U.S. patent application number 14/365934 was filed with the patent office on 2014-12-04 for expression of secretory iga antibodies in duckweed.
The applicant listed for this patent is Synthon Biopharmaceuticals B.V.. Invention is credited to Gerardus Joseph Andreas Ariaans, Guy De Roo, Declan Thomas Nolan, Jeff Regan, Frans van Dalen.
Application Number | 20140359902 14/365934 |
Document ID | / |
Family ID | 47504906 |
Filed Date | 2014-12-04 |
United States Patent
Application |
20140359902 |
Kind Code |
A1 |
Ariaans; Gerardus Joseph Andreas ;
et al. |
December 4, 2014 |
EXPRESSION OF SECRETORY IgA ANTIBODIES IN DUCKWEED
Abstract
Secretory IgA antibodies can be made by expression in a
stably-transformed duckweed.
Inventors: |
Ariaans; Gerardus Joseph
Andreas; (Nijmegen, NL) ; van Dalen; Frans;
(Nijmegen, NL) ; De Roo; Guy; (Nijmegen, NL)
; Nolan; Declan Thomas; (Nijmegen, NL) ; Regan;
Jeff; (Nijmegen, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Synthon Biopharmaceuticals B.V. |
Nijmegen |
|
NL |
|
|
Family ID: |
47504906 |
Appl. No.: |
14/365934 |
Filed: |
December 14, 2012 |
PCT Filed: |
December 14, 2012 |
PCT NO: |
PCT/EP2012/075673 |
371 Date: |
June 16, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61576727 |
Dec 16, 2011 |
|
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|
61576922 |
Dec 16, 2011 |
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Current U.S.
Class: |
800/288 |
Current CPC
Class: |
C07K 2317/13 20130101;
A61P 1/00 20180101; A61K 9/0053 20130101; A61P 19/02 20180101; C07K
2317/565 20130101; C07K 16/241 20130101; C07K 2317/76 20130101;
A61P 17/06 20180101; C12N 15/8258 20130101; A61K 2039/505 20130101;
C07K 2317/41 20130101; C07K 16/244 20130101; C07K 2317/24 20130101;
A61K 39/00 20130101; A61K 2039/54 20130101 |
Class at
Publication: |
800/288 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Claims
1. A method of producing a secretory IgA antibody, which comprises
expressing in a stably-transformed duckweed a fully-formed
secretory IgA antibody.
2. The method according to claim 1, wherein said stably-transformed
duckweed is a mature plant.
3. The method according to any of claims 1-2, wherein said duckweed
is selected from the group consisting of the stably-transformed
species of Lemna minor, Lemna minuscula, Lemna aequinoctialis, and
Lemna gibba, preferably L. minor.
4. The method according to any of claims 1-3, wherein said
expression occurs by culturing said duckweed on a liquid culture
media.
5. The method according to any of claims 1-4, wherein said
stably-transformed plant comprises DNA sequences encoding four
different promoters operably-linked to heavy chain, light chain,
J-chain, and secretory component polypeptides, respectively.
6. The method according to claim 5, wherein stably-transformed
plant further comprises DNA sequences encoding rice .alpha.-amylase
signal peptides operably-linked to said heavy chain, light chain,
J-chain, and secretory component polypeptides, respectively.
7. The method according to any of claims 1-6, wherein said
expressing step comprises expressing a plurality of secretory IgA
antibodies, wherein said plurality contains at least about 30% G0
glycans (preferably G0 glycans lacking Fuc and Xyl residues)
relative to the total amount of N-glycans in the population.
8. The method according to any of claims 1-6, wherein said
expressing step comprises expressing a plurality of secretory IgA
antibodies, wherein said plurality contains at least about 25%
high-mannose glycans (e.g., Man5, Man6, Man7, Man8, and/or Man9
glycans) relative to the total amount of N-glycans in the
population.
9. The method according to any of claims 7-8, wherein said G0
glycans (preferably G0 glycans lacking Fuc and Xyl residues) and
high-mannose glycans (e.g., Man5, Man6, Man7, Man8, and/or Man9
glycans) together are the majority of glycans present in the
plurality of secretory IgA antibodies, such as at least 70%
relative to the total amount of N-glycans.
10. The method according to any of claims 1-9, which further
comprises recovering said fully-formed secretory IgA antibody from
said duckweed.
11. The method according to claim 10, wherein said recovering step
comprises separating said duckweed from said liquid culture media
and extracting said fully-formed secretory IgA antibody from said
duckweed into an extraction media.
12. The method according to claim 11, which further comprises
purifying said recovered secretory IgA in said extraction media to
obtain a purified secretory IgA composition.
13. The method according to claim 12, wherein said purifying
comprises subjecting the secretory IgA to at least affinity
chromatography and size exclusion chromatography.
14. The method according to any of claims 1-4 and 7-13, further
comprising transforming a duckweed with a single expression vector
by Agrobacterium-mediated transformation in order to form said
stably-transformed duckweed, wherein said single expression vector
encodes heavy chain, light chain, J-chain, and secretory component
polypeptides.
15. The method according to claim 14, wherein said single
expression vector further encodes four different promoters
operably-linked to said heavy chain, light chain, J-chain, and
secretory component polypeptides, respectively.
16. The method according to claim 15, wherein said single
expression vector further encodes rice .alpha.-amylase signal
peptides operably-linked to said heavy chain, light chain, J-chain,
and secretory component polypeptides, respectively.
Description
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn.119(e) from U.S. provisional patent application Ser.
No. 61/576,727, filed Dec. 16, 2011, and Ser. No. 61/576,922, filed
Dec. 16, 2011; the entire contents of each provisional application
being incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The production of monoclonal antibodies and the development
of recombinant antibody technology have made antibodies a
clinically-important drug class. Many antibodies are on the market
for human therapy, and many more are in clinical development, for a
wide range of diseases, including autoimmunity and inflammation,
cancer, and infectious disease. Examples of currently marketed
antibodies include, among others, trastuzumab (HERCEPTIN.RTM.),
adalimumab (HUMIRA.RTM.) and natalizumab (TYSABRI.RTM.) in the
treatment of breast cancer, rheumatoid arthritis, and multiple
sclerosis, respectively.
[0003] IgG is the antibody class most widely adopted for
therapeutic antibodies. Currently most (if not all) commercial
therapeutic antibodies are of the IgG class. Production and
purification procedures for human IgG antibodies are well
established and reliably performed. See, e.g., Beyer et al.,
Journal of Immunology 346; 26-37 (2009).
[0004] In recent years, IgA antibodies have attracted increased
attention as potential therapeutics for infectious and malignant
diseases. Despite this, IgA antibodies have not been commercially
advanced. One of the main problems hampering work with IgA
antibodies is the lack of established methods for production and
purification. Clinical grade IgG antibodies are usually produced in
mammalian cell expression systems to achieve post-translational
modification and glycosylation patterns similar to humans. Vector
systems are commercially available and optimized for expression in
mammalian cells, e.g., in CHO cells--one of the most commonly used
mammalian expression systems. For the production of IgA antibodies,
however, expression vectors and cell lines have not been used
consistently. And despite several existing approaches to the
purification of IgA antibodies, no particular purification method
has achieved broad acceptance. See, e.g., 2009 Beyer.
[0005] Several different mammalian expression systems have been
used (at least on a research scale) to make IgA antibodies,
including (i) murine cell lines (e.g., Chintalacharuvu et al. Proc.
Natl. Acad. Sci. USA 94; 6364-6368 (June 1997), describing
anti-hapten mouse-human chimeric secretory IgA antibodies made by
expressing monomeric IgA in murine transfectomas and then
transfecting with human SC gene) and (ii) baby hamster kidney (BHK)
cell lines (e.g., Dechant et al. Blood 100(13); 4574-4580 (December
2002), describing anti-cancer chimeric monomeric IgA antibodies
made in BHK cells). Nevertheless, the literature seems to focus on
making IgA antibodies using CHO cell lines.
[0006] For example, one article describes making monomeric,
dimeric, and secretory anti-virus IgA antibodies in CHO cells.
Berdoz et al. Proc. Natl. Acad. Sci. USA 96; 3029-3034 (March
1999). In particular, this article describes generating single
clones to express each of the anti-virus monomeric IgA (mIgA clone
22), dimeric IgA (dIgA clone F), and secretory IgA (sIgA clone 6)
antibodies, and that clones expressed up to 20 ug of antibody per
1.times.10.sup.6 CHO cells in 24 hours. The article proposes that
the described CHO approach makes possible the production of large
quantities of secretory IgA for clinical applications, i.e.,
milligram quantities. Nonetheless, the same group later resorted to
a method of combining, in vitro, a dimeric IgA (expressed from CHO)
and a secretory chain to make fully assembled secretory IgAs. Favre
et al. (2003), Journal of Chromatography B, 786, pp 143-151.
[0007] Another article describes making anti-cancer monomeric IgA
antibodies in CHO cells. Dechant et al. Journal of Immunology 179;
2936-2943 (2007). The article describes that the disclosed CHO
system expressed 3 (IgA1) or 5 (IgA2) pg/cell/day. This article
further describes challenges in using the CHO system to express
IgAs, including a lack of homology between human and mouse receptor
systems, difficulties with large scale production and purification,
and obstacles in obtaining desired glycosylation. Still another
article describes the serum-free production and purification of
chimeric anti-cancer monomeric IgA antibodies in CHO cells. 2009
Beyer. This article describes that the disclosed CHO system
expressed 2.2 pg/cell/day, and that the disclosed CHO system
expressed 2 mg per week per roller bottle unit over 6 months (based
on data that is not shown). This article further describes that
difficulties in obtaining enough recombinant IgA material with
desired specificity for in vivo studies remained in 2009 because of
(among other reasons) a lack of established methods for production
and purification.
[0008] It has been proposed that plants expression systems have
potential advantages over traditional mammalian expression systems,
including lower production costs, the absence of animal pathogens,
rapid scalability, and the ability to fold complex proteins
accurately. See, e.g., Ma et al. Nat Rev Genet. 4; 794-805 (October
2003). Plant expression systems have been used for a variety of
recombinant proteins, but there is relatively little literature on
the expression of IgA antibodies in plant systems. Although one
article describes an anti-virus monomeric IgA made in maize
(Karnoup et al. Glycobiology 15(10); 965-981 (May 2005)), most
literature on this subject seems to be linked to one group, Planet
Biotechology Inc. This group describes in several articles an
anti-bacterial IgA-G hybrid antibody made in transgenic tobacco.
See, e.g., Ma et al. Science 268(5211), 716-719 (May 1995); Ma et
al. Nature Medicine 4(5); 601-606 (May 1998); Wycoff Current
Pharmaceutical Design 10(00); 1-9 (2004).
[0009] Specifically, the 1995 and 1998 Ma articles describe the
expression of an anti-Streptococcus mutans chimeric secretory IgA-G
hybrid antibody in tobacco. The hybrid antibody is described as
containing murine monoclonal kappa light chain, hybrid Ig A-G heavy
chain, murine J-chain, and rabbit secretory component. The antibody
was made by successive sexual crossing between four transgenic N.
tabacum plants and filial recombinants to form plant cells that
expressed all four protein chains simultaneously. The parent is
identified as the IgG antibody Guy's 13.
[0010] The 2004 Wycoff article also describes expressing in
transgenic tobacco the Guy's 13-based chimeric secretory IgA/G
hybrid antibody (called CaroRx in this article) and cites to the
1995 and 1998 Ma articles (among others). This article briefly
mentions that Planet Biotechnology Inc. had at some point
transformed plants with all four chains (heavy chain, light chain,
J-chain, and secretory component chain) simultaneously to produce a
sIgA that was fully human except for murine variable regions. But
this is based on unpublished results and is not further discussed.
Apparently, the CaroRx antibody was difficult to purify; the
affinity of Protein A for the murine Ig domain was too low and
Protein G was necessary for sufficient affinity chromatography.
Furthermore, the article states that several other chromatographic
media had shown little potential as purification steps for the
hybrid sIgA-G from tobacco leaf extracts. The article also
indicates that the authors were unable to control for human-like
glycosylation in tobacco, but that such was unlikely to be a
problem because people are exposed to plant glycans every day in
food without ill effect.
[0011] WO9949024, which lists Wycoff as an inventor, Planet
Biotechnology Inc. as the applicant, describes the use of the
variable regions of Guy's 13 to make a secretory antibody from
tobacco. The application contains only two examples--the first a
working example and the second a prophetic example. Working Example
1 describes the transient production of an anti-S. mutans (variable
regions from Guy's 13) in tobacco. The tobacco plant was
transiently transformed using particle bombardment of tobacco leaf
disks. Transgenic plants were then screened by Western blot "to
identify individual transformants expressing assembled human sIgA"
(p. 25). Prophetic Example 2 states that in a transformation system
for Lemna gibba (a monocot), bombardment of surface-sterilized leaf
tissue with DNA-coated particles "is much the same as with" tobacco
(a dicot). The prophetic example also stops at screening by
immunoblot analysis for antibody chains and assembled sIgA, and
states that the inventors "expect to find fully assembled
sIgA."
[0012] Despite the increased attention to plant expression systems
for antibodies and the use of IgA antibodies as therapeutic agents,
secretory IgA antibodies have not been commercially advanced.
Accordingly, it is desirable to have an expression system for the
efficient production and purification of therapeutic secretory IgA
antibodies.
SUMMARY OF THE INVENTION
[0013] The present invention relates to the discovery that duckweed
can serve as a useful system for the expression and production of
fully-formed secretory IgA antibodies. Accordingly, a first aspect
of the invention relates to a method of producing a secretory IgA
antibody, which comprises expressing in a stably-transformed
duckweed a fully-formed secretory IgA antibody. The duckweed that
is expressing the secretory IgA antibody can be a mature plant.
Once the stably-transformed duckweed is obtained, expression of the
secretory IgA antibody generally involves culturing the duckweed,
including growing mature plants. Subsequently the secretory IgA
antibody can be recovered, typically by extracting the antibody
from the tissue of the duckweed, and optionally purifying the
resulting antibody composition.
BRIEF DESCRIPTION OF THE SEQUENCES
[0014] SEQ ID NO:1 provides the amino acid sequence of a human K
light chain constant region (CL) (UniProtKB/Swiss-Prot database
entry P01834 (IGKC_HUMAN)).
[0015] SEQ ID NO:2 provides the amino acid sequence of a human
.lamda.1 light chain constant region (CL) (UniProtKB/Swiss-Prot
database entry P0CG04 (LAC1_HUMAN)).
[0016] SEQ ID NO:3 provides the amino acid sequence of a human
.lamda.2 light chain constant region (CL) (UniProtKB/Swiss-Prot
database entry P0CG05 (LAC2_HUMAN)).
[0017] SEQ ID NO:4 provides the amino acid sequence of a human
.lamda.3 light chain constant region (CL) (UniProtKB/Swiss-Prot
database entry P0CG06 (LAC3_HUMAN)).
[0018] SEQ ID NO:5 provides the amino acid sequence of a human
.lamda.6 light chain constant region (CL) (UniProtKB/Swiss-Prot
database entry P0CF74 (LAC6_HUMAN)).
[0019] SEQ ID NO:6 provides the amino acid sequence of a human
.lamda.7 light chain constant region (CL) (UniProtKB/Swiss-Prot
database entry A0M8Q6 (LAC7_HUMAN)).
[0020] SEQ ID NO:7 provides the amino acid sequence of a human IgA
.alpha.-1 heavy chain constant region
(C.alpha.1-C.alpha.2-C.alpha.3) (UniProtKB/Swiss-Prot database
entry P01876 (IGHA1_HUMAN)).
[0021] SEQ ID NO:8 provides the amino acid sequence of a human IgA
.alpha.-2 m(1)-allotype heavy chain constant region
(C.alpha.1-C.alpha.2-C.alpha.3) (UniProtKB/Swiss-Prot database
entry P01877 (IGHA2_HUMAN)).
[0022] SEQ ID NO:9 provides the amino acid sequence of a human IgA
.alpha.-2 m(2)-allotype heavy chain constant region
(Ca1-C.alpha.2-C.alpha.3). (UniProtKB/Swiss-Prot database entry
P01877 (IGHA2_HUMAN) with indicated modifications for allotype 2
variant).
[0023] SEQ ID NO:10 provides the amino acid sequence of a human IgA
.alpha.-2 (n)-allotype.
[0024] SEQ ID NO:11 provides the amino acid sequence of a human
J-chain (amino acids 23-159 UniProtKB/Swiss-Prot database entry
P01591).
[0025] SEQ ID NO:12 provides the amino acid sequence of a human
secretory component (amino acids 19-603 of UniProtKB/Swiss-Prot
database entry P01833 (PIGR_HUMAN), RCSB Protein Data Bank
structure 2OCW).
[0026] SEQ ID NO:13 provides the amino acid sequence for a signal
peptide (heavy chain secretion signal).
[0027] SEQ ID NO:14 provides the amino acid sequence of the heavy
chain variable region of infliximab (cA2).
[0028] SEQ ID NO:15 provides the DNA sequence for the infliximab
heavy chain IgA2m(n) optimized for maize.
[0029] SEQ ID NO:16 provides the amino acid sequence for a signal
peptide (light chain secretion signal).
[0030] SEQ ID NO:17 provides the amino acid sequence of the light
chain variable region of infliximab (cA2).
[0031] SEQ ID NO:18 provides the DNA sequence for the infliximab
light chain optimized for maize.
[0032] SEQ ID NO:19 provides the amino acid sequence of a natural
signal peptide (secretion signal) for a human J-chain (amino acids
1-22 UniProtKB/Swiss-Prot database entry P01591).
[0033] SEQ ID NO:20 provides the DNA sequence for a J-chain
optimized for maize.
[0034] SEQ ID NO:21 provides the amino acid sequence for a signal
peptide (SC-chain secretion signal).
[0035] SEQ ID NO:22 provides the DNA sequence for an SC-chain
optimized for maize.
[0036] SEQ ID NO:23 provides the amino acid sequence for a rice
.alpha.-amylase signal peptide (secretion signal).
[0037] SEQ ID NO:24 provides the amino acid sequence of the heavy
chain variable region of adalimumab (D2E7).
[0038] SEQ ID NO:25 provides the amino acid sequence of the light
chain variable region of adalimumab (D2E7).
[0039] SEQ ID NO:26 provides the DNA sequence for a J-chain
optimized for Lemna.
[0040] SEQ ID NO:27 provides the DNA sequence for an SC-chain
optimized for Lemna.
