Method for producing monoclonal antibodies

Abstract

Provided herein, inter alia, are compositions and methods relating to the production of monoclonal antibodies and / or functional fragments thereof that exhibit reduced proteolysis during fermentation, isolation and purification.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 389,472 filed on July 15, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Incorporation by Reference into the Sequence Listing

[0004] This application is submitted together with a sequence listing in electronic format. The sequence listing is provided as a file named NB41904-WO-PCT.xml created on June 30, 2023, and the file size is 19,451 bytes. The information in the electronic sequence listing is incorporated by reference in its entirety. Technical Field

[0005]

[0013] Provided herein, inter alia, are methods for producing antibodies and antigen-binding fragments thereof having one or more advantages, such as, but not limited to, reduced proteolytic degradation. Background Art

[0006] The therapeutic agent based on antibody has been successfully used to treat a variety of diseases, including cancer and autoimmune / inflammatory conditions. Antibodies also work in diagnosis and other medical devices. Due to the limitation of the amount of host cell protein production (mainly human embryonic kidney cell (HEK) and Chinese hamster ovary (CHO) cell), the poor thermal stability of the antibody manufactured, and the sensitivity to proteolytic cutting during fermentation, processing and purification, the large-scale manufacture of antibodies has been hindered. Therefore, it is necessary to be used to produce an improved alternative technology for the biotherapeutic agent based on antibody, which causes the improvement of manufacturability. Ideally, these improved technologies will result in higher antibody yields, thereby being conducive to large-scale production with cost and volume target, which will enable them to be used as traditional therapeutic agents and diagnosis, and can also be used as local and oral products for human and animal health.

[0007] The subject matter disclosed herein addresses these needs and provides additional benefits. Summary of the invention

[0008] In particular, a recombinant cell is provided herein, comprising a) a heterologously expressed barley alpha-amylase subtilisin inhibitor (BASI) polypeptide; and b) a heterologously expressed monoclonal antibody or a functional fragment thereof. In some embodiments, the antibody or a functional fragment thereof is a therapeutic antibody or a functional fragment thereof. In some of any embodiments disclosed herein, the cell is a bacterial, fungal, yeast, plant or mammalian cell. In some embodiments, the cell is a Trichodermareesei cell or an Aspergillus niger cell. In some embodiments, the cell is a Bacillus subtilis cell. In some embodiments, the cell is a Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cell. In some of any embodiments disclosed herein, the BASI polypeptide comprises the amino acid sequence of SEQ ID NO: 1. In some of any embodiments disclosed herein, the antibody or a functional fragment thereof exhibits less proteolytic degradation than an antibody or a functional fragment thereof that is not heterologously co-expressed with a BASI polypeptide. In some embodiments, the functional fragment is selected from the group consisting of: a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a disulfide-linked Fv (dsFv), a Fd fragment, a Fd' fragment, a single-chain Fv (scFv), a single-chain Fab (scFab), a diabody, an anti-idiotypic (anti-Id) antibody, or an antigen-binding fragment of any one thereof.

[0009] In other aspects, provided herein is a fermentation broth comprising any of the recombinant cells disclosed herein.

[0010] In a further aspect, provided herein is a method for reducing proteolysis of a heterologously expressed monoclonal antibody or a functional fragment thereof, the method comprising culturing a recombinant cell under suitable conditions for producing a heterologously expressed antibody or a functional fragment thereof and a barley alpha-amylase subtilisin inhibitor (BASI) polypeptide, the recombinant cell comprising a) a heterologously expressed BASI polypeptide; and b) a heterologously expressed monoclonal antibody or a functional fragment thereof. In some embodiments, the method further comprises isolating the antibody or a functional fragment thereof. In some of any embodiments disclosed herein, the antibody or a functional fragment thereof is a therapeutic antibody or a functional fragment thereof. In some embodiments, the cell is a bacterial, fungal, yeast, mammalian, or plant cell. In some embodiments, the cell is a Trichoderma reesei cell or an Aspergillus niger cell. In some embodiments, the cell is a Bacillus subtilis cell. In some embodiments, the cell is a Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cell. In some of any embodiments disclosed herein, the BASI polypeptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the antibody or a functional fragment thereof exhibits less proteolytic degradation than an antibody or a functional fragment thereof that is not heterologously co-expressed with a BASI polypeptide. In some of any of the embodiments disclosed herein, the functional fragment is selected from the group consisting of: a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a disulfide-linked Fv (dsFv), a Fd fragment, a Fd' fragment, a single-chain Fv (scFv), a single-chain Fab (scFab), a diabody, an anti-idiotypic (anti-Id) antibody, or an antigen-binding fragment of any one thereof.

[0011] In another aspect, provided herein is a method for reducing proteolysis of a recombinantly expressed monoclonal antibody or a functional fragment thereof, the method comprising isolating the recombinantly expressed antibody or a functional fragment thereof in the presence of an exogenously added barley alpha-amylase subtilisin inhibitor (BASI) polypeptide. In some embodiments, the antibody or a functional fragment thereof is a therapeutic antibody or a functional fragment thereof. In some of any embodiments disclosed herein, the BASI polypeptide is recombinantly expressed in bacteria, fungi, yeast, mammals, or plant cells. In some embodiments, the BASI polypeptide is recombinantly expressed in Bacillus subtilis cells or Aspergillus niger cells. In some of any embodiments disclosed herein, the BASI polypeptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the monoclonal antibody or a functional fragment thereof is recombinantly expressed in bacteria, fungi, yeast, mammals, or plant cells. In some embodiments, the monoclonal antibody or a functional fragment thereof is recombinantly expressed in Trichoderma reesei cells or Aspergillus niger cells. In some embodiments, the monoclonal antibody or a functional fragment thereof is recombinantly expressed in Bacillus subtilis cells. In some embodiments, the monoclonal antibody or a functional fragment thereof is recombinantly expressed in Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells. In some of any of the embodiments disclosed herein, the antibody or its functional fragment exhibits less proteolytic degradation and / or increased intact protein yield compared to the antibody or its functional fragment isolated in the absence of exogenously added BASI polypeptide. In some embodiments, the functional fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a disulfide-linked Fv (dsFv), a Fd fragment, a Fd' fragment, a single-chain Fv (scFv), a single-chain Fab (scFab), a diabody, an anti-idiotypic (anti-Id) antibody, or an antigen-binding fragment of any one thereof.

[0012] Each of the aspects and embodiments described herein can be used together unless explicitly or clearly excluded from the context of an embodiment or aspect.

