US20150010544A1

US20150010544A1 - Compounds and methods for treating inflammatory diseases

Info

Publication number: US20150010544A1
Application number: US14/365,867
Authority: US
Inventors: Gerardus Joseph Andreas Ariaans; Frans Van Dalen; Declan Thomas Nolan
Original assignee: Synthon Biopharmaceuticals BV
Current assignee: Byondis BV
Priority date: 2011-12-16
Filing date: 2012-12-14
Publication date: 2015-01-08
Also published as: WO2013087913A1; US20150166649A1; WO2013087912A1; EP2791171A1; EP2791169B1; EP2791172A1; US20140356357A1; US9573996B2; WO2013087914A1; US20140359902A1; US9580501B2; WO2013087911A1; EP2791172B1; EP2791170A1; EP2791169A1

Abstract

A monoclonal secretory IgA antibody, which binds to and neutralizes human p40 (the p40 subunit of IL-12 and IL-23). The secretory antibody is useful in treating a variety of inflammatory conditions in humans.

Description

This application claims the benefit of priority under 35 U.S.C. §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.

FIELD OF THE INVENTION

The present invention relates to monoclonal secretory IgA antibodies, which bind to and neutralize human p40 (the p40 subunit common to IL-12 and IL-23). The antibodies are useful in treating inflammatory diseases in humans, including by oral administration.

BACKGROUND OF THE INVENTION

Inflammation represents a key event of many diseases, such as psoriasis, inflammatory bowel diseases, rheumatoid arthritis, asthma, multiple sclerosis, atherosclerosis, cystic fibrosis, and sepsis. Inflammatory cells, such as neutrophils, eosinophils, basophils, mast cells, macrophages, endothelial cells, and platelets, respond to inflammatory stimuli and foreign substances by producing bioactive mediators. These mediators act as autocrines and paracrines by interacting with many cell types to promote the inflammatory response. There are many mediators that can promote inflammation, such as cytokines and their receptors, adhesion molecules and their receptors, antigens involved in lymphocyte activation, and IgE and its receptors.
Cytokines, for example, are soluble proteins that allow for communication between cells and the external environment. The term cytokines includes a wide range of proteins, such as lymphokines, monokines, interleukins, colony stimulating factors, interferons, tumor necrosis factors, and chemokines. Cytokines serve many functions, including controlling cell growth, migration, development, and differentiation, and mediating and regulating immunity, inflammation, and hematopoiesis. Even within a given function, cytokines can have diverse roles. For example, in the context of mediating and regulating inflammation, some cytokines inhibit the inflammatory response (anti-inflammatory cytokines), others promote the inflammatory response (pro-inflammatory cytokines). And certain cytokines fall into both categories, i.e., can inhibit or promote inflammation, depending on the situation. The targeting of proinflammatory cytokines to suppress their natural function, such as with antibodies, is a well-established strategy for treating various inflammatory diseases.
Interleukin 12 (IL-12) is a cytokine which promotes an inflammatory response and is involved in several inflammatory and autoimmune diseases or disorders. Binding IL-12 (p40) has been proposed as a strategy for providing various diagnostic and therapeutic effects. For example, U.S. Pat. No. 6,902,734 relates to anti-IL-12 antibodies and fragments thereof. In particular specific amino acid sequences for the heavy chain and light chain variable regions are disclosed. The antibody can be based, in theory, on any of the known Ig classes (“IgG, IgA, IgM, IgE, IgD, etc.”), but IgG is the identified class for the specific embodiments and only IgG is used in the examples. The anti-IL-12 antibody is suggested for treating a variety of conditions including rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, diabetes, and psoriasis. The antibody is normally administered by injection (subcutaneous, IV), though alternative routes are also presented including inhaled and oral.
The antibody Ustekinumab, which is believed to be covered by the above-mentioned U.S. Pat. No. 6,902,734, is sold as a formulated product under the brand name Stelara® (Centocor). Ustekinumab is a human IgG1 antibody that binds and neutralizes IL-12 and IL-23, more specifically the p40 subunit common to both of these interleukins. Ustekinumab is approved for treatment of psoriasis and is being studied for the treatment of Crohn's disease. The antibody formulation is administered by subcutaneous injection.
U.S. Pat. No. 6,914,128 relates to antibodies that bind IL-12. Several combinations of heavy chain and light chain variable regions are disclosed for use in an antibody or antigen binding portion. The heavy chain constant region can be of the IgG, IgM, IgA, or IgE class, but the preferred constant chain portions correspond to IgG1. The antibodies are suggested for treating rheumatoid arthritis, Crohn's disease, multiple sclerosis, insulin-dependent diabetes mellitus, and psoriasis. The antibody Briakinumab (J-695), which is believed to be covered by aforementioned patent U.S. Pat. No. 6,914,128, has been the subject of phase III clinical studies for the treatment of psoriasis.
In an attempt to avoid adverse events associated with systemic administration, another group, Avaxia Biologics Inc., describes a topical (e.g., oral or rectal) animal-dervied polyclonal anti-TNF composition that could be useful in treating inflammation of the digestive tract, such as inflammatory bowel disease. WO2011047328. The application generally states that preferably the polyclonal antibody composition is prepared by immunizing an animal with a target antigen, and the preferably the polyclonal antibody composition is derived from milk or colostrum with bovine colostrums being preferred (e.g., p. 14). The application also generally states that the animal derived polyclonal antibodies could be specific for (among other targets) other inflammatory cytokines (e.g., pp. 6-7). This application describes working examples in which cows were immunized with murine TNF and the colostrum was collected post-parturition to generate bovine polyclonal anti-TNF antibodies (designated as AVX-470). The uses of animal-derived antibodies and polyclonal antibodies, however, are undesirable.
IgA molecular forms have been proposed as treatments for various diseases, most notably as treatments for pollen allergies, as treatments against pathogens, and as treatments for cancer.
For example, two articles proposed the use of an anti-S. mutans antigen I/II secretory IgA-G hybrid antibody. Ma et al. “Generation and Assembly of Secretory Antibodies in Plants.” Science 268(5211), 716-719 (May 1995); Ma et al. “Characterization of a Recombinant Plant Monoclonal Secretory Antibody and Preventive Immunotherapy in Humans.” Nature Medicine 4(5); 601-606 (May 1998). The hybrid antibody contains 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 antibody (the source of the antigen binding regions, is identified as the IgG antibody Guy's 13. The group proposes that although sIgA may provide an advantage over IgG in the mucosal environment, such is not always the case (1998 Ma at p. 604, right column).
A related article identifies the anti-streptococcal antigen I/II secretory IgA-G hybrid antibody, which was derived from Guy's 13 IgA, as CaroRx. Wycoff. “Secretory IgA Antibodies from Plants.” Current Pharmaceutical Design 10(00); 1-9 (2004). Planet Biotechnology Inc. This related article states that the CaroRx antibody was designed to block adherence to teeth of the bacteria that causes cavities. 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 not a problem because people are exposed to plant glycans every day in food without ill effect.
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 SA I/III (variable region from Guy's 13) in tobacco. The tobacco plant was 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.”
It is desirable to have alternative antibody treatments for IL-12-related inflammatory diseases that preferably avoid the disadvantages of current systemic and previously-proposed topical treatments of inflammation.