[0041] SEQ ID NO:28 provides the DNA sequence for the adalimumab
heavy chain IgA1 optimized for Lemna.
[0042] SEQ ID NO:29 provides the DNA sequence for the adalimumab
light chain .kappa. optimized for Lemna.
[0043] SEQ ID NO:30 provides the amino acid sequence of the heavy
chain variable region of ustekinumab (CTNO-1275).
[0044] SEQ ID NO:31 provides the amino acid sequence of the light
chain variable region of ustekinumab (CTNO-1275).
[0045] SEQ ID NO:32 provides a complete Lemna-optimized UKB-SA1
heavy chain IgA1 DNA (including DNA encoding signal peptide SEQ ID
NO:23).
[0046] SEQ ID NO:33 provides a complete Lemna-optimized UKB-SA1
light chain DNA (including DNA encoding signal peptide SEQ ID
NO:23).
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] FIGS. 1A and 1B show the amino acid sequences of various
human antibody light chain subtypes and allotypes. FIG. 1A shows
the amino acid sequence of a human .kappa. light chain constant
region (C.sub.L) (UniProtKB/Swiss-Prot P01834) (SEQ ID NO:1); FIG.
1B shows the amino acid sequences of a human .lamda. light chain
constant region (C.sub.L) allotypes (UniProtKB/Swiss-Prot P0CG04,
P0CG05, P0CG06, P0CG74, and A0M8Q6; SEQ ID NOS:2 to 6).
[0048] FIG. 2 shows the amino acid sequences of the constant
regions of human IgA1 (SEQ ID NO:7), IgA2 m(1) (SEQ ID NO:8), IgA2
m(2) (SEQ ID NO:9), and IgA2(n) (SEQ ID NO:10) antibody a heavy
chains;
[0049] FIG. 3 shows the amino acid sequence of a human J-chain
(a.a. 23-159 of UniProtKB/Swiss-Prot entry P01591, SEQ ID
NO:11).
[0050] FIG. 4 shows the amino acid sequence of a human secretory
component (a.a. 19-603 of UniProtKB/Swiss-Prot database entry
P01833 [PIGR_HUMAN], SEQ ID NO:12).
[0051] FIG. 5 shows the structure of the SIgA vector constructs of
Reference Example 1.
[0052] FIG. 6 shows reducing and non reducing gels of an
anti-TNF.alpha. SIgA of Example 2. The label "A" shows non-reducing
SDS-PAGE analysis demonstrating expression of complete SIgA while
the label "B" shows reducing SDS-PAGE analysis.
[0053] FIGS. 7A through 7C show the structure of vector constructs
SynA01 (FIG. 7A), SynA02 (FIG. 7B) and SynA03 (FIG. 7C) used for
expression of an anti-p40 (anti-IL12/23) SIgA in Lemna in Example
4.
[0054] FIG. 8 shows reducing and non reducing gels of an anti-p40
(anti-IL-12/23) SIgA in Example 5. The A gel shows non-reducing
SDS-PAGE analysis demonstrating expression of complete SIgA. The B
gel shows reducing SDS-PAGE analysis.
[0055] FIG. 9 shows the degradation of an anti-p40 (anti-IL-12/23)
SIgA in Example 7 compared to ustekinumab (IgG1) and to colostral
secretory IgA in simulated intestinal fluid (SIF). Gel A compares
the SynA01-WT antibody of the invention (UKB-SA1) and gel B
compares the SynA01-G0 antibody of the invention (UKB-SA1g0).
DETAILED DESCRIPTION OF THE INVENTION
[0056] The present invention is based on the discovery that
secretory IgA antibodies can be advantageously expressed by a
stably-transformed duckweed. Though secretory IgA ("SIgA")
antibodies are large and complex, duckweed can nonetheless be
stably-transformed to express the fully-formed SIgA antibody.
Further, the duckweed expression system lends itself to the
recovery of the fully-formed SIgA antibody. Because good expression
rates can be obtained and the antibody can be subsequently
isolated/recovered, the use of stably-transformed duckweed permits
the production of SIgA antibodies in bench-scale, clinical-scale,
and/or commercial-scale amounts. In contrast, other plant
expression systems, namely tobacco and lettuce, gave very poor
expression of SIgA and essentially no recoverable yield.
Surprisingly, transient CHO expression systems also gave very low
titers of SIgA expression. The discovery that a duckweed expression
system can effectively express a fully-formed SIGA antibody is thus
a surprising and beneficial advance.
[0057] The antibodies that can be expressed by duckweed according
to the present invention are essentially any secretory IgA
antibody. An antibody, as is well known, has a basic structural
unit that consists of two heavy chain proteins (heavy chains) and
two light chain proteins (light chains), which are bound together
by non-covalent and covalent (e.g., disulfide bonds) interactions
into a single unit. The heavy and light chains have N-terminal
variable regions and C-terminal constant regions. The variable
regions of the light and heavy chains together form an "antigen
binding region." Because the antibody has two heavy and light
chains, the antibody has two antigen binding regions.
[0058] Antibodies are classified based on the heavy chain constant
region, e.g., classified as IgG, IgA, IgM, IgE, IgD, etc. In
nature, the heavy chain constant regions of the various classes are
produced by different genes: the IgA class heavy chains are
uniquely encoded-for by .alpha. genes, the IgG class heavy chains
by .gamma. genes, and so forth. The heavy chain constant regions
also impart the various classes with differences in their
physio-chemical properties, their isotypic antigentic determinants,
and/or in their biological function. Lefranc et al., The
Immunoglobulin FactsBook, Academic Press 2001, Chapter 2, (ISBN
0-12-441351-X). The constant region of the IgA heavy chains
(C.alpha.) typically have three domains that are referred to as
C.alpha.1, C.alpha.2, and C.alpha.3. Unlike the heavy chain
constant region, the light chain constant region is not used for
classification. In humans, for example, all classes use one of two
types of light chain constant regions, namely the C.kappa. (kappa)
or C.lamda. (lambda) type. The amino acid sequences of human kappa
(SEQ ID NO:1) and several lambda light chain constant regions (SEQ
ID NOS:2-6) are provided in FIGS. 1A and 1B, respectively. The
definition and structure of antibodies are well known to workers
skilled in this art, such as described in, e.g., Alberts, B. et
al., Molecular Biology of the Cell 3.sup.rd Edition, Chapter 23,
Garland Publishing Inc., New York, N.Y., 1994, and Nezlin, R., The
Immunoglobulins. Structure and Function (1998) Academic Press (ISBN
0-12-517970-7).
[0059] The C-terminal section of two IgA antibodies, i.e.,
C-terminal ends of the Ca3 region (also referred to as the "tail
piece"), can be joined together via a J-chain to form a dimer.
Dimeric IgA has four antigen binding regions; two from each IgA
monomer. Typically the four antigen binding regions (and their
CDRs) are identical for reasons such as ease of manufacture. But
the antigen binding regions can, in certain circumstances, be
different, e.g., different CDRs binding different epitopes on the
same antigen or event different antigens (such as in the case of
bispecific antibodies). Typically the CDRs of the four antigen
binding regions are identical. A secretory chain, sometimes called
a secretory component or SC-chain, can be attached to the dimeric
IgA antibody. The SC-chain provides increased resistance to
proteolysis especially in the intestinal tract. The SC-bound
dimeric IgA is referred to herein as "secretory IgA" or "SIgA."
Interestingly, in mammals such as humans, SIgA formation requires
the cooperation of two cell types: the plasma cells to produce
dimeric IgA and epithelial cells to add the SC-chain onto the
dimeric IgA post-secretion from the plasma cells. See Pabst (2012)
Nat Rev Immunol. 2012 12:821-832 for a recent review on this
subject. But surprisingly, in the present invention, a single
transformed duckweed plant cell can produce and assemble a
fully-formed SIgA.
[0060] Heavy chain constant regions that qualify as an IgA-class
antibody are well known in the art. Generally the amino acid
sequence of the heavy chain constant regions of an IgA, regardless
of how it is produced (e.g., naturally or recombinantly),
corresponds to an amino acid sequence encoded for by an
.alpha.-gene. In addition, IgA antibodies have characteristic
antigenic determinants unique to IgA-class antibodies and different
from the antigenic determinants of other classes of antibodies,
such as IgG-class antibodies (see, e.g., Nezlin, R., The
Immunoglobulins. Structure and Function (1998) Academic Press (ISBN
0-12-517970-7); Lefranc et al., The Immunoglobulin FactsBook,
Academic Press 2001, Chapter 2, (ISBN 0-12-441351-X)). Furthermore,
IgA antibodies are the only isotype that is known to specifically
bind to the Fc.alpha.R (see, e.g., Alberts, B. et al., Molecular
Biology of the Cell 3.sup.rd Edition, Chapter 23, Garland
Publishing Inc., New York, N.Y., 1994; Lefranc et al., The
Immunoglobulin FactsBook, Academic Press 2001, Chapter 2, (ISBN
0-12-441351-X)).
[0061] Accordingly, for purposes of the present invention, the
terms "IgA antibody," "monomeric IgA," "dimeric IgA" and "SIgA"
each refers to antibodies that contain the heavy chain constant
regions of an IgA class of immunoglobulin, e.g., which corresponds
to an amino acid sequence that can be encoded for by .alpha. genes
and which react with an antibody specific for the IgA-class heavy
chain. The amino acid sequence "corresponds" in that it is
identical to, or contains only minor variations
(insertions/deletions/substitutions) from, an amino acid sequence
produced by any a gene, an individual human's IgA heavy chain
sequence, or a human IgA heavy chain consensus sequence. Indeed,
variations can and do exist in the amino acid sequence of the IgA
heavy chain constant region without moving such antibodies outside
of the IgA class. For clarity, because the heavy chain sequence is
determinative of the Ig class, a recombinant antibody containing
the IgA heavy chain constant regions and further containing the
antigen binding regions encoded for by DNA sequences obtained from
a known IgG antibody is still an "IgA antibody." On the other hand,
a secretory IgA antibody modified to replace the C.alpha.2 heavy
chain constant domain (encoded for by the IgA-specific
.alpha.-gene) with a C.gamma.2 heavy chain constant domain (encoded
for by the IgG-specific .gamma.-gene) is not an IgA antibody, and
is instead a hybrid IgA/IgG antibody. Such a hybrid is not within
the scope of the terms "monomeric IgA," "dimeric IgA," and "SIgA"
antibodies, and thus is not a secretory IgA antibody according to
the invention.
[0062] Minor variations of the heavy chain constant regions are
permitted only to the extent that the overall antibody class,
framework, and functionality of SIgA is maintained; e.g., J-chain
binds to monomers and SC-chain binds to the dimeric structure and
provides proteolysis resistance. Such variations include
conservative substitutions. Exemplary conservative substitutions
are shown in Table 1. The amino acids in the same block in the
second column and preferably in the same line in the third column
may, for example, be substituted for each other.
TABLE-US-00001 TABLE 1 ALIPHATIC Non-polar G A P I L V
Polar-uncharged C S T M N Q Polar-charged D E K R AROMATIC H F W
Y
[0063] Typical minor variations of the constant regions from the
normal or naturally-occurring sequence involve only conservative
changes to the amino acid sequence using the recognized
substitutions, insertions and/or deletions. Generally, the
variations (substitutions, insertions, and/or deletions) of a
constant domain of the heavy or light chain means no more than 10
and usually no more than 5 amino acid additions, deletions, and/or
substitutions (either naturally-occurring or
genetically-engineered), in any C.alpha.1, C.alpha.2, or C.alpha.3
domain in comparison to a normal IgA constant domain. The sum of
these minor variations in the constant domains of the SIgA antibody
of the invention is usually less than 20 amino acids
(acid/deletions/substitutions) and often less than 10 or less than
5.
[0064] Accordingly, at a minimum, SIgA includes any recognized
amino acid sequence that is generally accepted as being within the
IgA class. For example, information on the structure and function
of IgA can be found in Snoeck et al., Vet. Res. 37; 455-467 (2006)
and "Mucosal immune defense: Immunoglobulin A", C. S. Kaetzel ed.,
Springer, New York (2007) ISBN 978-0-387-72231-3. Electronic
databases, such as RCSB Protein Data Bank, can also establish a
known IgA sequence or portion/domain thereof. The constant domains
contained in the SIgA antibodies of the invention can be human,
non-human, or a combination of these. Preferred are mammalian
constant regions. Most preferred are human constant regions.
[0065] In humans there are two recognized IgA subclasses: IgA1 and
IgA2 which differ in the hinge section between the C.alpha.1 and
C.alpha.2 domains of the heavy chain. In IgA1 this hinge section is
relatively long and in nature typically O-glycosylated. In IgA2 the
hinge section is relatively short and in nature lacks
glycosylation. Both IgA1 and IgA2 SIgA antibodies are usually
present in mucosal secretions. In humans, the IgA2 subclass has
three known allotypes: IgA2m(1), IgA2m(2) and IgA2m(n). Unlike the
subclasses, only one specific allotype will be found in a normal
healthy individual. The m(1) allotype is strongly prevalent in the
Caucasian population (98%) and varies between 23% and 96% for other
populations. The m(2) allotype has a high prevalence in populations
of African and Asian descent (50-70%). The m(n) allotype--which is
considered to be a hybrid of the m(1) and m(2) allotypes--has been
reported to be genetically possible, but has not been actually
observed in any population. See Chintalacharuvu et al., Journal of
Immunology 152, 5299-5304 (1994). Accordingly, SIgA antibodies of
the present invention preferably contain human IgA heavy chain
constant regions of the IgA1 or IgA2 sub-types, including IgA2m(1),
IgA2m(2) and IgA2m(n) allotypes, and combinations thereof (e.g.,
one constant domain from an IgA1 and another constant domain from
an IgA2).
[0066] Typically, the SIgA antibodies of the invention comprise the
C.alpha.1 domain, the hinge section, and the -C.alpha.2-C.alpha.3
domains of an IgA antibody (with or without minor variations),
including a human IgA1 and/or IgA2 antibody. In this embodiment,
the Cal domain, the hinge section, and the -C.alpha.2-C.alpha.3
domains can be of an IgA1, an IgA2m(1) allotype, an IgA2m(2)
allotype, or a combination thereof. Amino acid sequences of a human
IgA1 heavy chain constant region (SEQ ID NO:7), a human IgA2m(1)
heavy chain constant region (SEQ ID NO:8), a human IgA2m(2) heavy
chain constant region (SEQ ID NO:9), and a human IgA2(n) heavy
chain constant region (corresponding to the C.alpha.1 and C.alpha.2
regions of an IgA2m(2) and the C.alpha.3 region of an IgA2m(1))
(SEQ ID NO:10) are respectively shown in FIG. 2.
[0067] A modified, shortened, or removed linker/hinge section
between C.alpha.1 and C.alpha.2 in IgA1 has been reported to
increase resistance against proteases (for example see Senior et
al., J. Immunol. 2005; 174: 7792-7799). Such can be incorporated
into the SIgA antibodies of the present invention.
[0068] The J-chain is a protein that attaches to the tail piece of
a monomeric IgA to join two monomeric IgAs to form a dimer. The
J-chain is normally of mammalian origin, such as human, murine,
rat, rabbit, sheep, cow, or goat origin, but is preferably of human
origin. An example of the amino acid sequence of a human J-chain is
set forth in FIG. 3 (SEQ ID NO:11). Usually, the sequence of the
mammalian J-chain is the same as the naturally-occurring sequence,
but it can be subject to minor variations as described above for
constant regions generally, e.g., up to 10 amino acid insertions,
substitutions, or deletions. The minor variations do not
significantly alter the function of the J-chain, and in particular
the ability to join two monomeric IgA antibodies to form a dimer
and to enable attachment of the SC-chain.
[0069] The secretory component, also referred to as "SC" or
"SC-chain," is a protein that binds to the dimeric IgA framework
and imparts increased resistance against proteolysis upon the
antibody to which it is bound. Generally, the secretory component
is of mammalian origin, such as human, murine, rat, rabbit, sheep,
cow, or goat origin, but is preferably of human origin. The SC is
the result of cleavage of the Polymeric IgA-receptor (PIGR) which
usually occurs at a specific position. Some variation can occur in
the position of the cleavage resulting in variant forms of SC.
Usually, the sequence of the mammalian-derived SC-chain is the same
as the naturally-occurring sequence, but it can be subject to minor
variations as described above for constant regions generally, e.g.,
up to 10 amino acid insertions, substitutions, or deletions. The
minor variations do not significantly alter the function of the
secretory component, e.g., the ability to stabilize the SIgA
against proteolysis. An example of the amino acid sequence of a
human secretory component is set forth in FIG. 4 (SEQ ID
NO:12).
[0070] The antigen binding region binds to and neutralizes a target
antigen. Typically, the antigen binding region comprises a heavy
and light chain variable region pair, each containing hypervariable
regions (CDRs, which directly interact with the antigen) and the
supporting framework regions. The CDRs in each heavy and light
chain variable region are separated from each other and from the
constant domain by the framework regions, which serve to maintain
the CDRs in the correct binding conformation. In general each
variable part of an immunoglobulin heavy or light chain contains 3
different CDRs and four framework regions. For a more detailed
description of antibody antigen binding regions, see for example C.
A. Janeway et al., "Immunobiology" 6.sup.th Edition, Chapter 3, pp
110-115; Garland Science Publishing, New York, 2005 (ISBN
0815341016). Regarding framework regions in particular, see for
example WO92/22653 (discussing that although framework regions do
not directly interact with antigen, framework regions can influence
binding of the CDRs with antigen, such as binding strength and/or
downstream events).
[0071] In embodiments, the antigen binding region can target
bacteria, viruses, and other pathogens; pollen and other allergens;
cancer targets (e.g., tumor-specific antigens; tumor-associated
antigens); and proinflammatory mediators (among other targets). In
some embodiments, the antigen binding regions of the SIgAs of the
invention bind to and neutralize a proinflammatory cytokine or a
receptor thereof. These cytokines and receptors include TNF-alpha
(preferably soluble TNF-alpha), IFN-gamma, IFN-alpha, GM-CSF,
CXCL10/IP-10, IL-1-beta, IL-1-alpha, IL-4, IL-5, IL-6, IL-12,
IL-13, IL-17A, IL-18, IL-20, IL-22, IL-23, IL-1 receptor, IL-2
receptor, IL-4 receptor, IL-5 receptor, IL-6 receptor, and IL-17
receptor.