[0013] Throughout the specification, various patents, patent applications, and other types of publications (e.g., journal articles, electronic database entries, etc.) are cited. The disclosures of all patents, patent applications, and other publications cited herein are hereby incorporated by reference in their entirety for all purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1Depicted are DNAs used to introduce assembled cassettes at (A and B) the I1 locus and (C and D) the 1A locus. For each strain, A or B was combined with C or D for transformation. The DNA pairs depicted in AD each introduced HC and LC for one antibody at one locus. A and D introduced only the antibody expression cassette. B also introduced the BASI expression cassette. C also introduced the accessory protein cassette.

[0015] Figure 2 Graph depicting the positive effect of BASI co-expression on the titer (size exclusion chromatography (SEC), right axis) of a 150 kDa antibody species expressed in a Trichoderma reesei (BASI 1 SP1) strain. Protein A-based and SEC-based titers of a BASI-negative strain (sp1) appear to reach a maximum early on and then decline as the fermentation run progresses.

[0016] Figure 3 A 4%-12% SDS-PAGE reducing gel is depicted. The first panel on the left is a molecular weight marker in kd, the second panel on the left shows samples stored at -20°C, the third panel on the left shows samples stored at 5°C for 7 days, and the right panel shows samples stored at 5°C for 28 days. Lanes labeled 1 = no BASI; 2 = 0.5 g BASI filtrate / g broth; 3 = 1 g BASI filtrate / g broth; and 4 = 3 g BASI filtrate / g broth.

[0017] Figure 4 A 4%-12% SDS-PAGE reducing gel is depicted. The first panel on the left is a molecular weight marker in kDa, the second panel on the left shows samples stored at -20°C, the third panel on the left shows samples stored at 22°C for 7 days, and the right panel shows samples stored at 22°C for 28 days. Lanes labeled 1 = no BASI, 2 = 0.5 g BASI filtrate / g broth, 3 = 1 g BASI filtrate / g broth, and 4 = 3 g BASI filtrate / g broth.

[0018] Figure 5 A 4%-12% SDS-PAGE reducing gel is depicted. The upper frame depicts samples from complex medium and the lower frame depicts samples from defined medium. The gel images are arranged in the same order for each picture. Description from left to right: left lane = molecular weight marker; the second lane from the left shows samples stored at -20°C; the third and fourth lanes from the left are samples stored at 5°C with and without the addition of BASI, respectively; the fifth and sixth lanes from the left are samples stored at 22°C with and without the addition of BASI, respectively.

[0019] Figure 64%-12% SDS-PAGE reducing gel is depicted. Left gel image = molecular weight marker. Gel images are grouped in boxes by storage temperature. Left box from -20°C, middle box from 5°C, and right box from 22°C. None = sample with no BASI added; 1 = sample with 5g BASI filtrate / 10g UFC; 2 = sample with 20g BASI filtrate / 10g UFC.

[0020] Figure 7 4%-12% SDS-PAGE reducing gel is depicted. Gel image on the left = molecular weight marker. Gel images of samples are grouped by storage temperature and time. The left ones are from -20°C. Group 2 from the left are from 22°C after 2 days of storage. Group 3 from the left are from 5°C after 4 days of storage. Groups on the right are from 5°C for 10 days of storage. Sample descriptions: None = no BASI added; M = 1 g BASI / kg UFC; L = 0.25 g BASI / kg UFC; and H = 1.9 g BASI / kg UFC.

[0021] Figure 8 4%-12% SDS-PAGE reducing gels are depicted. The gel on the left shows samples from pH 5.5 fermentations without and with BASI added taken at fermentation times of 40h, 70h, 91h, 116h and 140h. The gel on the right shows comparable samples from pH 6.5 fermentations.

[0022] Fig. 9 Depicted is a 4%-12% SDS-PAGE reducing gel of samples after 15 days storage at 10°C compared to a -20°C reference. Left gel image = molecular weight marker; center gel image = sample without BASI addition; right gel image = sample with BASI addition.

[0023] Fig. 10A and 10B The reduced gel is depicted. Fig. 10A Depicted is a 4%-12% SDS-PAGE reducing gel comparing samples taken during recovery with samples of fermentation broth. Lane 1 = purified antibody; 2 = end of fermentation broth supernatant; 3 = cooled broth supernatant; 4 = transferred broth supernatant; 5 = treated broth supernatant; 6 = cell separation filtrate; 7 = filtrate at the beginning of ultrafiltration; 8 = ultrafiltration concentrate. Fig. 10B Depicted is a 4%-12% SDS-PAGE reducing gel comparing UF concentrate stored for 1 month at 10°C to -20°C.

[0024] Fig.11Depicted is a non-reducing SDS-PAGE gel comparing samples of antibody B from UFC 20200577 formulated as 1 g antibody / L. Samples are from before (lanes 2, 4, 6, 8; stored at -20°C) and after (lanes 3, 5, 7, 9) storage for 56 days at 4°C. Lane 1 contains a protein standard prestained with Novex Sharp, and lane 10 contains a purified antibody standard expressed by CHO, 0.5 ug. Lanes 2 and 3 show samples stored in formulation A (100 mM Bis-Tris, pH 5.8) with a residual band density of 7.1%. Lanes 4 and 5 show samples stored in formulation B (100 mM Bis-Tris, pH 5.8+3 g / kg BASI) with a residual band density of 45.2%. Lanes 6 and 7 show samples stored in formulation C (100 mM Bis-Tris, pH 5.8 + 2.3% arginine-HCl), with a residual band density of 27.9%. Lanes 8 and 9 show samples stored in formulation D (100 mM Bis-Tris, pH 5.8 + 2.3% arginine-HCl + 3 g / L BASI), with a residual band density of 79.8%.

[0025] Fig.12 A non-reducing SDS-PAGE gel is depicted, which compares samples of antibody B from UFC 20208053 formulated as 1 g antibody / L. The samples are from before (lanes 2, 4, 6, 8; stored at -20°C) and after (lanes 3, 5, 7, 9) storage for 25 days at 22°C. Lane 1 contains a protein standard prestained with Novex Sharp, and lane 10 contains a purified antibody standard expressed in CHO, 0.5ug. Lanes 2 and 3 show samples stored in formulation A (100mM Bis-Tris, pH 5.8), with a residual band density of 0.7%. Lanes 4 and 5 show samples stored in formulation B (100mM Bis-Tris, pH 5.8+3g / kg BASI), with a residual band density of 7.6%. Lanes 6 and 7 show samples stored in formulation C (100 mM Bis-Tris, pH 5.8 + 2.3% arginine-HCl), with a residual band density of 4.6%. Lanes 8 and 9 show samples stored in formulation D (100 mM Bis-Tris, pH 5.8 + 2.3% arginine-HCl + 3 g / L BASI), with a residual band density of 22.6%.

