(12) United States Patent (10) Patent No.: US 9,573.996 B2 Ariaans Et Al

USOO9573996 B2

(12) United States Patent (10) Patent No.: US 9,573.996 B2 Ariaans et al. (45) Date of Patent: Feb. 21, 2017

(54) MONOCLONAL ANTIBODIES TO HUMAN (56) References Cited PROINFLAMMLATORY CYTOKNES AND METHODS FOR TREATING U.S. PATENT DOCUMENTS NFLAMMLATORY DISEASES 4,771,002 A 9, 1988 Gelvin 5,075,236 A 12/1991 Yone et al. (71) Applicant: Synthon Biopharmaceuticals B.V., 5,428, 147 A 6/1995 Barker et al. Nijmegen (NL) 5,605,690 A 2f1997 Jacobs et al. 5,656,272 A 8, 1997 Le et al. 5,919,452 A 7/1999 Le et al. (72) Inventors: Gerardus Joseph Andreas Ariaans, 6,040.498 A 3/2000 Stomp et al. Nijmegen (NL); Frans van Dalen, 6,090,382 A 7/2000 Salfeld et al. Nijmegen (NL); Declan Thomas 6,451,982 B1 9, 2002 Chou et al. 6,790.444 B2 9, 2004 Le et al. Nolan, Nijmegen (NL) 6,902,724 B1 6/2005 Parekh et al. 6,902,734 B2 6/2005 Giles-Komar et al. (73) Assignee: Synthon Biopharmaceuticals B.V., 6,914,128 B1 7/2005 Salfeld et al. Nijmegen (NL) 7,012,135 B2 3/2006 Athwal et al. 7,147,854 B2 12/2006 Ye 7,161,064 B2 1/2007 Stomp et al. (*) Notice: Subject to any disclaimer, the term of this 7,176,024 B2 2/2007 Branson et al. patent is extended or adjusted under 35 7,186,820 B2 3/2007 Athwal et al. U.S.C. 154(b) by 0 days. 7,622,573 B2 11/2009 Dickey et al. 7,632,983 B2 12/2009 Dickey et al. 7,883,704 B2 2/2011 Salfeld et al. (21) Appl. No.: 14/365.940 7,884,264 B2 2/2011 Dickey et al. 8,034,916 B2 10/2011 Dickey et al. (22) PCT Filed: Dec. 14, 2012 2009 OO60921 A1 3/2009 Dickey et al. 2009, 0214528 A1 8, 2009 Dorai et al. (86). PCT No.: PCT/EP2012/075670 2010/0209966 A1 8, 2010 Everett et al. 2012/0190.004 A1 7/2012 Parsons et al. S 371 (c)(1), 2014/0356357 A1 12/2014 Ariaans et al. 2014/03599.02 A1 12/2014 Ariaans et al. (2) Date: Jun. 16, 2014 2015.OO 10544 A1 1/2015 Ariaans et al.

(87) PCT Pub. No.: WO2013/087911 FOREIGN PATENT DOCUMENTS PCT Pub. Date: Jun. 20, 2013 EP O449769 B1 12/1993 EP O932415 A1 8, 1999 (65) Prior Publication Data EP 0602126 B1 3, 2003 US 2015/O166649 A1 Jun. 18, 2015 (Continued)

OTHER PUBLICATIONS Related U.S. Application Data Chan AC, et al. Nature Reviews Immunology. 10(5):301-316, May (60) Provisional application No. 61/576,727, filed on Dec. 5, 2010.* 16, 2011, provisional application No. 61/576,922, Worledge KL, et al. Digestive Diseases and Sciences. 45(12): 2298 filed on Dec. 16, 2011. 2305, Dec. 1, 2000.* Berdoz J, et al. Proc. Natl. Acad. Sci. 96(6):3029-3034, Mar. 16, (51) Int. Cl. 1999.* C07K 6/24 (2006.01) Ma J K-C et al. Science. 268:716–719, May 5, 1995.* CI2N 5/82 (2006.01) Paul, WE. Fundamental Immunology, 3rd ed. Raven Press, NY. A6 IK 9/00 (2006.01) Chap. 9, pp. 292-295, 1993.* Rudikoff S. et al. Proc. Natl. Acad. Sci. USA, 7.9:1979-1983, 1982.* A6 IK 39/00 (2006.01) Colman, PM. Research in Immunology, Elsevier, NY, 145(1):33-36, (52) U.S. Cl. 1994.* CPC ...... C07K 16/241 (2013.01); A61K 9/0053 (Continued) (2013.01); C07K 16/244 (2013.01): CI2N 15/8258 (2013.01); A61 K 39/00 (2013.01); Primary Examiner — Robert Landsman A6 IK 2039/505 (2013.01); A61 K 2039/54 (74) Attorney, Agent, or Firm — Sterne Kessler Goldstein (2013.01); C07K 2317/13 (2013.01); C07K & Fox 231 7/24 (2013.01); C07K 2317/41 (2013.01); C07K 231 7/565 (2013.01); C07K 2317/76 (57) ABSTRACT (2013.01) A monoclonal secretory IgA antibody, which binds to and (58) Field of Classification Search neutralizes a human proinflammatory cytokine or which CPC ...... A61K 9/0053: A61K 39/00; A61 K binds to and blocks a human proinflammatory cytokine 2309/505; A61K 2309/54; C07K 16/241: receptor. The secretory IgA antibody is useful in treating a C07K 2317/13; C07K 2317/24: CO7K variety of inflammatory diseases in humans. 2317/41; C07K 2317/76; C07K 2317/565 See application file for complete search history. 24 Claims, 29 Drawing Sheets US 9,573.996 B2 Page 2

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U.S. Patent Feb. 21, 2017 Sheet 2 of 29 US 9,573.996 B2

FIGURE 2A LPAOVAFTPYAPEPGSTCRLREYYDOTAOMCCSKCSPGOHAKVFCTKTSDTVCDSCEDST 1 O 2O 3O 4 O 5 O 6 O YTOLWNWVPECLSCGSRCSSDOVE TOACTREONRTCTCRPGWYCALSKOEGCRLCAPLRK

19 O 2 OO 21 O 22 O 23 O FIGURE 2B FR1 | --CDR1-- FR2 | ----CDR2-- EVELEESGGGLVOPGGSMKLSCVASGFIFSNHWMNWVROSPEKGLEWVAEIRSKSINSAT ------...... l 1 O 2O 3O 4 O 5 O 6 O

...... FR3 |-CDR3-- FR4 HYAESVKGRFTISRDDSKSAVYLOMTDLRTEDTGVYYCSRNYYGSTYDYWGOGTTL TVSS ...... 7 O 8O 9 O 1 OO 11 O 12 O

FIGURE 2C FRI --CDRL--- FR2 |-CDR2 DILLTQSPAILSVSPGERVSFSCRASOFVGSSIHWYOORTNGSPRLLIKYASESMSGIPS ...... 1. 1 O 2O 3O 4 O 5 O 6 O FR3 | --CDR3-| FR4 RFSGSGSGTDFTLSINTVESEDIADYYCOOSHSWPFTFGSGTNLEVK ------...... O 8O 9 O 1 OO 1. Of

FIGURE 2D FR1 | --CDR1-- FR2 | ----CDR2-- EVOLVESGGGLVOPGRSLRLS CAASGFTFDDYAMHWVROAPGKGLEWVSATWINSGHIDY ...... 1. 1 O 2O 3O 4 O 5 O 6 O FR3 |-CDR3-- FR4 ADSVEGRETISRDNAKN SLY LOMNSLRAEDTAVYYCAKVSYILSTASSLDYWGOGTLVTVSS ------...... | O 8O 9 O 1 OO 11 O 121

FIGURE 2E FR1 | --CDR1--- FR2 |-CDR2 DIQMTQSPSSLSASVGDRVTITCRASOGIRNYLAWYOOKPGKAPKLLIYAASTLOSGVPS ...... 1. 1 O 2O 3O 4 O 5 O 6 O FR3 | --CDR3- FR4 RFSGSGSGTDFTLTISSLOPEDVATYYCORYNRAPYTFGOGTKVEIK ...... O 8O 9 O 1 OO 1. Of U.S. Patent Feb. 21, 2017 Sheet 3 of 29 US 9,573.996 B2

FIGURE 2F FR1 | --CDR1-- ER2 | ----CDR2-- OVOLVESGGGVVOPGRSLRLSCAASGFIFSSYAMHWVROAPGNGLEWVAFMSYDGSNKKY 8 × 8 ...... 1. 1 O 2O 3O 4 O 5 O 6 O FR3 | ------CDR3----- FR4 ADSVKGRETISRDNSKNTLY LOMINSLRAEDTAVYYCARDRGIAAGGNYYYYGMDWWGOGTTWTVSS 8 × 8 ...... 7 O 8O 9 O OO O 2O 26

FIGURE 2G FR1 | --CDR1--- FR2 |-CDR2 EIVLTOSPATLSLSPGERATLSCRASOSVYSYLAWYOOKPGOAPRILLIYDASNRATGIPA ------| ...... l 1 O 2O 3O 4 O 5 O 6 O FR3 | --CDR3-- FR4 RFSGSGSGTDFTLTISSLEPEDFAVYYCOORSNWPPFTFGPGTKVDIK 8 × 8 ...... O 8O 9 O 1 OO 1 O 8

FIGURE 2H FR1 |--CDR1-- FR2 | ----CDR2-- EVOLVESGGGLVOPGGSLRLSCAASGYVFTDYGMNWVROAPGKGLEWMGWINTYIGEPIY ------...... l 1 O 2O 3O 4 O 5 O 6 O FR3 | --CDR3- ER4 ADSVKGRFTFSLDTSKSTAYLOMNSLRAEDTAVYYCARGYRSYAMDYWGOGTLVTVSS ...... 7 O 8O 9 O 1 OO 11 O 118

FIGURE 2 FR1 | --CDR1--- FR2 |-CDR2 DIOMTOSPSSLSASVGDRVTITCKASONVGTNVAWYOOKPGKAPKALIYSASFLYSGVPY 8 × 8 ...... 1. 1 O 2O 3O 4 O 5 O 6 O FR3 | --CDR3- FR4 RFSGSGSGTDFTL TISSLOPEDFATYYCOOYNIYPLTFGOGTKVEIK 8 × 8 ...... O 8O 9 O 1 OO 1. Of U.S. Patent Feb. 21, 2017 Sheet 4 of 29 US 9,573.996 B2

FIGURE 3A

CL K RTVAAPSVFIFPPSDEOLKSGTASV VCLLNNFYPREAKVQWKVDNALOSGNSOESVTEOD 8 × 8 + 8 + 8 | ...... 1 1 O 2O 3O 4 O 5 O 6 O

SKDSTYSLSSTLTLSKADYEKHKVYACEVTHOGLSSPVTKSFNRGEC ------| ...... 7 O 8O 9 O 1 OO 1. Of

FIGURE 3B

CL W1 GOPKANPTVTLFPPSSEELOANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSK 22 GOPKAAPSVTLFPPSSEELOANKATLVCLTSDFYPGAVTVAWKADSSPVKAGVETT W3 GOPKAAPSVTLFPPSSEELOANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSK 26 GOPKAAPSVTLFPPSSEELOANKATLVCLISDFYPGAVKVAWKADGSPVNTGVETTTPSK W7 GOPKAAPSVTLFPPSSEELOANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSK ------| ...... 1 1 O 2O 3O 4 O 5 O 6 O

OSNNKYAASSYLSLTPEQWKSHRSYSCOVTHEGSTVEKTVAPTECS OSNNKYAASSYLSLTPEQWKSHRSYSCOVTHEGSTVEKTVAPTECS OSNNKYAASSYLSLTPEOWKSHKSYSCOVTHEGSTVEKTVAPTECS OSNNKYAASSYLSLTPEQWKSHRSYSCOVTHEGSTVEKTVAPAECS OSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAECS 8 × 8 + 8 + 8 | ...... 7 O 8O 9 O 1 OO 106 U.S. Patent Feb. 21, 2017 Sheet 5 Of 29 US 9,573.996 B2

FIGURE 4

OEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRI IVPLNNREN ISDPTSPLRTR 8 × 8 + 8 + 8 | ...... 1 1 O 2O 3 O 40 5 O 6 O FVYHLSDLCKKCDPTEVELDNOIVTATOSNICDEDSATETCYTYDRNKCYTAVVPLVYGG

ETKMVETALTPDACYPD 8 × 8 + 8 + 8 | ...... 13 O 137 U.S. Patent Feb. 21, 2017 Sheet 6 of 29 US 9,573.996 B2

FIGURES

KSPIFGPEEVNSVEGNSVSITCYYPPTSVNRHTRKYWCROGARGGCITLISSEGYVSSKY A w w | ...... 1 1 O 2O 3 O 4 O 5 O 6 O AGRANLTNFPENGTFVVNIAOLSODDSGRYKCGLGINSRGLSFDVSLEVSOGPGLLNDTK

55 O 5 6 O 57 O 58 O 585 U.S. Patent Feb. 21, 2017 Sheet 7 Of 29 US 9,573.996 B2

U.S. Patent Feb. 21, 2017 Sheet 8 of 29 US 9,573.996 B2

FIGURE 7

slgA-> is 250 is 150 100 SCH). 75 HC Š 50 37 LC& JC 25 2O 15 U.S. Patent Feb. 21, 2017 Sheet 9 Of 29 US 9,573.996 B2

FIGURE 8

slgA-> 250 ... 250 150 150

100 100 75 SC-e 75 HC-D 50 50 37 37 25 2O 15 15 10 10 U.S. Patent Feb. 21, 2017 Sheet 10 of 29 US 9,573.996 B2

FIGURE 9

Log concentration (nM) U.S. Patent Feb. 21, 2017 Sheet 11 of 29 US 9,573.996 B2

FIGURE 10

anti-TNF-a slgA anti-TNF-O slgA Colostral SlgA (adalimumab variable part) (infliximab variable part)

C C C F is . . . CD- F EEEto SS38 SS5:(s a?... dEEEEEE to 2S33S S fid- to S33S5

s U.S. Patent Feb. 21, 2017 Sheet 12 of 29 US 9,573.996 B2

FIGURE 11

110

100

50 U.S. Patent Feb. 21, 2017 Sheet 13 Of 29 US 9,573.996 B2

FIGURE 12

110

100 E 90 E : E 80 E.

O 15 28 Time (h) of exposure to TNFO, U.S. Patent Feb. 21, 2017 Sheet 14 of 29 US 9,573.996 B2

FIGURE 13

1 O U.S. Patent Feb. 21, 2017 Sheet 15 Of 29 US 9,573.996 B2

FIGURE 14

Treatment PBS ADB-SA1g Adalimumab Oral Oral Subcutaneous Mini endoscopy Dayt=15 U.S. Patent Feb. 21, 2017 Sheet 16 of 29 US 9,573.996 B2

FIGURE 15

U.S. Patent Feb. 21, 2017 Sheet 17 Of 29 US 9,573.996 B2

FIGURE 16

U.S. Patent Feb. 21, 2017 Sheet 18 Of 29 US 9,573.996 B2

FIGURE 17A FR1 | --CDR1-- FR2 | ----CDR2-- EVOLVOSGAEVKKPGE SLKISCKGSGYSFTTYWLGWVROMPGKGLDWIGIMSPVDSDIRY 8 × 8 + 8 + 8 | ...... 1 1 O 2O 3 O 40 5 O 6 O FR3 | --CDR3-- FR4 SPSFOGOVTMSVDKSITTAYLOWNSLKASDTAMYYCARRRPGOGYFDFWGOGTLVTVSS 8 × 8 + 8 + 8 | ...... 7 O 8O 9 O 1 OO 11 O 119

FIGURE 17C FR1 | CDR1 FR2 | ----CDR2-- OVOLVESGGGVVOPGRSLRLSCAASGFTFSSYGMHWVROAPGKGLEWVAFIRYDGSNKYY 8 × 8 + 8 + 8 | ...... 1 1 O 2O 3 O 40 5 O 6 O FR3 | CDR3 FR4 ADSVKGRFTISRDNSKNTLY LOMNSLRAEDTAVYYCKTHGSHDNWGOGTMVTVSS 8 × 8 + 8 + 8 | ...... 7 O 8O 9 O 1 OO 11 O 115

FIGURE 17D FR1 | ----CDR1---- FR2 |-CDR2 OSVLTOPPSVSGAPGORVTISCSGSRSNIGSNTVKWYOOLPGTAPKLLIYYNDORPSGVP 8 × 8 + 8 + 8 | ...... 1 1 O 2O 3 O 40 5 O 6 O FR3 | ---CDR3--- FR4 DRFSGSKSGTSASLAITGLOAEDEADYYCOSYDRYTHPALLFGTGTKVTVL 8 × 8 + 8 + 8 | ...... 7 O 8O 9 O 1 OO 111 U.S. Patent Feb. 21, 2017 Sheet 19 Of 29 US 9,573.996 B2

FIGURE 18A

TnOS H-chain Lmubq promoter Alpha amylase signal Sec \ \\ \ | Lmubq intron ADH1 \ \ \ / Alpha amylase signal Seq Rbcs leader \ \ | / Amaspnmas \ \\ \ \ ? / L-chain | /// / Tinos Laubu promoter /.../ - Laubq intron Alpha amylase signal seq

SC-chain

TnOS

See is /N SpUbc promoter e . s w SpUbq intron

N N \ \ \ Alpha amylase signal seq. \ J-chain RB Tnos U.S. Patent Feb. 21, 2017 Sheet 20 Of 29 US 9,573.996 B2

FIGURE 18B

Tnos L-chain Alpha amylase signal Seq 8. \ SpUbiq promoter Lm Ubiq intron \ \ \ SpUbq Intron Lm Ubc promoter \ \ \ \ w X Tnos & X , \ \ \ \ \ H-chain \ X 8, 8. f FucT forward Alpha amylase signal seq \\ \,y \,\ \ \ \\\ \ | xyT forward \ y \ ', 8, 8. \ ADH1 \, \, \ y \\ \\ f : XyTreverse RbcSleader FucTrewerse AmasPmas Y.

Laubq Intron 8 ---- y- --- Alpha amylase signal Seq

SC-chain

Tnos

SpUbq promoter

SpUbq Intron \ Alpha amylase signal seq. y v RB \ J-chain w Tnos U.S. Patent Feb. 21, 2017 Sheet 21 of 29 US 9,573.996 B2

FIGURE 18C

Tnos Lmuba promoter H-chain \ | Limbo Int Alpha amylase signal seq \ W\ f C O ADH1 \ \ \ | / Alpha amylase signal seq RbcS leader y\ \\ % \ %\ | / / / / L-chain AmasPmas x \ X \, x \, %\, \ ! / / 8 / A.OCS \ \\\, \, \ \ \ | f / / / / / / Tnos Aocs w / ? Laubq promoter AOCS sy - Laubq intron pNOS Alpha amylase signal seq Npt S- x. SC-chain pAg7 SynA03 Tnos - 24902 bp - e--- LB SpUbq promoter

Nptill SpUbq intron s Alpha amylase signal seq.

J-chain ORiv TnOS U.S. Patent Feb. 21, 2017 Sheet 22 of 29 US 9,573.996 B2

FIGURE 19

slgA->

SCH) HC-e-

LC&JC-> U.S. Patent Feb. 21, 2017 Sheet 23 Of 29 US 9,573.996 B2

FIGURE 20

S - $ S. s$ s s S ustickinurnab Colostral-SigA SynAC1-WT 8 S SS. S 395 5 S 3C O 3 ( 12 S

S $s ses s 8 ustekin unab Colost al-SigA SynaO2-GO es S

& as O is 15 3 is 5 3 5 5 33 2. U.S. Patent Feb. 21, 2017 Sheet 24 of 29 US 9,573.996 B2

FIGURE 21A

22 20 E 18 S 16 S 14 S. 12 - 10 & 8 CN 6 4 2 O O 0.1 1.0 10 Concentration antibody (ug/ml)

FIGURE 21B

2120 g 100 80 9 60 & t - 40- E N 20 & O I E. E. SS U.S. Patent Feb. 21, 2017 Sheet 25 Of 29 US 9,573.996 B2

FIGURE 21C

120 100

FIGURE 21D

250

200

150

100

50 U.S. Patent Feb. 21, 2017 Sheet 26 of 29 US 9,573.996 B2

FIGURE 22

25OOOO

200000

15OOOO

1OOOOO

5OOOO

U.S. Patent Feb. 21, 2017 Sheet 27 of 29 US 9,573.996 B2

FIGURE 23 Time (hour) SynAO 1 a IgG a SynAO1a SynAO1b Oral Oral Intra peritoneal Oral

0.3

4.5

24 U.S. Patent Feb. 21, 2017 Sheet 28 Of 29 US 9,573.996 B2

FIGURE 24 SynAO1, Oral IgG, Oral

U.S. Patent Feb. 21, 2017 Sheet 29 Of 29 US 9,573.996 B2

FIGURE 25A

on 8 35 6 4 o 2.