SUMMARY OF THE INVENTION

The present invention relates to monoclonal secretory IgA antibodies, which bind to and neutralize human p40 (the p40 subunit common to IL-12 and IL-23), and their use in treating inflammatory diseases in humans.
In embodiments, the antibody can be a chimeric antibody, a humanized antibody, or a human antibody. The antibody can contain a human secretory chain and a human J-chain. The antibody can be a human secretory IgA1 antibody. The antibody can contain CDR sequences that are identical to the CDR sequences of an antibody selected from the group consisting of ustekinumab and briakinumab, or a variation of one of the foregoing. The antibody can contain CDR sequences that are identical to the CDR sequences of ustekinumab.
In an embodiment, the antibody can contain heavy chain constant regions having the sequence of SEQ ID NO:5, light chain constant regions having the sequence of SEQ ID NO:9, a human secretory chain, a human J-chain, heavy chain variable regions having the sequence of SEQ ID NO:1, and light chain variable regions having the sequence of SEQ ID NO:2.
Another aspect of the present invention relates to a composition containing a plurality of the secretory IgA antibodies. In embodiments, substantially all N-glycans in the plurality of antibodies lack fucose and xylose residues. In embodiments, the plurality of 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. In embodiments, the a plurality of antibodies 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. In embodiments, 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 antibodies, such as at least 70% of the total amount of N-glycans in the plurality of antibodies.
Another aspect of the present invention relates to pharmaceutical compositions containing the secretory IgA antibodies, which can be adapted for oral administration and can be used to treat an inflammatory disease in a human.
Another aspect of the present invention relates to methods for treating an inflammatory disease in a human, which includes administering an anti-inflammatory effective amount of the secretory IgA antibodies (or compositions) to a human in need thereof, preferably orally administering the antibodies (compositions). The inflammatory disease can be selected from inflammatory bowel disease (including Crohn's disease and ulcerative colitis), psoriasis, psoriatic arthritis, ankylosing spondylitis, and multiple sclerosis.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 provides the amino acid sequence of the heavy chain variable region of ustekinumab (CTNO-1275).
SEQ ID NO:2 provides the amino acid sequence of the light chain variable region of ustekinumab (CTNO-1275).
SEQ ID NO:3 provides the amino acid sequence of the heavy chain variable region of briakinumab (J-695, ABT-874).
SEQ ID NO:4 provides the amino acid sequence of the light chain variable region of briakinumab (J-695, ABT-874).
SEQ ID NO:5 provides the amino acid sequence of a human IgA α-1 heavy chain constant region (Cα1-Cα2-Cα3) (UniProtKB/Swiss-Prot database entry P01876 (IGHA1_HUMAN)).
SEQ ID NO:6 provides the amino acid sequence of a human IgA α-2 m(1)-allotype heavy chain constant region (Cα1-Cα2-Cα3) (UniProtKB/Swiss-Prot database entry P01877 (IGHA2_HUMAN)).
SEQ ID NO:7 provides the amino acid sequence of a human IgA α-2 m(2)-allotype heavy chain constant region (Cα1-Cα2-Cα3). (UniProtKB/Swiss-Prot database entry P01877 (IGHA2_HUMAN) with indicated modifications for allotype 2 variant).
SEQ ID NO:8 provides the amino acid sequence of a human IgA α-2 (n)-allotype.
SEQ ID NO:9 provides the amino acid sequence of a human κ light chain constant region (CL) (UniProtKB/Swiss-Prot database entry P01834 (IGKC_HUMAN)).
SEQ ID NO:10 provides the amino acid sequence of a human λ1 light chain constant region (CL) (UniProtKB/Swiss-Prot database entry P0CG04 (LAC1_HUMAN)).
SEQ ID NO:11 provides the amino acid sequence of a human λ2 light chain constant region (CL) (UniProtKB/Swiss-Prot database entry P0CG05 (LAC2_HUMAN)).
SEQ ID NO:12 provides the amino acid sequence of a human λ3 light chain constant region (CL) (UniProtKB/Swiss-Prot database entry P0CG06 (LAC3_HUMAN)).
SEQ ID NO:13 provides the amino acid sequence of a human λ6 light chain constant region (CL) (UniProtKB/Swiss-Prot database entry P0CF74 (LAC6_HUMAN)).
SEQ ID NO:14 provides the amino acid sequence of a human λ7 light chain constant region (CL) (UniProtKB/Swiss-Prot database entry A0M8Q6 (LAC7_HUMAN)).
SEQ ID NO:15 provides the amino acid sequence of a human J-chain (amino acids 23-159 UniProtKB/Swiss-Prot database entry P01591).
SEQ ID NO:16 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).
SEQ ID NO:17 provides the amino acid sequence for a signal peptide (the rice α-amylase secretion signal).
SEQ ID NO:18 provides a complete lemna-optimized UKB-SA1 heavy chain IgA1 DNA (including DNA encoding signal peptide SEQ ID NO:17).
SEQ ID NO:19 provides a complete lemna-optimized UKB-SA1 light chain DNA (including DNA encoding signal peptide SEQ ID NO:17).
SEQ ID NO:20 provides a complete lemna-optimized human J-chain DNA (including DNA encoding signal peptide SEQ ID NO:17).
SEQ ID NO:21 provides a complete lemna-optimized human SC-chain DNA (including DNA encoding signal peptide SEQ ID NO:17).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequences of the human IgA1, IgA2 m(1), IgA2 m(2), and IgA2(n) antibody α heavy chain constant regions (SEQ ID NOS:5-8).

FIGS. 2A through 2D shows amino acid sequences of various p40 (the p40 subunit of IL12 and IL23) binding regions. FIG. 2A shows the amino acid sequence of the heavy chain variable region of the antibody ustekinumab (CTNO-1275) (SEQ ID NO:1); FIG. 2B shows the amino acid sequence of the light chain variable region of the antibody ustekinumab (CTNO-1275) (SEQ ID NO:2); FIG. 2C shows the amino acid sequence of the heavy chain variable region of the antibody briakinumab (J-695, ABT-874) (SEQ ID NO:3); FIG. 2D shows the amino acid sequence of the light chain variable region of the antibody briakinumab (J-695, ABT-874) (SEQ ID NO:4).

FIGS. 3A and 3B show the amino acid sequences of various human antibody light chain subtypes and allotypes. FIG. 3A shows the amino acid sequence of a human κ light chain constant region (C_L) (UniProtKB/Swiss-Prot P01834) (SEQ ID NO:9); FIG. 3B shows the amino acid sequences of a human λ light chain constant region (C_L) allotypes (UniProtKB/Swiss-Prot P0CG04, P0CG05, P0CG06, P0CG74, and A0M8Q6; SEQ ID NOS:10-14).

FIG. 4 shows the amino acid sequence of a human J-chain (a.a. 23-159 of UniProtKB/Swiss-Prot entry P01591, SEQ ID NO:15).

FIG. 5 shows the amino acid sequence of a human secretory component (a.a. 19-603 of UniProtKB/Swiss-Prot database entry P01833, SEQ ID NO:16).

FIGS. 6A through 6C show the structure of vector constructs SynA01 (FIG. 6A), SynA02 (FIG. 6B) and SynA03 (FIG. 6C) used for expression of an anti-p40 (anti-IL12/23) SIgA in Lemna in Example 1.

FIG. 7 shows reducing and non reducing gels of an anti-p40 (anti-IL-12/23) SIgA with ustekinumab variable regions, produced in Lemna. The A gel shows non-reducing SDS-PAGE analysis demonstrating expression of complete SIgA. The B gel shows reducing SDS-PAGE analysis.

FIG. 8 shows the degradation of an anti-p40 (anti-IL-12/23) SIgA with ustekinumab variable regions and expressed in Lemna compared to ustekinumab (IgG1) and to colostral SIgA 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).

FIGS. 9A and 9B show the results of inhibition of IL-12/23 (p40) production by LPS-stimulated Dendritic Cells from Examples 5A and 5B, respectively. In FIG. 9A, the open circle is SynA01 (UKB-SA1); the closed circle is SynA02 (UKB-SA1g0); the diamond is Colostral SIgA; and the square is ustekinumab.

FIGS. 9C and 9D show the results of inhibition of production of IFNγ by co-cultured Dendritic Cells and T-Cells from Examples 5C and 5D, respectively.

FIG. 10 shows the serum concentrations of SynA02 (UKB-SA1g0) after oral (circle) and intravenous (square) administration as described in Example 6.

FIG. 11 shows in vivo imaging of fluorescent labeled SynA01 (UKB-SA1) and ustekinumab antibodies in the thoracic and abdominal region of mice as described in Example 7.

FIG. 12 shows immunohistochemistry on cryo-sections of mice distal colon after administration of SynA01 (UKB-SA1) and ustekinumab antibodies as described in Example 8.