[0026] Fig.13Depicted is a non-reducing SDS-PAGE gel comparing samples of antibody A from UFC 20200874 formulated to 1 g antibody / L. Samples are from before (lanes 2, 4, 6, 8; stored at -20°C) and after (lanes 3, 5, 7, 9) storage at 22°C for 25 days. Lane 1 contains a protein standard prestained with Novex Sharp, and lane 10 contains a purified antibody standard expressed by CHO, 0.5 ug. Lanes 2 and 3 show samples stored in formulation C (100 mM Bis-Tris, pH 5.8 + 2.3% arginine-HCl), with a residual band density of 0%. Lanes 4 and 5 show samples stored in formulation D (100 mM Bis-Tris, pH 5.8 + 2.3% arginine-HCl + 3 g / L BASI), with a residual band density of 14.6%. Lanes 6 and 7 show samples stored in formulation A (100 mM Bis-Tris, pH 5.8) with a residual band density of 0%. Lanes 8 and 9 show samples stored in formulation B (100 mM Bis-Tris, pH 5.8 + 3 g / kg BASI) with a residual band density of 1.6%. DETAILED DESCRIPTION

[0027] Conventional monoclonal antibody production in mammalian derived cells such as Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells is limited by the titer of the product that can be produced by these cells in a single fermentation. Producing monoclonal antibodies in non-mammalian cells such as cell expression platforms based on filamentous fungi has the potential to significantly improve the overall titer and manufacturability of monoclonal antibodies for large-scale and / or industrial-scale production. However, during the fermentation, separation and purification of antibodies, the presence of a large amount of proteolytic degradation of recombinantly expressed monoclonal antibodies or their functional fragments limits these non-mammalian expression platforms.

[0028] As will be described in more detail herein, the inventors of the present application have surprisingly discovered that a protein inhibitor isolated from barley, namely barley alpha-amylase subtilisin inhibitor (BASI), substantially reduces the amount of proteolytic degradation of recombinantly expressed monoclonal antibodies in non-mammalian cell expression systems when co-expressed with the antibody or when added exogenously to the fermentation medium during production. Thus, in the compositions and methods disclosed herein, the use of BASI substantially increases the titer of intact monoclonal antibodies produced in these non-mammalian systems by significantly reducing proteolysis of recombinantly expressed monoclonal antibodies.

[0029] I. Definitions

[0030] As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or completely synthetic, such as recombinantly produced, including any fragment thereof containing at least a portion of the variable region of an immunoglobulin molecule that retains the binding specificity of a full-length immunoglobulin. Thus, antibodies include any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody combining site). Antibodies include antibody fragments. Thus, as used herein, the term antibody includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intrabodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above. The antibodies provided herein include members of any immunoglobulin class (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b).

[0031] As used herein, an "antibody fragment" or "antigen binding fragment" of an antibody refers to any portion of a full-length antibody that is smaller than the full length but contains at least a portion of the variable region of the antibody that binds to an antigen (e.g., one or more CDRs and / or one or more antibody combining sites) and thus retains at least a portion of the binding specificity and specific binding ability of the full-length antibody. Thus, an antigen binding fragment refers to an antibody fragment that contains an antigen binding portion that binds to the same antigen as the antibody from which the antibody fragment was derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetic, e.g., recombinantly produced derivatives. Antibody fragments are included in antibodies. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, e.g., Methods in Molecular Biology, Vol. 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; pp. 3-25, Kipriyanov). Fragments can include multiple chains linked together, such as by disulfide bridges and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (such as by replacing the corresponding region), produces immunospecific binding to an antigen (i.e., exhibits at least or at least about 10 7 -10 8 M -1 Ka) antibodies.

[0032] As used herein, "therapeutic antibodies" refers to any antibody or antigen-binding fragment thereof administered for the treatment of animals (including humans). Such antibodies can be prepared by any known method for producing polypeptides, and therefore include but are not limited to recombinantly produced antibodies, synthetically produced antibodies, and therapeutic antibodies extracted from cells or tissues and other sources. Therapeutic antibodies isolated or produced from any source may be heterogeneous in length, or differ in post-translational modifications such as glycosylation (i.e., carbohydrate content). The heterogeneity of therapeutic antibodies may also vary depending on the source of the therapeutic antibodies. Therefore, reference to therapeutic antibodies refers to heterogeneous populations produced or separated. When a homogeneous preparation is desired, it will be stated as such. Reference to therapeutic antibodies herein refers to their monomers, dimers, or other polymer forms, as the case may be.

[0033] As used herein, "neutralizing antibody" is any antibody or antigen-binding fragment thereof that binds to a pathogen and interferes with the ability of the pathogen to infect cells and / or cause disease in a subject. Examples of neutralizing antibodies are neutralizing antibodies that bind to viruses, bacteria, and fungal pathogens. Typically, the neutralizing antibodies provided herein bind to the surface of the pathogen. In examples where the pathogen is a virus, the neutralizing antibodies that bind to the virus typically bind to proteins on the surface of the virus. Depending on the type of virus, the surface protein may be a capsid protein (e.g., a capsid protein of a non-enveloped virus) or a viral envelope protein (e.g., a viral envelope protein of an enveloped virus). In some instances, the protein is a glycoprotein. The ability of a virus to inhibit viral infectivity can be measured, for example, by in vitro neutralization assays (such as, for example, plaque reduction assays performed using Vero host cells).

[0034] As used herein, "conventional antibodies" refer to antibodies containing two heavy chains (which may be represented as H and H') and two light chains (which may be represented as L and L') and two antibody combining sites, wherein each heavy chain may be a full-length immunoglobulin heavy chain or any functional region thereof that retains antigen binding ability (e.g., heavy chains include but are not limited to VH chains, VH-CH1 chains, and VH-CH1-CH2-CH3 chains), and each light chain may be a full-length light chain or any functional region thereof (e.g., light chains include but are not limited to VL chains and VL-CL chains). Each heavy chain (H and H') is paired with one light chain (L and L', respectively).

[0035] As used herein, a "full-length antibody" is an antibody having two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region, such as human antibodies naturally produced by antibody-secreting B cells and antibodies produced synthetically with the same domains.

[0036] As used herein, when referring to an antibody fragment derived from another antibody (such as a monoclonal antibody), the phrase "derived from" refers to the engineering of antibody fragments (e.g., Fab, F(ab'), F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments) that retain the binding specificity of the original antibody. Such fragments can be derived by a variety of methods known in the art, including but not limited to enzymatic cleavage, chemical cross-linking, recombinant means, or a combination thereof. In general, the derived antibody fragments share the same or substantially the same heavy chain variable region (VH) and light chain variable region (VL) of the parent antibody, such that the antibody fragment and the parent antibody bind to the same epitope.