O &E. ScN as

FIGURE 25B

Treatment PBS SynAO1 SynAO2 Ustekinumab Oral Oral Oral Subcutaneous Mini s endoscopy Day t=15 US 9,573.996 B2 1. 2 MONOCLONAL ANTIBODIES TO HUMAN cally and thus are often associated with unwanted side PROINFLAMMATORY CYTOKNES AND effects, which can include one or more of for example, METHODS FOR TREATING infusion reactions and immunogenicity, hypersensitivity NFLAMMLATORY DISEASES reactions, immunosuppression and infections, heart prob lems, liver problems, and others. Additionally the Suppres This application claims the benefit of priority under 35 sion of the target cytokines at non-diseased parts of the body U.S.C. S 119(e) from U.S. provisional patent application Ser. can lead to unwanted effects. No. 61/576,727, filed Dec. 16, 2011, and Ser. No. 61/576, In an attempt to reduce side effects associated with 922, filed Dec. 16, 2011; the entire contents of each provi systemic treatment and to eliminate the inconvenience and sional application being incorporated herein by reference. 10 expense of infusions, an article proposed an oral anti-TNF therapy that could be useful in treating Crohn's disease. FIELD OF THE INVENTION Worledge et al. “Oral Administration of Avian Tumor Necro sis Factor Antibodies Effectively Treats Experimental Coli The present invention relates to monoclonal secretory IgA tis in Rats.” Digestive Diseases and Sciences 45(12); 2298 antibodies that bind to and neutralize one or more human 15 2305 (December 2000). This article describes immunizing proinflammatory cytokines, or bind to and block a human hens with recombinant human TNF and an adjuvant, frac receptor of a human proinflammatory cytokine. The anti tionating polyclonal yolk antibody (IgY, which in chickens bodies are useful in treating inflammatory diseases in is the functional equivalent to IgG), and administering the humans. unformulated polyclonal IgY (diluted in a carbonate buffer to minimize IgYacid hydrolysis in the stomach) to rats in an BACKGROUND OF THE INVENTION experimental rodent model of colitis. The rats were treated with 600 mg/kg/day of the polyclonal IgY. The uses of Inflammation represents a key event of many diseases, animal antibodies and polyclonal antibodies, however, are Such as psoriasis, inflammatory bowel diseases, rheumatoid undesirable. arthritis, asthma, multiple Sclerosis, atherosclerosis, cystic 25 In a similar attempt to avoid adverse events associated fibrosis, and sepsis. Inflammatory cells. Such as neutrophils, with systemic administration, another group, Avaxia Bio eosinophils, basophils, mast cells, macrophages, endothelial logics Inc., describes a topical (e.g., oral or rectal) animal cells, and platelets, respond to inflammatory stimuli and derived polyclonal anti-TNF composition that could be foreign Substances by producing bioactive mediators. These useful in treating inflammation of the digestive tract, such as mediators act as autocrines and paracrines by interacting 30 inflammatory bowel disease. WO2011047328. The applica with many cell types to promote the inflammatory response. tion generally states that preferably the polyclonal antibody There are many mediators that can promote inflammation, composition is prepared by immunizing an animal with a Such as cytokines and their receptors, adhesion molecules target antigen, and the preferably the polyclonal antibody and their receptors, antigens involved in lymphocyte acti composition is derived from milk or colostrum with bovine Vation, and IgE and its receptors. 35 colostrums being preferred (e.g., p. 14). The application also Cytokines, for example, are soluble proteins that allow for generally states that the animal derived polyclonal antibod communication between cells and the external environment. ies could be specific for (among other targets) other inflam The term cytokines includes a wide range of proteins, such matory cytokines (e.g., pp. 6-7). This application describes as lymphokines, monokines, interleukins, colony Stimulat working examples in which cows were immunized with ing factors, interferons, tumor necrosis factors, and chemok 40 murine TNF and the colostrum was collected post-parturi ines. Cytokines serve many functions, including controlling tion to generate bovine polyclonal anti-TNF antibodies cell growth, migration, development, and differentiation, (designated as AVX-470). The uses of animal-derived anti and mediating and regulating immunity, inflammation, and bodies and polyclonal antibodies, however, are undesirable. hematopoiesis. Even within a given function, cytokines can IgA molecular forms have been proposed as treatments have diverse roles. For example, in the context of mediating 45 for various diseases, most notably as treatments for pollen and regulating inflammation, Some cytokines inhibit the allergies, as treatments against pathogens, and as treatments inflammatory response (anti-inflammatory cytokines), oth for cancer. ers promote the inflammatory response (pro-inflammatory For example, one article describes anti-AmbCI (a rag cytokines). And certain cytokines fall into both categories, weed pollen antigen) humanized monomeric IgA and i.e., can inhibit or promote inflammation, depending on the 50 dimeric IgA antibodies made in murine cells (NSO and Sp2/0 situation. The targeting of proinflammatory cytokines to cells). The dimeric IgA contains a mouse J-chain. The article Suppress their natural function, such as with antibodies, is a proposes that the antibodies may be applied to a mucosal well-established strategy for treating various inflammatory surface or the lower airway to inhibit entry of allergenic diseases. molecules across the mucosal epithelium and therefore to Many inflammatory diseases are treated by targeting 55 prevent the development of allergic response. Sun et al. proinflammatory cytokines with antibodies. Most (if not all) “Human IgA Monoclonal Antibodies Specific for a Major of the anti-proinflammatory cytokine antibodies currently on Ragweed Pollen Antigen.” Nature Biotechnology 13, 779 the market, and those currently in clinical trials, are of the 786 (1995). IgG class. See, for example, Nature Reviews, Vol. 10, pp. Several other articles propose the use of IgA antibodies as 301-316 (2010): Nature Medicine, vol. 18, pp. 736-749 60 a defense against pathogens. (2012): Nature Biotechnology, vol. 30, pp. 475-477 (2012); Two articles proposed the use of an anti-streptococcal Anti-Inflammatory & Anti-Allergy Agents in Medicinal antigen I/II secretory IgA-G hybrid antibody. Ma et al. Chemistry, vol. 8, pp. 51-71 (2009): F1000.com/Reports/ “Generation and Assembly of Secretory Antibodies in Biology/content/1/70, F1000 Biology Reports, 1:70 (2009): Plants.” Science 268(5211), 716–719 (May 1995); Ma et al. mAbs 4:1, pp. 1-3 (2012); mAbs 3:1, pp. 76-99 (2011): 65 “Characterization of a Recombinant Plant Monoclonal clinicaltrials.gov (generally), and clinicaltrialsregister.eu/ Secretory Antibody and Preventive Immunotherapy in (generally). These IgG antibodies are administered systemi Humans.” Nature Medicine 4(5): 601-606 (May 1998). The US 9,573.996 B2 3 4 hybrid antibody contains murine monoclonal kappa light in BHK cells cotransfected with human J-Chain and/or chain, hybrid Ig A-G heavy chain, murine J-Chain, and human secretory component (Vidarsson et al., Journal of rabbit secretory component. The antibody was made by Immunology 166; 6250-6256 (2001)); (4) anti-Pseudomonas Successive sexual crossing between four transgenic N. taba aeruginosa O6 lipopolysaccharide chimeric mouse/human cum plants and filial recombinants to form plant cells that mIgA1 made in CHO cells (Preston et al. Infection and expressed all four protein chains simultaneously. The parent Immunity 66(9): 4137-4142 (September 1998)); (5) anti antibody (the Source of the antigen binding regions, is Plasmodium mIgA made in CHO cells (Pleass et al. Blood identified as the IgG antibody Guy's 13. The group proposes 102(13): 4424-4429 (December 2003)) (which states that that although SIgA may provide an advantage over IgG in the unlike their parental mouse IgG antibodies, the mIgA anti mucosal environment, such is not always the case (1998 Ma 10 at p. 604, right column). bodies failed to protect against parasitic challenge in vivo); A related article identifies the anti-streptococcal antigen and (5) anti-Helicobacter pylori urease subunit A SIgA and I/II secretory IgA-G hybrid antibody, which was derived dIgA (Berdoz et al. Molecular Immunology 41 (10); 1013 from Guy's 13 IgA, as CaroRX. Wycoff. “Secretory IgA 1022 (August 2004)). Antibodies from Plants. Current Pharmaceutical Design 15 For a review article discussing passive and active protec 10(00): 1-9 (2004). Planet Biotechnology Inc. This related tion against pathogens at mucosal surfaces, see Corthésy. article states that the CaroRx antibody was designed to block “Recombinant Immunoglobulin A: Powerful Tools for Fun adherence to teeth of the bacteria that causes cavities. damental and Applied Research.” Trends in Biotechnology Apparently, the CaroRX antibody was difficult to purify; the 20(2): 65-71 (February 2002). affinity of Protein A for the murine Ig domain was too low Still other articles propose the use of IgA antibodies as a and protein G was necessary for Sufficient affinity chroma treatment for cancer. tography. Furthermore, the article states that several other For example, one article describes a Phase Ia trial of a chromatographic media had shown little potential as puri muring anti-transferrin receptor IgA antibody (Brooks et al. fication steps for the hybrid sIgA-G from tobacco leaf "Phase Ia Trial of Murine Immunoglobulin A Antitransferrin extracts. The article also indicates that the authors were 25 Receptor Antibody 42/6.” Clinical Cancer Research 1 (11): unable to control for human-like glycosylation in tobacco, 1259-1265 (November 1995)). Another article describes a but that such was not a problem because people are exposed human anti-Ep-CAM mIgA made in BHK (baby hamster to plant glycans every day in food without ill effect. kidney) cells (Huls et al. “Antitumor Immune Effector WO9949024, which lists Wycoff as an inventor, Planet Mechanisms Recruited by Phase Display-Derived Fully Biotechnology Inc. as the applicant, describes the use of the 30 variable regions of Guy's 13 to make a secretory antibody Human IgG1 and IgA1 Monoclonal Antibodies.” Cancer from tobacco. The application contains only two Research 59; 5778-5784 (November 1999)). Still another examples—the first a working example and the second a article describes an anti-HLA Class II chimeric mIgA anti prophetic example. Working Example 1 describes the tran body made in BHK cells (Dechant et al. “Chimeric IgA sient production of an anti-S. mutans SA I/III (variable 35 Antibodies Against HLA Class II Effectively Trigger Lym region from Guy's 13) in tobacco. The tobacco plant was phoma Cell Killing.” Blood 100(13): 4574-4580 (December transformed using particle bombardment of tobacco leaf 2002)). Yet other articles describe anti-EGFR mIgA or dIgA disks. Transgenic plants were then screened by Western blot antibodies made in CHO, including Dechant et al. “Effector “to identify individual transformants expressing assembled Mechanisms of Recombinant IgA Antibodies Against Epi human sIgA” (p. 25). Prophetic Example 2 states that in a 40 dermal Growth Factor Receptor.” Journal of Immunology transformation system for Lenna gibba (a monocot), bom 179; 2936-2943 (2007), Beyer et al. “Serum-Free Produc bardment of surface-sterilized leaf tissue with DNA-coated tion and Purification of Chimeric IgA Antibodies.” Journal particles “is much the same as with tobacco (a dicot). The of Immunology 346; 26-37 (2009) (stating that as of 2009, prophetic example also stops at Screening by immunoblot IgA antibodies have not been commercially explored for analysis for antibody chains and assembled SIgA, and states 45 problems including lack of production and purification that the inventors “expect to find fully assembled sIgA.” methods), and Lohse et al. “Recombinant Dimeric IgA Another article proposed the use of an anti-RSV glyco Antibodies Against the Epidermal Growth Factor Receptor protein FIgA antibodies (mIgA, dIgA, and SIgA). Berdozet Mediate Effective Tumor Cell Killing” Journal of Immunol al. “In vitro Comparison of the Antigen-Binding and Sta ogy 186: 3770-3778 (February 2011). bility Properties of the Various Molecular Forms of IgA 50 For a review article on anti-cancer IgA antibodies, see antibodies Assembled and Produced in CHO Cells.” Proc. Dechant et al. “IgA antibodies for Cancer Therapy.'Critical Natl. Acad. Sci. USA96: 3029-3034 (March 1999). The sIgA Reviews in Oncology/Hematology 39; 69-77 (2001); states antibody was made in CHO cells sequentially transfected that compared with infectious diseases, the role of IgA in with chimeric heavy and light chains, human J-Chain, and cancer immunotherapy is even less investigated). human secretory component, respectively. Single clones 55 It is desirable to have alternative antibody treatments for were generated to express the mIgA (clone 22), the dIgA inflammatory diseases that preferably avoid the disadvan (clone F), and the sIgA (clone 6) (p. 3031). tages of current systemic and previously-proposed topical Still other articles proposed, for example: (1) anti-HSV treatments of inflammation. mIgA made in maize (Karnoup et al. Glycobiology 15(10); 965-981 (May 2005)) (which states that at that time there 60 SUMMARY OF THE INVENTION had been little Success in the application of IgA class antibodies to therapeutic use because of the difficulty in The present invention relates to monoclonal secretory IgA producing the dimeric form in mammalian cells at economic antibodies that bind to and neutralize a human proinflam levels); (2) anti-C. difficile toxin A chimeric mouse-human matory cytokine, or bind to and block a human receptor of monomeric and dimeric IgA made in CHO cells (Stubbe et 65 a human proinflammatory cytokine, and the use of Such al. Journal of Immunology 164; 1952-1960 (2000)); (3) secretory IgA antibodies in treating inflammatory diseases in anti-N. meningitidis chimeric IgA antibodies were produced humans. US 9,573.996 B2 5 6 An aspect of the present invention relates to a monoclonal SEQ ID NO:3 provides the amino acid sequence of the secretory IgA antibody that binds to and neutralizes a human light chain variable region of infliximab (cA2). proinflammatory cytokine. SEQ ID NO:4 provides the amino acid sequence of the In embodiments, the antibody can be, for example a heavy chain variable region of adalimumab (D2E7). chimeric antibody, a humanized antibody, or a human anti SEQ ID NO:5 provides the amino acid sequence of the body. The antibody can contain a human secretory chain and light chain variable region of adalimumab (D2E7). a human J-chain. The antibody can be a human secretory SEQ ID NO:6 provides the amino acid sequence of the IgA1 antibody. The human proinflammatory cytokine can be heavy chain variable region of golimumab. selected from, for example, TNFC. (soluble), interferon SEQ ID NO:7 provides the amino acid sequence of the gamma, interferon alpha, GM-CSF, CXCL10/IP-10, IL-1B. 10 light chain variable region of golimumab. IL-1C, IL-4, IL-5, IL-6, IL-12; IL-13, IL-17A, IL-18, IL-20, SEQ ID NO:8 provides the amino acid sequence of the IL-22, and IL-23. The antibody can contain CDR sequences heavy chain variable region of certolizumab pegol. that are identical to the CDR sequences of an antibody SEQ ID NO:9 provides the amino acid sequence of the selected from adalimumab, infliximab, golimumab, certoli 15 light chain variable region of certolizumab pegol. Zumab pegol, oZoralizumab, Sifalimumab, canakinumab, SEQ ID NO:10 provides the amino acid sequence of a gevokizumab, pascolizumab, reslizumab, mepolizumab, human IgA C-1 heavy chain constant region (CC1-CC2 sirukumab, olokizumab, ustekinumab, briakinumab, traloki Co.3) (UniProtKB/Swiss-Prot database entry P01876 numab, anrukinzumab, lebrikizumab, secukinumab, ixeki (IGHA1 HUMAN)). Zumab, and fezakinumab. SEQ ID NO: 11 provides the amino acid sequence of a Another aspect of the present invention relates to a human IgA C-2 m (1)-allotype heavy chain constant region composition containing a plurality of the secretory IgA (Co. 1-Co.2-CO3) (UniProtKB/Swiss-Prot database entry antibodies. In embodiments, Substantially all N-glycans in PO1877 (IGHA2 HUMAN)). the plurality of antibodies lack fucose and xylose residues. SEQ ID NO:12 provides the amino acid sequence of a In embodiments, the plurality of antibodies contains at least 25 human IgA C-2 m (2)-allotype heavy chain constant region about 30% G0 glycans (preferably G0 glycans lacking Fuc (CO. 1-CO2-CO3). (UniProtKB/Swiss-Prot database entry and Xyl residues) relative to the total amount of N-glycans P01877 (IGHA2 HUMAN) with indicated modifications in the population. In embodiments, the a plurality of anti for allotype 2 variant). bodies ac contains at least about 25% high-mannose glycans SEQ ID NO:13 provides the amino acid sequence of a (e.g., Man5, Manó, Man7, Man8, and/or Man 9 glycans) 30 human IgA C-2 (n)-allotype. relative to the total amount of N-glycans in the population. SEQ ID NO:14 provides the amino acid sequence of a In embodiments, the G0 glycans (preferably G0 glycans human K light chain constant region (C) (UniProtKB/ lacking Fuc and Xyl residues) and high-mannose glycans Swiss-Prot database entry P01834 (IGKC HUMAN)). (e.g., Man5, Manó, Man7, Man8, and/or Man 9 glycans) SEQ ID NO:15 provides the amino acid sequence of a together are the majority of glycans present in said plurality 35 human 1 light chain constant region (C) (UniProtKB/ of antibodies, such as at least 70% of the total amount of Swiss-Prot database entry P0CG04 (LAC1 HUMAN)). N-glycans in said plurality of antibodies. SEQ ID NO:16 provides the amino acid sequence of a Another aspect of the present invention relates to phar human M2 light chain constant region (C) (UniProtKB/ maceutical compositions containing the Secretory IgA anti Swiss-Prot database entry P0CG05 (LAC2 HUMAN)). bodies, which can be adapted for oral administration and can 40 SEQ ID NO:17 provides the amino acid sequence of a be used to treat an inflammatory disease in a human. human M3 light chain constant region (C) (UniProtKB/ Another aspect of the present invention relates to methods Swiss-Prot database entry P0CG06 (LAC3 HUMAN)). for treating an inflammatory disease in a human, which SEQ ID NO:18 provides the amino acid sequence of a includes administering an anti-inflammatory effective human 6 light chain constant region (C) (UniProtKB/ amount of the secretory IgA antibodies (or compositions) to 45 Swiss-Prot database entry P0CF74 (LAC6 HUMAN)). a human in need thereof, preferably orally administering the SEQ ID NO:19 provides the amino acid sequence of a antibodies (compositions). The inflammatory disease can be human 7 light chain constant region (C) (UniProtKB/ selected from rheumatoid arthritis, inflammatory bowel dis Swiss-Prot database entry AOM8Q6 (LAC7 HUMAN)). ease (including Crohn's disease and ulcerative colitis), pso SEQ ID NO:20 provides the amino acid sequence of a riasis, psoriatic arthritis, ankylosing spondylitis, juvenile 50 human J-chain (amino acids 23-159 UniProtKB/Swiss-Prot idiopathic arthritis, uveitis, asthma, Alzheimer's disease, database entry P01591). multiple Sclerosis, type II diabetes, systemic sclerosis, lupus SEQ ID NO:21 provides the amino acid sequence of a nephritis, and allergic rhinitis. human secretory component (amino acids 19-603 of Uni Another aspect of the present invention relates to a ProtKB/Swiss-Prot database entry P01833 (PIGR HU monoclonal Secretory IgA antibody that binds to and blocks 55 MAN), RCSB Protein Data Bank structure 20OW). a human proinflammatory cytokine receptor selected from SEQ ID NO:22 provides the amino acid sequence of a IL-1R, IL-2R, IL-4R, IL-5R, IL-6R, and IL-17R. natural signal peptide (secretion signal) for a human J-chain (amino acids 1-22 UniProtKB/Swiss-Prot database entry BRIEF DESCRIPTION OF THE SEQUENCES PO1591). 60 SEQ ID NO:23 provides the amino acid sequence for a SEQ ID NO:1 provides the amino acid sequence of the signal peptide (heavy chain secretion signal). soluble TNF-C. receptor of etanercept (extracellular part of SEQ ID NO:24 provides the amino acid sequence for a tumor necrosis factor receptor 2: amino acids 23-257 of signal peptide (light chain secretion signal). UniProtKB/Swiss-Prot database entry P20333 SEQ ID NO:25 provides the amino acid sequence for a (TNR1B. HUMAN)). 65 signal peptide (SC-chain secretion signal). SEQ ID NO:2 provides the amino acid sequence of the SEQ ID NO:26 provides the amino acid sequence for a heavy chain variable region of infliximab (cA2). rice C.-amylase signal peptide (secretion signal). US 9,573.996 B2 7 8 SEQ ID NO:27 provides the DNA sequence for the (SEQ ID NO:14); FIG. 3B shows the amino acid sequences infliximab heavy chain IgA2m(n) optimized for maize. of a human w light chain constant region (C) allotypes SEQ ID NO:28 provides the DNA sequence for the (UniProtKB/Swiss-Prot POCG04, P0CG05, P0CG06, infliximab heavy chain IgA1 and light chain K optimized for P0CG74, and AOM8Q6; SEQ ID NOS:15 to 19). HEK. 5 FIG. 4 shows the amino acid sequence of a human J-chain SEQ ID NO:29 provides the DNA sequence for the (a.a. 23-159 of UniProtKB/Swiss-Prot entry P01591, SEQ infliximab light chain optimized for maize. ID NO:20). SEQ ID NO:30 provides the DNA sequence for SC- and FIG. 5 shows the amino acid sequence of a human J-chains optimized for HEK. secretory component (a.a. 19-603 of UniProtKB/Swiss-Prot SEQ ID NO:31 provides the DNA sequence for the 10 database entry P01833 PIGR HUMANI, SEQ ID NO:21). adalimumab heavy chain IgA1 optimized for Lenna. FIG. 6 shows the structure of the SIgA vector constructs SEQ ID NO:32 provides the DNA sequence for the of Example 1. adalimumab light chain K optimized for Lenna. SEQ ID NO:33 provides the DNA sequence for a J-chain FIG. 7 shows reducing and non reducing gels of an optimized for maize. 15 anti-TNFC. SIgA of the invention, having infliximab variable SEQID NO:34 provides the DNA sequence for a J-chain regions and being produced in mammalian HEK-293F cells. optimized for Lenna. The label “A” shows non-reducing SDS-PAGE analysis SEQ ID NO:35 provides the DNA sequence for an demonstrating expression of complete SIgA while the label SC-chain optimized for maize. “B” shows reducing SDS-PAGE analysis. SEQ ID NO:36 provides the DNA sequence for an FIG. 8 shows reducing and non reducing gels of an SC-chain optimized for Lenna. anti-TNFC. SIgA of the invention, having adalimumab vari SEQ ID NO:37 provides the amino acid sequence of the able regions and being produced in Lenna. The label “A” heavy chain variable region of ustekinumab (CTNO-1275). shows non-reducing SDS-PAGE analysis demonstrating SEQ ID NO:38 provides the amino acid sequence of the expression of complete SIgA while the label “B” shows light chain variable region of ustekinumab (CTNO-1275). 25 reducing SDS-PAGE analysis. SEQ ID NO:39 provides the amino acid sequence of the FIG. 9 shows binding curves of antibodies to TNFC. The heavy chain variable region of briakinumab (J-695, ABT closed circle represents a SIgA of the invention having 874). infliximab variable regions, square represents infliximab, SEQ ID NO:40 provides the amino acid sequence of the and diamond represents colostral secretory IgA. light chain variable region of briakinumab (J-695, ABT 30 FIG. 10 shows the degradation of an anti-TNFO. SIgA 874). according to the invention having infliximab variable SEQ ID NO:41 provides a complete lemma-optimized regions and being produced in mammalian cells, and an UKB-SA1 heavy chain IgA1 DNA (including DNA encod anti-TNFC, SIgA according to the invention having adalim ing signal peptide SEQ ID NO:26). umab variable regions and being produced in Lenna, com SEQ ID NO:42 provides a complete lemma-optimized 35 pared to colostral SIgA in simulated intestinal fluid (SIF). UKB-SA1 light chain DNA (including DNA encoding sig FIG. 11 shows TNFC-induced disturbance of Caco-2 nal peptide SEQ ID NO:26). monolayer integrity as measured by transepithelial electrical resistance (TER). BRIEF DESCRIPTION OF THE DRAWINGS FIG. 12 shows the restoration of TNFC.-induced distur 40 bance of Caco-2 monolayer integrity as measured by TER. FIG. 1 shows the amino acid sequences of the constant FIG. 13 represents the colitis scoring of DSS-induced regions of human IgA1 (SEQ ID NO:10), IgA2 m.1) (SEQ inflammatory bowel disease in C57BL/6 mice with and ID NO:11), IgA2 m.2) (SEQID NO:12), and IgA2(n) (SEQ without treatment. ID NO:13) antibody a heavy chains: FIG. 14 is a representative depiction of mini-endoscopic FIGS. 2A through 2I show the amino acid sequence of 45 pictures of the colitis score at day t—15 for a mouse from various TNF-C. binding regions. FIG. 2A shows the amino each test group. acid sequence of the soluble TNF-C. receptor of etanercept FIG. 15 shows the effects of subcutaneous application of (SEQ ID NO:1); FIG. 2B shows the amino acid sequence of etanercept and of oral application of ADB-SA1g on gripping the heavy chain variable region of infliximab (cA2) (SEQID strength of a transgenic TNFC-overexpressing mice that NO:2); FIG. 2C shows the amino acid sequence of the light 50 spontaneously develop arthritis (TNF^'-mice). chain variable region of infliximab (cA2) (SEQ ID NO:3); FIG. 16 shows the effects of subcutaneous application of FIG. 2D shows the amino acid sequence of the heavy chain etancercept and of oral application of ADB-SA1 g on variable region of adalimumab (D2E7) (SEQ ID NO:4): arthritic score of hind paws of transgenic TNFC-overex FIG. 2E shows the amino acid sequence of the light chain pressing mice which spontaneously develop arthritis variable region of adalimumab (D2E7) (SEQ ID NO:5): 55 (TNFA?'-mice). FIG. 2F shows the amino acid sequence of the heavy chain FIGS. 17A through 17D shows amino acid sequences of variable region of golimumab (SEQ ID NO:6): FIG. 2G various p40 (the p40 subunit of IL12 and IL23) binding shows the amino acid sequence of the light chain variable regions. FIG. 17A shows the amino acid sequence of the region of golimumab (SEQ ID NO:7); FIG. 2H shows the heavy chain variable region of the antibody ustekinumab amino acid sequence of the heavy chain variable region of 60 (CTNO-1275) (SEQID NO:37); FIG. 17B shows the amino certolizumab pegol (SEQ ID NO: 8); FIG. 2I shows the acid sequence of the light chain variable region of the amino acid sequence of the light chain variable region of antibody ustekinumab (CTNO-1275) (SEQID NO:38): FIG. certolizumab pegol (SEQ ID NO:9). 17C shows the amino acid sequence of the heavy chain FIGS. 3A and 3B show the amino acid sequences of variable region of the antibody briakinumab (J-695, ABT various human antibody light chain subtypes and allotypes. 65 874) (SEQ ID NO:39): FIG. 17D shows the amino acid FIG. 3A shows the amino acid sequence of a human K light sequence of the light chain variable region of the antibody chain constant region (C) (UniProtKB/Swiss-Prot PO1834) briakinumab (J-695, ABT-874) (SEQ ID NO:40). US 9,573.996 B2 9 10 FIGS. 18A through 18C show the structure of vector regions (SEQID NOS:15-19) are provided in FIGS. 3A and constructs SynAO1 (FIG. 18A), Syn A02 (FIG. 18B) and 3B, respectively. In nature, the heavy chain constant regions SynAO3 (FIG. 18C) used for expression of anti-IL12/23 of the various classes are produced by different genes: the SIgA in Lenna in Example 1. IgA class heavy chains are uniquely encoded-for by C. genes, FIG. 19 shows reducing and non reducing gels of an the IgG class heavy chains by Y genes, and so forth. The anti-IL-12/23 SIgA according to the invention, having heavy chain constant regions also impart the various classes ustekinumab variable regions and being produced in Lenna. with differences in their physio-chemical properties, their The Agel shows non-reducing SDS-PAGE analysis dem isotypic antigentic determinants, and/or in their biological onstrating expression of complete SIgA. The Bigel shows function. Lefranc et al., The Immunoglobulin FactsBook, reducing SDS-PAGE analysis. 10 Academic Press 2001, Chapter 2, (ISBN 0-12-441351-X). FIG. 20 shows the degradation of an anti-IL-12/23 SIgA The constant region of an IgAheavy chain (Ca) typically has according to the invention having ustekinumab variable three domains that are referred to as Co.1, CO2, and CO3, a regions and being expressed in Lenna compared to usteki short hinge section between Co.1 and CO2, and a short tail numab (IgG1) and to colostral secretory IgA in simulated piece at the C-terminal end of CO3. The definition and intestinal fluid (SIF). Gel A compares the SynAO1-WT 15 structure of antibodies are well known to workers skilled in antibody of the invention (UKB-SA1) and gel B compares this art, such as described in, e.g., Alberts, B. et al., Molecu the SynA01-GO antibody of the invention (UKB-SA1g0). lar Biology of the Cell 3" Edition, Chapter 23, Garland FIGS. 21A and 21B show the results of inhibition of Publishing Inc., New York, N.Y., 1994, and Nezlin, R., The IL-12/23 (p40) production by LPS-stimulated Dendritic Immunoglobulins. Structure and Function (1998) Academic Cells from Examples 15A and 15B, respectively. In FIG. Press (ISBN 0-12-517970-7). 21A, the open circle is SynaC)1 (UKB-SA1); the closed The C-terminal section of two IgA antibodies, i.e., the tail circle is SynA02 (UKB-SA1g0); the diamond is colostral pieces at the C-terminal ends of the CC3 region, can be secretory IgA, and the square is ustekinumab. joined together via a J-chain to form a dimer. Dimeric IgA FIGS. 21C and 21D show the results of inhibition of has four antigen binding regions; two from each IgA mono production of IFNY by co-cultured dendritic cells and 25 mer. Typically the four antigen binding regions (and their T-Cells from Examples 15C and 15D, respectively. complementarity determining regions or “CDRs) are iden FIG. 22 shows the serum concentrations of SynaO2 tical for reasons such as ease of manufacture. But the antigen (UKB-SA1g0) after oral (circle) and intravenous (square) binding regions can, in certain circumstances, be different, administration as described in Example 16. e.g., different CDRs binding different epitopes on the same FIG. 23 shows in vivo imaging of fluorescent labeled 30 antigen or event different antigens (such as in the case of Syna01 (UKB-SA1) and ustekinumab antibodies in the bispecific antibodies). Typically the CDRs of the four anti thoracic and abdominal region of mice as described in gen binding regions are identical. A secretory chain, some Example 17. times called a secretory component or SC-chain, can be FIG. 24 shows immunohistochemistry on cryo-sections of attached to the dimeric IgA antibody. The SC-chain provides mice distal colon after administration of SynaO1 (UKB 35 increased resistance to proteolysis especially in the intestinal SA1) and ustekinumab antibodies as described in Example tract. The SC-bound dimeric IgA is referred to herein as 18. “secretory IgA” or “SIgA.” FIGS. 25A and 25B show the efficacy of anti-IL12/23 Heavy chain constant regions that qualify as an IgA-class SIgA in in vivo animal models of IBD as described in antibody are well known in the art. Generally the amino acid Example 19. 40 sequence of the heavy chain constant regions of an IgA, regardless of how it is produced (e.g., naturally or recom DETAILED DESCRIPTION OF THE binantly), corresponds to an amino acid sequence encoded INVENTION for by an O-gene. In addition, IgA antibodies have charac teristic antigenic determinants unique to IgA-class antibod The present invention relates to monoclonal secretory IgA 45 ies and different from the antigenic determinants of other antibodies that bind to and neutralize a human proinflam classes of antibodies, such as IgG-class antibodies (see, e.g., matory cytokine, or bind to and block a human receptor of Nezlin, R., The Immunoglobulins. Structure and Function a human proinflammatory cytokine, and the use of Such (1998) Academic Press (ISBN 0-12-517970-7); Lefranc et secretory IgA antibodies in treating inflammatory diseases in al., The Immunoglobulin FactsBook, Academic Press 2001, humans. 50 Chapter 2, (ISBN 0-12-441351-X)). Furthermore, IgA anti As is well known, the basic structural unit of an antibody bodies are the only isotype that is known to specifically bind consists of two heavy chain proteins (heavy chains) and two to the FcC.R (see, e.g., Alberts, B. et al., Molecular Biology light chain proteins (light chains), which are bound together of the Cell 3" Edition, Chapter 23, Garland Publishing Inc., by non-covalent and covalent (e.g., disulfide bonds) inter New York, N.Y., 1994: Lefranc et al., The Immunoglobulin actions into a single unit. The heavy and light chains have 55 FactsBook, Academic Press 2001, Chapter 2, (ISBN 0-12 N-terminal variable regions and C-terminal constant 441351-X)). regions. The variable regions of the light and heavy chains Accordingly, for purposes of the present invention, the together form an “antigen binding region.” Because the terms “IgA antibody,” “monomeric IgA,” “dimeric IgA and antibody has two heavy and light chains, the antibody has “SIgA each refers to antibodies that contain the heavy two antigen binding regions. 60 chain constant regions of an IgA class of immunoglobulin, Antibodies are classified based on the heavy chain con e.g., which corresponds to an amino acid sequence that can stant region, e.g., classified as IgG, IgA, IgM, IgE, Ig|D, etc. be encoded for by a genes and which react with an antibody The light chain constant region is not used for classification. specific for the IgA-class heavy chain. The amino acid In humans, for example, all classes use one of two types of sequence "corresponds' in that it is identical to, or contains light chain constant regions, namely the CK (kappa) or CW 65 only minor variations (insertions/deletions/Substitutions) (lambda) type. The amino acid sequences of human kappa from, an amino acid sequence produced by any a gene, an (SEQ ID NO:14) and several lambda light chain constant individual human’s IgA heavy chain sequence, or a human US 9,573.996 B2 11 12 IgAheavy chain consensus sequence. Indeed, variations can In humans there are two recognized IgA Subclasses: IgA1 and do exist in the amino acid sequence of the IgA heavy and IgA2 which differ in the hinge section between the Co.1 chain constant region without moving such antibodies out and CO2 domains of the heavy chain. In IgA1 this hinge side of the IgA class. Examples of Such variations can be section is relatively long and in nature typically O-glycosy found in various genomic databases such as browser. lated. In IgA2 the hinge section is relatively short and in 1000genomes.org/index.html and ensembl.org/index.html. nature lacks glycosylation. Both IgA1 and IgA2 SIgA anti For clarity, because the heavy chain sequence is determina bodies are usually present in mucosal secretions. In humans, tive of the Ig class, a recombinant antibody containing the the IgA2 Subclass has three known allotypes: IgA2m.(1), IgA heavy chain constant regions and further containing the IgA2m (2) and IgA2m(n). Unlike the Subclasses, only one antigen binding regions encoded for by DNA sequences 10 specific allotype will be found in a normal healthy indi obtained from a known IgG antibody is still an “IgA vidual. The m1) allotype is strongly prevalent in the Cau antibody.” On the other hand, a secretory IgA antibody casian population (98%) and varies between 23% and 96% modified to replace the CO2 heavy chain constant domain for other populations. The m(2) allotype has a high preva (encoded for by the IgA-specific C-gene) with a Cy2 heavy lence in populations of African and Asian descent (50-70%). chain constant domain (encoded for by the IgG-specific 15 The mon) allotype which is considered to be a hybrid of the y-gene) is not an IgA antibody, and is instead a hybrid m(1) and m(2) allotypes—has been reported to be geneti IgA/IgG antibody. Such a hybrid is not within the scope of cally possible, but has not been actually observed in any the terms “monomeric IgA,” “dimeric IgA and “SIgA' population. See Chintalacharuvu et al., Journal of Immunol antibodies, and thus is not a secretory IgA antibody accord ogy 152, 5299-5304 (1994). Accordingly, SIgA antibodies of ing to the invention. the present invention preferably contain human IgA heavy Minor variations of the heavy chain constant regions are chain constant regions of the IgA1 or IgA2 sub-types, permitted only to the extent that the overall antibody class, including IgA2m.(1), IgA2m (2) and IgA2m(n) allotypes, and framework, and functionality of SIgA is maintained; e.g., combinations thereof (e.g., one constant domain or hinge J-chain binds to monomers and SC-chain binds to the section from an IgA1 and another constant domain or hinge dimeric structure and provides proteolysis resistance. Such 25 section from an IgA2). variations include conservative Substitutions. Exemplary Typically, the SIgA antibodies of the invention comprise conservative substitutions are shown in Table 1. The amino the Co.1 domain, the hinge section, and the -CO2-CO3 acids in the same block in the second column and preferably domains and tail section of an IgA antibody (with or without in the same line in the third column may, for example, be minor variations), including a human IgA1 and/or IgA2 substituted for each other. 30 antibody. In this embodiment, the Cal domain, the hinge section, and the -CO2-CO3 domains can be of an IgA1, an TABLE 1. IgA2m.(1) allotype, an IgA2m (2) allotype, or a combination thereof. Amino acid sequences of a human IgA1 heavy chain ALIPEHATIC Non-polar GAP constant region (SEQID NO:10), a human IgA2m.(1) heavy I LV Polar-uncharged C S TM 35 chain constant region (SEQID NO:11), a human IgA2m (2) NQ heavy chain constant region (SEQID NO:12), and a human Polar-charged DE IgA2(n) heavy chain constant region (corresponding to the KR Co.1 and CO2 regions of an IgA2m (2) and the CC3 region AROMATIC HF WY of an IgA2m.(1)) (SEQID NO:13) are respectively shown in 40 FIG 1. Typical minor variations of the constant regions from the A modified, shortened, or removed linker/hinge section normal or naturally-occurring sequence involve only con between Co.1 and CO2 in IgA1 has been reported to increase servative changes to the amino acid sequence using the resistance against proteases (for example see B. W. Senior et recognized substitutions, insertions and/or deletions. Gen al., J. Immunol. 2005; 174: 7792-7799). Such can be incor erally, the variations (substitutions, insertions, and/or dele 45 porated into the SIgA antibodies of the present invention. tions) of a constant domain of the heavy or light chain The J-chain is a protein that attaches to the tail piece of a involve no more than 10 and usually no more than 5 amino monomeric IgA to join two monomeric IgAS to form a acid additions, deletions, and/or Substitutions (either natu dimer. The J-chain is normally of mammalian origin, such as rally-occurring or genetically-engineered), in any CO1, human, murine, rat, rabbit, sheep, cow, or goat origin, but is CO2, or CC3 domain or hinge or tail sections in comparison 50 preferably of human origin. An example of the amino acid to a normal IgA constant domain. The Sum of these minor sequence of a human J-chain is set forth in FIG. 4 (SEQ ID variations in the constant domains of the SIgA antibody of NO:20). Usually, the sequence of the mammalian-derived the invention is usually less than 20 amino acids (acid/ J-chain is the same as the naturally-occurring sequence, but deletions/substitutions) and often less than 10 or less than 5. it can be subject to minor variations as described above for Accordingly, at a minimum, SIgA includes any recog 55 constant regions generally, e.g., up to 10 amino acid inser nized amino acid sequence that is generally accepted as tions, Substitutions, or deletions. The minor variations do not being within the IgA class. For example, information on the significantly alter the function of the J-chain, and in par structure and function of IgA can be found in Snoeck et al., ticular the ability to join two monomeric IgA antibodies to Vet. Res. 37:455-467 (2006) and “Mucosal immune defense: form a dimer and to enable attachment of the SC-chain. Immunoglobulin A', C. S. Kaetzel ed., Springer, New York 60 The secretory component, also referred to as “SC or (2007) ISBN 978-0-387-72231-3. Electronic databases, “SC-chain,” is a protein that binds to the dimeric IgA such as RCSB Protein Data Bank, can also establish a framework and imparts increased resistance against pro known IgA sequence or portion/domain thereof. The con teolysis upon the antibody to which it is bound. Generally, stant domains contained in the SIgA antibodies of the the Secretory component is of mammalian origin, Such as invention can be human, non-human, or a combination of 65 human, murine, rat, rabbit, sheep, cow, or goat origin, but is these. Preferred are mammalian constant regions. Most preferably of human origin. The SC is the result of cleavage preferred are human constant regions. of the Polymeric IgA-receptor (PIGR) which usually occurs US 9,573.996 B2 13 14 at a specific position. Some variation can occur in the matory cytokine receptor of 107 M or lower, preferably position of the cleavage resulting in variant forms of SC. 10 M, 10 M, 109 M, 10 M or 10M or lower. Usually, the sequence of the mammalian-derived SC-chain is the same as the naturally-occurring sequence, but it can be In embodiments, secretory IgA antibodies of the present Subject to minor variations as described above for constant invention neutralize a proinflammatory cytokine to which it regions generally, e.g., up to 10 amino acid insertions, is bound. For the present invention, the term “neutralizes' Substitutions, or deletions. The minor variations do not means inhibits/reduces the effect of the cytokine to some significantly alter the function of the secretory component, degree, such as by at least 30%, at least 35%, at least 40%, e.g., the ability to stabilize the SIgA against proteolysis. An and at least 45%. Typically the inhibition/reduction in the example of the amino acid sequence of a human secretory 10 effect of the cytokine at least 50%. In these embodiments, component is set forth in FIG. 5 (SEQ ID NO:21). Secretory IgA antibodies of the present invention bind to the secretory IgA antibodies of the present invention pref and neutralize a human proinflammatory cytokine or bind to erably inhibit/reduce the proinflammatory effect of a and block a human receptor of a human proinflammatory cytokine to which it is bound by at least 50%, such as by at cytokine. In a preferred embodiment, the antigen binding 15 least 55%, at least 60%, at least 65%, and at least 70%, and region of a SIgA according to the invention comprises a more preferably by at least 75%, at least 80%, at least 85%, heavy and light chain variable region pair, each containing at least 90%, at least 95%, and at least 98%. hypervariable regions (CDRs, which directly interact with the proinflammatory cytokine) and the Supporting frame In embodiments, SIgA antibodies of the present invention work regions. The CDRs in each heavy and light chain blocks a proinflammatory cytokine receptor to which it is variable region are separated from each other and from the bound. For the present invention, the term “blocks' means constant domain by the framework regions, which serve to inhibits/reduces the effect of the receptor to some degree, maintain the CDRs in the correct binding conformation. In such as by at least 30%, at least 35%, at least 40%, and at general each variable part of an immunoglobulin heavy or least 45%. Typically the inhibition/reduction in the effect of light chain contains 3 different CDRs and four framework 25 the receptor at least 50%. In these embodiments, the secre regions. For a more detailed description of antibody antigen tory IgA antibodies of the present invention preferably binding regions, see for example C. A. Janeway et al., inhibit/reduce the proinflammatory effect of a receptor to “Immunobiology' 6" Edition, Chapter 3, pp 110-115; Gar which it is bound by at least 50%, such as by at least 55%, land Science Publishing, New York, 2005 (ISBN at least 60%, at least 65%, and at least 70%, and more 0815341016). Regarding framework regions in particular, 30 preferably by at least 75%, at least 80%, at least 85%, at least see for example WO92/22653 (discussing that although 90%, at least 95%, and at least 98%. framework regions do not directly interact with antigen, framework regions can influence binding of the CDRs with The amino acid sequence of the antigen binding region, antigen, such as binding strength and/or downstream and in particular the CDRs thereof, is determined by the events). 35 epitope to which it binds. The amino acid sequence of the In these embodiments, the antigen binding regions of the antigen binding region can be novel or can be obtained from SIgAs of the invention bind to and neutralize the proinflam existing anti-proinflammatory-cytokine antibodies. Methods matory cytokine or bind to and block a human receptor of a for obtaining novel antigen binding sequences are well human proinflammatory cytokine. Preferably, the proinflam known in the art. See for example Mary A. Ritter and matory cytokine or receptor is selected from TNF-alpha 40 Heather M. Ladyman (Eds.) “Monoclonal antibodies: pro (preferably soluble TNF-alpha), IFN-gamma, IFN-alpha, duction, engineering, and clinical application'. Cambridge GM-CSF, CXCL10/IP-10, IL-1-beta, IL-1-alpha, IL-4, IL-5, University Press (1995), ISBN 0521425034, Zhiqiang An IL-6, IL-12, IL-13, IL-17A, IL-18, IL-20, IL-22, IL-23, IL-1 “Therapeutic Monoclonal Antibodies. From Bench to receptor, IL-2 receptor, IL-4 receptor, IL-5 receptor, IL-6 Clinic” Wiley, New York (2009), ISBN: 04701 17915, and receptor, and IL-17 receptor. In embodiments, the proin 45 Christopher Dean and Philip Shepherd (Ed.) "Monoclonal flammatory cytokine is selected from IFN-gamma, IFN Antibodies: A Practical Approach’ Oxford University Press, alpha, GM-CSF, CXCL10/IP-10, IL-1-beta, IL-1-alpha, USA (2000), ISBN 0199637229. IL-4, IL-5, IL-6, IL-13, IL-17A, IL-18, IL-20, and IL-22. Secretory IgA antibodies of the present invention specifi There are many therapeutic antibodies in clinical trials or cally and preferentially bind with high affinity to a corre 50 on the market that function to neutralize proinflammatory sponding human proinflammatory cytokine or human recep cytokines, or to block proinflammatory cytokines receptors, tor of a human proinflammatory cytokine. A variety of to treat various inflammatory diseases, and the antigen protocols for performing binding, competitive binding, or binding regions (and CDRS thereof) of these known anti immuno-radiometric assays to determine the specific bind bodies are suitable for use in the sectretory IgA antibodies of ing capability of an antibody are well known in the art (see 55 the present invention. Examples of Such known antibodies, for example Maddox et al., J. Exp. Med. 158, 1211-1226, along with the neutralized cytokines and inflammatory dis 1993). Such immunoassays typically involve the formation eases treated, are summarized in the following Table 2. See, of complexes between the specific protein and its antibody for example, Nature Reviews, vol. 10, pp. 301-316 (2010); and the measurement of complex formation (e.g., binding to Nature Medicine, Vol. 18, pp. 736-749 (2012): Nature Bio or unbinding from the specific protein). Generally, the 60 technology, vol. 30, pp. 475-477 (2012); Anti-Inflammatory affinity of secretory IgA antibodies of the invention is at least & Anti-Allergy Agents in Medicinal Chemistry, Vol. 8, pp. two-fold, at least 10-fold, at least 50-fold, at least 100-fold, 51-71 (2009): F1000.com/Reports/Biology/content/1/70, or at least 1000-fold or greater than the affinity of the F1000 Biology Reports, 1:70 (2009); landesbioscience.com/ antibodies for a non-specific protein such as, for instance, journals/mabs/ReichertMABS4-1.pdf, mAbs 4:1, pp. 1-3 BSA or casein. Typically the secretory IgA antibodies of the 65 (2012); landesbioscience.com/journals/mabs/article/13895/, present invention exhibit a binding affinity constant (K) mAbs 3:1, pp. 76-99 (2011); clinicaltrials.gov (generally), with respect to the proinflammatory cytokine or proinflam and clinicaltrialsregister.eu/ (generally). US 9,573.996 B2 15 16 TABLE 2 Proinflammatory Cytokine Target Antibody Inflammatory Disease TNF-alpha adalimumab (Humira; Abbott); infliximab Rheumatoid arthritis (RA); (Remicade; Janssen); golimumab (Simponi; inflammatory bowel disease (IBD, Centocor); certolizumab pegol (Cimzia; UCB); which includes Crohn's disease Ozoralizumab (Pfizer); ATN-192 (PF-05230905; and ulcerative colitis); psoriasis; Pfizer); ART621 (Arana). psoriatic arthritis; ankylosing spondylitis; juvenile idiopathic arthritis (JIA): uveitis; (eosinophilic allergic) asthma; and/or Alzheimer's disease. FN-gamma AMG 811 (Amgen). Psoriasis. FN-alpha sifallimumab (MedImmune). Psoriasis. GM-CSF MOR103 (MorphoSys AG). RA; multiple sclerosis (MS). CXCL1OJIP-10 MDX-1100 (Medarex). RA; IBD. L-1-beta Canakinumab (Ilaris; Novartis); rilonacept RA; JLA: type II diabetes: uveitis; (Arcalyst; Regeneron); gevokizumab; (XOMA). Systemic Sclerosis (scleroderma). L-1-alpha TrueHuman RA-18C3 (XBiotech). Psoriasis. L-4 pascolizumab (PDL). (Eosinophilic allergic) asthma. L-5 reslizumab (Cephalon); mepolizumab (GSK); (Eosinophilic allergic) asthma. SCH55700 (Celltech). L-6 sirukumab (Centocor); olokizumab (UCB); PF- RA; IBD; lupus nephritis. 04236921 (Pfizer); C326 (Avidia). L-12/23 (p40) ustekinumab (Stelara; Janssen); briakimumab BD, psoriasis; psoriatic arthritis; (Abbott). ankylosing spondylitis; MS. L-13 QAX576 (Novartis); tralokinumab BD; (eosinophilic allergic) (MedImmune); anrukinZumab (Pfizer); asthma; allergic rhinitis. lebrikizumab (Genentech): IMA-026 (Pfizer). L-17A secukinumab (Novartis); ixekizumab (Eli Lilly). RA; IBS; psoriasis; psoriatic arthritis; ankylosing spondylitis; uveitis; MS; (eosinophilic allergic) asthma. L-18 GSK1070806 (GSK). BD; type II diabetes. L-20 NN8226 (Novo Nordisk). RA; psoriasis. L-22 fezakinumab (Pfizer). RA; psoriasis. L-23 (p19) MK-3222 (Merck); CNTO-1959 (Centocor): Psoriasis: IBD. AMG-139 (Amgen). L-1 receptor GSK1827771 (GSK); AMG-108 (Amgen). RA L-2 receptor daclizumab (Zenapax: Roche); basiliximab BD; psoriasis; JIA: uveitis; MS; (Simulect; Novartis). (eosinophilic allergic) asthma. L-4 receptor AMG-317 (Amgen). (Eosinophilic allergic) asthma. L-5 receptor benralizumab (MedImmune). (Eosinophilic allergic) asthma. L-6 receptor tocilizumab (Actemra: Roche); ALX-0061 RA; ankylosing spondylitis; JLA. (Ablynx); sarilumab (Regeneron). L-17 receptor brodalumab (Amgen). RA; IBD; psoriasis; (eosinophilic allergic) asthma, psoriatic arthritis.