[0072] Secretory IgA antibodies specifically and preferentially
bind with high affinity to a corresponding antigen, such as a human
proinflammatory cytokine. A variety of protocols for performing
binding, competitive binding, or immuno-radiometric assays to
determine the specific binding capability of an antibody are well
known in the art (see for example Maddox et al, J. Exp. Med. 158,
1211-1226, 1993). Such immunoassays typically involve the formation
of complexes between the specific protein and its antibody and the
measurement of complex formation (e.g., binding to or unbinding
from the specific protein). Generally, the affinity of secretory
IgA antibodies of the invention is at least two-fold, at least
10-fold, at least 50-fold, at least 100-fold, or at least 1000-fold
or greater than the affinity of the antibodies for a non-specific
protein such as, for instance, BSA or casein. Typically the
secretory IgA antibodies of the present invention exhibit a binding
affinity constant (K.sub.D) with respect to the antigen of
10.sup.-7M or lower, preferably 10.sup.-8M, 10.sup.-9 M,
10.sup.-10M, 10.sup.-11 M, or 10.sup.-12M or lower.
[0073] Generally the secretory IgA antibodies of the present
invention neutralize the antigen to which it is bound. For the
present invention, the term "neutralizes" means inhibits/reduces
the effect of the antigen to some degree, such as by at least 30%,
at least 35%, at least 40%, and at least 45%. Typically the
inhibition/reduction in the effect of the antigen at least 50%. For
example, in some embodiments, the secretory IgA antibodies of the
present invention inhibit/reduce the effect of an antigen to which
it is bound by at least 55%, at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, or at least 98%.
[0074] Secretory IgA antibodies of the invention can be non-human
antibodies, chimeric antibodies, humanized antibodies, human
antibodies, or other combinations of human and non-human
sequences/regions. A chimeric antibody is an antibody having an
antigen binding region (CDRs and framework) originating from a
first species (typically a mouse) and heavy chain constant regions
originating from a second species (typically a human). Exemplary
chimeric antibodies are described in Knight, D. M, et al. (1993)
Mol. Immunol. 30:1443-1453; WO 92/16553). A humanized antibody is a
human antibody onto which non-human (typically murine) CDRs have
been grafted. In the humanized antibody, certain human supporting
framework amino acid residues can be replaced with their
counterparts from the non-human parent antibody. Such an antibody
containing certain non-human framework residues is still a
humanized antibody. See, e.g., Carter et al. (1992) Proc Natl Acad
Sci USA. 1992 89(10):4285-9. In the humanized antibodies, the
sequence of the supporting framework into which the non-human CDRs
are grafted can be obtained from any human isotype/class,
preferentially from IgG or IgA, and may be modified to improve the
properties thereof (e.g., antigen binding and/or downstream
effects). A human antibody is fully-human, containing only human
constant and variable regions, i.e., having only human heavy and
light chains (derivable from human genomic sequences by
naturally-occurring recombination and mutation processes, consensus
sequences, phage display selection of specificities with the
highest binding affinities for a given target, etc.). Likewise, a
non-human antibody contains only non-human constant and variable
regions, i.e., having only non-human heavy and light chains.
Exemplary fully human antibodies are described in pub no WO97/29131
and references cited therein.
[0075] The SIgA antibodies of the present invention are monoclonal
antibodies. A "monoclonal antibody" refers to a population or
collection of antibodies that are substantially identical because
they were all produced by clones of a single cell. For the present
invention, a monoclonal SIgA is a SIgA containing monoclonal
monomeric IgA antibodies, a J-Chain, and an SC-chain that were all
produced by clones of a single cell.
[0076] The method of the present invention expresses a SIgA
antibody as described above in a stably-transformed duckweed.
Generally, a genetically modified duckweed is a known expression
system for producing various proteins (see U.S. Pat. No.
6,040,498), including for the production of monoclonal antibodies
(see U.S. Pat. No. 7,632,983). Duckweed is the common name for the
members of the monocotyledonous family Lemnaceae. The five known
genera and 38 species of Lemnaceae are all small, free-floating,
fresh-water plants whose geographical range spans the entire globe:
genus Lemna (L. aequinoctialis, L. disperma, L. ecuadoriensis, L.
gibba, L. japonica, L. minor, L. minuscula, L. obscura, L.
perpusilla, L. tenera, L. trisulca, L. turionifera, L. valdiviana);
genus Spirodela (S. intermedia, S. polyrrhiza); genus Wolffia (Wa.
angusta, Wa. arrhiza, Wa. australina, Wa. borealis, Wa.
brasiliensis, Wa. columbiana, Wa. elongata, Wa. globosa, Wa.
microscopica, Wa. neglecta) genus Wolfiella (Wl. caudata, Wl.
denticulata, Wl. gladiata, Wl. hyalina, Wl. lingulata, Wl. repunda,
Wl. rotunda, and Wl. neotropica), and genus Landoltia (L.
punctata). For clarity, the term "duckweed` as used in the present
invention includes the foregoing species, genetically modified
variants thereof (e.g., modified to control glycosylation,
secretion, etc.), and any other genera or species of Lemnaceae, if
they exist, optionally in a genetically modified form. Typically
the genus Lemna is preferred, especially the species L. minor and
L. gibba in natural or genetically modified forms. Also, the use of
the term "duckweed," or any genus or species thereof, is meant to
include individual plant cell(s), nodules, as well as whole plants
including mature plants having root and fronds, unless otherwise
indicated by context or express statement.
[0077] The stably-transformed duckweed of the present invention
means that the nucleic acid sequences and/or genes needed to
produce the desired SIgA have been introduced into the genome of a
duckweed by transgenesis. This modification is considered a stable
transformation because the nucleic acid introduced to the duckweed
have integrated into the genome and are capable of being inherited
by the progeny thereof, more particularly, by the progeny of
multiple successive generations. Transformant lines are isolated by
applying a selective pressure, resistance to which is encoded in
the construct used for transgenesis Transformed duckweed lines
carrying the exogenous DNA of interest do not necessarily express
the SIgA of interest due to, for example, transgene silencing and
epigenetic control of gene expression; however screening of
different lines allows for the isolation of lines expressing the
SIgA of interest at desired levels. This genetic modification
causes the duckweed to express the desired SIgA antibody during its
otherwise normal metabolic activity. The term "express" and its
grammatical variants refers to the biosynthesis of the SIgA
antibody, which includes the transcription, translation, and
assembly of the antibody by the duckweed. Thus, expressing a SIgA
antibody in a stably-transformed duckweed entails providing the
stably-transformed duckweed with an environment that supports
metabolic activity and/or growth for a sufficient period of time to
produce the SIgA antibody. Generally this environment comprises
providing light (natural and/or artificial) and a liquid medium
typically based on water. Providing this environment is often
referred to as "culturing" the duckweed. Methods of culturing
duckweed including the media, supplements (if any), conditions,
etc., are known in the art and have been disclosed in, e.g., U.S.
Pat. Nos. 6,040,498; 7,161,064; and 7,632983; and references cited
therein, respectively.
[0078] Culturing of transgenic duckweed of the invention can be
performed in transparent vials, flask, culture bags, or any other
container capable of supporting growth using defined media. In some
embodiments of the invention large scale growth of duckweed,
necessary to achieve industrial production levels, is carried out
in bioreactor tailor-designed for growth of duckweed. In a
preferred embodiment, duckweed bioreactors, which can be inoculated
aseptically, support aseptic growth of duckweed. In even more
preferred embodiments, a bioreactor can be directly connected to
harvest bag to separate the media from the plant material, either
of which can then be piped into downstream purification steps.
Suitable bioreactors, methods/devices to inoculate them
aseptically, and aseptic harvest bags are described in U.S. Pat.
No. 7,176,024 to Branson et al. or in US application 2010/209966 To
Everett et al.
[0079] In embodiments, the SIgA of the invention are expressed in
auxotrophic duckweed requiring specific supplements for growth.
This property allows for effective containment of the transgenic
duckweed. Auxotrophic duckweed can be created by RNAi
downregulation of expression of metabolic enzyme such as threonine
deaminase. Methods of making such auxotrophic duckweed, which
require supplementation of Isoleucine for growth are described in
Ngyen et al. (2012) Transgenic res 21: 1071-83.
[0080] The SIgA antibody expressed by the stably-transformed
duckweed is fully-formed. This means that the antibody is intact,
properly-folded, and functional (e.g., binds to its target
antigen). It does not exclude, however, the formation of non-intact
SIgA antibodies, such as monomeric IgA, dimeric IgA, or fragments
thereof, or the formation of incorrectly folded SIgA antibodies.
Rather, so long as at least some of the complete, intact, and
properly folded SIgA antibody is expressed, then the transformed
duckweed has produced fully-formed SIgA. Preferably, the
stably-transformed duckweed expresses more SIgA antibodies than
monomeric IgA antibodies, based on a mole basis. Likewise, the
stably-transformed duckweed preferably expresses more SIgA
antibodies than dimeric IgA antibodies (i.e., dimers devoid of
SC-chain). Generally the stably-transformed duckweed expresses
sufficient amounts of the fully-formed SIgA antibody that the SIgA
antibody is at least 1% of the total soluble protein (TSP), often
at least 2%, and typically at least 5%. In practice, the expressed
SIgA antibodies generally comprise 5 to 25% TSP. In terms of
weight, the stably-transformed duckweed generally expresses at
least 50 mg of SIgA antibody per kg of wet biomass, up to 500 mg
per each kg of wet biomass. In general 12% TSP equates to about 1.0
gram of antibody.
[0081] In a preferred embodiment, the culturing of the
stably-transformed duckweed amounts to growing a clone population
of stably-transformed duckweed plants, each expressing the same
fully-formed SIgA antibody. The plants double their population in
approximately every 30 to 60 hours. The plants grow on the surface
of a relatively still (not circulating or turbulent) water-based
media and are provided with light source and a CO.sub.2 source,
such as air. In this way, a large biomass can be accumulated which
has expressed the fully-formed SIgA antibody, and thus large
amounts of the antibody can be produced.
[0082] Once the stably-transformed duckweed has expressed the
fully-formed SIgA antibody, the duckweed preferably secretes the
antibody. For purposes of the present invention, "secretion" means
translocation of a polypeptide across the plasma membrane of the
duckweed cell to region in between the cell membrane and the cell
wall (known as the apoplast) and/or to the cell wall itself (where
the secreted sIgA remains associated with the cell wall). The SIgA
secreted into the apoplast (and/or cell wall) generally does not
cross the cell wall and diffuse into the liquid media (or culture
media) in any detectable amounts. Typically, less than 1% of the
fully-formed SIgA antibody crosses the cell wall and enters the
liquid media/external environment.
[0083] Following expression of the fully-formed SIgA antibody,
recovery of the antibody from the duckweed and/or the culture media
is often desired. The first step, generally, is to separate the
SIgA antibody from the duckweed. Typically, the fully-formed SIgA
antibody is retained within the duckweed's apoplast and/or the
duckweed's cell wall. Separation in this case generally requires
extraction.
[0084] Extraction of secreted SIgAs typically involves a
homogenization step to disrupt the plant material and allow for
release of the secreted SIgA from the apoplast and/or cell wall
into the homogenization buffer, which is also referred to as
extraction buffer or extraction media. Homogenization buffers and
techniques are known in the art. Small scale homogenization can be
performed manually, such as by using mortar-and-pestle crushing,
and the like. Larger scale homogenization is preferably performed
using a mechanical mixer, typically a high shear mixer such as a
Silverson 275 UHS mixer, or similar apparatuses. The buffer is
typically an aqueous phosphate buffer composition though such is
not required. The buffer may contain additional functional
ingredients as is known in the art. For example, to reduce
proteolysis by metallated proteases, EDTA may be added to the
extraction buffer, typically in amounts from 1 to 20 mM, including
5 to 10 mM. Also, one or more anti-oxidants, such as ascorbic acid,
sodium metabisulfite, benzyl alcohol, benzoic acid, and the like,
may be added during the homogenization process. Homogenization is
generally followed by centrifugation and filtration to obtain a
buffer solution that contains the SIgA antibodies and other soluble
proteins.
[0085] Homogenization is often followed by clarification; a step
that seeks to remove certain naturally abundant impurities
including host cell proteins such as RuBiSco, as well as
non-proteinaceous impurities, such as tannins. This is usually
achieved by acidic precipitation. For example, clarification can be
performed by adjusting the pH of the filtrated homogenate to 4.5,
followed by centrifugation (such as for 30 min at 12000),
neutralization to pH 7.4, and an additional filtration step. In a
preferred embodiment, pH adjustments are performed using 1 M citric
acid pH 1.5, or 1M sodium acetate for acidification and 2M
tris-base for neutralization, though other suitable pH adjusting
agents can also be used instead of or in addition to such agents.
Filtration is performed as known in the art, often by using a 0.22
.mu.m filter.
[0086] The recovery of the SIgA antibodies from duckweed may end
with the extraction buffer or the clarified material. However, for
some uses, purification of the antibody is desired. Purification
can be performed using known methods and techniques and generally
comprises subjecting the clarified material to affinity
chromatography (AC), size exclusion chromatography (SEC), and
optional polishing steps. For efficiency, AC usually precedes SEC,
though such is not required.
[0087] Methods of using affinity chromatography (AC) as a
purification step to remove contaminant proteins and impurities are
known in the art and are described in "Process Scale Purification
of Antibodies" (2009), Edited by U. Gottschalk, J. Wiley and son,
Hoboken, N.J., and references cited therein. Usually the SIgA
antibody is bound to the affinity resin material while one or more
impurities are not bound. The conditions are modified and the
previously bound SIgA antibody is eluted from the column. The
opposite can also be performed with the desired antibody passing
though and the impurity or impurities being bound to the column.
The light chain constant region can be the affinity target. Useful
affinity columns include KappaSelect and Capto L from GE Healthcare
Life Sciences (Piscataway, N.J., USA). When KappaSelect is used,
the addition of MgCl.sub.2 is often advantageous. The use of
Protein A as an AC column is usually avoided. Another kind of AC
step is known as IMAC (immobilized copper affinity chromatography).
IMAC can be used as the sole AC step or in combination with more
traditional AC steps. When used, IMAC is often carried out first.
If the crude antibody composition, such as the clarified material,
contains EDTA, then it is advantageous to use CuSO.sub.4 to
neutralize the negative effects of EDTA, which interferes which the
IMAC purification process. Often IMAC is used for small to medium
scale purification of SIgA where the amounts are less than 10 g,
typically less than 5 grams.
[0088] Methods of using SEC for purification of monoclonal
antibodies are known in the art. In general, SEC allows the
separation of fully assembled SIgAs of interest from lower
molecular forms (such a monomer of IgA, J-chain and SC-chain, or
combinations thereof). Furthermore, SEC also permits a buffer
change, such as, for example, the reformulation of the SIgA of
interest into a new desired buffer. Suitable columns include, for
example, a Sephacryl S300 HR column.
[0089] Other purification steps can be employed as well. For
example, ion exchange chromatography (IEX) can be useful for
removing colored impurities associated with the plant material.
Methods and techniques for performing IEX chromatographic
purification of antibodies are known in the art and are described,
e.g., in Graf et al. (1994) Bioseparation, vol. 4, no. 1 pages
7-20, or in "Process scale purification of antibodies (2009) Edited
by U. Gottschalk, ed. J. Wiley and son, Hoboken, N.J., and
references cited therein. Often IEX, such as anion exchange
chromatography (AEX) or cation exchange chromatography (CEX), is
performed before IMAC or other AC step is employed, but is not
limited thereto and can be employed at other points of the
purification and/or can be employed multiple times with the same or
different exchange resin (e.g., AEX and subsequently CEX). In some
embodiments an AEX column such as DOWEX 1.times.2 is employed,
often before the AC column.
[0090] Further polishing/purification steps can be added, as is
known in the art. For example, after any and/or each purification
step (e.g., chromatography step) an ultrafiltration (UF) step can
be performed. Typically, a UF step is performed at or near the end
of the polishing phase in order to increase purity and/or change
the buffer or concentration of antibody in the buffer.
[0091] The SIgA antibodies are often sufficiently recovered so as
to be "isolated" or in an isolated form. As used herein, the terms
"isolate," "isolating" and "isolation" refer to separating the
antibody from the host cells and native host cell proteins. The
extent of separation is generally at least 50%, but is frequently
at least 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% (w/w).
Isolation is thus related to purification and is generally achieved
by completion of the recovery/extraction step, clarification,
and/or capture steps described above. Preferably the antibody in
isolated form has removed, or been separated from, at least 90%,
more typically at least 99% (w/w) of the host cell proteins of the
original composition. Typically, about 20% of the expressed sIgA
can be recovered, e.g., about 25-100 mg per each kg of wet biomass.
An isolated form of the SIgA antibody can have a concentration of 1
mg/ml or greater, often 2-10 mg/ml or greater.
[0092] For some embodiments, especially where a pharmaceutical use
is ultimately intended for the antibody, low amounts of incomplete
SIgA antibodies are often desirable. This applies to isolated as
well as non-isolated or crude forms of the SIgA antibodies. For
instance, compositions where the amount of dimer IgA (no SC-chain)
is less than 50%, more desirably less than 25%, and often less than
10%, based on the amount of SIgA antibodies in the composition, are
often preferred. For clarity, where a composition contained 10 mg
of SIgA, the amount of non-SC-chain dimeric IgA would preferably be
less than 1 mg, i.e., less than 10% the amount of the SIgA
antibody. The same is true for monomeric IgAs: the content of IgA
monomers is desirably less than 50% the amount of SIgA's, more
desirably less than 25% and often less than 10%. In some
embodiments, the combined amount of dimer IgA (i.e., no SC-chain)
and monomer IgA is less than 25% of the amount of SIgA in the
composition, often less than 10%, and even less than 5%. The above
amounts apply to both isolated and non-isolated forms of SIgA
compositions. Accordingly, the low relative amounts of incomplete
SIgA can be a result of the expression system (native
low-production of incomplete SIgA), the result of separation and/or
purification that removes incomplete SIgAs, or both.
[0093] Purified SIgA compositions are also useful. A purified
composition contains SIgA antibodies in an amount of at least 85%,
often at least 90%, more often at least 95%, and preferably at
least 97%, 98%, or 99%, based on the total soluble protein content.
The purified composition can be a solid, such as a lyophilized
product, or a liquid. A typical liquid form contains no solids,
e.g., no insoluble cell wall materials, and is often based on water
as the main or sole solvent and optionally containing salts, pH
adjusting agents, or buffers. A purified liquid composition
generally contains the SIgA antibody in a concentration of 50
.mu.g/ml or more, often at least 100 .mu.g/ml, preferably at least
1 mg/ml.