[0037] As used herein, "parent antibody" or "source antibody" refers to the antibody from which antibody fragments (eg, Fab, F(ab'), F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments) are derived.

[0038] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants typically contain chemically active surface groups of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics.

[0039] As used herein, "chimeric polypeptide" refers to a polypeptide containing portions from at least two different polypeptides or two non-contiguous portions from a single polypeptide. Thus, a chimeric polypeptide generally includes an amino acid residue sequence from all or part of one polypeptide and an amino acid sequence from all or part of another different polypeptide. The two portions may be linked directly or indirectly, and may be linked by peptide bonds, other covalent bonds, or other non-covalent interactions of sufficient strength to maintain the integrity of a substantial portion of the chimeric polypeptide under equilibrium conditions and physiological conditions (such as in isotonic pH 7 buffered saline). For purposes herein, chimeric polypeptides include those containing all or part of an antibody or functional fragment thereof and / or a BASI polypeptide linked to another polypeptide, such as a multimerization domain, a heterologous immunoglobulin constant domain or framework region, a diagnostic or therapeutic polypeptide, or a secretion enhancing polypeptide.

[0040] As used herein, "fusion protein" is a polypeptide engineered to contain a sequence of amino acids corresponding to two different polypeptides, which are linked together, such as by expressing the fusion protein from a vector containing two nucleic acids encoding the two polypeptides, which are in close proximity to each other along the length of the vector, such as adjacent. In general, the fusion protein provided herein refers to a polypeptide containing a polypeptide having the amino acid sequence of an antibody or its antigen-binding fragment and a polypeptide or peptide having the amino acid sequence of a heterologous polypeptide or peptide, such as, for example, a diagnostic or therapeutic polypeptide. Therefore, a fusion protein refers to a chimeric protein containing two or more proteins or peptides directly or indirectly connected by a peptide bond, or parts from two or more proteins or peptides. The two molecules can be adjacent in the construct, or separated by a linker or spacer polypeptide. The spacer can encode a polypeptide that changes the properties of the polypeptide (such as solubility or intracellular transport).

[0041] As used herein, a "linker" or "spacer" peptide refers to a short sequence of amino acids that connects two polypeptide sequences (or nucleic acids encoding such amino acid sequences). A "peptide linker" refers to a short sequence of amino acids that connects two polypeptide sequences. Examples of polypeptide linkers are linkers that connect a peptide transduction domain to an antibody or linkers that connect two antibody chains in a synthetic antibody fragment (such as a scFv fragment). Linkers are well known, and any known linker can be used in the provided methods. An exemplary polypeptide linker is a (Gly-Ser) amino acid sequence in which some Glu or Lys residues are dispersed to increase solubility. Other exemplary linkers are described herein; any of these and other known linkers can be used with the provided compositions and methods.

[0042] As used herein, "antibody hinge region" or "hinge region" refers to a polypeptide region naturally present in the heavy chain of γ, δ and α antibody isotypes, between the CH1 and CH2 domains that have no homology with other antibody domains. This region is rich in proline residues and confers flexibility to IgG, IgD and IgA antibodies, thereby allowing the two "arms" of the Fab portion (each arm containing an antibody combining site) to be movable so that they present various angles relative to each other when binding antigen. This flexibility allows the Fab arms to move so that the antibody combining sites are aligned to interact with epitopes on the cell surface or other antigens. The two interchain disulfide bonds in the hinge region stabilize the interaction between the two heavy chains. In some embodiments provided herein, the synthetically produced antibody fragments contain one or more hinge regions, for example, for promoting stability by interaction between two antibody chains. The hinge region is an example of a dimerization domain.

[0043] As used herein, "humanized antibody" refers to an antibody that has been modified to include a "human" sequence of amino acids so that administration to humans does not cause an immune response. Humanized antibodies typically contain complementary determining regions (CDRs) derived from immunoglobulins of non-human species, while the remainder of the antibody molecule is primarily derived from human immunoglobulins. Methods for preparing such antibodies are known. For example, the DNA encoding a monoclonal antibody can be altered by recombinant DNA technology to encode an antibody in which the amino acid composition of the non-variable region is based on that of a human antibody. Methods for identifying such regions are known, including computer programs designed to identify variable and non-variable regions of immunoglobulins.

[0044] As used herein, an "Ig domain" is a domain recognized by those skilled in the art, characterized by a structure known as an immunoglobulin (Ig) fold, which contains two beta-pleated sheets, each of which contains antiparallel beta strands of amino acids connected by a loop. The two beta sheets in the Ig fold are clamped together by hydrophobic interactions and conserved intrachain disulfide bonds. The individual immunoglobulin domains within an antibody chain can be further distinguished based on function. For example, the light chain contains one variable region domain (VL) and one constant region domain (CL), while the heavy chain contains one variable region domain (VH) and three or four constant region domains (CH). Each of the VL, CL, VH, and CH domains is an example of an immunoglobulin domain.

[0045] As used herein, "variable domain" or "variable region" is a specific Ig domain of an antibody heavy chain or light chain, which contains an amino acid sequence that varies between different antibodies. Each light chain and each heavy chain has a variable region domain, VL and VH, respectively. The variable domain provides antigen specificity and is therefore responsible for antigen recognition. Each variable region contains CDRs that are part of an antigen binding site domain and a framework region (FR).

[0046] As used herein, "antigen binding domain", "antigen binding site", "antigen combining site" and "antibody combining site" are used synonymously to refer to domains within an antibody that recognize and physically interact with a cognate antigen. A natural, conventional full-length antibody molecule has two conventional antigen binding sites, each of which contains a portion of the heavy chain variable region and a portion of the light chain variable region. A conventional antigen binding site contains loops connecting antiparallel beta strands within the variable region domain. An antigen binding site may contain other portions of a variable region domain. Each conventional antigen binding site contains three hypervariable regions from the heavy chain and three hypervariable regions from the light chain. Hypervariable regions are also called complementarity determining regions (CDRs).

[0047] As used herein, "hypervariable region", "HV", "complementarity determining region" and "CDR" and "antibody CDR" are used interchangeably to refer to one of the multiple parts within each variable region that together form the antigen binding site of the antibody. Each variable region domain contains three CDRs, designated CDR1, CDR2 and CDR3. These three CDRs are non-contiguous along the linear amino acid sequence, but are adjacent in the folded polypeptide. The CDRs are located in the loops connecting the parallel strands of the beta sheets of the variable domain. As described herein, those skilled in the art understand and can identify CDRs and framework regions based on Kabat or Chothia numbering (see, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, the disclosures of which are incorporated herein by reference).

