WO2011116387A1 - Production of aglycosylated monoclonal antibodies in ciliates - Google Patents

Abstract

The invention is directed to a methods for generating aglycosylated antibodies in ciliates. The invention also relates to aglycosylated antibodies generated using the methods described herein, methods for using such antibodies and to ciliates useful for producing such antibodies.

Description

PRODUCTION OF AGLYCOSYLATED

MONOCLONAL ANTIBODIES IN CILIATES

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 61/315,542, filed on March 19, 2010, which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The invention relates to recombinant antibody production and, in particular, methods and compositions for the production of aglycosylated antibodies in ciliates.

BACKGROUND OF THE INVENTION

[0003] Monoclonal antibodies (mAbs) are useful in a broad range of therapeutic applications, including, but not limited to the treatment or prevention of infectious disease and multiple forms of neoplasia. With more than 20 licensed recombinant antibodies now available and hundreds more in various stages of clinical development the world market for recombinant antibodies is estimated to be $29 billion in 2010 (Walter (2007)). To date a large majority of therapeutic recombinant antibodies are manufactured in mammalian cell lines - most notably, Chinese hamster ovary (CHO) cells - and all are of the IgG class. Recombinant mAbs are typically expensive (in the range of $l,000-$2,500 per dose), and significant production issues related to post-translation modification (in particular N-glycosylation).

[0004] Although efforts have been made to control the efficacy and safety of cell- line derived biologies, the potential for contamination remains (e.g., viral contamination) in mammalian cell culture remains problematic. To these ends, there is a desire to broaden the production of therapeutic monoclonal antibodies to microbial systems where the risk of viral contamination is greatly minimized through the use of cells with GRAS designation and the elimination of media components that have the potential to harbor mammalian viruses.

[0005] Because microbial systems typically fail to N-glycosylate proteins (e.g., bacteria), or introduce highly branched immunogenic N-glycans (e.g., yeast), mAb production has been almost exclusively restricted to mammalian cell culture and has led to the development of glyco-engineered mammalian (Shields et al., (2002)) and microbial (Li et al., (2006)) cells that exhibit desired glycan modifications. Nevertheless, even in mammalian cells glycosylation of mAbs is imprecise and results in a heterogeneous mix of glycoforms in a given production run, with each glycoform potentially associated with varying degrees of efficacy.

[0006] Currently there are two general strategies for the production of

aglycosylated mAbs; expression in host cells that lack the ability to modify proteins with N- linked glycans (e.g., bacteria), and mutation of the N-linked consensus (N-X-S/T) site on the mAb heavy chain. Neither option is ideal since it is difficult to produce correctly folded tetrameric antibodies in bacteria and mAb mutation has the potential to exacerbate adverse immunogenic responses in the host.

[0007] Therefore, there is a need for a microbial expression platform capable of producing correctly folded native mAbs in a homogenous form lacking glycan modifications. This invention addresses this need and provides a marked improvement over existing systems by offering significant cost, safety and productivity advantages over mammalian cell culture.

SUMMARY OF THE INVENTION

[0008] The invention relates to the production of antibodies (e.g., IgG class immunoglobulins) lacking an asparagine-linked glycan on the heavy chain (aglycosylated IgGs) in ciliates. In particular, the invention relates to the surprise finding that Tetrahymena thermophila fails to glycosylate wild-type heavy-chain protein and therefore offers a method to produce aglycosylated IgG molecules that do not contain engineered mutations.

[0009] In one aspect, the invention relates to a method for producing an aglycosylated antibody in a ciliate, the method comprising: a) transforming the ciliate with a nucleic acid construct encoding the antibody, b) culturing the ciliate and expressing the antibody, and c) isolating the antibody.

[0010] In some embodiments, the nucleic acid construct is a vector, a plasmid, a cosmid, a chromosome or minichromosome, a transposon, a ribosomal DNA or any combination thereof.

[0011] In some embodiments, the ciliate is Tetrahymena thermophila. In some embodiments, the ciliate is Tetrahymena pyriformis. In still further embodiments, the nucleic acid construct encoding the antibody further comprises a nucleic acid sequence encoding a signal peptide such that translation of the nucleic acid construct results in the production of a polypeptide comprising a signal peptide operably linked to the antibody. [0012] In some embodiments, the signal peptide is an Ichthyophthirius multifiliis signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 6.

[0013] In some embodiments, the antibody comprises an antibody light chain sequence. In some embodiments, the antibody comprises an antibody heavy chain sequence. In still further embodiments, the antibody comprises an antibody light chain sequence wherein the heavy chain sequence comprises an asparagine-linked glycan consensus site.

[0014] In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a mouse antibody. In some embodiments, the antibody is a chimeric antibody. In still further embodiments, the antibody is a humanized antibody.

[0015] In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgM antibody. In some embodiments, the antibody is an IgD antibody. In some embodiments, the antibody is an IgE antibody. In some embodiments, the antibody is an IgA antibody. In some embodiments, the antibody is a tetrameric antibody. In some embodiments, the tetrameric antibody comprises two light chains and two heavy chains. In some embodiments, the antibody is a multispecific antibody. In some embodiments, the multispecific antibody is a bispecific antibody.

[0016] In some embodiments, the antibody comprises an amino acid sequence consisting essentially of SEQ ID NO: 1. In some embodiments, the antibody comprises an amino acid sequence consisting essentially of SEQ ID NO: 2. In still further embodiments, the antibody comprises an amino acid sequence consisting essentially of SEQ ID NO: 3. In some embodiments, the antibody comprises an amino acid sequence consisting essentially of SEQ ID NO: 4. In further embodiments, the antibody comprises an amino acid sequence of any of SEQ ID NOs: 8-129.

[0017] In some embodiments, the antibody is a therapeutic antibody. In some embodiments, the antibody specifically binds a cytokine.

[0018] In some embodiments, the antibody specifically binds an inflammatory molecule. In some embodiments, the antibody specifically binds a growth factor. In some embodiments, the antibody specifically binds a growth factor receptor. In some embodiments, the antibody specifically binds an oncogene. In some embodiments, the antibody specifically binds an agriculturally related polypeptide. In some embodiments, the antibody specifically binds an antibody. In some embodiments, the antibody specifically binds a prophylactic vaccine, a therapeutic vaccine.

[0019] In another aspect, the invention relates to an aglycosylated antibody produced by a method comprising: a) transforming the ciliate with a nucleic acid construct encoding the antibody, b) culturing the ciliate and expressing the antibody, and c) isolating the antibody.

[0020] In another aspect, the invention relates to a ciliate cell capable of producing an aglycosylated antibody. In some embodiments, the ciliate has been genetically- engineered to express an aglycosylated antibody. In some embodiments, the antibody comprises an asparagine-linked glycan consensus site.

[0021] These and other aspects of the invention will be apparent to those of ordinary skill in the art in view of the following detailed description and examples.

BRIEF DESCRIPTION OF THE FIGURES

[0022] Figure 1. Figure 1 shows a schematic illustration of an expression construct designed for production of aglycosylated monoclonal antibodies in Tetrahymena thermophila. Abbreviations: Pr, Promoter; Tr, Terminator; NE02, neomycin resistance cassette.

[0023] Figure 2. Figure 2 shows murine anti-transferrin IgGi light (Fig. 2A) and heavy chain amino acid amino acid sequence (Fig. 2B). The Ichthyophthirius multifiliis immobilization antigen variant B protein signal peptide sequence is underlined in both sequences. The predicted asparagine glycan attachment site in the heavy chain is shown in bold and is underlined and italicized.

[0024] Figure 3. Figure 3 shows that T. thermophila derived murine IgGi mAb is correctly folded and functional. Fig. 3A shows SDS-PAGE analysis of purified anti-human transferring mAb resolved under reducing and non-reducing conditions. Under reducing conditions light and heavy chains resolve at the expected masses of 25 and 50 kDa, respectively. Under non-reducing conditions a majority of light and heavy chains resolve as a complex >150 kDa. Fig. 3B shows that anti-transferrin antibody specifically precipitates antigen (transferrin) whereas control non-specific mAb does not. [0025] Figure 4. Figure 4 shows T. thermophila derived anti-human transferrin IgGi mAb is aglycosylated. Shown is MALDI-TOF mass spectrometry analysis of purified mAb. Apparent and predicted mass of glycosylated heavy chain is indicated.

[0026] Figure 5. Figure 5 shows humanized anti-transferrin IgGi light (Fig. 5A) and heavy chain (Fig. 5B) amino acid sequence. The Ichthyophthirius multifiliis

immobilization antigen variant B protein signal peptide sequence is underlined. The predicted asparagine glycan attachment site in the heavy chain is shown in bold and is underlined and italicized.

[0027] Figure 6. Figure 6 shows that humanized mAb forms a >150 kDa complex under non-reducing (NR) conditions anti-human IgG Western analysis of humanized anti- transferrin IgGi purified from Tetrahymena (Fig. 6A). Under reducing conditions (R) light and heavy chains resolve at the expected masses of 25 and 50 kDa, respectively. Purified humanized mAb was used as a primary antibody in a western analysis of 1 μg antigen

(transferrin) and non-related protein (BSA). Purified mAb specifically detects antigen and is therefore functional (Fig. 6B). Conconavalin A based Western shows lack of detection of BSA (aglycosylated negative control), detection of yeast derived enterokinase (glycosylated positive control) and lack of detection of humanized anti-transferrin heavy chain (Fig. 6C). This result indicates that the humanized heavy chain is not modified with an N-linked glycan. Abbreviations: R, reducing; NR, non-reducing; yEK, yeast derived recombinant enterokinase; hlgG, humanized anti-transferrin IgGi.