[0094] Recombinant production of sIgAs in duckweed requires
transformation of duckweed by transgenesis. Stable transgenesis in
duckweed can be obtained by different techniques as described in
U.S. Pat. Nos. 6,040,498 and 7,161,064 to Stomp et al. Briefly
stable duckweed transgenesis can be achieved by DNA-coated particle
bombardment, electroporation, and Agrobacterium spp.-mediated
transformation. Preferably, transgenesis of duckweed is performed
by using A. tumefaciens-mediated transformation. Briefly,
Agrobacterium-mediated transformation is carried out by
dedifferentiating fully grown duckweed plants or tissues,
preferably tissues of meristematic origin, into calli. Callus
induction is carried out by growing duckweed in medium containing
plant growth regulators and supplements. Calli can/will
re-differentiate into organized nodules. Both nodules or calli can
be infected with Agrobacterium, according to the procedure
described in U.S. Pat. Nos. 6,040,498 and 7,161,064 to Stomp et al.
Regeneration of plants from infected calli/nodules and concomitant
selection for transformants by applying the desired selective
pressure results in the isolation of transgenic duckweed lines
carrying the exogenous DNA of interest.
[0095] Construct for expression of SIgAs, to be used for
transformation of duckweed, can be produced by using standard
techniques for example, the techniques described in Sambrook &
Russell, Molecular Cloning: A Laboratory Manual, 3.sup.rd Edition,
Cold Spring Harbor Laboratory, NY (2001) and Ausubel et al, Current
Protocols in Molecular Biology (Greene Publishing Associates and
Wiley Interscience, NY (1989)). Vectors for transformation of
duckweed have been described elsewhere, such as in U.S. Pat. Nos.
6,040,498 and 7,161,064 to Stomp et al. Preferably, an A.
tumefaciens binary vector (generated, for example, by standard
cloning in E. coli) is used to first transform A. tumefaciens; the
transgenic line obtained can then be employed to transform
duckweed. Preferably, such vectors contain multiple resistance
genes, to allow for selection in bacteria and in duckweed. Genes
for bacterial selection are known in the art. Suitable resistance
genes for selection in plants have been described in U.S. Pat. Nos.
6,040,498 and 7,161,064 to Stomp et al., and include gentamycin and
kanamycin.
[0096] For expression of SIgAs, multiple transformations can be
performed with separate vectors including different cassettes
coding for the J-chain, the SC-chain, the antibody H chain and L
chain. In a preferred embodiment, a single expression vector is
used for transformation that contains 4 cassettes each encoding for
one of the structural subunit of the sIgA (namely, H chain, L
chain, SC-chain and J-chain). Construction of expression vectors
containing multiple expression cassette for antibody expression
have been described in U.S. Pat. No. 7,632,983 to Dickey et al.
[0097] In embodiments, expression of the cassettes is driven by
individual promoters, even when the cassettes are located in the
same vector for single-step transformation. Examples of suitable
promoter can be found in U.S. Pat. No. 4,771,002 to Stanton, U.S.
Pat. No. 5,428,147 to Barker et al., U.S. Pat. No. 7,622,573 &
U.S. Pat. No. 8,034,916 to Dickey et al., disclosures of which are
incorporated herein by references. Most preferably, a different
promoter is used for each expression cassette (such as the chimeric
A. tumefaciens octopine and mannopine synthase promoter, the L.
minor polyubiquitin promoter (LmUbq), Lemna aequinoctialis
polyubiquitin promoter (LaUbq) and Spirodela polyrrhiza
polyubiquitin promoter (SpUbq)). In a preferred embodiment, the
expression vector includes cassettes coding for all 4 of the SIgA
components, i.e. J-chain, SC-chain, H-chain and L-chain. In an even
more preferred embodiment, each of the 4 cassettes is driven by a
different promoter. In some embodiments, an inducible promoter(s)
can be used. See, e.g., U.S. Pat. No. 7,632,983 to Dickey et al.
Examples of such inducible promoters include heat shock gene
promoters, cold-induced promoters, drought-inducible gene
promoters, pathogen-inducible gene promoters, wound-inducible gene
promoters, and light/dark-inducible gene promoters, promoters from
genes induced by abscissic acid, auxins, cytokinins, and
gibberellic acid.
[0098] In an advantageous embodiment, the vectors used for
expression include, 5' of the coding sequence of the expression
cassette, a signal peptide sequence placed in frame with the
N-terminal portion of the protein of interest. Thus, each of these
DNA sequences are "operably linked" to each other. The term
"operably linked" means multiple nucleotide sequences that are
placed in a functional relationship with each other. Generally,
operably-linked DNA sequences are contiguous, and where necessary
to join two protein coding regions, in reading frame. Such signal
peptide sequence interacts with a receptor protein on the membrane
of the endoplasmic reticulum (ER) to direct the translocation of
the growing polypeptide chain across the membrane and into the
endoplasmic reticulum for secretion. Presence of the signal peptide
sequence ensures efficient secretion. This signal peptide is
generally cleaved from the precursor polypeptide to produce a
mature polypeptide lacking the signal peptide. Suitable signal
peptide include the Arabidopsis thaliana chitinase signal peptide,
the Oryza sativa .alpha.-amylase signal peptide, or any other
suitable duckweed signal peptide sequences, as described in U.S.
Pat. No. 7,632,983 to Dickey et al. In a most preferred embodiment,
the sequence of the signal peptide used in the O. sativa
.alpha.-amylase signal peptide. In some embodiments of the present
invention, the secreted SIgAs are retained within the apoplast (the
region between the plasma membrane and the cell wall) and/or
associated with the cell wall.
[0099] Other suitable nucleotide sequences including enhancers, 5'
leader sequences, such as the leader sequence of L. gibba
ribulose-bis-phosphate carboxylase small subunit 5B gene, 3' UTR
sequences, introns, enhancers, "ACC" and "ACA" trinucleotides to be
introduced directly upstream of the translation initiation codon of
the nucleotide sequence of interest can be used to improve
expression as disclosed in the art and in U.S. Pat. Nos. 6,040,498
and 7,161,064 to Stomp et al as well as U.S. Pat. No. 7,622,573
& U.S. Pat. Nos. 8,034,916 7,632,983 to Dickey et al,
disclosures of which are all incorporated by reference herein.
[0100] The expression from the transgenic lines obtained can also
be improved by optimizing the codon distribution of the encoded
proteins for expression in duckweed. Duckweed-preferred codons, as
used herein, refers to codons that have a frequency of codon usage
in duckweed of greater than 17%. Likewise the codons can be
optimized for expression in L. minor or L. gibba. In each case the
codons have a frequency of codon usage of greater than 17%.
Duckweed and Lemna ssp. codon optimization is known in the art and
is carried out, e.g. as described in U.S. Pat. No. 7,632,983 to
Dickey et al.
[0101] Another option is to modify the glycosylation profile of the
duckweed. It is recognized that antibodies having more than one
glycosylation site can have the same glycan species attached to
each glycosylation site, or can have different glycan species
attached to different glycosylation sites. In this manner,
different patterns of glycan attachment yield different glycoforms
of a glycoprotein. Monomeric IgA1 antibodies have two conserved
N-glycosylation sites (per chain): one on the CH2 region and one on
the tailpiece. Monomeric IgA2 antibodies have an additional two or
three N-glycosylation sites (per chain). Furthermore, the J-chain
of dimeric IgA has one conserved N-glycosylation site, and the
secretory component of secretory IgA has 7 conserved
N-glycosylation sites. The stably-transformed duckweed can express
secretory IgA antibodies having various glycan patterns, including
for example, aglycosylated sIgAs and sIgAs having glycan patterns
native to plant, mammalian (human), or insect cells.
[0102] The terms "N-glycan(s)" and "N-linked glycan(s)" are used
interchangeably and refer to an N-linked oligosaccharide, e.g., one
that is or was attached by an N-acetylglucosamine (GlcNAc) residue
linked to the amide nitrogen of an asparagine residue in a protein.
The predominant sugars found on glycoproteins are glucose (Glu),
galactose (Gal), mannose (Man), fucose (Fuc), N-acetylgalactosamine
(GalNAc), N-acetylglucosamine (GlcNAc), and sialic acid (e.g.,
N-acetyl-neuraminic acid (NeuAc)). The processing of the sugar
groups occurs co-translationally in the lumen of the ER and
continues in the Golgi apparatus for N-linked glycoproteins.
[0103] For the purposes of the present invention, the term "G2
glycan," "G2 glycan species," and "G2 glycan structure" are used
interchangeably and refer to an N-linked glycan having the
GlcNAc2Man3GlcNAc2Gal2 structure, in which two terminal galactose
(Gal) sugar residues are present. For the purposes of the present
invention, the term "G1 glycan," "G1 glycan species," and "G1
glycan structure" are used interchangeably and refer to an N-linked
glycan having the GlcNAc2Man3GlcNAc2Gal structure, in which only
one terminal galactose (Gal) sugar residue is present. For the
purposes of the present invention, the term "G0 glycan," "G0 glycan
species," and "G0 glycan structure" are used interchangeably and
refer to an N-linked glycan having the GlcNAc2Man3GlcNAc2
structure, in which no terminal galactose (Gal) sugar residues are
present.
[0104] For the purposes of the present invention, the term
"high-mannose glycan," high-mannose glycan species," and
"high-mannose glycan structure" are used interchangeably and refer
to an N-linked glycan having the GlcNAc2ManX structure, wherein X
is a whole number greater than three, such as 5, 6, 7, 8, or 9. For
the purposes of the present invention, the term "Man5 glycan," Man5
glycan species," and "Man5 glycan structure" are used
interchangeably and refer to an N-linked glycan having the
GlcNAc2Man5 structure. The same is applicable for the terms Man6
glycan (species; glycan structure), Man7 glycan (species; glycan
structure), Man8 glycan (species; glycan structure), Man 9 glycan
(species; glycan structure), etc.
[0105] In mammals, naturally-occurring N-glycans contain a fucose
(Fuc) residue attached to the GlcNAc2Man3 core structure by an
.alpha.1,6 linkage. In plants, naturally-occurring N-glycans
contain a fucose (Fuc) residue attached to the GlcNAc2Man3 core
structure by an .alpha.1,3 linkage and further contain a xylose
(Xyl) residue attached to the GlcNAc2Man3 core structure by a
.beta.1,2 linkage. For the purposes of the present invention, a G0
glycan containing the mammalian .alpha.1,6-linked Fuc residue
attached to the GlcNAc2Man3 core structure is referred to as a
"G0F<6> glycan." For the purposes of the present invention, a
G0 glycan containing the plant .alpha.1,3-linked Fuc residue
attached to the GlcNAc2Man3 core structure is referred to as a
"G0F<3> glycan," a G0 glycan containing the plant
.beta.1,2-linked Xyl residue attached to the GlcNAc2Man3 core
structure is referred to herein as a "G0X glycan," and a G0 glycan
containing each of the plant .alpha.1,3-linked Fuc residue and the
plant .beta.1,2-linked Xyl residue attached to the GlcNAc2Man3 core
structure is referred to herein as a "G0XF<3> glycan." In an
embodiment, the invention relates to a secretory IgA antibody, or a
population of secretory IgA antibodies, in which substantially all
N-glycans lack Fuc and Xyl residues.
[0106] The stably-transformed duckweed can also contain a genetic
modification that alters the glycan profiles. For example, the
N-glycans of the SIgA can be expressed with reduced levels of
fucose and xylose residue, preferably less than 10%, more
preferably less than 1%. This modification from natural glycan
profile can be achieved by several techniques, including knocking
out endogenous .alpha.1,3-fucosyltransferase (FucT) and
.beta.1,2-xylosyltransferase (XylT), or otherwise inhibiting their
transcription, translation into protein or enzymatic activity. In a
preferred embodiment, the duckweed is transformed with at least one
recombinant nucleotide construct that provides for the inhibition
of expression of .alpha.1,3-fucosyltransferase (FucT) and
.beta.1,2-xylosyltransferase (XylT) in a plant. In a more preferred
embodiment, these constructs triggers RNA interference targeting
the mRNAs of .alpha.1,3-fucosyltransferase (FucT) and
.beta.1,2-xylosyltransferase (XylT). In an even more preferred
embodiment, the construct is a RNA hairpin construct. These methods
for altering the N-glycosylation pattern of proteins in duckweed
are known in the art and are described in U.S. Pat. No. 7,884,264
to Dickey et al. The use of the RNA hairpin construct can be
advantageous for obtaining a glycan profile where at least 30% of
the N-glycans are G0 glycans lacking Fuc and Xyl residues and/or
where the combination of G0 glycans lacking Fuc and Xyl plus
high-mannose glycans are at least 70% relative to the total amount
of N-glycans in the plurality of secretory IgA antibodies.
[0107] Accordingly, the present invention also relates to a method
of expressing a plurality of secretory IgA antibodies containing
multiple N-glycans, such as two or more different N-glycans, using
any of the above-mentioned techniques. Generally, the plurality of
secretory IgA antibodies contains at least about 30% G0 glycans
(preferably G0 glycans lacking Fuc and Xyl residues) relative to
the total amount of N-glycans in the population. Alternatively, or
in addition thereto, the plurality of secretory IgA antibodies can
contain at least about 25% high-mannose glycans (e.g., Man5, Man6,
Man7, Man8, and/or Man9 glycans) relative to the total amount of
N-glycans in the population. Often the G0 glycans (preferably G0
glycans lacking Fuc and Xyl residues) and high-mannose glycans
(e.g., Man5, Man6, Man7, Man8, and/or Man9 glycans) together are
the majority of glycans present in the plurality of secretory IgA
antibodies, such as at least 70% relative to the total amount of
N-glycans in the plurality of secretory IgA antibodies.
[0108] The glycoform of an antibody, and the nature of glycan
species, can be determined by measuring the glycosylation profile
thereof. The term "glycosylation profile" means the characteristic
fingerprint of the representative N-glycan species that have been
released from an antibody, either enzymatically or chemically, and
then analyzed for their carbohydrate structure, for example, using
LC-HPLC, or MALDI-TOF MS, and the like. See, for example, the
review in Current Analytical Chemistry, Vol. 1, No. 1 (2005), pp.
28-57." For more information on glycosylation of therapeutic
antibodies, see, e.g., Fernandes et al., Eur. Biopharm. Rev.,
Summer 2005, pp. 106-110; Jefferis, Nature Reviews/Drug Discovery,
vol. 8, March 2009, pp. 226-234.
[0109] The duckweed lines, which produce the SIgAs, can be
cryopreserved for indefinite periods of time to ensure maintenance
of lines having specific desired characteristic and to protect them
from genetic modifications that may occur over time. Methods of
cryopreservation of duckweed are known in the art and are described
in US patent application US20120190004 to Parson et al.
Reference Example 1
Transient Expression of an Anti-TNF-.alpha. Secretory IgA Antibody
in Plants
[0110] a) Preparation of cDNA Constructs
[0111] The amino acid sequences of a suitable leader sequence
(e.g., secretion signal, SEQ ID NO:13), the heavy chain variable
region of infliximab (SEQ ID NO:14), and the human .alpha.2(n) IgA
heavy chain constant region (Chintalacharuvu et al., Journal of
Immunology, 1994, 152, 5299-5304; SEQ ID NO:10) were joined
together. Cleavage of the signal sequence (a.a. 1-19) corresponded
to the predicted cleavage site using the SignalP program (available
on the world wide web at .cbs.dtu.dk/services/SignalP).
[0112] The resulting amino acid sequence was back-translated into a
cDNA sequence optimized for expression in maize (Zea mays) (SEQ ID
NO:15) (see Liangjiang Wang and Marilyn J. Roossinck, "Comparative
analysis of expressed sequences reveals a conserved pattern of
optimal codon usage in plants." Plant Mol Biol (2006)
61:699-710).
[0113] Similarly the cDNA sequence for the light chain of the
construct was obtained by joining the sequences of a suitable
secretion signal (SEQ ID NO:16), the light chain variable region of
infliximab (SEQ ID NO:17), and the human .kappa. Ig light chain
constant region (SEQ ID NO:1), and back-translating the obtained
amino acid sequence into a cDNA sequence optimized for expression
in maize (Zea mays) (SEQ ID NO:18).
[0114] The cDNA sequence for the human J-chain was obtained by
joining the amino acid sequences of secretion signal (SEQ ID NO:19)
and J-chain sequence (SEQ ID NO:11), both obtained from
UniprotKB/Swiss-Prot database entry P01591, and back-translating
the obtained amino acid sequence into a cDNA sequence optimized for
expression in maize (Zea mays) (SEQ ID NO:20).
[0115] The cDNA sequence for the SC-chain was obtained by joining
the amino acid sequences of a suitable secretion signal (SEQ ID
NO:21) and SC-chain sequence (SEQ ID NO:12, amino acids 19-603 of
UniprotKB/Swiss-Prot database entry P01833), and back-translating
the obtained amino acid sequence into a cDNA sequence optimized for
expression in maize (Zea mays) (SEQ ID NO:22). The used secretion
signal sequence was derived from the natural SC secretion signal by
the addition of codons for two extra amino acids in order to obtain
more favorable splicing sites for the construction of plasmid
vectors.
[0116] The cDNA's of the four constructs (the heavy chain, the
light chain, the J-Chain and the SC-Chain) were obtained from a
commercial source.
b) Vector Construction and Cloning Strategy
[0117] The cDNAs for the heavy chain of the construct (HC) and of
the J-chain (JC) were ligated into the pGA15 and pGA14 plasmid
vectors, respectively, using Pacl and Ascl restriction sites. After
transfer to E. coli K12 XL10 gold and expansion, the constructs
were each transferred to separate pRAP plasmids using Ncol and Kpnl
restriction sites, resulting in the expression cassettes
35S:HC:Tnos and 35S:JC:Tnos. The pRAP plasmids containing the
expression cassettes were transferred to and expanded in E. coli
K12 DH10B.
[0118] The cDNAs for the light chain (LC) and of the SC-chain (SC)
were ligated into the pGA14 and pGA15 plasmid vectors,
respectively, using Pacl and Ascl restriction sites. After transfer
to E. coli K12 XL10 gold and expansion, the constructs were each
transferred into separate pTR2 plasmids using Ncol and Xbal
restriction sites, resulting in the expression cassettes
TR1'TR2':LC:T35S and TR1'TR2':SC:T35S. The pTR2 plasmids containing
the expression cassettes were transferred to and expanded in E.
coli K12 DH10B.