FIGS. 13A and 13B show the efficacy of an anti-p40 (anti-IL12/23) SIgA in in vivo animal models of IBD as described in Example 9.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to monoclonal secretory IgA antibodies that bind to and neutralize human p40 (the p40 subunit common to IL-12 and IL-23).
As is well known, the basic structural unit of an antibody 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.
Antibodies are classified based on the heavy chain constant region, e.g., classified as IgG, IgA, IgM, IgE, IgD, etc. 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 Cκ (kappa) or Cλ (lambda) type. The amino acid sequences of human kappa (SEQ ID NO:9) and lambda (SEQ ID NOS:10-14) light chain constant regions are provided in FIGS. 3A and 3B, respectively. 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 α genes, the IgG class heavy chains by γ 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 an IgA heavy chain (Cα) typically has three domains that are referred to as Cα1, Cα2, and Cα3, a short hinge section between Cα1 and Cα2, and a short tail piece at the C-terminal end of Cα3. 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^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).
The C-terminal section of two IgA antibodies, i.e., the tail pieces at the C-terminal ends of the Cα3 region, 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 complementarity determining regions or “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.”
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 α-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 FcαR (see, e.g., Alberts, B. et al., Molecular Biology of the Cell 3^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)).
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 α 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. Examples of such variations can be found in various genomic databases such as browser.1000genomes.org/index.html and ensembl.org/index.html. 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 Cα2 heavy chain constant domain (encoded for by the IgA-specific α-gene) with a Cγ2 heavy chain constant domain (encoded for by the IgG-specific γ-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.
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 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

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 involve 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 Cα1, Cα2, or Cα3 domain or hinge or tail sections 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.
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.
In humans there are two recognized IgA subclasses: IgA1 and IgA2 which differ in the hinge section between the Cα1 and Cα2 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 or hinge section from an IgA1 and another constant domain or hinge section from an IgA2).
Typically, the SIgA antibodies of the invention comprise the Cal domain, the hinge section, and the -Cα2-Cα3 domains and tail section of an IgA antibody (with or without minor variations), including a human IgA1 and/or IgA2 antibody. In this embodiment, the Cα1 domain, the hinge section, and the -Cα2-Cα3 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:5), a human IgA2m(1) heavy chain constant region (SEQ ID NO:6), a human IgA2m(2) heavy chain constant region (SEQ ID NO:7), and a human IgA2(n) heavy chain constant region (corresponding to the Cα1 and Cα2 regions of an IgA2m(2) and the Cα3 region of an IgA2m(1)) (SEQ ID NO:8) are respectively shown in FIG. 1.
A modified, shortened, or removed linker/hinge section between Cα1 and Cα2 in IgA1 has been reported to increase resistance against proteases (for example see B. W. Senior et al., J. Immunol. 2005; 174: 7792-7799). Such can be incorporated into the SIgA antibodies of the present invention.
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. 4 (SEQ ID NO:15). Usually, the sequence of the mammalian-derived 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.
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. 5 (SEQ ID NO:16).
The antigen binding region comprises a heavy and light chain variable region pair, each containing hypervariable regions (CDRs, which directly interact with the p40) 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^thEdition, 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).
The antigen binding regions of the secretory IgA antibodies of the present invention bind to human p40. Usually, though not necessarily, the antigen binding is specific to the p40 subunit of IL-12 (and IL-23); meaning that it binds preferentially and with high affinity. A variety of protocols for 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. Generally the secretory IgA antibodies of the present invention exhibit a preference or specificity for the human p40, such that the affinity 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 its affinity for binding to a non-specific polypeptide such as, for instance, BSA or casein. Typically the secretory antibody of the present invention exhibits a binding affinity constant (K_D) with respect to the p40, of 10⁻⁷M or lower, preferably 10⁻⁸M, 10⁻⁹M, 10⁻¹⁰M, 10⁻¹¹M, or 10⁻¹²M or lower.
In embodiments, secretory IgA antibodies of the present invention neutralize a human p40 (IL-12/23) to which it is bound. For the present invention, the term “neutralizes” means inhibits/reduces the effect of the cytokine 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 cytokine at least 50%. In these embodiments, the secretory IgA antibodies of the present invention preferably inhibit/reduce the proinflammatory effect of a human p40 (IL-12/23) to which it is bound by at least 50%, such as by at least 55%, at least 60%, at least 65%, and at least 70%, and more preferably by at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 98%.
The amino acid sequence of the antigen binding region, and in particular the CDRs thereof, is determined by the epitope to which it binds. The amino acid sequence of the antigen binding region can be novel or can be obtained from existing anti-p40 antibodies. Methods for obtaining novel antigen binding sequences are well known in the art. See for example Mary A. Ritter and Heather M. Ladyman (Eds.) “Monoclonal antibodies: production, engineering, and clinical application”, Cambridge University Press (1995), ISBN 0521425034, Zhiqiang An “Therapeutic Monoclonal Antibodies: From Bench to Clinic” Wiley, New York (2009), ISBN: 0470117915, and Christopher Dean and Philip Shepherd (Ed.) “Monoclonal Antibodies: A Practical Approach” Oxford University Press, USA (2000), ISBN 0199637229.
In embodiments, secretory IgA antibodies of the invention comprise CDR sequences that are identical to the CDR sequences of an antibody selected from ustekinumab and briakinumab. The CDR sequences for these antibodies are identified below in Table 2. Of course, the CDR sequences for these and other known antibodies can be obtained from other patent and non-patent literature, and commercially-available antibodies can be purchased and sequenced.

TABLE 2

Pair No	Parental antibody	CDR1	CDR2	CDR3

1	Ustekinumab -heavy chain variable	26-35	50-66	99-108
	region (SEQ ID NO: 1) (FIG. 2A)
	Ustekinumab -light chain variable	24-34	50-55	89-107
	region (SEQ ID NO: 2) (FIG. 2B)
2	Briakinumab -heavy chain variable	31-35	50-66	99-104
	region (SEQ ID NO: 3) (FIG. 2C)
	Briakinumab -light chain variable	22-35	51-57	90-101
	region (SEQ ID NO: 4) (FIG. 2D)