[0028] Figure 7. Figure 7A shows the amino acid sequence of recombinant enterokinase expressed in Tetrahymena. The Ichthyophthirius multifiliis immobilization antigen variant B protein signal peptide sequence is underlined. Three potential N-linked glycan sites are bold, underlined and italicized. Figure 7B shows that recombinant

enterokinase secreted from Tetrahymena is a glycoprotein as judged by glycan removal following PNGaseF treatment.

[0029] Figure 8. Figure 8 shows that MALDI-TOF analysis of purified recombinant enterokinase derived from Tetrahymena reveals that a majority of the protein has each of three potential N-linked glycan sites occupied while the remainder has 2 of the 3 glycan sites occupied. Predicted occupancy and apparent and predicted mass of glycosylated enzyme are indicated. [0030] Figure 9. Figure 9A-C shows the nucleic acid sequence of the mouse anti- human transferrin IgGl expression cassette into the ribosomal DNA vector for expression as set forth in Example 1. The sequence is flanked by two Notl sites that were used for cloning into the ribosomal DNA vector.

DETAILED DESCRIPTION OF THE INVENTION

[0031] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued U.S. patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

[0032] Definitions

[0033] All scientific and technical terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent or later-developed techniques which would be apparent to one of skill in the art. In addition, in order to more clearly and concisely describe the subject matter which is the invention, the following definitions are provided for certain terms which are used in the specification and appended claims.

[0034] As used herein, the term "ciliates" means eukaryotes belonging to the kingdom Chromalveolata, the superphylum Alveolata, and the phylum Ciliophora. Ciliates are complex protozoa characterized by the presence of cilia on their cell surfaces and dimorphic nuclei consisting of a macronucleus and one or more micronuclei.

[0035] As used herein, "Tetrahymena spp." refers to ciliate protozoa in the family of Tetrahymenidae. Exemplary Tetrahymena spp. include, but are not limited to,

T. thermophila and T. pyriformis.

[0036] As used herein, the term "aglycosylated" as used in reference to an antibody, means that at least one site which is normally glycosylated in a natural host {e.g. , a human or a mouse) or in a host cell {e.g. , a human cell or a mouse cell) is not glycosylated when the antibody is expressed in the ciliate. In some embodiments, the term as used in reference to an antibody, means that the protein is not modified by asparagine-linked glycosylation.

[0037] As used herein, the term "mAb" refers to a monoclonal antibody.

[0038] As used herein, the terms "increase" and "decrease" mean, respectively, to cause an increase or decrease of at least 5%, as determined by a method and sample size that achieves statistically significance (i.e., p < 0.1).

[0039] As used herein, the term "statistically significant" means having a probability of less than 10% under the relevant null hypothesis (i.e., p < 0.1).

[0040] As used herein, the recitation of a numerical range for a variable is intended to convey that the invention may be practiced with the variable equal to any of the values within that range. Thus, for a variable that is inherently discrete, the variable can be equal to any integer value within the numerical range, including the end-points of the range. Similarly, for a variable that is inherently continuous, the variable can be equal to any real value within the numerical range, including the end-points of the range. As an example, and without limitation, a variable which is described as having values between 0 and 2 can take the values 0, 1 or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, . . . , 0.9, 0.99, 0.999, or any other real values > 0 and≤ 2, if the variable is inherently continuous.

[0041] As used herein, unless specifically indicated otherwise, the word "or" is used in the inclusive sense of "and/or" and not the exclusive sense of "either/or."

[0042] As used herein and in the appended claims, the use of singular forms of words, and the use of the singular articles "a," "an" and "the," are intended to include and not exclude the use of a plurality of the referenced term unless the content clearly dictates otherwise.

[0043] General Considerations

[0044] The present invention relates to the unexpected finding that ciliates produce a correctly assembled functional IgG mAb following co-expression of light and heavy chain genes. In certain aspects, the invention relates to the production of aglycosylated antibodies in ciliates. In other aspects, the invention relates to the production of IgG class immunoglobulins lacking an asparagine-linked glycan on the heavy chain (aglycosylated IgGs). In certain aspects, the invention also relates to methods for producing aglycosylated antibody (e.g., IgG) molecules that do not contain engineered mutations at one or more glycosylation sites on the antibody.

[0045] In one aspect, the methods described herein are useful for producing a desired antibody in aglycosylated form, the method comprising expressing one or more nucleic acid sequences encoding an antibody in a ciliate. In some embodiments, the methods and compositions described herein can be used to produce aglycosylated antibodies (e.g., IgG immunoglobulin) in a ciliate by co-expression of the immunoglobulin light and heavy chain genes. In some embodiments, the methods described herein can be used to produce aglycosylated antibodies that retain an unmodified asparagine-linked glycan consensus site in the heavy chain. In some embodiments, the methods described herein can be used to produce aglycosylated antibodies comprising a native heavy chain asparagine-linked glycan consensus site. One advantage of this invention is the application of microbial-based capabilities for the production of aglycosylated therapeutic antibodies. In certain aspects, the methods described herein do not require modification of an expression cassette design comprising a nucleic acid sequence encoding an antibody or mutation of the heavy chain N- linked site, to produce aglycosylated antibody.

[0046] As used herein, the term "antibody" is intended to embrace naturally produced antibodies, recombinantly produced antibodies, chimeric antibodies, humanized antibodies, human antibodies, synthetic antibodies, intrabodies, recombinantly produced antibodies, antibodies subjected to in vitro or in-vivo alterations, monoclonal antibodies, and polyclonal antibodies, as well as antibody fragments such as a heavy chain, a light chain, a Fab fragment, a F(ab')₂ fragment, a Fv fragment, and a single-chain Fv fragment (scFv), disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, and derivatives or epitope- binding fragments of any of the above.

[0047] As used herein, the term "chimeric antibody" refers to antibody molecules wherein a portion of the heavy and/or light chain is derived from a particular species or belongs to a particular antibody class or subclass, while the remainder of the chain(s) is derived from another species or belongs to another antibody class or subclass. As used herein, the "humanized" antibody refers to a chimeric antibody wherein at least one portion of the primary sequence is modified to reduce non-human sequences and/or to increase sequences corresponding to those found in human antibodies. Methods for humanizing non-human antibodies, generating chimeric antibodies and aligning antibody sequences are well known in the art.

[0048] Antibodies can elicit their therapeutic benefits by linking high-specificity for antigen to immune responses via conserved regions on their Fc domains. Some therapeutic mAbs, for example those used in cancer treatment, rely on antibody-dependent and/or complement-dependent cytotoxicity (ADCC or CDC) for their effect. These processes can be initiated through engagement of the Fc domain of IgG with the Fc gamma receptor (FcyR) on cells of the host immune system. This interaction can, in turn, depend on the presence and composition of a single N-linked glycan moiety on asparagine 297 of the Ig heavy chain (Jefferis (2009); Shantha Raju (2008)). Removal of the Fc glycan by point mutation (Shields et al, (2001)), enzymatic deglycosylation (Mimura et al, (2001)), or expression in host cells that lack the ability to post-translationally modify proteins {e.g., E. coli) (Mazor et al, (2007)), has been shown to result in reduced binding to FcyR family members.

[0049] While a majority of therapeutic applications require mAbs to engage the immune system, in a number of indications, effector functions, such as ADCC and CDC are not required, and would optimally be avoided for mAb efficacy. Examples of conditions where it can be advantageous to generate antibodies lacking an N-linked glycan moiety on an asparagine of the Ig heavy chain include, but are not limited to, autoimmune disorders and transplant rejection therapies where antibody-mediated depletion of target cells is unwarranted, or where engagement of the Fey receptor is deleterious. For example, the therapeutic treatment of acute rejection of allografts with a glycosylated anti-CD3 mAb results in excessive cytokine production (and potential toxic shock) caused by simultaneous engagement of the T cell receptor complex and FcyR (Friend et al, (1999)). By contrast, an aglycosylated anti-CD3 mAb maintains its immunosuppressive potency free of the toxicity brought on by the glycosylated form (Friend et al, (1999)). Similarly, clinical efficacy of anti-CD4 mAbs that have shown promise for the Otreatment of rheumatoid arthritis and other autoimmune disorders are hampered by FcyR-mediated depletion of CD4 cells resulting from ADCC initiated by glycosylated antibodies (Reddy et al, (2000); Isaacs et al, (1996)). Further, Fc variants lacking N-glycans are capable of engaging the immune system and aglycosylated antibodies can be useful for therapeutic strategies that include target cell depletion (Sazinsky et al, (2008)). Thus, in some embodiments, the methods described herein provide an improved method for manufacturing therapeutic antibodies as compared to existing methods which otherwise result in the production of glycosylated mAbs that suffer from batch-to-batch glycoform heterogeneity.

[0050] The methods described herein for producing aglycosylated antibodies can be used to produce antibodies from a variety of sources, and species origins, including, but not limited to murine, humanized and human sources. This finding is surprising, in part because, numerous reports have shown that T. thermophila is capable of producing both native and recombinant glycosylated proteins. Further, it has been reported that when these proteins are produced in Tetrahymena, that are decorated with primitive high-mannose N- glycan structures that display limited heterogeneity comprising MansGlcNAc₂,

Man₄GlcNAc₂ and Man₃GlcNAc₂ structures (Taniguchi et ah, (1985); Becker and Rusing (2003); Weide et ah, (2006)). In some embodiments, the methods for producing

aglycosylated antibodies described herein do not depend on the type of signal peptide used to direct secretion of the aglycosylated antibodies. In some embodiments, a signal peptide having the sequence MGSKFNILIILIISLFINELRA (SEQ ID NO: 6) can be fused to the N- terminal end of an antibody sequence, including, but not limited to a light chain sequence or a heavy chain sequence, so as to direct secretion of the aglycosylated antibodies according to the methods described herein. In certain embodiments, the aglycosylated antibodies produced according to the methods described herein can be further modified with N-linked glycans either during secretion, after secretion, upon isolation or purification.