[0119] The light chain expression cassette TR1'TR2':LC:T35S was
transferred to the pRAP vector containing the heavy chain
expression cassette 35S:HC:Tnos using HindIII restriction sites,
and transferred to and expanded in E. coli K12 DH10B. Finally, the
combined cassettes containing HC and LC
(35S:HC:Tnos:TR1'TR2':LC:T35S) were transferred to a pBIN+
expression vector using AscI and PacI restriction sites. This pBIN+
vector containing the combined HC and LC cassettes was transferred
to Agrobacterium tumefaciens strain MOG101 using electroporation.
Similarly the cassettes containing JC and SC
(35S:JC:Tnos:TR1'TR2':SC:T35S) were combined in a pBIN+ expression
vector. This pBIN+ vector containing the combined JC and SC
cassettes was transferred to Agrobacterium tumefaciens strain
MOG101 using electroporation.
[0120] The two transfected A. tumefaciens strains were used in
combination for transient plant transformation and expression of
the full SIgA construct. For vector information see also van
Engelen et al., Plant Molecular Biology 1994, 26: 1701-1710.
Information on AlMV leader sequence: van der Vossen et al., Nucleic
Acids Research 1993, 21: 1361-1367. pBIN+ is described in: van
Engelen et al., Transgenic Research 1995, 4: 288-290. (In the
referenced literature sources: pRAP35=pCPO31). By way of example,
FIG. 5 shows a schematic representation of the building of the
pBIN+ vector containing the combined HC and LC cassettes.
c) Transient Expression in Tobacco Plants
[0121] The youngest fully expanded leaves of six week old tobacco
plants were infiltrated with a mixture of the two A. tumefaciens
strains by placing a 2-ml syringe (without needle) containing the
bacterial cell suspension at the lower side of a leaf and gently
pressing the suspension into the leaf. The infiltrated area was
usually clearly visible. Expression took 4-6 days with optimum
levels at days 5-6.
[0122] The leaves were frozen at -80.degree. C., crushed and 2 ml
extraction buffer per gram of leaf material was added. The
extraction buffer was PBS pH=7.4; 0.02% Tween-20 (v/v); 2% polyclar
AT (v/v); and 1% inhibitor cocktail (v/v). The suspension was
homogenized with an Ultra Turrax (Janke & Kunkel). Solid
material was removed by centrifugation (15 minutes, 10.000 g). The
solid material was extracted twice more with extraction buffer, the
same as above except the inhibitor cocktail was replaced with 10 mM
PMSF. The combined extracts were stored at -20.degree. C. Using
SDS-page and immunoblotting, formation of a complete SIgA could be
shown, but the predominant antibody expressed was the monomeric
IgA. The expression level for the IgA antibodies (including all
molecular forms) was less than 0.15 mg/kg of tobacco leaves. The
majority of the expressed IgA was monomeric IgA, and sIgA was
effectively not recoverable.
d) Transient Expression in Lettuce
[0123] The SIgA construct was transiently expressed in lettuce by
vacuum infiltration using the same vectors as for expression in
tobacco (see: Negrouk et al., Highly efficient transient expression
of functional recombinant antibodies in lettuce, Plant Science
2005, 169, 433-438). Full grown crops of lettuce Lactuca sativa L.
(Oak Leaf lettuce) and vacuum infiltrated with a mixture of the two
A. tumefaciens strains and harvested 3-5 days after infiltration.
Formation of complete SIgA was not certain, and in any event was
expressed at insubstantial or trace levels; i.e., <0.01 mg/kg of
lettuce and recovery was not possible.
Example 2
Stable Expression of Anti-TNF-.alpha. Secretory IgA Antibody
(ADB-SA1g) in Lemna
[0124] Anti-TNF-.alpha. SIgA was produced in Lemna by stable
insertion of DNA coding for the following proteins: The amino acid
sequence of the heavy chain consisted of the rice amylase secretion
signal (SEQ ID NO:23) attached to the N-terminal amino acid of the
heavy chain variable region of adalimumab (anti-TNF-.alpha. IgG1,
Humira .RTM., CAS number 331731-18-1) (SEQ ID NO:24) attached to
the N-terminal amino acid of a human IgA1 heavy chain constant
region (SEQ ID NO:7). The amino acid sequence of the light chain of
the Anti-TNF-.alpha. SIgA combines the rice amylase secretion
signal (SEQ ID NO:23) with the TNF-.alpha. binding light chain
variable region of adalimumab (SEQ ID NO:25) and the human
.kappa.-light chain constant region (SEQ ID NO:1). The amino acid
sequence of the J-chain consisted of the rice amylase secretion
signal (SEQ ID NO:23) attached to the N-terminal amino acid of the
human J-chain (SEQ ID NO:11). The amino acid sequence of the
SC-chain consisted of the rice amylase secretion signal (SEQ ID
NO:23) attached to the N-terminal amino acid of the human SC-chain
(SEQ ID NO:12). The produced SIgA, having IgA1 heavy chain constant
region, human .kappa.-light chain constant region, and adalimumab
heavy and light chain variable regions, combined with human J-chain
and SC-chain is referred to as ADB-SA1. ADB-SA1g specifically
refers to material produced in Lemna with modified N-glycosylation
(i.e. G0 glycosylation lacking fucose and xylose)
[0125] Genes were designed for each of the four components with
Lemna minor preferred codon usage (63-67% GC content). Tables with
suitable preferred codon use in Lemnaceae can be found in PCT
application WO2005/035768 and in relevant references contained
therein. The synthetic genes also contained the rice
.alpha.-amylase signal sequence (GenBank M24286; SEQ ID NO:23)
fused to the 5' end of their coding sequences. Restriction
endonuclease sites were added to allow cloning into A. tumefaciens
binary vectors. Design of DNA sequences and vector construction was
performed by Biolex Therapeutics, Inc., Pittsboro, N.C., USA. DNA
sequences were produced by DNA2.0 (Menlo Park, Calif., USA).
[0126] The ADB-SA1g antibody was expressed in Lemna minor by
transfection via an A. tumefaciens binary vector containing DNA
sequences encoding all four of the SIgA components: J-chain (SEQ ID
NO:26), SC-chain (SEQ ID NO:27), H-chain (SEQ ID NO:28) and L-chain
(SEQ ID NO:29). To prepare this vector, independent expression
cassettes were created containing a promoter and also DNA sequences
encoding the protein and terminator for the J-chain, SC-chain,
H-chain and L-chain. The H chain was fused to the modified chimeric
octopine and mannopine synthase promoter with Lemna gibba 5' leader
from ribulose bis-phosphatecarboxylase small subunit-1. The
L-chain, SC-chain and J-chain genes were fused to high expression
Lemnaceae Ubiquitin promoters L. minor polyubiquitin promoter
(LmUbq), Lemna aequinoctialis polyubiquitin promoter (LaUbq) and
Spirodela polyrrhiza polyubiquitin promoter (SpUbq), respectively.
Sequences of these promoters have been disclosed in PCT application
WO2007/124186. These expression cassettes were then cloned into a
single A. tumefaciens transformation vector EC2.2 (a modification
of the A. tumefaciens binary vector pBMSP3, which is a derivative
of pBINPLUS. See Ni, M., Cui, D., Einstein, J., Narasimhulu, S.,
Vergara, C. E., and Gelvin, S. B., Plant J. 7, 661-676, (1995), van
Engelen, Transgenic Res. 4:288-290 (1995), and Gasdaska et al.,
Bioprocessing J., 3:50-56 (2003)), with the appropriate restriction
sites to create the final transformation vector SynB02. This vector
also contained the gentamicin acetyltransferase-3-I gene (aacC1)
which confers resistance to gentamicin and allows for selection of
transgenic L. minor lines.
[0127] SynB02 was used to create an additional transformation
vector to generate a glycan optimized version of anti-TNF-.alpha.
SIgA, having G0 glycosylation lacking fucose and lacking xylose. A
chimeric hairpin RNA was used to silence endogenous L. minor mRNAs
encoding .alpha.-1,3-fucosyltransferase (Fuct1, GenBank DQ789145)
and .beta.-1,2-xylosyltransferase (Xylt1, GenBank DQ789146). A DNA
sequence for this chimeric RNAi molecule was fused to the high
expression SpUbq promoter and subsequently moved into the SynB02
vector creating the final transformation vector SynB03. Further
details on production of glycan optimized proteins in Lemnaceae can
be found in PCT applications WO2007/084672, WO2007/084922,
WO2007/084926 and in Cox, K. M., Nature Biotechnology 2006, 12:
1591-1597.
[0128] Lemna minor strain 8627 was transfected with vector SynB03,
and glycosylation modified Lemna minor strain XF04 was transfected
with vector SynB02. Once transformed plants were regenerated
(approximately three months), single plants were harvested from the
antibiotic selection plates and propagated separately in liquid
growth media, without selection antibiotic, for further screening
and characterization. Thus several hundred individual transgenic
plant lines from each construct were generated. Independent
transgenic lines were harvested and clonally propagated in
individual harvest jars. For screening of transgenic lines, clonal
lines were preconditioned for 1 week at light levels of 150 to 200
.mu.mol/m.sup.2s in vented plant growth vessels containing SH
medium (Schenk R. U. et al., Can. J. Biol. 1972, 50: 199-204)
without sucrose. Fifteen to twenty preconditioned fronds were then
placed into vented containers containing fresh SH medium, and
allowed to grow for two weeks. Tissue samples from each line were
collected and frozen for analysis.
[0129] To determine SIgA expression, frozen tissue samples were
homogenized and centrifuged, and the supernatant was removed and
screened by an ELISA method using sheep anti-human IgA secretory
chain (AbD Serotec catalog #5111-4804--1:1000 dilution) coated
plates to capture the SIgA antibody. The samples were then detected
using a goat anti-human kappa light chain HRP conjugated antibody
(Sigma catalog #A7164--1:2000 dilution). The highest-expressing
lines from this primary screening were then grown again for two
weeks in small research vessels under the optimal growth
conditions. The resulting tissue was harvested and the ELISA was
performed to determine the percent of the total soluble protein
that is the expressed SIgA antibody (ADB-SA1g). The results are
summarized in Table 2.
TABLE-US-00002 TABLE 2 # of lines Highest Construct Glycosylation
screened Expression level SynB02 G0 glycosylation 55 16.2% TSP
SynB03 G0 glycosylation 227 13.6% TSP
[0130] Results of non-reducing and reducing SDS-PAGE analyses of
purified material obtained from Lemna transfected with construct
SynB02 are shown in FIG. 6. ADB-SA1g can be isolated from lemna
culture and purified as described in Example 3.
Example 3
Isolation and Purification of Anti-TNF-.alpha. Secretory IgA
Antibody ADB-SA1g from Lemna
[0131] Biomass from transgenic Lemna expressing anti TNF-.alpha.
SIgA, having variable regions that are the amino acid sequence of
the variable regions (antigen binding regions) of adalimumab, was
homogenized in 50 mM Sodium phosphate, 0.3M Sodium chloride, buffer
pH 7.4, at a buffer to tissue ratio of 4:1. An acid precipitation
step was performed on the crude extract to remove rubisco and other
plant proteins by adjusting the extract to pH 4.5 using 1M Sodium
acetate, pH 2.5. The precipitate was removed by centrifugation of
the material at 14,000.times.g for 30 minutes at 4.degree. C. The
supernatant was adjusted to pH 7.4 and loaded on DOWEX (Dowex
1.times.2 anion exchange resin) to remove colored impurities. The
flow-through fraction containing anti TNF-.alpha. SIgA was 0.22
.mu.m was filtered prior to chromatography using affinity
chromatography and Size Exclusion Chromatography.
[0132] Affinity purification: A KappaSelect (GE Healthcare prod.
Nr. 17-5458-03) column was prepared according to manufacturer
instructions (28-9448-22 AA). The column was equilibrated with 5 cv
of TBS buffer (50 mM Tris, 0.15M Sodium chloride, pH 7.4). The
supernatant was loaded on the column. Approximately 5 mg sIgA/ml
resin was loaded on columns of up to 1 L KappaSelect. Non-binding
material was washed from the column with 5 cv TBS buffer. The
product was eluted from the column using 10 cv of 25 mM Sodium
Acetate, pH 6.6 buffer containing 3.5 M MgCl.sub.2. The fractions
containing the secretory proteins were pooled and immediately
diluted 4-fold using TBS buffer (50 mM Tris, 0.15M Sodium chloride,
pH 7.4). MgCl.sub.2 was replaced by buffer exchange with at least
10 volumes of TBS buffer using a Pall Centramate cassette
ultrafiltration system and a Pellicon 2 Mini Filter (Millipore
prod.Nr. P2C005C01-PLCCC 5K, regenerated cellulose) with 5 kDa
cut-off.
[0133] SEC purification: Material obtained by KappaSelect
chromatography was further purified on a Sephacryl S300 HR column.
The column was equilibrated with 2 cv of PBS buffer at a flow rate
of 1.0 ml/min. The KappaSelect eluate was concentrated 3-4.times.
using ultra filtration using a Pellicon 2 Mini Filter (Millipore
prod.Nr. P2C005C01-PLCCC 5K, regenerated cellulose) with 5 kDa
cut-off.
[0134] Typically, a concentration of 3-5 mg/ml and feed volume of
50 ml was used for a 1L column. The feed was applied using an AKTA
purifier at 1.0 ml/min. Elution was performed with at least 2
column volumes of PBS buffer at room temperature and a flow rate of
1.0 ml/min. Fractions were collected and sufficiently pure
fractions were pooled.
Example 4
Stable Expression of Anti-IL-12/23 Secretory IgA Antibody Based on
Ustekinumab (UKB-SA1) in Lemna
a) Construction of Vectors
[0135] Synthetic genes were designed for each of the 4 different
protein chains of an anti-IL-12/23 secretory IgA. The amino acid
sequence of the heavy chain consisted of the rice .alpha.-amylase
secretion signal (SEQ ID NO:23) joined to the N-terminal amino acid
of the variable part of the heavy chain of anti-IL12/23 IgG1
antibody Ustekinumab (Stelara.RTM., CAS number 815610-63-O, SEQ ID
NO:30) which in turn is joined to the N-terminal amino acid of the
constant part of a human IgA1 heavy chain (SEQ ID NO:7). The amino
acid sequence of the light chain consisted of the rice
.alpha.-amylase secretion signal (SEQ ID NO:23) joined to the
N-terminal amino acid of the light chain sequence of Ustekinumab
(CAS number 815610-63-0), which combines an anti-IL-12/23 binding
variable part (SEQ ID NO:31) with a human .kappa.-light chain
constant part (SEQ ID NO:1). The SC-chain consisted of the rice
.alpha.-amylase secretion signal (SEQ ID NO:23) joined to the
N-terminal amino acid of the amino acid sequence of amino acids 19
to 603 of the human polymeric immunoglobulin receptor disclosed in
UniProtKB/Swiss-Prot database entry P01833 (SEQ ID NO:12). The
J-chain sequence consisted of the rice .alpha.-amylase secretion
signal (SEQ ID NO:23) joined to the N-terminal amino acid of the
amino acid sequence of amino acids 23 to 159 of the human sequence
disclosed in UniProtKB/Swiss-Prot database entry P01591 (SEQ ID
NO:11).
[0136] Genes were designed for each of the four components with
Lemna minor preferred codon usage (63-67% GC content). Tables with
suitable preferred codon use in Lemnaceae can be found in PCT
application WO2005/035768 and in relevant references contained
therein. Restriction endonuclease sites were added to allow cloning
into A. tumefaciens binary vectors. Design of DNA sequences and
vector construction was performed by Biolex Therapeutics, Inc.,
Pittsboro, N.C., USA. DNA sequences were produced by DNA2.0 (Menlo
Park, Calif., USA).
[0137] The anti-IL-12/23 SIgA (UKB-SA1) was expressed in Lemna
minor by transfection via an Agrobacterium tumefaciens binary
vector containing DNA sequences encoding all four of the SIgA
components: J-chain, SC-chain, H-chain and L-chain. To prepare this
vector independent expression cassettes were created containing
promoter, DNA sequences encoding the protein and terminator for the
J-chain (SEQ ID NO:26), SC-chain (SEQ ID NO:27), H-chain (SEQ ID
NO:32) and L-chain (SEQ ID NO:33). The H chain was fused to the
modified chimeric octopine and mannopine synthase promoter with
Lemna gibba 5' leader from ribulose bis-phosphatecarboxylase small
subunit-1. The L-chain, SC-chain and J-chain genes were fused to
high expression Lemnaceae Ubiquitin promoters L. minor
polyubiquitin promoter (LmUbq), Lemna aequinoctialis polyubiquitin
promoter (LaUbq) and Spirodela polyrhiza polyubiquitin promoter
(SpUbq), respectively. Sequences of these promoters have been
disclosed in PCT application WO2007/124186. These expression
cassettes were then cloned into a single A. tumefaciens
transformation vector EC2.2 (a modification of the A. tumefaciens
binary vector pBMSP3, which is a derivative of pBINPLUS. See Ni,
M., Cui, D., Einstein, J., Narasimhulu, S., Vergara, C. E., and
Gelvin, S. B. Plant J. 7, 661-676, (1995), van Engelen Transgenic
Res. 4:288-290 (1995), and Gasdaska et al., Bioprocessing J.,
3:50-56 (2003)), with the appropriate restriction sites to create
the final transformation vector SynA01 (FIG. 7A). This vector also
contained the gentamicin acetyltransferase-3-I gene (aacC1) which
confers resistance to gentamicin and allows for selection of
transgenic L. minor lines, and was used to produce UKB-SA1 with
wild-type (unmodified) N- and O-glycosylation.
[0138] SynA01 was used to create additional transformation vectors
to generate a glycan optimized version of UKB-SA1, further
identified as UKB-SA1g0. UKB-SA1g0 has G0 glycosylation and lacks
fucose and xylose. The term "G0 glycosylation" (also referred to as
"G0") is a term of art indicating a particular type of human
N-glycosylation defined by the lack of any galactose residues. See,
e.g., Fernandes et al., "Demonstrating Comparability of Antibody
Glycosylation during Biomanufacturing," Eur. Biopharm. Rev., Summer
2005, pp. 106-110; Jefferis, "Glycosylation as a strategy to
improve antibody-based therapeutics," Nature Reviews/Drug
Discovery, vol. 8, March 2009, pp. 226-234. A chimeric hairpin RNA
was used to silence endogenous L. minor mRNAs encoding
.alpha.-1,3-fucosyltransferase (Fuct1, GenBank DQ789145) and
.beta.-1,2-xylosyltransferase (Xylt1, GenBank DQ789146). A DNA
sequence for this chimeric RNAi molecule was fused to the high
expression SpUbq promoter and subsequently moved into the SynA01
vector creating the final transformation vector SynA02 (FIG. 7B).