In still other embodiments, antigen binding regions (or CDRs) of secretory IgA antibodies of the invention are obtained from other and/or novel anti-p40 antibodies. Methods for obtaining antibodies against specific antigens are well known in the art and can be used to obtain suitable p40-binding variable regions. See, for example, Mary A. Ritter and Heather M. Ladyman (Eds.) “Monoclonal antibodies: production, engineering, and clinical application”, Cambridge University Press (1995), ISBN 0521425034, Zhiqiang An “Therapeutic Monoclonal Antibodies: From Bench to Clinic” Wiley, New York (2009), ISBN: 0470117915, and Christopher Dean and Philip Shepherd (Ed.) “Monoclonal Antibodies: A Practical Approach” Oxford University Press, USA (2000), ISBN 0199637229.
Secretory IgA antibodies of the invention can be non-human antibodies, chimeric antibodies, humanized antibodies, human antibodies, or other mixes of human and non-human sequences/regions. See, e.g., Yamashita et al. Cytotechnology 55: 55-60 (2007). 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). 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., WO92/22653. 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, etc.). Likewise, a non-human antibody contains only non-human constant and variable regions, i.e., having only non-human heavy and light chains.
The secretory IgA antibodies of the invention may contain additional atoms, moieties, molecules, and/or modications beyond the dimeric IgA, J-chain, and SC-chain. For example, the secretory IgA antibodies of the invention may be PEGylated or glycosylated (or aglycosylated) in various orientations and/or amounts. The location, attachment, amount, and structure of attached glycans found in naturally occurring antibodies shows substantial variability and mainly depends on the source of the glycoprotein (i.e., the type of cell producing the glycoprotein), but is also influenced by growing conditions (i.e., feed and environmental conditions). The secretory IgA antibodies of the present invention are not limited to any specific form of glycosylation and specifically include non-glycosylated proteins, partially or fully deglycosylated proteins, variants obtained by genetic or other manipulation of the glycosylation system of the producing cell, and variants with chemically or enzymatically modified glycans. The secretory IgA antibodies of the invention can be glycoproteins with glycosylation patterns native to plant, mammalian (human), or insect cells. Additionally the antibodies of the invention may be conjugated with (fluorescent) markers or therapeutic agents, etc. (see, e.g., Lash, A. “Making the case for antibody-drug conjugates;” In Vivo: The Business & Medicine Report; vol. 28, No. 11, pp. 32-39 (December 2010) (www.ElsevierBI.com).
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 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.
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 GlcNAc2Man3GlcNAc2Ga12 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 GlcNAc2Man3GlcNAc2Ga1 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.
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.
In mammals, naturally-occurring N-glycans contain a fucose (Fuc) residue attached to the GlcNAc2Man3 core structure by an α1,6 linkage. In plants, naturally-occurring N-glycans contain a fucose (Fuc) residue attached to the GlcNAc2Man3 core structure by an α1,3 linkage and further contain a xylose (Xyl) residue attached to the GlcNAc2Man3 core structure by a β1,2 linkage. For the purposes of the present invention, a G0 glycan containing the mammalian α1,6-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 α1,3-linked Fuc residue attached to the GlcNAc2Man3 core structure is referred to as a “G0F<3> glycan,” a G0 glycan containing the plant β1,2-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 α1,3-linked Fuc residue and the plant β1,2-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.
The present invention also relates to a composition comprising a plurality of secretory IgA antibodies containing multiple N-glycans, such as two or more different N-glycans. In embodiments, 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. In embodiments, the plurality of secretory IgA antibodies 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. In embodiments, 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.
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.
The SIgA antibodies of the present invention are preferably 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. Preferably, a monoclonal SIgA contains monomeric IgA antibodies, a J-Chain, and an SC-chain that were all produced by a clone of a single cell.
The antibodies of the present invention are often isolated or in an isolated form. As used herein, the terms “isolate,” “isolating” and “isolation” refer to separating the antibody from its production environment. 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). When the SIgA antibody of the present invention is produced in a cell, which is the typical process, the separation refers to separating the antibody from the host cells and native host cell proteins. Isolation is thus related to purification. Preferably the antibody of the present invention 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.
Various compositions that contain the secretory IgA antibodies of the present invention, whether in an isolated form or not, are also contemplated as being part of the present invention. For instance, compositions that contain low amounts of incomplete secretory IgA antibodies are often desirable. With respect to the amount of secretory IgA antibodies in the composition, the amount of dimer IgA (no SC-chain) is desirably less than 50%, more desirably less than 25% and often less than 10%. Thus in a composition that contained 10 mg of secretory IgA, the amount of non-SC-chain dimeric IgA would preferably be less than 1 mg, i.e., less than 10%. The same is true for monomeric IgA: the content of IgA monomers is desirably less than 50% the amount of secretory IgA, 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 secretory IgA in the composition, often less than 10%, and even less than 5%. The above amounts apply to both isolated and non-isolated forms of secretory IgA compositions. Accordingly, the low relative amounts of incomplete secretory IgA can be a result of the expression system (native low-production of incomplete secretory IgA), the result of some separation or purification that removes incomplete secretory IgA antibodies, or both.
Purified secretory IgA compositions are also useful. A purified secretory IgA (composition) contains a secretory IgA antibody of the present invention 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 secretory IgA antibody of the invention in a concentration of 50 μg/ml or more, often at least 100 μg/ml, preferably at least 1 mg/ml.
Production of Proteins of the Present Invention
The SIgA antibodies of the invention can be produced using recombinant techniques. Although several expression systems are known, including CHO, HEK, yeast, tobacco, etc., the use of duckweed as the host cell has been found to be advantageous for the production of SIgAs. Other plant host cells, namely tobacco and lettuce, tend to give very low expression rates of the desired SIgA and typically render impractical a measurable recovery of the antibody. Similarly, CHO cells also tend to give low results. HEK generally have higher titers than CHO cells, but have certain production and regulatory disadvantages. Accordingly, duckweed is, surprisingly, a convenient host cell for expressing secretory IgAs of the present invention.
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. hyalin, 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.
Recombinant production of sIgAs in duckweed requires transformation of duckweed, either transiently or stably. For production purposes, a stable transformation, wherein the nucleic acid sequences and/or genes needed to produce the desired SIgA have been operably introduced into the genome of a duckweed, is typically preferred. 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.
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, 3rd 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.
For expression of SIgAs, multiple transformations can be performed with separate vectors including different cassette coding for the J-chain, the SC-chain, the antibody H chain and L chain. In a preferred embodiment, a single 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 vectors containing multiple expression cassette for antibody expression have been described in U.S. Pat. No. 7,632,983 to Dickey et al.
In one embodiment, expression of the cassettes is driven by individual promoters. Examples of suitable promoter can be found in US patents 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, four different promoters are 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 even more preferred embodiment, each of the 4 cassettes is driven by a different promoter. In a different embodiment, the constructs are driven by, 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, as described in U.S. Pat. No. 7,632,983 to Dickey et al.
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. 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 from the cell. Presence of the signal peptide sequence ensures efficient secretion into the extracellular space. 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 α-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 α-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. In other embodiments, the polypeptide diffuses across the cell wall of the plant host cell into the external environment/media.
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. No. 8,034,916 U.S. Pat. No. 7,632,983 to Dickey et al, disclosures of which are all incorporated by reference herein.
For example, the expression from the transgenic lines obtained can 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.
Another option is to modify the glycosylation profile of the duckweed. 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 α1,3-fucosyltransferase (FucT) and β1,2-xylosyltransferase (XylT), or otherwise inhibiting their transcription of the gene/expression 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 α1,3-fucosyltransferase (FucT) and β1,2-xylosyltransferase (XylT) in a plant. In a more preferred embodiment, these constructs triggers RNA interference targeting the mRNAs of α1,3-fucosyltransferase (FucT) and β1,2-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.
Once the transformed duckweed is obtained, the genetic modification will cause 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. Generally this entails providing an environment to keep the duckweed alive and/or to promote growth; e.g., 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 7,632,983; and references cited therein, respectively.
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.
