum samples 30 with greater than 4 Jug/ml of ADL can be tested with a larger with F1-labeled TNF-C. amount of ADL-F1 to address the competition of circulating Results ADL with ADL-F1. FIG. 19 shows the separation of the anti-human IgG bound Fl-ADL complex from the free Fl-ADL due to the mobility TABLE 5 35 shift of the high molecular weight complex. As seen in panels Patient Serum Levels of ADA and ADL c to h, the retention time of the fluorescent peak shifted from Measured by the Mobility Shift Assay 10.1 min to 7.3-9.5 min. The more the anti-human IgG is added in the reaction mixture, the less the free ADL remains Subjects Sex Age ADL Level ADA in the chromatogram and the more the immunocomplex is (n) (M/F) (Mean) (ig/ml) Positive formed (h to c). The retention time for the internal control is 40 Healthy 100 38.62 18-62 O.76 1.00 O 13.5 min. Control (37.1) FIG. 20 shows the dose-response curve of the fluorescent IBD 114 S1.63 20-69 10.80 + 17.80 0-4 g/ml ADL: Patient (39.9) 4 of 42 (9.52%) peak shift caused by the addition of anti-human IgG. Increas Treated ing the concentration of anti-human IgG reduces the ratio of with ADL free ADL to internal control due to the formation of the 45 immunocomplex. The assay sensitivity is 10 ng/ml of anti human IgG. The internal control “Fl-BioCyt” corresponds to Conclusions an Alexa Fluor R. 488-biocytin (BioCyt) which combines the The mobility shift assay format used for measuring HACA/ green-fluorescent Alexa Fluor R. 488 fluorophore with biotin IFX is a homogeneous assay without the coating of antigens and an aldehyde-fixable primary amine (lysine) (Invitrogen 50 to a solid Surface, and without multiple washing and incuba Corp.; Carlsbad, Calif.). tion steps like a typical ELISA. This assay can be applied to FIG. 21 shows the separation of the ADL bound TNF-C-Fl measure ADA and anti-TNF drugs. The sensitivity of the complex from the free TNF-C-F1 due to the mobility shift of assay (in g/ml range) is higher for both ADA and ADL the high molecular weight complex. As seen in panels candi, measurement with patient serum compared to ELISA meth the retention time of the fluorescent peak shifted from 11.9 55 ods (in mg/ml range). Healthy control serum samples did not min to 6.5-10.5 min. The more the ADL is added in the cause mobility shift of the Fl-labeled ADL, and 4.3% of the reaction mixture, the less the free TNF-C-F1 peak remains in patients treated with ADL were found to have ADA by this the chromatogram and the more the immuno-complex is assay. In particular, 9.52% of patients with 0.4/ml ADL were formed. found to have ADA in this assay. Furtherevaluation of normal FIG.22 shows the dose-response curves of the TNF-C-Fl 60 samples and patient samples with higher concentrations of peak shift caused by the addition of ADL. Based on the added ADL-F1 will be done. Although healthy control serum ADL, the detection limit is 10 ng/mL of ADL in serum. samples caused mobility shift of the Fl-labeled TNF-C., which Table 4 shows that serum samples from 100 healthy sub may have been due to the presence of soluble free receptor of jects and 114 IBD patients treated with ADL were analyzed TNF-C., the average of ADL inpatients treated with ADL was for ADA and ADL levels using the mobility shift assay of the 65 much higher (10.8 vs. 0.76 ug/ml). Early detection of ADA present invention. All 100 healthy subject samples had ADA and monitoring of ADL drug level while the patient is receiv levels below the limit of detection (no shift of the free Fl ing ADL treatment will allow the physician to optimize the US 8,865,417 B2 57 58 dosing of ADL or switch to another anti-TNF-C. drug when HPLC, and dividing this number by the resultant integration appropriate and, thereby, optimizing the overall management of the area-under-the-curve for the labeled-TNFC. peak from of the patient’s disease. the plot. The labeled-TNFC. is known a priori. Step 2: Determining Level of Human Anti-Chimeric Anti Example 7 body, HACA. In one exemplary embodiment, an anti-TNFC. drug, e.g., Determining the Concentration Levels of REMICADETM, is labeled with a fluorophore, e.g., Alexa, REMICADETM and Human Anti-Drug Antibodies wherein the fluorophore can be detected by, either or both of the visible and fluorescent spectra. The labeled anti-TNFC. This example describes a method for determining the lev 10 drug is incubated with human serum in a liquid phase reaction els of Anti-TNFC. Drugs, e.g. REMICADETM (infliximab), in to allow any HACA present in the serum to bind. The labeled a serum sample as well as for determining the levels of a anti-TNFC. drug can also be incubated with known amounts human anti-drug antibody, e.g. a human anti-chimeric anti of an anti-IgG antibody or pooled positive patient serum in a body (HACA) to REMICADETM (infliximab). liquid phase reaction to create a standard curve. Following Step 1: Determining Concentration Level of REMI 15 incubation, the samples are loaded directly onto a size exclu CADETM (Infliximab) in