In embodiments, a secretory IgA antibody according to 982, for mepolizumab see CAS Registry No. 196078-29-2 the invention comprises CDR sequences that are identical to (entered STN 24 Oct. 1997), for sirukumab see CAS Reg the CDR sequences of an antibody selected from adalim 45 istry No. 1194585-53-9 (entered STN 1 Dec. 2009), for umab, infliximab, golimumab, certolizumab pegol, oZorali olokizumab see CAS Registry No. 1007223-17-7 (entered Zumab, sifalimumab, canakinumab, gevokizumab, pascoli STN 10 Mar. 2008), forustekinumab see CAS Registry No. Zumab, reslizumab, mepolizumab, sirukumab, olokiZumab, 815610-63-0 (entered STN 18 Jan. 2005) and EP1309692, ustekinumab, briakinumab, tralokinumab, anrukinZumab, for briakinumab see CAS Registry No. 339308-60-0 (en lebrikizumab, secukinumab, ixekizumab, and fezakinumab. 50 tered STN 4 Jun. 2001), for tralokinumab see CAS Registry The CDR sequences for these antibodies are publicly No. 1044515-88-9 (entered STN 29 Aug. 2008) and available. For example, for adalimumab see WO8907452, WO2005007699, for anrukinzumab see CAS Registry No. for infliximab see CAS Registry No. 170277-31-3 (entered 910649-32-0 (entered STN 18 Oct. 2006), for lebrikizumab STN 17 Nov. 1995), forgolimumab see CAS Registry No. see CAS Registry No. 953400-68-5 (entered STN14 Nov. 476181-74-5 (entered STN 13 Dec. 2002) and 55 2007), for secukinumab see EP1776142, for ixekizumab see WO2007028106, for certolizumab pegol see CAS Registry CAS Registry No. 1143503-69-8 (entered STN 7 May No. 428863-50-7 (entered STN 12 Jun. 2002), for ozorali 2009), and for fezakinumab see CAS Registry No. 1007106 Zumab see CAS Registry No. 1167985-17-2 (entered STN 86-6 (entered STN 7 Mar. 2008). Of course, these are not 24 Jul. 2009), for sifalimumab see CAS Registry No. necessarily the only source for the CDR sequences, e.g., 1006877-43-3 (entered STN 6 Mar. 2008) and 60 some CDR sequences can be obtained from other patent and WO2012162367, for canakinumab see CAS Registry No. non-patent literature, and commercially-available antibodies 914613-48-2 (entered STN 4 Dec. 2006) and EP1313769, can be purchased and sequenced. for gevokizumab see CAS Registry No. 1129435-60-4 (en For example, a secretory IgA antibody according to the tered STN 30 Mar. 2009) and WO2009086003, for pascoli invention can comprise CDR sequences that are identical to Zumab see CAS Registry No. 331243-22-2 (entered STN 13 65 the CDR sequences of an antibody selected from golim Apr. 2001), for reslizumab see CAS Registry No. 241473 umab, certolizumab pegol, oZoralizumab, sifalimumab, 69-8 (entered STN 25 Sep. 1999) and U.S. Pat. No. 6,451, canakinumab, gevokizumab, pascolizumab, reslizumab, US 9,573.996 B2 17 18 mepolizumab, sirukumab, olokizumab, briakinumab, tral antibody is fully-human, containing only human constant okinumab, anrukinzumab, lebrikizumab, secukinumab, and variable regions, i.e., having only human heavy and ixekizumab, and fezakinumab. As another example, a secre light chains (derivable from human genomic sequences by tory IgA antibody according to the invention can comprise naturally-occurring recombination and mutation processes, CDR sequences that are identical to the CDR sequences of 5 consensus sequences, etc.). Likewise, a non-human antibody an antibody selected from oZoralizumab, sifalimumab, contains only non-human constant and variable regions, i.e., canakinumab, gevokizumab, pascolizumab, reslizumab, having only non-human heavy and light chains. mepolizumab, sirukumab, olokizumab, tralokinumab, The secretory IgA antibodies of the invention may contain anrukinZumab, lebrikizumab, secukinumab, ixekizumab, additional atoms, moieties, molecules, and/or modifications and fezakinumab. 10 beyond the dimeric IgA, J-chain, and SC-chain. For In embodiments a secretory IgA antibody according to the example, the secretory IgA antibodies of the invention may invention comprises CDR sequences that are identical to the be PEGylated or glycosylated (or aglycosylated) in various CDR sequences of an antibody selected from daclizumab, orientations and/or amounts. The location, attachment, basiliximab, benralizumab, tocilizumab, sarilumab, amount, and structure of attached glycans found in naturally brodalumab, and omalizumab. The CDR sequences for these 15 occurring antibodies shows Substantial variability and antibodies are publicly available. For example, for dacli mainly depends on the source of the glycoprotein (i.e., the Zumab see CAS Registry No. 152923-56-3 (entered STN 10 type of cell producing the glycoprotein), but is also influ Feb. 1994) and EP932415, for basiliximab see CAS Registry enced by growing conditions (i.e., feed and environmental No. 179045-86-4 (entered STN 1 Aug. 1996) and conditions). The secretory IgA antibodies of the present EP0449769, for benralizumab see CAS Registry No. invention are not limited to any specific form of glycosy 104.451 1-01-4 (entered STN 29 Aug. 2008), for tocilizumab lation and specifically include non-glycosylated proteins, see CAS Registry No. 375823-41-9 (entered STN 17 Dec. partially or fully deglycosylated proteins, variants obtained 2001) and EP0783893, for sarilumab see CAS Registry No. by genetic or other manipulation of the glycosylation system 1189541-98-7 (entered STN 22 Oct. 2009), for brodalumab of the producing cell, and variants with chemically or see CAS Registry No. 1174395-19-7 (entered STN 17 Aug. 25 enzymatically modified glycans. The Secretory IgA antibod 2009), and for omalizumab see CAS Registry No. 242138 ies of the invention can be glycoproteins with glycosylation 07-4 (entered STN 28 Sep. 1999) and EP0602126. Of patterns native to plant, mammalian (human), or insect cells. course, these are not necessarily the only source for the CDR Additionally the antibodies of the invention may be conju sequences, e.g., Some CDR sequences can be obtained from gated with (fluorescent) markers or therapeutic agents, etc. other patent and non-patent literature, and commercially 30 (see, e.g., Lash, A. "Making the case for antibody-drug available antibodies can be purchased and sequenced. conjugates.” In Vivo: The Business & Medicine Report; vol. In still other embodiments, antigen binding regions of 28, No. 11, pp. 32-39 (December 2010) Elsevier Business secretory IgA antibodies of the invention are obtained from Intelligence). other and/or novel anti-proinflammatory cytokine (or anti It is recognized that antibodies having more than one proinflammatory cytokine receptor) antibodies. Methods for 35 glycosylation site can have the same glycan species attached obtaining antibodies against specific antigens are well to each glycosylation site, or can have different glycan known in the art and can be used to obtain suitable inflam species attached to different glycosylation sites. In this matory cytokine-binding variable regions. See for example manner, different patterns of glycan attachment yield differ Mary A. Ritter and Heather M. Ladyman (Eds.) “Monoclo ent glycoforms of a glycoprotein. Monomeric IgA1 anti nal antibodies: production, engineering, and clinical appli 40 bodies have two conserved N-glycosylation sites (per cation’, Cambridge University Press (1995), ISBN chain): one on the CH2 region and one on the tailpiece. 0521425034, Zhiqiang An “Therapeutic Monoclonal Anti Monomeric IgA2 antibodies have an additional two or three bodies. From Bench to Clinic” Wiley, New York (2009), N-glycosylation sites (per chain). Furthermore, the J-chain ISBN: 04701 17915, and Christopher Dean and Philip Shep of dimeric IgA has one conserved N-glycosylation site, and herd (Ed.) "Monoclonal Antibodies: A Practical Approach” 45 the secretory component of secretory IgA has 7 conserved Oxford University Press, USA (2000), ISBN 0199637229. N-glycosylation sites. Secretory IgA antibodies of the invention can be non The terms “N-glycan(s) and “N-linked glycan(s) are human antibodies, chimeric antibodies, humanized antibod used interchangeably and refer to an N-linked oligosaccha ies, human antibodies, or other mixes of human and non ride, e.g., one that is or was attached by an N-acetylglu human sequences/regions. See, e.g., Yamashita et al. 50 cosamine (GlcNAc) residue linked to the amide nitrogen of Cytotechnology 55: 55-60 (2007). A chimeric antibody is an an asparagine residue in a protein. The predominant Sugars antibody having an antigen binding region (CDRS and found on glycoproteins are glucose (Glu), galactose (Gal), framework) originating from a first species (typically a mannose (Man), fucose (Fuc), N-acetylgalactosamine (Gal mouse) and heavy chain constant regions originating from a NAc), N-acetylglucosamine (GlcNAc), and sialic acid (e.g., second species (typically a human). A humanized antibody 55 N-acetyl-neuraminic acid (NeuAc)). The processing of the is a human antibody onto which non-human (typically Sugar groups occurs co-translationally in the lumen of the murine) CDRs have been grafted. In the humanized anti ER and continues in the Golgi apparatus for N-linked body, certain human Supporting framework amino acid glycoproteins. residues can be replaced with their counterparts from the For the purposes of the present invention, the term "G2 non-human parent antibody. Such an antibody containing 60 glycan.” “G2 glycan species,” and "G2 glycan structure are certain non-human framework residues is still a humanized used interchangeably and refer to an N-linked glycan having antibody. See, e.g., WO92/22653. In the humanized anti the GlcNAc2Man3GlcNAc2Gal2 structure, in which two bodies, the sequence of the Supporting framework into terminal galactose (Gal) Sugar residues are present. For the which the non-human CDRs are grafted can be obtained purposes of the present invention, the term “G1 glycan.” from any human isotype/class, preferentially from IgG or 65 “G1 glycan species,” and “G1 glycan structure are used IgA, and may be modified to improve the properties thereof interchangeably and refer to an N-linked glycan having the (e.g., antigen binding and/or downstream effects). A human GlcNAc2Man3GlcNAc2Gal structure, in which only one US 9,573.996 B2 19 20 terminal galactose (Gal) Sugar residue is present. For the Current Analytical Chemistry, Vol. 1, No. 1 (2005), pp. purposes of the present invention, the term "G0 glycan.” 28-57. For more information on glycosylation of therapeu “G0 glycan species,” and “G0 glycan structure are used tic antibodies, see, e.g., Fernandes et al., Eur: Biopharm. interchangeably and refer to an N-linked glycan having the Rev. Summer 2005, pp. 106-110; Jefferis, Nature Reviews/ GlcNAc2Man3GlcNAc2 structure, in which no terminal Drug Discovery, vol. 8, March 2009, pp. 226-234. galactose (Gal) Sugar residues are present. The SIgA antibodies of the present invention are prefer For the purposes of the present invention, the term “high ably monoclonal antibodies. A "monoclonal antibody” refers mannose glycan.” high-mannose glycan species,” and to a population or collection of antibodies that are substan “high-mannose glycan structure' are used interchangeably tially identical because they were all produced by clones of and refer to an N-linked glycan having the GlcNAc2ManX 10 a single cell. For the present invention, a monoclonal SIgA structure, wherein X is a whole number greater than three, is a SIgA containing monoclonal monomeric IgA antibodies. such as 5, 6, 7, 8, or 9. For the purposes of the present Preferably, a monoclonal SIgA contains monomeric IgA invention, the term “Man5 glycan.” Man5 glycan species.” antibodies, a J-Chain, and an SC-chain that were all pro and “Man5 glycan structure' are used interchangeably and duced by a clone of a single cell. refer to an N-linked glycan having the GlcNAc2Man5 15 The antibodies of the present invention are often isolated structure. The same is applicable for the terms Manóglycan or in an isolated form. As used herein, the terms "isolate.” (species; glycan structure), Man7 glycan (species; glycan "isolating and "isolation” refer to separating the antibody structure), Man8 glycan (species; glycan structure), Man 9 from its production environment. The extent of separation is glycan (species; glycan structure), etc. generally at least 50%, but is frequently at least 70%, 80%, In mammals, naturally-occurring N-glycans contain a 90%. 95%, 98%, 99%, 99.5%, or 99.9% (w/w). When the fucose (Fuc) residue attached to the GlcNAc2Man3 core SIgA antibody of the present invention is produced in a cell, structure by an C.1.6 linkage. In plants, naturally-occurring which is the typical process, the separation refers to sepa N-glycans contain a fucose (Fuc) residue attached to the rating the antibody from the host cells and native host cell GlcNAc2Man3 core structure by an C.1.3 linkage and further proteins. Isolation is thus related to purification. Preferably contain a xylose (Xyl) residue attached to the 25 the antibody of the present invention in isolated form has GlcNAc2Man3 core structure by a B1.2 linkage. For the removed, or been separated from, at least 90%, more typi purposes of the present invention, a G0 glycan containing cally at least 99% (w/w) of the host cell proteins of the the mammalian C.1.6-linked Fuc residue attached to the original composition. GlcNAc2Man3 core structure is referred to as a “GOF<6> Various compositions that contain the secretory IgA anti glycan.” For the purposes of the present invention, a GO 30 bodies of the present invention, whether in an isolated form glycan containing the plant C.1.3-linked Fuc residue attached or not, are also contemplated as being part of the present to the GlcNAc2Man3 core structure is referred to as a invention. For instance, compositions that contain low “GOF<3> glycan, a G0 glycan containing the plant B1.2- amounts of incomplete secretory IgA antibodies are often linked Xyl residue attached to the GlcNAc2Man3 core desirable. With respect to the amount of secretory IgA structure is referred to herein as a “GOX glycan.” and a GO 35 antibodies in the composition, the amount of dimer IgA (no glycan containing each of the plant C.1.3-linked Fuc residue SC-chain) is desirably less than 50%, more desirably less and the plant B1.2-linked Xyl residue attached to the than 25% and often less than 10%. Thus in a composition GlcNAc2Man3 core structure is referred to herein as a that contained 10 mg of secretory IgA, the amount of “GOXF<3> glycan.” In an embodiment, the invention relates non-SC-chain dimeric IgA would preferably be less than 1 to a secretory IgA antibody, or a population of secretory IgA 40 mg, i.e., less than 10%. The same is true for monomeric IgA: antibodies, in which substantially all N-glycans lack Fuc and the content of IgA monomers is desirably less than 50% the Xyl residues. amount of secretory IgA, more desirably less than 25% and The present invention also relates to a composition com often less than 10%. In some embodiments, the combined prising a plurality of Secretory IgA antibodies containing amount of dimer IgA (i.e., no SC-chain) and monomer IgA multiple N-glycans, such as two or more different N-gly 45 is less than 25% of the amount of secretory IgA in the cans. In embodiments, the plurality of secretory IgA anti composition, often less than 10%, and even less than 5%. bodies contains at least about 30% G0 glycans (preferably The above amounts apply to both isolated and non-isolated G0 glycans lacking Fuc and Xyl residues) relative to the forms of Secretory IgA compositions. Accordingly, the low total amount of N-glycans in the population. In embodi relative amounts of incomplete secretory IgA can be a result ments, the plurality of secretory IgA antibodies contains at 50 of the expression system (native low-production of incom least about 25% high-mannose glycans (e.g., Man5. Manó. plete secretory IgA), the result of some separation or puri Man7. Man8, and/or Man9 glycans) relative to the total fication that removes incomplete secretory IgA antibodies, amount of N-glycans in the population. In embodiments, G0 or both. glycans (preferably G0 glycans lacking Fuc and Xyl resi Purified secretory IgA compositions are also useful. A dues) and high-mannose glycans (e.g., Man5. Manó, Man7. 55 purified secretory IgA (composition) contains a secretory Man8, and/or Man9 glycans) together are the majority of IgA antibody of the present invention in an amount of at glycans present in the plurality of Secretory IgA antibodies, least 85%, often at least 90%, more often at least 95%, and such as at least 70% relative to the total amount of N-glycans preferably at least 97%, 98%, or 99%, based on the total in the plurality of secretory IgA antibodies. soluble protein content. The purified composition can be a The glycoform of an antibody, and the nature of glycan 60 Solid, Such as a lyophilized product, or a liquid. A typical species, can be determined by measuring the glycosylation liquid form contains no Solids, e.g., no insoluble cell wall profile thereof. The term “glycosylation profile' means the materials, and is often based on water as the main or sole characteristic fingerprint of the representative N-glycan spe Solvent and optionally containing salts, pH adjusting agents, cies that have been released from an antibody, either enzy or buffers. A purified liquid composition generally contains matically or chemically, and then analyzed for their carbo 65 the secretory IgA antibody of the invention in a concentra hydrate structure, for example, using LC-HPLC, or MALDI tion of 50 g/ml or more, often at least 100 g/ml, preferably TOF MS, and the like. See, for example, the review in at least 1 mg/ml. US 9,573.996 B2 21 22 Production of Proteins of the Present Invention applying the desired selective pressure results in the isola The SIgA antibodies of the invention can be produced tion of transgenic duckweed lines carrying the exogenous using recombinant techniques. Although several expression DNA of interest. systems are known, including CHO, HEK, yeast, tobacco, Construct for expression of SIgAs, to be used for trans etc., the use of duckweed as the host cell has been found to formation of duckweed, can be produced by using standard be advantageous for the production of SIgAs. Other plant techniques for example, the techniques described in Sam host cells, namely tobacco and lettuce, tend to give very low brook & Russell, Molecular Cloning: A Laboratory Manual, expression rates of the desired SIgA and typically render 3rd Edition, Cold Spring Harbor Laboratory, NY (2001) and impractical a measurable recovery of the antibody. Simi Ausubel et al. Current Protocols in Molecular Biology larly, CHO cells also tend to give low results. HEK generally 10 (Greene Publishing Associates and Wiley Interscience, NY have higher titers than CHO cells, but have certain produc (1989)). Vectors for transformation of duckweed have been tion and regulatory disadvantages. Accordingly, duckweed described elsewhere, such as in U.S. Pat. Nos. 6,040,498 and is, Surprisingly, a convenient host cell for expressing secre 7,161,064 to Stomp et al. Preferably, an A. tumefaciens tory IgAs of the present invention. binary vector (generated, for example, by standard cloning Generally, a genetically modified duckweed is a known 15 in E. coli) is used to first transform A. tumefaciens; the expression system for producing various proteins (see U.S. transgenic line obtained can then be employed to transform Pat. No. 6,040.498), including for the production of mono duckweed. Preferably, such vectors contain multiple resis clonal antibodies (see U.S. Pat. No. 7,632,983). Duckweed tance genes, to allow for selection in bacteria and in duck is the common name for the members of the monocotyle weed. Genes for bacterial selection are known in the art. donous family Lemnaceae. The five known genera and 38 Suitable resistance genes for selection in plants have been species of Lemnaceae are all Small, free-floating, fresh described in U.S. Pat. Nos. 6,040,498 and 7,161,064 to water plants whose geographical range spans the entire Stomp et al., and include gentamycin and kanamycin. globe: genus Lemma (L. aequinoctialis, L. disperma, L. For expression of SIgAS, multiple transformations can be ecuadoriensis, L. gibba, L. japonica, L. minor, L. minuscula, performed with separate vectors including different cassette L. Obscura, L. perpusilla, L. tenera, L. trisulca, L. turi 25 coding for the J-chain, the SC-chain, the antibody H chain Onifera, L. valdiviana); genus Spiro dela (S. intermedia, S. and L chain. In a preferred embodiment, a single vector is polyrrhiza); genus Wolffia (Wa. angusta, Wa. arrhiza, Wa. used for transformation that contains 4 cassettes each encod australina, Wa. borealis, Wa. brasiliensis, Wa. Columbiana, ing for one of the structural subunit of the SiGA (namely, H Wa. elongata, Wa. globosa, Wa. microscopica, Wa. neglecta) chain, L chain, SC-chain and J-chain). Construction of genus Wolfiella (WI. caudata, WI. denticulata, WI. gladiata, 30 vectors containing multiple expression cassette for antibody WI. hyalina, WI. lingulata, WI. repunda, WI. rotunda, and expression have been described in U.S. Pat. No. 7,632,983 WI. neotropica), and genus Landoltia (L. punctata). For to Dickey et al. clarity, the term "duckweed’ as used in the present invention In one embodiment, expression of the cassettes is driven includes the foregoing species, genetically modified variants by individual promoters. Examples of suitable promoter can thereof (e.g., modified to control glycosylation, secretion, 35 be found in U.S. Pat. No. 4,771,002 to Stanton, U.S. Pat. No. etc.), and any other genera or species of Lemnaceae, if they 5,428,147 to Barker et al., U.S. Pat. No. 7,622,573 & U.S. exist, optionally in a genetically modified form. Typically Pat. No. 8,034,916 to Dickey et al., disclosures of which are the genus Lenna is preferred, especially the species L. minor incorporated herein by references. Most preferably, four and L. gibba in natural or genetically modified forms. Also, different promoters are used for each expression cassette the use of the term "duckweed,” or any genus or species 40 (such as the chimeric A. tumefaciens octopine and man thereof, is meant to include individual plant cell(s), nodules, nopine synthase promoter, the L. minor polyubiquitin pro as well as whole plants including mature plants having root moter (Lmubq), Lenna aequinoctialis polyubiquitin pro and fronds, unless otherwise indicated by context or express moter (Lal Jbq) and Spirodella polyrrhiza polyubiquitin Statement. promoter (SpUbq). In a preferred embodiment, the expres Recombinant production of SIgAS in duckweed requires 45 sion vector includes cassettes coding for all 4 of the SIgA transformation of duckweed, either transiently or stably. For components, i.e. J-chain, SC-chain, H-chain and L-chain. In production purposes, a stable transformation, wherein the even more preferred embodiment, each of the 4 cassettes is nucleic acid sequences and/or genes needed to produce the driven by a different promoter. In a different embodiment, desired SIgA have been operably introduced into the the constructs are driven by, heat shock gene promoters, genome of a duckweed, is typically preferred. Stable trans 50 cold-induced promoters, drought-inducible gene promoters, genesis in duckweed can be obtained by different techniques pathogen-inducible gene promoters, wound-inducible gene as described in U.S. Pat. Nos. 6,040,498 and 7,161,064 to promoters, and light/dark-inducible gene promoters, pro Stomp et al. Briefly stable duckweed transgenesis can be moters from genes induced by abscissic acid, auxins, cyto achieved by DNA-coated particle bombardment, electropo kinins, and gibberellic acid, as described in U.S. Pat. No. ration, and Agrobacterium spp.-mediated transformation. 55 7,632,983 to Dickey et al. Preferably, transgenesis of duckweed is performed by using In an advantageous embodiment, the vectors used for A. tumefaciens-mediated transformation. Briefly, Agrobac expression include, 5' of the coding sequence of the expres terium-mediated transformation is carried out by dediffer sion cassette, a signal peptide sequence placed in frame with entiating fully grown duckweed plants or tissues, preferably the N-terminal portion of the protein of interest. Such signal tissues of meristematic origin, into calli. Callus induction is 60 peptide sequence interacts with a receptor protein on the carried out by growing duckweed in medium containing membrane of the endoplasmic reticulum (ER) to direct the plant growth regulators and Supplements. Calli can/will translocation of the growing polypeptide chain across the re-differentiate into organized nodules. Both nodules or calli membrane and into the endoplasmic reticulum for secretion can be infected with Agrobacterium, according to the pro from the cell. Presence of the signal peptide sequence cedure described in U.S. Pat. Nos. 6,040,498 and 7,161,064 65 ensures efficient secretion into the extracellular space. This to Stomp et al. Regeneration of plants from infected calli/ signal peptide is generally cleaved from the precursor poly nodules and concomitant selection for transformants by peptide to produce a mature polypeptide lacking the signal US 9,573.996 B2 23 24 peptide. Suitable signal peptide include the Arabidopsis transcription, translation, and assembly of the antibody by thaliana chitinase signal peptide, the Oryza sativa C.-amy the duckweed. Generally this entails providing an environ lase signal peptide, or any other Suitable duckweed signal ment to keep the duckweed alive and/or to promote growth; peptide sequences, as described in U.S. Pat. No. 7,632.983 e.g., providing light (natural and/or artificial) and a liquid to Dickey et al. In a most preferred embodiment, the medium typically based on water. Providing this environ sequence of the signal peptide used in the O. sativa C.-amy ment is often referred to as “culturing the duckweed. lase signal peptide. In some embodiments of the present Methods of culturing duckweed including the media, invention, the secreted SIgAS are retained within the apo Supplements (if any), conditions, etc., are known in the art plast, the region between the plasma membrane and the cell and have been disclosed in, e.g., U.S. Pat. Nos. 6,040.498: wall. In other embodiments, the polypeptide diffuses across 10 7,161,064; and 7,632983; and references cited therein, the cell wall of the plant host cell into the external environ respectively. ment/media. Culturing of transgenic duckweed of the invention can be Other Suitable nucleotide sequences including enhancers, performed in transparent vials, flask, culture bags, or any 5' leader sequences, such as the leader sequence of L. gibba other container capable of Supporting growth using defined ribulose-bis-phosphate carboxylase small subunit 5B gene, 15 media. In some embodiments of the invention large scale 3' UTR sequences, introns, enhancers, “ACC” and “ACA growth of duckweed, necessary to achieve industrial pro trinucleotides to be introduced directly upstream of the duction levels, is carried out in bioreactor tailor-designed for translation initiation codon of the nucleotide sequence of growth of duckweed. In a preferred embodiment, duckweed interest can be used to improve expression as disclosed in bioreactors, which can be inoculated aseptically, Support the art and in U.S. Pat. Nos. 6,040,498 and 7,161,064 to aseptic growth of duckweed. In even more preferred Stomp et all as well as U.S. Pat. No. 7,622,573 & U.S. Pat. embodiments, a bioreactor can be directly connected to Nos. 8,034,916 7,632,983 to Dickey et al, disclosures of harvest bag to separate the media from the plant material, which are all incorporated by reference herein. either of which can then be piped into downstream purifi For example, the expression from the transgenic lines cation steps. Suitable bioreactors, methods/devices to inocu obtained can be improved by optimizing the codon distri 25 late them aseptically, and aseptic harvest bags are described bution of the encoded proteins for expression in duckweed. in U.S. Pat. No. 7,176,024 to Branson et al. or in US Duckweed-preferred codons, as used herein, refers to application 2010/209966 To Everett et al. codons that have a frequency of codon usage in duckweed Following expression of the fully formed SIgA antibody, of greater than 17%. Likewise the codons can be optimized recovery of the antibody from the duckweed and/or the for expression in L. minor or L. gibba. In each case the 30 culture media is often desired. The first step, generally, is to codons have a frequency of codon usage of greater than separate the SIgA antibody from the duckweed. If the 17%. Duckweed and Lenna ssp. codon optimization is antibody is secreted and diffuses into the culture media, then known in the art and is carried out, e.g. as described in U.S. a simple filter can separate the crude antibody product from Pat. No. 7,632,983 to Dickey et al. the duckweed. Typically, however, the fully formed SIgA Another option is to modify the glycosylation profile of 35 antibody is retained within the duckweed’s apoplast. Sepa the duckweed. The stably transformed duckweed can also ration in this case generally requires extraction. contain a genetic modification that alters the glycan profiles. Extraction of secreted SIgAS typically involves a homog For example, the N-glycans of the SIgA can be expressed enization step to disrupt the plant material and allow for with reduced levels of fucose and xylose residue, preferably release of the secreted SIgA from the apoplast into the less than 10%, more preferably less than 1%. This modifi 40 homogenization buffer; also referred to as extraction buffer cation from natural glycan profile can be achieved by several or extraction media. Homogenization buffers and techniques techniques, including knocking out endogenous C.1.3-fuco are known in the art. Small scale homogenization can be syltransferase (FucT) and B1.2-xylosyltransferase (XylT), or performed manually, Such as by using mortar-and-pestle otherwise inhibiting their transcription of the gene/expres crushing, and the like. Larger scale homogenization is sion or enzymatic activity. In a preferred embodiment, the 45 preferably performed using a mechanical mixer, typically a duckweed is transformed with at least one recombinant high shear mixer such as a Silverson 275 UHS mixer, or nucleotide construct that provides for the inhibition of similar apparatuses. The buffer is typically an aqueous expression of C. 1,3-fucosyltransferase (FucT) and B1.2-xy phosphate buffer composition though Such is not required. losyltransferase (XylT) in a plant. In a more preferred The buffer may contain additional functional ingredients as embodiment, these constructs triggers RNA interference 50 is known in the art. For example, to reduce proteolysis by targeting the mRNAs of C.1.3-fucosyltransferase (FucT) and metallated proteases, EDTA may be added to the extraction B1.2-xylosyltransferase (XylT). In an even more preferred buffer, typically in amounts from 1 to 20 mM, including 5 to embodiment, the construct is a RNA hairpin construct. 10 mM. Also, one or more anti-oxidants, such as ascorbic These methods for altering the N-glycosylation pattern of acid, Sodium metabisulfite, benzyl alcohol, benzoic acid, and proteins in duckweed are known in the art and are described 55 the like, may be added during the homogenization process. in U.S. Pat. No. 7,884.264 to Dickey et al. The use of the Homogenization is generally followed by centrifugation and RNA hairpin construct can be advantageous for obtaining a filtration to obtain a buffer solution that contains the SIgA glycan profile where at least 30% of the N-glycans are GO antibodies and other soluble proteins. glycans lacking Fuc and Xyl residues and/or where the To remove some of the unwanted soluble proteins, combination of G0 glycans lacking Fuc and Xyl plus high 60 homogenization is often followed by clarification; a step that mannose glycans are at least 70% relative to the total amount seeks to remove certain naturally abundant impurities of N-glycans in the plurality of Secretory IgA antibodies. including (host cell proteins). Such as RuBiSco, as well as Once the transformed duckweed is obtained, the genetic non-proteinaceous impurities, such as tannins. This is usu modification will cause the duckweed to express the desired ally achieved by acidic precipitation. For example, clarifi SIgA antibody during its otherwise normal metabolic activ 65 cation can be performed by adjusting the pH of the filtrated ity. The term “express' and its grammatical variants refers to homogenate to 4.5, followed by centrifugation (such as for the biosynthesis of the SIgA antibody, which includes the 30 min at 12000), neutralization to pH 7.4, and an additional US 9,573.996 B2 25 26 filtration step. In a preferred embodiment, pH adjustments (e.g., AEX and Subsequently CEX). In some embodiments are performed using 1 M citric acid pH 1.5, or 1M sodium an AEX column such as DOWEX 1X2 is employed, often acetate for acidification and 2M tris-base for neutralization, before the AC column. though other Suitable pH adjusting agents can also be used Further polishing/purification steps can be added, as is instead of or in addition to such agents. Filtration is per known in the art. For example, after any and/or each formed as known in the art, often by using a 0.22 Lum filter. purification step (chromatography step) an ultrafiltration The recovery of the SIgA antibodies from duckweed may (UF) step can be performed. Typically, a UF step is per end with the extraction buffer or the clarified material. formed at or near the end of the polishing phase in order to However, for some uses, purification of the antibody is increase purity and/or change the buffer or concentration of desired. Purification can be performed using known methods 10 antibody in the buffer. and techniques and generally comprises subjecting the clari The SIgA antibodies are often sufficiently recovered so as fied material to affinity chromatography (AC), size exclu to be "isolated' or in an isolated form. Isolation is thus sion chromatography (SEC), and optional polishing steps. related to purification and is generally achieved by comple For efficiency, AC usually precedes SEC, though such is not tion of the recovery/extraction step, clarification, and/or required. 