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. If the antibody is secreted and diffuses into the culture media, then a simple filter can separate the crude antibody product from the duckweed. Typically, however, the fully formed SIgA antibody is retained within the duckweed's apoplast. Separation in this case generally requires extraction.
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 into the homogenization buffer; 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.
To remove some of the unwanted soluble proteins, 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 μm filter.
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.
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 MgCl2 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 add CuSO4 to the column in order to remove 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.
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.
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) “Ion exchange resins for the purification of monoclonal antibodies from animal cell culture” 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 1X2 is employed, often before the AC column.
Further polishing/purification steps can be added, as is knownin the art. For example, after any and/or each purification step (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.
The SIgA antibodies are often sufficiently recovered so as to be “isolated” or in an isolated form. Isolation is thus related to purification and is generally achieved by completion of the recovery/extraction step, clarification, and/or capture steps described above.
Pharmaceutical Compositions
The SIgA antibodies of the invention can be used in various pharmaceutical compositions. Typically the pharmaceutical composition comprises the antibody and at least one pharmaceutically acceptable excipient. The pharmaceutical compositions can be solid, semi-solid, or liquid. Generally the pharmaceutical composition is adapted for a particular route of administration. For example, the pharmaceutical composition can be adapted for oral administration, rectal administration, buccal administration, topical administration, etc. Preferably, the pharmaceutical composition is adapted for oral administration.
Pharmaceutical compositions for administering SIgA antibodies via topical administration include powders, creams, ointments, gels, lotions, solutions and suspensions (including mouth washes). The excipient carrier is often aqueous, oil, or polymer based, each optionally in the form of an emulsion or microemulsion. The term “topical administration” includes, for example, optical administration (e.g., via a cream/ointment) and administration to the skin (e.g., at an inflamed joint).
Pharmaceutical compositions for administering the antibody via oral administration include solid oral dosage forms such as tablets, capsules, enteric coated forms thereof, lozenges, and films, as well as liquid dosage forms including solutions, suspensions, liquid filled capsules, and mouth washes. Tablets can be soluble tablets, dispersible tablets, effervescent tablets, chewable tablets, lyophilized tablets, coated tablets (e.g., sugar-coated or enteric-coated), and modified release tablets. Capsules include hard gelatin capsules that can be filled with powder, pellets, granules, small tablets, or mini-tablets, or solutions or emulsions or combinations and can be coated for enteric or modified release. Soft capsules are also contemplated and are more typically filled with liquids, gels or dispersions, but are not limited thereto. Granules can be effervescent granules, coated granules (e.g., sugar-coated or enteric-coated), and modified release granules. Although the SIgA antibody of the present invention is preferably administered orally, it should be understood that such administration may be considered to be a topical administration to the GI tract. Likewise, a suppository or rectal injection may also be used to topically trat the intestines. The use of an oral dosage form to treat gastrointestinal disease(s) using the sIgA of the present invention is a specific aspect of the present invention.
Pharmaceutical compositions for administering the SIgA antibody via parenteral administration are typically liquid. Water is commonly used as a main excipient, although other pharmaceutically-acceptable liquids such as ethanol, glycerol, ethyl oleate, Myglyol, benzyol oleate, castor oil, MCT, benzyl alcohol isopropyl myristate can be used alone or in combination with water or each other. Aqueous compositions that contain no other excipients are also contemplated, and can be prepared from lyophilized, amorphous, or crystalline compounds. Often the injectable composition, which can be for subcutaneous, IM, or IV injection, contains isotonizing agents. An injectable solution or suspension is typically sterile, as are all liquid pharmaceutical dosage forms.
An overview of dosage forms can be found in Ansel's Pharmaceutical Dosage forms and Drug Delivery Systems. 9^thed. L. V. Allan, N. G. Popovitch, H. C. Ansel, 2010 Lippincott, ISBN: 978-0781779340; Formularium der Nederlandse Apothekers. 2004 WINAp ISBN 90-70605-75-9; Recepteerkunde, G. K. Bolhuis, Y. Bouwman-Boer, F. Kadir en J. Zuiderma, 2005 WINAp ISBN 90-70605-65-1; and Apothekenrezeptur und-defektur. Deutscher Apotheker Verlag Stuttgart 1986 ISBN 3-7692-1092-1. See also U.S. Pat. No. 7,147,854 for a description of topical preparations for delivering IL-8 antibodies to treat skin inflammatory disease such as psoriasis.
The pharmaceutical composition generally contains about 0.01 to 1000 mg of the antibody per dose, depending in part upon the dosage form employed. The dose can be, for example, fixed or variable (e.g, based on body weight) Pharmaceutically-acceptable excipients are known in the art and include diluents, carriers, fillers, binders, lubricants, disintegrants, glidants, colorants, pigments, taste masking agents, sweeteners, plasticizers, and any acceptable auxiliary substances such as absorption enhancers, penetration enhancers, surfactants, co-surfactants, preservatives, anti-oxidants and specialized oils. Specific to the field of biopharmaceutical proteins are excipients intended to stabilize proteins and cryo-protectants to provide protection during freeze-drying. Suitable excipient(s) are selected based in part on the dosage form, the intended mode of administration, the intended release rate, and manufacturing reliability. Non-limiting examples of commonly used excipients include polymers, waxes, calcium phosphates, sugars (e.g., trehalose, sucrose, or mannitol), buffers (such as phosphate, acetate, citrate, histidine, or glycine based buffers at pH between 5 and 7.5), salts (e.g., NaCl or NaEDTA), polysorbate 20, polysorbate 80, human albumin, dextran, and benzyl alcohol.
Treatments
As used herein, the term “treat” or “treatment” means the application or administration of a SIgA antibody of the invention, alone or as part of a composition, to a patient with the purpose to cure, heal, alleviate, improve or prevent an inflammatory disease. The term “inflammatory disease” means a condition associated with symptoms of inflammation, which may be caused by external factors, such as infectious disease, or by internal dysfunctions, such as an autoimmune disease. In this context, the terms disease, disorder, syndrome, condition, and the like are used interchangeably. In embodiments, the SIgA antibodies of the present invention are useful in the topical treatment of inflammatory diseases in humans, e.g., local administration to the site of inflammation, such as orally or rectally. Preferably, the SIgA antibodies of the present invention are useful in the oral treatment of inflammatory diseases.
As used herein, an amount of the SIgA of the present invention effective to treat an inflammatory disease, or a “therapeutically effective amount,” refers to an amount of the antibody which is effective beyond that which is expected in the absence of such treatment.
As used herein, the term “patient” is intended to include humans and non-human animals. The term “non-human animals” includes all vertebrates, e.g., non-mammals (such as chickens, amphibians, reptiles) and mammals, such as non-human primates, sheep, dog, cow, horse, pig, etc. In a preferred instance, the patient is human.
The SIgA antibodies of the present invention are generally useful in treating inflammatory diseases in a human. Specific targets of inflammation include inflammatory bowel disease (including Crohn's disease and ulcerative colitis), psoriasis, psoriatic arthritis, ankylosing spondylitis, and multiple sclerosis.
In a preferred embodiment, the SIgA antibodies of the present invention are orally administered to treat inflammation, inflammatory diseases or disorder, and/or autoimmune disorders of the gut. It should be understood that “treating” in this context includes prophylactic treatment as well as symptomatic treatment. The amount administered to be effective for prophylactic or symptomatic treatment can be determined by routine skill and experimentation for the given patient and condition and will often be in the range of 0.1 mg to 1000 mg per day. The constant regions of the SIgA antibody preferably are the same as, or closely resemble, the normal or natural constant regions of IgA found in the species of patient to be treated as such is believed to minimize the patient's immune response to antibody. The antibody can be administered by itself, such as by a capsule containing the lyophilized protein or as a plant extract added to food, etc., or, in a pharmaceutical composition such as a tablet or capsule.
In other embodiments, the SIgA antibodies of the present invention targets include skin disorders such as psoriasis, acne ectopica (hidradenitis suppurativa), oral aphtha (aphthous stomatitis), bullous and cicatricial pemphigoid, mucocutaneous symptoms of Behcet's Disease, dermatomyositis, erythema annulare centrifugum, skin manifestations of graft-versus-host-disease, non-infectious (non-caseating) granulomatous skin diseases, including granuloma annulare, granuloma cheilitis, and granulomatous rosacea. Additionally, inflammation and/or disorders of the eye can also be a target of the SIgA antibodies of the present invention. The SIgA antibody of the invention can be administered systemically such as via injection and in some circumstances orally. Preferably the antibody can be administered locally by topical application (lotion or ophthalmic compositions, etc.) or by local injection near the affected area; e.g., subcutaneous injection or an injection in the eye.
Additional conditions include disorders mediated by an excess IL-12 or IL-23 as recited in U.S. Pat. No. 6,902,724 and U.S. Pat. No. 6,914,128. Such IL-12 or IL-23 mediated disorders include rheumatoid arthritis, systemic lupus erythematosus (SLE), pyoderma gangrenosum, oral lichen planus oral or orofacial Crohn's disease, and sarcoidosis, but is not limited thereto.