[0051] In certain embodiments, the aglycosylated antibodies described herein can be a full-length antibodies having a structure generally corresponding to the natural biological form of an antibody found in nature, including variable and constant regions. In certain embodiments, the aglycosylated antibody can have the amino acid sequence of a

heterologous antibody is not naturally occurring. For example, the aglycosylated antibody can have a amino acid sequence that differs from that of a corresponding native (wild-type) or parent antibody sequence at one or more one amino acid positions. Such differences can comprise an insertion, a deletion, a substitution or any combination thereof. In some embodiments, the aglycosylated antibody can be a variant of a naturally occurring or non- naturally occurring antibody, wherein the aglycosylated antibody comprises a substantially similar activity {e.g., substantially the same target binding activity), as the naturally occurring or non-naturally occurring antibody. In some embodiments, the variant aglycosylated antibody can have at least about 60% amino acid identity with the naturally occurring or non- naturally occurring antibody, at least about 70% amino acid identity with the naturally occurring or non-naturally occurring antibody, at least about 80% amino acid identity with the naturally occurring or non-naturally occurring antibody, at least about 90% amino acid identity with the naturally occurring or non-naturally occurring antibody, or at least about 95% amino acid identity with the naturally occurring or non-naturally occurring antibody.

[0052] The methods described herein can be used to produce aglycosylated antibodies of all immunoglobulin classes, including but not limited to, IgM, IgG, IgD, IgE, IgA, as well as their subclasses, including, but not limited to IgGl, IgG2, IgG3, IgG4, IgAl and IgA2). In certain embodiments, the aglycosylated antibody can be a protein comprising a one or more polypeptides encoded by an immunoglobulin gene (e.g., an IgG gene). The aglycosylated antibodies described herein can be from any source or derivation, including but not limited to a mouse IgG gene, a human IgG gene, a chimeric IgG gene or a humanized IgG gene. The aglycosylated antibodies described herein may specifically bind to one or more FcRs. Where one or more variable regions are present in the aglycosylated antibodies described herein, the antibody may also specifically bind to an antigen. As used herein, the term "specific binding" can refer to an interaction having an association rate constant in the range of at least lxl 0⁴ M^"1 s^"1. Such rate constants can be measured by BiaCore according to the methods set forth in Katsamba et al., Anal Biochem. 2006 May 15;352(2):208-21.

[0053] The antibodies of the invention may be monospecific, bispecific, trispecific or of greater multi specificity. Multispecific antibodies can specifically bind to different epitopes of an antigen or can specifically bind to epitopes on separate antigens.

[0054] The antibodies of the invention include derivatives that are modified, e.g. the covalent attachment of any type of molecule to the antibody. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non- classical amino acids.

[0055] In certain embodiments, the aglycosylated antibodies described herein can be tetramer antibodies and may comprise two identical pairs of two Ig chains, each pair having one light chain and one heavy chain. In certain embodiments, each light chain can comprise VL and CL immunoglobulin domains. In certain embodiments, each heavy chain can comprise VH and C5 immunoglobulin domains. In certain embodiments, the antibodies described herein can be encoded by kappa (K) and lambda (λ) light chain genetic loci, or fragments thereof, and heavy chain genetic loci, or fragments thereof, which include constant region genes mu (μ), delta (δ), gamma (γ), epsilon (ε), and alpha (a) for the IgM, IgD, IgG, IgE, and IgA isotypes, respectively.

[0056] In certain embodiments, the aglycosylated antibodies described herein can have sequence identity or sequence similarity with antibodies from transgenic animals. For example, the aglycosylated antibody can have a sequence of a fully human antibody generated from transgenic mice harboring human heavy and light chain loci. Fully human antibodies obtained from transgenic mice are described by Green et al., (1994), Lonberg et al., (1994), and Taylor et al., (1994). Fully human antibodies useful for the methods described herein can also be antibodies generated by phage display technology, by

chromosomal transfection methods, or in vitro by activated B cells (McCafferty et al., (1990); Johnson and Chiswell (1993); U.S. Patent Nos. 5,567,610 and 5,229,275).

[0057] In certain embodiments, the aglycosylated antibodies described herein can camelized single domain antibodies (see e.g., Muyldermans et al., 2001, Trends Biochem. Sci. 26:230).

[0058] The aglycosylated antibodies described herein can also be antibodies that are linked one or more agents having one or more effector functions or pharmacokinetic properties. In certain embodiments, the linkage can be synthetic in nature, e.g., via chemical conjugation, or via recombinant expression, e.g., a fusion polypeptide is formed. For example, in certain embodiments, the linked polypeptide can be a polypeptide useful for isolating or purifying a linked antibody (e.g., a tag such as a FLAG-tag, Strep-tag, glutathione S transferase, maltose binding protein (MBP) or a His-tag). Agents that can be linked to the aglycosylated antibodies described herein include, but are not limited to, various enzymes, such as but not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Agents that can be linked to the aglycosylated antibodies described herein include, but are not limited to, various prosthetic groups, such as but not limited to streptavidin/biotin and avidin/biotin. Agents that can be linked to the aglycosylated antibodies described herein include, but are not limited to, fluorescent materials, such as but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine,

dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Agents that can be linked to the aglycosylated antibodies described herein include, but are not limited to, luminescent materials, such as but not limited to, luminol; bioluminescent materials, such as but not limited to, luciferase, luciferin, and aequorin.

[0059] In certain embodiments, the linked polypeptide can be, but is not limited to, a ligand for a receptor, an extracellular domain of a receptor, a variable region of a heavy Ig chain, a toxin, a radioisotope or a chemotherapeutic.

[0060] The aglycosylated antibodies described herein can also be subjected to in vitro alterations, for example, by treatment with enzymes or chemicals (e.g., proteases or molecules are useful for linking (e.g., conjugating) the antibodies described herein to a toxin, a chemotherapeutic, a radioisotope, a fluorescent labels (e.g., FITC), enzymatic labels (e.g., alkaline phosphatase), a chemiluminescent label, a biotinyl group, an avidin group, or polypeptide epitope, a sugar, a lipid, a fat, a metal, a synthetic polymer, or any combination thereof.

[0061] In certain embodiments, the aglycosylated antibodies described herein can be labeled with a detectable moiety. Suitable labels include, but are not limited to, radioisotopes, such as 34S, 14C, 1251, 3H, and 1311, and fluorescent labels such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, phycoerythrin, Texas Red, and the like. Techniques for labeling with radioisotopes and fluorophores, as well as other molecules, are known to the art.

[0062] Antibody Production in Ciliates

[0063] In one aspect, the methods described herein relate to the production of an aglycosylated antibody, or fragment thereof in ciliates, the method comprising (a) transforming a ciliate with an nucleic acid comprising a nucleotide sequence encoding the pantibody; (b) culturing the ciliate to produce the antibody; and (c) isolating the antibody. In some embodiments, the ciliate is transformed with a single nucleic acid encoding both light chain and heavy chain antibody sequences. In some embodiments, the ciliate is transformed with separate nucleic acids encoding light chain and heavy chain antibody sequences. In some embodiments, the ciliate is transformed with a nucleic acid encoding a fragment of an antibody. The antibody encoded by the nucleic acid can be from any source, either naturally occurring or non-naturally occurring. Further, the antibody encoded by the nucleic acid can be an antibody from any organism, such that the antibody produced according to the methods described herein is an aglycosylated antibody, or fragment thereof, comprising amino acid sequences having identity to antibodies from said species. Thus, in certain embodiments, the antibody can be a human antibody, or fragment thereof, a mouse antibody, or fragment thereof, a chimeric antibody, or a humanized antibody. In some embodiments, the

aglycosylated antibody can be a therapeutically useful antibody.

[0064] The aglycosylated antibodies produced according to the methods described herein can be of any size. In certain embodiments, the antibody has a molecular weight between about lkDa to about lOkDa, between about lOkDa to about 20kDa, between about 20kDa to about 50kDa, between about 50kDa to about lOOkDa, between about lOOkDa to about 200kDa, between about 200kDa to about 400kDa, between about 400kDa to about 800kDa, between about 800kDa to about l,500kDa, or greater than l,500kDa. In certain embodiments, the aglycosylated antibody comprises at least 10, at least 15, at least 20, at least 30, at least 40, at least 50 or at least 100 amino acids.

[0065] In one aspect, the aglycosylated antibodies produced according to the methods described herein have at least about 60%, at least about 70%>, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% or more amino acid identity to known mouse, human, humanized or chimeric antibodies

[0066] Ciliates Useful in the Invention

[0067] The methods described herein may be practiced with a variety of different ciliates, including, but not limited to, c T. thermophila and T. pyriformis. Ciliates are a large and diverse eukaryotic phylum whose members display a structural and functional complexity comparable to that of higher metazoan cells (Frankel (2000)), and include over 7,000 species with 11 major subdivisions.

[0068] Tetrahymena spp. are amenable to genetic manipulation, can be grown on a large scale and have a doubling time of 1.5-3 hrs. Unlike T. thermophila, which has an optimal growth temperature of 35°C, the optimal growth temperature for T. pyriformis is lower having a maximal growth temperature of 34°C. Cells reach high-density in a short time on a variety of inexpensive media and can be expanded for growth in bioreactors up to several thousand liters in size (Hellenbroich et ah, (1999); de Coninck et ah, (2000)). These cells can achieve high cell density (- 50 g/cell dry cell weight) at scale (Kiy and Tiedke (1992)) and can be grown in a variety of inexpensive media including animal-free cGMP compliant veggietone media. Methods for reliable transformation, along with robust inducible promoters for driving high-level expression are also available for this system (Shang et al., (2002)). Methods for transformation, along with robust, inducible promoters for driving high- level gene expression have recently been described for this system (Bruns and Cassidy- Hanley (2000); Gaertig and Kapler (2000); Shang et al, (2002); Boldrin et al, (2006)).