Also the neomycin phosphotransferase II gene (NPTII) was moved into
SynA01 replacing aacC1 to produce transformation vector SynA03
(FIG. 7C). This exchange allows for kanamycin selection instead of
gentamicin selection of transgenic glycan optimized L. minor lines.
Further details on procedures for production of glycan optimized
proteins in Lemnaceae can be found in PCT applications
WO2007/084672, WO2007/084922, WO2007/084926 and in Cox, K. M.,
Nature Biotechnology 2006, 12: 1591-1597.
b) Transformation of and Expression Using Lemna
[0139] Lemna transformation vectors SynA01, SynA02 and SynA03 were
transvected into Agrobacterium tumefaciens strain C58Z707 (Hepburn
et al., J. Gen. Microbiol. 1985, 131: 2961-2969) by
electroporation. Agrobacterium colonies were selected using
gentamycin (SynA01 and SynA02) or kanamycin (SynA03) and analyzed
for the presence of the appropriate binary vector using a PCR based
assay. A single colony was selected for each transformation vector
and taken forward into L. minor transformation process (as
follows).
[0140] Partially dedifferentiated Lemna tissue (Lemna minor strain
8627) was incubated with Agrobacterium harboring the expression
cassette plasmid by briefly dipping the tissue into the solution.
The tissue was then placed on co-cultivation plates for two days in
continuous light at 25.degree. C. Following co-cultivation, the
tissue was transferred to antibiotic selection plates and returned
to continuous light at 25.degree. C. The tissue was transferred
weekly to fresh antibiotic selection plates. Cefotaxime was
included in the antibiotic selection plates to eradicate the
Agrobacterium. Gentamicin was included in these plates to select
for transgenic tissue obtained from vectors SynA01 and SynA02 where
there is a selectable marker gene included in the transferred
genetic cassette which confers gentamycin resistance. Kanamycin was
included in plates to select for transgenic tissue from vector
SynA03.
[0141] UKB-SA1 with unmodified wild-type (WT) glycosylation was
obtained by transfection of Lemna minor strain 8627 (Biolex
Therapeutics Inc.) with vector SynA01. UKB-SA1g0, having G0
glycosylation lacking fucose and lacking xylose, was obtained by
transfecting Lemna minor strain 8627 with vector SynA02, or by
transfection of the N-glycosylation modified Lemna minor strain
XF04 (Biolex Inc.) with vector SynA03. Once transformed plants were
regenerated (approximately three months) single plants were
harvested from the antibiotic selection plates and propagated
separately in liquid growth media, without selection antibiotic,
for further screening and characterization. Thus several hundred
individual transgenic plant lines from each construct were
generated. Independent transgenic lines were harvested and clonally
propagated in individual harvest jars. For screening of transgenic
lines, clonal lines were preconditioned for 1 week at light levels
of 150 to 200 .mu.mol/m2s in vented plant growth vessels containing
SH medium (Schenk R. U. et al., Can. J. Biol. 1972, 50: 199-204)
without sucrose. Fifteen to twenty preconditioned fronds were then
placed into vented containers containing fresh SH medium, and
allowed to grow for two weeks. Tissue samples from each line were
collected and frozen for analysis.
[0142] To determine SIgA expression, frozen tissue samples were
homogenized, centrifuged and the supernatant was removed and
screened by an ELISA method using sheep anti-human IgA secretory
chain (AbD Serotec catalog #5111-4804--1:1000 dilution) coated
plates to capture the SIgA antibody. The samples were then detected
using a goat anti-human kappa light chain HRP conjugated antibody
(Sigma catalog #A7164--1:2000 dilution). The highest lines from
this primary screening were then grown again for two weeks in small
research vessels under the optimal growth conditions, the resulting
tissue was harvested and the ELISA was performed to determine the
percent of the total soluble protein that the SIgA antibody is
expressed. The results are summarized in Table 3.
TABLE-US-00003 TABLE 3 # of lines Highest Construct Product
screened Expression level SynA01 in UKB-SA1 262 8.5% TSP Lemna
Minor 8627 (WT glycosylation) SynA02 in UKB-SA1g0 164 15.1% TSP
Lemna Minor 8627 (G0 N-glycosylation) SynA03 in UKB-SA1g0 452 11.8%
TSP Lemna Minor XF04 (G0 N-glycosylation)
Example 5
Isolation and Purification of UKB-SA1 and UKB-SA1g0 Secretory IgA
Antibody from Lemna
[0143] Biomass from transgenic Lemna expressing UKB-SA1 or
UKB-SA1g0, having variable regions that are the amino acid sequence
of the variable regions
[0144] (antigen binding regions) of Ustekinumab, was homogenized in
50 mM Sodium phosphate, 0.3M Sodium chloride, buffer pH 7.4, at a
buffer to tissue ratio of 4:1. An acid precipitation step was
performed on the crude extract to remove the enzyme RuBisCo and
other plant proteins by adjusting the extract to pH 4.5 using 1M
Sodium acetate, pH 2.5. The precipitate was removed by
centrifugation of the material at 14,000.times.g for 30 minutes at
4.degree. C. The supernatant was adjusted to pH 7.4 and filtered to
0.22 .mu.m prior to IMAC chromatography.
[0145] IMAC purification: A Chelating Sepharose FF (GE Healthcare
prod. Nr. 17-0575-01) column was prepared according to manufacturer
instructions (28-4047-39 AC). The column was charged with 3-5
column volumes (cv) of 0.1M Copper sulfate. Excess copper was
washed with 3-5 cv of double distilled water. The column was
equilibrated with 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, pH
4.0 buffer; 3-5 cv PBS buffer with 0.5M Imidazole; and 3-5 cv PBS
buffer.
[0146] The supernatant was loaded on the column. Approximately 3.8
mg SIgA/ml resin was loaded on columns of up to 350 ml Chelating
Sepharose. Non-binding material was washed from the column with 10
cv PBS buffer, 10 cv of 0.1M Sodium acetate, pH 4.0 buffer, and 10
cv PBS buffer. The product was eluted from the column using 10 cv
of PBS buffer containing 0.075 M Imidazole (a gradient of 0-0.075 M
imidazole was also used but did not lead to improved results). The
fractions containing the secretory IgA antibodies were pooled.
[0147] The column was regenerated by removing copper using a 0.2M
EDTA, 0.3M sodium chloride solution, followed by treatment with
0.1N NaOH.
[0148] SEC purification: Material obtained by IMAC chromatography
was further purified on a Sephacryl S300 HR column. The column was
equilibrated with 2 column volumes of PBS buffer at a flow rate of
0.5 ml/min. The IMAC eluate was concentrated 3.times. using ultra
filtration (e.g., using a 5- or 30 kDa regenerated cellulose
(hydrosart) membrane) by using, for example, a spin filter
(Vivaspin 15R) or a UF cassette (Sartorius 305 144 59 01 E).
Typically, a concentration of 5-7 mg/ml and feed volume of 15-20 ml
was used for a 360 ml column. The feed was applied using an AKTA
purifier at 0.5 ml/min. Elution was performed with at least 2
column volumes of PBS buffer at room temperature and a flow rate of
0.5 ml/min. Fractions were collected and sufficiently pure
fractions were pooled.
[0149] Results of non-reducing and reducing SDS-PAGE analyses of
purified material obtained from Lemna transfected with construct
SynA01 are shown in FIG. 8 as gels A and B, respectively.
[0150] Some batches of material produced by the purification method
described above were further purified using affinity chromatography
using CaptureSelect human IgA (BAC).
[0151] Using an Akta Explorer 10 system a column loaded with 30.9
ml Capture Select IgA (BAC) was equilibrated using 3 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 of 2.5 ml/min. After a wash
step with 5 CV of 20 mM Tris buffer pH 7.0 at 5 ml/min the product
was eluted with 5 CV of 20 mM Tris buffer pH 7.0 containing 3.5 M
MgCl.sub.2 at 5 ml/min.
[0152] The eluate was first dialyzed twice against 5 L 20 mM Tris
buffer pH 7.0 using snakeskin 10 kDa dialysis tubing for at least 2
hours, followed by dialysis against 5 L PBS puffer pH 7.4 for at
least 2 hours. The solution was concentrated using a stirred cell
(Amicon 8200, Millipore, overhead pressure 30 Psi) with 30 kDa
regenerated cellulose membrane filter (Millipore) to a final
concentration of approximately 1 mg/ml. The obtained product was
further purified using SEC purification as described previously.
Glycosylation modified product UKB-SA1g0 was purified using the
same method.
[0153] No substantial differences were observed in purification of
the two glycosylation forms, wild-type UKB-SA1 and G0
N-glycosylated UKB-SA1g0.
Example 6
Binding of UKB-SA1 and UKB-SA1g0 to IL12
[0154] The binding of purified anti-IL-12/23 SIgA, with variable
regions taken from Ustekinumab and produced in Lemna as in Examples
11 and 12, to IL-12 was determined. The binding of both wild type
glycosylation (UKB-SA1) and G0-glycosylation (UKB-SA1g0) products
were determined in comparison to Ustekinumab (Stelara.RTM.) and
colostral SIgA. Plates were coated with IL-12 (Abcam, AB52086) 1
.mu.g/ml. Detection of bound UKB-SA1/UKB-SA1g0 (secretory IgA) and
Ustekinumab (IgG1) antibodies was performed using a 1:1500 fold
dilution of anti human kappa chain antibody (Abbiotec, cat. no.
250987), 100 .mu.l per well, for one hour at RT, washing 3 times,
30 seconds with 200 .mu.l PBS/0.05% Tween with shaking, followed by
incubation with a 1:1500 fold dilution of donkey anti mouse HRP
conjugated (Emelca biosciences, MS3001), 100 .mu.l per well, for
one hour at RT.
[0155] The UKB-SA1, UKB-SA1g0 and Ustekinumab antibodies all bound
with high affinity to IL-12 under the conditions of this assay. For
UKB-SA1 and UKB-SA1g0 antibodies, binding occurred independent of
the type of glycosylation. Colostral SIgA had minimal to no binding
to IL-12.
Example 7
Proteolytic Stability of UKB-SA1 and UKB-SA1g0
[0156] The stability of UKB-SA1 and UKB-SA1g0 with antigen binding
regions having the amino acid sequence of Ustekinumab antigen
binding regions (i.e., the variable heavy and light chains),
produced in Lemna, was determined in simulated intestinal fluid
(SIF, 0.05M phosphate buffer pH 6.8 containing 10 mg/ml
pancreatin). Both the form with wild-type Lemna glycosylation
obtained with vector SynA01, and the G0 glycosylation variant
obtained with vector SynA02 were analysed. Stability was compared
to Ustekinumab (IgG1) and to aspecific human colostral SIgA which
was purified to contain only kappa light chains and .alpha.-1 heavy
chains.
[0157] 90 .mu.l of a 1 mg/ml solution of the material to be tested
was added to 810 .mu.l of SIF at 37.degree. C. 50 .mu.l samples
were drawn at T=0, 5, 15, 30, 60 and 120 min and immediately frozen
in liquid nitrogen. Samples were analyzed by non-reducing SDS-Page
gel electrophoresis. Briefly; 17 .mu.l of a 0.15M solution of
iodoacetamide in LDS sample buffer was added to each of the frozen
samples. Samples were thawed and applied to Criterion Tris-HCl gel
(12.5%, 18 well, 30 .mu.l comb (Biorad, 345-0015). After
electrophoresis gels were treated with Krypton protein stain and
analyzed. Stability of the samples was qualitatively assessed
visually. Results are shown in FIG. 9 where gel A is the WT
glycosylation (UKB-SA1, labeled SynA01-WT in FIG. 9) and gel B is
G0 glycosylation (UKB-SA1g0, labeled SynA02-G0 in FIG. 9).
[0158] Both glycosylation forms (WT and G0) of Lemna produced
anti-IL12/23 SIgA exhibited a stability that was comparable to
natural human colostral SIgA. The IgG1 antibody Ustekinumab
degraded under these conditions at such a high rate that detection
at T=0 was not possible.
[0159] Each of the patents, patent applications, and journal
articles mentioned above are incorporated herein by reference in
their entirety. The invention having been described it will be
obvious that the same may be varied in many ways and all such
modifications are contemplated as being within the scope of the
invention as defined by the claims appended hereto.
Sequence CWU 1
1
331107PRTHomo sapiens 1Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90
95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 2106PRTHomo
sapiens 2Gly Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro
Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys
Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp
Lys Ala Asp Gly Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr
Lys Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser
Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser
Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys
Thr Val Ala Pro Thr Glu Cys Ser 100 105 3106PRTHomo sapiens 3Gly
Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 1 5 10
15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser
Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys
Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu
Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln
Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro
Thr Glu Cys Ser 100 105 4106PRTHomo sapiens 4Gly Gln Pro Lys Ala
Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu
Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe
Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 35 40
45 Ala Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln
Trp Lys 65 70 75 80 Ser His Lys Ser Tyr Ser Cys Gln Val Thr His Glu
Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser
100 105 5106PRTHomo sapiens 5Gly Gln Pro Lys Ala Ala Pro Ser Val
Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys
Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala
Val Lys Val Ala Trp Lys Ala Asp Gly Ser Pro 35 40 45 Val Asn Thr
Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70
75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr
Val 85 90 95 Glu Lys Thr Val Ala Pro Ala Glu Cys Ser 100 105
6106PRTHomo sapiens 6Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu
Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr
Leu Val Cys Leu Val Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr
Val Ala Trp Lys Ala Asp Gly Ser Pro 35 40 45 Val Lys Val Gly Val
Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala
Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 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 100 105 7353PRTHomo
sapiens 7Ala Ser Pro Thr Ser Pro Lys Val Phe Pro Leu Ser Leu Cys
Ser Thr 1 5 10 15 Gln Pro Asp Gly Asn Val Val Ile Ala Cys Leu Val
Gln Gly Phe Phe 20 25 30 Pro Gln Glu Pro Leu Ser Val Thr Trp Ser
Glu Ser Gly Gln Gly Val 35 40 45 Thr Ala Arg Asn Phe Pro Pro Ser
Gln Asp Ala Ser Gly Asp Leu Tyr 50 55 60 Thr Thr Ser Ser Gln Leu
Thr Leu Pro Ala Thr Gln Cys Leu Ala Gly 65 70 75 80 Lys Ser Val Thr
Cys His Val Lys His Tyr Thr Asn Pro Ser Gln Asp 85 90 95 Val Thr
Val Pro Cys Pro Val Pro Ser Thr Pro Pro Thr Pro Ser Pro 100 105 110
Ser Thr Pro Pro Thr Pro Ser Pro Ser Cys Cys His Pro Arg Leu Ser 115
120 125 Leu His Arg Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser Glu Ala
Asn 130 135 140 Leu Thr Cys Thr Leu Thr Gly Leu Arg Asp Ala Ser Gly
Val Thr Phe 145 150 155 160 Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala
Val Gln Gly Pro Pro Glu 165 170 175 Arg Asp Leu Cys Gly Cys Tyr Ser
Val Ser Ser Val Leu Pro Gly Cys 180 185 190 Ala Glu Pro Trp Asn His
Gly Lys Thr Phe Thr Cys Thr Ala Ala Tyr 195 200 205 Pro Glu Ser Lys
Thr Pro Leu Thr Ala Thr Leu Ser Lys Ser Gly Asn 210 215 220 Thr Phe
Arg Pro Glu Val His Leu Leu Pro Pro Pro Ser Glu Glu Leu 225 230 235
240 Ala Leu Asn Glu Leu Val Thr Leu Thr Cys Leu Ala Arg Gly Phe Ser
245 250 255 Pro Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln Glu
Leu Pro 260 265 270 Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu
Pro Ser Gln Gly 275 280 285 Thr Thr Thr Phe Ala Val Thr Ser Ile Leu
Arg Val Ala Ala Glu Asp 290 295 300 Trp Lys Lys Gly Asp Thr Phe Ser
Cys Met Val Gly His Glu Ala Leu 305 310 315 320 Pro Leu Ala Phe Thr
Gln Lys Thr Ile Asp Arg Leu Ala Gly Lys Pro 325 330 335 Thr His Val
Asn Val Ser Val Val Met Ala Glu Val Asp Gly Thr Cys 340 345 350 Tyr
8340PRTHomo sapiens 8Ala Ser Pro Thr Ser Pro Lys Val Phe Pro Leu
Ser Leu Asp Ser Thr 1 5 10 15 Pro Gln Asp Gly Asn Val Val Val Ala
Cys Leu Val Gln Gly Phe Phe 20 25 30 Pro Gln Glu Pro Leu Ser Val
Thr Trp Ser Glu Ser Gly Gln Asn Val 35 40 45 Thr Ala Arg Asn Phe
Pro Pro Ser Gln Asp Ala Ser Gly Asp Leu Tyr 50 55 60 Thr Thr Ser
Ser Gln Leu Thr Leu Pro Ala Thr Gln Cys Pro Asp Gly 65 70 75 80 Lys
Ser Val Thr Cys His Val Lys His Tyr Thr Asn Pro Ser Gln Asp 85 90
95 Val Thr Val Pro Cys Pro Val Pro Pro Pro Pro Pro Cys Cys His Pro
100 105 110 Arg Leu Ser Leu His Arg Pro Ala Leu Glu Asp Leu Leu Leu
Gly Ser 115 120 125 Glu Ala Asn Leu Thr Cys Thr Leu Thr Gly Leu Arg
Asp Ala Ser Gly 130 135 140 Ala Thr Phe Thr Trp Thr Pro Ser Ser Gly
Lys Ser Ala Val Gln Gly 145 150 155 160 Pro Pro Glu Arg Asp Leu Cys
Gly Cys Tyr Ser Val Ser Ser Val Leu 165 170 175 Pro Gly Cys Ala Gln
Pro Trp Asn His Gly Glu Thr Phe Thr Cys Thr 180 185 190 Ala Ala His
Pro Glu Leu Lys Thr Pro Leu Thr Ala Asn Ile Thr Lys 195 200 205 Ser
Gly Asn Thr Phe Arg Pro Glu Val His Leu Leu Pro Pro Pro Ser 210 215
220 Glu Glu Leu Ala Leu Asn Glu Leu Val Thr Leu Thr Cys Leu Ala Arg
225 230 235 240 Gly Phe Ser Pro Lys Asp Val Leu Val Arg Trp Leu Gln
Gly Ser Gln 245 250 255 Glu Leu Pro Arg Glu Lys Tyr Leu Thr Trp Ala
Ser Arg Gln Glu Pro 260 265 270 Ser Gln Gly Thr Thr Thr Phe Ala Val
Thr Ser Ile Leu Arg Val Ala 275 280 285 Ala Glu Asp Trp Lys Lys Gly
Asp Thr Phe Ser Cys Met Val Gly His 290 295 300 Glu Ala Leu Pro Leu
Ala Phe Thr Gln Lys Thr Ile Asp Arg Leu Ala 305 310 315 320 Gly Lys
Pro Thr His Val Asn Val Ser Val Val Met Ala Glu Val Asp 325 330 335
Gly Thr Cys Tyr 340 9340PRTHomo sapiens 9Ala Ser Pro Thr Ser Pro
Lys Val Phe Pro Leu Ser Leu Asp Ser Thr 1 5 10 15 Pro Gln Asp Gly
Asn Val Val Val Ala Cys Leu Val Gln Gly Phe Phe 20 25 30 Pro Gln
Glu Pro Leu Ser Val Thr Trp Ser Glu Ser Gly Gln Asn Val 35 40 45
Thr Ala Arg Asn Phe Pro Pro Ser Gln Asp Ala Ser Gly Asp Leu Tyr 50
55 60 Thr Thr Ser Ser Gln Leu Thr Leu Pro Ala Thr Gln Cys Pro Asp
Gly 65 70 75 80 Lys Ser Val Thr Cys His Val Lys His Tyr Thr Asn Ser
Ser Gln Asp 85 90 95 Val Thr Val Pro Cys Arg Val Pro Pro Pro Pro
Pro Cys Cys His Pro 100 105 110 Arg Leu Ser Leu His Arg Pro Ala Leu
Glu Asp Leu Leu Leu Gly Ser 115 120 125 Glu Ala Asn Leu Thr Cys Thr
Leu Thr Gly Leu Arg Asp Ala Ser Gly 130 135 140 Ala Thr Phe Thr Trp
Thr Pro Ser Ser Gly Lys Ser Ala Val Gln Gly 145 150 155 160 Pro Pro
Glu Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser Ser Val Leu 165 170 175
Pro Gly Cys Ala Gln Pro Trp Asn His Gly Glu Thr Phe Thr Cys Thr 180
185 190 Ala Ala His Pro Glu Leu Lys Thr Pro Leu Thr Ala Asn Ile Thr
Lys 195 200 205 Ser Gly Asn Thr Phe Arg Pro Glu Val His Leu Leu Pro
Pro Pro Ser 210 215 220 Glu Glu Leu Ala Leu Asn Glu Leu Val Thr Leu
Thr Cys Leu Ala Arg 225 230 235 240 Gly Phe Ser Pro Lys Asp Val Leu
Val Arg Trp Leu Gln Gly Ser Gln 245 250 255 Glu Leu Pro Arg Glu Lys
Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro 260 265 270 Ser Gln Gly Thr
Thr Thr Tyr Ala Val Thr Ser Ile Leu Arg Val Ala 275 280 285 Ala Glu
Asp Trp Lys Lys Gly Glu Thr Phe Ser Cys Met Val Gly His 290 295 300
Glu Ala Leu Pro Leu Ala Phe Thr Gln Lys Thr Ile Asp Arg Met Ala 305
310 315 320 Gly Lys Pro Thr His Ile Asn Val Ser Val Val Met Ala Glu
Ala Asp 325 330 335 Gly Thr Cys Tyr 340 10340PRTHomo sapiens 10Ala
Ser Pro Thr Ser Pro Lys Val Phe Pro Leu Ser Leu Asp Ser Thr 1 5 10
15 Pro Gln Asp Gly Asn Val Val Val Ala Cys Leu Val Gln Gly Phe Phe
20 25 30 Pro Gln Glu Pro Leu Ser Val Thr Trp Ser Glu Ser Gly Gln
Asn Val 35 40 45 Thr Ala Arg Asn Phe Pro Pro Ser Gln Asp Ala Ser
Gly Asp Leu Tyr 50 55 60 Thr Thr Ser Ser Gln Leu Thr Leu Pro Ala
Thr Gln Cys Pro Asp Gly 65 70 75 80 Lys Ser Val Thr Cys His Val Lys
His Tyr Thr Asn Ser Ser Gln Asp 85 90 95 Val Thr Val Pro Cys Arg
Val Pro Pro Pro Pro Pro Cys Cys His Pro 100 105 110 Arg Leu Ser Leu
His Arg Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser 115 120 125 Glu Ala
Asn Leu Thr Cys Thr Leu Thr Gly Leu Arg Asp Ala Ser Gly 130 135 140
Ala Thr Phe Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val Gln Gly 145
150 155 160 Pro Pro Glu Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser Ser
Val Leu 165 170 175 Pro Gly Cys Ala Gln Pro Trp Asn His Gly Glu Thr
Phe Thr Cys Thr 180 185 190 Ala Ala His Pro Glu Leu Lys Thr Pro Leu
Thr Ala Asn Ile Thr Lys 195 200 205 Ser Gly Asn Thr Phe Arg Pro Glu
Val His Leu Leu Pro Pro Pro Ser 210 215 220 Glu Glu Leu Ala Leu Asn
Glu Leu Val Thr Leu Thr Cys Leu Ala Arg 225 230 235 240 Gly Phe Ser
Pro Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln 245 250 255 Glu
Leu Pro Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro 260 265
270 Ser Gln Gly Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg Val Ala
275 280 285 Ala Glu Asp Trp Lys Lys Gly Asp Thr Phe Ser Cys Met Val
Gly His 290 295 300 Glu Ala Leu Pro Leu Ala Phe Thr Gln Lys Thr Ile
Asp Arg Leu Ala 305 310 315 320 Gly Lys Pro Thr His Val Asn Val Ser
Val Val Met Ala Glu Val Asp 325 330 335 Gly Thr Cys Tyr 340
11137PRTHomo sapiens 11Gln Glu Asp Glu Arg Ile Val Leu Val Asp Asn
Lys Cys Lys Cys Ala 1 5 10 15 Arg Ile Thr Ser Arg Ile Ile Arg Ser
Ser Glu Asp Pro Asn Glu Asp 20 25 30 Ile Val Glu Arg Asn Ile Arg
Ile Ile Val Pro Leu Asn Asn Arg Glu 35 40 45 Asn Ile Ser Asp Pro
Thr Ser Pro Leu Arg Thr Arg Phe Val Tyr His 50 55 60 Leu Ser Asp
Leu Cys Lys Lys Cys Asp Pro Thr Glu Val Glu Leu Asp 65 70 75 80 Asn
Gln Ile Val Thr Ala Thr Gln Ser Asn Ile Cys Asp Glu Asp Ser 85 90
95 Ala Thr Glu Thr Cys Tyr Thr Tyr Asp Arg Asn Lys Cys Tyr Thr Ala
100 105 110 Val Val Pro Leu Val Tyr Gly Gly Glu Thr Lys Met Val Glu
Thr Ala 115 120 125 Leu Thr Pro Asp Ala Cys Tyr Pro Asp 130 135
12585PRTHomo sapiens 12Lys Ser Pro Ile Phe Gly Pro Glu Glu Val Asn
Ser Val Glu Gly Asn 1 5 10 15 Ser Val Ser Ile Thr Cys Tyr Tyr Pro
Pro Thr Ser Val Asn Arg His 20 25 30 Thr Arg Lys Tyr Trp Cys Arg
Gln Gly Ala Arg Gly Gly Cys Ile Thr 35 40 45 Leu Ile Ser Ser Glu
Gly Tyr Val Ser Ser Lys Tyr Ala Gly Arg Ala 50 55 60 Asn Leu Thr
Asn Phe Pro Glu Asn Gly Thr Phe Val Val Asn Ile Ala 65 70 75 80 Gln
Leu Ser Gln Asp Asp Ser Gly Arg Tyr Lys Cys Gly Leu Gly Ile 85 90
95 Asn Ser Arg Gly Leu Ser Phe Asp Val Ser Leu Glu Val Ser Gln Gly
100 105 110 Pro Gly Leu Leu Asn Asp Thr Lys Val
Tyr Thr Val Asp Leu Gly Arg 115 120 125 Thr Val Thr Ile Asn Cys Pro
Phe Lys Thr Glu Asn Ala Gln Lys Arg 130 135 140 Lys Ser Leu Tyr Lys
Gln Ile Gly Leu Tyr Pro Val Leu Val Ile Asp 145 150 155 160 Ser Ser
Gly Tyr Val Asn Pro Asn Tyr Thr Gly Arg Ile Arg Leu Asp 165 170 175
Ile Gln Gly Thr Gly Gln Leu Leu Phe Ser Val Val Ile Asn Gln Leu 180
185 190 Arg Leu Ser Asp Ala Gly Gln Tyr Leu Cys Gln Ala Gly Asp Asp
Ser 195 200 205 Asn Ser Asn Lys Lys Asn Ala Asp Leu Gln Val Leu Lys
Pro Glu Pro 210 215 220 Glu Leu Val Tyr Glu Asp Leu Arg Gly Ser Val
Thr Phe His Cys Ala 225 230 235 240 Leu Gly Pro Glu Val Ala Asn Val
Ala Lys Phe Leu Cys Arg Gln Ser 245 250 255 Ser Gly Glu Asn Cys Asp
Val Val Val Asn Thr Leu Gly Lys Arg Ala 260 265 270 Pro Ala Phe Glu
Gly Arg Ile Leu Leu Asn Pro Gln Asp Lys Asp Gly 275 280 285 Ser Phe
Ser Val Val Ile Thr Gly Leu Arg Lys Glu Asp Ala Gly Arg 290 295 300
Tyr Leu Cys Gly Ala His Ser Asp Gly Gln Leu Gln Glu Gly Ser Pro 305
310 315 320 Ile Gln Ala Trp Gln Leu Phe Val Asn Glu Glu Ser Thr Ile
Pro Arg 325 330 335 Ser Pro Thr Val Val Lys Gly Val Ala Gly Gly Ser
Val Ala Val Leu 340 345 350 Cys Pro Tyr Asn Arg Lys Glu Ser Lys Ser
Ile Lys Tyr Trp Cys Leu 355 360 365 Trp Glu Gly Ala Gln Asn Gly Arg
Cys Pro Leu Leu Val Asp Ser Glu 370 375 380 Gly Trp Val Lys Ala Gln
Tyr Glu Gly Arg Leu Ser Leu Leu Glu Glu 385 390 395 400 Pro Gly Asn
Gly Thr Phe Thr Val Ile Leu Asn Gln Leu Thr Ser Arg 405 410 415 Asp
Ala Gly Phe Tyr Trp Cys Leu Thr Asn Gly Asp Thr Leu Trp Arg 420 425
430 Thr Thr Val Glu Ile Lys Ile Ile Glu Gly Glu Pro Asn Leu Lys Val
435 440 445 Pro Gly Asn Val Thr Ala Val Leu Gly Glu Thr Leu Lys Val
Pro Cys 450 455 460 His Phe Pro Cys Lys Phe Ser Ser Tyr Glu Lys Tyr
Trp Cys Lys Trp 465 470 475 480 Asn Asn Thr Gly Cys Gln Ala Leu Pro
Ser Gln Asp Glu Gly Pro Ser 485 490 495 Lys Ala Phe Val Asn Cys Asp
Glu Asn Ser Arg Leu Val Ser Leu Thr 500 505 510 Leu Asn Leu Val Thr
Arg Ala Asp Glu Gly Trp Tyr Trp Cys Gly Val 515 520 525 Lys Gln Gly
His Phe Tyr Gly Glu Thr Ala Ala Val Tyr Val Ala Val 530 535 540 Glu
Glu Arg Lys Ala Ala Gly Ser Arg Asp Val Ser Leu Ala Lys Ala 545 550
555 560 Asp Ala Ala Pro Asp Glu Lys Val Leu Asp Ser Gly Phe Arg Glu
Ile 565 570 575 Glu Asn Lys Ala Ile Gln Asp Pro Arg 580 585
1319PRTArtificial SequenceAmino acid sequence for a signal peptide
(heavy chain secretion signal). 13Met Glu Leu Gly Leu Cys Trp Val
Phe Leu Val Ala Ile Leu Glu Gly 1 5 10 15 Val Gln Cys
14120PRTArtificial SequenceAmino acid sequence of the heavy chain
variable region of infliximab (cA2). 14Glu Val Lys Leu Glu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Lys Leu Ser
Cys Val Ala Ser Gly Phe Ile Phe Ser Asn His 20 25 30 Trp Met Asn
Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala
Glu Ile Arg Ser Lys Ser Ile Asn Ser Ala Thr His Tyr Ala Glu 50 55
60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ala
65 70 75 80 Val Tyr Leu 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 Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser Ser 115 120
151440DNAArtificial SequenceDNA sequence for infliximab heavy chain
IgA2m(n) optimized for maize. 15atggagctgg gcctgtgctg ggtgttcctg
gtggccatcc tcgagggcgt ccagtgcgag 60gtgaagctcg aggagtccgg cggcggcctg
gtccagcctg gcggctccat gaagctgtcc 120tgcgtggcct ccggcttcat
cttctccaac cactggatga actgggtgcg ccagtccccc 180gagaagggcc
tcgagtgggt ggccgagatc cgctccaagt ccatcaactc cgccacccac
240tacgccgagt ccgtcaaggg caggttcacc atctcccgcg acgactccaa
gtccgccgtg 300tacctccaga tgaccgacct gcgcaccgag gacaccggcg
tgtactactg ctcccgcaac 360tactacggct ccacctacga ctactggggc
cagggcacca ccctgaccgt gtcctccgct 420agccccacct cccccaaggt
gttccccctg tccctggact ccacccccca ggacggcaac 480gtggtggtgg
cctgcctggt ccagggcttc ttcccccagg agcccctgtc cgtcacctgg
540agcgagtccg gccagaacgt gaccgcccgc aacttccccc cctcccagga
cgcttccggc 600gacctgtaca ccacctcctc ccagctgacc ctgcccgcta
cccagtgccc cgacggcaag 660tccgtgacct gccacgtgaa gcactacacc
aactccagcc aggacgtgac cgtgccctgc 720cgcgtgcctc ctccccctcc
ctgctgccac cccaggctgt ccctgcacag gcccgctctc 780gaggacctgc
tgctgggctc cgaggccaac ctgacctgca ccctgaccgg cctgagggac
840gcctccggcg ctaccttcac ctggaccccc tcctccggca agtccgctgt
ccaaggccca 900cctgagcgtg atctctgcgg ctgctactcc gtgtccagcg
tgctgcccgg ctgcgcccag 960ccctggaacc acggcgagac cttcacctgc
accgccgctc accccgagct taagaccccc 1020ctgaccgcca acatcaccaa
gtccggcaac accttccgcc ccgaggtgca cctgctgccc 1080cctccctccg
aggagctggc cctgaacgag ctggtgaccc tgacctgcct cgccaggggc
1140ttctccccga aggacgtgct tgtgcgctgg ctccagggct cccaggagct
gccccgcgag 1200aagtacctga cctgggcctc caggcaggag ccttcccagg
gcaccaccac cttcgccgtg 1260acctccatcc tgagggtggc cgccgaggac
tggaagaagg gcgacacctt cagctgcatg 1320gtgggtcacg aggctctgcc
cctggccttc acccaaaaga ccatcgaccg cctggctggc 1380aagcccaccc
acgtgaacgt gtccgtggtg atggccgagg tggacggcac ctgctactga
14401622PRTArtificial SequenceAmino acid sequence for a signal
peptide (light chain secretion signal). 16Met Lys Tyr Leu Leu Pro
Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala
Met Ala 20 17107PRTArtificial SequenceAmino acid sequence of the
light chain variable region of infliximab (cA2). 17Asp Ile Leu Leu
Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg
Val Ser Phe Ser Cys Arg Ala Ser Gln Phe Val Gly Ser Ser 20 25 30
Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35
40 45 Lys Tyr Ala Ser Glu Ser Met Ser Gly Ile Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Thr
Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser
His Ser Trp Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Asn Leu Glu
Val Lys 100 105 18711DNAArtificial SequenceDNA sequence for
infliximab light chain optimized for maize. 18atgaagtacc tgctgcccac
cgctgctgct ggcctgctgc tgctcgctgc ccagcccgct 60atggccgaca tcctgctgac
ccagtccccc gccatcctgt ccgtgtcccc aggcgagcgc 120gtgtccttct
cctgccgcgc ctcccagttc gtgggctcct ccatccactg gtatcagcag
