WO2015091130A1

WO2015091130A1 - Method for improving the recombinant production of soluble fusion polypeptides

Info

Publication number: WO2015091130A1
Application number: PCT/EP2014/077137
Authority: WO
Inventors: Erhard Kopetzki; Sebastian Neumann; Peter Maier; Peter Michael HUELSMANN; Stefan Lorenz
Original assignee: F. Hoffmann-La Roche Ag; Hoffmann-La Roche Inc.
Priority date: 2013-12-20
Filing date: 2014-12-10
Publication date: 2015-06-25

Abstract

Herein is reported a method for recombinantly producing a polypeptide in soluble form comprising the steps of transfecting a eukaryotic cell with a nucleic acid encoding the polypeptide, whereby the polypeptide has been modified (compared to the wild-type polypeptide) by the introduction of one or more artificial glycosylation sites, cultivating the transfected cell in a cultivation medium, and recovering the polypeptide from the cultivation medium, whereby the yield (determined after one purification step) of monomeric polypeptide is increased by at least 100 % compared to the wild-type polypeptide.

Description

Method for improving the recombinant production of soluble fusion

polypeptides

The current invention is in the field of recombinant polypeptide production. Herein is reported a method for improving the recombinant production of a fusion polypeptide in soluble form whereby the improvement is achieved by the introduction of one or more artificial glycosylation sites, e.g. in the peptidic linker, in the fusion polypeptide.

Background of the Invention

For the development of vaccines, e.g. directed against viral or bacterial pathogens, or for the induction of tolerance, e.g. against non-pathogenic organisms or for the prevention/reduction of allergic reactions or auto-immune diseases, the selection of the proper antigenic polypeptide and the presentation of the antigenic polypeptide is of importance.

In recent years the use of so called vehicles, such as e.g. antibodies, for targeted delivery is constantly increasing, e.g. for delivering antigenic polypeptides to antigen-presenting cells (APCs) especially dendritic cells (DCs). For this purpose fusion polypeptides comprising a targeting entity, such as e.g. an antibody, and an antigenic polypeptide entity are required.

For generating an efficient cellular immunicity directed against pathogens often the concomittant action of multiple T-cell epitope containing antigenic polypeptides is required. These antigenic polypeptides are normally derived from highly conserved regions of the pathogen, such as e.g. the gag, pol, env and nef proteins of the human immunodeficiency virus (HIV).

The genetic conjugation of an artificial multi-antigenic polypeptide entity that comprises a multitude of (different) antigenic polypeptides to an antibody chain (N- and/or C-terminal) often results in drawbacks during the recombinant production, such as an impaired solubility and the formation of aggregates. In addition to that, low expression levels are sometimes observed. This might be due to the fact that the antigenic polypeptides are deprived from their natural surrounding increasing non-specific interactions. It has been tried to counteract these non-specific interactions by using hydrophilic peptidic linkers, such as e.g. GS-linkers to connect the individual antigenic polypeptides but without much success.

By using bacteria-derived N- and/or O-glycosylated natural peptidic linkers the solubility of antibody-antigenic polypeptide fusion polypeptides can be improved and the non-specific interactions resulting in the formation of aggregates and precipitates can be reduced. These fusion polypeptides generally are stablized in solution by using high concentrations of chaotropic agents, such as e.g. arginine, guanidinium hydrochloride, urea, thiourea, butanole, and ethanole. Additionally the use of non-human bacteria-derived peptidic linkers results in the risk of inducing an immunogenic response against those linkers, which is not desireable. Further due to a non-homogenic glycosylation of the glycosylation sites in the bacteria-derived peptidic linkers, especially of the O-glycosylation sites, a very heterogeneous product is formed. This may present a major hurdle with respect to chemistry, manufacturing, and control (CMC) requirements to be met by a therapeutic polypeptide, as a high degree of heterogeneous glycosylation together with the inability to remove the glycosylation in a quantitative manner, may lead to a situation, which does not allow analysis of the protein backbone identity and integrity and production of the active pharmaceutical ingredient (API) in consistent.

Summary of the Invention

It has been found that by the introduction of additional glycosylation sites the solubility of a recombinantly produced polypeptide can be increased. This is suitable for the recombinant production of polypeptides that tend to form soluble and insoluble aggregates.

It has further been found that by using/introducing of short glycine-serine-based peptidic linkers comprising defined N-glycosylation sites for conjugating of polypeptides, such as e.g. antigenic polypeptides to antibody polypeptide chains, the expression, purification and solubility of the fusion polypeptides could be significantly improved.

A lower product complexity was observed and also the accessability by standard analytical methods was improved. Without being bound by this theory this improvement might be based on the fact that deglycosylation of N-glycans can be done in a more quantitative manner (in contrast to O-glycans).

One aspect as reported herein is a method for recombinantly producing a polypeptide in soluble form comprising the following steps: a) transfecting a eukaryotic cell with a nucleic acid encoding the polypeptide, whereby the polypeptide has been modified (compared to the wild-type polypeptide) by the introduction of one or more artificial glycosylation sites, b) cultivating the transfected cell in a cultivation medium, and c) recovering the polypeptide from the cultivation medium, whereby the yield (determined after one purification step) is increased by at least 100 % compared to the wild-type polypeptide.

In one embodiment the glycosylation site is an N-glycosylation site.

In one embodiment the yield is the yield of monomeric polypeptide.

In one embodiment the polypeptide is a fusion polypeptide comprising (at least) a first part and a second part conjugated to each other by a peptidic linker.

In one embodiment the first part and the second part of the fusion polypeptide are originally (before being fused together) not parts of the same polypeptide, i.e. they do not originate/are not derived from the same polypeptide.

In one embodiment the first part is a targeting entity and the second part is a biologically active entity.

In one embodiment the first part is an antibody or antibody fragment and the second part is a biologically active entity.

In one embodiment the first part is an antibody or antibody fragment and the second part is an (multi-)antigenic polypeptide.

In one embodiment the glycosylation site is in a peptidic linker.

In one preferred embodiment each peptidic linker comprises one N-glycosylation site. In one embodiment the glycosylation site is selected from the group comprising NGT (SEQ ID NO: 122), NWT (SEQ ID NO: 123), NST (SEQ ID NO: 124) and NDT (SEQ ID NO: 125).

In one preferred embodiment the peptidic linker is selected from the group comprising GGGSGGNGTGGGGSGG (SEQ ID NO: 26), GGGGSGGNGTGGGGSGG (SEQ ID NO: 27), GGSGGGNWTG (SEQ ID NO: 28), GGSGGGGSGGGNSTG (SEQ ID NO: 29), GGGGSGGGNSTGG (SEQ ID NO: 30), GGSGGGNDTG (SEQ ID NO: 31), and GGSGGGNGTG (SEQ ID NO: 32). In one embodiment the polypeptide is recovered from the cleard cultivation supernatant after cell separation (e.g. using centrifugation at 1000 x g for 3 min.) and filtration.

In one embodiment the yield is increased by at least 250 %.

In one embodiment the artificial glycosylation site has been introduced by making one, or two or three sequential amino acid changes (mutations) into the amino acid sequence of the polypeptide (to generate an artificial glycosylation site sequon), or by fusing a peptidic linker containing a glycosylation site to the polypeptide.

In one embodiment the fusion polypeptide is a fusion polypeptide comprising three or more entities, whereby each entity is fused at most at each terminus to a further entity by a peptidic linker, whereby in each of the peptidic linkers an artificial glycosylation site (an artificial glycosylation site sequon) is introduced.

One aspect as reported herein is the use of one or more artificial glycosylation sites for the increase of the yield of recombinantly produced soluble polypeptide.

In one embodiment the yield is of recombinantly produced soluble monomeric polypeptide.

Detailed Description of the Invention

DEFINITIONS:

The term "amino acid" denotes the group of carboxy a-amino acids, which directly or in form of a precursor can be encoded by a nucleic acid. The individual amino acids are encoded by nucleic acids consisting of three nucleotides, so called codons or base-triplets. Each amino acid is encoded by at least one codon. This is known as "degeneration of the genetic code". The term "amino acid" as used within this application denotes the naturally occurring carboxy a-amino acids comprising alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gin, Q), glutamic acid

(glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V). Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) aromatic: Trp, Tyr, Phe.

The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')₂; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.

The term "artificial glycosylation site" denotes a glycosylation site that is normally not present in a polypeptide and which has been introduced into the polypeptide by using molecular biological methods for the modification of the encoding nucleic acid. An artificial glycosylation site can be introduced into a polypeptide, for example, i) by making one, or two or three sequential amino acid changes

(mutations) into the amino acid sequence of the polypeptide to generate an artificial glycosylation site (an artificial glycosylation site sequon), or ii) if the polypeptide is a fusion polypeptide comprising at least two entities fused together by a peptidic linker by introducing an artificial glycosylation site (an artificial glycosylation site sequon) into the peptidic linker (or in each of the peptidic linkers if the fusion polypeptide comprises three or more entities each fused together by a peptidic linker), or iii) by fusing a glycosylation-tag to the polypeptide.

The term„biologically active entity" as used herein refers to an organic molecule, e.g. a biological macromolecule such as a peptide, protein, glycoprotein, nucleoprotein, mucoprotein, lipoprotein, synthetic polypeptide or protein, that causes a biological effect when administered in or to artificial biological systems, such as bioassays using cell lines and viruses, or in vivo to an animal, including but not limited to birds or mammals, including humans. This biological effect can be but is not limited to enzyme inhibition or activation, binding to a receptor or a ligand, either at the binding site or circumferential, signal triggering or signal modulation. Biologically active entities are without limitation for example immunoglobulins, or hormones, or cytokines, or growth factors, or receptor ligands, or agonists or antagonists, or cytotoxic agents, or antiviral agents, or imaging agents, or enzyme inhibitors, enzyme activators or enzyme activity modulators such as allosteric substances, or (multi-)antigenic polypeptides.

The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG₂, IgG₃, IgG₄, IgA_ls and IgA₂. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, δ, ε, γ, and μ, respectively.

A "complete antibody heavy chain" comprises a variable domain (variable region) (generally the amino terminal portion), which comprises binding regions that are able to interact with an antigen, and a constant region (generally the carboxyl terminal portion). The constant region of the heavy chain mediates the binding of the antibody i) to cells bearing a Fc gamma receptor (FcyR), such as phagocytic cells, or ii) to cells bearing the neonatal Fc receptor (FcRn) also known as Brambell receptor. It also mediates the binding to some factors including factors of the classical complement system such as component (Clq). Depending on the amino acid sequence of the constant region of the heavy chains, antibodies (immunoglobulins) are divided in the classes: IgA, IgD, IgE, IgG, and IgM. Some of these classes are further divided into subclasses (isotypes), i.e. IgG in IgGl, IgG2, IgG3, and IgG4, or IgA in IgAl and IgA2. An "expression cassette" refers to a nucleic acid construct that contains the necessary regulatory elements, such as promoter and polyadenylation site, for expression of at least the contained encoding nucleic acid in a cell. An "expression vector" is a nucleic acid providing all required elements for the expression of the contained expression cassette(s) in a (host) cell. Typically, an expression vector comprises a prokaryotic plasmid propagation unit, e.g. for E. coli, comprising an origin of replication, and a selectable marker, an eukaryotic selection marker, and one or more expression cassettes for the expression of the structural gene(s) of interest each comprising a promoter, a structural gene, and a transcription terminator including a polyadenylation signal. Gene expression is usually placed under the control of a promoter, and such a structural gene is said to be "operably linked to" the promoter. Similarly, a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter. The term "Fc-region" herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc-regions and variant Fc-regions. In one embodiment, a human IgG heavy chain Fc-region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc-region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc-region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat, E.A. et al, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91 -3242.