[0069] The methods described herein may also be practiced with a variety of different Tetrahymena strains, including, but not limited to strains SB 1969, SB210-E, CU428.2, CU427.4, IA171, IA104, B2086.2, B2-688a, B1868, C3 368.1, C3 491 lb, and D1968-3a. Further, the methods described herein may also be practiced with a variety of Tetrahymena strains having different Mic genotypes, including, but not limited the following Mic genotypes: chxl-l/chxl-1; gall-l/gall-1; mprl-l/mprl-1; fat4-l/fat4-l; cdaAl- 1/cdaAl-l; wild type B; wild type B2; Wild type C3; and wild type D. The methods described herein may also be practiced with Tetrahymena strains having different mating types, including, but not limited to MT I, MT II, MT III, MT IV, MT V, MT VI, and MT, VII.

[0070] In certain embodiments, the aglycosylated antibodies described herein can be isolated or purified upon secretion from a ciliate. Ciliates engage in regulated secretion of proteins stored in cortical secretory organelles (granules), which are discharged in a stimulus- dependent or regulated fashion (Turkewitz et al, (2000); Turkewitz (2004)). In Tetrahymena spp., these dense core granules are termed mucocysts. Each Tetrahymena spp. cell contains numerous mucocysts docked at the plasma membrane. Upon stimulation, the discharge of the mucocyst contents occurs in a rapid and synchronous manner (Satir (1977)). Ciliates, such as Tetrahymena, also have a constitutive secretory pathway through which many secretory proteins are released. Mucocyst discharge can be triggered with appropriate secretory stimuli to release mucocyst-targeted heterologous proteins into the extracellular space in association with the proteinaceous mucocyst gel. Regulated secretion can depend on the level of the stimulus, and can be an all-or-none phenomenon with, in some cases, large amounts of protein being released within a short period of time (on the order of milliseconds). Regulated secretion can be triggered by the presence of chemical mediators known as secretagogues. For example, such mediators can cause increased levels of intracellular calcium (Ca²⁺), which, in turn, trigger fusion of cortical granules with the plasma membrane resulting in a release of the granule contents into the surrounding extracellular space.

Examples of secretagogues useful in the invention include, but are not limited to, dibucaine, Alcian blue, elevated NaCl, sucrose and Ca²⁺ ionophores. For example, treatment of

Tetrahymena spp. cells with dibucaine, or other secretagogues, results in rapid fusion of mucocyst membranes with the plasma membrane, and discharge of the granule contents into the extracellular space (Turkewitz et al., (2000); Turkewitz (2004); Maihle and Satir (1986)). Regulated secretion can also be triggered by secretory stimuli other than secretagogues. Examples of such secretory stimuli useful in the invention include, but are not limited to, treatment with mechanical shock, cross-linking of surface antigens, and electroshock (e.g., electroporation).

[0071] Nucleic Acids Encoding the Polypeptides Described Herein

[0072] Any methods known to the art may be can be used to prepare nucleic acid sequences encoding the aglycosylated antibodies described herein. Suitable methods include, but are not limited to, PCR, ligation, recombination, site-directed mutagenesis (Carter et al., Nucleic Acids Res.. 13.:4431 (1985); Kunkel et al, Proc. Natl. Acad. Sci. USA, 82:488 (1987)), saturation mutagenesis (U.S. Patent Nos. 6,171 ,820, 6,562,594 and 6,764,835), PCR mutagenesis (Higuchi, in PCR Protocols, pp.177- 183 (Academic Press. 1990)), cassette mutagenesis (Wells et al., Gene. 34:315 (1985)), synthetic ligation reassembly (U.S. Patent Nos. 6,537,776 and 6,605,449). Methods useful to prepare nucleic acids encoding variant antigen binding sites, e.g., in antibodies, are disclosed, for instance, in U.S. published application 20030219752.

[0073] Vectors

[0074] Heterologous nucleic acids encoding the aglycosylated antibodies described herein can be introduced into the ciliate host on an expression vector. In some embodiments, the vector can be a vector that is capable of integrating into the host's genome. As used herein, the term "heterologous" means, with respect to two or more genetic or protein sequences, that the sequences do not occur in the same physical relation to each other in nature and/or do not naturally occur within the same genome or protein. For example, a genetic construct may include a coding sequence which is operably joined to one or more regulatory sequences, or to one or more other coding sequences, and these sequences are considered heterologous to each other if they are not operably joined in nature and/or they are not found in the same relation in a genome in nature. Similarly, a protein may include a first polypeptide sequence which is joined by a standard peptide bond to a second polypeptide sequence, and these sequences are considered heterologous to each other if they are not found in the same relation in any protein or proteome in nature. As used herein, the term "vector" means any genetic construct, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable transferring nucleic acids between cells. Vectors may be capable of one or more of replication, expression, and insertion or integration, but need not possess each of these capabilities. Thus, the term includes cloning, expression, homologous recombination, and knock-out vectors. For example, expression vectors capable of homologous recombination with a highly expressed gene that is endogenous to the protozoan host, such as a P-tubulin gene are known in the art. As used herein, the term "endogenous" means, with respect to a genetic or protein sequence, that the sequence occurs naturally in the same physical relation to a specified sequence, or occurs naturally in a specified cell or genome. For example, a genetic construct may include a coding sequence which is operably joined to one or more regulatory sequences, and the regulatory sequences are considered endogenous if they are operably joined to the coding sequence in nature, and/or they are found in the same relation in a genome in nature. Similarly, a protein that occurs naturally in a specified cell type or species, is considered endogenous to that cell or species.

[0075] Methods for creating mitotically stable ciliate transformants, for example, by integration of a heterologous gene by homologous DNA recombination, are known in the art. Methods for generating ciliates having targeted gene knockouts by homologous DNA recombination are also known in the art (Bruns and Cassidy-Hanley (2000); Hai et al., (2000) 514-531; Gaertig et al., (1999); Cassidy-Hanley et al., (1997)). The somatic macronucleus or the generative micronucleus can be transformed in alternation. For example, sterile transformants, which may provide improved safety parameters, can be obtained with macronucleus transformation.

[0076] There are a number of suitable vectors suitable for transformation of ciliates known in the art. Such vectors include but are not limited to replacement vectors, ribosomal DNA vectors, and ribosomal DNA-based vectors.

[0077] Replacement vectors accomplish DNA-mediated transformation by replacing or altering endogenous genes using homologous recombination. Integration of the heterologous nucleic acid into the host's genome at the targeted site is accomplished via homologous recombination involving a double crossover event with the vector containing the heterologous nucleic acid. An example of an expression vector useful for genomic incorporation of a heterologous nucleic acid by replacement is one that includes a

heterologous coding sequence flanked by portions of the endogenous BTU1 gene of T.

thermophyla. [0078] A replacement vector can include a 5' region, followed by a heterologous coding region, followed by a 3' region, wherein at least a portion of each of the 5' and 3' regions is complementary to 5' and 3' regions on an endogenous gene of the host, to allow for genomic integration of the heterologous coding region via homologous recombination. The 5' and 3' regions of the vector can also comprise regulatory elements, such as a promoter and a terminator. The necessary regulatory elements can also be supplied by the endogenous gene into which the heterologous coding region integrates. Suitable regulatory regions include, but are not limited to, promoters, termination sequences, signal peptides and proprotein domains involved in the expression and secretion of proteins. For example, such regulatory elements can provide efficient heterologous expression of proteins in ciliates under control of promoters and/or terminators which are derived from genes in ciliates. Such vectors can comprise naturally occurring promoters and/or terminators from proteins secreted at a high level in ciliates. The expression of recombinant polypeptides in ciliates can be driven by strong promoters, pre/pro sequences and terminators. In some embodiments, the promoters and/or terminators can be selected from proteins secreted at a high level independent of the cell-cycle in ciliates (US Patent Application 2006/0127973;

WO2003/078566). Inducible promoters from ciliate genes have also been described that allow robust expression of foreign genes. For example, heat-inducible promoters of the heat shock protein family of ciliates are also suitable for use with the methods described herein. Suitable heat shock promoters from ciliates are known in the art (see WO2007/006812).

[0079] Alternatively, a heterologous nucleic acid encoding the aglycosylated antibodies described herein transformed into a ciliate can be maintained extrachromosomally on an autonomous plasmid. An expression vector maintained as an extrachromosomal element can be a ribosomal DNA-based vector containing an ori from ciliate (e.g., T.

thermophila) ribosomal DNA, which is known to support extrachromosomal replication. Such a vector can further comprise a 5' regulatory region from an endogenous ciliate gene containing a promoter region operably linked to the heterologous coding region and, optionally, a 3' regulatory region from the same or a different ciliate gene. For example, regulatory regions from ciliate genes in such vectors can include, but are not limited to, regulatory regions from genes such as HHFI, rpl29, BTU1, BTU2, SerFB, and actin.