15 capture steps described above. Methods of using affinity chromatography (AC) as a Pharmaceutical Compositions purification step to remove contaminant proteins and impu The SIgA antibodies of the invention can be used in rities are known in the art and are described in Process Scale various pharmaceutical compositions. Typically the pharma Purification of Antibodies (2009), Edited by U. Gottschalk, ceutical composition comprises the antibody and at least one J. Wiley and son, Hoboken, N.J., and references cited pharmaceutically acceptable excipient. The pharmaceutical therein. Usually the SIgA antibody is bound to the affinity compositions can be solid, semi-solid, or liquid. Generally resin material while one or more impurities are not bound. the pharmaceutical composition is adapted for a particular The conditions are modified and the previously bound SIgA route of administration. For example, the pharmaceutical antibody is eluted from the column. The opposite can also be composition can be adapted for oral administration, rectal performed with the desired antibody passing though and the 25 administration, buccal administration, topical administra impurity or impurities being bound to the column. The light tion, etc. Preferably, the pharmaceutical composition is chain constant region can be the affinity target. Useful adapted for oral administration. affinity columns include KappaSelect and Capto L from GE Pharmaceutical compositions for administering SIgA anti Healthcare Life Sciences (Piscataway, N.J., USA). When bodies via topical administration include powders, creams, KappaSelect is used, the addition of MgCl2 is often advan 30 ointments, gels, lotions, Solutions and Suspensions (includ tageous. The use of Protein A as an AC column is usually ing mouth washes). The excipient carrier is often aqueous, avoided. Another kind of AC step is known as IMAC oil, or polymer based, each optionally in the form of an (immobilized copper affinity chromatography). IMAC can emulsion or microemulsion. The term “topical administra be used as the sole AC step or in combination with more tion' includes, for example, optical administration (e.g., via traditional AC steps. When used, IMAC is often carried out 35 a cream/ointment) and administration to the skin (e.g., at an first. If the crude antibody composition, such as the clarified inflamed joint). material, contains EDTA, then it is advantageous to add Pharmaceutical compositions for administering the anti CuSO4 to the column in order to remove EDTA, which body via oral administration include Solid oral dosage forms interferes which the IMAC purification process. Often Such as tablets, capsules, enteric coated forms thereof, IMAC is used for small to medium scale purification of 40 lozenges, and films, as well as liquid dosage forms including SIgA where the amounts are less than 10 g, typically less Solutions, Suspensions, liquid filled capsules, and mouth than 5 grams. washes. Tablets can be soluble tablets, dispersible tablets, Methods of using SEC for purification of monoclonal effervescent tablets, chewable tablets, lyophilized tablets, antibodies are known in the art. In general, SEC allows the coated tablets (e.g., Sugar-coated or enteric-coated), and separation of fully assembled SIgAs of interest from lower 45 modified release tablets. Capsules include hard gelatin cap molecular forms (such a monomer of IgA, J-chain and Sules that can be filled with powder, pellets, granules, Small SC-chain, or combinations thereof). Furthermore, SEC also tablets, or mini-tablets, or Solutions or emulsions or com permits a buffer change. Such as, for example, the reformu binations and can be coated for enteric or modified release. lation of the SIgA of interest into a new desired buffer. Soft capsules are also contemplated and are more typically Suitable columns include, for example, a Sephacryl S300 50 filled with liquids, gels or dispersions, but are not limited HR column. thereto. Granules can be effervescent granules, coated gran Other purification steps can be employed as well. For ules (e.g., Sugar-coated or enteric-coated), and modified example, ion exchange chromatography (IEX) can be useful release granules. Although the SIgA antibody of the present for removing colored impurities associated with the plant invention is preferably administered orally, it should be material. Methods and techniques for performing IEX chro 55 understood that Such administration may be considered to be matographic purification of antibodies are known in the art a topical administration to the GI tract. Likewise, a Supposi and are described, e.g., in Graf et al. (1994) “Ion exchange tory or rectal injection may also be used to topically trat the resins for the purification of monoclonal antibodies from intestines. The use of an oral dosage form to treat gastro animal cell culture' BioSeparation, Vol. 4, no. 1 pages 7-20, intestinal disease(s) using the SIgA of the present invention or in “Process scale purification of antibodies (2009) Edited 60 is a specific aspect of the present invention. by U. Gottschalk, ed. J. Wiley and son, Hoboken, N.J., and Pharmaceutical compositions for administering the SIgA references cited therein. Often IEX, such as anion exchange antibody via parenteral administration are typically liquid. chromatography (AEX) or cation exchange chromatography Water is commonly used as a main excipient, although other (CEX), is performed before IMAC or other AC step is pharmaceutically-acceptable liquids Such as ethanol, glyc employed, but is not limited thereto and can be employed at 65 erol, ethyl oleate, Myglyol, benzyol oleate, castor oil, MCT, other points of the purification and/or can be employed benzyl alcohol isopropyl myristate can be used alone or in multiple times with the same or different exchange resin combination with water or each other. Aqueous composi US 9,573.996 B2 27 28 tions that contain no other excipients are also contemplated, as chickens, amphibians, reptiles) and mammals, such as and can be prepared from lyophilized, amorphous, or crys non-human primates, sheep, dog, cow, horse, pig, etc. In a talline compounds. Often the injectable composition, which preferred instance, the patient is human. can be for Subcutaneous, IM, or IV injection, contains The SIgA antibodies of the present invention are generally isotonizing agents. An injectable solution or Suspension is 5 useful in treating inflammatory diseases in humans. Specific typically sterile, as are all liquid pharmaceutical dosage targets include rheumatoid arthritis, inflammatory bowel forms. disease (including Crohn's disease and ulcerative colitis), An overview of dosage forms can be found in Ansel's psoriasis, psoriatic arthritis, ankylosing spondylitis, juvenile Pharmaceutical Dosage forms and Drug Delivery Systems. idiopathic arthritis, uveitis, asthma, Alzheimer's disease, 9" ed. L. V. Allan, N. G. Popovitch, H. C. Ansel, 2010 10 Lippincott, ISBN: 978-0781779340, Formularium der Ned multiple Sclerosis, type II diabetes, systemic Sclerosis, lupus erlandse Apothekers. 2004 WINAp ISBN 90-70605-75-9, nephritis, and allergic rhinitis. The present invention will be Recepteerkunde, G. K. Bolhuis, Y. Bouwman-Boer, F. Kadir further illustrated in the following non-limiting examples. en J. Zuiderma, 2005 WINAp ISBN 90-70605-65-1; and ANTI-TNF-C. SIGA EXAMPLES Apothekenrezeptur und-defektur. Deutscher Apotheker Ver 15 lag Stuttgart 1986 ISBN 3-7692-1092-1. See also U.S. Pat. No. 7,147,854 for a description of topical preparations for Example 1 delivering IL-8 antibodies to treat skin inflammatory disease Such as psoriasis. Transient Expression of an Anti-TNF-C. Secretory The pharmaceutical composition generally contains about Protein Based on Infliximab (IIB-SA2n) in Plants 0.01 to 1000 mg of the antibody per dose, depending in part upon the dosage form employed. The dose can be, for a) Preparation of cDNA Constructs example, fixed or variable (e.g., based on body weight) The amino acid sequences of a Suitable leader sequence Pharmaceutically-acceptable excipients are known in the art (e.g., secretion signal, SEQ ID NO:23), the heavy chain and include diluents, carriers, fillers, binders, lubricants, 25 variable region of infliximab (SEQID NO:2), and the human disintegrants, glidants, colorants, pigments, taste masking Cl2(n) IgA heavy chain constant region (Chintalacharuvu et agents, Sweeteners, plasticizers, and any acceptable auxil al., Journal of Immunology, 1994, 152, 5299-5304; SEQID iary Substances such as absorption enhancers, penetration N0:13) were joined together. Cleavage of the signal enhancers, Surfactants, co-surfactants, preservatives, anti sequence (a.a. 1-19) corresponded to the predicted cleavage oxidants and specialized oils. Specific to the field of biop 30 site using the SignalP program. This program is available harmaceutical proteins are excipients intended to stabilize online via the site of the Center for Biological Sequence proteins and cryo-protectants to provide protection during Analysis at the Technical University of Denmark. freeze-drying. Suitable excipient(s) are selected based in The resulting amino acid sequence was back-translated part on the dosage form, the intended mode of administra into a cDNA sequence optimized for expression in maize (Z. tion, the intended release rate, and manufacturing reliability. 35 mays) (SEQ ID NO:27) (see Liangjiang Wang and Marilyn Non-limiting examples of commonly used excipients J. Roossinck. “Comparative analysis of expressed include polymers, waxes, calcium phosphates, Sugars (e.g., sequences reveals a conserved pattern of optimal codon trehalose. Sucrose, or mannitol), buffers (such as phosphate, usage in plants.” Plant Mol Biol (2006) 61:699-710). acetate, citrate, histidine, or glycine based buffers at pH Similarly the cDNA sequence for the light chain of the between 5 and 7.5), salts (e.g., NaCl or NaEDTA), polysor 40 construct was obtained by joining the sequences of a Suitable bate 20, polysorbate 80, human albumin, dextran, and ben secretion signal (SEQ ID NO:24), the light chain variable Zyl alcohol. region of infliximab (SEQ ID NO:3), and the human K Ig Treatments light chain constant region (SEQ ID NO:14), and back As used herein, the term “treat' or “treatment’ means the translating the obtained amino acid sequence into a cDNA application or administration of a SIgA antibody of the 45 sequence optimized for expression in maize (Zea mays) invention, alone or as part of a composition, to a patient with (SEQ ID NO:29). the purpose to cure, heal, alleviate, improve or prevent an The cDNA sequence for the human J-chain was obtained inflammatory disease. The term “inflammatory disease' by joining the amino acid sequences of secretion signal means a condition associated with Symptoms of inflamma (SEQ ID NO:22) and J-chain sequence (SEQ ID NO:20), tion, which may be caused by external factors, such as 50 both obtained from UniprotKB/Swiss-Prot database entry infectious disease, or by internal dysfunctions, such as an PO1591, and back-translating the obtained amino acid autoimmune disease. In this context, the terms disease, sequence into a cDNA sequence optimized for expression in disorder, syndrome, condition, and the like are used inter maize (Z. mays) (SEQ ID NO:33). changeably. In embodiments, the SIgA antibodies of the The cDNA sequence for the SC-chain was obtained by present invention are useful in the topical treatment of 55 joining the amino acid sequences of a suitable secretion inflammatory diseases in humans, e.g., local administration signal (SEQ ID NO:25) and SC-chain sequence (SEQ ID to the site of inflammation, Such as orally or rectally. NO:21, amino acids 19-603 of UniprotKB/Swiss-Prot data Preferably, the SIgA antibodies of the present invention are base entry P01833), and back-translating the obtained amino useful in the oral treatment of inflammatory diseases. acid sequence into a cDNA sequence optimized for expres As used herein, an amount of the SIgA of the present 60 sion in maize (Z. mays) (SEQID NO:35). The used secretion invention effective to treat an inflammatory disease, or a signal sequence was derived from the natural SC Secretion “therapeutically effective amount,” refers to an amount of signal by the addition of codons for two extra amino acids the antibody which is effective beyond that which is in order to obtain more favorable splicing sites for the expected in the absence of Such treatment. construction of plasmid vectors. As used herein, the term “patient' is intended to include 65 The cDNAs of the four constructs (the heavy chain, the humans and non-human animals. The term “non-human light chain, the J-Chain and the SC-Chain) were obtained animals' includes all vertebrates, e.g., non-mammals (such from a commercial source. US 9,573.996 B2 29 30 b) Vector Construction and Cloning Strategy SIgA comprises IgA(2)(n) heavy chain constant region, The cDNAs for the heavy chain of the construct (HC) and human K light chain constant region, and the heavy and light of the J-chain (JC) were ligated into the pGA15 and pGA14 chain variable regions of infliximab, and is referred to herein plasmid vectors, respectively, using Pacl and Ascl restriction as IIB-SA2n sites. After transfer to E. coli K12 XL 10 gold and expansion, d) Transient Expression in Lettuce the constructs were each transferred to separate pRAP The SIgA construct was transiently expressed in lettuce plasmids using Nicol and Kpnl restriction sites, resulting in by vacuum infiltration using the same vectors as for expres the expression cassettes 35S:HC:Tnos and 35S:JC:Tnos. sion in tobacco (see: Negrouk et al., Highly efficient tran The pRAP plasmids containing the expression cassettes sient expression of functional recombinant antibodies in were transferred to and expanded in E. coli K12 DH 10B. 10 lettuce. Plant Science 2005, 169, 433-438). Full grown The cDNAs for the light chain (LC) and of the SC-chain crops of lettuce Lactuca sativa L. (oak leaf lettuce) and (SC) were ligated into the pGA14 and pGA15 plasmid vacuum infiltrated with a mixture of the two A. tumefaciens vectors, respectively, using PacI and AscI restriction sites. strains and harvested 3-5 days after infiltration. Formation of After transfer to E. coli K12 XL 10 gold and expansion, the complete SIgA could be shown, but the product was not constructs were each transferred into separate pTR2 plas 15 isolated or purified. mids using Nicol and Xbal restriction sites, resulting in the expression cassettes TR1TR2':LC:T35S and TR1TR2':SC: Example 2 T35S. The pTR2 plasmids containing the expression cas settes were transferred to and expanded in E. coli K12 Expression of an Anti-TNF-C. Secretory Protein DH1 OB. Based on Infliximab (JIB-SA2n) in Mammalian The light chain expression cassette TR1 "TR2':LC:T35S Cells was transferred to the pRAP vector containing the heavy chain expression cassette 35S:HC:Tnos using HindIII 2a: Transient Expression of IIB-SA2n restriction sites, and transferred to and expanded in E. coli (1) Vector Generation: K12 DH1 OB. Finally, the combined cassettes containing HC 25 cDNAs encoding SIgA heavy chain (HC), light chain and LC (35S:HC:Tnos: TR1'TR2':LC:T35S) were trans (LC), J-chain, and SC-chain were cut from the pGA14 and ferred to a pBIN+ expression vector using AscI and Pad pGA15 plasmids described in Example 1. The excised HC restriction sites. This pEBIN+ vector containing the combined and LC sequences were PCR amplified and cloned into HC and LC cassettes was transferred to Agrobacterium pMQR-hIgG1 (Neo) to give pMQR-Zmo.2-iib (Neo) and tumefaciens strain MOG101 using electroporation. Simi 30 pMQR-Zmk-iib (Neo) vectors. Similarly, the excised J-chain larly the cassettes containing JC and SC (35S:JC:Tnos: and SC-chain were PCR amplified and cloned into pMQR TR1TR2':SC:T35S) were combined in a pBIN+ expression kappa (Hygro) to give the pMQR-Zm (Hygro) and pMQR vector. This pBIN+ vector containing the combined JC and ZmSC (Hygro) vectors. The pMQR-hIgG1 (Neo) and SC cassettes was transferred to A. tumefaciens strain pMQR-kappa (Hygro) vectors are based on the pcDNA3 MOG101 using electroporation. 35 (Invitrogen) backbone, which was modified to receive any The two transfected A. tumefaciens strains were used in combination of Ig V- and C-region “cassettes”. This was combination for transient plant transformation and expres performed by Subcloning well-defined genomic Ig gene sion of the full SIgA construct. For vector information see segments into pcDNA3, after which silent mutations were also van Engelen et al., Plant Molecular Biology 1994, 26: introduced flanking the V- and C-regions to create restric 1701-1710. Information on AlMV leader sequence: van der 40 tions sites allowing for the exchange of V- and C-gene Vossen et al, Nucleic Acids Research 1993, 21: 1361-1367. segments. Other occurrences of these specific introduced pBIN+ is described in: van Engelen et al., Transgenic restriction sites were removed from the vector backbone to Research 1995, 4: 288-290. (In the referenced literature ensure proper exchange of V and C regions on unique sources: pRAP35=p0PO31). By way of example, FIG. 6 restriction sites. In short, the BSmI site was removed by shows a schematic representation of the building of the 45 exchanging the original BSmI sequence (5'-TGCATTC-3) pBIN+ vector containing the combined HC and LC cas present in the vector into 5'-TGCAAAC-3". Furthermore the SetteS. MunI/MfeI and HindIII sites were destroyed by restriction c) Transient Expression in Tobacco Plants digestion followed by end-filling and re-ligation. The youngest fully expanded leaves of six week old Large-scale production of each of the four expression tobacco plants were infiltrated with a mixture of the two A. 50 vectors for transfection was performed using Maxiprep-kits tumefaciens strains by placing a 2-ml syringe (without (Sigma). needle) containing the bacterial cell Suspension at the lower (2) Cell Transfection and Culture: side of a leaf and gently pressing the Suspension into the leaf. Transient SIgA producing CHO-S and HEK-293F cells The infiltrated area was usually clearly visible. Expression (Invitrogen) and HEK293-EBNA1 (ATCC CRL-10852) took 4-6 days with optimum levels at days 5-6. 55 were made as follows: Invitrogen derived cell types were The leaves were frozen at -80° C., crushed and 2 ml used as host for transfection using FreeStyle Max or Amaxa extraction buffer per gram of leaf material was added. The Nucleofection. HEK293-EBNA1 was used for large scale extraction buffer was PBS pH=7.4; 0.02% Tween-20 (v/v); (101) transient polymer transfection. To obtain a complete 2% polyclar AT (v/v); and 1% inhibitor cocktail (v/v). The SIgA-producing transfectant, all 4 different vectors were Suspension was homogenized with an Ultra Turrax (Janke & 60 transferred simultaneously into each of these cell types Kunkel). Solid material was removed by centrifugation (15 (CHO-S, HEK293F, and HEK203-EBNA1). minutes, 10.000 g). The solid material was extracted twice (3) FreeStyle MAX Transfection more with extraction buffer, the same as above except the To transfect 30x10 CHO-S and HEK-293F cells (sepa inhibitor cocktail was replaced with 10 mM PMSF. The rately), 9,375 ug of each vector was used. SIgA production combined extracts were stored at -20°C. Using SDS-page 65 was performed by culturing cells in a 200 ml shake flask at and immunoblotting, formation of a complete SIgA could be 37° C., 130 rpm and 8% CO during 120 hours in respec shown, but the product was not isolated or purified. The tively CHO-S FreeStyle and 293F FreeStyle medium. The US 9,573.996 B2 31 32 cell conditions and transfection efficiency were determined mately 15 L of culture medium containing 440 mg of by GUAVA ViaCount and Expresse Pro analysis to guaran complete SIgA (according to ELISA analysis) was obtained tee the optimal cell growth conditions in between 0.5x10° to from the expanded 2D5 cells. The SIgA comprises IgA(1) 2.0x10 cells/ml. Each time the cells had to be diluted the heavy chain constant region, human K light chain constant volume was increased and if necessary transferred to a 500 region, and the heavy and light chain variable regions of ml baffled shake flask. infliximab; and is referred to herein as “IIB-SA1. IIB-SA1 CHO-S/SIgA 24 hours post transfection: Transfection can be isolated from culture medium and purified as efficiency; 25.04% and cell viability: 91.59%. described in Example 3b. HEK-293F/SIgA 24 hours post transfection: Transfection efficiency; 25.52% and cell viability: 80.36%. 10 Example 3 (4) Amaxa Transfection To transfect 1x10 Invitrogen's CHO-S or HEK-293F Purification of Anti-TNF-C. Secretory IgA cells 1 ug of each vector was used. SIgA production was Antibodies (IIB-SA2n and IIB-SA1) from performed by static culturing cells in a T25 flask at 37° C. Mammalian Cell Culture and 5% CO during 96 hours. At 96 hours post transfection 15 cell Suspensions fractions were taken for production analy 3a–Affinity and SEC (IIB-SA2n) S1S. The Supernatant obtained from the transient expression of CHO-S/SIgA 24 hours post transfection: Transfection IIB-SA2n in HEK-293-EBNA1 cells using polymer trans efficiency: 54.27% and cell viability: 94.19%. fection (experiment 2a(5)) was purified using affinity chro HEK-293F/SIgA 24 hours post transfection: Transfection matography with an anti-IgA lama antibody fragment immo efficiency: 61.57% and cell viability; 68.15%. bilized on Sepharose (CaptureSelect human IgA, BAC, (5) Polymer Transfections product code 2880) followed by SEC. Transfections of HEK293-EBNA1 cells were performed (1) Affinity Chromatography: essentially as described by Durocher at al. Nucleic Acids Anti-human IgA matrix (Capture Select) was poured into Res. 30. E9 (2002) (see also Morlot C. et al., Acta Crystal 25 a XK16/20 column (16x200 mm) in 20% ethanol in MilliO logr Sect F Struct Biol Cryst Commun. 63, 689-91 (2007)). water at a flow rate of 15 ml/min. After adjusting the position The day prior to transfection, the cells were routinely diluted of the upper flow adaptor, the column was equilibrated in 4-5 times in fresh Freestyle medium (without further addi PBS buffer pH 7.4. The HEK-cell culture medium was tion of FCS or G418) to a density of 0.3x10/ml. The next filtered to remove large cell debris and subsequently loaded day transfection mixtures were prepared. For transfection of 30 on the pre-equilibrated anti-human IgA column with a flow 1 liter of cell culture 500 ug of high quality DNA was diluted rate of 4.5 ml/min. After washing with 10 column volumes in 25 ml OptiMEM medium (Invitrogen) 1 ml of a 1 mg/ml PBS pH 7.4, the active secretory IgA antibody was isocrati stock solution of linear 25 kDa PEI (Polysciences) was cally eluted from the column with 5 column volumes of added to the mixture and immediately vortexed for 10 citrate/arginine/NaCl buffer pH 2.5, followed by immediate seconds. After 10 minutes for incubation the DNA:PEI 35 neutralization to pH 7.7 with a 1 M Tris stock solution. mixture was added to the cells. Cells or conditioned medium (2) SEC Chromatography: were routinely harvested six days post transfection. Culture In the second step, the adjusted pool from the first step was performed at 10 L scale. The anti-TNF-C. secretory IgA was concentrated 4-fold down to 5 ml using a Vivaspin 15R antibody, IIB-SA2n, in the supernatant of this culture can be device (30 kDa cut-off filter) and loaded on a prepacked isolated and purified as described in Example 3a. 40 Superdex 200 16/60 GL column equilibrated with PBS pH 2B: Stable Expression in HEK293F of IIB-SA1 7.4. The sample loading volume was 4% of the bed volume (1) Vector Construction and the flow rate was 1.5 ml/min. This size exclusion gel cDNA encoding the heavy chain variable region of inflix filtration step resulted in fractionation of SIgA and mono imab was cut from the previously obtained vector pMQR meric IgA according to their molecular weights. The elution Zmo.2-iib (Neo) (see Example 2a(1)). This cDNA was fused 45 fractions containing SIgA or monomeric IgA as determined into a pMQR vector containing the human genomic DNA by non-reducing SDS-PAGE were pooled separately and sequence encoding the IgA C1 heavy chain constant region subsequently concentrated 10-fold using a Vivaspin 15R to give vector pMQR-huC.1-iib (Neo). Similarly, the light device (30 kDa cut-off filter). chain variable region of infliximab was cut from pMQR Samples of substantially pure IIB-SA2n and the corre Zmk-iib (Neo) and transferred to a pMQR vector containing 50 sponding monomeric anti-TNF-C. IgA were collected and the human genomic DNA sequence encoding the K-light characterized. chain constant region to give vector pMQR-huk-iib (Neo). 3b Jacalin Affinity and SEC (JIB-SA1) The two vectors pMQR-huC.1-iib (Neo) and pMQR-huk-iib Culture medium obtained from HEK-293F clonal cell line (Neo) were fused into a single vector pMQR-hulgA1-iib 2D5 expressing IIB-SA1 (see Example 2b) was purified (Neo) (SEQ ID NO:28). 55 using Jacalin affinity chromatography followed by SEC. Previously obtained vectors pMQR-ZmJ (Hygro) and (1) Jacalin Affinity Chromatography pMQR-ZmSC (Hygro) were also combined into a single 500 ml culture medium was filtered through a 0.45/0.2 um vector PMQR-ZmJ/SC (Hygro) (SEQ ID NO:30). Sartopore 2 filter. A Jacalin column was prepared by packing (2) Transfection 15 ml of immobilized Jacalin resin (Pierce art nr. 20395) in HEK-293F cells were transformed with both of the two 60 an omnifit 15 mm glass column. The column was equili vectors described above using polymer transfection as brated using 5 column volumes of PBS buffer pH 7.4. described in experiment 2a(5). Cells were grown in neomy Culture medium was loaded at a flow rate of 3 ml/min. after cin and hygromycin containing medium. After recovery, washing with 5 column volumes of PBS buffer pH7.4 at 5 cells were grown on semisolid medium with neo/hygromy ml/min followed by elution of bound material with 8 column cin and clones were selected using Clonepix analysis and 65 volumes of PBS buffer pH7.4 containing 0.5M galactose. selection. A suitable clone (2D5) was selected. Clone 2D5 The fractions of the eluate containing SIgA were pooled was expanded under various conditions and scales. Ulti (detection by UV OD280 measurement). The pooled eluate US 9,573.996 B2 33 34 (64 ml) was concentrated to a Volume of 5 ml using Vivaspin chain, H-chain and L-chain. The H chain was fused to the filters. Measurement of SIgA content using ELISA showed modified chimeric octopine and mannopine synthase pro that only appr. 30% was bound to the Jacalin, the remainder moter with Lenna gibba 5' leader from ribulose bis-phos being in the flow-through fraction. phatecarboxylase small subunit-1. The L-chain, SC-chain (2) SEC Chromatography and J-chain genes were fused to high expression Lemnaceae The concentrated eluate was placed in a Superloop and Ubiquitin promoters L. minor polyubiquitin promoter loaded on a Sephacryl S300 column (GE-Healthcare art.nr. (Lmubq), Lenna aequinoctialis polyubiquitin promoter 17-1167-01) with a flow rate of 1 ml/min. The product was (Lal Jbq) and Spirodella polyrrhiza polyubiquitin promoter eluted using 1.5 column volumes of PBS buffer (50 mM (SpUbq), respectively. Sequences of these promoters have phosphate pH 7.4) containing 0.5Marginine. The chromato 10 been disclosed in PCT application WO2007/124186. These gram (UV OD280 detection) showed three overlapping expression cassettes were then cloned into a single A. peaks. Fractions were analysed by SDS-PAGE to identify tumefaciens transformation vector EC2.2 (a modification of the SIgA containing fraction. The fractions containing the the A. tumefaciens binary vector p3MSP3, which is a product were pooled. Reducing and non-reducing SDS derivative of pBINPLUS. See Ni, M., Cui, D., Einstein, J., PAGE analyses of the purified product, IIB-SA1, are shown 15 Narasimhulu, S., Vergara, C. E., and Gelvin, S. B., Plant J. in FIG. 7. 7, 661-676, (1995), van Engelen, Transgenic Res. 4:288-290 (1995), and Gasdaska et al., Bioprocessing J., 3:50-56 Example 4 (2003)), with the appropriate restriction sites to create the final transformation vector SynB02. This vector also con Stable Expression of Anti-TNF-C. Secretory IgA tained the gentamicin acetyltransferase-3-I gene (aacC1) Antibody (ADB-SA1g) in Lenna which confers resistance to gentamicin and allows for selec tion of transgenic L. minor lines. Anti-TNF-C. SIgA was produced in Lenna by stable SynB02 was used to create an additional transformation insertion of DNA coding for the following proteins: The vector to generate a glycan optimized version of anti-TNF-C. amino acid sequence of the heavy chain consisted of the 25 SIgA, having G0 glycans lacking fucose and Xylose resi Oryza sativa (rice) amylase secretion signal (SEQ ID dues. A chimeric hairpin RNA was used to silence endog NO:26) attached to the N-terminal amino acid of the heavy enous L. minor mRNAS encoding C-1,3-fucosyltransferase chain variable region of adalimumab (anti-TNF-C. IgG1. (Fuct1, GenBank DQ789.145) and B-1,2-xylosyltransferase Humira R, CAS number 331731-18-1) (SEQ ID NO:4) (Xylt1, GenBank DQ789146). A DNA sequence for this attached to the N-terminal amino acid of a human IgA1 30 chimeric RNAi molecule was fused to the high expression heavy chain constant region (SEQ ID NO:10). The amino SpUbq promoter and subsequently moved into the SynB02 acid sequence of the light chain of the Anti-TNF-C. SIgA vector creating the final transformation vector SynB03. combines the rice C.-amylase secretion signal (SEQ ID Further details on production of glycan optimized proteins in NO:26) with the TNF-C. binding light chain variable region Lemnaceae can be found in PCT applications WO2007/ of adalimumab (SEQID NO:5) and the human K-light chain 35 084672, WO2007/084922, WO2007/084926 and in Cox, K. constant region (SEQID NO:14). The amino acid sequence M., Nature Biotechnology 2006, 12: 1591-1597. of the J-chain consisted of the rice C.-amylase secretion L. minor strain 8627 was transfected with vector SynB03, signal (SEQ ID NO:26) attached to the N-terminal amino and glycosylation modified L. minor strain XF04 was trans acid of the human J-chain (SEQID NO:20). The amino acid fected with vector SynB02. Once transformed plants were sequence of the SC-chain consisted of the rice amylase 40 regenerated (approximately three months), single plants secretion signal (SEQID NO:26) attached to the N-terminal were harvested from the antibiotic selection plates and amino acid of the human SC-chain (SEQ ID NO:21). The propagated separately in liquid growth media, without selec produced SIgA, having IgA1 heavy chain constant region, tion antibiotic, for further screening and characterization. human K-light chain constant region, and adalimumab heavy Thus several hundred individual transgenic plant lines from and light chain variable regions, combined with human 45 each construct were generated. Independent transgenic lines J-chain and SC-chain is referred to as ADB-SA1. were harvested and clonally propagated in individual harvest Genes were designed for each of the four components jars. For screening of transgenic lines, clonal lines were with Lenna minor preferred codon usage (63-67% GC preconditioned for 1 week at light levels of 150 to 200 content). Tables with suitable preferred codon use in Lem umol/m’s in vented plant growth vessels containing SH naceae can be found in PCT application WO2005/035768 50 medium (Schenk R. U. et al., Can. J. Biol. 1972, 50: and in relevant references contained therein. The synthetic 199-204) without sucrose. Fifteen to twenty preconditioned genes also contained the rice O-amylase signal sequence fronds were then placed into vented containers containing (GenBank M24286: SEQ ID NO:26) fused to the 5' end of fresh SH medium, and allowed to grow for two weeks. their coding sequences. Restriction endonuclease sites were Tissue samples from each line were collected and frozen for added to allow cloning into A. tumefaciens binary vectors. 55 analysis. Design of DNA sequences and vector construction was To determine SIgA expression, frozen tissue samples performed by Biolex Therapeutics, Inc., Pittsboro, N.C., were homogenized and centrifuged, and the Supernatant was USA. DNA sequences were produced by DNA2.0 (Menlo removed and screened by an ELISA method using sheep Park, Calif., USA). anti-human IgA secretory chain (AbD Serotec catalog The ADB-SA1g antibody was expressed in L. minor by 60 #51 11-4804-1:1000 dilution) coated plates to capture the transfection via an A. tumefaciens binary vector containing SIgA antibody. The samples were then detected using a goat DNA sequences encoding all four of the SIgA components: anti-human kappa light chain HRP conjugated antibody J-chain (SEQ ID NO:34), SC-chain (SEQ ID NO:36), (Sigma catalog #A7164-1:2000 dilution). The highest-ex H-chain (SEQ ID NO:31) and L-chain (SEQID NO:32). To pressing lines from this primary Screening were then grown prepare this vector, independent expression cassettes were 65 again for two weeks in Small research vessels under the created containing a promoter and also DNA sequences optimal growth conditions. The resulting tissue was har encoding the protein and terminator for the J-chain, SC vested and the ELISA was performed to determine the US 9,573.996 B2 35 36 percent of the total soluble protein that is the expressed SIgA formed with at least 2 column volumes of PBS buffer at antibody (ADB-SA1g). The results are summarized in Table room temperature and a flow rate of 1.0 ml/min Fractions 3. were collected and sufficiently pure fractions were pooled.