In one embodiment the antibody has an Fc-region of the human subclass IgG4 or of the human subclass IgGl, IgG2, or IgG3. In one embodiment the Fc-region is of a human antibody of the subclass IgGl with mutations L234A and L235A (numbering according to Kabat (see e.g. Kabat, E.A., et al, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of

Health, Bethesda, MD (1991), NIH Publication 91-3242, Vols. 1-3). In another embodiment the Fc-region is of a human antibody of the subclass IgGl with mutations L234A, L235A, and P329G (numbering according to Kabat). In one embodiment the Fc-region is of a human antibody of the subclass IgG4 with mutations S228P and L235E. While IgG4 shows reduced Fey receptor (FcyRIIIa) binding, antibodies of other IgG subclasses show strong binding. However Pro238, Asp265, Asp270, Asn297 (loss of Fc-region carbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, or/and His435 are residues which, if altered, provide also reduced Fey receptor binding (see e.g. Shields, R.L., et al, J. Biol. Chem. 276 (2001) 6591-6604; Lund, J., et al, FASEB J. 9 (1995) 115-119; Morgan, A., et al, Immunology 86 (1995)

319-324; EP 0 307 434).

"Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity

(ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation. In one embodiment the antibody of the fusion polypeptide has no effector function (is deprived of Fc-region effector function).

The terms "full length antibody", "intact antibody", and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc- region as defined herein.

The term "glycosylated" or grammatical equivalents thereof denote that the respective polypeptide comprises at least one saccharide residue covalently linked to an amino acid of the amino acid backbone of the polypeptide. The term "N- glycosylation site" (sequon) denotes an N-glycosylation site amino acid motifs comprising the amino acid sequence asn-X-thr, asn-X-ser, or asn-X-cys, wherein X can be any amino acid residues but not proline (pro, P).

The term„HIV-derived antigenic polypeptide" denotes a polypeptide that is a fragment of a HIV polypeptide gag, env or pol.

The high HIV-1 mutation rates hamper the identification of an efficient vaccination strategy. Since 1994, the French National Agency for AIDS Research (ANRS) has studied conserved regions of HIV-1 for use as antigens in immunotherapeutic vaccine clinical trials. Five immunogenic peptides were identified for inclusion in the ANRS HIV-LIPO-5 vaccine. The vaccine is comprised of five peptides, each covalently linked at their C-terminal ends to a palmitoyl-lysylamide moiety: Gag (17-35); Gag (253-284); Nef (66-97); Nef (116-145); Pol (325-355). In clinical testing it was found that doses of 50, 150 and 500 μg were able to elicit HlV-specific sustained CD 8+ and CD4+ T-cell responses in healthy adults and several boosts of low doses of HIV-LIPO-5 vaccine could be safely administered.

See e.g. Cobb, A., et al, J. Immunol. Methods. 365 (2011) 27-37; Salmon-Ceron, D., et al, AIDS 24 (2010) 2211-2223; WO 2000/075181; WO 2010/104761;

WO 2008/097866; US 61/332,465; WO 2010/104748; WO 2010/104747; PCT/US2011/22147; US 61/450480; WO 2010/009346; US 61/466,292.

The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

An "isolated" antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS- PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman, S. et al, J. Chromatogr. B 848 (2007) 79-87. An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

The term "monomeric" denotes a form of a polypeptide that is not associated with other polypeptide molecules resulting in non-naturally occurrig assemblies. The term includes homodimers, homotrimers and higher order homooligomers of the polypeptide if this form is the naturally occurring form of the polypeptide. The term also includes homodimers of asymmetric molecules, e.g. of asymmetric Fc- region fusion polypeptides. In this case the asymmetric Fc-region fusion polypeptide is treated as single polypeptide, although a homodimer of two asymmetric Fc-region fusion polypeptides essentially comprises two not fused Fc- region heavy chain polypeptides and two fused Fc-region heavy chain polypeptides. An asymmetric Fc-region fusion polypeptide is treated according to the definition used herein as homodimer. The term "nucleic acid" denotes a polymeric molecule consisting of the individual nucleotides (also called bases) a, c, g, and t (or u in RNA), for example, DNA or R A or modifications thereof. This polynucleotide molecule can be a naturally occurring polynucleotide molecule or a synthetic polynucleotide molecule or a combination of one or more naturally occurring polynucleotide molecules with one or more synthetic polynucleotide molecules. Also encompassed by this definition are naturally occurring polynucleotide molecules in which one or more nucleotides are changed (e.g. by mutagenesis), deleted, or added. A nucleic acid can either be isolated, or integrated in another nucleic acid, e.g. in an expression cassette, a plasmid, or the chromosome of a host cell. A nucleic acid is likewise characterized by its nucleic acid sequence consisting of individual nucleotides.

To a person skilled in the art procedures and methods are well known to convert an amino acid sequence, e.g. of a polypeptide, into a corresponding nucleic acid sequence encoding this amino acid sequence. Therefore, a nucleic acid is characterized by its nucleic acid sequence consisting of individual nucleotides and likewise by the amino acid sequence of a polypeptide encoded thereby.

"Operably linked" refers to a juxtaposition of two or more components, wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a promoter and/or enhancer are operably linked to a coding sequence, if it acts in cis to control or modulate the transcription of the linked sequence. Generally, but not necessarily, the DNA sequences that are "operably linked" are contiguous and, where necessary to join two protein encoding regions such as a secretory leader and a polypeptide, contiguous and in (reading) frame. The term "peptide linker" denotes amino acid sequences of natural and/or synthetic origin. They consist of a linear amino acid chain wherein the 20 naturally occurring amino acids are the monomeric building blocks. The peptide linker has a length of from 1 to 50 amino acids, in one embodiment between 1 and 28 amino acids, in a further embodiment between 2 and 25 amino acids. The peptide linker may contain repetitive amino acid sequences or sequences of naturally occurring polypeptides.

In one embodiment the peptide linker is rich in glycine, glutamine, and/or serine residues. These residues are arranged e.g. in small repetitive units of up to five amino acids, such as GS (SEQ ID NO: 21), GGS (SEQ ID NO: 22), GGGS (SEQ ID NO: 23), and GGGGS (SEQ ID NO: 24). This small repetitive unit may be repeated for one to five times. At the amino- and/or carboxy-terminal ends of the multimeric unit up to six additional arbitrary, naturally occurring amino acids may be added. Other synthetic peptidic linkers are composed of a single amino acid, which is repeated between 10 to 20 times and may comprise at the amino- and/or carboxy-terminal end up to six additional arbitrary, naturally occurring amino acids. All peptidic linkers can be encoded by a nucleic acid molecule and therefore can be recombinantly expressed. As the linkers are themselves peptides, the polypeptide connected by the linker are connected to the linker via a peptide bond that is formed between two amino acids.

In one embodiment the peptidic linkers are based independently from each other on the amino acid sequences GS (SEQ ID NO: 21), GGS (SEQ ID NO: 22), GSG (SEQ ID NO: 34), GGGS (SEQ ID NO: 23), GGGSGGGS (SEQ ID NO: 110), GGGSGGGSGGGS (SEQ ID NO: 111), GGGSGGGSGGGSGGGS (SEQ ID NO: 112), GGGSGGGSGGGSGGGSGGGS (SEQ ID NO: 113), GGGGS (SEQ ID NO: 24), GGGGSGGGGS (SEQ ID NO: 114), GGGGSGGGGSGGGGS (SEQ ID NO: 115), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 116),

GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 117), and GCGGSGGGGSGGGGS (SEQ ID NO: 118) in which one or more artificial N- glycosylation sites (sequons) are introduced.

The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject., A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term "soluble form of a polypeptide" or short "soluble form" denotes that the polypeptide is after recombinant production in a eukaryotic cell secreted to the cultivation medium and remains in solution in the cultivation medium, i.e. the secreted polypeptide does not form aggregate or the like that can be separated from the cultivation medium by physical operations, such as e.g. filtration or centrifugation. Thus, the soluble form of a polypeptide can be recovered from the cleared cultivation supernatant after the cell culture has been processed by centrifugation and/or filtration. The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt, T.J. et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y. (2007), page 91) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano, S. et al, J. Immunol. 150 (1993) 880-887; Clackson, T. et al, Nature 352 (1991) 624-628).

The term "vector", as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

The term "wild-type polypeptide" denotes a (parent) polypeptide that has not been modified by the introduction of artificial glycosylation sites. A wild-type polypeptide differs from the polypeptide with artificial glycosylation sites only with respect to the introduced glycosylation sites. For example, if the polypeptide is a fusion polypeptide comprising a first entity and a second entity fused together by a peptidic linker and the artificial glycosylation site has been introduced in the peptidic linker (e.g. GGGSXXXGGGS) then the wild-type polypeptide is the identical polypeptide (with respect to the amino acid sequence) except that the artificial glycosylation site is not present (e.g. GGGSGGGSGGGS). If, for example, the artificial glycosylation site has been introduced by fusing a glycosylation site comprising short moiety to the polypeptide (e.g. an opsin-tag) then the wild-type polypeptide is the polypeptide without the fused additional moiety.

THE CURRENT INVENTION

It has been found that by the introduction of one or more (artificial) glycosylation sites in a fusion polypeptide the recombinant production thereof can be improved. By the introduction of one or more (artificial) glycosylation site(s) the recombinant production of the polypeptide in a mammalian cell can be improved by improving the solubility of the fusion polypeptide. This method is especially suited e.g. for polypeptides which are poorly or not at all expressed in mammalian cells or secreted into the culture medium or which tend to aggregate non-specifically. In one preferred embodiment the introduced glycosylation site is in the peptidic linker connecting the two polypeptide entities of a fusion-polypeptide. In one preferred embodiment the two polypeptide entities of the fusion-polypetide are not derived/ do not originate from the same polypeptide.

N-glycosylation is the most frequent post translational modification of secreted polypeptides. N-linked carbohydrates are classified in three main groups depending on their core structure:

high-mannose oligosaccharides,

complex oligosaccharides, and

hybrid oligosaccharides.

The polypeptide-linked/conjugated carbohydrates are associated with different functions which can be different for each polypeptide:

folding,

stability,

protection from protease degradation,

protection from aggregation by shielding/neutralisation of hydrophobic patches on the surface of the polypeptide, and

covering of potentially immunogenic epitopes/neoepitopes.

The N-glycosylation can influence

pharmacokinetic properties (in vivo half-life), and

pharmacodynamic properties (tissue distribution) of a polypeptide. This depends on the terminal sugar residues of carbohydrate together with specific cell-surface receptors (e.g. asialoglycoprotein receptor or mannose receptor).

Furthermore, N-linked carbohydrates play an important role in the biological activity of polypeptides (e.g. ADCC and CDC of antibodies). I. antibody-polypeptide fusion polypeptides

ID_tt con_srcu

Example: Fusion-polypeptides comprising an antibody part and HIV-derived polypeptides

It has been foun_{iiidd lkt}d_se _pe_pcn_e u that by introducing new (artificial) glycosylation sites in each of the peptidic linker connecting the parts of a fusion polypeptide the yield of recombinantly produced soluble fusion polypeptide can be increased.

Exemplary fusion polypeptide comprise an antibody part (anti-CD40 antibody) and HIV-derived polypeptides (gag 17-35=red; gag 253-284=orange; nef 116- 145=green; nef 66-97=dark green; pol 325-355=blue).