[0080] For example, ciliates can be transformed with a ribosomal DNA vector (Tondravi and Yao (1986); Yu and Blackburn (1989)). The shuttle vector pXS76 allows insertion of transgenes downstream of a cadmium-inducible promoter from the MTT1 metallothionein gene of T. thermophila via homologous recombination and selection in paromomycin. Alternatively, inserts can be introduced into high copy number ribosomal DNA vectors (such as pD5H8) under control of the cadmium-inducible MTT1 promoter. The pD5H8 vector takes advantage of a biological feature of ciliates {e.g., T. thermophila) in which the ribosomal cistrons become amplified to extraordinarily high copy numbers following conjugation. A ribosomal DNA-based vector can be a circular vector that contains a 5' non-translated sequence comprising two or more ori sequences from Tetrahymena spp. ribosomal DNA. A nucleic acid fragment containing a heterologous coding region, for example a selectable marker or transgene, can also be added to the vector. The vector can further comprise a 5' untranslated region of a ciliate gene and a 3' untranslated region of a ciliate gene, inserted upstream and downstream of the selectable marker and/or the transgene. Methods for transformation, along with robust, inducible promoters for driving high-level gene expression have recently been described for this system (Bruns and Cassidy-Hanley (2000); Gaertig and Kapler (2000); Shang et al, (2002); Boldrin et al, (2006)).

[0081] Sequence variations within the origins of replication of ribosomal DNA from wild-type B- and C3- strains of T. thermophila convey a replicative advantage to the C3- form in B/C3 heterozygotes. Although both B- and C3- forms of ribosomal DNA are initially present in the macronucleus in approximately equal amounts, within 30 fissions only the C3 variant remains (Pan et al, (1982); Orias et al, (1988)). pIC19-based shuttle vectors containing the C3 origin of replication have been used as high-copy number vectors for the delivery of foreign DNA to Tetrahymena spp. (Yu and Blackburn (1989)).

[0082] Although such vectors can become unstable and be lost within about 50 to about 80 generations, micronuclear versions of the C3 ribosomal DNA is accurately processed (to form a palindrome) following introduction into T. thermophila B cell lines. The micronuclear version is maintained as a stable linear chromosome over many generations (Bruns et al, (1985)). Functional transgenes can be inserted into the 3'-nontranscribed spacer (3 '-NTS) of such vectors with no effect on ribosomal DNA processing. Within 6-10 generations, recombinant molecules can comprise 50-100% of the total ribosomal DNA complement, with as many as 18,000 copies of the transgene per cell (Blomberg et al, (1997)). The use of this approach enables an increase in the number of cloned genes in transformed cell lines by orders of magnitude and leads to increased expression at the protein level. For example, the use of ribosomal DNA-based vectors in combination with the MTT1 promoter can be used to drive expression a protein of interest (Lin et al, (2002)). Similarly, pD5H8 ribosomal DNA-based vectors (Blomberg et al., (1997)) can be used to boost expression of proteins by at least 3-10 fold compared with trans formants in which respective transgenes are integrated at somatic gene loci. Other vectors suitable for use with the methods described here include vectors comprising a ribosomal DNA sequence. Such vectors can replicate at high copy numbers and can be used to deliver a heterologous DNA sequence to Tetrahymena spp. for purposes of RNA expression.

[0083] Transformation.

[0084] A nucleic acid encoding the aglycosylated antibodies described herein can be introduced into ciliates using established protocols or any method known to one skilled in the art. Transformation of ciliates can be achieved by microinjection (Tondravi and Yao (1986)), electroporation (Gaertig and Gorovsky (1992)), or biolistically (Cassidy-Hanley et al., (1997)). As used herein, the term "transform" means to introduce into a cell an exogenous nucleic acid or nucleic acid analog which replicates within that cell, that encodes a polypeptide sequence which is expressed in that cell (with or without integration into the genome of the cell), and/or that is integrated into the genome of that cell so as to affect the expression of a genetic locus within the genome. Accordingly, "transform" is used to embrace all of the various methods of introducing such nucleic acids or nucleic acid analogs, including, but not limited to the methods referred to in the art as transformation, transfection, transduction, or gene transfer, and including techniques such as microinjection, DEAE- dextran-mediated endocytosis, calcium phosphate coprecipitation, electroporation, liposome- mediated transfection, ballistic injection, viral-mediated transfection, and the like.

[0085] Thus, in some embodiments, ciliate cells can be transformed with a nucleic acid encoding the aglycosylated antibodies described herein by particle bombardment (also known as biolistic transformation) (Cassidy-Hanley et al., (1997)). Particle bombardment transformation can be achieved by several ways. For example, inert or biologically active particles can be propelled at cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the chimeric gene. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Other variations of particle bombardment, now known or hereafter developed, can also be used. [0086] Microcarrier bombardment can also be used to transform ciliate cells by means of DNA-loaded gold particles (US Pat. No. 6,087,124; European Pat. EP 847 444; WO 1998/001572). In this approach, microcarrier bombardment with DNA-coated gold is used as a means of introducing foreign genes into ciliates. In some embodiments, microcarrier bombardment can be used to transform ciliates and introduce genes into the (germline) micronucleus

[0087] Methods for selection of transformed cells harboring a nucleic acid encoding a aglycosylated antibodies described herein are known in the art. For example, the vector can further comprise a selectable cassette marker to permit selection for transformed cells (e.g., a neo 2 cassette) (Gaertig et al., (1994)). Selection of transformants can be achieved by growing the cultured ciliates in a medium which allows only the transformants to survive. Suitable selection agents include antibiotics which will kill most all non- transformants but allow transformants (which also possess an antibiotic resistance gene) to survive. A number of antibiotic-resistance markers are known in the art. Any known antibiotic-resistance marker can be used to transform and select transformed host cells in accordance with the present invention. For example, selection of the transformants can be performed by means of a resistance marker such as a point mutation in the 17s ribosomal DNA, which confers resistance to paromomycin, can allow for selection of ribosomal DNA transformants (Spangler and Blackburn (1985); Bruns et al., (1985)). Other methods include the use of a mutant cell line that allows targeting of genes to the beta tubulin- 1 locus of T. thermophila by homologous recombination, and allows efficient selection of transformed cell lines by growth in the microtubule-stabilizing agent (taxol) (U.S. Pat. No. 6,846,481). Another method for selection of transformed cells harboring foreign genes is to insert full length coding regions into the pD5HA vector (Cowan et al., (2005)). In this method, transcription is driven by the inducible MTT1 promoter. Once cells have been transformed with the pD5HA vector selection of positive transformants is determined by paramomycin resistance (e.g., cell growth in media containing the drug). Presence of the transgene is then verified by PCR and then induced with cadmium chloride to over-express the recombinant gene product.

[0088] Many other selectable marker systems are known in the art. Selectable marker genes that confer resistance or tolerance to a normally toxic selection agent allow only successfully transfected cells to survive in the presence of the selection agent, and are referred to as positive selectable markers. Examples of positive selectable marker genes and their corresponding selection agents are: aminoglycoside phosphotransferase (APH) and G418; dihydrofolate reductase (DHFR) and methotrexate (Mtx); hygromycin-B- phosphotransferase (HPH) and hygromycin-B; xanthine-guanine phosphoribosyltransferase (XGPRT) and mycophenolic acid; and adenosine deaminase (ADA) and 9- -D-xylofuranosyl adenine (Xyl-A). In another example of a positive selectable marker system, thymidine kinase (TK) and aminopterin (included, e.g., in hypoxanthine-aminopterin-thymidine (HAT) medium) can be used in cells that are initially thymidine kinase deficient (tk^~). The aminopterin will normally kill tk^~ cells and, therefore, only successful TK transfectants will survive. Selectable marker genes that confer sensitivity or susceptibility to a normally nontoxic selection agent cause only successfully transfected cells to die in the presence of the selection agent, and are referred to as negative selectable markers. An example of a negative selectable marker system is thymidine kinase (TK) and gancyclovir. Phenotypic selectable marker genes permit selection based upon morphological or biochemical traits rather than cell death or survival. In some cases, the phenotypic marker is detectable only in the presence of an additional selection agent. An example of a phenotypic selectable marker system is β-galactosidase (lacZ) and X-gal.

[0089] Conditions Treatable with the Antibodies Described Herein

[0090] The antibodies described herein can be used to treat or prevent a condition or disease in subject (e.g., a human subject or a non-human animal subject) by administering the antibody which is at risk of, e.g., prone to having a disease, prior to the onset of the condition and so prevent or inhibit one or more symptoms of that condition. In certain embodiments, the antibodies described herein can be administered after clinical manifestation of a disease in a human or non-human animal to inhibit or treat the disease. In some embodiments, the condition or disease can be a autoimmune, immunological, infectious, inflammatory, neurological, or neoplastic disease, e.g., cancer. The antibodies described herein can also be use to treat a human or non-human animal having an infection, or to passively immunize a human or non-human animal form infection.