a Sample. sion column. Binding of the autoantibodies to the labeled In one exemplary embodiment, TNFC. is labeled with a anti-TNFC. drug results in a leftward shift of the peak com fluorophore (e.g. Alexaaz), wherein the fluorophore can be pared to labeled drug alone. The concentration of HACA detected by, either or both of the visible and fluorescent present in the serum sample can then be compared to the spectra. The labeled TNFC. is incubated with human serum in standard curve and controls. a liquid phase reaction to allow the anti-TNFC. drug present in SE-HPLC analysis of HACA levels in patient serum. the serum to bind. The labeled TNFC. can also be incubated Purified REMICADETM was labeled with a fluorophore. with known amounts of the anti-TNFC. drug in a liquid phase Labeled REMICADETM was incubated with different dilu reaction to create a standard curve. Following incubation, the tions of pooled HACA-positive serum or diluted patient samples are loaded directly onto a size exclusion column. 25 serum for one hour at room temperature. Samples of 100 uL Binding of the anti-TNFC. drug to the labeled TNFC. results in Volume were analyzed by size-exclusion chromatography on a leftward shift of the peak compared to labeled TNFC. alone. an HPLC system. Fluorescence label detection was used to The concentration of the anti-TNFC. drug present in the serum monitor the free labeled REMICADETM and the bound sample can then be compared to the standard curve and con labeled REMICADETM immuno-complex based on their trols. 30 retention times. The ratio of bound and free labeled REMI SE-HPLC Analysis of REMICADETM (Infliximab) Levels CADETM was used to determine the HACA level as described in Patient Serum. below. Human recombinant TNFC. was labeled with a fluoro Mobility Shift Assay Procedure to Measure HACA in phore, Alexa Fluor R. 488, according to the manufactureR's Serum. instructions. Labeled TNFC. was incubated with different 35 The principle of this assay is based on the mobility shift of amounts of REMICADETM or patient serum for one hour at the complex of an anti-drug antibody, e.g. HACA, with room temperature. Samples of 100 u, volume were analyzed Alexa,-labeled REMICADETM relative to free Alexaaz by size-exclusion chromatography on an HPLC system. Fluo labeled REMICADETM, on size exclusion-high performance rescence label detection was used to monitor the free labeled liquid chromatography (SE-HPLC) due to the increase in TNFC. and the bound labeled TNFC. immuno-complex based 40 molecular weight of the complex. The chromatography is on their retention times. Serum REMICADETM levels were performed in an Agilent- 1200 HPLC System, using a Bio-Sep calculated from the standard curve. 300x7.8 mm SEC-3000 column (Phenomenex) with a The following equations are relevant to this assay: molecular weight fractionating range of 5,000-700,000 and a labeled-TNFC+REMICADETM-> (labeled mobile phase of 1xPBS, pH 7.3, at a flow-rate of 0.5-1.0 TNFa. REMICADETM). Equation I 45 mL/min with fluorescence label detection, e.g. UV detection at 650 nm. In front of the Agilent-1200 HPLC System with a REMICADE"lithout labeled TNF resent (labeled Bio-Sep 300x7.8 mm SEC-3000 column is a analytical pre TNFC REMICADETM). Equation II column which is a BioSep 75x7.8 mm SEC-3000. A 100 uL REMICADETM)=(labeled-TNFC. sample Volume is loaded onto the column for each analysis. REMICADETM)/(labeled-TNFolx|la 50 The complex of HACA and labeled REMICADETM complex beled-TNFC. Equation III is formed by incubating serum from a REMICADETM-treated In Step 1, a known amount of the labeled-TNFC. is con patient and labeled REMICADETM in the 1xPBS, pH 7.3, tacted with a REMICADETM-containing serum sample. The elution buffer at room temperature for 1 hour before SE labeled-TNFC. and the REMICADETM form a complex, (la HPLC analysis. beled-TNFa. REMICADETM). See Equation I. 55 The following equations are relevant to this assay: Because almost all of the REMICADETM will form a complex REMICADETM-labeled-REMICADETM-HACA-e with the labeled-TNFO, the concentration of REMICADETM (REMICADETM-HACA)+(Labeled present before introduction of the labeled-TNFC. is equal to REMICADETM-HACA). Equation IV the measured concentration of labeled |REMICADETM/IREMICADETM. HACA]= TNFC: REMICADETM, See Equation II. The concen 60 labeled-REMICADETM/Labeled tration level of REMICADETM is calculated by multiplying REMICADETM-HACA) Equation V the ratio of (label-TNFa. REMICADETM). 1/|labeled TNFC) by labeled-TNFC), See Equation III. The ratio, (la (HACA)=|REMICADETM-HACA)+|labeled bel-TNFa. REMICADETM). 