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2) GGGGSGGNGTGGGGSGG

3) GGSGGGNWTG

4) GGSGGGGSGG

5) GGGGSGGGGSGGGGSG

6) GGGGSGGGGSGG

1) GSGGGGSGG 4 13 mg/L 3 a

2) GGGGSGGNGTGGGGSGG

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5) GGGGSGGGGSGG

C9 1) GGGSGGNGTGGGGSGG 7 20 mg/L 8 m

2) GGGGSGGNGTGGGGSGG

3) GGSGGGNWTG

4) GGSGGGGSGGGNSTG

5) GGGGSGGNGTGGGGSGG

6) GGGGSGGGNSTGG

7) GGSGGGNDTG

CIO 1) GGGSGGNGTGGGGSGG 7 20 mg/L 9 m

2) GGGGSGGNGTGGGGSGG

3) GGSGGGNGTG

4) GGSGGGGSGGGNSTG

5) GGGGSGGNGTGGGGSGG

6) GGGGSGGGNSTGG

7) GGSGGGNDTG

Cl l 1) GGGSGGNGTGGGGSGG 7 23 mg/L 10 a/

2) GGGGSGGNGTGGGGSGG mJ

3) GGSGGGNGTG f

4) GGSGGGGSGGGNSTG

5) GGGGSGGNGTGGGGSGG

6) GGGGSGGGNSTGG

7) GGSGGGNDTG

C12 1) GGGSGGGGSGG 0 4 mg/L 1 1 a

2) GGGSGGGGSGG

3) GGSGGGG

4) GGSGGGGSGG

5) GGGGSGGGGSGGGGSG

6) GGGGSGGGGSGG

It can be seen that depending on the number of artificial glycosylation sites in the peptidic linkers the distribution of aggregates / monomer / fragments changes.

Example: Fusion-polypeptides comprising an antibody part and GFP

Exemplary fusion-polypeptides comprising an antibody and a GFP moiety were constructed.

A GlySer-linker (GGGGSGGGGSG; SEQ ID NO: 01) was used to fuse either i) a eGFP moiety (enhanced green fluorescent protein; SEQ ID NO: 02), or ii) a emGFP moiety (emerald green fluorescent protein; SEQ ID NO: 03), or iii) a tagGFP moiety (SEQ ID NO: 04) to the C-terminal ends of the heavy chains (HCs) of an anti-IGF-lR antibody of the subclass IgGl (HC: SEQ ID NO: 05; LC: SEQ ID NO: 06). The different heavy chain-fusions have an amino acid sequence of SEQ ID NO: 07, 08, and 09.

Secretion of the antibody-fusion-polypeptides could not be detected at all in the cell culture supernatants of transiently transfected HEK293 cells using Western Blotting and/or a protein A based High-Performance Liquid Chromatography (HPLC) assay. Also biologically active GFP could not be monitored by its bio luminescent properties (GFP-specific fluorescence). But the fusion-polypeptides could be detected in the cell pellet fraction by Western Blot analysis. The results are shown in the following table.

Table 1

< 1 μg/ml = be ow limit of detection Without being bound by this theory GFP is a cytoplasmic monomeric protein with a tendency to form (weak) dimers. Thus, the wild-type GFP is not destined "from nature" (the natural Aequorea victoria jellyfish cell) to be secreted, and, therefore, the GFP molecule must be made suitable for secretion due to incompatibility with the mammalian cell secretion machinery. In order to provide secreted GFP containing fusion-polypeptides a polypeptide derived from the human light-sensitive membrane-bound G protein-coupled opsin receptor found in photoreceptor cells of the retina has been included further in the fusion-polypeptide, i.e. as glycosylation-tag. With this glycosylation-tag additional (artificial) N-glycosylation sites are introduced in the fusion-polypeptide. The opsin receptor derived polypeptide was fused directly, i.e. without intervening linker peptide, to the C-terminus of the GFP moiety. The opsin-derived polypeptide (NGTEGPNFYVPFSNATGVV; opsin-tag; SEQ ID NO: 10) comprises two N- glycosylation site motifs: the NGT motif and the NAT motif (general N- glycosylation site motif: NxS/T; Asn followed by any amino acid except Pro, followed by either Ser or Thr). Transient expression of the fusion-polypeptides comprising the opsin glycosylation tag (SEQ ID NO: 11) in HEK293 cells resulted in secretion of the fusion-polypeptide. The results are shown in the following table.

Table 2

< 1 μg/m

From SDS-PAGE analysis (band broadening) it can be seen that the secreted fusion-polypeptides contain additional carbohydrates.

The antibody-GFP-opsin-tag-fusion-polypeptides were purified with a two-step procedure, in particular, a protein A affinity chromatography followed by a size exclusion chromatography. The functionality of the antibody moiety within the fusion-polypeptide was demonstrated by binding to the IGF-IR receptor protein using surface plasmon resonance (BIAcore) and internalization into cells via receptor mediated endocytosis on cells over-expressing IGF-IR by FACS and/or confocal microscopy. The functionally of the GFP moiety was shown due to its green fluorescence characteristics.

Variant Fc-regions

In certain embodiments, one or more amino acid modifications may be introduced into the Fc-region of an antibody in the fusion polypeptide provided herein, thereby generating an antibody comprising a variant Fc-region. The variant Fc-region may comprise a human Fc-region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4

Fc-region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.

In certain embodiments, the fusion polypeptide according to the invention contemplates a variant Fc-region that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the fusion polypeptide in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the fusion polypeptide lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch, J.V. and Kinet, J.P., Annu. Rev. Immunol. 9

(1991) 457-492. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in US 5,500,362 (see, e.g. Hellstrom, I. et al, Proc. Natl. Acad. Sci. USA 83 (1986) 7059-7063; and Hellstrom, I. et al, Proc. Natl. Acad. Sci. USA 82 (1985) 1499-1502); US 5,821,337 (see Bruggemann, M. et al, J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96^® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes, R. et al, Proc. Natl. Acad. Sci. USA 95 (1998) 652-656. Clq binding assays may also be carried out to confirm that the fusion polypeptide is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro, H. et al, J. Immunol. Methods 202 (1996) 163-171; Cragg, M.S. et al, Blood 101 (2003) 1045-1052; and Cragg, M.S. and M.J. Glennie, Blood 103 (2004) 2738- 2743). FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al, Int.

Immunol. 18 (2006: 1759-1769).

Antibody-containing fusion polypeptides with reduced effector function include those comprising an antibody with substitution of one or more of Fc-region residues 238, 265, 269, 270, 297, 327 and 329 (US 6,737,056). Such Fc-region mutants include Fc-region mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc-region mutant with substitution of residues 265 and 297 to alanine (US 7,332,581).

Certain Fc-region variants with improved or diminished binding to FcRs are described (see, e.g., US 6,737,056; WO 2004/056312; and Shields, R.L. et al, J. Biol. Chem. 276 (2001) 6591-6604). In certain embodiments, an fusion polypeptide comprises an antibody variant which comprises an Fc-region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc- region (EU numbering of residues). In some embodiments, alterations are made in the Fc-region that result in altered

(i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US 6,194,551, WO 99/51642, and Idusogie, E.E. et al, J. Immunol. 164 (2000) 4178-4184.

Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer, R.L. et al, J. Immunol. 117 (1976) 587-593, and Kim, J.K. et al, J. Immunol. 24 (1994) 2429-2434), are described in US 2005/0014934. Those antibodies comprise an Fc-region with one or more substitutions therein which improve binding of the Fc-region to FcRn. Such Fc-region variants include those with substitutions at one or more of Fc-region residues: 238, 256, 265, 272, 286,

303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc-region residue 434 (US 7,371,826).

See also Duncan, A.R. and Winter, G., Nature 322 (1988) 738-740; US 5,648,260; US 5,624,821; and WO 94/29351 concerning other examples of Fc-region variants. II. polypeptide-polypeptide fusion polypeptides

Example: Neurotrophic proteins

An exemplary neurotrophic protein is brain-derived neurotropic factor (BDNF). i) Glycosylation-tag

Human wild-type pre-pro-BDNF comprising a C-terminal T7-His6-tag (MASMTGGQQMG-HHHHHH; used for affinity purification; SEQ ID NO: 12) fused via a GSG-linker was expressed in HEC69 cells (pre-pro-BDNF-T7-His6; SEQ ID NO: 13). The amino acid sequence of mature BDNF does not contain an N-glycosylation site motif and is therefore not N-glycosylated. Mature BDNF is obtained in low yields of a few μg/ml only. The low expression yield could not be improved by optimization of the gene codon usage, or by removal of potential protease cleavage sites (pre-pro-BDNF(-RGR)-T7-His6), or by exchanging the native BDNF signal sequence by a signal sequence of a well expressed antibody (MGWSCIILFL VATATGVHS; SEQ ID NO: 14), or by an exchange of the BDNF pre-pro-segment with that of NGF. The results are shown in the following table (normalized to concentration of maturated BDNF).

Table 3

In order to improve the (secretion) yield of BDNF (artificial) glycosylation sites were introduced into the BDNF molecule using opsin-glycosylation-tags of different length: 16 amino acid residues (NGTEGPNFYVPFSNAT; SEQ ID NO: 15), 19 amino acid residues (NGTEGPNFYVPFSNATGVV; SEQ ID NO: 10), and 20 amino acid residues (NGTEGPNFYVPFSNATGVVR; SEQ ID NO: 16). Using this modification the expression yield (normalized to concentration of maturated BDNF) could be improved. The results are shown in the following table (normalized to concentration of maturated BDNF). Table 4

* : exceeded linear range of quantification

Surprisingly, the BDNF variants comprising a glycosylation-tag had also an improved biological activity when compared to native non-glycosylated BDNF (CHO-TrkB-luciferase reporter gene assay). The results are shown in the following table.

Table 5

Further possible glycosylation-tags are AAANGTGGA (one N glycosylation site motif; SEQ ID NO: 17), ANITVNITV (two N-glycosylation site motifs; SEQ ID NO: 18), and NATGADNGTGAS (two N-glycosylation site motifs; SEQ ID NO: 19). ii) Introduced N-glycosylation site motif(s)

In order to improve the (secretion) yield of BDNF artificial N-glycosylation sites were introduced into the BDNF amino acid sequence. The artificial N- glycosylation site can be introduced e.g. by point mutation to the BDNF amino acid sequence (N-glycosylation-site motif: Asn-Xxx-Ser/Thr; Xxx any amino acid except Pro). The corresponding encoding nucleic acid sequences were prepared and transiently expressed in HEK293 cells. The numbering of the mutations is based on the amino acid sequence of mature BDNF (SEQ ID NO: 25). The secreted BDNF variants were analyzed for expression/secretion level, degree of N-glycosylation (deduced from migration as band with approx. 3-5 kDa per introduced and used N- glycosylation site increased MW (when compared with the non-glycosylated BDNF reference) by immunoblotting analysis), and functionality/receptor binding via a CHO-TrkB-luciferase reporter gene assay. The results are shown in the following tables (expression yield and biological activities normalized to concentration of maturated BDNF). Table 6

n.d.: not determined -: no glycosylation

+: partial glycosylation

++: full glycosylation

active; BDNF activity comparable to the activity of unglycosylated mature wild- type BDNF expressed in E. coli, refolded and purified

Inactive: no activity determined

The functionality/biological activity of glycosylated BDNF variants was also shown in a dorsal root ganglion (DRG) assay. The activity determined in the DRG assay was comparable to non-glycosylated wild-type BDNF. RECOMBINANT METHODS AND COMPOSITIONS

Fusion polypeptides may be produced using recombinant methods and compositions, e.g., as described in US 4,816,567. In one embodiment, an isolated nucleic acid encoding the fusion polypeptide as described herein is provided. In a further embodiment, a vector (e.g., expression vector) comprising such nucleic acid is provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the fusion polypeptide, or (2) a vector comprising a nucleic acid that encodes an amino acid sequence comprising one fusion polypeptide chain and an amino acid sequence that encodes the other fusion polypeptide chain, or (3) a first vector comprising a nucleic acid that encodes the first fusion polypeptide chain and a second vector comprising a nucleic acid that encodes the second fusion polypeptide chain. In one embodiment, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid/myeloid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of making a fusion polypeptide as reported herein is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the fusion polypeptide, as provided above, under conditions suitable for expression of the fusion polypeptide, and optionally recovering the fusion polypeptide from the host cell (or host cell culture medium).