[0091] Other conditions that can be treated with the antibodies described herein include, but are not limited to, congestive heart failure (CHF), vasculitis, rosecea, acne, eczema, myocarditis and other conditions of the myocardium, systemic lupus erythematosus, diabetes, spondylopathies, synovial fibroblasts, and bone marrow stroma; bone loss; Paget's disease, osteoclastoma; multiple myeloma; breast cancer; disuse osteopenia; malnutrition, periodontal disease, Gaucher's disease, Langerhans' cell histiocytosis, spinal cord injury, acute septic arthritis, osteomalacia, Cushing's syndrome, monoostotic fibrous dysplasia, polyostotic fibrous dysplasia, periodontal reconstruction, and bone fractures; sarcoidosis; multiple myeloma; osteolytic bone cancers, breast cancer, lung cancer, kidney cancer and rectal cancer; bone metastasis, bone pain management, and humoral malignant

hypercalcemia, ankylosing spondylitisa and other spondyloarthropathies; transplantation rejection, viral infections, fungal infections, or bacterial infections. In some embodiments, the Fc variants of the present invention may be used to treat conditions including but not limited to hematologic neoplasias and neoplastic- like conditions for example, Hodgkin's lymphoma; non-Hodgkin's lymphomas (Burkitt's lymphoma, small lymphocytic lymphoma/chronic lymphocytic leukemia, mycosis fungoides, mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, hairy cell leukemia and

lymphoplasmacytic leukemia), tumors of lymphocyte precursor cells, including B-cell acute lymphoblastic leukemia/lymphoma, and T-cell acute lymphoblastic leukemia/lymphoma, thymoma, tumors of the mature T and NK cells, including peripheral T-cell leukemias, adult T-cell leukemia/T-cell lymphomas and large granular lymphocytic leukemia, Langerhans cell histocytosis, myeloid neoplasias such as acute myelogenous leukemias, including AML with maturation, AML without differentiation, acute promyelocyte leukemia, acute

myelomonocytic leukemia, and acute monocytic leukemias, myelodysplastic syndromes, and chronic myeloproliferative disorders, including chronic myelogenous leukemia, tumors of the central nervous system, e.g., brain tumors (glioma, neuroblastoma, astrocytoma,

medulloblastoma, ependymoma, and retinoblastoma), solid tumors (nasopharyngeal cancer, basal cell carcinoma, pancreatic cancer, cancer of the bile duct, Kaposi's sarcoma, testicular cancer, uterine, vaginal or cervical cancers, ovarian cancer, primary liver cancer or endometrial cancer, and tumors of the vascular system (angiosarcoma and

hemagiopericytoma), osteoporosis, hepatitis, HIV, AIDS, spondylarthritis, rheumatoid arthritis, inflammatory bowel diseases (IBD), sepsis and septic shock, Crohn's Disease, psoriasis, schleraderma, graft versus host disease (GVHD), allogenic islet graft rejection, hematologic malignancies, such as multiple myeloma (MM), myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML), inflammation associated with tumors, peripheral nerve injury or demyelinating diseases.

[0092] In certain embodiments, the antibodies described herein can be part of a formulation. In some embodiments, the concentration of the antibodies described herein in the formulation may vary from about 0.1 to 100 weight %. In some embodiments, the concentration of the antibody administered to a subject is a therapeutically effective amount alone or in a formulation. As used herein, the term therapeutically effective amount refers to an amount of the antibodies described herein which produces the effects for which it is administered. In certain embodiments, the therapeutically effective amount can also be dependent on the physiological condition of the subject or whether the purpose of the administration is therapeutic or prophylactic.

[0093] Delivery of the antibodies described herein, alone or in formulation, can be in the form of a sterile aqueous solution, a nebulizer, topical administration, spray or any other in a liposome vehicle or any other method of delivery known in the art. Administration of the antibodies described herein can be performed by any method known in the art, including, but not limited to oral administration, subcutaneous administration, intravenous administration, intranasal administration, intraotical administration, transdermal

administration, topical administration, intraperitoneal administration, intramuscular administration, intrapulmonary administration, by inhalation, vaginal administration, parenteral administration, rectal administration, or intraocular administration.

[0094] The following examples illustrate the present invention, and are set forth to aid in the understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter.

EXAMPLES

[0095] Example 1: Production of a murine Aglycosylated Monoclonal Antibody in Ciliates

[0096] The following example describes a method for producing of a murine aglycosylated anti-human transferrin IgGi monoclonal antibody in ciliates. This molecule is meant to be representative of immunoglobulins, and the methods described herein are applicable to the production of any aglycosylated immunoglobulin subtypes such as IgG2, IgG3 and IgG4 as well as the production of any aglycosylated immunoglobulin, including, but not limited to, antibodies having fully human sequences. Non-limiting examples of immunoglobulins that can be produced in an aglycosylated form according to the methods described herein are shown in Table 1.

[0097] Murine anti-human transferrin light and heavy chain genes were codon- optimized for expression in Tetrahymena with each predicted mature fragment fused in- frame to a 22 amino acid signal peptide derived from the immobilization antigen variant B protein of Ichthyophthirius multifiliis that directed each fragment to the constitutive secretion pathway (Figures 1 and 2). Chemically synthesized genes were then cloned into a mAb expression vector, as shown in Figure 1. The expression vector is designed to co-express light and heavy chains under the control of the tightly regulated powerful metallothionein promoters MTT5 and MTT1, respectively. The use of different, though equally effective, promoters that are induced with the same agent is a specific design consideration that minimizes recombination events that can lead to the loss of one of the antibody transgenes in Tetrahymena.

[0098] A Notl fragment containing the entire light and heavy chain gene expression cassette was then cloned into the same site of the ribosomal DNA vector pD5H8. Due to the formation and amplification of palindromic ribosomal DNA chromosomes (9000 copies per cell) during macronuclear development in Tetrahymena, ribosomal DNA vector- based expression can result in Tetrahymena strains harboring 18,000 copies of a transgene on recombinant ribosomal DNA chromosomes. The pD5H8 vector containing the dual expression construct was introduced into T thermophila strain CU428 by biolistic methods and transformants selected by growth in 1 X Neff media (2.5 g/L proteose peptone, 2.5 g/L yeast extract, 5 g/L dextrose and 33 μΜ FeCl₃) containing paromomycin. Drug-resistant transformants were grown in 1 X Neff media to a cell density of approximately 5xl0⁵ cells/ml and induced to express both light and heavy chain genes by the addition of 2 μg/ml CdCl₂. Twelve hours following induction, spent culture medium was harvested by centrifugation and secreted mAb purified by Protein A chromatography.

[0099] Figure 3A shows that the light and heavy chains resolve at the expected mass of approximately 25 (light chain) and 50 kDa (heavy chain) under reducing conditions, and that the subunits can form complex tetramers under non-reducing conditions.

Furthermore, full-length antibody is functional as determined by a human transferrin pulldown assay (Figure 3B). Mass spectrometry analysis of the purified light and heavy chain indicate that each has an apparent mass that is equal to the predicted mass of aglycosylated subunits(Figure 4). This results shows that the heavy chain is not modified by the addition of an N-Glycan. This is an unexpected result as Tetrahymena have been previously shown to efficiently modify glycoproteins with N-linked glycans that generally display minimal heterogeneity of a primitive core glycan ranging from Man₂GlcNAc₂ to MansGlcNAc₂ (Taniguchi et al, (1985); Weide et al, (2006)). [00100] Example 2: Production of a humanized Aglycosylated Monoclonal Antibody in Ciliates

[00101] The following example describes a method for producing of a humanized aglycosylated anti-human transferrin IgGi monoclonal antibody in T. thermophila. This molecule is meant to be representative of immunoglobulins, and the methods described herein are applicable to the production of any aglycosylated immunoglobulin subtypes such as IgG2, IgG3 and IgG4 as well as the production of any aglycosylated immunoglobulin, including, but not limited to antibodies having fully human sequences. Non-limiting examples of immunoglobulins that can be produced in an aglycosylated form according to the methods described herein are shown in Table 1.

[00102] The murine mAb light and heavy chains shown in Figure 2 were humanized following variable domain CDR grafting and replacement of light and heavy chain constant domains with those from human IgGi (Figure 5). As with the murine mAb, both light and heavy chain genes were directed to the secretory pathway via in-frame fusions with 22 amino acid signal peptide derived from the immobilization antigen variant B protein of Ichthyophthirius multifiliis. The light and heavy chain genes were chemically synthesized and cloned into a mAb expression vector and subsequently a Tetrahymena ribosomal DNA vector as described in Example 1. T. thermophila transformants were generated as described in Example 1. Drug-resistant transformants were grown in 1 X Neff media to a cell density of approximately 5xl0⁵ cells/ml and induced to express both light and heavy chain genes by the addition of 2 μg/ml CdCl₂. Twelve hours following induction, spent culture medium was harvested by centrifugation and secreted mAb purified by Protein A chromatography. Figure 6 shows that purified humanized anti-transferrin resolves as a complex of light and heavy chains under non-reducing conditions (Fig. 6A) and is functional as judged by an ability to specifically detect transferrin antigen but not a non-related protein (Fig. 6B). Analysis of humanized mAb glycosylation was carried out by Conconavalin A (ConA) western analysis. ConA is a lectin that specifically binds glycan moieties containing terminal mannose residues such as those known to decorate Tetrahymena glycoprotein's. Figure 6 (Fig. 6B) shows that a ConA western did not detect purified humanized IgG heavy chain indicating that the humanized mAb is not modified with an N-linked glycan.

[00103] Example 3: Production of a recombinant glycoprotein in Ciliates [00104] To demonstrate the propensity of ciliates to produce secreted recombinant protein modified with N- linked glycans the gene encoding the catalytic subunit of bovine enterokinase, a serine protease, was chemically synthesized and cloned into a Tetrahymena expression vector. The construct design included the same 22 amino acid signal peptide derived from the immobilization antigen variant B protein of Ichthyophthirius multifiliis used in mAb production to direct the recombinant protein to the Tetrahymena secretory pathway (Figure 7A). Transformed T. thermophila cell lines were generated as described in Examples 1 and 2 and recombinant enterokinase expressed following growth of cells to a density of approximately 5xl0⁵ cells/ml in 1 X Neff media and induction by the addition of 2 μg/ml CdCl₂. Twenty-four hours following induction, spent culture medium was harvested by centrifugation and secreted enterokinase purified by a combination of heparin and soybean trypsin inhibitor chromatography. Purified enterokinase was shown to be active on both fluorescently-labeled peptide and protein substrates (not shown).

[00105] Figure 7 (Fig. 7B) shows that enterokinase purified from Tetrahymena is a glycosylated protein as judged by PNGaseF treatment. Furthermore, mass analysis of the purified enzyme indicates that a majority of the purified protein has each of 3 potential N- linked glycan sites modified with an N-glycan and the remainder 2 of 3 sites occupied with N-glycans (Figure 8). Masses are based on analysis of N-linked glycans derived from recombinant enterokinase that were shown to exclusively contain Man3GlcNAc₂ structures with a mass of 910.327 dalton per glycan.