EXAMPLES

The present invention will be further illustrated in the following non-limiting examples.

Example 1

Stable Expression of Anti-IL-12/23 Secretory Protein Based on Ustekinumab (UKB-SA1) in Lemna

a) Construction of Vectors
Synthetic genes were designed for each of the 4 different protein chains of an anti-p40 (anti-IL-12/23) secretory IgA. The amino acid sequence of the heavy chain consisted of the rice α-amylase secretion signal (SEQ ID NO:17) joined to the N-terminal amino acid of the variable part of the heavy chain of anti-IL12/23 IgG1 antibody Ustekinumab (Stelara®, CAS number 815610-63-0, SEQ ID NO:1) which in turn is joined to the N-terminal amino acid of the constant part of a human IgA1 heavy chain (SEQ ID NO:5). The amino acid sequence of the light chain consisted of the rice α-amylase secretion signal (SEQ ID NO:17) joined to the N-terminal amino acid of the light chain sequence of ustekinumab (CAS number 815610-63-0), which combines an anti-p40 (anti-IL-12/23) binding variable part (SEQ ID NO:2) with a human κ-light chain constant part (SEQ ID NO:9). The SC-chain consisted of the rice α-amylase secretion signal (SEQ ID NO:17) 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:16). The J-chain sequence consisted of the rice α-amylase secretion signal (SEQ ID NO:17) 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:15).
Genes were designed for each of the four components with L. 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).
The anti-p40 (anti-IL-12/23) SIgA (UKB-SA1) was expressed in L. minor by transfection via an A. 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:20), SC-chain (SEQ ID NO:21), H-chain (SEQ ID NO:18) and L-chain (SEQ ID NO:19). The H chain was fused to the modified chimeric octopine and mannopine synthase promoter with L. gibba 5′ leader from ribulose bis-phosphate carboxylase (RuBisCo) 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), L. 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. 6A). This vector also contained the gentamycin acetyltransferase-3-I gene (aacC1) which confers resistance to gentamycin and allows for selection of transgenic L. minor lines, and was used to produce UKB-SA1 with wild-type (unmodified) N- and O-glycosylation.
SynA01 was used to create additional transformation vectors to generate a glycan optimized version of UKB-SA1, further identified as UKB-SA1g0. A chimeric hairpin RNA was used to silence endogenous L. minor mRNAs encoding α-1,3-fucosyltransferase (Fuct1, GenBank DQ789145) and β-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. 6B). Also the neomycin phosphotransferase II gene (NPTII) was moved into SynA01 replacing aacC1 to produce transformation vector SynA03 (FIG. 6C). 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
Lemna transformation vectors SynA01, SynA02 and SynA03 were transvected into A. 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).
Partially dedifferentiated Lemna tissue (L. 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° C. Following co-cultivation, the tissue was transferred to antibiotic selection plates and returned to continuous light at 25° 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.
UKB-SA1 with unmodified wild-type (WT) glycosylation was obtained by transfection of L. minor strain 8627 (Biolex Therapeutics Inc.) with vector SynA01. UKB-SA1g0, having G0 glycans lacking fucose and lacking xylose, was obtained by transfecting L. minor strain 8627 with vector SynA02, or by transfection of the N-glycosylation modified L. 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 μmol/m2·s 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.
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 3

		# of	Highest
		lines	Expression
Construct	Product	screened	level

SynA01 in	UKB-SA1 (WT glycosylation)	262	8.5% TSP
L. minor 8627
SynA02 in	UKB-SA1g0 (G0 N-glycans)	164	15.1% TSP
L. minor 8627
SynA03 in	UKB-SA1g0 (G0 N-glycans)	452	11.8% TSP
L. minor XF04

Example 2

Isolation and Purification of UKB-SA1 and UKB-SA1g0 Secretory IgA Antibody from Lemna

Biomass from transgenic Lemna expressing UKB-SA1 or UKB-SA1g0, having variable regions that are the amino acid sequence of the variable regions (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 ribulose bis-phosphate carboxylase (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×g for 30 minutes at 4° C. The supernatant was adjusted to pH 7.4 and filtered to 0.22 μm prior to IMAC chromatography.
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.
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.
The column was regenerated by removing copper using a 0.2M EDTA, 0.3M sodium chloride solution, followed by treatment with 0.1N NaOH.
SEC purification: Material obtained by IMAC 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 0.5 ml/min. The IMAC eluate was concentrated 3x 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.
Results of non-reducing and reducing SDS-PAGE analyses of purified material obtained from Lemna transfected with construct SynA01 are shown in FIG. 7 as gels A and B, respectively.
Some batches of material produced by the purification method described above were further purified using affinity chromatography using CaptureSelect human IgA (BAC).
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 MgCl₂at 5 ml/min.
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.
No substantial differences were observed in purification of the two glycosylation forms, wild-type UKB-SA1 and UKB-SA1g0.

Example 3

Binding of UKB-SA1 and UKB-SA1g0 to IL12

The binding of purified anti-IL-12/23 SIgA, with variable regions taken from ustekinumab and produced in Lemna as in Examples 1 and 2, to IL-12 was determined. The binding of both UKB-SA1 and UKB-SA1g0 products were determined in comparison to ustekinumab (STELARA®) and colostral SIgA. Plates were coated with IL-12 (Abcam, AB52086) 1 μg/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 μl per well, for one hour at RT, washing 3 times, 30 seconds with 200 μl 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 μl per well, for one hour at RT.
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 4

Proteolytic Stability of UKB-SA1 and UKB-SA1g0

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 α-1 heavy chains.
90 μl of a 1 mg/ml solution of the material to be tested was added to 810 μl of SIF at 37° C. 50 μl 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 μl 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 μl 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. 8 where gel A is UKB-SA1 (labeled SynA01-WT in FIG. 8) and gel B is UKB-SA1g0, labeled SynA02-G0 in FIG. 8).
Both UKB-SA1 and UKB-SA1g0 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.

Example 5A

Inhibition of IL-12/23 in Dendritic Cells

To demonstrate inhibition of endogenous produced IL12/IL23 (p40) by UKB-SA1 or UKB-SA1g0 versus ustekinumab (IgG1) or non-specific SIgA (polyclonal colostral SIgA), in vitro experiments were performed with dendritic cells and T cells. Monocytic cells were isolated from human donor blood and stimulated with lipopolysccharide (LPS) and the cells were differentiated into mature dendritic cells. The stimulation with LPS and resulting differentiation was performed in the presence of a SIgA antibody of the invention, Ustekinumab, polyclonal colostral SIgA, or without any added antibody (i.e., LPS only).
In more detail, the experiments were performed as follows. Human dendritic cells (DCs) were obtained from isolated monocytes from buffy coats. The adherent monocytes were cultured in synthetic X-VIVO 15 medium (Lonza, Cat. No. BE04-418Q) supplemented with 2% of AB human serum (Sigma, Cat. No. H4522), 450 U/ml GM-CSF (Miltenyi Biotec, Cat. No. 130-093-867) and 300 U/ml IL-4 (Miltenyi Biotec, Cat. No. 130-093-924) as growth and differentiation factors, respectively, for 6 days to obtain immature DCs. After one week, immature DCs (5×10³) were activated for 24 hours with 1 μg/ml LPS (Invivogen, Cat. No. tlrl-pelps) in the presence of different concentrations UKB-SA1, UKB-SA1g0, or control antibodies to induce DC maturation. IL12/IL23 concentration in culture medium was measured by ELISA according to the manufactures' instructions (BD OptEIA, Cat. No. 555171).
The results are represented in FIG. 9A where open circle is UKB-SA1; closed circle is UKB-SA1g0; Diamond is Colostral SIgA; and Square is ustekinumab. Data are means (+/−SEM) of 3 experiments. Both UKB-SA1 and UKB-SA1g0 inhibited endogenous produced p40/IL12/IL23 by dendritic cells. Both UKB-SA1 and UKB-SA1g0 had a similar efficacy and potency as ustekinumab in this regard.