For recombinant production of a a fusion polypeptide as reported herein, nucleic acid encoding the fusion polypeptide, e.g., as described above, are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid may be readily synthesized using conventional procedures. Suitable host cells for cloning or expression of the fusion polypeptide-encoding vector include prokaryotic or eukaryotic cells described herein. Eukaryotic microbes such as filamentous fungi or yeast are suitable cloning and/or expression hosts for fusion polypeptide-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of a fusion polypeptide with a partially or fully human glycosylation pattern (see Gerngross, T.U., Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al, Nat.

Biotech. 24 (2006) 210-215).

Suitable host cells for the expression of glycosylated fusion polypeptide are also derived from multicellular organisms (invertebrates and vertebrates). Invertebrate cells include insect cells. Numerous baculo viral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plants and plant cell cultures can also be utilized as hosts (see, e.g., US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants)) including cells with an engineered humanized glycosylation pathway.

Vertebrate cells may also be used as hosts. For example, mammalian cells/cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cells/cell lines are monkey kidney cells (CV1) transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham, F.L. et al, J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells

(BHK); mouse Sertoli cells (TM4 cells as described, e.g., in Mather, J.P., Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3 A); human lung cells (W138); human liver cells (Hep_G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather, J.P. et al, Annals N.Y. Acad. Sci. 383 (1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR^" CHO cells (Urlaub, G. et al, Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NSO and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2004), pp. 255- 268. Description of the Figures

Figure 1 SE-HPLC of reference compounds

Figure 2 SE-HPLC of contract No. 3.

Figure 3 SE-HPLC of contract No. 4.

Figure 4 SE-HPLC of contract No. 5.

Figure 5 SE-HPLC of contract No. 6.

Figure 6 SE-HPLC of contract No. 7.

Figure 7 SE-HPLC of contract No. 8.

Figure 8 SE-HPLC of contract No. 9.

Figure 9 SE-HPLC of contract No. 10.

Figure 10 SE-HPLC of contract No. 11.

Figure 11 SE-HPLC of contract No. 12.

Figure 12 SDS-gels of constructs No. 3 to 8.

Figure 13 SDS-gels of constructs No. 9 to 11.

Figure 14 SDS-gel of construct No. 12.

The following examples, figures and sequences are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

Materials and Methods

Recombinant DNA techniques

Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The molecular biological reagents were used according to the manufacturer's instructions.

Gene and oligonucleotide synthesis

Desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into an E. coli plasmid for propagation/amplification. The DNA sequences of subcloned gene fragments were verified by DNA sequencing. Alternatively, short synthetic DNA fragments were assembled by annealing chemically synthesized oligonucleotides and/or via PCR. The respective oligonucleotides were prepared by metabion GmbH (Planegg-Martinsried, Germany).

Protein determination:

Samples were analyzed by OD280 using a UV spectrophotometer to determine the protein concentration in solution. The extinction coefficient required for this was calculated from the amino acid sequence according to Pace (Pace et al., Protein Science 4 (1995) 2411-2423).

Size exclusion high performane liquid chromatogrpahy:

Size-exclusion chromatography (SE-HPLC) was performed on TSK-Gel300SWXL or Superdex 200 columns with a 0.2 M potassium phosphate buffer, comprising

0.25 M KCl, pH 7.0 as mobile phase in order to determine the content of different species (e.g. monomeric and aggregated species) in the samples.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis:

Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (reducing and non-reducing) was performed to analyze the purity of protein A affinity purified polypeptides with regard to product-related degradation products and unrelated impurities.

Reagents

All commercial chemicals, antibodies and kits were used as provided according to the manufacturer's protocol if not stated otherwise.

Example 1

Generation of recombinant expression vectors a) Generation of vectors for the expression of anti-CD40 antibody heavy chain HIV fusion polypeptide containing two HIV-derived peptides and a human IgG4 constant region variant (S228P, L235E and "hole" mutation)

The anti-CD40 antibody heavy chain HIV fusion gene encoding an immunoglobulin heavy chain comprising a variable domain and the human IgG4 constant region (VH, CHI , hinge, CH2, CH3), a first peptidic linker, a first HIV- derived polypeptide, a second peptidic linker and a second HIV-derived polypeptide was assembled by fusing DNA fragments coding for the respective element. The human IgG4 constant region contains the SPLE and "hole" mutation.

The anti-CD40 antibody heavy chain with the mutations S228P and L235E and the "hole" mutation has the following amino acid sequence: EVQLVESGGG LVQPGGSLKL SCATSGFTFS DYYMYWVRQA PGKGLEWVAY

INSGGGSTYY PDTVKGRFTI SRDNAKNTLY LQMNSLRAED TAVYYCARRG LPFHAMDYWG QGTLVTVSS

ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV

HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFEGGPSV FLFPPKPKDT LMI SRTPEVT CVWDVSQED

PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK

CKVSNKGLPS S I EKTI SKAK GQPREPQVCT LPPSQEEMTK NQVSLSCAVK

GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGS FFLVSRL TVDKSRWQEG

NVFSCSVMHE ALHNHYTQKS LSLSLGK (SEQ ID NO: 119).

The expression vector also comprised an origin of replication from the vector pUC18, which allows replication of this plasmid in E. coli, and a beta-lactamase gene which confers ampicillin resistance in E. coli.

The transcription unit of anti-CD40 antibody heavy chain HIV fusion gene comprises the following functional elements in 5' to 3' direction:

the immediate early enhancer and promoter from the human cytomegalovirus (P-CMV),

a human heavy chain immunoglobulin 5 '-untranslated region (5 'UTR), a nucleic acid encoding a murine immunoglobulin heavy chain signal sequence,

a nucleic acid encoding an anti-CD40 heavy chain variable (VH) domain,

a nucleic acid encoding a human IgG4 constant region with the mutations S228P, L235E and "hole" mutation,

- a nucleic acid encoding the a peptidic linker,

a nucleic acid encoding a first HIV-derived polypeptide,

a nucleic acid encoding a second peptidic linker,

a nucleic acid encoding a second HIV-derived polypeptide, optionally a nucleic acid encoding a third peptidic linker, and the bovine growth hormone polyadenylation sequence (BGH pA). b) Generation of vectors for the expression of anti-CD40 antibody heavy chain HIV fusion polypeptide containing three HIV-derived peptides and a human IgG4 constant region variant (S228P, L235E and "hole" mutation)

The anti-CD40 antibody heavy chain HIV fusion gene encoding an immunoglobulin heavy chain comprising a variable domain and the human IgG4 constant region (VH, CHI , hinge, CH2, CH3), a first peptidic linker, a first HIV- derived polypeptide, a second peptidic linker, a second HIV-derived polypeptide, a third peptidic linker and a third HIV-derived polypeptide was assembled by fusing DNA fragments coding for the respective element. The human IgG4 constant region contains the SPLE and "hole" mutations.

The anti-CD40 antibody heavy chain with the mutations S228P and L235E and the "hole" mutation has the following amino acid sequence:

EVQLVESGGG LVQPGGSLKL SCATSGFTFS DYYMYWVRQA PGKGLEWVAY INSGGGSTYY PDTVKGRFTI SRDNAKNTLY LQMNSLRAED TAVYYCARRG LPFHAMDYWG QGTLVTVSS

ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFEGGPSV FLFPPKPKDT LMI SRTPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS S I EKTI SKAK GQPREPQVCT LPPSQEEMTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGS FFLVSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK

(SEQ ID NO: 119).

The transcription unit of the anti-CD40 antibody heavy chain HIV fusion gene comprises the following functional elements in 5' to 3' direction:

the immediate early enhancer and promoter from the human cytomegalovirus (P-CMV), a human heavy chain immunoglobulin 5 '-untranslated region (5 'UTR), a nucleic acid encoding a murine immunoglobulin heavy chain signal sequence,

a nucleic acid encoding an anti-CD40 antibody heavy chain variable (VH) domain,

a nucleic acid encoding a human IgG4 constant region with the mutations S228P and L235E and the "hole" mutation,

a nucleic acid encoding a first peptidic linker,

a nucleic acid encoding a first HIV-derived polypeptide,

- a nucleic acid encoding a second peptidic linker,

a nucleic acid encoding a second HIV-derived polypeptide, a nucleic acid encoding a third peptidic linker,

a nucleic acid encoding a third HIV-derived polypeptide,

optionally a nucleic acid encoding a fourth peptidic linker, and - the bovine growth hormone polyadenylation sequence (BGH p A). c) Generation of vectors for the expression of anti-CD40 antibody heavy chain HIV fusion polypeptide containing two HIV-derived peptides and a human IgG4 constant region variant (S228P, L235E and "knob" mutation)

The anti-CD40 antibody heavy chain HIV fusion gene encoding an immunoglobulin heavy chain comprising a variable domain and the human IgG4 constant region (VH, CHI , hinge, CH2, CH3), a first peptidic linker, a first HIV- derived polypeptide, a second peptidic linker and a second HIV-derived polypeptide was assembled by fusing DNA fragments coding for the respective element. The human IgG4 constant region contains the SPLE and "knob" mutations.

The anti-CD40 antibody heavy chain with mutations S228P and L235E has the "knob" mutation has following amino acid sequence:

ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFEGGPSV FLFPPKPKDT LMI SRTPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS S I EKTI SKAK GQPREPQVYT LPPCQEEMTK NQVSLWCLVK GFYPSDIAVE WESNGPENNY KTTPPVLDSD GS FFLYSRLT VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK

(SEQ ID NO: 120).

a nucleic acid encoding an anti-CD40 antibody heavy chain variable (VH) domain,

a nucleic acid encoding a human IgG4 constant region with the mutations S228P and L235E and the "knob" mutation,

a nucleic acid encoding a first peptidic linker,

a nucleic acid encoding a first HIV-derived polypeptide,

a nucleic acid encoding a second peptidic linker,

a nucleic acid encoding a second HIV-derived polypeptide, optionally a nucleic acid encoding a third peptidic linker, and the bovine growth hormone polyadenylation sequence (BGH pA). d) Generation of vectors for the expression of anti-CD40 antibody heavy chain HIV fusion polypeptide containing three HIV-derived peptides and a human IgG4 constant region variant (S228P, L235E and "knob" mutation)

The anti-CD40 antibody heavy chain HIV fusion gene encoding an immunoglobulin heavy chain comprising a variable domain and the human IgG4 constant region (VH, CHI , hinge, CH2, CH3), a first peptidic linker, a first HIV- derived polypeptide, a second peptidic linker, a second HIV-derived polypeptide, a third peptidic linker and a third HIV-derived polypeptide was assembled by fusing DNA fragments coding for the respective element. The human IgG4 constant region contains the SPLE and "knob" mutations. The anti-CD40 antibody heavy chain with mutations S228P and L235E has the "knob" mutation has following amino acid sequence:

ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSWT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFEGGPSV FLFPPKPKDT LMI SRTPEVT CVWDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RWSVLTVLH QDWLNGKEYK CKVSNKGLPS S I EKTI SKAK GQPREPQVYT LPPCQEEMTK NQVSLWCLVK

GFYPSDIAVE WESNGPENNY KTTPPVLDSD GS FFLYSRLT VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK

(SEQ ID NO: 120).

a nucleic acid encoding an anti-CD40 antibody heavy chain variable (VH) domain,

a nucleic acid encoding a human IgG4 constant region with the mutations S228P and L235E and the "knob",

a nucleic acid encoding a first peptidic linker,

a nucleic acid encoding a first HIV-derived polypeptide,

- a nucleic acid encoding a second peptidic linker,

a nucleic acid encoding a second HIV-derived polypeptide, a nucleic acid encpding a third peptidic linker,

a nucleic acid encoding a third HIV-derived polypeptide,

optionally a nucleic acid encoding a fourth peptidic linker, and the bovine growth hormone polyadenylation sequence (BGH pA). e) Generation of vector for the expression of the anti-CD40 antibody light chain

The anti-CD40 antibody light chain encoding gene comprising a variable domain and the human kappa constant region (VL, CL) was assembled by fusing a DNA fragment coding for the respective element.