Example 4: Antibodies that can be produced using the methods of the invention

[00106] Antibodies that can be produced using the methods of the invention include aglycosylated versions of the antibodies listed in Table 1.

Table 1. Non-limiting examples of antibodies that can be produced according to the methods described herein

t iix iaii

hemoglobinuria

[00107] Exemplary chimeric antibody amino acid sequences suitable for use with the methods described herein, include, but are not limited to, the antibodies and antibody sequences listing Table 2.

Table 2: Exemplary chimeric antibody amino acid sequences suitable for use with the methods described herein

SQSITC VAHPASSTKVDKKIEPRPKSCDKTHTCPPCPAPE

LLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEV

KFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLH

QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY

TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQG VFSCSV

MHEALHNHYTQKSLSLSPGK

(SEQ ID NO: 8)

>1TXV:L ReoPro-like antibody Light Chain 1

DILMTQSPSSMSVSLGDTVSITCHASQGISSNIGWLQQKP

GKSFMGLIYYGTNLVDGVPSRFSGSGSGADYSLTISSLDS

EDFADYYCVQYAQLPYTFGGGTKLEIKRADAAPTVSIFPP

SSEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQNGV

LNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATH

KTSTSPIVKSFNRNEC

(SEQ ID NO: 9)

>1TXV:L ReoPro-like antibody Light Chain 2

DILMTQSPSSMSVSLGDTVSITCHASQGISSNIGWLQQKP

GKSFMGLIYYGTNLVDGVPSRFSGSGSGADYSLTISSLDS

EDFADYYCVQYAQLPYTFGGGTKLEIKRADAAPTVSIFPP

SSEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQNGV

LNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATH

KTSTSPIVKSFNRNEC

(SEQ ID NO: 10)

basilixima Chimeric IgGlK >1MIM:H Anti-CD25 antibody heavy CHIMERIC chain 1 b QLQQSGTVLARPGASVKMSCKASGYSFTRYWMHWIKQ

RPGQGLEWIGAIYPGNSDTSYNQKFEGKAKLTAVTSAST

AYMELSSLTHEDSAVYYCSRDYGYYFDFWGQGTTLTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV

S WNSGALT SGVHTFPAVLQS SGLYSLS S WTVP S S SLGTQ

TYICNVNHKPSNTKVDKRVEPPKSCDKTHTCPPCPAPELL

GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKF

NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQD

WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL

PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN

YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGK

(SEQ ID NO: 11) >1MIM:H Anti-CD25 antibody heavy CHIMERIC chain 2

QLQQSGTVLARPGASVKMSCKASGYSFTRYWMHWIKQ

RPGQGLEWIGAIYPGNSDTSYNQKFEGKAKLTAVTSAST

AYMELSSLTHEDSAVYYCSRDYGYYFDFWGQGTTLTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV

S WNSGALT SGVHTFPAVLQS SGLYSLS S WTVP S S SLGTQ

TYIC VNHKPSNTKVDKRVEPPKSCDKTHTCPPCPAPELL

GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKF

NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQD

WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL

PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE N

YKTTPPVLDSDGSFFLYSKLTVDKSRWQQG VFSCSVM

HEALHNHYTQKSLSLSPGK

(SEQ ID NO: 12)

>1MIM:L Anti-CD25 antibody light CHIMERIC chain 1

QIVSTQSPAIMSASPGEKVTMTCSASSSRSYMQWYQQKP

GTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEA

EDAATYYCHQRSSYTFGGGTKLEIKRTVAAPSVFIFPPSD

EQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQ

ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGE

(SEQ ID NO: 13)

>1MIM:L Anti-CD25 antibody light CHIMERIC chain 2

QIVSTQSPAIMSASPGEKVTMTCSASSSRSYMQWYQQKP

GTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEA

EDAATYYCHQRSSYTFGGGTKLEIKRTVAAPSVFIFPPSD

EQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQ

ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

GLSSPVTKSFNRGE

(SEQ ID NO: 14) cetuximab Chimeric IgGlK >Anti-EGFR heavy chain 1

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQ

SPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQV

FFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVT

VS AASTKGPS VFPLAP S SKST SGGTAALGCLVKDYFPEPV

T VS WNSGALT SGVHTFPAVLQS SGLYSLS S WTVPS S SLG

TQTYIC VNHKPSNTKVDKRVEPKSPKSCDKTHTCPPCP

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHED

PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTV

LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ

VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ

PE YKTTPPVLDSDGSFFLYSKLTVDKSRWQQG VFSC

SVMHEALHNHYTQKSLSLSPGK

(SEQ ID NO: 15)

>Anti-EGFR heavy chain 2

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQ

SPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQV

FFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVT

VS AASTKGPS VFPLAP S SKST SGGTAALGCLVKDYFPEPV

TVS WNSGALT SGVHTFPAVLQS SGLYSLS S WTVPS S SLG

TQTYICNVNHKPSNTKVDKRVEPKSPKSCDKTHTCPPCP

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHED

PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTV

LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ

VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ

PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC

SVMHEALHNHYTQKSLSLSPGK

(SEQ ID NO: 16)

>Anti-EGFR light chain 1

DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTN

GSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIA

DYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDE

QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE

SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG

LSSPVTKSFNRGA

(SEQ ID NO: 17)

>Anti-EGFR light chain 2 DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTN

GSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIA

DYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDE

QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE

SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG

LSSPVTKSFNRGA

(SEQ ID NO: 18) rituximab Chimeric IgGlK >Mouse-Human chimeric Anti-CD20 Heavy Chain 1

QAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWV

KQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSS

STAYMQLSSLTSEDSAVYFCARWYYSNSYWYFDVWGT

GTTVTVSGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV

T VS WNSGALT SGVHTFPAVLQS SGLYSLS S WTVPS S SLG

TQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPE

LLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEV

KFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLH

QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY

TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN

NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV

MHEALHNHYTQKSLSLSPGK

(SEQ ID NO: 19)

>Mouse-Human chimeric Anti-CD20 Heavy Chain 2

QAYLQQSGAELVRPGASVKMSCKASGYTFTSYNMHWV

KQTPRQGLEWIGAIYPGNGDTSYNQKFKGKATLTVDKSS

STAYMQLSSLTSEDSAVYFCARWYYSNSYWYFDVWGT

GTTVTVSGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV

TVS WNSGALT SGVHTFPAVLQS SGLYSLS S WTVPS S SLG

TQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPE

LLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEV

KFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLH

QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY

TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN

NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV

MHEALHNHYTQKSLSLSPGK

(SEQ ID NO: 20)

>Mouse-Human chimeric Anti-CD20 Light Chain 1

QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKP GSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAE

DAATYYCQQWSFNPPTFGAGTKLELKRTVAAPSVFIFPPS

DEQLKSGTASWCLL NFYPREAKVQWKVDNALQSGNS

QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH

QGLSSPVTKSFNR

(SEQ ID NO: 21)

>Mouse-Human chimeric Anti-CD20 Light Chain 2

QIVLSQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKP

GSSPKPWIYAPSNLASGVPARFSGSGSGTSYSLTISRVEAE

DAATYYCQQWSFNPPTFGAGTKLELKRTVAAPSVFIFPPS

DEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNS

QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH

QGLSSPVTKSFNR

(SEQ ID NO: 22)

[00108] Exemplary humanized antibody amino acid sequences suitable for use with the methods described herein, include, but are not limited to, the antibodies and antibody sequences listing Table 3.

Table 3 : Exemplary humanized antibody amino acid sequences suitable for use with the methods described herein

>lcz8_H|Fab-12 variant Y0317|||VH-CH1 (VH(1-

[00109] Exemplary human antibody amino acid sequences suitable for use with the methods described herein, include, but are not limited to, the antibodies and antibody sequences listing Table 4.

Table 4: Exemplary human antibody amino acid sequences suitable for use with the methods described herein

[00110] Exemplary antibodies that can be produced using the methods described herein include but are not limited to, antibodies that specifically bind to one or more cytokines, inflammatory molecules, growth factors, their receptors, and oncogene products or portions thereof. Examples of cytokines, inflammatory molecules, growth factors, their receptors, and oncogene products include, but are not limited to, e.g., alpha- 1 antitrypsin, Angiostatin, Antihemolytic factor, antibodies (including an antibody or a functional fragment or derivative thereof selected from: Fab, Fab', F(ab)2, Fd, Fv, ScFv, diabody, tribody, tetrabody, dimer, trimer or minibody), angiogenic molecules, angiostatic molecules,

Apolipopolypeptide, Apopolypeptide, Asparaginase, Adenosine deaminase, Atrial natriuretic factor, Atrial natriuretic polypeptide, Atrial peptides, Angiotensin family members, Bone Morphogenic Polypeptide (BMP-1 , BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP- 8a, BMP-8b, BMP-10, BMP- 15, etc.); C-X-C chemokines (e.g., T39765, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP- 10, GCP-2, NAP-4, SDF-1 , PF4, MIG), Calcitonin, CC chemokines (e.g., Monocyte chemoattractant polypeptide- 1 , Monocyte chemoattractant polypeptide-2, Monocyte chemoattractant polypeptide-3, Monocyte inflammatory polypeptide- 1 alpha, Monocyte inflammatory polypeptide- 1 beta, RANTES, 1309, R83915, R91733, HCC1 , T58847, D31065, T64262), CD40 ligand, C-kit Ligand, Ciliary Neurotrophic Factor, Collagen, Colony stimulating factor (CSF), Complement factor 5a, Complement inhibitor, Complement receptor 1 , cytokines, (e.g., epithelial Neutrophil Activating Peptide-78, GRO alpha/MGSA, GRO beta , GRO gamma , MIP-1 alpha , MIP-1 delta, MCP-1),

deoxyribonucleic acids, Epidermal Growth Factor (EGF), Erythropoietin ("EPO", representing a preferred target for modification by the incorporation of one or more non- natural amino acid), Exfoliating toxins A and B, Factor IX, Factor VII, Factor VIII, Factor X, Fibroblast Growth Factor (FGF), Fibrinogen, Fibronectin, G-CSF, GM-CSF,