TABLE 3 Example 6 Hightest Construct Glycosylation # of lines screened Expression level Binding of Anti-TNF-C. SIgA IIB-SA1 to TNF-C. SynB02 GO glycans 55 16.2% TSP Binding of IIB-SA1 (infliximab binding head) after puri SynB03 GO glycans 227 13.6% TSP 10 fication as described in Example 3b, to TNF-C. was deter mined using ELISA according to the following procedure. Results of non-reducing and reducing SDS-PAGE analy 96-Well plates were incubated overnight at 4°C. with 100 ses of purified material obtained from Lenna transfected ul/well of 0.5 g/ml TNF-C. in PBS pH7.4. After emptying with construct SynB02 are shown in FIG.8. ADB-SA1 g can the wells were incubated for 2 hours at RT with 200 ul/well be isolated from Lemma culture and purified as described in 15 of PBS pH7.4 containing 1% BSA and 0.05% Tween-20. Example 5. After washing (room temperature) with 300 ul/well of PBS/0.05% Tween-20 three times for 30 seconds, shaking, Example 5 the plates were used. The wells were incubated with samples, 1 hour at 37° C., 100 ul/well. The wells were washed (room temperature) with PBS/0.05% Tween-20 Isolation and Purification of Anti-TNF-C. Secretory three times for 30 seconds, shaking, 300 ul/well. The wells IgA Antibody ADB-SA1g from Lemna were incubated with 100 ul/well of 0.5 g/ml HRP labeled anti-human kappa light chain antibody (Sigma, A7164) for Biomass from transgenic Lenna expressing anti TNF-C. 1 h at RT, and then washed (room temperature) with PBS/ SIgA, having variable regions that are the amino acid 25 0.05% Tween-20 three times for 30 seconds, shaking, 300 sequence of the variable regions (antigenbinding regions) of ul/well. adalimumab, was homogenized in 50 mM Sodium phos For detection, the wells were incubated with TMB (Tebu phate, 0.3M Sodium chloride, buffer pH 7.4, at a buffer to Bio), 10 minutes at room temperature, 100 ul/well. The tissue ratio of 4:1. An acid precipitation step was performed reaction was stopped with 0.3 M sulphuric acid, 100 ul/well, on the crude extract to remove ribuloase bis-phosphate 30 and the OD at 450 nm was measured with well correction at carboxylase (RuBisCo) and other plant proteins by adjusting 630 nm. Measurement of IIB-SA1 from expression in mam the extract to pH 4.5 using 1M Sodium acetate, pH 2.5. The malian cells (Example 2b) showed strong TNF-C. affinity at precipitate was removed by centrifugation of the material at a level comparable to that of infliximab as shown in FIG. 9. 14,000xg for 30 minutes at 4° C. The supernatant was Note that the closed circle represents the SIgA of the adjusted to pH 7.4 and loaded on DOWEX (DoweX 1X2 35 invention (IIB-SA1), square represents Infliximab, and dia anion exchange resin) to remove colored impurities. The mond represents Colostral SIgA. Equilibrium constants of flow-through fraction containing anti TNF-C. SIgA was 0.22 8.1x10' and 2.4x10' for IIB-SA1 and for Infliximab um was filtered prior to chromatography using affinity itself (REMICADE(R), respectively, were calculated. This chromatography and Size Exclusion Chromatography. shows that the transfer of the TNF-C. binding variable part of Affinity purification: A KappaSelect (GE Healthcare prod. 40 IgG1 antibody infliximab to a secretory IgA does not sub Nr. 17-5458-03) column was prepared according to manu stantially alter affinity (taking the variation and discrimina facturer instructions (28-9448-22 AA). The column was tory power of the experimental setup into account), and that equilibrated with 5 column volumes (cv) of TBS buffer (50 the obtained product is fully functional. mM Tris, 0.15M Sodium chloride, pH 7.4). The supernatant was loaded on the column Approximately 5 mg SIgA/ml 45 Example 7 resin was loaded on columns of up to 1 L. KappaSelect. Non-binding material was washed from the column with 5 Proteolytic Stability of Anti-TNF-C. SIgAs cv TBS buffer. The product was eluted from the column (IIB-SA1 and ADB-SA1g) using 10 cv of 25 mM Sodium Acetate, pH 6.6 buffer containing 3.5 M MgCl2. The fractions containing the 50 The stability of IIB-SA1 (Example 3b), and of ADB secretory proteins were pooled and immediately diluted SA1g (Example 4, from SynB03) was determined in simu 4-fold using TBS buffer (50 mM Tris, 0.15M Sodium lated intestinal fluid (SIF, 0.05M phosphate buffer pH 6.8 chloride, pH 7.4). MgCl, was replaced by buffer exchange containing 10 mg/ml pancreatin). Stability was compared to with at least 10 volumes of TBS buffer using a Pall Cen a human colostral SIgA which was purified to contain only tramate cassette ultrafiltration system and a Pellicon 2 Mini 55 kappa light chains and C-1 heavy chains. Filter (Millipore prod.Nr. P2C005C01-PLCCC 5K, regen 150 ul of a 0.14 mg/ml solution of the material to be tested erated cellulose) with 5 kDa cut-off. (i.e., IIB-SA1, ADB-SA1g, or human colostral SIgA (kappa SEC purification: Material obtained by KappaSelect chro light chain, C-1 heavy chain) was added to 550 ul of matography was further purified on a Sephacryl 5300 HR pre-heated SIF at 37° C. 100 ul samples were drawn at T=0, column. The column was equilibrated with 2 cv of PBS 60 5, 15, 30, 60 and 120 min, added to 25 ul protease inhibitor buffer at a flow rate of 1.0 ml/min. The KappaSelect eluate cocktail (Roche, 1169749800) followed by immediate freez was concentrated 3-4x using ultra filtration using a Pellicon ing in liquid nitrogen. Samples were analyzed by non 2 Mini Filter (Millipore prod.Nr. P2C005C01-PLCCC 5K, reducing SDS-Page gel electrophoresis. Briefly; 57.5 ul of a regenerated cellulose) with 5 kDa cut-off. 0.08M solution of iodoacetamide in LDS sample buffer was Typically, a concentration of 3-5 mg/ml and feed Volume 65 added to each of the frozen samples. Samples were thawed of 50 ml was used for a 1 L column. The feed was applied and applied to Criterion Tris-HC1 gel (12.5%, 18 well, 30 ul using an AKTA purifier at 1.0 ml/min. Elution was per comb (Biorad, 345-0015). After electrophoresis gels were US 9,573.996 B2 37 38 treated with Krypton protein stain and analyzed. Stability of was replaced by 20 ng/ml TNF-C. as indicated above, and the the samples was qualitatively assessed visually. Results are polarized Caco-2 cell monolayers were sensitized for shown in FIG. 10. respectively 15 or 28 hours prior to addition of SIgAs The two forms of anti-TNF-C. SIgA, i.e., IIB-SA1 (labeled (specifically IIB-SA1 or ADB-SA1 g). After sensitization, as infliximab variable part in FIG. 10) and ADB-SA1g 5 Caco-2 cell were exposed to SIgAs for 24 hours and TER (labeled as adalimumab variable part in FIG. 10) each was monitored. showed a stability which was slightly less than that of the Results: colostral SIgA. The results are shown in FIG. 12. Black bars represent exposure to TNF-C. only, striped bars represent addition of Example 8a 10 ADB-SA1 g after indicated time of exposure to TNF-C. dotted bars represent addition of IIB-SA1 after indicated Caco-2 Cell Based Assays time of exposure to TNF-C. Statistical analysis using ANOVA followed by Dunnett's Multiple Comparison test, The aims of Caco-2 cell experiments were: (a) to evaluate ADB-SA1g and IIB-SA1 were compared to TNF-C. and had in vitro the capacity of anti-TNF-C. SIgA’s to block the 15 a p<0.001 (indicated by *** in FIG. 12). Data are means of biological activity of TNF-C. and (b) to study transport over 4 independent replicates Snapwell inserts for each condition. an epithelial cell monolayer. As expected, no effect was seen when adding the SIgA Caco-2 Cell Culture Conditions and Exposure to TNF-C. compounds at t=15 h, a time-point when the TER is not yet Human colonic adenocarcinoma epithelial Caco-2 cells affected. At a later time-point (28 hours), which is known to (HTB 37, American Type Tissue Collection) were grown in dramatically affect TER (see e.g., Dongmei Ye, et al., Am J C-DMEM consisting of DMEM-Glutamax (Life Technolo Physiol Gastrointest Liver Physiol 290: pp. 496-504, 2006), gies) supplemented with 10% fetal calf serum (FCS), 1% application of the SIgA compound in the apical compart non essential amino acids, 10 mM HEPES, 0.1% transferrin ment resulted in marked recovery of the TER measured 24 and 1% streptomycin/penicillin Cells cultivated to 80% hours later. confluency were seeded on Snapwell filters (diameter, 12 25 Conclusion: mm, pore size, 0.4 mm; Corning Costar) at a density of These data Suggests that delayed apical neutralization of 0.4x10 cells/cm. The formation of a polarized Caco-2 cell the effect of TNF-C. on polarized Caco-2 cell monolayers can monolayer at week 3 was established by morphology (laser lead to recovery, as long as the SIgA’s are delivered within scanning confocal microscopy) and monitoring of the tran 28 hours post-cytokine exposure. sepithelial electrical resistance (TER: 300-350 S2xcm) 30 Tracking of the Bio-Availability of Fluorescent ADB using a Millicell-ERS apparatus (Millipore). After formation SA1g Applied in the Apical Compartment of Polarized of the Caco-2 cell monolayer, the apical compartment Caco-2 Cell Monolayers medium was replaced by culture medium lacking FCS. One ADB-SA1g was labeled with indocyanine Cy5 using the hour later, the medium lacking FCS was then replaced by antibody labeling kit (General Electrics Healthcare), follow medium containing 10 or 20 ng/ml of TNF-C. The polarized 35 ing the procedure provided by the kits manufacturer. One Caco-2 cell monolayers were sensitized with the TNF-C. for microgram of labeled ADB-SA1 g diluted in 500 microliters 28 hours and the integrity of the monolayer was monitored of FCS-free Caco-2 cell culture medium was added to the by measurement of the TER. apical compartment and incubated overnight. Snapwell Effect of SIgAs on Polarized Caco-2 Cell Monolayer inserts were washed twice with PBS prior to fixation over Sensitized by TNF-C. Applied Apically 40 night with 5 ml of 4% paraformaldehyde at 4° C. After Caco-2 cell monolayers were established as described washing with PBS, filters were permeabilized with 0.2% above. For each Snapwell insert, 10 ng TNF-C. was mixed Triton X-100, and non-specific binding sites were blocked with an equimolar, or a 10-fold molar excess, of an antibody with 5% FCS in PBS. Inserts were incubated with rabbit IIB-SA1 or ADB-SA1g to a final volume of 40 microliters anti-human Zonula occludens-1 (ZO-1) (1/200 dilution), phosphate-buffered saline (PBS). The TNF and antibody 45 washed in PBS prior to addition of secondary Ab (goat mixture was incubated for 20 minutes at ambient tempera anti-rabbit IgG conjugated with Alexa Fluor 647; 1/100 ture followed by dilution with 460 microliters of culture dilution). After PBS washing, filters were then incubated medium lacking FCS. The medium in the apical compart with 4,6-diamidino-2-phenylindole (DAPI) at a concentra ment was replaced by the TNF and antibody mixture. The tion of 100 ng/ml in PBS (Invitrogen) for 30 min. Filters Caco-2 cell monolayers were sensitized for 28 hours and the 50 were recovered from their holders, and mounted in integrity of the monolayer was monitored by measurement Vectashield (Vector Laboratories) for observation using a of the TER Zeiss LSM 710 Meta confocal microscope (Carl Zeiss, Results: Germany) equipped with a 40x objective (Cellular Imaging As shown in FIG. 11 for the 10-fold molar excess assay, Facility platform, Lausanne University, Switzerland) and taking the medium condition as 100%, the drop in TER 55 processed using Zeiss ZEN 2009 light software. values induced by TNF-C. could be completely compensated Results: by the presence of either anti-TNF-C. SIgA preparation. The Because 3D image reconstitution does not allow to finely data are a mean of 6 independent replicates for each con pinpoint the location of the SIgA in contact with the Caco-2 dition. Statistical analysis using ANOVA followed by Dun cell monolayer, reliable tracking was carried out by analyZ nett's Multiple Comparison test found a p<0.001 for the 60 ing Successive confocal plans. Cell sections were selected at ADB-SA1g and IIB-SA1 results (indicated with *** in FIG. the bottom of microVilli, at the level of tight junctions, in the 11). nuclear periphery, and at the level of the nucleus. Proper Effect of SIgAs on Polarized Caco-2 Cell Monolayer visualization was ensured by simultaneous staining with Sensitized by TNF-C. Applied Apically: Possible Recovery anti-ZO-1 antibodies and DAPI. ADB-SA1g was localized after Apical Delivery. 65 on the cell surface. The antibody was detected in the form of Polarized Caco-2 cell monolayers were established as dense fluorescent spots within the cell cytosol, most likely in described above. The medium in the apical compartment endocytotic/micropinocytic compartments. US 9,573.996 B2 39 40 Conclusion: system an analysis of the status of disease was performed. This indicates that intracellular distribution may serve as Briefly, a mini-endoscope (1.9 mm outer diameter) was second line of defense and that ADB-SA1g could eventually introduced via the anus and the colon was carefully inflated neutralize internalized TNF-C. with an air pump. Endoscopic pictures obtained allow the monitoring and grading of inflammation. Thereafter, endo Example 8b scopic scoring of five parameters from 0-3 (1; translucent structure, 2.; granularity, 3: fibrin, 4; Vascularity, and 5; Stool) Localization of Anti-TNF SIgA ADB-SA1g in resulting in the overall score from 0 (no change) to 15 In-Vivo Animal Models of IBD (severe colitis) was performed. Typically, after one week 10 exposure to DSS the symptoms are so strong that mice need In an animal model of IBD, disease was induced in female a resting period to recover. At day t-16 the experiment was C57Bl/6 mice, 8 weeks of age, by topical sensibilization terminated and clinical score analyzed. (day 1) with 0.5% TNBS in acetonitril (4:1), followed by a The results are depicted in FIG. 13. Statistical analysis rectal challenge of 2.5% TNBS in 100% Ethanol on day 4 using ANOVA followed by Dunnett's Multiple Comparison Animals were treated orally 3 times with ADB-SA1 g (100 15 test was performed comparing all groups to PBS control ug/mouse): 2 days before topical sensibilization (day -1) group. The ADB-SA1g was significant having a p<0.001 and on day 1 and 4. At day 6, when disease symptoms (indicated as *** in FIG. 13). n=7 mice per group. Oral became apparent, animals were sacrificed and distal colon treatment with ADB-SA1g significantly inhibited DSS-in was collected for histological analyses. Fresh frozen tissue duced inflammatory bowel disease in C57BL/6 mice. In was fixed in 4% paraformaldehyde and washed 3 times with contrast, Subcutaneous treatment with Adalimumab was not wash buffer (1xPBS+2% BSA). Unspecific binding sites effective, even though both antibodies used the same binding were blocked with protein block reagent, washed 3 times head (variable region). and incubated with primary antibody over night at 4° C. In FIG. 14 a representative mini-endoscopic picture of the After 3 wash steps, tissue slides were incubated with sec colitis score at day t—15 is depicted. ondary antibody for 4 hours at 4°C., washed 3 times and 25 incubated with streptavidin-Alexa488 for 1 hour at 4° C. Example 10 Finally tissue slides were mounted with Vectashield mount ing medium. Primary antibodies used were: sc-20656 (hu Efficacy of Anti-TNF SIgA ADB-SA1 g in an man SC chain) rabbit (Santa Cruz) 1:200, CD31 (Dendritic In-Vivo Animal Model of RA cells) rat antibody (Biolegend) 1:200, CD326 (EpCam) rat 30 antibody (Biolegend) 1:200, CD11c (Dendritic cells) arm The therapeutic efficacy of oral ADB-SA1 g was evaluated Hamster (Biolegend) 1:200 and M-cells ULEX-1: lectin in a rheumatoid arthritis (RA) model based on a TNFC.- FITC directly conjugated (Sigma-Aldrich) 1:200. Secondary overexpressing transgenic mouse model which spontane antibodies used were: donkey-anti-rabbit Dylight 549 mm ously develops arthritis. (red) (Dianova) for sc-20565, goat-anti-rat biotin antibody 35 TNF driven spondylitis mouse (TNF^'") male mice (BD Pharmingen) 1:500+Streptavidin-Alexa488 nm (green) (available via BiomedCode Hellas SA, heterozygous for the 1:200 for CD31 and CD326, and goat-anti-arm. Hamster mouse TNFC. mutation (maintained in a C57BL/6J genetic biotin antibody (Caltag) 1:500+Streptavidin Alexa488 mm background) were crossed with C57BL/6J females. Their for CD11c. heterozygous offspring used in this study were identified by Results: 40 tail DNA genotyping which was further confirmed by the To demonstrate the presence of ADB-SA1g, antibody phenotype of these animals which exhibit a ruffled fur coat. sc-20656 was used. This antibody binds to the human Mixed sex mice were used in this study. secretory chain, but not to mouse secretory chain. Positive From the fourth week of age and onwards, the mice staining for ADB-SA1 g was found in epithelial cells (posi received daily (Monday-Friday) by oral gavage either drink tive for CD 326) and in dendritic cells (positive for CD11c-- 45 ing water, buffer (vehicle), ADB-SA1 g (5 mg/kg), or sub and CD31+). Surprisingly, no co-localization of ADB-SA1g cutaneously every other day (Mon-Wed-Fri) etanercept (10 with M-cells was found under the above described experi mg/kg). mental conditions. Body weight and grip strength measurements were Conclusion: recorded weekly for each mouse. All TNF^' mice This data confirms the “tracking of the bio-availability” 50 showed normal body weight gain when compared to wild results from Example 8a and indicates that intracellular type littermate control mice. Statistical analysis using distribution may serve as second line of defense and that ANOVA followed by Newman-Keuls Multiple Comparison ADB-SA1g could eventually neutralize internalized TNF-C. Test was performed comparing all groups to TNF^'" vehicle treated group (n=6-8 per group, ** p<0.01, *** Example 9 55 p<0.001). At 11 weeks of age, the vehicle-treated TNF^'" mice displayed significantly reduced grip strength in com Efficacy of Anti-TNF SIgA ADB-SA1g in In-Vivo parison to wt littermate control mice and etanercept-treated Animal Models of IBD TNF' mice. Mice treated orally with ADB-SA1g and mice treated Subcutaneously with etanercept displayed com C57BL/6 mice were pre-treated orally 5 times a week (not 60 parable, statistically-significant increased grip strength in weekend) with PBS control. ADB-SA1 g (100 ug/mouse), (FIG. 15). or 3 times per week subcutaneous with Adalimumab (100 At 11 weeks of age, when the control TNF^' group ug/mouse). Start of pre-treatment is t-0. At day t—7 mice had obvious signs of arthritic pathology, mice from all were treated with 2% dextran sulfate sodium (DSS) in groups were sacrificed and tissue samples from ankle joints drinking water to induce inflammation symptoms in intes 65 were collected and processed for histopathological analysis. tine. First signs of inflammation can be seen at day 10-12, Ankle joints were assessed histopathologically. The hind together with declined body weight. With mini-endoscopic joints of the TNF^' mice were scored for synovial US 9,573.996 B2 41 42 hyperplasia, existence of inflammatory foci, cartilage was performed by Biolex Therapeutics, Inc., Pittsboro, N.C., destruction, and bone erosion using the scoring scale USA. DNA sequences were produced by DNA2.0 (Menlo detailed below: Park, Calif., USA). 0 normal The anti-IL-12/23 SIgA (UKB-SA1) was expressed in L. 1-mild inflammation in periarticular tissue and/or mild minor by transfection via an A. tumefaciens binary vector oedema containing DNA sequences encoding all four of the SIgA 2 moderate inflammation and pannus formation with components: J-chain, SC-chain, H-chain and L-chain. To Superficial cartilage and bone destruction prepare this vector independent expression cassettes were 3 marked inflammation with pannus formation and mod created containing promoter, DNA sequences encoding the erate cartilage and bone destruction (depth to middle 10 protein and terminator for the J-chain (SEQ ID NO:34), Zone) SC-chain (SEQ ID NO:36), H-chain (SEQ ID NO:41) and 4-severe inflammation with pannus formation and L-chain (SEQ ID NO:42). The H chain was fused to the marked cartilage and bone destruction (depth to tide modified chimeric octopine and mannopine synthase pro mark). moter with L. gibba 5' leader from ribulose bis-phosphate Histopathological analysis of the ankle joints revealed 15 carboxylase (RuBisCo) small subunit-1. The L-chain, SC that the vehicle treated TNF^' mice displayed severe chain and J-chain genes were fused to high expression signs of arthritis. Statistical analysis using ANOVA followed Lemnaceae Ubiquitin promoters L. minor polyubiquitin by Newman-Keuls Multiple Comparison Test was per promoter (Lmubq), L. aequinoctialis polyubiquitin pro formed comparing all groups to TNF^' vehicle treated moter (Lal Jbq) and Spirodella polyrhiza polyubiquitin pro group (n=6-8 per group, * p-0.05, *p,0.01). Etanercept moter (SpUbq), respectively. Sequences of these promoters treated animals displayed significantly decreased signs of have been disclosed in PCT application WO2007/124186. pathology. Mice treated orally with ADB-SA1g also showed These expression cassettes were then cloned into a single A. decreased joint inflammation when compared to vehicle tumefaciens transformation vector EC2.2 (a modification of treated TNF^' mice with an effect comparable to sc the A. tumefaciens binary vector p3MSP3, which is a etanercept treated animals (FIG. 16). 25 derivative of pBINPLUS. See Ni, M., Cui, D., Einstein, J., Narasimhulu, S., Vergara, C. E., and Gelvin, S. B. Plant J. 7, ANTI-p40 SIGA EXAMPLES 661-676, (1995), van Engelen Transgenic Res. 4:288-290 (1995), and Gasdaska et al., Bioprocessing J., 3:50-56 Example 11 (2003)), with the appropriate restriction sites to create the 30 final transformation vector SynAO1 (FIG. 18A). This vector Stable Expression of Anti-IL-12/23 Secretory also contained the gentamycin acetyltransferase-3-I gene Protein Based on Ustekinumab (UKB-SA1) in (aacC1) which confers resistance to gentamycin and allows Lenna for selection of transgenic L. minor lines, and was used to produce UKB-SA1 with wild-type (unmodified) N- and a) Construction of Vectors 35 O-glycosylation. Synthetic genes were designed for each of the 4 different Syn A01 was used to create additional transformation protein chains of an anti-IL-12/23 secretory IgA. The amino vectors to generate a glycan optimized version of UKB acid sequence of the heavy chain consisted of the rice SA1, further identified as UKB-SA1g0. A chimeric hairpin C.-amylase secretion signal (SEQ ID NO:26) joined to the RNA was used to silence endogenous L. minor mRNAs N-terminal amino acid of the variable part of the heavy chain 40 encoding C-1,3-fucosyltransferase (Fuct1, GenBank of anti-IL12/23 IgG1 antibody Ustekinumab (Stelara R, DQ7891.45) and B-1,2-xylosyltransferase (Xylt1, GenBank CAS number 815610-63-0, SEQID NO:37) which in turn is DQ789146). A DNA sequence for this chimeric RNAi joined to the N-terminal amino acid of the constant part of molecule was fused to the high expression SpUbq promoter a human IgA1 heavy chain (SEQ ID NO:10). The amino and subsequently moved into the SynaO1 vector creating the acid sequence of the light chain consisted of the rice 45 final transformation vector SynaC2 (FIG. 18B). Also the C.-amylase secretion signal (SEQ ID NO:26) joined to the neomycin phosphotransferase II gene (NPTII) was moved N-terminal amino acid of the light chain sequence of Usteki into Syna01 replacing aacC1 to produce transformation numab (CAS number 815610-63-0), which combines an vector SynaC3 (FIG. 6C). This exchange allows for anti-IL-12/23 binding variable part (SEQID NO:38) with a kanamycin selection instead of gentamicin selection of human K-light chain constant part (SEQ ID NO:14). The 50 transgenic glycan optimized L. minor lines. Further details SC-chain consisted of the rice C-amylase secretion signal on procedures for production of glycan optimized proteins in (SEQ ID NO:26) joined to the N-terminal amino acid of the Lemnaceae can be found in PCT applications WO2007/ amino acid sequence of amino acids 19 to 603 of the human 084672, WO2007/084922, WO2007/084926 and in Cox, K. polymeric immunoglobulin receptor disclosed in Uni M., Nature Biotechnology 2006, 12: 1591-1597. ProtKB/Swiss-Prot database entry P01833 (SEQID NO:21). 55 b) Transformation of and Expression Using Lenna The J-chain sequence consisted of the rice C.-amylase secre Lenna transformation vectors SynAO1, SynAO2 and tion signal (SEQ ID NO:26) joined to the N-terminal amino Syn A03 were transvected into A. tumefaciens strain acid of the amino acid sequence of amino acids 23 to 159 of C58Z707 (Hepburn et al., J. Gen. Microbiol. 1985, 131: the human sequence disclosed in UniProtKB/Swiss-Prot 2961-2969) by electroporation. Agrobacterium colonies database entry P01591 (SEQ ID NO:20). 