1/|labeled-TNFC), is REMICADETM-HACA). Equation VI obtained by integrating the area-under-the curve for the (la 65 |REMICADETM. HACA)=|REMICADETMIx bel-TNFC REMICADETM), peak, from a plot of signal |labeled-REMICADETM. HACA/labeled intensity as a function of elution time from the size exclusion REMICADETM) Equation VII US 8,865,417 B2 59 60 |labeled-REMICADETM. HACA)=|labeled multiplied by 0.25, which is equal to 9.4 ng labeled REMICADETM)x|labeled REMICADETM-HACA. Using Equation VI, the total REMICADETM-HACA)/(labeled-REMI amount of HACA equals the sum of 9.4ng and 7.5 ng, which CADETM) Equation VIII equals 16.9 ng HACA. The 16.9 ng HACA was present in 4 uL of sample. The HACA was 16.9 ng/4 uL, which equals (REMICADETM-REMICADETMI 4.23 g/ml. Using Equation IX, the effective amount of HACA) Equation IX REMICADETM is equal to 7.5ug/ml REMICADETM, deter Determining the Concentration Levels of Human Anti mined from Step 1, minus 4.23 ug/ml HACA, determined TNFC. Drug Antibodies, e.g. HACA. from Step 2. In this exemplary calculation, the effective A known concentration of Labeled-REMICADETM is 10 REMICADETM was equal to 3.27 g/ml. added to a serum sample. HACA forms a complex with either REMICADETM or Labeled-REMICADETM, See Equation Example 8 IV. The REMICADETM) is determined in Step 1 above. By integrating the area-under-the-curve for the labeled Determining the Concentration Levels of REMICADETM. HACA and dividing this number by 15 HUMIRATM and Human Anti-Drug Antibodies the resultant integration for the area-under-the-curve for the free Labeled-REMICADETM, the ratio of labeled This example describes a method for determining the lev REMICADETM-HACAcomplex to labeled-REMICADETM) els of HUMIRATM in a serum sample as well as for determin is obtained. The ratio of REMICADETM) to ing the levels of human anti-human antibodies (HAHA). REMICADETM-HACA) is equal to the ratio of Ila Step 1: Determining Concentration Level of HUMIRATM beled-REMICADETM) to labeled-REMICADETM-HA in a Sample. CA). See Equation V. Because HACA equilibrates In one exemplary embodiment, TNFC. is labeled with a and forms a complex with both REMICADETM and Labeled fluorophore (e.g. Alexa), REMICADETM, the total amount of HACA equals the sum of wherein the fluorophore can be detected by, either or both the amount of REMICADETM-HACA and the amount 25 of the visible and fluorescent spectra. The labeled TNFC. is of labeled-REMICADETM-HACA, See Equation VI. incubated with human serum in a liquid phase reaction to Because the ratio of REMICADETM) tO allow the anti-TNFC. drug present in the serum to bind. The REMICADETM-HACAcomplex is equal to the ratio of la labeled TNFC. can also be incubated with known amounts of beled-REMICADETM) tO labeled the anti-TNFC. drug in a liquid phase reaction to create a REMICADETM-HACA), both the REMICADETM 30 standard curve. Following incubation, the samples are loaded HACA) complex and the |labeled-REMICADETM directly onto a size exclusion column. Binding of the anti HACA) are determined by multiplying the ratio of the TNFC. drug to the labeled TNFC. results in a leftward shift of |labeled-REMICADETM-HACA)/|labeled-REMI the peak compared to labeled TNFC. alone. The concentration CADETM) by, respectively, the concentration amount of of the anti-TNFC. drug present in the serum sample can then REMICADETM, determined in Step 1, and the concentration 35 be compared to the standard curve and controls. amount of labeled-REMICADETM, known a priori, See Equa SE-HPLC Analysis of HUMIRATM Levels in Patient tions VII and VIII. Therefore, the total amount of HACA Serum. equals the sum of (1) the REMICADETM), from step 1, Human recombinant TNFC. was labeled with a fluoro multiplied by labeled-REMICADETM-HACA).I/Ila phore, Alexa Fluor R. 488, according to the manufacturers beled-REMICADETM), and (2) the labeled REMICADETM), 40 instructions. Labeled TNFC. was incubated with different known a priori, multiplied by labeled amounts of HUMIRATM or patient serum for one hour at REMICADETM-HACA).I/|labeled-REMICADETM). room temperature. Samples of 100 u, volume were analyzed Determining the effective concentration levels of REMI by size-exclusion chromatography on an HPLC system. Fluo CADETM. rescence label detection was used to monitor the free labeled Because HACA complexes with REMICADETM, the 45 TNFC. and the bound labeled TNFC. immuno-complex based effective amount of REMICADETM available in a serum on their retention times. Serum HUMIRATM levels were cal sample is the amount of REMICADETM, measured from Step culated from the standard curve. 1, minus the amount of HACA, measured from Step 2, See The following equations are relevant to this assay: Equation IX. labeled-TNFC+HUMIRATM-> (labeled Exemplary Calculation. 