Example 5B

Inhibition of IL-12/23 in Dendritic Cells

Similar to above, inhibition of IL-12/23 in dendritic cells was measured but at a fixed concentration of the antibody. Human dendritic cells (DCs) were obtained from isolated monocytes from buffy coats. The adherent monocytes were cultured in synthetic X-VIVO 15 medium (Lonza, Cat. No. BE04-418Q) supplemented with 2% of AB human serum (Sigma, Cat. No. H4522), 450 U/ml GM-CSF (130-093-867, Miltenyi Biotec) and 300 U/ml IL-4 (Miltenyi Biotec, Cat. No. 130-093-924) as growth and differentiation factors, respectively, for 6 days to obtain immature DCs. After one week, immature DCs (5×10³) were activated for 24 hours with 1 μg/ml LPS (Invivogen, Cat. No. tlrl-pelps) in the presence of 10 μg/ml UKB-SA1, UKB-SA1g0, or control antibodies to induce DC maturation. IL12/IL23 concentration in culture medium was measured by ELISA according to the manufactures' instructions (BD OptEIA, Cat. No. 555171).
The results are represented in FIG. 9B. Both UKB-SA1 (labeled SynA01 in FIG. 9B) and UKB-SA1g0 (labeled SynA02 in FIG. 9B) inhibited endogenous produced p40/IL12/IL23 by dendritic cells to the same extent as ustekinumab.

Example 5C

Inhibition of IL-12/23 in Dendritic Cells and T cells

To determine if other “intrinsic” properties were affected by the antibodies, dendritic cells were co-cultured with T-cells. The crosstalk between these cells, which might result in a change of phenotype of the dendritic cells, or cytokine secretion profile of the T-cells was examined. DCs were stimulated with LPS for 6 h; afterwards DCs were washed twice in PBS and in the presence of 10 μg/ml of UKB-SA1, UKB-SA1g0 or control antibodies cultured with allogeneic T cells. The allo-response was tested in a mixed lymphocyte reaction; allogeneic T cells (10⁵cells) were co-cultured in triplicate with differently treated DCs (5*10³) in a 96-well round bottom plate, in 200 μl of medium (X-VIVO 15+2% HS). For T helper polarisation T lymphocytes supernatants were collected at day 4 and T helper specific cytokine IFNγ was analysed by ELISA (BD OptEIA, Cat. No. 555142), according to the manufacturers' instructions.
The results are represented in FIG. 9C. UKB-SA1 (labeled SynA01 in FIG. 9C) and UKB-SA1g0 (labeled SynA02 in FIG. 9C) blocked the IFNγ secretion by co-cultured DCs and T-cells to the same extent as ustekinumab, indicating an inhibition of proinflammatory T helper-1 cells.

Example 5D

Inhibition of IL-12/23 in Dendritic Cells and T cells

Similar to above, the antibodies were applied in fixed concentrations to the DC and T cells. DCs were stimulated with LPS for 6 h; afterwards DCs were washed twice in PBS and in the presence of 10 μg/ml of UKB-SA1, UKB-SA1g0, or control antibodies cultured with allogeneic T cells. The allo-response was tested in a mixed lymphocyte reaction; allogeneic T cells (10⁵cells) were co-cultured in triplicate with differently treated DCs (5*10³) in a 96-well round bottom plate, in 200 μl of medium (X-VIVO 15+2% HS). For T helper polarisation T lymphocytes supernatants were collected at day 4 and T helper specific cytokine IL-10 was analysed by ELISA (eBioscience Cat. No. 88-7106-88), according to the manufacturers' instructions.
The results are represented in FIG. 9D. UKB-SA1 (labeled SynA01 in FIG. 9D) and UKB-SA1g0 (labeled SynA02 in FIG. 9D) had no effect on the IL-10 secretion by co-cultured DCs and T-cells.

Example 6

Pharmacokinetics of UKB-SA1g0

Serum concentrations of UKB-SA1g0 after oral and intravenous administration were determined. UKB-SA1g0 was administered to C57Bl/6 mice (200 μg for intravenous, 400 μg oral) and blood samples were taken by orbital bleeding at different time points. UKB-SA1g0 was measured by an in-house constructed ELISA. A goat anti-human kappa light chain antibody (SouthernBiotech, Cat. No. SBA 2060-02) was used for coating of the plates, and a goat anti-human IgA antibody (SouthernBiotech, Cat. No. SBA 2050-05) for detection. The minimum detection limit of the ELISA is 5 ng/ml.
The results are represented in FIG. 10 wherein circle shows oral administration and square shows intravenous administration. Dotted line at 5 ng/ml depicts the detection limited of the ELISA used for quantification. Data are mean+/−SD of 2 representative mice. UKB-SA1g0 (labeled SynA02 in FIG. 10) is rapidly cleared from circulation after intravenous injection and very little (if any) exposure was measured after oral administration.

Example 7

In Vivo Administration of SIgA and IgG Anti-IL12/23 Pharmacokinetics

Using in vivo imaging of fluorescent labeled antibodies, images of thoracic and abdominal region of mice can reveal distribution of the antibody over time. UKB-SA1 was conjugated with either Alexa Flour 647 (Molecular probes), or DyLight 755 (ThermoFisher) according manual instructions. The dye can be detected as a fluorescent signal with the Maestro in vivo imaging system (Cambridge Research & Instrumentation). Fluorescent conjugated antibodies (100 μg/mice) were administered orally or intraperitoneally. Mice were anaesthetized with Xylamine/Ketamine before examination with the Maestro system in order to immobilize animals during picture taking procedure. Then mice were analyzed and pictures of the distribution of the fluorescence conjugated UKB-SA1 were taken at different time points indicated at the experimentally setup. After that background fluorescence were subtracted by software.
The results are represented in FIG. 11 wherein the first three columns (^a) correspond to the DyLight 775 conjugate and the fourth column (^b) is the UKB-SA1 Alexa Flour 647 conjugate. First hours after oral administration fluorescent conjugated antibodies UKB-SA1 (labeled SynA01 in FIG. 11) and IgG1 ustekinumab (labelled IgG in FIG. 11) are present in thoracic area of mice, probably the stomach, whereas after 24 hours a clear signal of UKB-SA1, but not IgG1, is present in the abdominal region (intestine) of the mice. Intraperitoneal administration results in a diffuse thoracic and abdominal signal after 24 hours.

Example 8

Distribution In Vivo after Oral Administration

Immunohistochemistry on cryo-sections of mice distal colon, 48 hours after oral administration of antibody were assessed. Antibodies (100 μg/mouse) were administered orally and after 48 hours animals were sacrificed and intestinal tract isolated. Cry-section were stained with anti-SC chain antibody (Santa Cruz, Cat. No. sc20656) for detection of antibodies containing a human secretory chain. This antibody does not cross-react with mouse secretory chain. As secondary antibody goat-anti-rabbit conjugated with Cy3-red antibody (Jackson Immunoresearch, Cat. No. 111-166-045) was used.
The results are represented in FIG. 12 where the left side is UKB-SA1 (labeled SynA01 in FIG. 12) and the right side is IgG1 ustekinumab (labeled IgG in FIG. 12). UKB-SA1 is present in cells of the sub mucosal layer of the distal colon as indicated by the bright spots in the vicinity of the white arrows. No IgG1 is indicated as being present in the distal colon cells.

Example 9

Efficacy of UKB-SA1 and UKB-SA1g0 in In-Vivo Animal Models of IBD

Heparinized blood was obtained from healthy donors (buffy coats). PBMC's were isolated from buffy coats by Ficoll-Paque 1.077 g/ml (PAA Laboratories GmbH) density centrifugation and cells were washed with 1×PBS before reconstitution. NOD.CB17-Prkdcscid/J γc−/− (NOD-SCID-IL2 receptor deficient) mice 4 to 8 weeks of age were reconstituted with 30×10⁶human cells. Three weeks after reconstitution animals were pre-treated orally 5 times a week (not in weekend) with PBS control, UKB-SA1 (100 μg/mouse), UKB-SA1g0 (100 μg/mouse), or 3 times per week subcutaneous with Ustekinumab (100 μg/mouse). Start of pre-treatment is t=0. At day t=7 reconstituted mice were treated with 2% dextran sulfate sodium (DSS) in drinking water to induce inflammation symptoms in intestine. First signs of inflammation can be seen at day 10-12, together with declined body weight. With mini-endoscopic system an analysis of the status of disease was performed. Briefly, a mini-endoscope (1.9 mm outer diameter) was introduced via the anus and the colon was carefully inflated with an air pump. Endoscopic pictures obtained are of high quality and allow the monitoring and grading of inflammation. Thereafter, endoscopic scoring of five parameters from 0-3 (1; translucent structure, 2; granularity, 3; fibrin, 4; vascularity, and 5; stool) resulting in the overall score from 0 (no change) to 15 (severe colitis) was performed. Typically, after one week exposure to DSS the symptoms are so strong that mice need a resting period to recover. At day t=16 the experiment was terminated and clinical score analyzed.
The results are depicted in FIGS. 13A and B. Oral treatment with UKB-SA1 (labeled SynA01 in FIGS. 13A and 13B) and UKB-SA1g0 (labeled SynA02 in FIGS. 13A and 13B), as well as subcutaneous treatment with Ustekinumab, significantly inhibited DSS-induced inflammatory bowel disease in humanized-scid mice. Statistical analysis: ANOVA followed by Dunnett's Multiple Comparison test, all groups were compared to PBS control group. * p<0.05, *** p<0.001, n=7 mice per group. FIG. 13B depicts a representative mini-endoscopic picture of the colitis score at day t-15.