The anti-CD40 antibody light chain has the following amino acid sequence:

DIQMTQSPSS LSASVGDRVT ITCSASQGIS NYLNWYQQKP GKAVKLLIYY

TSILHSGVPS RFSGSGSGTD YTLTISSLQP EDFATYYCQQ FNKLPPTFGG

GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG

LSSPVTKSFN RGEC

(SEQ ID NO: 121).

The transcription unit of the anti-CD40 antibody kappa light chain comprises the following functional elements in 5' to 3' direction:

the immediate early enhancer and promoter from the human cytomegalovirus (P-CMV) including intron A,

- a human heavy chain immunoglobulin 5 '-untranslated region (5 'UTR), a nucleic acid encoding a murine immunoglobulin heavy chain signal sequence,

a nucleic acid encoding an anti-CD40 light chain variable (VL) domain, a nucleic acid encoding a human Ig-kappa constant region, and - the bovine growth hormone polyadenylation sequence (BGH pA). f) Summary of the constructs

Table 8 construct composition i) LC and ii) HC each listed in N- to contains new

ID C-terminal direction glycosylation sites

C3 i) anti-CD40 antibody light chain (2)

ii) anti-CD40 antibody "hole" heavy chain

peptidic linker (GSGGGGSGG)

HIV-derived peptide (AIFQSSMTKI

LEPFR QNPD IVIYQYMDDL Y)

peptidic linker (GGGGSGGNGTGGGGSGG) yes

HIV-derived peptide (VGFPVTPQVP

LRPMTYKAAV DLSHFLKEKG GL)

peptidic linker (GGSGGGNWTG) yes iii) anti-CD40 antibody "knob" heavy chain

peptidic linker (GGSGGGGSGG)

HIV-derived peptide (EKIRLRPGGK

K YKLKHIV)

peptidic linker (GGGGSGGGGSGGGGSG)

HIV-derived peptide (NPPIPVGEIY

KRWIILGLNK IVRMYSPTSI LD)

peptidic linker (GGGGSGGGGSGG)

HIV-derived peptide (HTQGYFPDWQ

NYTPGPGVRY PLTFGWLYKL)

C4 i) anti-CD40 antibody light chain (4)

ii) anti-CD40 antibody "hole" heavy chain

peptidic linker (GSGGGGSGG)

HIV-derived peptide (AIFQSSMTKI

LEPFRKQNPD IVIYQYMDDL Y)

peptidic linker (GGGGSGGNGTGGGGSGG) yes

HIV-derived peptide (VGFPVTPQVP

LRPMTYKAAV DLSHFLKEKG GL)

peptidic linker (GGSGGGNWTG) yes iii) anti-CD40 antibody "knob" heavy chain

peptidic linker (GGSGGGGSGG)

HIV-derived peptide (EKIRLRPGGK

KKYKLKHIV)

peptidic linker (GGGGSGGNGTGGGGSGG) yes

HIV-derived peptide (NPPIPVGEIY

KRWIILGLNK IVRMYSPTSI LD)

peptidic linker (GGGGSGGGGSGG)

HIV-derived peptide (HTQGYFPDWQ

NYTPGPGVRY PLTFGWLYKL)

peptidic linker (GGSGGGNDTG yes

construct composition i) LC and ii) HC each listed in N- to contains new

ID C-terminal direction glycosylation sites

C7 i) anti-CD40 antibody light chain (2)

ii) anti-CD40 antibody "hole" heavy chain

peptidic linker (GSGGGGSGG)

HIV-derived peptide (AIFQSSMTKI

LEPFR QNPD IVIYQYMDDL Y)

peptidic linker (GGGGSGGGGSGG)

HIV-derived peptide (VGFPVTPQVP

LRPMTYKAAV DLSHFLKE G GL)

iii) anti-CD40 antibody "knob" heavy chain

peptidic linker (GGSGGGGSGG)

HIV-derived peptide (EKIRLRPGGK

K YKLKHIV)

peptidic linker (GGGGSGGNGTGGGGSGG) yes

HIV-derived peptide (NPPIPVGEIY

KPvWIILGLNK IVRMYSPTSI LD)

peptidic linker (GGGGSGGGGSGG)

HIV-derived peptide (HTQGYFPDWQ

NYTPGPGVRY PLTFGWLYKL)

peptidic linker (GGSGGGNDTG) yes

C8 i) anti-CD40 antibody light chain (0)

(control) ii) anti-CD40 antibody "hole" heavy chain

peptidic linker (GSGGGGSGG)

HIV-derived peptide (AIFQSSMTKI

LEPFRKQNPD IVIYQYMDDL Y)

peptidic linker (GGGGSGGGGSGG)

HIV-derived peptide (VGFPVTPQVP

LRPMTYKAAV DLSHFLKEKG GL)

iii) anti-CD40 antibody "knob" heavy chain

peptidic linker (GGSGGGGSGG)

HIV-derived peptide (EKIRLRPGGK

KKYKLKHIV)

peptidic linker (GGGGSGGGGSGGGGSG)

HIV-derived peptide (NPPIPVGEIY

KRWIILGLNK IVRMYSPTSI LD)

peptidic linker (GGGGSGGGGSGG)

HIV-derived peptide (HTQGYFPDWQ

NYTPGPGVRY PLTFGWLYKL) construct composition i) LC and ii) HC each listed in N- to contains new

ID C-terminal direction glycosylation sites

C9 i) anti-CD40 antibody light chain (V)

ii) anti-CD40 antibody "hole" heavy chain

peptidic linker (GGGSGGNGTGGGGSGG) yes

HIV-derived peptide (AIFQSSMTKI

LEPFR QNPD IVIYQYMDDL Y)

peptidic linker (GGGGSGGNGTGGGGSGG) yes

HIV-derived peptide (VGFPVTPQVP

LRPMTYKAAV DLSHFLKEKG GL)

peptidic linker (GGSGGGNWTG) yes iii) anti-CD40 antibody "knob" heavy chain

peptidic linker (GGSGGGGSGGGNSTG) yes

HIV-derived peptide (EKIRLRPGG

K YKLKHIV)

peptidic linker (GGGGSGGNGTGGGGSGG) yes

HIV-derived peptide (NPPIPVGEIY

KPvWIILGLNK IVRMYSPTSI LD)

peptidic linker (GGGGSGGGNSTGG) yes

HIV-derived peptide (HTQGYFPDWQ

NYTPGPGVRY PLTFGWLYKL)

peptidic linker (GGSGGGNDTG yes

CIO i) anti-CD40 antibody light chain (?)

ii) anti-CD40 antibody "hole" heavy chain

peptidic linker (GGGSGGNGTGGGGSGG) yes

HIV-derived peptide (AIFQSSMTKI

LEPFRKQNPD IVIYQYMDDL Y)

peptidic linker (GGGGSGGNGTGGGGSGG) yes

HIV-derived peptide (VGFPVTPQVP

LRPMTYKAAV DLSHFLKEKG GL)

peptidic linker (GGSGGGNGTG) yes iii) anti-CD40 antibody "knob" heavy chain

peptidic linker (GGSGGGGSGGGNSTG) yes

HIV-derived peptide (EKIRLRPGGK

KKYKLKHIV)

peptidic linker (GGGGSGGNGTGGGGSGG) yes

HIV-derived peptide (NPPIPVGEIY

KRWIILGLNK IVRMYSPTSI LD)

peptidic linker (GGGGSGGGNSTGG) yes

HIV-derived peptide (HTQGYFPDWQ

NYTPGPGVRY PLTFGWLYKL)

Deotidic linker (GGSGGGNDTG yes construct composition i) LC and ii) HC each listed in N- to contains new

ID C-terminal direction glycosylation sites

Cl l i) anti-CD40 antibody light chain (7)

ii) anti-CD40 antibody "hole" heavy chain

peptidic linker (GGGSGGNGTGGGGSGG) yes

HIV-derived peptide (VGFPVTPQVP

LRPMTYKAAV DLSHFLKE G GL)

peptidic linker (GGGGSGGNGTGGGGSGG) yes

HIV-derived peptide (AIFQSSMTKI

LEPFRKQNPD IVIYQYMDDL Y)

peptidic linker (GGSGGGNGTG) yes iii) anti-CD40 antibody "knob" heavy chain

peptidic linker (GGSGGGGSGGGNSTG) yes

HIV-derived peptide (EKIRLRPGG

K YKLKHIV)

peptidic linker (GGGGSGGNGTGGGGSGG) yes

HIV-derived peptide (NPPIPVGEIY

KRWIILGLNK IVRMYSPTSI LD)

peptidic linker (GGGGSGGGNSTGG) yes

HIV-derived peptide (HTQGYFPDWQ

NYTPGPGVRY PLTFGWLYKL)

peptidic linker (GGSGGGNDTG yes

C12 i) anti-CD40 antibody light chain (0)

ii) anti-CD40 antibody "hole" heavy chain

peptidic linker (GGGSGGGGSGG)

HIV-derived peptide (VGFPVTPQVP

LRPMTYKAAV DLSHFLKEKG GL)

peptidic linker (GGGSGGGGSGG)

HIV-derived peptide (AIFQSSMTKI

LEPFRKQNPD IVIYQYMDDL Y)

peptidic linker (GGSGGGG)

iii) anti-CD40 antibody "knob" heavy chain

peptidic linker (GGSGGGGSGG)

HIV-derived peptide (EKIRLRPGGK

KKYKLKHIV)

peptidic linker (GGGGSGGGGSGGGGSG)

HIV-derived peptide (NPPIPVGEIY

KRWIILGLNK IVRMYSPTSI LD)

peptidic linker (GGGGSGGGGSGG)

HIV-derived peptide (HTQGYFPDWQ

NYTPGPGVRY PLTFGWLYKL) Example 2

Cloning and recombinant production

The nucleic acid encoding DNA fragments of the heavy and light chain variable domains (VH and VL) of the anti-CD40 antibody were synthesized by Geneart AG and cloned in the respective expression vectors encoding for the human kappa light chain constant region or a human IgG4 constant region variant (SPLE, knob or hole mutation), respectively. Cloning of all constructs was verified by sequencing.

Transient expression of the antibody fusion polypeptides in HEK293 cells was performed in F17-medium (Invitrogen) with 293 free trans fection reagent (Novagen) (2 x 1 L for each target; cotransfection of light chain and heavy chain vectors). After seven days cultivation supernatant was harvested and stored until purification at reduced temperature (-80 °C).

General information regarding the recombinant expression of human immunoglobulins in e.g. HEK293 cells is given in: Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203.

Example 3

Purification of recombinant fusion polypeptides

The antibody-fusion polypeptide-containing culture supematants were filtered and the antibody titer determined via protein A affinity HPLC. Subsequently the antibody-fusion constructs were purified by one chromatographic step. The antibody-fusion polypeptides were captured by affinity chromatography using HiTrap MabSelectSuRe (GE Healthcare) equilibrated with 1.0 M arginine, pH 8.0. Unbound proteins were removed by washing with equilibration buffer, and the fusion polypeptide was recovered with 1.0 M arginine, pH 1.8, and immediately after elution neutralized to pH 7.0 with 2 M Tris/HCl.

The protein concentrations of the antibody-fusion polypeptides were determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence.

Purity and integrity of the antibody-fusion polypeptides were analyzed by SDS- PAGE (NuPAGE 4-12% Bis-Tris Gel, Invitrogen) in the presence and absence of a reducing agent (5 mM 1 ,4-dithiotreitol) and staining with Simply Blue Safe Stain (Invitrogen). Aggregate content of the fusion polypeptide preparations was determined by high- performance SEC using a TSK G3000 SWXL analytical size-exclusion column (Tosoh Bioscience GmbH GE Healthcare) using 2 x PBS, pH 7.4 as running buffer.