50 TNFC HUMIRATM) Equation X In patient JAG on V10, the REMICADETM was deter mined to be 7.5 g/ml. See FIG.16c. This result was obtained HUMIRATM)=(labeled-TNFC HUMIRA). Equation XI by following Step 1 and using Equitions I-III.7.5 g/ml equals 30 ng/4 L. Since 4 ul of sample was used in the measure (HUMIRATM)=(label-TNFa. HUMIRATM), labeled-TNFox|labeled-TNFC. Equation XII ment in Step 2, a total of 30.0 ng of REMICADETM was 55 present in the sample analyzed. The ratio of labeled In Step 1, a known amount of the labeled-TNFC. is con REMICADETM-HACAL/I labeled-REMICADETM) for tacted with a HUMIRATM-containing serum sample. The patient JAG on V10 was 0.25, See FIG. 16b. The labeled labeled-TNFC. and the HUMIRATM form a complex, (la REMICADETM introduced into the sample was 37.5 ng/100 beled-TNFC HUMIRATM). See Equation X. Because uL. Since 1000, of the labeled-REMICADETM was used in 60 almost all of the HUMIRATM will form a complex with the the measurement in Step 2, a total of 37.5 ng of labeled labeled-TNFO, the HUMIRATM present before introduction REMICADETM was present in the sample analyzed. Using of the labeled-TNFO is equal to the measured (labeled Equation VII, the total amount of TNFa. HUMIRATM)). See Equation XI. The HU REMICADETM-HACA was 30 ng multiplied by 0.25, MIRATM is calculated by multiplying the ratio of (label which is equal tO 7.5 ng labeled 65 TNFa. HUMIRATM). 1/|Labeled-TNFC) by labeled REMICADETM. HACA. Using Equation VIII, the total TNFC). See Equation XII. By integrating the area-under-the amount of labeled-REMICADETMHACAcomplex was 37.5ng curve for the labeled-TNFC. and the area-under-the-curve for US 8,865,417 B2 61 62 the (labeled-TNFC HUMIRATM) and dividing the |labeled-HUMIRATM-HAHA]=|labeled-HU resultant integration for (labeled-TNFa. HUMIRATM). MIRATM)x|labeled-HUMIRATM-HAHAconnplex by the resultant integration for the labeled-TNFC., the ratio of labeled-HUMIRATM) Equation XVII (label-TNFa. HUMIRATM) to labeled-TNFC) is obtained. The labeled-TNFC. is known a priori. HUMIRATM) effective-annount=HUMIRATM-HAHA) Equation XVIII Step 2: Determining Level of Human Anti-Human Anti body, e.g. HAHA. Calculation for Step 2: A known concentration of labeled In one exemplary embodiment, an anti-TNFC. drug, e.g., HUMIRATM is added to a serum sample. HAHA forms a HUMIRATM, is labeled with a fluorophore, e.g., Alexa, complex with either HUMIRATM or Labeled-HUMIRATM, wherein the fluorophore can be detected by, either or both of 10 See EquationXIII. The HUMIRATM is determined in Step 1 the visible and fluorescent spectra. The labeled anti-TNFC. as described above. By integrating the area-under-the-curve drug is incubated with human serum in a liquid phase reaction for the Labeled-HUMIRATM HAHA and the area-un to allow any HAHA present in the serum to bind. The labeled der-the-curve for the Labeled-HUMIRATM and dividing the anti-TNFC. drug can also be incubated with known amounts resultant integration for the Labeled of an anti-IgG antibody or pooled positive patient serum in a 15 HUMIRATM HAHA. by the resultant integration for the liquid phase reaction to create a standard curve. Following Labeled-HUMIRATM, the ratio of the Labeled incubation, the samples are loaded directly onto a size exclu HUMIRATM-HAHA) to Labeled-HUMIRATM) is sion column. Binding of the autoantibodies to the labeled obtained. The ratio of the HUMIRATM to the anti-TNFC. drug results in a leftward shift of the peak com HUMIRATM-HAHAcomplex is equal to the ratio of the La pared to labeled drug alone. The concentration of HAHA beled-HUMIRATM) tO the Labeled present in the serum sample can then be compared to the HUMIRATM-HAHA). See Equation XIV. Because standard curve and controls. HAHA equilibrates and forms a complex with both SE-HPLC Analysis of HAHA Levels in Patient Serum. HUMIRA and Labeled-HUMIRATM, the total amount of Purified HUMIRATM was labeled with a fluorophore. HAHA equals the sum of the amount of Labeled HUMIRATM was incubated with different dilutions 25 of pooled HAHA-positive serum or diluted patient serum for HUMIRATM-HAHA. and the Labeled one hour at room temperature. Samples of 100 uL volume HUMIRATM HAHA. See Equation XV. Because the were analyzed by size-exclusion chromatography on an ratio of HUMIRATM to HUMIRATM-HAHA) is HPLC system. Fluorescence label detection was used to equal to the ratio of Labeled-HUMIRA to Labeled monitor the free labeled HUMIRATM and the bound labeled 30 HUMIRATM HAHA), the concentration of both the HUMIRATM immuno-complex based on their retention |HUMIRATM-HAHA., and the Labeled-HUMIRATM times. The ratio of bound and free labeled HUMIRATM was HAHA are determined by multiplying the ratio of the used to determine the HAHA level as described below. |Labeled-HUMIRAHAHA/|Labeled-HUMIRA) by Mobility Shift Assay Procedure to Measure HAHA in the HUMIRATM, determined in Step 1, and the Labeled Serum. 