[0048] As used herein, "framework regions" (FR) are domains within the antibody variable region domain that are located within the beta sheets; FR regions are relatively more conserved in terms of their amino acid sequences than are hypervariable regions.

[0049] As used herein, "constant region" or "constant domain" is a domain in an antibody heavy chain or light chain that contains an amino acid sequence that is relatively more conservative than the amino acid sequence of the variable region domain. In a conventional full-length antibody molecule, each light chain has a single light chain constant region (CL) domain, and each heavy chain contains one or more heavy chain constant region (CH) domains, which include CH1, CH2, CH3 and CH4. Full-length IgA, IgD and IgG isotypes contain CH1, CH2, CH3 and hinge regions, while IgE and IgM contain CH1, CH2, CH3 and CH4. The CH1 and CL domains extend the Fab arm of the antibody molecule, thereby facilitating the interaction with the antigen and the rotation of the antibody arm. The antibody constant region can play an effector function, such as but not limited to clearing antigens, pathogens and toxins to which the antibody specifically binds, for example, by interacting with various cells, biomolecules and tissues.

[0050] As used herein, a functional region of an antibody is a portion of an antibody that contains at least the VH, VL, CH (eg, CH1, CH2 or CH3), CL or hinge region domains of the antibody, or at least a functional region thereof.

[0051] As used herein, "specific binding" or "immunospecific binding" with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with a cognate antigen through non-covalent interactions between the antibody combining sites of the antibody and the antigen. Affinity constants can be determined by standard kinetic methods of antibody reactions, e.g., immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol [Biotechnol] 11:54; Englebienne (1998) Analyst. [Analyst] 123:1599), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art (see, e.g., Paul, ed., Fundamental Immunology [Basic Immunology], 2nd ed., Raven Press, New York [New York Raven Press], pp. 332-336 (1989)). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (e.g., BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335). Typically, antibodies or antigen-binding fragments thereof provided herein that immunospecifically bind to an epitope do not cross-react with other antigens or cross-react with significantly (at least 10-100-fold) lower affinity for such antigens. Antibodies or antigen-binding fragments that immunospecifically bind to a particular epitope can be identified, for example, by immunoassays such as radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), surface plasmon resonance, or other techniques known to those skilled in the art. An antibody or antigen binding fragment thereof that immunospecifically binds to an epitope is typically an antibody or antigen binding fragment thereof that binds to the epitope with a higher binding affinity than any cross-reactive epitope, as determined using experimental techniques (such as, but not limited to, immunoassays, surface plasmon resonance, or other techniques known to those skilled in the art). Immunospecific binding to an isolated protein (i.e., a recombinantly produced protein) does not necessarily mean that the antibody will exhibit the same immunospecific binding. Such measurements and properties are different. The affinity of an antibody or antigen binding fragment to the presented antigen can be determined. For purposes herein, when describing affinity or related terms, a target, such as an isolated protein, will be identified.

[0052] As used herein, "Fc" or "Fc region" or "Fc domain" refers to a polypeptide containing the constant region of an antibody heavy chain, excluding the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgE, or the last three constant region immunoglobulin domains of IgE and IgM. Optionally, the Fc domain may include all or part of the flexible hinge at the N-terminus of these domains. For IgA and IgM, Fc may include a J chain. For an exemplary Fc domain of IgG, Fc contains immunoglobulin domains Cγ2 and Cγ3, and optionally, all or part of the hinge between Cγ1 and Cγ2. The boundaries of the Fc region may vary, but typically include at least a portion of the hinge region. In addition, Fc also includes any allele or species variant or any variant or modified form, such as any variant or modified form that alters binding to FcR or alters Fc-mediated effector functions.

[0053] As used herein, "tag" or "epitope tag" refers to an amino acid sequence typically added to the N-terminus or C-terminus of a polypeptide (such as an antibody provided herein). The inclusion of a tag fused to a polypeptide can facilitate polypeptide purification and / or detection. Typically, a tag or tag polypeptide refers to a polypeptide that has enough residues to provide an epitope recognized by an antibody or can be used for detection or purification, but is short enough so that it does not interfere with the activity of the chimeric polypeptide to which it is attached. The tag polypeptide is typically unique enough so that the antibody that specifically binds to it does not substantially cross-react with the epitope in the polypeptide to which it is attached. Suitable tag polypeptides generally have at least 5 or 6 amino acid residues, and are generally between about 8-50 amino acid residues, typically between 9-30 residues. The tag can be connected to one or more chimeric polypeptides in a multimer and allows detection of the multimer or recovery of the multimer from a sample or mixture. Such tags are well known and can be easily synthesized and designed. Exemplary tag polypeptides include those used for affinity purification, and include His tags; influenza hemagglutinin (HA) tag polypeptide and its antibody 12CA5 (Field et al. (1988) Mol. Cell. Biol. [Molecular and Cellular Biology] 8: 2159-2165); c-myc tag and its 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies (see, e.g., Evan et al. (1985) Molecular and Cellular Biology [Molecular and Cellular Biology] 5: 3610-3616); and herpes simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al. (1990) Protein Engineering [Protein Engineering] 3: 547-553). Antibodies used to detect antibodies with epitope tags are typically referred to herein as secondary antibodies.

[0054] As used herein, "polypeptide" refers to two or more amino acids covalently linked. The terms "polypeptide" and "protein" are used interchangeably herein.

[0055] As used herein, "peptide" refers to a polypeptide of 2 to about 40 amino acids in length.

[0056] "Barley alpha-amylase / subtilisin inhibitor" or "BASI" is a dual-headed inhibitor of alpha-amylase and serine proteases of the subtilisin family from barley. In some embodiments, BASI comprises the amino acid sequence of SEQ ID NO: 1. In other embodiments, BASI may have at least about 50% identity to the mature peptide of SEQ ID NO: 1, or it may comprise a sequence having at least about 50% identity to residues 67-96 of SEQ ID NO: 1. The identity may specifically be at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 1 or to residues 67-96 of SEQ ID NO: 1.

[0057] As used herein, "amino acid" is an organic compound containing an amino group and a carboxylic acid group. A polypeptide contains two or more amino acids. For the purposes of this article, the amino acids contained in the antibodies provided include twenty naturally occurring amino acids, non-natural amino acids, and amino acid analogs (e.g., amino acids in which the α-carbon has a side chain). As used herein, the amino acids present in the various amino acid sequences of the polypeptides presented herein are identified according to their well-known three-letter or single-letter abbreviations. The nucleotides present in various nucleic acid molecules and fragments are named with the standard single-letter nomenclature conventionally used in the art.