Table 9: average yields of soluble fusion polypeptide per liter culture supernatant depending on the number of artificial glycosylation sites in the peptidic linker

Example 4

Generation of antibody expression plasmids a) Generation of the antibody expression plasmids for the parental human anti- human IGF-1R antibody

The gene segments encoding the human kappa light (Vk) and heavy chain variable regions (VH) were joined to the gene segments encoding the human kappa light chain constant region (Ck) or the human gamma- 1 heavy chain constant region (CH1-Hinge-CH2-CH3), respectively. Both antibody chain genes were expressed from two separate expression plasmids including the genomic exon-intron structure of the antibody genes. The amino acid sequence of the mature (without signal sequence) heavy and light chain of anti-human IGF-1R antibody are shown in SEQ ID NO: 05 and SEQ ID NO: 06.

The expression of antibody chains is controlled by a shortened intron A-deleted immediate early enhancer and promoter from the human cytomegalovirus (HCMV) including a human heavy chain immunoglobulin 5 '-untranslated region (5'-UTR), a murine immunoglobulin heavy chain signal sequence, and the polyadenylation signal from bovine growth hormone (BGH pA). The expression plasmids also contain an origin of replication and a β-lactamase gene from the vector pUC18 for plasmid amplification in Escherichia coli (see Kopetzki, E., et al., Virol. J. 5 (2008) 56; Ji, C, et al, J. Biol. Chem. 284 (2009) 5175-5185). b) Generation of the anti-transferrin receptor antibody light chain expression plasmid

In order to obtain a light chain for the anti-transferrin-receptor antibody, a light chain gene was chemically synthesized coding for the murine immunoglobulin heavy chain signal sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 14), the

VL variable domain of the rat-anti-murine transferrin receptor and the human Vkappa light chain constant region. The amino acid sequence of the VL domain of the rat antibody was obtained from Boado, R.J., et al., Biotechnol. Bioeng. 102 (2009) 1251-1258). The amino acid sequence of the chimeric rat/human anti- transferrin receptor antibody light chain is shown in SEQ ID NO: 80 .

Example 5

Generation of antibody-GFP-fusion polypeptide expression plasmids a) Generation of the expression plasmids for the anti-IGF-lR antibody-GFP fusion-polvpeptides All anti-human IGF-1R antibody heavy chain-GFP-fusion-polypeptide encoding genes were assembled by fusing a chemically synthesized DNA fragment coding for the respective GFP variant and a glycine-serine linker consisting of 2 Gly₄Ser repeats and a further Gly (heavy chain...LSPG--gggsggggsg— GFP) to the 3' end of the anti-IGF-lR antibody heavy chain gene coding for a slightly truncated human gamma- 1 heavy chain constant region (removal of the last natural amino acid Lys).

The amino acid sequence of the anti-IGF-lR antibody heavy chain eGFP, emGPF and tagGFP fusion protein is shown in SEQ ID NO: 07, SEQ ID NO: 08 and SEQ ID NO: 09, respectively.

The antibody heavy and light chain genes were expressed from two separate expression plasmids including the genomic exon-intron structure of antibody genes. b) Generation of expression plasmids for anti-IGF-lR antibody heavy chain- eGFP-opsin-tag-fusion-polypeptides

The expression plasmids for the transient expression of the anti-IGF-lR antibody heavy chain-GFP-opsin-tag-fusion-polypeptides in HEK293 cells was derived from the expression vectors described above. They differ only in the DNA segment encoding the GFP-opsin-tag where upon the 19 amino acid peptide (NGTEGPNF Y VPF SN ATG V V ; opsin(M); SEQ ID NO: 10) was fused directly to the C-terminal end of a respective GFP. As example, the amino acid sequence of the anti-IGF-lR antibody heavy chain-eGFP-opsin(M)-tag-fusion-polypeptide is shown in SEQ ID NO: 33.

Example 6

Generation of BDNF expression plasmids a) Generation of the expression plasmid for wild-type pre-pro-BDNF

The DNA segment coding for human pre-pro-BDNF gene was prepared by chemical synthesis and inserted into the basic expression vector described above.

For this purpose, the pre-pro-BDNF gene was ligated with the CMV -promoter at its 5 '-end and with the bovine growth hormone polyadenylation sequence at its 3'- end.The amino acid sequence of the wild type pre-pro-BDNF protein is shown in SEQ ID NO: 20. b) Generation of the expression plasmids for BDNF variants

In order to obtain optimal production yield several BDNF variants were constructed (see Table below):

in some variants the wild-type BDNF signal sequence (pre-segment) was exchanged by an signal sequence which is derived from a highly expressed murine immunoglobulin heavy chain antibody

(MG WS CIILFL V AT ATG VHS) ;

in some variants the codon usage of the encoding BDNF gene was exchanged to an optimized codon usage in the pro-segment and/or in the mature part of BDNF; the BDNF genes with optimized codon usages were obtained by backtranslation of the amino respective acid sequence using algorithms from

Geneart (see e.g. Fath, S., et al, PLOS One 6 (2011) el7596); in some variants a T7-His6-tag (SEQ ID NO: 12) was used, since it is generally believed to enhance protein expression (see e.g. Luan, C.H., et al, Genome Res. 14 (2004) 2102-2110);

in most BDNF variants a His6-tag was included in order to simplify sample preparation/purification;

in some variants the last three C-terminal amino acids, the RGR motif of mature BDNF, was deleted, since it might function as cryptic protease cleavage site for proteases like furin or other PC convertases;

in another variant the signal sequence and pro-segment of BDNF was exchanged by the corresponding amino acid sequence of human NGF, since it was published that this variation improves expression for another neurotrophin (Iwane et al., Appl. Microbiol. Biotechnol. 41 (1994) 225-232).

Table 10

1 : C-terminal RGR-motif of BDNF deleted (deltaRGR).

2: Signal sequence derived from a highly expressed murine immunoglobulin heavy chain antibody (MGWSCIILFLVATATGVHS) as defined by the amino acid sequence shown in SEQ ID NO: 14.

3: Signal sequence and pro-fragment of BDNF exchanged by corresponding sequence from human NGF c) Generation of the expression plasmids for pre-pro(IgA)-BDNF and pre- proflgA; His6) variants, partially including a His6-tag N-terminally of mature BDNF within the pro-segment

The pre-pro-(IgA)-BDNF gene codes for a pro-polypeptide variant wherein the naturally occurring furin cleavage site (RVRR) was replaced by the engineered IgA protease cleavage site with the sequence GSVVAPPAP (see Table below).

In addition, in some variants a R54A point mutation was introduced into the pro- polypeptide of BDNF to destroy a putative protease cleavage site (see Mowla, S.J., et al., J. Biol. Chem. 276 (2001) 12660-12666). Furthermore, in some variants also a removable His6-tag was included N-terminally of mature BDNF within the pro- fragment. This tag simplifies protein purification of the pro(IgA; His6)-BDNF variant protein. Upon final in vitro protein maturation with IgA protease the pro(IgA; His6 fragment) is removed and, thus, a potential risk of immunogenicity is avoided. The expression plasmids for the transient expression of the pre-pro(IgA)-BDNF and pre-pro(IgA; His6)-BDNF variant genes/proteins in HEK293 cells are derived from the expression vector described above which encodes for the pre-pro- BDNF(-RGR)-T7-His6 protein. They differentiate in the following characteristics:

Table 11

The numbering of the R54A mutation is based on the amino acid sequence of wildtype pre-pro-BDNF (SEQ ID NO: 20) d) Generation of the expression plasmids for isoelectric point engineered BDNF variants Previously some in E. coli expressed and refolded BDNF variants have been described with an engineered lowered IEP. The pre-pro-BDNF(-RGR)-T7-His6 gene was mutated accordingly and the mutant BDNF genes obtained transiently expressed in HEK293 cells (see Table below). In addition, a myc-tagged BDNF variant was constructed, since (1) the myc-tag (EQKLISEEDL; SEQ ID NO: 90) introduces a net charge difference of about -3 and (2) the myc-tag is of human origin and thus supposed to be less immunogenic.

Table 12

-RGD and (deltaRGR); deletion of the last 3 C-terminal amino acids of mature BDNF;

Numbering of amino acid mutations starts at the first amino acid of mature BDNF;

The IEP of the matured BDNF(-RGR) variants were calculated using the protein statistic program pepstats from the European Molecular Biology Open Software Suite (EMBOSS). e) Generation of the expression plasmids for BDNF variants with additional glycosylation sites

BDNF variants were generated which harbor (1) a C -terminal tag containing glycosylation sites or (2) one engineered glycosylation site within the matured BDNF moiety.

The glycosylation tags were deduced from published sequences (e. g. Meder, D., et al, J. Cell Biol. 168 (2005) 303-313; Bulbarelli, A., et al, J. Cell Sci. 115 (2002) 1689-1702; Perlman, S., et al, J. Clin. Endocrinol. Metab. 88 (2003) 3227-3235; WO 2002/002597) and putative N-glycosylation sites were predicted with an artificial neuronal network (NetNglyc server; http ://www.cbs .dtu. dk/services/NetNGlyc/) .

For introduction of N-glycosylation sites within the maturated BDNF moiety the sequence was inspected for the presence of asparagins, serins or threonins within the matured BDNF sequence. Then, based on the three-dimensional protein structure of human BDNF (lbnd; http://www.rcsb.org") all non-surface localized

Asn, Ser or Thr residues were excluded. For the remaining surface exposed Asn, Ser and Thr residues the adjacent amino acid residues were identified in order to engineer a putative N-glycosylation site (consensus motif: N-X-(S/T), X=any amino acid except Pro) by site-directed mutagenesis. The amino acid position of these putative engineered N-glycosylation sites were used to identify the corresponding amino acids in the structurally and functionally homologous neurotrophins NGF and NT-3 by sequence alignment and protein 3D-structure comparisons. Amino acid positions were excluded that are expected to be part of the neurotrophin::p75NTR or neutrophin::Trk(A, B) interaction interface based on homologous receptor: :ligand crystal structures (e.g. 3buk, 3ij2 and 2ifg). Selected mutations for surface-exposed putative N-glycosylation sites outside the putative BDNF-TrkB/p75 interaction interfaces are mentioned in the Table below (second column).

The expression plasmids for the transient expression of N-glycosylated BDNF variant proteins in HEK293 cells are derived from the expression vector coding for the BDNF variant pre-pro-BDNF(-RGR)-T7-His6 (-RGR: a truncated mature wild- type BDNF wherein the last 3 C-terminal amino acids RGR are deleted; a T7-tag and a His6-tag is attached to the truncated C-terminus of BDNF via a GSG-linker). The BDNF segments, tags, glycolinkers and mutations introduced for the generation of additional artificial N-glycosylation sites are presented following table.

Table 13

SEQ ID NOs for amino acid mutations introduced by site-directed mutagenesis into mature BDNF(deltaRGR) are exemplarily shown for the BDNF variants pre-pro- BDNF(-RGR; M61T)-T7-His6 and pre-pro-BDNF(-RGR; R81N)-T7-His6. f) Generation of the expression plasmids for BDNF variants containing combined mutations for the introduction of multiple (two or more) N- glycosylation sites and an IgA cleavage site

In order to generate BDNF variants with multiple N-glycosylation sites some of the additional N-glycosylation sites identified in previous experiments were combined. The starting construct pre-pro(IgA)-BDNF(-RGR)-His6 variant polypeptide is characterized by an IgA protease cleavage site instead of the native furin site within the pro-segment, a C-terminally truncated mature BDNF (deletion of the last 3 amino acids RGR) and a C-terminal His6-tag. The desired mutations and tags were introduced/attached as shown in the Table below.

Table 14

opsn - s g) Generation of the expression plasmids for BDNF variants containing combined mutations for the introduction of multiple (two or more) N- glycosylation sites

In order to generate BDNF variants with multiple N-glycosylation sites some of the additional N-glycosylation sites identified in previous experiments were combined. For this purpose the pre-pro-BDNF(-RGR)-T7-His6 variant protein characterized by a C-terminally truncated mature BDNF (deletion of the last 3 amino acids RGR) and a C-terminal T7-His6-tag was used as starting material. The desired mutations were inserted as shown in the Table below.