35 HUMIRATM, known a priori, respectively, See Equations The principle of this assay is based on the mobility shift of XVI and XVII. Because HAHA complexes with the antibody, e.g. HAHA, bound Alexa7-labeled HUMIRATM, the effective amount of HUMIRATM available HUMIRATM complex versus free Alexa,-labeled in a serum sample is the amount of HUMIRA, measured from HUMIRATM on size exclusion-high performance liquid chro Step 1, minus the amount of HAHA, measured from Step 2. matography (SE-HPLC) due to the increase in molecular 40 See Equation XVIII. weight of the complex. The chromatography is performed in Exemplary Calculation. an Agilent-1200 HPLC System, using a Bio-Sep300x7.8 mm In patient SL03246013, see FIG. 25, the HUMIRATM SEC-3000 column (Phenomenex) with a molecular weight was determined to be 16.9 ug/ml, see FIG. 25. This result was fractionating range of 5,000-700,000 and a mobile phase of obtained by following Step 1 and using Equtions X-XII. 16.9 1xRBS, pH 7.3, at a flow-rate of 0.5-1.0 mL/min with fluo 45 ug/ml equals 67.6 ng/4 uL. Since 4 LL of sample was used in rescence label detection, e.g. UV detection at 650 nm. In front the measurement in Step 2, a total of 67.6ng of HUMIRATM of the Agilent-1200 HPLC System with a Bio-Sep 300x7.8 was present in the sample analyzed. The ratio of labeled mm SEC-3000 column is a analytical pre-column which is a HUMIRATM-HAHAL/|labeled-HUMIRATM for BioSep 75x7.8 mm SEC-3000. A 100 uL sample volume is patient SL03246013 was 0.055, see FIG. 25. The labeled loaded onto the column for each analysis. A 100 uL sample 50 HUMIRATM introduced into the sample was 37.5 ng/100 uL. Volume is loaded onto the column for each analysis. The Since 100 uL of the labeled-HUMIRATM was used in the HAHA bound labeled HUMIRATM complex is formed by measurement in Step 2, a total of 37.5 ng of labeled-HU incubating serum from a HUMIRA-treated patient and MIRATM was present in the sample analyzed. Using Equation labeled HUMIRATM in the 1xPBS, pH 7.3, elution buffer at XVI, the total amount of HUMIRATM-HAHA was 67.6 room temperature for 1 hour before SE-HPLC analysis. 55 ng multiplied by 0.055, which is equal to 3.71 ng labeled HUMIRATM-labeled-HUMIRATM-HAHA-> HUMIRATMHAHA. Using Equation XVII, the total (HUMIRATM-HAHA).--(labeled amount of labeled-HUMIRATM HAHA was 37.5 ng HUMIRATMHAHA). Equation XIII multiplied by 0.055, which is equal to 2.06 ng labeled HUMIRATM HAHA. Using Equation XV, the total (HUMIRATM/IHUMIRATM-HAHA]=|labeled 60 amount of HAHA equals the sum of 3.71 ng and 2.06 ng, HUMIRATM/labeled-HUMIRA. HAHA. Equation XIV which equals 5.77 ng HAHA. The 5.77 ng HAHA was present in 4 uL of sample. The HAHA was 5.77 ng/4 uL, which HAHA]=HUMIRATM HAHAH-labeled equals 1.44 ug/ml. Using Equation XVIII, the effective HUMIRATMHAHA. Equation XV amount of HUMIRATM is equal to 16.99 g/ml HUMIRATM, (HUMIRATM-HAHA]=|HUMIRATM)x|labeled 65 determined from Step 1, minus 1.44 ug/ml HAHA, deter HUMIRATMHAHA)/(labeled-HU mined from Step 2. In this exemplary calculation, the effec MIRATM) Equation XVI tive HUMIRATM was equal to 15.46 ug/ml. US 8,865,417 B2 63 64 Example 9 The ratio of the labeled anti-TNFC. drug, e.g. REMI CADETM or HUMIRATM, to the labeled internal control is Determining the Amount of a Complex of HACA or obtained by integrating the areas-under-the-curve for both the HAHA with Either REMICADETM, labeled anti-TNFC. drug and the labeled internal control and Labeled-REMICADETM, HUMIRA, or then dividing the resultant integration for the labeled anti Labeled-HUMIRA TNFC. drug by the resultant integration for the labeled inter nal control. This example describes a method for determining the The ratio of (labeled-anti-TNFC. Drug amount of a complex of HACA or HAHA with either REMI Autoantibody).I/internal control is obtained by inte CADETM, Labeled-REMICADETM, HUMIRA, or Labeled 10 grating the area-under-the curve for the (labeled-anti-TNFC. HUMIRATM with reference to an internal Standard. drug Autoantibody), peak from aplot of signal intensity By using an internal control, e.g. Biocytin-Alexa 488, as a function of elution time from the size exclusion HPLC, serum artifacts and variations from one experiment to another and dividing this number by the resultant integration of the experiment can be identified and properly analyzed. The area-under-the-curve for the internal control peak from the amount of internal control, e.g. Biocytin-Alexa 488, is from 15 plot. In some embodiments, the labeled anti-TNFC. drug is about 50 to about 200pg per 100 uL analyzed. labeled REMICADETM. In some other embodiments, the Fluorophore (FI)-labeled HUMIRATM was incubated with labeled anti-TNFC. drug is labeled HUMIRATM. patient serum to form the immunocomplex. A Fl-labeled Small peptide, e.g. Biocytin-Alexa 488, was included as an Example 11 internal control in each reaction. In one instance, different amounts of anti-human IgG were used to generate a standard Determining the Ratio of Free and Complexed curve to determine the serum HAHA levels. In another Labeled TNFC. instance, titrated pooled positive patient serum that has been calibrated with purified HAHA was used to generate a stan This example describes a method for determining the dard curve to determine the serum HAHA levels. In yet 25 amount of a complex of labeled-TNFC. with either REMI another instance, the method described in Example 7 was CADETM or HUMIRATM with reference to an internal stan used to generate a standard curve to determine the serum dard. HAHA levels. Free labeled HUMIRA was separated from the By using an internal control, e.g. Biocytin-Alexa 488, antibody bound complex based on its molecular weight by serum artifacts and variations from one experiment to another size-exclusion