180cgcaccaacg gctcccccag gctgctgatc aagtacgcct ccgagtccat
gtccggcatc 240ccgtccaggt tctccggctc cggcagcggc accgacttca
ccctgtccat caacaccgtg 300gagtccgagg acatcgccga ctactactgc
cagcagtccc actcctggcc cttcaccttc 360ggctccggca ccaacctcga
ggtgaagcgt acggtggctg ccccctccgt gttcatcttc 420cccccctccg
acgagcagct gaagtccggc accgcctccg ttgtctgcct ccttaacaac
480ttctaccccc gcgaggccaa ggtccagtgg aaggtggaca acgccctcca
gtccggcaac 540tcccaggagt ccgtcaccga gcaggactcc aaggacagca
cctactccct gtcctccacc 600ctgaccctgt ccaaggccga ctacgagaag
cacaaggtgt acgcctgcga ggtgacccac 660cagggcctgt cctcccccgt
caccaagtcc ttcaaccgcg gcgagtgctg a 7111922PRTHomo sapiens 19Met Lys
Asn His Leu Leu Phe Trp Gly Val Leu Ala Val Phe Ile Lys 1 5 10 15
Ala Val His Val Lys Ala 20 20480DNAArtificial SequenceDNA sequence
for J-chain optimized for maize. 20atgaagaacc acctgctgtt ctggggcgtg
ctggccgtgt tcatcaaggc cgtgcacgtg 60aaggctcagg aggatgagcg catcgtcctc
gtggacaaca agtgcaagtg cgcccgcatc 120acctcccgca tcatccgctc
ctccgaggac cccaacgagg acatcgtgga gcgcaacatc 180cgcatcatcg
tgcccctgaa caaccgcgag aacatctccg accccacctc ccccctgagg
240acccgcttcg tgtaccacct gtccgacctg tgcaagaagt gcgaccccac
cgaggtggag 300ctggacaacc agatcgtgac cgccacccag tccaacatct
gcgacgagga ctccgccacc 360gagacctgct acacctacga ccgcaacaag
tgctacaccg ccgtggtgcc cctggtgtac 420ggcggcgaga ccaagatggt
ggagaccgcc ctgacccccg acgcttgcta ccccgactga 4802120PRTArtificial
SequenceAmino acid sequence for a signal peptide (SC chain
secretion signal). 21Met Ala Met Leu Leu Phe Val Leu Thr Cys Leu
Leu Ala Val Phe Pro 1 5 10 15 Ala Ile Ser Thr 20
221818DNAArtificial SequenceDNA sequence for SC-chain optimized for
maize. 22atggctatgc tgctgttcgt gctgacctgc ctgctggccg tgttccccgc
catctccacc 60aagtccccca tcttcggccc cgaggaggtg aactccgtgg agggcaactc
cgtgtccatc 120acctgctact acccccccac ctccgtgaac cgccacacta
ggaagtactg gtgcaggcag 180ggcgctaggg gcggctgcat caccctgatc
tcctccgagg gctacgtttc tagcaagtac 240gctggcaggg ccaacctgac
caacttcccc gagaacggca ccttcgtggt gaacatcgcc 300cagctgtccc
aggacgactc cggccgctac aagtgcggcc tgggcatcaa ctccaggggc
360ctgtccttcg acgtgtccct cgaggtgtcc cagggcccag gcctgctgaa
cgacaccaag 420gtgtacaccg tggacctcgg tcgcaccgtc accatcaact
gccccttcaa gaccgagaac 480gcccagaagc gcaagtccct gtacaagcag
atcggcctgt accccgtgct ggtgatcgac 540tcctccggct acgtgaaccc
caactacacc ggccgcatcc gcctggacat ccagggcact 600ggccagctgc
tgttctccgt ggtgatcaac cagctgcgcc tgtccgatgc tggccaatac
660ctgtgccaag ctggcgacga ctccaactcc aacaagaaga acgccgacct
ccaggtgctg 720aagcccgagc ccgagctggt gtacgaggac ctgcgcggct
ccgtgacctt ccactgcgct 780ctgggccccg aggtggcgaa cgttgcgaag
ttcctctgca ggcagtcctc cggcgagaac 840tgcgacgtgg tggtgaacac
cctgggcaag cgcgctcccg ctttcgaggg ccgcatcctg 900ctgaaccccc
aggacaagga cggctccttc tccgtcgtca tcaccggcct gcgcaaggag
960gacgctggcc gctacctctg cggcgctcac tccgacggcc agctccagga
gggctccccc 1020atccaggcct ggcagctgtt cgtgaacgag gagtccacca
tccccaggtc ccccaccgtg 1080gtgaagggcg tggctggcgg ctccgtggct
gtgctgtgcc cctacaaccg caaggagtcc 1140aagtccatca agtactggtg
cctgtgggag ggcgctcaga acggcaggtg ccccctgctc 1200gtggactccg
agggctgggt gaaggcccag tacgagggca ggctgtccct gctcgaggag
1260cccggcaacg gcaccttcac cgtgatcctg aaccagctga cctcccgcga
cgccggcttc 1320tactggtgcc tcaccaacgg cgacaccctg tggaggacca
ccgtggagat caagatcatc 1380gagggcgagc ccaacctgaa ggtcccaggg
aacgtgaccg ctgtgctggg cgagaccctg 1440aaggtcccct gccacttccc
ctgcaagttc tcctcctacg agaagtactg gtgcaagtgg 1500aacaacaccg
gctgccaggc tctgccctcc caggacgagg gcccctccaa ggccttcgtg
1560aactgcgacg agaactcccg cctggtgtcc ctgaccctga acctggtgac
cagggccgac 1620gagggctggt actggtgcgg cgtgaagcag ggccacttct
acggcgagac cgccgctgtg 1680tacgtggccg tggaggagag gaaggccgct
ggctctaggg atgtttctct tgccaaggct 1740gacgctgccc ccgacgagaa
ggtgctggac tccggcttcc gcgagatcga gaacaaggcc 1800atccaggacc cccgctga
18182331PRTArtificial SequenceAmino acid sequence for rice
alpha-amylase signal peptide (secretion signal). 23Met Gln Val Leu
Asn Thr Met Val Asn Lys His Phe Leu Ser Leu Ser 1 5 10 15 Val Leu
Ile Val Leu Leu Gly Leu Ser Ser Asn Leu Thr Ala Gly 20 25 30
24121PRTArtificial SequenceAmino acid sequence of the heavy chain
variable region of adalimumab (D2E7). 24Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val 50 55
60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu
Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 25107PRTArtificial SequenceAmino acid sequence of the light
chain variable region of adalimumab (D2E7). 25Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn
Arg Ala Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105 26504DNAArtificial SequenceDNA sequence for J-chain
optimized for Lemna. 26atgcaggtcc tgaacacgat ggtcaacaag cacttcctct
ccctgtccgt cctcatcgtc 60ctcctcgggc tgagcagcaa cctcaccgcc ggccaggagg
acgagcgcat cgtcctcgtg 120gacaacaagt gcaagtgcgc gcgcatcacc
agccgcatca tccggtccag cgaggacccc 180aacgaggaca tcgtcgagcg
caacatccgc atcatcgtgc ccctgaacaa ccgcgagaac 240atctccgacc
cgacctcccc gctgaggacc cgcttcgtgt accacctgag cgacctctgc
300aagaagtgcg accccaccga ggtggaactc gacaaccaga tcgtcacggc
cacccagagc 360aacatctgcg acgaggactc cgccaccgag acgtgctaca
cctacgacag gaacaagtgc 420tacacggccg tcgtgcccct cgtgtacggc
ggggagacca agatggtcga gacggccctg 480accccggacg cgtgctaccc cgac
504271848DNAArtificial SequenceDNA sequence for SC-chain optimized
for Lemna. 27atgcaggtcc tgaacacgat ggtcaacaag cacttcctct ccctgtccgt
cctcatcgtc 60ctcctcgggc tgagcagcaa cctcaccgcc ggcaagtccc cgatcttcgg
gccggaggaa 120gtgaactcgg tggaggggaa ctccgtcagc atcacctgct
actatcctcc gacgagcgtg 180aaccggcaca cgaggaagta ctggtgcagg
caaggggcca gaggcgggtg catcaccctc 240atctccagcg aggggtacgt
ctcgtccaag tacgccggtc gcgccaacct cacgaacttc 300cccgagaacg
ggacgttcgt tgtgaacatc gcccagctgt cgcaggacga cagcggccgc
360tacaagtgtg gcctcgggat caacagccgc gggctctcct tcgacgtctc
gctggaggtg 420tcccagggcc ccggcctcct gaacgacacg aaagtgtaca
ccgttgacct cggccggacg 480gtcactatca actgcccgtt caagaccgag
aacgcccaga agcgcaagag cctctacaag 540cagatcggcc tctacccggt
cctggtgatc gactcctcgg gctacgtcaa cccgaactac 600accggccgca
tccggctgga catccagggg accgggcagc tgctcttctc cgtggtcatc
660aaccagctcc gcctgagcga cgccgggcag tacctgtgcc aggccgggga
cgatagcaac 720tccaacaaga agaacgccga cctccaagtg ctgaagcccg
agccggagct ggtctacgag 780gacctccgcg ggtccgtgac cttccactgc
gccctgggcc cggaggtcgc caacgtggcg 840aagttcctgt gccggcagtc
ctcgggtgag aactgcgacg tggtcgtgaa caccctcggg 900aagcgcgcgc
ccgccttcga gggccgcatc ctgctcaatc cccaggacaa ggacgggtcc
960ttcagcgtgg tcatcacggg cctccgcaag gaagacgcgg gccgctacct
ctgcggcgcc 1020cactcggacg gccagctcca ggaggggagc cccatccagg
cctggcagct gttcgtcaac 1080gaggagtcca cgatccccag aagccccacc
gtggttaagg gcgtcgccgg tggctccgtg 1140gcggtgctgt gcccgtacaa
ccgcaaggag agcaagtcca tcaagtactg gtgcctgtgg 1200gagggcgccc
agaacgggag atgcccgctc ctggtggact ccgaggggtg ggtgaaggcg
1260cagtacgagg gccgcctctc gctcctggag gagcccggca acggcacctt
caccgtcatc 1320ctcaaccagc tgacgtcgcg ggacgccggc ttctactggt
gcctcaccaa cggcgacacc 1380ctctggagga ccacggtgga gatcaagatc
atcgagggcg agccgaacct gaaagtccct 1440gggaacgtga ccgctgtcct
cggcgagacc ctgaaggtgc cctgccactt cccctgcaag 1500ttctccagct
acgagaagta ctggtgcaag tggaacaaca ccggctgtca ggccctcccc
1560tcgcaggacg aggggccgtc caaggcgttc gtgaactgcg acgagaacag
ccgcctcgtg 1620tccctcaccc tcaacctcgt gacccgcgcc gacgagggct
ggtactggtg cggggtgaag 1680cagggccact tctacgggga gaccgccgcg
gtctacgtgg cggtcgagga gcgcaaggcc 1740gctggctccc gcgacgtcag
cctggccaag gccgacgccg cgcccgacga gaaggtgctg 1800gactccgggt
tccgggagat cgagaacaag gccatccagg atccccgg 1848281515DNAArtificial
SequenceDNA sequence for adalimumab heavy chain IgA1 optimized for
Lemna. 28atgcaggtcc tgaacacgat ggtcaacaag cacttcctct ccctgtccgt
cctcatcgtc 60ctcctcgggc tgagcagcaa cctcaccgcc ggcgaggtcc agctggtgga
gtccggcggg 120ggcctggtcc agcccgggcg ctccctgcgc ctcagctgcg
cggccagcgg cttcaccttc 180gacgattacg ccatgcactg ggtcagacag
gccccgggga agggcctcga gtgggtgtcc 240gccatcactt ggaacagcgg
ccacatcgac tacgccgact ccgtggaggg gcggttcacg 300atcagccggg
acaacgccaa gaactccctg tacctgcaga tgaactccct cagagcggag
360gacactgcgg tctactattg cgccaaggtg agctacctga gcaccgcgtc
cagcttggac 420tactgggggc agggcaccct ggtgacggtg tcctcagcct
cgcccacgtc gccgaaggtc 480ttccccctca gcctctgctc gacgcagccc
gacgggaacg ttgtcatcgc ctgcctcgtg 540cagggcttct tcccgcagga
gccgctctcc gtgacctggt ccgagagcgg ccagggcgtt 600accgctcgga
acttcccgcc ctcccaggac gcgagcggcg atctctacac tacgtcctcg
660cagcttacgc tcccggcgac gcaatgcctc gccggcaaga gcgtgacgtg
ccacgtcaag 720cactacacca atccctcgca ggacgtgacc gtgccctgcc
cggtgccttc cacgcctccg 780acgccgagcc cgtccacgcc gccgacccct
tcgcccagct gctgtcaccc gcgcctctcc 840ctccaccggc ccgccctgga
ggacctgttg ctcgggagcg aggcgaacct cacctgcacg 900ctcacgggcc
tgcgcgacgc ctctggcgtt accttcacct ggacgccctc ttccgggaag
960agcgccgtgc agggcccgcc cgagagagac ctctgcggtt gctactccgt
gtcgagcgtg 1020ctcccggggt gcgcggagcc gtggaaccac gggaagacct
tcacctgcac cgcggcctac 1080cctgagtcca agacgcccct gaccgccacc
ctgtccaagt ccgggaacac cttccgcccg 1140gaggtgcact tgctgccacc
gcccagcgag gaactcgccc tgaacgagct ggtcacgctc 1200acctgcctgg
ctaggggctt ctcgcccaag gacgtgctgg tccgctggtt gcaaggcagc
1260caggagctgc cgcgcgagaa gtacctcacc tgggcctcta ggcaggagcc
ctcgcagggc 1320accactacgt tcgcggtgac ctccatcctc agggtcgccg
ctgaggactg gaagaaaggc 1380gacaccttct cctgcatggt cggccacgag
gcgctgcccc tcgccttcac ccagaagacc 1440atcgaccggc tggccgggaa
gcccacccac gtcaacgtca gcgtcgttat ggctgaggtt 1500gacgggacgt gctac
151529735DNAArtificial SequenceDNA sequence for adalimumab light
chain kappa optimized for Lemna. 29atgcaggtcc tgaacacgat ggtcaacaag
cacttcctct ccctgtccgt cctcatcgtc 60ctcctcgggc tgagcagcaa cctcaccgcc
ggcgacatcc agatgacgca gagcccgtcg 120agcctgtccg cctccgtcgg
ggacagggtc acgatcacct gcagagccag ccagggcatc 180cgcaactacc
tggcctggta tcaacagaag cccgggaagg ctccgaagct cctgatctac
240gcggccagca ccctccagag cggcgtcccg agccggttct ccggcagcgg
ctccggcacc 300gacttcaccc tcaccatcag ctcgctccag cccgaggacg
tcgccaccta ctactgccag 360aggtacaacc gcgcgccgta cacgttcggg
cagggcacga aggtggagat caagcgcacg 420gtggccgcgc ccagcgtctt
catcttcccg ccctcggacg agcagctgaa gtccggcact 480gcctccgtgg
tctgcctgct caacaacttc tacccccgcg aagccaaggt gcagtggaag
540gttgacaacg cgctccagag cgggaactcc caggagtccg tgaccgagca
ggactccaag 600gactccacct acagcctgtc cagcaccctc accctctcca
aggccgacta cgagaagcac 660aaggtgtacg cctgcgaggt gacccaccag
ggcctctcct ccccggtgac caagtccttc 720aaccgcggtg agtgc
73530119PRTArtificial SequenceAmino acid sequence of the heavy
chain variable region of ustekinumab (CTNO-1275). 30Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu
Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr 20 25 30
Trp Leu Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Asp Trp Ile 35
40 45 Gly Ile Met Ser Pro Val Asp Ser Asp Ile Arg Tyr Ser Pro Ser
Phe 50 55 60 Gln Gly Gln Val Thr Met Ser Val Asp Lys Ser Ile Thr
Thr Ala Tyr 65 70 75 80 Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr
Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Arg Arg Pro Gly Gln Gly Tyr
Phe Asp Phe Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser
115 31107PRTArtificial SequenceAmino acid sequence of the light
chain variable region of ustekinumab (CTNO-1275). 31Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35
40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Asn Ile Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 105 321509DNAArtificial SequenceLemna-optimized
complete SILA heavy chain IgA1 DNA (including signal peptide SEQ ID
NO26). 32atgcaggtcc tgaacacgat ggtcaacaag cacttcctct ccctgtccgt
cctcatcgtc 60ctcctcgggc tgagcagcaa cctcaccgcc ggcgaggtcc agctggtgca
gtccggcgcg 120gaggtcaaga agcctgggga atccctgaag atcagctgca
agggcagcgg gtacagcttc 180accacttact ggctcggctg ggtgcgccag
atgccgggca aggggctgga ctggatcggg 240atcatgtccc ccgtggactc
cgacatccgg tactccccga gcttccaggg ccaggtcact 300atgtccgtgg
acaagagcat caccacggcc tacttgcagt ggaactccct gaaggcgagc
360gataccgcga tgtactattg cgcgagacgg agacccgggc agggctactt
cgacttctgg 420gggcagggca ccctggtgac ggtgtcctca gcctcgccca
cgtcgccgaa ggtcttcccc 480ctcagcctct gctcgacgca gcccgacggg
aacgttgtca tcgcctgcct cgtgcagggc 540ttcttcccgc aggagccgct
ctccgtgacc tggtccgaga gcggccaggg cgttaccgct 600cggaacttcc
cgccctccca ggacgcgagc ggcgatctct acactacgtc ctcgcagctt
660acgctcccgg cgacgcaatg cctcgccggc aagagcgtga cgtgccacgt
caagcactac 720accaatccct cgcaggacgt gaccgtgccc tgcccggtgc
cttccacgcc tccgacgccg 780agcccgtcca cgccgccgac cccttcgccc
agctgctgtc acccgcgcct ctccctccac 840cggcccgccc tggaggacct
gttgctcggg agcgaggcga acctcacctg cacgctcacg 900ggcctgcgcg
acgcctctgg cgttaccttc acctggacgc cctcttccgg gaagagcgcc
960gtgcagggcc cgcccgagag agacctctgc ggttgctact ccgtgtcgag
cgtgctcccg 1020gggtgcgcgg agccgtggaa ccacgggaag accttcacct
gcaccgcggc ctaccctgag 1080tccaagacgc ccctgaccgc caccctgtcc
aagtccggga acaccttccg cccggaggtg 1140cacttgctgc caccgcccag
cgaggaactc gccctgaacg agctggtcac gctcacctgc 1200ctggctaggg
gcttctcgcc caaggacgtg ctggtccgct ggttgcaagg cagccaggag
1260ctgccgcgcg agaagtacct cacctgggcc tctaggcagg agccctcgca
gggcaccact 1320acgttcgcgg tgacctccat cctcagggtc gccgctgagg
actggaagaa aggcgacacc 1380ttctcctgca tggtcggcca cgaggcgctg
cccctcgcct tcacccagaa gaccatcgac 1440cggctggccg ggaagcccac
ccacgtcaac gtcagcgtcg ttatggctga ggttgacggg 1500acgtgctac
150933735DNAArtificial SequenceLemna-optimized complete SILA light
chain DNA (including signal peptide SEQ ID NO26). 33atgcaggtcc
tgaacacgat ggtcaacaag cacttcctct ccctgtccgt cctcatcgtc 60ctcctcgggc
tgagcagcaa cctcaccgcc ggcgacatcc agatgacgca gagcccgtcg
120agcctgtccg cctccgtcgg ggacagggtc acgatcacct gcagagccag
ccagggcatc 180tcgtcctggc tggcctggta tcaacagaag cccgagaagg
ctccgaagtc cctgatctac 240gcggccagct ccctccagag cggcgtcccg
agccggttct ccggcagcgg ctccggcacc 300gacttcaccc tcaccatcag
ctcgctccag cccgaggact tcgccaccta ctactgccag 360cagtacaaca
tctacccgta cacgttcggg cagggcacga agctggagat caagcgcacg
420gtggccgcgc ccagcgtctt catcttcccg ccctcggacg agcagctgaa
gtccggcact 480gcctccgtgg tctgcctgct caacaacttc tacccccgcg
aagccaaggt gcagtggaag 540gttgacaacg cgctccagag cgggaactcc
caggagtccg tgaccgagca ggactccaag 600gactccacct acagcctgtc
cagcaccctc accctctcca aggccgacta cgagaagcac 660aaggtgtacg
cctgcgaggt gacccaccag ggcctctcct ccccggtgac caagtccttc
720aaccgcggtg agtgc 735
* * * * *