Table 15

BDNF variant additional glycosylation sites SEQ ID (description of NO (aa construct features) sequence):

K25 T35 G62N, Q79T R81N

N N Y63G

pre-pro-BDNF(-RGR; X X X 70

K25N; T35N; Q79T)-T7- His6

Example 7

Generation of BDNF antibody fragment fusion-polypeptides expression plasmids a) Generation of the expression plasmids for the BDNF-Fab antibody heavy chain fusion-polypeptides

In order to obtain BDNF-(Gly₄Ser)_n-Fab(anti-IGF-lR antibody heavy chain) fusion-polypeptides, plasmids for transient expression in HEK293 cells were constructed which harbored a chemically synthesized DNA fragment of a CDS (coding DNS sequence) coding for polypeptides with the following characteristics: - the wild-type pre-pro-BDNF moiety deleted for the C-terminal RGR motif is fused at the C-terminus with a glycine -rich linker followed by a Fab heavy chain portion (VH-CH1) of the human anti-IGF-lR antibody and a C- terminal His6-tag; the glycine-rich linker consists of a (G4S)2-GG or a (G4S)4-GG or a (G4S)6- GG motif (see Table below).

Table 16

BDNF-antibody variant, linker Fab Tag SEQ ID (description of construct between fragment NO (aa features) BDNF (anti- sequence):

and Fab IGF-1R

fragment mAb)

pre-pro-BDNF(- (G₄S)₂- VL- His6 71

RGD) (G4S)2GG VL<IGF-1R>- GG Ckappa

Ck-His6

pre-pro-BDNF(- (G₄S)₄- VL- His6 72

RGD) (G4S)4GG VL<IGF-1R>- GG Ckappa

Ck-His6 BDNF-antibody variant, linker Fab Tag SEQ ID (description of construct between fragment NO (aa features) BDNF (anti- sequence):

and Fab IGF-1R

fragment mAb)

pre-pro-BDNF(- (G₄S)₆- VL- His6 73

RGD) (G4S)6GG VL<IGF-1R>- GG Ckappa

Ck-His6

pre-pro-BDNF(- (G₄S)₂- VH-CH1 His6 74

RGD) (G4S)2GG VH<IGF-1R>- GG

CH1-His6

pre-pro-BDNF(- (G₄S)₄- VH-CH1 His6 75

RGD) (G4S)4GG VH<IGF-1R>- GG

CH1-His6

pre-pro-BDNF(- (G₄S)₆- VH-CH1 His6 76

RGD) (G4S)6GG VH<IGF-1R>- GG

CH1-His6 b) Generation of the expression plasmids for the BDNF-Fab antibody light chain fusion-polypeptides

In order to obtain BDNF-(Gly₄Ser)_n-Fab(anti-IGF-lR antibody light chain) fusion- polypeptides, plasmids for transient expression in HEK293 cells were constructed which harbored a chemically synthesized DNA fragment of a CDS coding for polypeptides with the following characteristics: the wild-type pre-pro-BDNF moiety deleted for the C-terminal RGR motif is fused at the C-terminus with a glycine-rich linker followed by the Fab VL- Ckappa light chain domains of the human anti-IGF-lR antibody and a C- terminal His6 tag; the glycine-rich linker consists of a (G4S)2-GG or a (G4S)4-GG or a (G4S)6- GG motif (see Table above). c) Generation of the expression plasmid for the anti-IGF-lR antibody light chain

The native anti-IGF-lR antibody light chain was used for the generation anti-IGF- 1R based BDNF-Fab complexes. The generation of the anti-IGF-lR antibody light chain expression plasmid is described in example 1. d) Generation of the expression plasmid for the BDNF-Fab(anti-IGF-1R antibody heavy chain) fusion-polypeptides containing a negatively charged Gly-Asp linker

In order to obtain BDNF-(G3D)4-Fab(anti-IGF-1R) antibody heavy chain fusion- polypeptide (pre-pro-BDNF(-RGD)_(G3D)5 -G3 S_VL<IGF- 1 R>-Ck-His6), a plasmid for transient expression in HEK293 cells was constructed which harbored a chemically synthesized DNA fragment of a CDS coding for the polypeptide with the following characteristics:

the wild-type pre-pro-BDNF moiety deleted for the C-terminal RGR motif is fused at the C-terminus with a glycine-rich negatively charged linker followed by the Fab VH-CH1 heavy chain domains of the human anti-IGF- 1R antibody and a C-terminal His6-tag:

the glycine-rich negatively charged linker consists of the (G3D)4-GGGS motif. e) Generation of the expression plasmids for the BDNF-Fab(anti-IGF-1R antibody heavy chain) fusion proteins harboring an IgA cleavage site within the pro-BDNF segment, a negatively charged GlvAsp linker and multiple N- glycosylation sites

In order to obtain pro(IgA)-BDNF-(G3D)4-Fab(anti-IGF-lR antibody heavy chain fusion-polypeptides with multiple N-glycosylation sites, plasmids for transient expression in HEK293 cells were constructed which harbored a chemically synthesized DNA fragment of a CDS which code for polypeptides with the following characteristics:

the pre-pro(IgA)-BDNF moiety deleted for the C-terminal RGR motif of mature BDNF is fused at the C-terminus with the extended opsin-tag (NGTEGPNF Y VPF SN ATG V VR; opsin(L); SEQ ID NO: 16) followed by a negatively charged glycine-aspartic-acid-rich linker and a Fab heavy chain portion (VH-CH1, partially extended with the hinge-derived peptide EPKSC) of the human monoclonal antibody directed against human insulin-like growth factor 1 (IGF-1R) and a C-terminal His6-tag;

the negatively charged glycine-aspartic-acid-rich linker consists either the (G3D)4-GGGS motif or the (G2D)5-G2SG motif.

The details of the constructs are summarized in the Table below. Table 17

f) Generation of the expression plasmids for the BDNF-Fab(anti-transferrin receptor antibody heavy chain) fusion-polypeptides harboring an IgA cleavage site within the pro-BDNF segment, a negatively charged GlyAsp linker and multiple N-glycosylation sites

In order to obtain pro(IgA)-BDNF-(G3D)4-Fab(anti-TfR) antibody heavy chain fusion-polypeptides with a negatively charged GlyAsp linker and multiple N- glycosylation sites, plasmids for transient expression in HEK293 cells were constructed which harbored a chemically synthesized DNA fragment of a CDS which code for polypeptides with the following characteristics:

the pre-pro(IgA)-BDNF moiety deleted for the C-terminal RGR motif is fused at the C-terminus with the opsin(L)-tag followed by a negatively charged glycine-aspartic-acid-rich linker and a chimeric rat/human Fab heavy chain portion wherein the VH variable domain is derived from the rat 8D3 monoclonal antibody which is directed against the mouse transferrin receptor

(mTfR) and wherein the CHI domain is derived from human IgGland a C- terminal His6-tag; the amino acid sequence of VH domain of the antibody was obtained from Boado, R.J., et al., Biotechnol. Bioeng. 102 (2009) 1251- 1258); the negatively charged glycine-aspartic-acid-rich linker consists of the (G3D)4-GGGS motif;

in most variants the endogenous furin/PC convertase cleavage site between the pro-segment and the mature part of BDNF was exchanged by an IgA

5 protease cleavage site (GSVVAPPAP);

in some variants the truncated wild-type mature BDNF moiety (BDNF;

deltaPvGR) was exchanged by a truncated mature BDNF variant harboring an artificial R209N glycosylation site (BDNF(deltaRGR; R209N)).

The details of the constructs are summarized in the Table below.

10 Table 18

BDNF-antibody variant, His6- protease introduced linker origin of hinge SEQ (description of construct features) tag cleavage additional N- between Fab heavy EPKS ID NO site glycosylation BDNF and chain C (aa sites Fab fragment sequen fragment ce): opsin BDNF(A

-tag RGR)

moiety

pre-pro(IgA)-BDNF(-RGR) (G3D)4- His6 IgA none none (G3D)4- <TfR>8D3 yes 107 G3S VH<TfR>8D3-CHl-EPKSC- GGGS

His6

pre-pro(IgA)-BDNF(-RGR; His6 IgA opsin R81N GSG- <TfR>8D3 no 81 R81N) opsin(L)-(G3D)4- (L) opsin(L)- G3S_VH<TfR>8D3-CHl-His6 (G3D)4- GGGS

pre-pro(IgA)-BDNF(-RGR) (G3D)4- His6 IgA none none (G3D)4- <TfR>8D3 no 108 G3S VH<TfR>8D3-CHl-His6 GGGS

pre-pro(IgA)-BDNF(-RGR) (G3D)4- none IgA none None (G3D)4- <TfR>8D3 No 109 G3S VH<TfR>8D3-CHl GGGS

pre-pro(IgA)-BDNF(- His6 IgA opsin none GSG- <TfR>8D3 No 79 RGR) opsin(L)-(G3D)4- (L) opsin(L)- G3S VH<TfR>8D3-CHl-His6 (G3D)4- GGGS

Example 8

Generation of the expression plasmid for the BDNF-scFv fusion-polypeptides

In order to obtain a BDNF(G4S)3-scFvanti-IGF-lR antibody heavy chain-fusion- polypeptide, a plasmid for transient expression in HEK293 cells was constructed which harbored a chemically synthesized DNA fragment of a CDS which codes for a polypeptide with the following characteristics:

the wild-type pre-pro-BDNF moiety deleted for the C-terminal RGR motif is fused at the C-terminus with a (G4S)3 linker followed by a scFv moiety of the human anti-human IGF-1R antibody;

- the scFv moiety of the anti-human IGF-1R antibody is build up of a VL domain, followed by a (G4S)4-GG linker, a VH region and a His6-tag; the amino acid sequence of the pre-pro-BDNF(-RGR)_(G4S)3_scFv-His6<IGF- 1R> fusion protein is shown in SEQ ID NO: 78.

Example 9

Transient expression, purification and analytical characterization of polypeptides

Transient expression

The polypeptides were generated by transient transfection of HEK293 cells (human embryonic kidney cell line 293 -derived) cultivated in F17 Medium (Invitrogen Corp.). For transfection "293-Free" Transfection Reagent (Novagen) was used. The antibody and antibody fragment (Fab and scFv) comprising fusion-polypeptides were expressed from one, two or three different plasmids using an equimolar plasmid ratio upon transfection. Transfections were performed as specified in the manufacturer's instructions. The recombinant polypeptide-containing cell culture supernatants were harvested four to seven days after transfection. Supernatants were stored at reduced temperature until purification.

General information regarding the recombinant expression of human immunoglobulins in e.g. HEK293 cells is given e.g. in Meissner, P., et al, Biotechnol. Bioeng. 75 (2001) 197-203. Purification a) GFP fusion-polypeptides were purified using a two-step procedure including a protein A chromatography and a size exclusion chromatography on a Superdex 200™ column

GFP fusion-polypeptide containing culture supematants were filtered. Thereafter the GFP fusion-polypeptides were captured by affinity chromatography using HiTrap MabSelectSuRe (GE Healthcare) equilibrated with PBS (1 mM KH₂P0₄, 10 mM NaHP0₄, 137 mM NaCl, 2.7 mM KC1, pH 7.4). Unbound polypeptides were removed by washing with equilibration buffer, and the fusion-polypeptide was recovered with 0.1 M citrate buffer, pH 2.8. Immediately after elution the fractions were neutralized to pH 6.0 with 1 M Tris-base, pH 9.0.