Glucocerebrosidase, Gonadotropin, growth factors, Hedgehog polypeptides (e.g., Sonic, Indian, Desert), Hemoglobin, Hepatocyte Growth Factor (HGF), Hepatitis viruses, Hirudin, Human serum albumin, Hyalurin-CD44, Insulin, Insulin-like Growth Factor (IGF-I, IGF-II), interferons (e.g., interferon-alpha, interferon-beta, interferon-gamma, interferon-epsilon, interferon-zeta, interferon-eta, interferon-kappa, interferon-lambda, interferon-T, interferon- zeta, interferon-omega), glucagon- like peptide (GLP-1), GLP-2, GLP receptors, glucagon, other agonists of the GLP-1R, natriuretic peptides (ANP, BNP, and CNP), Fuzeon and other inhibitors of HIV fusion, Hurudin and related anticoagulant peptides, Prokineticins and related agonists including analogs of black mamba snake venom, TRAIL, RANK ligand and its antagonists, calcitonin, amylin and other glucoregulatory peptide hormones, and Fc fragments, exendins (including exendin-4), exendin receptors, interleukins (e.g., IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1 , IL-12, etc.), I-CAM-l/LFA-1 ,

Keratinocyte Growth Factor (KGF), Lactoferrin, leukemia inhibitory factor, Luciferase, Neurturin, Neutrophil inhibitory factor (NIF), oncostatin M, Osteogenic polypeptide, Parathyroid hormone, PD-ECSF, PDGF, peptide hormones (e.g., Human Growth Hormone), Oncogene products (Mos, Rel, Ras, Raf, Met, etc.), Pleiotropin, Polypeptide A, Polypeptide G, Pyrogenic exotoxins A, B, and C, Relaxin, Renin, ribonucleic acids, SCF/c-kit, Signal transcriptional activators and suppressors (p53, Tat, Fos, Myc, Jun, Myb, etc.), Soluble complement receptor 1, Soluble I-CAM 1, Soluble interleukin receptors (IL-1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15), soluble adhesion molecules, Soluble TNF receptor, Somatomedin, Somatostatin, Somatotropin, Streptokinase, Superantigens, e.g., Staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SEC3, SED, SEE), Steroid hormone receptors (such as those for estrogen, progesterone, testosterone, aldosterone, LDL receptor ligand and corticosterone), Superoxide dismutase (SOD), Toll-like receptors (such as Flagellin), Toxic shock syndrome toxin (TSST-1), Thymosin a 1, Tissue plasminogen activator, transforming growth factor (TGF- alpha, TGF- beta), Tumor necrosis factor beta (TNF beta), Tumor necrosis factor receptor (TNFR), Tumor necrosis factor- alpha (TNF alpha), transcriptional modulators (for example, genes and transcriptional modular polypeptides that regulate cell growth, differentiation and/or cell regulation), Vascular Endothelial Growth Factor (VEGF), viruslike particle, VLA-4/VCAM-1, Urokinase, signal transduction molecules, estrogen, progesterone, testosterone, aldosterone, LDL, corticosterone.

[00111] Further exemplary antibodies that can be produced using the methods described herein include but are not limited to, antibodies that specifically bind to one or more enzymes {e.g., industrial enzymes) or portions thereof. Examples of enzymes include, but are not limited to, amidases, amino acid racemases, acylases, dehalogenases,

dioxygenases, diarylpropane peroxidases, epimerases, epoxide hydrolases, esterases, isomerases, kinases, glucose isomerases, glycosidases, glycosyl transferases,

haloperoxidases, monooxygenases {e.g., p450s), lipases, lignin peroxidases, nitrile

hydratases, nitrilases, proteases, phosphatases, subtilisins, transaminase, and nucleases.

[00112] Further exemplary antibodies that can be produced using the methods described herein include but are not limited to, antibodies that specifically bind agriculturally related polypeptides such as insect resistance polypeptides {e.g., Cry polypeptides), starch and lipid production enzymes, plant and insect toxins, toxin-resistance polypeptides,

Mycotoxin detoxification polypeptides, plant growth enzymes {e.g., Ribulose 1,5- Bisphosphate Carboxylase/Oxygenase), lipoxygenase, and Phosphoenolpyruvate carboxylase.

[00113] Further exemplary antibodies that can be produced using the methods described herein include but are not limited to, antibodies that specifically bind antibodies, immunoglobulin domains of antibodies and their fragments. Examples of antibodies include, but are not limited to, antibodies, antibody fragments, antibody derivatives, Fab fragments, Fab' fragments, F(ab)2 fragments, Fd fragments, Fv fragments, single-chain Fv fragments (scFv), diabodies, tribodies, tetrabodies, dimers, trimers, and minibodies.

[00114] Further exemplary antibodies that can be produced using the methods described herein include but are not limited to, antibodies that bind to prophylactic vaccines or therapeutic vaccines. Examples of prophylactic vaccines or a therapeutic vaccines include, but are not limited to, polypeptides, polypeptide fragments, or carbohydrate antigens from infectious fungi (e.g., Aspergillus, Candida species) bacteria (e.g., E. coli, Staphylococci aureus)), or Streptococci (e.g. , pneumoniae); protozoa such as sporozoa (e.g., Plasmodia), rhizopods (e.g., Entamoeba) and flagellates (Trypanosoma, Leishmania, Trichomonas, Giardia, etc.); viruses such as (+) RNA viruses (examples include Poxviruses e.g., vaccinia; Picornaviruses, e.g., polio; Togaviruses, e.g., rubella; Flaviviruses, e.g., HCV; and

Coronaviruses), (-) RNA viruses (e.g., Rhabdoviruses, e.g., VSV; Paramyxovimses, e.g., RSV; Orthomyxovimses, e.g., influenza; Bunyaviruses; and Arenaviruses), dsDNA viruses (Reoviruses, for example), RNA to DNA viruses, e.g., Retroviruses, e.g., HIV and HTLV, and certain DNA to RNA viruses such as Hepatitis B

[00115] In yet another aspect, the methods described herein can used to generate one or more antibodies useful for passively immunizing a subject against a virus, the method comprising administering to the subject an effective amount of an aglycosylated peptide produced in a ciliate.

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Claims

WHAT IS CLAIMED IS:

A method for producing an aglycosylated antibody in a ciliate, the method comprising: a) transforming the ciliate with a nucleic acid construct encoding the antibody, b) culturing the ciliate and expressing the antibody, and c) isolating the antibody.

The method of claim 1 , wherein the nucleic acid construct is a vector, a plasmid, a cosmid, a chromosome or minichromosome, a transposon, a ribosomal DNA or any combination thereof.

The method of claim 1 , wherein the ciliate is Tetrahymena thermophila or

Tetrahymena pyriformis.

The method of claim 1 , wherein the nucleic acid construct encoding the antibody, further comprises a nucleic acid sequence encoding a signal peptide such that translation of the nucleic acid construct results in the production of a polypeptide comprising a signal peptide operably linked to the antibody.

The method of claim 4, wherein the signal peptide is an Ichthyophthirius multifiliis signal peptide.

The method of claim 4, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 6.

The method of claim 1 , wherein the antibody, comprises an antibody light chain sequence.

The method of claim 1 , wherein the antibody, comprises an antibody heavy chain sequence.

9. The method of claim 1, wherein the antibody, comprises an antibody light chain sequence, and wherein the heavy chain sequence comprises an asparagine-linked glycan consensus site.

10. The method of claim 1, wherein the antibody, is a human antibody.

11. The method of claim 1 , wherein the antibody, is a mouse antibody

12. The method of claim 1, wherein the antibody, is a chimeric antibody.

13. The method of claim 1, wherein the antibody, is a humanized antibody.

14. The method of claim 1, wherein the antibody is an IgG antibody.

15. The method of claim 1, wherein the antibody is an IgM, IgD, IgE, or IgA antibody.

16. The method of claim 1, wherein the antibody is a tetrameric antibody.

17. The method of claim 16, wherein the tetrameric antibody comprises two light chains and two heavy chains.

18. The method of claim 1, wherein the antibody is a multispecific antibody.

19. The method of claim 18, wherein the multispecific antibody is a bispecific antibody.

20. The method of claim 1, wherein the antibody, comprises an amino acid sequence consisting essentially of SEQ ID NO: 1.

21. The method of claim 1, wherein the antibody, comprises an amino acid sequence consisting essentially of SEQ ID NO: 2.

22. The method of claim 1, wherein the antibody, comprises an amino acid sequence consisting essentially of SEQ ID NO: 3.

23. The method of claim 1, wherein the antibody, comprises an amino acid sequence consisting essentially of SEQ ID NO: 4.

24. The method of claim 1, wherein the antibody, comprises an amino acid sequence of any of SEQ ID NOs: 8-129.

25. The method of claim 1, wherein the antibody, is a therapeutic antibody.

26. The method of claim 1, wherein the antibody, specifically binds a cytokine, an

inflammatory molecule, a growth factor, a growth factor receptor, an oncogene, an agriculturally related polypeptide, antibody, a prophylactic vaccine, a therapeutic vaccine, or any combination thereof.

27. An aglycosylated antibody produced by the method of claim 1.

28. A ciliate cell capable of producing an aglycosylated antibody.

29. The ciliate of claim 28, wherein the ciliate has been genetically-engineered to express an aglycosylated antibody.

30. The ciliate of claim 28, wherein the antibody comprising an asparagine-linked glycan consensus site.