chromatography. The ratio of free labeled 30 experiment can be identified and properly analyzed. The HUMIRA to an internal control from each sample was used to amount of internal control, e.g. Biocytin-Alexa 488, is from extrapolate the HAHA concentration from the standard about 1 to about 25 ng per 100 ul, analyzed. curve. A similar methodology was used to measure HUMIRA In one embodiment, the uncomplexed labeled TNFC. has levels in patient serum samples with labeled TNF-C. levels between about 50 ng and 150 ng in a sample. In certain The initial ratio of the Labeled-Drug, i.e. Labeled-REMI 35 instances, the amount of labeled-TNFC. is about 100.0 ng. CADETM or Labeled-HUMIRA, to the internal control is Fluorophore (FI)-labeled TNFC. was incubated with patient equal to 100. As depicted in FIGS. 23 and 24, when the ratio serum to form the immunocomplex. A Fl-labeled small pep of the Labeled-Drug to the internal control falls below 95, the tide, e.g. Biocytin-Alexa 488, was included as an internal labeled-drug is inferred to be complexed with an anti-Drug control in each reaction. A standard curve was created by binding compound, e.g. HACA, HAHA. The ratio of the 40 spiking in known concentrations of purified anti-TNFC. drug Labeled-drug to internal control is obtained by integrating and then extrapolating from the curve to determine the con the areas-under-the-curve for the Labeled-Drug and for the centration in units of ug/mL. internal control and then dividing the resultant integration for The initial ratio of the Labeled-TNFC. to the internal con the Labeled-Drug by the resultant integration for the internal trol is equal to 100. When the ratio of the Labeled-TNFC. to control. 45 the internal control falls below 95, the labeled-TNFO is inferred to be complexed with an anti-TNFO. drug, e.g. Remi Example 10 cadeTM, HumiraTM. The ratio of the Labeled-TNFC. to inter nal control is obtained by integrating the areas-under-the Determining the Ratio of Complexed Anti-TNFC. curve for the Labeled-TNFC. and for the internal control and Drugs to Uncomplexed Anti-TNFC. Drugs 50 then dividing the resultant integration for the Labeled-TNFC. by the resultant integration for the internal control. The ratio of the complexed anti-TNFC. drug to uncom plexed anti-TNFC. drug is obtained by integrating the areas Example 12 under-the-curve for both the complexed anti-TNFC. drug and the uncomplexed anti-TNFC. drug and then dividing the 55 Optimizing Anti-TNFC. Drug Therapy by Measuring resultant integration for the complexed anti-TNFC. drug by Anti-TNFC. Drug and/or Anti-Drug Antibody (ADA) the resultant integration for the uncomplexed anti-TNFC. Levels drug. In one embodiment, the uncomplexed anti-TNFC. drug is This example describes methods for optimizing anti REMICADETM having levels between about 0ng and 100ng 60 TNFC. drug therapy, reducing toxicity associated with anti in a sample. The amount of labeled-REMICADETM is about TNFC. drug therapy, and/or monitoring the efficacy of thera 37.5ng. peutic treatment with an anti-TNFC. drug by measuring the By using an internal control, e.g. Biocytin-Alexa 488, amount (e.g., concentration level) of anti-TNFC. drug (e.g., serum artifacts and variations from one experiment to another level of free anti-TNFO, therapeutic antibody) and/or anti experiment can be identified and properly analyzed. The 65 drug antibody (ADA) (e.g., level of autoantibody to the anti amount of internal control, e.g. Biocytin-Alexa 488, is from TNFC. drug) in a sample from a subject receiving anti-TNFC. about 50 to about 200pg per 100 uL analyzed. drug therapy. Accordingly, the methods set forth in the US 8,865,417 B2 65 66 present example provide information useful for guiding treat know of suitable higher or lower doses to which the current ment decisions, e.g., by determining when or how to adjust or course of therapy can be adjusted Such that drug therapy is modify (e.g., increase or decrease) the Subsequent dose of an optimized, e.g., a Subsequent dose that is at least about 1.5. 2. anti-TNFC. drug, by determining when or how to combine an 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, anti-TNFC. drug (e.g., at an increased, decreased, or same 25, 30, 35, 40, 45, 50, or 100-fold higher or lower than the dose) with one or more immunosuppressive agents such as current dose. methotrexate (MTX) or azathioprine, and/or by determining Scenario #3: Medium Level of Anti-TNFC. Drug with when or how to change the current course of therapy (e.g., Medium Level of ADA. switch to a different anti-TNFC. drug). Drug levels=5-20 ng/10 ul; ADA levels=0.5-10 ng/10 ul. For purposes of illustration only, the following scenarios 10 Patient samples having this profile include samples from provide a demonstration of how the methods of the present patient JMM on visit 10 (“V10') and patient J-L on visit 14 invention advantageously enable therapy to be optimized and (“V14). See, FIG. 16b. toxicity (e.g., side-effects) to be minimized or