Size exclusion chromatography on Superdex 200™ (GE Healthcare, Uppsala, Sweden) was used as second purification step. The size exclusion chromatography was performed in 50 mM histidine buffer, 0.15 M NaCl, pH 6.8. The recovered GFP fusion-polypeptides were stored at -80 °C. b) Histidine -tagged proteins were purified using a two-step protocol starting with an immobilized metal ion affinity chromatography (IMAC) and followed by a size exclusion chromatography on a Superdex 75TM column

The histidine -tagged polypeptide-containing culture supematants were adjusted with NaCl to a final NaCl concentration of 500 mM. The filtered culture supernatant was loaded onto a Ni-Sepharose™ 6 Fast Flow column pre- equilibrated with a NiA-buffer (50 mM TRIS, 300 mM NaCl, 5 mM imidazole containing an EDTA-free protease inhibitor cocktail tablet as specified in the manufacturer's instructions; EDTA-free Complete Mini Tablets; Roche Applied Science) at a flow of 1 ml/min using an AKTA explorer 100 system (GE Healthcare, Uppsala, Sweden). The column was washed with NiA-buffer until the UV reading reached back close to baseline. The histidine-tagged polypeptide was eluted with a 5 mM to 300 mM linear imidazole gradient in 50 mM TRIS and 500 mM NaCl, pH 8.0 in 10 column volumes.

Size exclusion chromatography on Superdex 75™ (GE Healthcare, Uppsala, Sweden) was used as second purification step. The size exclusion chromatography was performed in 50 mM histidine buffer, 0.15 M NaCl, pH 6.8. The eluted histidine-tagged proteins were stored at -80 °C. Analytical characterization

The protein concentrations of the purified polypeptides were determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and proper dimer formation of polypeptides were analyzed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1 ,4-dithiotreitol) and staining with Coomassie brilliant blue. Aggregate content of the Fc-fusion-polypeptide preparations was determined by high-performance SEC using a Superdex 200™ analytical size- exclusion column (GE Healthcare, Uppsala, Sweden). The integrity of the amino acid backbone of reduced polypeptides was verified by Nano Electrospray QTOF mass spectrometry after removal of N-glycans by enzymatic treatment with a combination of neuraminidase, O-glycanase and peptide-N-glycosidase F (Roche Applied Science, Mannheim, Germany).

Determination of the BDNF concentration in culture supernatants The concentration of wild-type BDNF, BDNF variants and BDNF containing fusion-polypeptides in culture supernatants was determined by semi-quantitative Western Blot analysis using recombinant human BDNF from Peprotech (catalog number: 450-02) as reference standard. A rabbit anti-BDNF antibody (Santa Cruz; catalog number: sc-20981) (first antibody) and a horseradish peroxidase conjugated sheep anti-rabbit antisera (diluted 1 :5000, Roche Diagnostics GmbH, Germany)

(secondary antibody) and enhanced chemiluminescence substrate (LUMI-Light plus Western Blotting substrate, Roche Diagnostics GmbH, Germany) was used for staining.

The concentration of BDNF fusion-polypeptides was also determined with a BDNF ELISA using the BDNF Emax® ImmunoAssay Kit from Promega (catalog number: G7610) according to the instructions of the supplier. Example 10

In vitro functional characterization

Determination of the biological activity of wild-type GFP and GFP-containing fusion-polypeptides The biological activity of purified wild-type GFP and GFP-containing fusion- polypeptides was monitored by its bioluminescent properties (GFP-specific fluorescence).

Determination of the BDNF binding affinity via surface plasmon resonance (SPR, BIAcore) Amine coupling of around 750 resonance units (RU) of a capturing system (capturing mAb specific for human IgG, Jackson Immunoresearch) was performed on a CM5 chip at pH 4.5 using an amine coupling kit according to the manufacturer's manual (supplied by GE Healthcare, Uppsala, Sweden). Human Fc- tagged TrkB (R&D Systems, catalog number: 688-TK-100) was captured at a concentration of 5 μg/ml. Excess binding sites were blocked by injecting a human

Fc mixture at a concentration of 1.25 μΜ (Biodesign, catalog number: 50175). Different concentrations of BDNF containing fusion-polypeptides ranging from 0.1 nM to 50 nM were passed with a flow rate of 10 μΕ/ηιίη through the flow cells at 298 K for 120 to 240 sec. The dissociation phase was monitored for up to 600 sec and triggered by switching from the sample solution to running buffer. The surface was regenerated by 1 min washing with a 100 mM phosphoric acid solution at a flow rate of 30 μΕ/ηιίη. For all experiments HBS-P+ buffer supplied by GE Healthcare was chosen (10 mM HEPES ((4-(2-hydroxyethyl)-l-piperazine ethanesulfonic acid)), pH 7.4, 150 mM NaCl, 0.05 % (v/v) Surfactant P20). Bulk refractive index differences were corrected for by subtracting the response obtained from a blank-coupled surface. Blank injections are also subtracted (double referencing).

The equilibrium dissociation constant (Kd), defined as ka/kd, was determined by analyzing the sensorgram curves obtained with several different concentrations, using BIAevaluation 4.1 software package. The fitting of the data followed a suitable binding model. Determination of the BDNF binding affinity via ELISA

The binding properties of BDNF-containing fusion proteins were determined with a TrkB ELISA. Maxisorb plates were coated with 1 μg/mL of a TrkB-Fc fusion (R&D Systems) in PBS overnight at 4 °C. After blocking the plate with PBSTC (PBS with 0.05 % Tween-20 and 2 % Chicken serum (Gibco)) for 1 h at RT and three washes with PBST, BDNF-containing fusion proteins or BDNF alone were added to the wells at concentrations of 15 to 500 ng/mL in PBSTC and incubated for 2 h at RT. After six washes, the wells were incubated with mouse-anti-BDNF antibody (clone 4F11.1A1, 1 μg/mL in PBSTC) and, after further washes, with anti-mouse-HRP antibody (1 : 10000 in PBSTC), both for 1 h at RT. After three washes with PBST, HRP activity was detected using ABTS substrate and photometric quantification at a wavelength of 492 nm.

Determination of the biological activity of BDNF containing fusion polypeptides via a TrkB reporter gene assay The biological activity of BDNF variants and BDNF-containing fusion polypeptides was determined with a TrkB-receptor-transfected CHO cell line containing a stably transfected luciferase reporter gene under the control of a SRE (serum-response element)-containing -promoter (CHO-Cl-fiTrkB/pSRE-Luc). The day before the experiments cell medium was changed from growth medium (Ham's F12 containing 10 % FCS, 2 mM L-glutamine, 300 μg/mL G418 and 3 μg/mL puromycin) to the same medium without FCS for starvation. The next day, 10⁵ cells were seeded per well of a 96-well plate in 50 μί medium. Then BDNF fusion proteins were added at concentrations between 0.02 nM and 115 nM, in 50 μΐ, medium. After incubation for 4 h at 37 °C, 7.5 % C0₂, cells were equilibrated for 30 min at RT and 100 μΐ_^ of BrightGlo Luciferase Assay reagent

(Promega) was added per well. Luminescence was read out after 5 minutes incubation using a Tecan plate reader (integration time 100 ms).

Example 11

In vivo pharmacokinetic and pharmacodynamic Determination of the biological activity of BDNF containing fusion proteins in a

SH-SY5Y neurite outgrowth assay

The biological activity of BDNF variants and BDNF-containing fusion proteins was determined with a neurite outgrowth assay using human SH-SY5Y neuroblastoma cells. Briefly, SH-SY5Y cells were plated in a 96-well plate at 4000 cells per well in normal growth medium (Ham's F12, 1 x non-essential amino acids (PAN), 10 % FCS, 2 mM L-glutamine, 1 x sodium pyruvate (PAN)) under addition of 10 μΜ retinoic acid (Sigma) to induce neuronal differentiation. After three days, medium was replaced with growth medium containing different concentrations of BDNF fusion proteins. After three additional days, cells were fixed using 4 % paraformaldehyde in PBS for lO min. at RT, washed, briefly permeabilized (0.1 % Triton-X-100), blocked with 1 % BSA in PBS and stained for anti-beta-tubulin immunoreactivity using the TuJl antibody (Covance) at a dilution of 1 : 1000 in PBS/1 % BSA, followed by three washes and incubation with a Alexa-

488-labeled anti-goat antibody (Invitrogen). Numbers of neurites were determined by fluorescence microscopy, evaluating one visual field per well.

Determination of the binding activity of an antibody/antibody fragment containing fusion proteins via FACS The binding activity of BDNF fusion proteins to the respective target receptors

(transferrin receptor, IGF-1R) was determined by FACS. Cells expressing the respective receptor (mouse transferrin receptor: MEF-1 mouse embryonal fibroblasts; IGF-1R: 3T3 fibroblasts stably transfected with human IGF-1R) were harvested from their growth media, washed with PBS, and resuspended in FACS buffer (PBS + 5 % FCS; 100 μΐ, containing 3 x 10⁵ cells per well of 96-well round- bottom plate). Primary antibody (depending on the BDNF fusion protein used, e.g., anti-human-Fab (Jackson ImmunoResearch) or anti-His6 antibody (Roche)) was added at 1-10 μg/mL and cells incubated for two hours on ice. After three washes with FACS buffer, bound antibody was detected using PE-labeled secondary antibody (Jackson ImmunoResearch, 1 :5000 - 1 : 10000) for one hour on ice. Cells were washed again and mean fluorescence measured on a FACS Canto cytometer (Becton-Dickinson) .

Claims

Patent Claims

A method for recombinantly producing a polypeptide in soluble form, characterized in comprising the following steps: a) transfecting a eukaryotic cell with a nucleic acid encoding the polypeptide, whereby the polypeptide has been modified (compared to the wild-type polypeptide) by the introduction of one or more artificial glycosylation sites selected from the group comprising NGT (SEQ ID NO: 122), NWT (SEQ ID NO: 123), NST (SEQ ID NO: 124) and NDT (SEQ ID NO: 125),

b) cultivating the transfected cell in a cultivation medium, and

c) recovering the polypeptide from the cultivation medium, whereby the yield (determined after one purification step) is increased by at least 100 % compared to the wild-type polypeptide.

The method according to claim 1 , characterized in that the glycosylation site is an N-glycosylation site.

The method according to any one of claims 1 to 2, characterized in that the yield is the yield of monomeric polypeptide.

The method according to any one of claims 1 to 3, characterized in that the polypeptide is a fusion polypeptide comprising at least a first part and a second part conjugated to each other by a peptidic linker.

The method according to any one of claims 1 to 4, characterized in that the glycosylation site is in a peptidic linker.

The method according to any one of claims 4 to 5, characterized in that each peptidic linker comprises one N-glycosylation site.

The method according to any one of claims 4 to 6, characterized in that the peptidic linker is selected from the group comprising GGGSGGNGTGGGGSGG (SEQ ID NO: 26), GGGGSGGNGTGGGGSGG (SEQ ID NO: 27), GGSGGGNWTG (SEQ ID NO: 28), GGSGGGGSGGGNSTG (SEQ ID NO: 29), GGGGSGGGNSTGG (SEQ ID NO: 30), GGSGGGNDTG (SEQ ID NO: 31), and GGSGGGNGTG (SEQ ID NO: 32).

8. The method according to any one of claims 1 to 7, charatcerized in that the polypeptide is recovered from the cleared cultivation supernatant after cell separation and filtration.

9. The method according to any one of claims 1 to 8, characterized in that the yield is increased by at least 250 %.

10. The method according to any one of claims 1 to 9, characterized in that the artificial glycosylation site has been introduced by making one, or two or three sequential amino acid changes (mutations) into the amino acid sequence of the polypeptide (to generate an artificial glycosylation site sequon), or by fusing a peptidic linker containing a glycosylation site to the polypeptide.

11. The method according to any one of claims 4 to 10, characterized in that the fusion polypeptide is a fusion polypeptide comprising three or more entities, whereby each entity is fused at most at each terminus to a further entity by a peptidic linker, whereby in each of the peptidic linkers an artificial glycosylation site (an artificial glycosylation site sequon) is introduced.

12. Use of one or more artificial glycosylation sites for the increase of the yield of recombinantly produced soluble polypeptide.

13. The use according to claim 12, characterized in that the yield is of recombinantly produced soluble monomeric polypeptide.