60 were selected using gentamycin (SynaO1 and SynaO2) or Genes were designed for each of the four components kanamycin (SynaO3) and analyzed for the presence of the with L. minor preferred codon usage (63-67% GC content). appropriate binary vector using a PCR based assay. A single Tables with suitable preferred codon use in Lemnaceae can colony was selected for each transformation vector and be found in PCT application WO2005/035768 and in rel taken forward into L. minor transformation process (as evant references contained therein. Restriction endonuclease 65 follows). sites were added to allow cloning into A. tumefaciens binary Partially dedifferentiated Lenna tissue (L. minor strain vectors. Design of DNA sequences and vector construction 8627) was incubated with Agrobacterium harboring the US 9,573.996 B2 43 44 expression cassette plasmid by briefly dipping the tissue into Example 12 the Solution. The tissue was then placed on co-cultivation plates for two days in continuous light at 25°C. Following Isolation and Purification of UKB-SA1 and co-cultivation, the tissue was transferred to antibiotic selec UKB-SA1g0 Secretory IgA Antibody from Lemna tion plates and returned to continuous light at 25°C. The tissue was transferred weekly to fresh antibiotic selection Biomass from transgenic Lenna expressing UKB-SA1 or plates. Cefotaxime was included in the antibiotic selection UKB-SA1g0, having variable regions that are the amino plates to eradicate the Agrobacterium. Gentamicin was acid sequence of the variable regions (antigen binding included in these plates to select for transgenic tissue regions) of Ustekinumab, was homogenized in 50 mM 10 Sodium phosphate, 0.3M Sodium chloride, buffer pH 7.4, at obtained from vectors SynaO1 and Syn A02 where there is a buffer to tissue ratio of 4:1. An acid precipitation step was a selectable marker gene included in the transferred genetic performed on the crude extract to remove the enzyme cassette which confers gentamycin resistance. Kanamycin ribulose bis-phosphate carboxylase (RuBisCo) and other was included in plates to select for transgenic tissue from plant proteins by adjusting the extract to pH 4.5 using 1M vector SynAO3. 15 Sodium acetate, pH 2.5. The precipitate was removed by UKB-SA1 with unmodified wild-type (WT) glycosylation centrifugation of the material at 14,000xg for 30 minutes at was obtained by transfection of L. minor strain 8627 (Biolex 4°C. The supernatant was adjusted to pH 7.4 and filtered to Therapeutics Inc.) with vector SynaC)1. UKB-SA1g0, hav 0.22 um prior to IMAC chromatography. ing G0 glycans lacking fucose and lacking Xylose, was IMAC purification: A Chelating Sepharose FF (GE obtained by transfecting L. minor strain 8627 with vector Healthcare prod. Nr. 17-0575-01) column was prepared Syna02, or by transfection of the N-glycosylation modified according to manufacturer instructions (28–4047-39 AC). L. minor strain XF04 (Biolex Inc.) with vector SynaO3. The column was charged with 3-5 column volumes (cv) of Once transformed plants were regenerated (approximately 0.1M Copper sulfate. Excess copper was washed with 3-5 cv three months) single plants were harvested from the antibi of double distilled water. The column was equilibrated with otic selection plates and propagated separately in liquid 25 3-5 cv of PBS buffer (50 mM Sodium phosphate, 0.15M Sodium chloride, pH 7.4); 3-5 cv of 0.1M Sodium acetate, growth media, without selection antibiotic, for further pH 4.0 buffer; 3-5 cv PBS buffer with 0.5M Imidazole; and screening and characterization. Thus several hundred indi 3-5 cV PBS buffer. vidual transgenic plant lines from each construct were The Supernatant was loaded on the column Approximately generated. Independent transgenic lines were harvested and 30 3.8 mg SIgA/ml resin was loaded on columns of up to 350 clonally propagated in individual harvestjars. For screening ml chelating Sepharose. Non-binding material was washed of transgenic lines, clonal lines were preconditioned for 1 from the column with 10 cv PBS buffer, 10 cv of 0.1M week at light levels of 150 to 200 umol/m’s in vented plant Sodium acetate, pH 4.0 buffer, and 10 cv PBS buffer. The growth vessels containing SH medium (Schenk R.U. et al., product was eluted from the column using 10 cv of PBS Can. J. Biol. 1972, 50: 199-204) without sucrose. Fifteen to 35 buffer containing 0.075 M Imidazole (a gradient of 0-0.075 twenty preconditioned fronds were then placed into vented M imidazole was also used but did not lead to improved containers containing fresh SH medium, and allowed to results). The fractions containing the Secretory IgA antibod grow for two weeks. Tissue samples from each line were ies were pooled. collected and frozen for analysis. The column was regenerated by removing copper using a 40 0.2M EDTA, 0.3M sodium chloride solution, followed by To determine SIgA expression, frozen tissue samples treatment with 0.1N NaOH. were homogenized, centrifuged and the Supernatant was SEC purification: Material obtained by IMAC chroma removed and screened by an ELISA method using sheep tography was further purified on a Sephacryl 5300 HR anti-human IgA secretory chain (AbD Serotec catalog column. The column was equilibrated with 2 cv of PBS #5111-4804-1:1000 dilution) coated plates to capture the 45 buffer at a flow rate of 0.5 ml/min. The IMAC eluate was SIgA antibody. The samples were then detected using a goat concentrated 3x using ultra filtration (e.g., using a 5- or 30 anti-human kappa light chain HRP conjugated antibody kDa regenerated cellulose (hydrosart) membrane) by using, (Sigma catalog #A7164-1:2000 dilution). The highest lines for example, a spin filter (Vivaspin 15R) or a UF cassette from this primary Screening were then grown again for two (Sartorius 305 14459 01 E). Typically, a concentration of weeks in Small research vessels under the optimal growth 50 5-7 mg/ml and feed volume of 15-20 ml was used for a 360 conditions, the resulting tissue was harvested and the ELISA ml column. The feed was applied using an AKTA purifier at was performed to determine the percent of the total soluble 0.5 ml/min Elution was performed with at least 2 column protein that the SIgA antibody is expressed. The results are volumes of PBS buffer at room temperature and a flow rate summarized in Table 4. of 0.5 ml/min Fractions were collected and sufficiently pure 55 fractions were pooled. TABLE 4 Results of non-reducing and reducing SDS-PAGE analy ses of purified material obtained from Lenna transfected # of Highest lines Expression with construct Syna01 are shown in FIG. 19 as gels A and Construct Product Screened level B, respectively. 60 Some batches of material produced by the purification SynAO1 in UKB-SA1 (WT glycosylation) 262 8.5% TSP method described above were further purified using affinity L. minor 8627 chromatography using CaptureSelect human IgA (BAC). SynAO2 in UKB-SA1g0 (GO N-glycans) 164 15.19% TSP L. minor 8627 Using an Akta Explorer 10 system a column loaded with SynAO3 in UKB-SA1g0 (GO N-glycans) 452 11.8% TSP 30.9 ml Capture Select IgA (BAC) was equilibrated using 3 L. piror XFO4 65 column volumes (CV) 20 mM Tris pH 7.0 buffer at a flow rate of 5 ml/min A solution containing 75.9 mg UKB-SA1 in Tris buffer pH 7.0 was loaded on the column at a flow rate US 9,573.996 B2 45 46 of 2.5 ml/min. After a wash step with 5 CV of 20 mM Tris comb (Biorad, 345-0015). After electrophoresis gels were buffer pH 7.0 at 5 ml/min the product was eluted with 5 CV treated with Krypton protein stain and analyzed. Stability of of 20 mM Tris buffer pH 7.0 containing 3.5 M MgCl, at 5 the samples was qualitatively assessed visually. Results are ml/min. shown in FIG. 20 where gel A is the UKB-SA1 (labeled The eluate was first dialyzed twice against 5 L 20 mM Tris SynAO1-WT in FIG. 20) and gel B is UKB-SA1g0 (labeled buffer pH 7.0 using snakeskin 10 kDa dialysis tubing for at SynAO2-G0 in FIG. 20). least 2 hours, followed by dialysis against 5 L PBS puffer pH Both UKB-SA1 and UKB-SA1g0 of exhibited a stability 7.4 for at least 2 hours. The Solution was concentrated using that was comparable to natural human colostral SIgA. The a stirred cell (Amicon 8200, Millipore, overhead pressure 30 IgG1 antibody ustekinumab degraded under these condi Psi) with 30 kDa regenerated cellulose membrane filter 10 tions at such a high rate that detection at T=0 was not (Millipore) to a final concentration of approximately 1 possible. mg/ml. The obtained product was further purified using SEC purification as described previously. Glycosylation modified Example 15A product UKB-SA1g0 was purified using the same method. No substantial differences were observed in purification 15 Inhibition of IL-12/23 in Dendritic Cells of the two glycosylation forms, wild-type UKB-SA1 and UKB-SA1g0. To demonstrate inhibition of endogenous produced p40/ IL12/IL23 by UKB-SA1 or UKB-SA1g0 versus usteki Example 13 numab (IgG1) or non-specific SIgA (polyclonal colostral SIgA), in vitro experiments were performed with dendritic Binding of UKB-SA1 and UKB-SA1g0 to IL12 cells and T cells. Monocytic cells were isolated from human donor blood and stimulated with lipopolysccharide (LPS) The binding of purified anti-IL-12/23 SIgA, with variable and the cells were differentiated into mature dendritic cells. regions taken from Ustekinumab and produced in Lenna as The stimulation with LPS and resulting differentiation was in Examples 11 and 12, to IL-12 was determined. The 25 performed in the presence of a SIgA antibody of the inven binding of both UKB-SA1 and UKB-SA1g0 products were tion, Ustekinumab, polyclonal colostral SIgA, or without determined in comparison to ustekinumab (STELARAR) any added antibody (i.e., LPS only). and colostral SIgA. Plates were coated with IL-12 (Abcam, In more detail, the experiments were performed as fol AB52086) 1 g/ml. Detection of bound UKB-SA1/UKB lows. Human dendritic cells (DCs) were obtained from SA1g0 (secretory IgA) and ustekinumab (IgG1) antibodies 30 isolated monocytes from buffy coats. The adherent mono was performed using a 1:1500 fold dilution of anti human cytes were cultured in synthetic X-VIVO 15 medium kappa chain antibody (Abbiotec, cat. no.250987), 100 ul per (Lonza, Cat. No. BE04-418Q) supplemented with 2% of AB well, for one hour at RT, washing 3 times, 30 seconds with human serum (Sigma, Cat. No. H4522), 450 U/ml GM-CSF 200 ul PBS/0.05% Tween with shaking, followed by incu (Miltenyi Biotec, Cat. No. 130-093-867) and 300 U/ml IL-4 bation with a 1:1500 fold dilution of donkey anti mouse 35 (Miltenyi Biotec, Cat. No. 130-093-924) as growth and HRP conjugated (Emelcabiosciences, MS3001), 100 ul per differentiation factors, respectively, for 6 days to obtain well, for one hour at RT. immature DCs. After one week, immature DCs (5x10) were The UKB-SA1, UKB-SA1g0 and ustekinumab antibodies activated for 24 hours with 1 lug/ml LPS (Invivogen, Cat. all bound with high affinity to IL-12 under the conditions of No. tirl-pelps) in the presence of different concentrations this assay. For UKB-SA1 and UKB-SA1g0 antibodies, 40 UKB-SA1, UKB-SA1g0, or control antibodies to induce DC binding occurred independent of the type of glycosylation. maturation. IL12/IL23 concentration in culture medium was Colostral SIgA had minimal to no binding to IL-12. measured by ELISA according to the manufactures instruc tions (BD OptEIA, Cat. No. 555171). Example 14 The results are represented in FIG. 21A where open circle 45 is UKB-SA1: closed circle is UKB-SA1g0; Diamond is Proteolytic Stability of UKB-SA1 and UKB-SA1g0 Colostral SIgA; and Square is ustekinumab. Data are means (+/-SEM) of 3 experiments. Both UKB-SA1 and UKB The stability of UKB-SA1 and UKB-SA1g0 with antigen SA1g0 inhibited endogenous produced p40/IL12/IL23 by binding regions having the amino acid sequence of usteki dendritic cells. Both UKB-SA1 and UKB-SA1g0 had a numab antigen binding regions (i.e., the variable heavy and 50 similar efficacy and potency as Ustekinumab in this regard. light chains), produced in Lenna, was determined in simu lated intestinal fluid (SIF, 0.05M phosphate buffer pH 6.8 Example 15B containing 10 mg/ml pancreatin). Both the form with wild type Lemma glycosylation obtained with vector SynaO1, and Inhibition of IL-12/23 in Dendritic Cells the G0 glycosylation variant obtained with vector SynaO2 55 were analysed. Stability was compared to ustekinumab Similar to above, inhibition of IL-12/23 in dendritic cells (IgG1) and to aspecific human colostral SIgA which was was measured but at a fixed concentration of the antibody. purified to contain only kappa light chains and C-1 heavy Human dendritic cells (DCs) were obtained from isolated chains. monocytes from buffy coats. The adherent monocytes were 90 ul of a 1 mg/ml solution of the material to be tested was 60 cultured in synthetic X-VIVO 15 medium (Lonza, Cat. No. added to 810 ul of SIF at 37° C. 50 ul samples were drawn BE04-418Q) supplemented with 2% of AB human serum at T=0, 5, 15, 30, 60 and 120 min and immediately frozen (Sigma, Cat. No. H4522), 450 U/ml GM-CSF (130-093-867, in liquid nitrogen. Samples were analyzed by non-reducing Miltenyi Biotec) and 300 U/ml IL-4 (Miltenyi Biotec, Cat. SDS-Page gel electrophoresis. Briefly; 17 Jul of a 0.15M No. 130-093-924) as growth and differentiation factors, solution of iodoacetamide in LDS sample buffer was added 65 respectively, for 6 days to obtain immature DCs. After one to each of the frozen samples. Samples were thawed and week, immature DCs (5x10) were activated for 24 hours applied to Criterion Tris-HC1 gel (12.5%, 18 well, 30 ul with 1 lug/ml LPS (Invivogen, Cat. No. tirl-pelps) in the US 9,573.996 B2 47 48 presence of 10 ug/ml UKB-SA1, UKB-SA1g0, or control administered to C57Bl/6 mice (200 g for intravenous, 400 antibodies to induce DC maturation. IL12/IL23 concentra ug oral) and blood samples were taken by orbital bleeding at tion in culture medium was measured by ELISA according different time points. UKB-SA1g0 was measured by an to the manufactures instructions (BD OptEIA, Cat. No. in-house constructed ELISA. A goat anti-human kappa light 555.171). The results are represented in FIG. 21B. Both UKB-SA1 chain antibody (Southern Biotech, Cat. No. SBA 2060-02) (labeled SynAO1 in FIG. 21B) and UKB-SA1g0 (labeled was used for coating of the plates, and a goat anti-human Syna02 in FIG. 21B) inhibited endogenous produced p40/ IgA antibody (Southern Biotech, Cat. No. SBA 2050-05) for IL12/IL23 by dendritic cells to the same extent as usteki detection. The minimum detection limit of the ELISA is 5 numab. ng/ml. 10 The results are represented in FIG. 22 wherein circle Example 15C shows oral administration and square shows intravenous administration. Dotted line at 5 ng/ml depicts the detection Inhibition of IL-12/23 in Dendritic Cells and T Cells limited of the ELISA used for quantification. Data are 15 mean+/-SD of 2 representative mice. UKB-SA1g0 (labeled To determine if other “intrinsic' properties were affected Syn A02 in FIG.22) is rapidly cleared from circulation after by the antibodies, dendritic cells were co-cultured with intravenous injection and very little (if any) exposure was T-cells. The crosstalk between these cells, which might measured after oral administration. result in a change of phenotype of the dendritic cells, or cytokine secretion profile of the T-cells was examined. DCs Example 17 were stimulated with LPS for 6 h; afterwards DCs were washed twice in PBS and in the presence of 10 g/ml of In Vivo Administration of SIgA and IgG UKB-SA1, UKB-SA1g0 or control antibodies cultured with Anti-IL12/23 Pharmacokinetics allogeneic T cells. The allo-response was tested in a mixed lymphocyte reaction; allogeneic T cells (10 cells) were 25 Using in vivo imaging of fluorescent labeled antibodies, co-cultured in triplicate with differently treated DCs (5*10) images of thoracic and abdominal region of mice can reveal in a 96-well round bottom plate, in 200 ul of medium distribution of the antibody over time. UKB-SA1 was con (X-VIVO 15+2% HS). For T helper polarisation T lympho jugated with either Alexa Flour 647 (Molecular probes), or cytes Supernatants were collected at day 4 and T helper DyLight 755 (ThermoFisher) according manual instruc specific cytokine IFNY was analysed by ELISA (BD 30 tions. The dye can be detected as a fluorescent signal with OptEIA, Cat. No. 555142), according to the manufacturers the Maestro in vivo imaging system (Cambridge Research & instructions. Instrumentation). Fluorescent conjugated antibodies (100 The results are represented in FIG. 21C. UKB-SA1 (la ug/mice) were administered orally or intraperitoneally. Mice beled Syn A01 in FIG. 21C) and UKB-SA1g0 (labeled were anaesthetized with Xylamine/Ketamine before exami SynAO2 in FIG. 21C) blocked the IFNY secretion by co 35 nation with the Maestro system in order to immobilize cultured DCs and T-cells to the same extent as Ustekinumab, animals during picture taking procedure. Then mice were indicating an inhibition of proinflammatory T helper-1 cells. analyzed and pictures of the distribution of the fluorescence conjugated UKB-SA1 were taken at different time points Example 15D indicated at the experimentally setup. After that background 40 fluorescence were subtracted by software. Inhibition of IL-12/23 in Dendritic Cells and T The results are represented in FIG. 23 wherein the first Cells three columns () correspond to the DyLight 775 conjugate and the fourth column () is the UKB-SA1 Alexa Flour 647 Similar to above, the antibodies were applied in fixed conjugate. First hours after oral administration fluorescent concentrations to the DC and T cells. DCs were stimulated 45 conjugated antibodies UKB-SA1 (labeled SynaC)1 in FIG. with LPS for 6 h; afterwards DCs were washed twice in PBS 23) and IgG1 Ustekinumab (labelled IgG in FIG. 23) are and in the presence of 10 ug/ml of UKB-SA1, UKB-SA1g0 present in thoracic area of mice, probably the stomach, or control antibodies cultured with allogeneic T cells. The whereas after 24 hours a clear signal of UKB-SA1, but not allo-response was tested in a mixed lymphocyte reaction; IgG1, is present in the abdominal region (intestine) of the allogeneic T cells (10 cells) were co-cultured in triplicate 50 mice. Intraperitoneal administration results in a diffuse tho with differently treated DCs (5*10) in a 96-well round racic and abdominal signal after 24 hours. bottom plate, in 200 ul of medium (X-VIVO 15+2% HS). For T helper polarisation T lymphocytes supernatants were Example 18 collected at day 4 and T helper specific cytokine IL-10 was analysed by ELISA (eBioscience Cat. No. 88-7106-88), 55 Distribution. In Vivo after Oral Administration according to the manufacturers instructions. The results are represented in FIG. 21D. UKB-SA1 Immunohistochemistry on cryo-sections of mice distal (labeled SynAO1 in FIG. 21D) and UKB-SA1g0 (labeled colon, 48 hours after oral administration of antibody were Syna02 in FIG. 21D) had no effect on the IL-10 secretion assessed. Antibodies (100 ug/mouse) were administered by co-cultured DCs and T-cells. 60 orally and after 48 hours animals were sacrificed and intes tinal tract isolated. Cry-section were stained with anti-SC Example 16 chain antibody (Santa Cruz, Cat. No. sc20656) for detection of antibodies containing a human secretory chain. This Pharmacokinetics of UKB-SA1g0 antibody does not cross-react with mouse secretory chain. 65 As secondary antibody goat-anti-rabbit conjugated with Serum concentrations of UKB-SA1g0 after oral and intra Cy3-red antibody (Jackson Immunoresearch, Cat. No. 111 venous administration were determined UKB-SA1g0 was 166-045) was used. US 9,573.996 B2 49 50 The results are represented in FIG. 24 where the left side 2 mm biopsy was tested with vehicle and the other with is UKB-SA1 (labeled Syn A01 in FIG. 24) and the right side UKB-SA1. From the other punch, one 2 mm biopsy as tested is IgG1 Ustekinumab (labeled IgG in FIG. 24). UKB-SA1 is with UKB-SA1g0 and the other with ustekinumab. UKB present in cells of the sub mucosal layer of the distal colon SA1 and UKB-SA1g0 were tested at equimolar concentra as indicated by the bright spots in the vicinity of the white tions of 14 Jug/ml and Ustekinumab was tested at 10 ug/ml. arrows. No IgG1 is indicated as being present in the distal The biopsies, medium, and antibody as appropriate were colon cells. combined in a 24 well plate (800 ul) for 8 days with the medium changed every 2-3 days. After 8 days of culture, the Example 19 skin biopsies were frozen in liquid nitrogen and then evalu 10 ated. Genes were evaluated by RTPCR: Beta-defensin 4, Efficacy of UKB-SA1 and UKB-SA1g0 in In-Vivo Keratin 16, Interleukin-10, and GAPDH (house-keeping Animal Models of IBD gene). Keratin 16 and B-Defensin 4 are proteins expressed in Heparinized blood was obtained from healthy donors epithelial tissue and related to skin diseases. Down-regula (buffy coats). PBMC's were isolated from buffy coats by 15 tion of these markers anticipate clinical efficacy of anti Ficoll-Paque 1.077 g/ml (PAA Laboratories GmbH) density inflammatory treatment in the clinic. Gene expression in centrifugation and cells were washed with 1xPBS before treated biopsies was normalized to house-keeping gene reconstitution. NOD.CB17-Prkdcscid/J Yc-/- (NOD-SCID GAPDH according to the formula 1,8e (Ct GADPH-Ct gene IL2 receptor deficient) mice 4 to 8 weeks of age were of interest)x10000 (Ct-cycle threshold time). After normal reconstituted with 30x10' human cells. Three weeks after ization, gene expression of gene of interest was compared to reconstitution animals were pre-treated orally 5 times a gene expression in vehicle-treated biopsies. week (not in weekend) with PBS control, UKB-SA1 (100 The results are represented in the table below: ug/mouse), UKB-SA1g0 (100 ug/mouse), or 3 times per week subcutaneous with Ustekinumab (100 ug/mouse). Start of pre-treatment is t—0. At day t—7 reconstituted mice were 25 Time of treated with 2% dextran sulfate sodium (DSS) in drinking evolution of the water to induce inflammation symptoms in intestine. First Patient No. lesion (months) SynAO1 SynAO2 Ustekinumab signs of inflammation can be seen at day 10-12, together 2 12 K16 ++ K16 - K16 = BF +++ BF : BF ------with declined body weight. With mini-endoscopic system an L10 --- L10 -- L10 ++ 30 analysis of the status of disease was performed. Briefly, a 5 12 K16 = K16 + K16 = mini-endoscope (1.9 mm outer diameter) was introduced via BF +++ BF +++ BF ---- the anus and the colon was carefully inflated with an air L10 - L10 - L10 = 4 6 K16 + K16 +++ K16 +++ pump. Endoscopic pictures obtained are of high quality and BF +++ BF -- BF ------allow the monitoring and grading of inflammation. There L10 + L10 + L1O = after, endoscopic scoring of five parameters from 0-3 (1: 35 3 3 K16 = K16 + K16 = translucent structure, 2.; granularity, 3., fibrin, 4, vascularity, BF : BF BF L10 ++ L10 + L1O = and 5; stool) resulting in the overall score from 0 (no 7 2 K16 +++ K16 +++ K16 +++ change) to 15 (severe colitis) was performed. Typically, after BF --- BF --- BF one week exposure to DSS the symptoms are so strong that L10 --- L10 --- L10 --- mice need a resting period to recover. At day t—16 the 40 6 1 K16 --- K16 ++ K16 -- BF BF +++ BF experiment was terminated and clinical score analyzed. L10 +++ L10 +++ L10 - The results are depicted in FIGS. 25A and B. Oral 1 1 K16 -- K16 - K16 - treatment with UKB-SA1 (labeled Syn A01 in FIGS. 25A BF BF +++ BF and 25B) and UKB-SA1g0 (labeled SynAO2 in FIGS. 25A L10 ++ L10 +++ L10 - 45 and 25B), as well as subcutaneous treatment with Usteki Legend: Beta-defensin 4 (BF4), Keratin 16 (K16), Interleukin 10 (IL-10) No regulation numab, significantly inhibited DSS-induced inflammatory (0-10% difference in gene expression) is “=”: Up-regulated (11-25% difference in gene expression) is +; 26-50% is ++; and >51% is +++, Down-regulated (11-25% difference in bowel disease in humanized-scid mice. Statistical analysis: gene expression) is -; 26-50% is -; and >51% is ---. ANOVA followed by Dunnett's Multiple Comparison test, all groups were compared to PBS control group. * p-0.05, Time of evolution of the lesion appears to be relevant for *** p<0.001, n=7 mice per group. FIG. 25B depicts a 50 the activity of antip40 antibodies as UKB-SA1, UKB representative mini-endoscopic picture of the colitis score at SA1g0, and ustekinumab have activities in patients, espe day t-15. cially those with less than 6 months of evolution of the lesion. Gene expression of Keratin 16 and Beta defensin 4 Example 20 (two) were reduced in young lesions (1-3 month) by 55 Syn A01, SynaO2 and ustekinumab, indicating reduced epi Efficacy of UKB-SA1 and UKB-SA1g0 in Human thelial activity and a treatment effect. Psoriatic Skin Biopsies In these young lesions, Interleukin-10 expression was increased only by UKB-SA1 and UKB-SA1g0, but not by The activity of SIgA anti-p40 on gene expression was ustekinumab, indicating an additional anti-inflammatory evaluated for a limited number of selected genes in skin 60 cytokine response of SIgA's compared to an IgG antibody. explants of psoriatic lesions in vitro. Psoriatic cutaneous Each of the patents, patent applications, and journal lesions can be maintained in culture up to 8 days without articles mentioned above are incorporated herein by refer significant changes in their psoriatic phenotype. Two punch ence in their entirety. The invention having been described biopsies of 4 mm were obtained from 7 psoriasis patients it will be obvious that the same may be varied in many ways (volunteers). Time of evolution of the lesion varied between 65 and all Such modifications are contemplated as being within 1 and 12 months. The two biopsies from each patient were the scope of the invention as defined by the claims appended cut in half making four 2 mm biopsies. From one punch, one hereto. US 9,573.996 B2 51