[0058] As used herein, "amino acid residue" refers to an amino acid formed when a polypeptide is chemically digested (hydrolyzed) at its peptide linkages. The amino acid residues described herein are generally in the "L" isomeric form. Residues in the "D" isomeric form can replace any L-amino acid residue as long as the polypeptide retains the desired functional properties. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxyl group present at the carboxyl terminus of a polypeptide. According to the standard polypeptide nomenclature described in J.Biol.Chem. [Journal of Biological Chemistry], 243:3557-59 (1968) and adopted in 37 C.FR §§1.821-1.822, abbreviations for amino acid residues are used throughout the text. All amino acid residue sequences represented by structural formulas herein have a left-to-right orientation in the conventional direction from the amino terminus to the carboxyl terminus. In addition, the phrase "amino acid residue" is defined to include natural, modified, non-natural and uncommon amino acids. In addition, a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or to an amino terminal group (such as NH2) or to a carboxyl terminal group (such as COOH). As used herein, "naturally occurring amino acids" refers to the 20 L-amino acids present in polypeptides.

[0059] As used herein, "stability" and "stable" refer to the resistance of the antibody in the formulation to aggregation, degradation (such as proteolytic degradation) or fragmentation (such as proteolytic mediated fragmentation) under given manufacturing, preparation, transportation and storage conditions. Antibodies with improved stability and resistance to degradation will maintain biological activity under given manufacturing, preparation, transportation and storage conditions. The stability of the antibody can be assessed by the degree of aggregation, degradation or fragmentation, as measured by high performance size exclusion chromatography (HPSEC), static light scattering (SLS), Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), urea unfolding (urea unfolding) technology, intrinsic tryptophan fluorescence, differential scanning calorimetry and / or ANS binding technology. The stability of the antibody can also be measured by the intensity of the band representing the full-length antibody species on the gel before and after the assessment process. The stability of the antibody can be compared with comparable molecules under the same conditions. The overall stability of the antibody can also be assessed by various immunoassays, including ELISA and radioimmunoassays performed using, for example, separated antigen molecules or cells expressing these antigen molecules.

[0060] As used herein, an "activity" or "functional activity" of a polypeptide (such as an antibody) refers to any activity exhibited by the polypeptide. Such activities can be determined empirically. Exemplary activities include, but are not limited to, the ability to interact with biomolecules (e.g., by antigen binding, DNA binding, ligand binding, or dimerization), enzymatic activity (e.g., kinase activity or proteolytic activity). For antibodies (including antibody fragments), activities include, but are not limited to, the ability to specifically bind to a specific antigen, the affinity of antigen binding (e.g., high or low affinity), the avidity of antigen binding (e.g., high or low affinity), the on-rate, the off-rate, the effector function (such as the ability to promote antigen neutralization or clearance), virus neutralization, and in vivo activity (such as the ability to prevent pathogen infection or invasion, or the ability to promote clearance, or the ability to penetrate specific tissues or body fluids or cells in the body), or improved manufacturability, thermal stability, or protease resistance. Activity can be assessed in vitro or in vivo using recognized assays such as ELISA, flow cytometry, surface plasmon resonance or equivalent assays that measure association or dissociation rates, immunohistochemistry and immunofluorescence histology and microscopy, cell-based assays, flow cytometry, and binding assays (e.g., panning assays). For example, for antibody polypeptides, activity can be assessed by measuring in vitro binding affinity, avidity, and / or binding coefficient (e.g., for association rate / dissociation rate) and other activities, or by measuring various effects in vivo (such as immune effects, e.g., antigen clearance, antibody penetration or localization into tissues, protection against disease (e.g., infection), serum or other body fluid antibody titers, or other assays well known in the art). The results of such assays that indicate that a polypeptide exhibits activity can be correlated with the in vivo activity of the polypeptide, where in vivo activity can be referred to as therapeutic activity or biological activity. The activity of the modified polypeptide can be any level of percentage of the activity of the unmodified polypeptide, including but not limited to 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500% or more of the activity compared to the unmodified polypeptide. Assays for determining the functionality or activity of modified (e.g., variant) antibodies are well known in the art.

[0061] As used herein, "exhibiting less proteolytic degradation" refers to the extent of proteolytic cleavage of an antibody or its functional fragment produced recombinantly (such as an antibody produced according to the methods provided herein, such as a therapeutic antibody or its functional fragment) during fermentation, separation, purification and / or storage compared to the same antibody not produced according to the methods provided herein. The antibody or its functional fragment produced according to the methods provided herein may exhibit any of the following degree of proteolytic degradation reduction: 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% compared to an antibody or its functional fragment not produced according to the methods disclosed herein. In other embodiments, the reduction in proteolytic degradation is at least about 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times or more. Determine the degree of proteolytic degradation of the test antibody or its functional fragment is well known in the art. Such determinations can be performed in vitro or in vivo. For example, the activity can be measured using the determinations described in the examples below.

[0062] As used herein, "nucleic acid" refers to at least two connected nucleotides or nucleotide derivatives linked together (typically by phosphodiester linkages), including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The term "nucleic acid" also includes analogs of nucleic acids, such as peptide nucleic acids (PNA), thiophosphate DNA, and other such analogs and derivatives or combinations thereof. Nucleic acids also include DNA and RNA derivatives, which contain, for example, nucleotide analogs or "backbone" bonds other than phosphodiester bonds, such as phosphotriester bonds, phosphoramidate bonds, phosphorothioate bonds, thioester bonds or peptide bonds (peptide nucleic acids). The term also includes equivalents, derivatives, variants and analogs of RNA or DNA made from nucleotide analogs, single-stranded (sense or antisense) nucleic acids and double-stranded nucleic acids. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine. For RNA, the uracil base is uridine.

[0063] As used herein, the "regulatory region" of a nucleic acid molecule means a cis-acting nucleotide sequence that has a positive or negative effect on the expression of an operably linked gene. The regulatory region includes nucleotide sequences that confer inducible (i.e., a substance or stimulant is required to increase transcription) expression of the gene. When the inducer is present or the concentration increases, gene expression can increase. The regulatory region also includes sequences that confer gene expression inhibition (i.e., a substance or stimulant reduces transcription). When the repressor is present or the concentration increases, gene expression can decrease. It is well known that regulatory regions affect, regulate or control many biological activities in vivo, including cell proliferation, cell growth and death, cell differentiation and immunomodulation. The regulatory region typically binds to one or more trans-acting proteins, resulting in an increase or decrease in gene transcription.