Example 10

Efficacy of UKB-SA1 and UKB-SA1g0 in Human Psoriatic Skin Biopsies

The activity of SIgA anti-p40 on gene expression was evaluated for a limited number of selected genes in skin explants of psoriatic lesions in vitro. Psoriatic cutaneous lesions can be maintained in culture up to 8 days without significant changes in their psoriatic phenotype. Two punch biopsies of 4 mm were obtained from 7 psoriasis patients (volunteers). Time of evolution of the lesion varied between 1 and 12 months. The two biopsies from each patient were cut in half making four 2 mm biopsies. From one punch, one 2 mm biopsy was tested with vehicle and the other with UKB-SA1. From the other punch, one 2 mm biopsy as tested with UKB-SA1g0 and the other with ustekinumab. UKB-SA1 and UKB-SA1g0 were tested at equimolar concentrations of 14 μg/ml and ustekinumab was tested at 10 μg/ml. The biopsies, medium, and antibody as appropriate were combined in a 24 well plate (800 μl) for 8 days with the medium changed every 2-3 days. After 8 days of culture, the skin biopsies were frozen in liquid nitrogen and then evaluated. Genes were evaluated by RTPCR: Beta-defensin 4, Keratin 16, Interleukin-10, and GAPDH (house-keeping gene). Keratin 16 and β-Defensin 4 are proteins expressed in epithelial tissue and related to skin diseases. Down-regulation of these markers anticipate clinical efficacy of anti-inflammatory treatment in the clinic. Gene expression in treated biopsies was normalized to house-keeping gene GAPDH according to the formula 1,8e (Ct GADPH-Ct gene of interest)×10000 (Ct=cycle threshold time). After normalization, gene expression of gene of interest was compared to gene expression in vehicle treated biopsies.
The results are represented in the table below:


	Time of
	evolution of the
Patient No.	lesion (months)	SynA01	SynA02	Ustekinumab

2	12	K16	++	K16	−	K16	=
		BF	+++	BF	=	BF	+++
		IL10	−−−	IL10	−−	IL10	++
5	12	K16	=	K16	+	K16	=
		BF	+++	BF	+++	BF	++
		IL10	−	IL10	−	IL10	=
4	6	K16	+	K16	+++	K16	+++
		BF	+++	BF	+	BF	+++
		IL10	+	IL10	+	IL10	=
3	3	K16	=	K16	+	K16	=
		BF	=	BF	−−	BF	−−
		IL10	++	IL10	+	IL10	=
7	2	K16	+++	K16	+++	K16	+++
		BF	−−−	BF	−−−	BF	−−−
		IL10	−−−	IL10	−−−	IL10	−−−
6	1	K16	−−−	K16	++	K16	−−
		BF	−−	BF	+++	BF	−−−
		IL10	+++	IL10	+++	IL10	−
1	1	K16	−−	K16	−	K16	−
		BF	−−	BF	+++	BF	−
		IL10	++	IL10	+++	IL10	−

Legend: Beta-defensin 4 (BF4), Keratin 16 (K16), Interleukin 10 (IL-10) No regulation (0-10% difference in gene expression) is “=”; Up-regulated (11-25% difference in gene expression) is +; 26-50% is ++; and >51% is +++. Down-regulated (11-25% difference in gene expression) is −; 26-50% is −−; and >51% is −−−.

Time of evolution of the lesion appears to be relevant for the activity of antip40 antibodies as UKB-SA1, UKB-SA1g0, and ustekinumab have activities in patients, especially those with less than 6 months of evolution of the lesion. Gene expression of Keratin 16 and Beta defensin 4 (two) were reduced in young lesions (1-3 month) by SynA01, SynA02 and ustekinumab, indicating reduced epithelial activity and a treatment effect.
In these young lesions, Interleukin-10 expression was increased only by UKB-SA1 and UKB-SA1g0, but not by ustekinumab, indicating an additional anti-inflammatory cytokine response of SIgA's compared to an IgG antibody.
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.

Claims

1. A monoclonal secretory IgA antibody, which binds to and neutralizes the p40 subunit of human IL-12 and human IL-23.

2. The antibody according to claim 1, wherein said antibody is a chimeric antibody.

3. The antibody according to claim 1, wherein said antibody is a humanized antibody or a human antibody.

4. The antibody according to any of claims 1-3, wherein said antibody comprises a human secretory chain and a human J-chain.

5. The antibody according to claim 4, wherein said human secretory chain has the sequence of SEQ ID NO:16 and said human J-chain has the sequence of SEQ ID NO:17.

6. The antibody according to any of claims 1-5, wherein said antibody is a human secretory IgA1 antibody.

7. The antibody according to any of claim 1-6, wherein said antibody comprises CDR sequences that are identical to the CDR sequences of an antibody selected from the group consisting of ustekinumab and briakinumab, or a variation of one of the foregoing.

8. The antibody according to any of claims 1-7, wherein said antibody comprises CDR sequences that are identical to the CDR sequences of ustekinumab.

9. The antibody according to claim 1, wherein said antibody comprises:

heavy chain constant regions having the sequence of SEQ ID NO:5;

light chain constant regions having the sequence of SEQ ID NO:9;

a human secretory chain, preferably having the sequence of SEQ ID NO:16;

a human J-chain, preferably having the sequence of SEQ ID NO:17;

heavy chain variable regions having the sequence of SEQ ID NO:1; and

light chain variable regions having the sequence of SEQ ID NO:2.

10. A secretory IgA composition, comprising a plurality of antibodies according to any of claims 1-9, wherein substantially all N-glycans in said plurality of antibodies lack fucose and xylose residues.

11. A secretory IgA composition, comprising a plurality of antibodies according to any of claims 1-9, wherein said plurality of 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.

12. A secretory IgA composition, comprising a plurality of antibodies according to any of claims 1-9, wherein said plurality of antibodies 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.

13. The composition according to any of claims 11-12, wherein 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 said plurality of antibodies.

14. The composition according to claim 13, wherein said G0 glycans and said high-mannose glycans together are at least 70% of the total amount of N-glycans in said plurality of antibodies.

15. A pharmaceutical composition, comprising the antibody according to any of claims 1-12, or the composition according to any of claims 13-14, and at least one pharmaceutically acceptable excipient.

16. The pharmaceutical composition according to claim 15, wherein said composition is adapted for oral administration.

17. The antibody according to any one of claims 1-12, or the composition according to any of claims 13-16, for use as a medicament for the treatment of an inflammatory disease in a human.

18. A method for treating an inflammatory disease in a human, which comprises administering an anti-inflammatory effective amount of the antibody according to any one of claims 1-12, or the composition according to any of claims 13-16, to a human in need thereof.

19. The method according to claim 18, wherein said administering comprises orally administering said antibody to said human.

20. The method according to any of claims 18-19, wherein the inflammatory disease is selected from the group consisting of inflammatory bowel disease (including Crohn's disease and ulcerative colitis), psoriasis, psoriatic arthritis, ankylosing spondylitis, and multiple sclerosis.