reduced based Patients receiving anti-TNFC. drug therapy and having this upon the level of anti-TNFC. drug (e.g., level of free anti particular profile should be treated with a different drug. As a TNFC. therapeutic antibody) and/or ADA (e.g., level of 15 non-limiting example, a patient on infliximab (IFX) therapy autoantibody to the anti-TNFC. drug) in a sample from a and having medium levels of IFX and ADA (i.e., HACA) subject receiving anti-TNFC. drug therapy. The levels of the should be switched to therapy with adalimumab (HU anti-TNFC. drug and ADA can be measured with the novel MIRATM). assays described herein. Scenario #4: Low Level of Anti-TNFC. Drug with High Scenario #1: High Level of Anti-TNFC. Drug with Low Level of ADA. Level of Anti-Drug Antibody (ADA). Drug levels=0-5 ng/10 ul; ADA levels—3.0-50 ng/10 ul. Drug levels=10-50 ng/10 ul; ADA levels=0.1-2 ng/10 ul. Patient samples having this profile include samples from all Patient samples having this profile include samples from patients on V14 in FIG. 16b. patients BAB and JAA on visit 10 (“V10'). See, FIG. 16b. Patients receiving anti-TNFC. drug therapy and having this Patients receiving anti-TNFC. drug therapy and having this 25 particular profile should be treated with a different drug. As a particular profile should be treated with immunosuppressive non-limiting example, a patient on infliximab (IFX) therapy drugs like azathioprine (AZA) along with the anti-TNFC. drug and having a low level of IFX with a high level of ADA (i.e., (e.g., infliximab). HACA) should be switched to therapy with adalimumab Scenario #2: Medium Level of Anti-TNFC. Drug with Low (HUMIRATTM). Level of ADA. 30 Although the foregoing invention has been described in Drug levels=5-20 ng/10 ul; ADA levels=0.1-2 ng/10 ul. Some detail by way of illustration and example for purposes Patient samples having this profile include samples from of clarity of understanding, one of skill in the art will appre patients DGO, JAG, and JJH on V10. See, FIG. 16b. ciate that certain changes and modifications may be practiced Patients receiving anti-TNFC. drug therapy and having this within the scope of the appended claims. In addition, each particular profile should be treated with immunosuppressive 35 reference provided herein is incorporated by reference in its drugs like azathioprine (AZA) along with a higher dose of the entirety to the same extent as if each reference was individu anti-TNFC. drug (e.g., infliximab). One skilled in the art will ally incorporated by reference.
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS: 3
<21 Oc SEO ID NO 1 <211 LENGTH: 233 <212> TYPE PRT <213> ORGANISM: Homo sapiens <22 Os FEATURE; <223> OTHER INFORMATION: soluble form of tumor necrosis factor-alpha (TNF-alpha)
<4 OOs SEQUENCE: 1
Met Ser Thr Glu Ser Met Ile Arg Asp Wall Glu Lieu. Ala Glu Glu Ala 1. 5 15
Lieu Pro Llys Llys Thir Gly Gly Pro Glin Gly Ser Arg Arg Cys Leul Phe 25 3 O
Luell Ser Lell Phe Ser Phe Luell Ile Wall Ala Gly Ala Thir Thir Leul Phe 35 4 O 45
Lieu. Lieu. His Phe Gly Wall Ile Gly Pro Glin Arg Glu Glu Phe Pro SO 55 60
Arg Asp Luell Ser Luell Ile Ser Pro Leu Ala Glin Ala Wall Arg Ser Ser 65 70 8O
Ser Arg Thir Pro Ser Asp Pro Wall Ala His Wall Wall Ala Asn. Pro 85 90 95
Glin Ala Glu Gly Glin Lieu Gln Trp Lieu. Asn Arg Arg Ala ASn Ala Lieu US 8,865,417 B2
- Continued
1OO 105 11 O Lieu Ala Asn Gly Val Glu Lieu. Arg Asp Asn Gln Lieu Val Val Pro Ser 115 12 O 125 Glu Gly Lieu. Tyr Lieu. Ile Tyr Ser Glin Val Lieu. Phe Lys Gly Glin Gly 13 O 135 14 O Cys Pro Ser Thr His Val Lieu. Lieu. Thr His Thr Ile Ser Arg Ile Ala 145 150 155 160 Val Ser Tyr Glin Thr Llys Val Asn Lieu. Lieu. Ser Ala Ile Llys Ser Pro 1.65 17O 17s Cys Glin Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu 18O 185 19 O Pro Ile Tyr Lieu. Gly Gly Val Phe Glin Lieu. Glu Lys Gly Asp Arg Lieu. 195 2OO 2O5 Ser Ala Glu Ile Asn Arg Pro Asp Tyr Lieu. Asp Phe Ala Glu Ser Gly 21 O 215 22O Glin Val Tyr Phe Gly Ile Ile Ala Leu 225 23 O
<210s, SEQ ID NO 2 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic antibody epitope of tumor necrosis factor alpha (TNF-alpha), residues 136-157
<4 OOs, SEQUENCE: 2 Tyr Ser Glin Val Lieu. Phe Lys Gly Glin Gly Cys Pro Ser Thr His Val 1. 15
Lieu. Lieu. Thir His Thir Ile
SEQ ID NO 3 LENGTH: 22 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic antibody epitope of tumor necrosis factor alpha (TNF-alpha), residues 164-185
SEQUENCE: 3 Tyr Glin Thr Llys Val Asn Lieu. Lieu. Ser Ala Ile Llys Ser Pro Cys Glin 1. 15 Arg Glu Thr Pro Glu Gly