[0064] The specific example of gene regulatory region is promoter and enhancer.Promoter is the sequence that is positioned at transcription or translation start site, is typically positioned at the 5' of translation start site.Promoter is usually positioned at 1Kb of translation start site, but can be positioned at more distant position, for example 2Kb, 3Kb, 4Kb, 5Kb or more distant, at most and include 10Kb.As is well known, enhancer can affect gene expression when being positioned at 5' or 3' of gene, or when being positioned at exon or intron or its part.Enhancer also can play a role at the considerable distance from gene, for example, at about 3Kb, 5Kb, 7Kb, 10Kb, 15Kb or more distant distance.

[0065] In addition to the promoter region, the regulatory region also includes, but is not limited to, sequences that promote translation, splicing signals for introns, maintaining the correct reading frame of the gene to allow in-frame translation of the mRNA, and stop codons, leader and fusion partner sequences, internal ribosome binding site (IRES) elements for generating polygenic or polycistronic messages, polyadenylation signals that provide for proper polyadenylation of the transcript of the gene of interest, and stop codons, and may optionally be included in an expression vector.

[0066] As used herein, "operably connected" with respect to a nucleic acid sequence, region, element or domain means that the nucleic acid regions are functionally related to each other. For example, a nucleic acid encoding a leader peptide can be operably connected to a nucleic acid encoding a polypeptide, whereby the nucleic acid can be transcribed and translated to express a functional fusion protein, wherein the leader peptide achieves secretion of the fusion polypeptide. In some cases, a nucleic acid encoding a first polypeptide (e.g., a leader peptide) is operably connected to a nucleic acid encoding a second polypeptide, and the nucleic acid is transcribed into a single mRNA transcript, but translation of the mRNA transcript can result in one of the two polypeptides being expressed. For example, an amber stop codon can be located between a nucleic acid encoding a first polypeptide and a nucleic acid encoding a second polypeptide, so that when introduced into a partially amber suppressed cell, the resulting single mRNA transcript can be translated to produce a fusion protein containing the first and second polypeptides, or can be translated to produce only the first polypeptide. In another example, a promoter can be operably connected to a nucleic acid encoding a polypeptide, whereby the promoter regulates or mediates transcription of the nucleic acid.

[0067] As used herein, "synthetic" with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or a polypeptide molecule that is produced by recombinant methods and / or by chemical synthesis methods.

[0068] As used herein, "produced by recombinant means using recombinant DNA methods" means expressing a protein encoded by a cloned DNA using well-known molecular biology methods.

[0069] As used herein, "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. The expression level of a polypeptide can be assessed using any method known in the art (including, for example, a method for determining the amount of a polypeptide produced from a host cell). Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.

[0070] As used herein, "host cell" is a cell for receiving, maintaining, propagating and amplifying a vector. Host cells can also be used to express polypeptides encoded by the vector. When the host cell divides, the nucleic acid contained in the vector is replicated, thereby amplifying the nucleic acid. In one example, the host cell is a genetic package that can be induced to express a variant polypeptide on its surface. In another example, the host cell is infected with a genetic package. For example, the host cell can be a phage display compatible host cell that can be transformed with a phage or phagemid vector and accommodates the packaging of a phage expressing a fusion protein containing a variant polypeptide.

[0071] As used herein, "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell. References to vectors include those vectors into which nucleic acids encoding polypeptides or fragments thereof can be introduced (typically by restriction digestion and ligation). References to vectors also include those vectors containing nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells to amplify nucleic acids or to express / display polypeptides encoded by nucleic acids. Vectors can be designed to achieve integration of genes or portions thereof into chromosomes of the genome. Vectors as artificial chromosomes are also envisioned, such as yeast artificial chromosomes and mammalian artificial chromosomes. The selection and use of such vectors are well known to those skilled in the art.

[0072] As used herein, "expression vector" includes vectors capable of expressing DNA operably linked to regulatory sequences (such as promoter regions) that enable expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, expression vectors refer to recombinant DNA or RNA constructs, such as plasmids, phages, recombinant viruses, or other vectors that cause expression of cloned DNA after being introduced into appropriate host cells. Suitable expression vectors are well known to those skilled in the art, and include those vectors that can be replicated in eukaryotic cells and / or prokaryotic cells and those vectors that remain free or are integrated into the host cell genome.

[0073] As used herein, "similarity" between two proteins or nucleic acids refers to the correlation between the amino acid sequence of a protein or the nucleotide sequence of a nucleic acid. Similarity can be based on the degree of identity between a residue sequence and the residues contained therein. Methods for assessing the degree of similarity between proteins or nucleic acids are known to those skilled in the art. For example, in a method for assessing sequence similarity, two amino acid or nucleotide sequences are compared in a manner that produces the maximum level of identity between sequences. "Identity" refers to the degree to which an amino acid or nucleotide sequence remains unchanged. The comparison of amino acid sequences, and to a certain extent the comparison of nucleotide sequences, can also consider conservative differences and / or frequent substitutions of amino acids (or nucleotides). Conservative differences are differences in the physicochemical properties of the residues involved. The comparison can be global (comparison of comparison sequences over the entire length of the sequence and including all residues) or local (comparison of a portion of the sequence including only the most similar one or more regions).

[0074] As used herein, when a polypeptide or nucleic acid molecule or region thereof contains or has "identity" or "homology" with another polypeptide or nucleic acid molecule or region, the two molecules and / or regions share greater than or equal to 40% or about 40% sequence identity, and typically greater than or equal to 50% or about 50% sequence identity, such as at least or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; if necessary, the exact percentage of identity can be specified. A nucleic acid molecule or region thereof that is identical or homologous to a second nucleic acid molecule or region can specifically hybridize with a nucleic acid molecule or region that is 100% complementary to the second nucleic acid molecule or region. Alternatively, the identity between two theoretical nucleotide or amino acid sequences or between a nucleic acid or polypeptide molecule and a theoretical sequence can be compared.