SEQUENCE LISTING

<16O is NUMBER OF SEO ID NOS: 42

<210s, SEQ ID NO 1 &211s LENGTH: 235 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amino acid sequence of the soluble TNFalpha receptor of etanercept (extracellular part of TNF receptor 2: amino acids 23-257 of UniProtKB/Swiss-Prot database entry P2O333 TNR1 HUMAN) .

<4 OOs, SEQUENCE: 1 Lieu Pro Ala Glin Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser 1. 5 1O 15 Thr Cys Arg Lieu. Arg Glu Tyr Tyr Asp Glin Thr Ala Gln Met Cys Cys 2O 25 3O Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr 35 4 O 45 Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu SO 55 6 O Trp Asn Trp Val Pro Glu. Cys Lieu Ser Cys Gly Ser Arg Cys Ser Ser 65 70 7s 8O Asp Glin Val Glu Thr Glin Ala Cys Thr Arg Glu Glin Asn Arg Ile Cys 85 90 95 Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Glin Glu Gly Cys 1OO 105 11 O Arg Lieu. Cys Ala Pro Lieu. Arg Lys Cys Arg Pro Gly Phe Gly Val Ala 115 12 O 125 Arg Pro Gly Thr Glu Thir Ser Asp Val Val Cys Llys Pro Cys Ala Pro 13 O 135 14 O Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 145 150 155 160 Glin Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala 1.65 17O 17s Val Cys Thir Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val 18O 185 19 O His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Glin Pro Thr 195 2OO 2O5 Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Lleu Pro Met Gly 21 O 215 22O Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp 225 23 O 235

<210s, SEQ ID NO 2 &211s LENGTH: 120 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amino acid sequence of the heavy chain variable region of infliximab (cA2). <4 OOs, SEQUENCE: 2 Glu Val Lys Lieu. Glu Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly 1. 5 1O 15 Ser Met Lys Lieu Ser Cys Val Ala Ser Gly Phe Ile Phe Ser Asn His 2O 25 3O

Trp Met Asn Trp Val Arg Glin Ser Pro Glu Lys Gly Lieu. Glu Trp Val 35 4 O 45 US 9,573.996 B2 53 - Continued

Ala Glu Ile Arg Ser Lys Ser Ile Asn. Ser Ala Thr His Tyr Ala Glu SO 55 6 O Ser Val Lys Gly Arg Phe Thir Ile Ser Arg Asp Asp Ser Lys Ser Ala 65 70 7s 8O Val Tyr Lieu Gln Met Thr Asp Leu Arg Thr Glu Asp Thr Gly Val Tyr 85 90 95 Tyr Cys Ser Arg Asn Tyr Tyr Gly Ser Thr Tyr Asp Tyr Trp Gly Glin 1OO 105 11 O Gly. Thir Thr Lieu. Thr Val Ser Ser 115 12 O

<210s, SEQ ID NO 3 &211s LENGTH: 107 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amino acid sequence of the light chain variable region of infliximab (cA2). <4 OOs, SEQUENCE: 3 Asp Ile Leu Lieu. Thr Glin Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1. 5 1O 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Glin Phe Val Gly Ser Ser 2O 25 3O Ile His Trp Tyr Glin Glin Arg Thr Asn Gly Ser Pro Arg Lieu. Lieu. Ile 35 4 O 45 Llys Tyr Ala Ser Glu Ser Met Ser Gly Ile Pro Ser Arg Phe Ser Gly SO 55 6 O Ser Gly Ser Gly Thr Asp Phe Thr Lieu Ser Ile Asn Thr Val Glu Ser 65 70 7s 8O Glu Asp Ile Ala Asp Tyr Tyr Cys Glin Glin Ser His Ser Trp Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Asn Lieu. Glu Val Lys 1OO 105

<210s, SEQ ID NO 4 &211s LENGTH: 121 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amino acid sequence of the heavy chain variable region of adalimumab (D2E7). <4 OOs, SEQUENCE: 4 Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Arg 1. 5 1O 15 Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 2O 25 3O Ala Met His Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Val 35 4 O 45 Ser Ala Ile Thir Trp Asn. Ser Gly His Ile Asp Tyr Ala Asp Ser Val SO 55 6 O

Glu Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Lys Asn. Ser Lieu. Tyr 65 70 7s 8O

Lieu. Glin Met Asn. Ser Lieu. Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Lys Val Ser Tyr Lieu. Ser Thr Ala Ser Ser Lieu. Asp Tyr Trp Gly 1OO 105 11 O US 9,573.996 B2 55 - Continued

Gln Gly Thr Lieu Val Thr Val Ser Ser 115 12 O

<210s, SEQ ID NO 5 &211s LENGTH: 107 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amino acid sequence of the light chain variable region of adalimumab (D2E7). <4 OOs, SEQUENCE: 5 Asp Ile Gln Met Thr Glin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1. 5 1O 15 Asp Arg Val Thir Ile Thr Cys Arg Ala Ser Glin Gly Ile Arg Asn Tyr 2O 25 3O Lieu Ala Trp Tyr Glin Glin Llys Pro Gly Lys Ala Pro Llys Lieu. Lieu. Ile 35 4 O 45 Tyr Ala Ala Ser Thr Lieu. Glin Ser Gly Val Pro Ser Arg Phe Ser Gly SO 55 6 O Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thir Ile Ser Ser Leu Gln Pro 65 70 7s 8O Glu Asp Wall Ala Thr Tyr Tyr Cys Glin Arg Tyr Asn Arg Ala Pro Tyr 85 90 95 Thr Phe Gly Glin Gly Thr Lys Val Glu Ile Llys 1OO 105

<210s, SEQ ID NO 6 &211s LENGTH: 126 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amino acid sequence of the heavy chain variable region of golimumab.

<4 OOs, SEQUENCE: 6 Glin Val Glin Lieu Val Glu Ser Gly Gly Gly Val Val Glin Pro Gly Arg 1. 5 1O 15 Ser Lieu. Arg Lieu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Tyr 2O 25 3O Ala Met His Trp Val Arg Glin Ala Pro Gly Asn Gly Lieu. Glu Trp Val 35 4 O 45 Ala Phe Met Ser Tyr Asp Gly Ser Asn Llys Llys Tyr Ala Asp Ser Val SO 55 6 O Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn. Ser Lys Asn. Thir Lieu. Tyr 65 70 7s 8O Lieu. Glin Met Asn. Ser Lieu. Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Ile Ala Ala Gly Gly Asn Tyr Tyr Tyr Tyr Gly 1OO 105 11 O

Met Asp Val Trp Gly Glin Gly Thr Thr Val Thr Val Ser Ser 115 12 O 125

<210s, SEQ ID NO 7 &211s LENGTH: 108 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amino acid sequence of the light chain variable region of golimumab. US 9,573.996 B2 57 - Continued

<4 OO > SEQUENCE: 7 Glu Ile Val Lieu. Thr Glin Ser Pro Ala Thr Lieu Ser Leu Ser Pro Gly 1. 5 1O 15 Glu Arg Ala Thr Lieu. Ser Cys Arg Ala Ser Glin Ser Val Tyr Ser Tyr 2O 25 3O Lieu Ala Trp Tyr Glin Glin Llys Pro Gly Glin Ala Pro Arg Lieu. Lieu. Ile 35 4 O 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly SO 55 6 O Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thir Ile Ser Ser Lieu. Glu Pro 65 70 7s 8O Glu Asp Phe Ala Val Tyr Tyr Cys Glin Glin Arg Ser Asn Trp Pro Pro 85 90 95 Phe Thr Phe Gly Pro Gly Thr Llys Val Asp Ile Lys 1OO 105

<210s, SEQ ID NO 8 &211s LENGTH: 118 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amino acid sequence of the heavy chain variable region of certolizumab pegol.

<4 OOs, SEQUENCE: 8 Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Val Glin Pro Gly Gly 1. 5 1O 15 Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Gly Tyr Val Phe Thir Asp Tyr 2O 25 3O Gly Met Asn Trp Val Arg Glin Ala Pro Gly Lys Gly Lieu. Glu Trp Met 35 4 O 45 Gly Trp Ile Asn Thr Tyr Ile Gly Glu Pro Ile Tyr Ala Asp Ser Val SO 55 6 O Lys Gly Arg Phe Thr Phe Ser Lieu. Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 7s 8O Lieu. Glin Met Asn. Ser Lieu. Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Arg Ser Tyr Ala Met Asp Tyr Trp Gly Glin Gly Thr 1OO 105 11 O

Leul Wall. Thir Wal Ser Ser 115

<210s, SEQ ID NO 9 &211s LENGTH: 107 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amino acid sequence of the light chain variable region of certolizumab pegol.

<4 OOs, SEQUENCE: 9 Asp Ile Gln Met Thr Glin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1. 5 1O 15

Asp Arg Val Thir Ile Thr Cys Lys Ala Ser Glin Asn Val Gly. Thir Asn 2O 25 3O

Val Ala Trp Tyr Glin Gln Llys Pro Gly Lys Ala Pro Lys Ala Lieu. Ile 35 4 O 45 US 9,573.996 B2 59 60 - Continued

Tyr Ser Ala Ser Phe Lieu. Tyr Ser Gly Wall Pro Tyr Arg Phe Ser Gly SO 55 6 O

Ser Gly Ser Gly Thr Asp Phe Thr Luell Thir Ile Ser Ser Lieu. Glin Pro 65 70 7s 8O

Glu Asp Phe Ala Thr Tyr Tyr Cys Glin Glin Asn Ile Tyr Pro Luell 85 90 95

Thir Phe Gly Glin Gly Thr Lys Val Glu Ile 1OO 105

<210s, SEQ ID NO 10 &211s LENGTH: 353 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 10

Ala Ser Pro Thir Ser Pro Llys Val Phe Pro Luell Ser Luell Ser Thir 1. 5 15

Glin Pro Asp Gly Asn. Wal Wall Ile Ala Luell Wall Glin Gly Phe Phe 25

Pro Glin Glu Pro Leu Ser Wall. Thir Trp Ser Glu Ser Gly Glin Gly Wall 35 4 O 45

Thir Ala Arg Asn Phe Pro Pro Ser Glin Asp Ala Ser Gly Asp Luell Tyr SO 55 6 O

Thir Thir Ser Ser Gln Lieu. Thir Lieu Pro Ala Thir Gln Cys Luell Ala Gly 65 70 8O

Ser Wall Thir Cys His Wall Lys His Tyr Thir Asn. Pro Ser Glin Asp 85 90 95

Wall Thir Wall Pro Cys Pro Val Pro Ser Thir Pro Pro Thir Pro Ser Pro 105 11 O

Ser Thir Pro Pro Thir Pro Ser Pro Ser His Pro Arg Luell Ser 115 12 O 125

Lell His Arg Pro Ala Lieu. Glu Asp Luell Luell Luell Gly Ser Glu Ala Asn 13 O 135 14 O

Lell Thir Thir Lieu. Thr Gly Lieu. Arg Asp Ala Ser Gly Wall Thir Phe 145 150 155 160

Thir Trp Thir Pro Ser Ser Gly Lys Ser Ala Wall Gln Gly Pro Pro Glu 1.65 17O 17s

Arg Asp Luell Cys Gly Cys Tyr Ser Wall Ser Ser Wall Lieu Pro Gly 185 19 O

Ala Glu Pro Trp Asn His Gly Lys Thir Phe Thir Cys Thr Ala Ala 195 2OO 2O5

Pro Glu Ser Thr Pro Leu. Thir Ala Thir Luell Ser Lys Ser Gly Asn 21 O 215 22O

Thir Phe Arg Pro Glu Val His Lieu. Luell Pro Pro Pro Ser Glu Glu Luell 225 23 O 235 24 O

Ala Luell Asn Glu Lieu Wall. Thir Lieu. Thir Cys Luell Ala Arg Gly Phe Ser 245 250 255

Pro Asp Wall Lieu Val Arg Trp Luell Glin Gly Ser Glin Glu Luell Pro 26 O 265 27 O

Arg Glu Lys Lieu. Thir Trp Ala Ser Arg Glin Glu Pro Ser Glin Gly 27s 28O 285

Thir Thir Thir Phe Ala Wall. Thir Ser Ile Luell Arg Wall Ala Ala Glu Asp 29 O 295 3 OO

Trp Gly Asp Thr Phe Ser Met Wall Gly His Glu Ala Luell 3. OS 310 315 32O US 9,573.996 B2 61 - Continued

Pro Lieu Ala Phe Thr Glin Llys Thir Ile Asp Arg Lieu Ala Gly Lys Pro 3.25 330 335 Thr His Val Asn Val Ser Val Val Met Ala Glu Val Asp Gly Thr 34 O 345 35. O Tyr

<210s, SEQ ID NO 11 &211s LENGTH: 34 O 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 11 Ala Ser Pro Thr Ser Pro Llys Val Phe Pro Leu Ser Lieu. Asp Ser Thir 1. 5 1O 15

Pro Glin Asp Gly Asn Val Val Val Ala Cys Lieu Val Glin Gly Phe Phe 2O 25 3O

Pro Glin Glu Pro Leu Ser Val Thir Trp Ser Glu Ser Gly Glin Asn Wall 35 4 O 45 Thir Ala Arg Asn. Phe Pro Pro Ser Glin Asp Ala Ser Gly Asp Lieu. Tyr SO 55 6 O Thir Thr Ser Ser Glin Lieu. Thir Lieu Pro Ala Thr Glin Cys Pro Asp Gly 65 70 7s Lys Ser Val Thr Cys His Val Llys His Tyr Thr Asn Pro Ser Glin Asp 85 90 95

Val Thr Val Pro Cys Pro Val Pro Pro Pro Pro Pro Cys Cys His Pro 1OO 105 11 O

Arg Lieu. Ser Lieu. His Arg Pro Ala Lieu. Glu Asp Lieu Lleu Lieu. Gly Ser 115 12 O 125 Glu Ala Asn Lieu. Thir Cys Thr Lieu. Thr Gly Lieu. Arg Asp Ala Ser Gly 13 O 135 14 O Ala Thr Phe Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val Glin Gly 145 150 155 160

Pro Pro Glu Arg Asp Lieu. Cys Gly Cys Tyr Ser Val Ser Ser Val Luell 1.65 17O 17s

Pro Gly Cys Ala Glin Pro Trp Asn His Gly Glu Thr Phe Thr Cys Thir 18O 185 19 O Ala Ala His Pro Glu Lieu Lys Thr Pro Leu. Thir Ala Asn Ile Thr 195 2OO 2O5

Ser Gly Asn Thr Phe Arg Pro Glu Val His Leu Lleu Pro Pro Pro Ser 21 O 215 22O Glu Glu Lieu Ala Lieu. Asn. Glu Lieu Val Thir Lieu. Thir Cys Lieu Ala Arg 225 23 O 235 24 O

Gly Phe Ser Pro Lys Asp Val Lieu Val Arg Trp Lieu. Glin Gly Ser Glin 245 250 255

Glu Lieu Pro Arg Glu Lys Tyr Lieu. Thir Trp Ala Ser Arg Glin Glu Pro 26 O 265 27 O

Ser Glin Gly Thr Thr Thr Phe Ala Val Thr Ser Ile Lieu. Arg Val Ala 27s 28O 285

Ala Glu Asp Trp Llys Lys Gly Asp Thir Phe Ser Cys Met Val Gly His 29 O 295 3 OO

Glu Ala Lieu Pro Lieu Ala Phe Thr Glin Llys Thir Ile Asp Arg Lieu. Ala 3. OS 310 315 32O

Gly Llys Pro Thr His Val Asn Val Ser Val Val Met Ala Glu Val Asp 3.25 330 335 US 9,573.996 B2 63 - Continued