What is claimed is: (c) detecting the first labeled complex and the second 1. A method for detecting the presence or level of an labeled complex, thereby detecting the presence or level autoantibody to an anti-TNFC. drug in a sample, the method 55 of a non-neutralizing form of the autoantibody when comprising: both the first labeled complex and the second labeled (a) contacting a labeled anti-TNFC. drug and labeled TNFC. complex are present, and detecting the presence or level with the sample to form a first labeled complex between of a neutralizing form of the autoantibody when only the the labeled anti-TNFC. drug, the labeled TNFC., and the second labeled complex is present. autoantibody and a second labeled complex between the 60 2. The method of claim 1, wherein the sample is serum. labeled anti-TNFC. drug and the autoantibody, wherein the labeled anti-TNFC. drug and the labeled TNFC.com 3. The method of claim 1, wherein the anti-TNFC. drug is a prise different labels; member selected from the group consisting of infliximab, (b) subjecting the first labeled complex and the second etanercept, adalimumab, certolizumab pegol, and combina labeled complex to size exclusion chromatography to is tions thereof. separate the first labeled complex and the second labeled 4. The method of claim 1, wherein the autoantibody is a complex; and member selected from the group consisting of a human anti US 8,865,417 B2 69 70 mouse antibody (HAMA), a human anti-chimeric antibody TNFC. comprise different labels, and wherein the second (HACA), a human anti-humanized antibody (HAHA) and labeled complex does not include the autoantibody; combinations thereof. (e) subjecting the second labeled complex to a second size exclusion chromatography to separate the second 5. The method of claim 1, wherein the size exclusion chro labeled complex from free labeled TNFC.; and matography is size exclusion-high performance liquid chro (f) detecting the second labeled complex, thereby detecting matography (SE-HPLC). the presence or level of a neutralizing form of the 6. The method of claim 1, wherein the labeled anti-TNFC. autoantibody. drug is a fluorophore-labeled anti-TNFC. drug. 12. The method of claim 11, wherein the sample is serum. 7. The method of claim 6, wherein the fluorophore is 13. The method of claim 11, wherein the anti-TNFC. drug is Selected from the group consisting of a polymethine dye, a 10 a member selected from the group consisting of infliximab, pthalocyanine dye, a cyanine dye, a xanthene dye, a fluorene etanercept, adalimunab, certolizumab pegol, and combina dye, a rhodamine dye, a coumarin dye, and a fluorescein dye. tions thereof. 8. The method of claim 1, wherein the labeled TNFO is a 14. The method of claim 11, wherein the autoantibody is a member selected from the group consisting of a human anti fluorophore-labeled TNFO. 15 9. The method of claim 8, wherein the fluorophore is mouse antibody (HAMA), a human anti-chimeric antibody Selected from the group consisting of a polymethine dye, a (HACA), a human anti-humanized antibody (HAHA), and pthalocyanine dye, a cyanine dye, a xanthene dye, a fluorene combinations thereof. dye, a rhodamine dye, a coumarin dye, and a fluorescein dye. 15. The method of claim 11, wherein the first and/or the 10. The method of claim 1, wherein the sample is obtained Second size exclusion chromatography is size exclusion-high from a subject receiving therapy with the anti-TNFC drug. performance liquid chromatography (SE-HPLC). 11. A method for detecting the presence or level of an 16. The method of claim 11, wherein the labeled anti autoantibody to an anti-TNFC. drug in a sample, the method TNFC. drug is a fluorophore-labeled anti-TNFC. drug. comprising: 17. The method of claim 16, wherein the fluorophore is Selected from the group consisting of a polymethine dye, a (a) contacting a labeled anti-TNFC. drug with the sample to 25 firm a first labeled complex between the labeled anti pthalocyanine dye, a cyanine dye, a xanthene dye, a fluorene TNFC. drug and the autoantibody; dye, a rhodamine dye, a coumarin dye, and a fluorescein dye. (b) subjecting the first labeled complex to a first size exclu 18. The method of claim 11, wherein the labeled TNFO is Sion chromatography to separate the first labeled com a fluorophore-labeled TNFO. plex from free labeled anti-TNFC. drug; 19. The method of claim 18, wherein the fluorophore is (c) detecting the first labeled complex, thereby detecting 30 Selected from the group consisting of a polymethine dye, a the presence or level of the autoantibody; pthalocyanine dye, a cyanine dye, a xanthene dye, a fluorene (d) contacting labeled TNFC. with the first labeled complex dye, a rhodamine dye, a coumarin dye, and a fluorescein dye. from step (c) to form a second labeled complex between 20. The method of claim 11, wherein the sample is obtained the labeled anti-TNFC. drug and the labeled TNFO, from a subject receiving therapy with the anti-TNFC drug. wherein the labeled anti-TNFC. drug and the labeled ck ck ck ck *k UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 8,865,417 B2 Page 1 of 1 APPLICATIONNO. : 14/046863 DATED : October 21, 2014 INVENTOR(S) : Singh et al. It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:
In the Claims:
In Column 69, line 26, Claim 11: please delete “firm and insert --form--.
Signed and Sealed this Third Day of November, 2015 74-4-04- 2% 4 Michelle K. Lee Director of the United States Patent and Trademark Office