Gly Thr Cys Tyr 34 O

<210s, SEQ ID NO 12 &211s LENGTH: 34 O 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 12 Ala Ser Pro Thr Ser Pro Llys Val Phe Pro Leu Ser Lieu. Asp Ser Thr 1. 5 1O 15 Pro Glin Asp Gly Asn Val Val Val Ala Cys Lieu Val Glin Gly Phe Phe 2O 25 3O Pro Glin Glu Pro Leu Ser Val Thir Trp Ser Glu Ser Gly Glin Asn Val 35 4 O 45 Thir Ala Arg Asn. Phe Pro Pro Ser Glin Asp Ala Ser Gly Asp Lieu. Tyr SO 55 6 O Thir Thr Ser Ser Glin Lieu. Thir Lieu Pro Ala Thr Glin Cys Pro Asp Gly 65 70 7s 8O Lys Ser Val Thr Cys His Val Llys His Tyr Thr Asn Ser Ser Glin Asp 85 90 95 Val Thr Val Pro Cys Arg Val Pro Pro Pro Pro Pro Cys Cys His Pro 1OO 105 11 O Arg Lieu. Ser Lieu. His Arg Pro Ala Lieu. Glu Asp Lieu Lleu Lieu. Gly Ser 115 12 O 125 Glu Ala Asn Lieu. Thr Cys Thr Lieu. Thr Gly Lieu. Arg Asp Ala Ser Gly 13 O 135 14 O Ala Thr Phe Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val Glin Gly 145 150 155 160 Pro Pro Glu Arg Asp Lieu. Cys Gly Cys Tyr Ser Val Ser Ser Val Lieu. 1.65 17O 17s Pro Gly Cys Ala Glin Pro Trp Asn His Gly Glu Thr Phe Thr Cys Thr 18O 185 19 O Ala Ala His Pro Glu Lieu Lys Thr Pro Lieu. Thir Ala Asn. Ile Thir Lys 195 2OO 2O5 Ser Gly Asn Thr Phe Arg Pro Glu Val His Leu Lleu Pro Pro Pro Ser 21 O 215 22O Glu Glu Lieu Ala Lieu. Asn. Glu Lieu Val Thir Lieu. Thir Cys Lieu Ala Arg 225 23 O 235 24 O Gly Phe Ser Pro Lys Asp Val Lieu Val Arg Trp Lieu. Glin Gly Ser Glin 245 250 255 Glu Lieu Pro Arg Glu Lys Tyr Lieu. Thir Trp Ala Ser Arg Glin Glu Pro 26 O 265 27 O Ser Glin Gly Thr Thr Thr Tyr Ala Val Thr Ser Ile Lieu. Arg Val Ala 27s 28O 285 Ala Glu Asp Trp Llys Lys Gly Glu Thr Phe Ser Cys Met Val Gly His 29 O 295 3 OO

Glu Ala Lieu Pro Lieu Ala Phe Thr Glin Llys Thir Ile Asp Arg Met Ala 3. OS 310 315 32O

Gly Llys Pro Thr His Ile Asin Val Ser Val Val Met Ala Glu Ala Asp 3.25 330 335 Gly Thr Cys Tyr 34 O US 9,573.996 B2 65 - Continued

<210s, SEQ ID NO 13 &211s LENGTH: 34 O 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 13 Ala Ser Pro Thr Ser Pro Llys Val Phe Pro Leu Ser Lieu. Asp Ser Thr 1. 5 1O 15 Pro Glin Asp Gly Asn Val Val Val Ala Cys Lieu Val Glin Gly Phe Phe 2O 25 3O Pro Glin Glu Pro Leu Ser Val Thir Trp Ser Glu Ser Gly Glin Asn Val 35 4 O 45 Thir Ala Arg Asn. Phe Pro Pro Ser Glin Asp Ala Ser Gly Asp Lieu. Tyr SO 55 6 O Thir Thr Ser Ser Glin Lieu. Thir Lieu Pro Ala Thr Glin Cys Pro Asp Gly 65 70 7s 8O Lys Ser Val Thr Cys His Val Llys His Tyr Thr Asn Ser Ser Glin Asp 85 90 95 Val Thr Val Pro Cys Arg Val Pro Pro Pro Pro Pro Cys Cys His Pro 1OO 105 11 O Arg Lieu. Ser Lieu. His Arg Pro Ala Lieu. Glu Asp Lieu Lleu Lieu. Gly Ser 115 12 O 125 Glu Ala Asn Lieu. Thir Cys Thr Lieu. Thr Gly Lieu. Arg Asp Ala Ser Gly 13 O 135 14 O Ala Thr Phe Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val Glin Gly 145 150 155 160 Pro Pro Glu Arg Asp Lieu. Cys Gly Cys Tyr Ser Val Ser Ser Val Lieu. 1.65 17O 17s Pro Gly Cys Ala Glin Pro Trp Asn His Gly Glu Thr Phe Thr Cys Thr 18O 185 19 O Ala Ala His Pro Glu Lieu Lys Thr Pro Lieu. Thir Ala Asn. Ile Thir Lys 195 2OO 2O5 Ser Gly Asn Thr Phe Arg Pro Glu Val His Leu Lleu Pro Pro Pro Ser 21 O 215 22O Glu Glu Lieu Ala Lieu. Asn. Glu Lieu Val Thir Lieu. Thir Cys Lieu Ala Arg 225 23 O 235 24 O Gly Phe Ser Pro Lys Asp Val Lieu Val Arg Trp Lieu. Glin Gly Ser Glin 245 250 255 Glu Lieu Pro Arg Glu Lys Tyr Lieu. Thir Trp Ala Ser Arg Glin Glu Pro 26 O 265 27 O Ser Glin Gly Thr Thr Thr Phe Ala Val Thr Ser Ile Lieu. Arg Val Ala 27s 28O 285 Ala Glu Asp Trp Llys Lys Gly Asp Thir Phe Ser Cys Met Val Gly His 29 O 295 3 OO Glu Ala Lieu Pro Lieu Ala Phe Thr Glin Llys Thir Ile Asp Arg Lieu Ala 3. OS 310 315 32O Gly Llys Pro Thr His Val Asn Val Ser Val Val Met Ala Glu Val Asp 3.25 330 335 Gly Thr Cys Tyr 34 O

<210s, SEQ ID NO 14 &211s LENGTH: 107 212. TYPE: PRT <213> ORGANISM: Homo sapiens US 9,573.996 B2 67 - Continued

<4 OOs, SEQUENCE: 14 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1. 5 1O 15 Glin Lieu Lys Ser Gly Thr Ala Ser Val Val Cys Lieu. Lieu. Asn. Asn. Phe 2O 25 3O Tyr Pro Arg Glu Ala Lys Val Glin Trp Llys Val Asp Asn Ala Lieu. Glin 35 4 O 45 Ser Gly Asn. Ser Glin Glu Ser Val Thr Glu Glin Asp Ser Lys Asp Ser SO 55 6 O Thr Tyr Ser Leu Ser Ser Thr Lieu. Thir Leu Ser Lys Ala Asp Tyr Glu 65 70 7s 8O Lys His Llys Val Tyr Ala Cys Glu Val Thr His Glin Gly Lieu. Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu. Cys 1OO 105

<210s, SEQ ID NO 15 &211s LENGTH: 106 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 15 Gly Glin Pro Lys Ala Asn Pro Thr Val Thr Lieu Phe Pro Pro Ser Ser 1. 5 1O 15 Glu Glu Lieu. Glin Ala Asn Lys Ala Thr Lieu Val Cys Lieu. Ile Ser Asp 2O 25 3O Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro 35 4 O 45 Val Lys Ala Gly Val Glu Thir Thr Llys Pro Ser Lys Glin Ser Asn. Asn SO 55 6 O Lys Tyr Ala Ala Ser Ser Tyr Lieu Ser Lieu. Thr Pro Glu Glin Trp Llys 65 70 7s 8O Ser His Arg Ser Tyr Ser Cys Glin Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu. Cys Ser 1OO 105

<210s, SEQ ID NO 16 &211s LENGTH: 106 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 16 Gly Glin Pro Lys Ala Ala Pro Ser Val Thr Lieu Phe Pro Pro Ser Ser 1. 5 1O 15 Glu Glu Lieu. Glin Ala Asn Lys Ala Thr Lieu Val Cys Lieu. Ile Ser Asp 2O 25 3O

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 35 4 O 45

Val Lys Ala Gly Val Glu Thir Thr Thr Pro Ser Lys Glin Ser Asn Asn SO 55 6 O

Lys Tyr Ala Ala Ser Ser Tyr Lieu Ser Lieu. Thr Pro Glu Glin Trp Llys 65 70 7s 8O

Ser His Arg Ser Tyr Ser Cys Glin Val Thr His Glu Gly Ser Thr Val 85 90 95

Glu Lys Thr Val Ala Pro Thr Glu. Cys Ser US 9,573.996 B2 69 70 - Continued

1OO 105

<210s, SEQ ID NO 17 &211s LENGTH: 106 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 17

Gly Glin Pro Lys Ala Ala Pro Ser Wall Thir Luell Phe Pro Pro Ser Ser 1. 5 15

Glu Glu Lieu. Glin Ala Asn Lys Ala Thir Luell Wall Cys Lell Ile Ser Asp 2O 25

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Ala Asp Ser Ser Pro 35 4 O 45

Ala Lys Ala Gly Val Glu Thir Thr Thir Pro Ser Lys Glin Ser Asn Asn SO 55 6 O

Llys Tyr Ala Ala Ser Ser Tyr Lieu Ser Luell Thir Pro Glu Glin Trp Lys 65 70

Ser His Llys Ser Tyr Ser Cys Glin Wall Thir His Glu Gly Ser Thir Wall 85 90 95

Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 1OO 105

<210s, SEQ ID NO 18 &211s LENGTH: 106 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 18

Gly Glin Pro Lys Ala Ala Pro Ser Wall Thir Luell Phe Pro Pro Ser Ser 1. 5 15

Glu Glu Lieu. Glin Ala Asn Lys Ala Thir Luell Wall Cys Lell Ile Ser Asp 2O 25

Phe Tyr Pro Gly Ala Val Lys Val Ala Trp Ala Asp Gly Ser Pro 35 4 O 45

Val Asn Thr Gly Val Glu Thir Thr Thir Pro Ser Lys Glin Ser Asn Asn 6 O

Llys Tyr Ala Ala Ser Ser Tyr Lieu Ser Luell Thir Pro Glu Glin Trp Lys 65 70 7s

Ser His Arg Ser Tyr Ser Cys Glin Wall Thir His Glu Gly Ser Thir Wall 85 90 95

Glu Lys Thr Val Ala Pro Ala Glu Cys Ser 1OO 105

<210s, SEQ ID NO 19 &211s LENGTH: 106 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 19

Gly Glin Pro Lys Ala Ala Pro Ser Wall Thir Luell Phe Pro Pro Ser Ser 1. 5 15

Glu Glu Lieu. Glin Ala Asn Lys Ala Thir Luell Wall Cys Lell Wall Ser Asp 2O 25 3O

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Ala Asp Gly Ser Pro 35 4 O 45

Val Llys Val Gly Val Glu Thir Thr Pro Ser Lys Glin Ser Asn Asn SO 55 6 O US 9,573.996 B2 71 - Continued

Lys Tyr Ala Ala Ser Ser Tyr Lieu Ser Lieu. Thr Pro Glu Glin Trp Llys 65 70 7s 8O Ser His Arg Ser Tyr Ser Cys Arg Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Ala Glu. Cys Ser 1OO 105

<210s, SEQ ID NO 2 O &211s LENGTH: 137 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 2O Glin Glu Asp Glu Arg Ile Val Lieu Val Asp Asn Lys Cys Lys Cys Ala 1. 5 1O 15 Arg Ile Thir Ser Arg Ile Ile Arg Ser Ser Glu Asp Pro Asn. Glu Asp 2O 25 3O Ile Val Glu Arg Asn. Ile Arg Ile Ile Val Pro Lieu. Asn. Asn Arg Glu 35 4 O 45 Asn Ile Ser Asp Pro Thr Ser Pro Leu Arg Thr Arg Phe Val Tyr His SO 55 6 O Lieu. Ser Asp Lieu. Cys Llys Lys Cys Asp Pro Thr Glu Val Glu Lieu. Asp 65 70 7s 8O Asn Glin Ile Val Thr Ala Thr Glin Ser Asn. Ile Cys Asp Glu Asp Ser 85 90 95 Ala Thr Glu Thir Cys Tyr Thr Tyr Asp Arg ASn Lys Cys Tyr Thr Ala 1OO 105 11 O Val Val Pro Leu Val Tyr Gly Gly Glu Thir Lys Met Val Glu. Thir Ala 115 12 O 125 Lieu. Thr Pro Asp Ala Cys Tyr Pro Asp 13 O 135

<210s, SEQ ID NO 21 &211s LENGTH: 585 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 21 Lys Ser Pro Ile Phe Gly Pro Glu Glu Val Asn Ser Val Glu Gly Asn 1. 5 1O 15 Ser Val Ser Ile Thr Cys Tyr Tyr Pro Pro Thr Ser Val Asn Arg His 2O 25 3O Thir Arg Llys Tyr Trp Cys Arg Glin Gly Ala Arg Gly Gly Cys Ile Thr 35 4 O 45 Lieu. Ile Ser Ser Glu Gly Tyr Val Ser Ser Lys Tyr Ala Gly Arg Ala SO 55 6 O

Asn Lieu. Thir Asn Phe Pro Glu Asn Gly Thr Phe Val Val Asn Ile Ala 65 70 7s 8O

Glin Lieu. Ser Glin Asp Asp Ser Gly Arg Tyr Lys Cys Gly Lieu. Gly Ile 85 90 95

Asn Ser Arg Gly Lieu. Ser Phe Asp Val Ser Lieu. Glu Val Ser Glin Gly 1OO 105 11 O

Pro Gly Lieu. Lieu. Asn Asp Thr Llys Val Tyr Thr Val Asp Lieu. Gly Arg 115 12 O 125

Thr Val Thir Ile Asn Cys Pro Phe Llys Thr Glu Asn Ala Glin Lys Arg 13 O 135 14 O US 9,573.996 B2 73 74 - Continued

Lys Ser Luell Glin Ile Gly Luell Pro Wall Lell Wall Ile Asp 145 150 155 160

Ser Ser Gly Wall Asn Pro Asn Thir Gly Arg Ile Arg Luell Asp 1.65 17O 17s

Ile Glin Gly Thir Gly Glin Lell Luell Phe Ser Wall Wall Ile Asn Glin Luell 18O 185 19 O

Arg Luell Ser Asp Ala Gly Glin Tyr Luell Glin Ala Gly Asp Asp Ser 195

Asn Ser Asn Asn Ala Asp Luell Glin Wall Lell Pro Glu Pro 21 O 215

Glu Luell Wall Glu Asp Lell Arg Gly Ser Wall Thir Phe His Ala 225 23 O 235 24 O

Lell Gly Pro Glu Wall Ala Asn Wall Ala Lys Phe Lell Arg Glin Ser 245 250 255

Ser Gly Glu Asn Asp Wall Wall Wall Asn Thir Lell Gly Lys Arg Ala 26 O 265 27 O

Pro Ala Phe Glu Gly Arg Ile Luell Luell Asn Pro Glin Asp Asp Gly 27s 285

Ser Phe Ser Wall Wall Ile Thir Gly Luell Arg Glu Asp Ala Gly Arg 29 O 295 3 OO

Tyr Luell Gly Ala His Ser Asp Gly Glin Luell Glin Glu Gly Ser Pro 3. OS 310 315

Ile Glin Ala Trp Glin Lell Phe Wall Asn Glu Glu Ser Thir Ile Pro Arg 3.25 330 335

Ser Pro Thir Wall Wall Gly Wall Ala Gly Gly Ser Wall Ala Wall Luell 34 O 345 35. O

Pro Tyr Asn Arg Glu Ser Ser Ile Tyr Trp Luell 355 360 365

Trp Glu Gly Ala Glin Asn Gly Arg Pro Luell Lell Wall Asp Ser Glu 37 O 375

Gly Trp Wall Ala Glin Glu Gly Arg Luell Ser Lell Luell Glu Glu 385 390 395 4 OO

Pro Gly Asn Gly Thir Phe Thir Wall Ile Luell ASn Glin Lell Thir Ser Arg 4 OS 415

Asp Ala Gly Phe Trp Luell Thir Asn Gly Asp Thir Luell Trp Arg 42O 425 43 O

Thir Thir Wall Glu Ile Ile Ile Glu Gly Glu Pro Asn Luell Wall 435 44 O 445

Pro Gly Asn Wall Thir Ala Wall Luell Gly Glu Thir Lell Wall Pro 450 45.5 460

His Phe Pro Phe Ser Ser Glu Lys Trp Trp 465 470 48O

Asn Asn Thir Gly Cys Glin Ala Luell Pro Ser Glin Asp Glu Gly Pro Ser 485 490 495

Ala Phe Wall Asn Asp Glu Asn Ser Arg Lell Wall Ser Luell Thir SOO 505 51O

Lell Asn Luell Wall Thir Ala Asp Glu Gly Trp Trp Gly Wall 515 525

Glin Gly His Phe Tyr Gly Glu Thir Ala Ala Wall Wall Ala Wall 53 O 535 54 O

Glu Glu Arg Ala Ala Gly Ser Arg Asp Wall Ser Lell Ala Ala 5.45 550 555 560 US 9,573.996 B2 75 76 - Continued Asp Ala Ala Pro Asp Glu Lys Val Lieu. Asp Ser Gly Phe Arg Glu Ile 565 st O sts Glu Asn Lys Ala Ile Glin Asp Pro Arg 58O 585

<210s, SEQ ID NO 22 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 22 Met Lys Asn His Lieu Lleu Phe Trp Gly Val Lieu Ala Val Phe Ile Llys 1. 5 1O 15 Ala Wal His Val Lys Ala 2O

<210s, SEQ ID NO 23 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amino acid sequence for a signal peptide (heavy chain secretion signal). <4 OOs, SEQUENCE: 23 Met Glu Lieu. Gly Lieu. Cys Trp Val Phe Lieu Val Ala Ile Lieu. Glu Gly 1. 5 1O 15 Val Glin Cys

<210s, SEQ ID NO 24 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amino acid sequence for a signal peptide (light chain secretion signal). <4 OOs, SEQUENCE: 24 Met Lys Tyr Lieu Lleu Pro Thr Ala Ala Ala Gly Lieu. Lieu. Lieu. Lieu Ala 1. 5 1O 15

Ala Glin Pro Ala Met Ala 2O

<210s, SEQ ID NO 25 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amino acid sequence for a signal peptide (SC chain secretion signal).

<4 OOs, SEQUENCE: 25 Met Ala Met Leu Lleu Phe Val Lieu. Thr Cys Lieu. Leu Ala Val Phe Pro 1. 5 1O 15

Ala Ile Ser Thr 2O

<210s, SEQ ID NO 26 &211s LENGTH: 31 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amino acid sequence for rice alpha-amylase signal peptide (secretion signal).

US 9,573.996 B2 97 - Continued

Thir Lieu Val Thir Wal Ser Ser 115

<210s, SEQ ID NO 38 &211s LENGTH: 107 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amino acid sequence of the light chain variable region of ustekinumab (CTNO-1275). <4 OOs, SEQUENCE: 38

Asp Ile Gln Met Thr Glin Ser Pro Ser Ser Lieu. Ser Ala Ser Wall Gly 1. 5 1O 15

Asp Arg Val Thir Ile Thr Cys Arg Ala Ser Glin Gly Ile Ser Ser Trp 2O 25

Lell Ala Trp Tyr Glin Gln Llys Pro Glu Lys Ala Pro Lys Ser Luell Ile 35 4 O 45

Ala Ala Ser Ser Lieu. Glin Ser Gly Val Pro Ser Arg Phe Ser Gly SO 55 6 O

Ser Gly Ser Gly Thr Asp Phe Thr Lieu. Thir Ile Ser Ser Luell Glin Pro 65 70 7s 8O

Glu Asp Phe Ala Thr Tyr Tyr Cys Glin Glin Tyr Asn Ile Pro 85 90 95

Thir Phe Gly Glin Gly Thr Llys Lieu. Glu Ile Llys 1OO 105

<210 SEQ ID NO 39 &211s LENGTH: 115 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Amino acid sequence of the heavy chain variable region of briakinumab (CJ-695, ABT-874). <4 OOs, SEQUENCE: 39

Glin Val Glin Lieu Val Glu Ser Gly Gly Gly Val Val Glin Pro Gly Arg 1. 5 1O 15

Ser Lieu. Arg Lieu. Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser 2O 25

Gly Met His Trp Val Arg Glin Ala Pro Gly Lys Gly Lell Glu Trp Wall 35 4 O 45

Ala Phe Ile Arg Tyr Asp Gly Ser Asn Llys Tyr Tyr Ala Asp Ser Wall SO 55 6 O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn. Ser Lys Asn Thir Luell Tyr 65 70 7s

Lell Gln Met Asn. Ser Lieu. Arg Ala Glu Asp Thr Ala Wall Tyr 85 90 95

Thr His Gly Ser His Asp Asn Trp Gly Glin Gly Thir Met Wall Thir 1OO 105 11 O

Wall Ser Ser 115

<210s, SEQ ID NO 4 O &211s LENGTH: 112 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: OTHER INFORMATION: Amino acid sequence of the light chain variable region of briakinumab (J-695, ABT-874).

US 9,573.996 B2 101 102 - Continued ttct Cotgca tdtcggc.ca caggcgctg. CCC ct cqcct t cacccagala gaccatcgac 144 O cggctggc.cg ggaagcc.cac ccacgtcaac git cagcgt.cg titatggctgaggttgacggg 15OO acgtgctac 1509

SEQ ID NO 42 LENGTH 735 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Lemna-optimized complete SILA light chain DNA (including signal peptide SEQ ID NO: 26).

<4 OOs, SEQUENCE: 42 atgcaggit co togalacacgat gigt caacaag cact tcc tot c cctdtc.cgt. cct catcgt.c 6 O

CtcCtcgggc tigagcagcaa cct caccgcc ggcga catcC agatgacgca gagcc.cgt.cg 12 O agcctgtc.cg cct cogtcgg ggaCaggg.tc acgat cacct gcagagc.ca.g cCagggcatc 18O tcgt.cctggc tiggcctggta t caacagaag CCC gaga agg citc.cgaagtic cctgat citac 24 O gcggc.cagct c cct coagag cggcgtc.ccg agc.cggttct CC9gcagcgg citc.cggcacc 3OO gactitcaccc ticaccatcag citcgct coag ccc.gaggact tcqccaccita c tact.gc.ca.g 360 cagtacaa.ca totaccc.gta cacgttcggg Cagggcacga agctggagat caa.gc.gcacg 42O

c cagogtc.tt catct tcc.cg ccct cqgacg agcagctgaa gtc.cggCact 48O gcct cogtgg tctgcc tigct caacaact to taccc.ccg.cg aagccaaggt gCagtggaag 54 O gttgacaacg. c9ctCcagag C9ggaactCC Caggagt CC tgaCC gagca ggact CCaag 6OO gact coacct acagoctdtc cagcaccctic accct ct coa aggc.cgact a cgaga agcac 660 alaggtgtacg Cctgcgaggit gacccaccag ggcct ct cot CCC cqgtgac caagt ccttic 72 O alacc.gcggtg agtgc 73

The invention claimed is: 40 5. The antibody according to claim 1, wherein said 1. A monoclonal Secretory IgA antibody, which binds to proinflammatory cytokine is TNF-C. and wherein said anti and neutralizes a human proinflammatory cytokine, wherein body neutralizes TNF-C. by at least 50%. said antibody comprises: 6. The antibody according to claim 5, wherein said (a) a light chain constant region having the sequence of proinflammatory cytokine is TNF-C. and wherein said anti SEQ ID NO:14, a heavy chain variable region having the sequence of SEQ 45 body neutralizes TNF-C. by least 98%. ID NO:4, and 7. A pharmaceutical composition comprising a therapeu a light chain variable region having the sequence of SEQ tically effective amount of a monoclonal Secretory IgA ID NO:5; or antibody, which binds to and neutralizes a human proin (b) a light chain constant region having the sequence of flammatory cytokine, wherein said antibody comprises: SEQID NO: 14, a heavy chain variable region having (a) a light chain constant region having the sequence of the sequence of SEQ ID NO:37, and SEQ ID NO:14, a light chain variable region having the sequence of SEQ a heavy chain variable region having the sequence of SEQ ID NO: 38. ID NO:4; and 2. The antibody according to claim 1, wherein said a light chain variable region having the sequence of SEQ antibody comprises: 55 (a) a light chain constant region having the sequence of ID NO:5; or SEQ ID NO:14: (b) a light chain constant region having the sequence of (b) a heavy chain variable region having a sequence of SEQ ID NO: 14, SEQ ID NO:4; and a heavy chain variable region having the sequence of SEQ (c) a light chain variable region having a sequence of SEQ 60 ID NO:37, and ID NO:5. a light chain variable region having the sequence of SEQ 3. The antibody according to claim 1, wherein said ID NO:38; and antibody comprises a human secretory chain having the at least one pharmaceutically acceptable excipient. sequence of SEQ ID NO:21. 8. The composition of claim 7, wherein the composition 4. The antibody according to claim 1, wherein said 65 comprises about 0.01 to 1000 mg of the antibody. antibody comprises a human J-chain having the sequence of 9. The composition of claim 7, wherein the composition SEQ ID NO:20. comprises a plurality of secretory IgA antibodies. US 9,573.996 B2 103 104 10. The composition of claim 9, wherein said plurality of the sequence of SEQ ID NO: 37, and a light chain secretory IgA antibodies contains at least about 30% GO variable region having the sequence of SEQID NO:38: glycans. and 11. The composition of claim 10, wherein said G0 glycans (c) a human secretory chain having the sequence of SEQ ID NO:21, or minor variations thereof, wherein the lack fucose and Xylose residues. 5 minor variations are no more than 10 conservative 12. The composition of claim 7, wherein said plurality of amino acid additions, deletions, or Substitutions, and secretory IgA antibodies contains at least at least about 25% wherein said amino acid insertions, Substitutions, or dele high-mannose glycans. tions do not significantly alter the ability of the secre 13. The composition of claim 12, wherein said high tory chain to stabilize secretory IgA proteolysis. mannose glycans are selected from the group consisting of 10 21. A monoclonal secretory IgA antibody, which binds to Man5, Manó, Man7, Man8, and Man9. and neutralizes a human proinflammatory cytokine, wherein 14. The composition of claim 9, wherein said plurality of said antibody comprises: secretory IgA antibodies contains at least 70% of G0 glycans (a) a light chain constant region having the sequence of and high-mannose glycans relative to the total amount of SEQ ID NO:14, 15 a heavy chain variable region having the sequence of SEQ N-glycans in the plurality of secretory IgA antibodies. ID NO:4, and a light chain variable region having the 15. The composition of claim 7, wherein the composition sequence of SEQ ID NO:5; or can be administered to a human by oral, rectal, buccal, (b) a light chain constant region having the sequence of topical, parenteral, or Subcutaneous administration. SEQID NO: 14, a heavy chain variable region having 16. The composition of claim 7, wherein the composition the sequence of SEQ ID NO: 37, and a light chain comprises less than 10% of dimer IgA. variable region having the sequence of SEQID NO:38: 17. The composition of claim 16, wherein the composi and tion comprises less than 10% of monomeric IgA. (c) a human J-chain having the sequence of SEQ ID 18. A monoclonal secretory IgA antibody, which binds to NO:20, or minor variations thereof, wherein the minor and neutralizes a human proinflammatory cytokine, wherein 25 variations are no more than 10 conservative amino acid said antibody comprises: additions, deletions, or Substitutions, and (a) a heavy chain variable region having the sequence of wherein said amino acid insertions, Substitutions, or dele SEQID NO:4 and a light chain variable region having tions do not significantly alter the ability of the J-chain the sequence of SEQ ID NO:5; or to join two monomeric IgA antibodies to form a dimer (b) a heavy chain variable region having the sequence of 30 and to enable attachment of the secretory chain. SEQID NO:37 and a light chain variable region having 22. A method for treating an inflammatory disease in a the sequence of SEQ ID NO:38. human, which comprises administering an anti-inflamma 19. A monoclonal secretory IgA antibody, which binds to tory effective amount of a monoclonal Secretory IgA anti and neutralizes a human proinflammatory cytokine, wherein body which binds to and neutralizes a human proinflamma said antibody comprises: 35 tory cytokine, wherein said antibody comprises: (a) a light chain constant region having the sequence of (a) a light chain constant region having the sequence of SEQ ID NO:14, or minor variations thereof, a heavy SEQ ID NO:14: chain variable region having the sequence of SEQ ID a heavy chain variable region having the sequence of SEQ NO:4, and a light chain variable region having the ID NO:4; and sequence of SEQ ID NO:5; or 40 a light chain variable region having the sequence of SEQ (b) a light chain constant region having the sequence of ID NO:5; or SEQ ID NO: 14, or minor variations thereof, a heavy (b) a light chain constant region having the sequence of chain variable region having the sequence of SEQ ID SEQ ID NO: 14; NO: 37, and a light chain variable region having the a heavy chain variable region having the sequence of SEQ sequence of SEQ ID NO:38; and 45 ID NO:37; and wherein the minor variations are no more than 10 con a light chain variable region having the sequence of SEQ servative amino acid additions, deletions, or Substitu ID NO:38. tions. 23. The method according to claim 22, wherein said 20. A monoclonal secretory IgA antibody, which binds to administering is oral, rectal, buccal, topical, parenteral, or and neutralizes a human proinflammatory cytokine, wherein 50 Subcutaneous. said antibody comprises: 24. The method according to claim 23, wherein said (a) a light chain constant region having the sequence of disease is selected from the group consisting of rheumatoid SEQ ID NO:14, arthritis, inflammatory bowel disease, Crohn's disease, a heavy chain variable region having the sequence of SEQ ulcerative colitis, psoriasis, psoriatic arthritis, ankylosing ID NO:4, and a light chain variable region having the 55 spondylitis, juvenile idiopathic arthritis, uveitis, asthma, sequence of SEQ ID NO:5; or Alzheimer's disease, multiple Sclerosis, type II diabetes, (b) a light chain constant region having the sequence of systemic Sclerosis, lupus nephritis, and allergic rhinitis. SEQID NO: 14, a heavy chain variable region having k k k k k