[0075] Sequence "identity" itself has a recognized meaning in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using public techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of a molecule. (See, e.g., Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M. and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1993.) New York, 1991). Although there are a variety of methods to measure the identity between two polynucleotides or polypeptides, the term "identity" is well known to those skilled in the art (Carrillo, H. and Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0076] Sequence identity along the full length comparison of two polynucleotides or polypeptides refers to the percentage of identical nucleotides or amino acid residues along the full length of the molecule. For example, if polypeptide A has 100 amino acids and polypeptide B has 95 amino acids, which are identical to amino acids 1-95 of polypeptide A, then polypeptide B has 95% identity when sequence identity is compared along the full length of polypeptide A compared to the full length of polypeptide B. Alternatively, the sequence identity between polypeptide A and polypeptide B can be compared along a region of each polypeptide (such as a similar region of 20 amino acids). In this case, if polypeptides A and B have 20 identical amino acids along the region, the sequence identity of these regions is 100%. Alternatively, sequence identity can be compared along the length of another molecule compared to the region of the molecule. Alternatively, the sequence identity between polypeptide A and polypeptide B can be compared along polypeptides of the same length, but with amino acid substitutions, such as conservative amino acid substitutions or non-conservative amino acid substitutions. As described below, and as known to those skilled in the art, various programs and methods for assessing identity are known to those skilled in the art. High levels of identity, such as 90% or 95% identity, can be readily determined without software.

[0077] Whether any two nucleic acid or polypeptide molecules have nucleotide sequences that are at least or about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% "identical" can be determined using a known computer algorithm such as the "FASTA" program, using, for example, the default parameters in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J. et al. (1984) Nucleic Acids Research 12(I):387), BLASTP, BLASTN, FASTA (Altschul, S. F. et al. (1990) J. Molec. Biol. 215:403; Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego). Diego [San Diego Academic Press], 1994 and Carrillo et al. (1988) SIAM J Applied Math [SIAM Journal of Applied Mathematics] 48:1073). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include the DNAStar "MegAlign" program (Madison, Wisconsin) and the University of Wisconsin Genetics Computer Group (UWG) "Gap" program (Madison, Wisconsin). The percent homology or identity of proteins and / or nucleic acid molecules can be determined, for example, by comparing sequence information using the GAP computer program (e.g., Needleman et al. (1970) J. Mol. Biol. [Journal of Molecular Biology] 48:443, as revised by Smith and Waterman ((1981) Adv. Appl. Math. [Advances in Applied Mathematics] 2:482). In short, the GAP program defines similarity as the number of similar aligned symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences.Default parameters for the GAP program may include: (1) a unary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, as described in Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap; and (3) no penalty for end gaps.

[0078] As used herein, "modification" refers to the modification of the amino acid sequence of a polypeptide or the nucleotide sequence in a nucleic acid molecule, and includes deletion, insertion and substitution of amino acids and nucleotides, respectively. Methods for modifying polypeptides are routine methods of those skilled in the art, such as by using recombinant DNA methods.

[0079] As used herein, "substitution" refers to replacing one or more nucleotides or amino acids in a native nucleic acid or polypeptide sequence, a target nucleic acid or polypeptide sequence, a wild-type nucleic acid or polypeptide sequence, or other nucleic acid or polypeptide sequence with an alternative nucleotide or amino acid without changing the length of the molecule (described in terms of the number of residues). Thus, one or more substitutions in a molecule do not change the number of amino acid residues or nucleotides of the molecule. Substitution mutations compared to a particular polypeptide can be represented by the number of amino acid residues along the length of the polypeptide sequence.

[0080] As used herein, a "label" or "detectable moiety" is a detectable marker (e.g., a fluorescent molecule, a chemiluminescent molecule, a bioluminescent molecule, a contrast agent (e.g., a metal), a radionuclide, a chromophore, a detectable peptide, or an enzyme that catalyzes the formation of a detectable product) that can be directly or indirectly attached or linked to a molecule or associated with a molecule and can be detected in vivo and / or in vitro. The detection method can be any method known in the art, including known in vivo and / or in vitro detection methods (e.g., imaging by visual inspection, magnetic resonance (MR) spectroscopy, ultrasound signals, X-rays, gamma-ray spectroscopy (e.g., positron emission tomography (PET) scans, single photon emission computed tomography (SPECT)), fluorescence spectroscopy, or absorption). Indirect detection refers to measuring physical phenomena such as energy or particle emission or absorption of atoms, molecules, or compositions that are directly or indirectly bound to a detectable moiety (e.g., detecting a labeled secondary antibody or antigen-binding fragment thereof that is bound to a primary antibody).

[0081] As used herein, an "isolated" or "purified" polypeptide or protein (e.g., an isolated antibody or antigen-binding fragment thereof) or a biologically active portion thereof (e.g., an isolated antigen-binding fragment) is substantially free of cellular material or other contaminating proteins from the cell or tissue from which the protein was derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. A preparation can be determined to be substantially free of readily detectable impurities if the preparation appears to be free of readily detectable impurities, or is sufficiently pure that further purification does not detectably alter the physical and chemical properties of the substance (such as enzymatic activity and biological activity), as determined by standard analytical methods used by those skilled in the art to assess purity (such as thin layer chromatography (TLC), gel electrophoresis, and high performance liquid chromatography (HPLC)). Methods for purifying a compound to produce a substantially chemically pure compound are known to those skilled in the art. However, a substantially chemically pure compound may be a mixture of stereoisomers. In this case, further purification may increase the specific activity of the compound. As used herein, a "cell extract" or "lysate" refers to a preparation or fraction made from lysed or disrupted cells.

[0082] As used herein, "oxygen uptake rate" or "OUR" refers to the rate of oxygen consumption in a bioreactor and is defined as mmolO2 / Lbroth / h (Buckland et al., 1985).

[0083] As used herein, the phrase "dissolved oxygen" or "DO %" refers to the amount of oxygen dissolved in the broth and is expressed as a percentage of air saturation.

[0084] As used herein, "carbon dioxide evolution rate" or "CER" refers to the rate at which carbon dioxide is produced in a bioreactor and is defined as mmol CO2 / Lbroth / h (Buckland et al., 1985).

[0085] As used herein, "elapsed fermentation time" or "EFT" refers to the number of hours that have elapsed since the bioreactor was inoculated with a seed culture.

[0086] As used herein, "seed culture" refers to a liquid culture used to accumulate biomass required to start a production culture.

[0087] As used herein, "dry cell weight" or "DCW" refers to grams of dry weight per kilogram of broth, and will be used as a measure of biomass accumulation.

[0088] As used herein, the term "biomass" refers to the total mass of a recombinant host cell strain (eg, a fungal strain) in a defined volume.

[0089] As used herein, the phrase "specific sugar feed rate" or "Qs" refers to the sugar feed rate divided by the amount of biomass in the fermentor.

[0090] Certain ranges are presented herein with the term "about" preceding a numerical value. The term "about" is used herein to provide textual support for the exact number that follows it and for numbers that are close or approximate to the number that follows the term. In determining whether a number is close to or approximate to a specifically stated number, the close or approximate unstated number may be a number that provides a substantial equivalent of the specifically stated number in the context in which it is presented. For example, with respect to a numerical value, the term "about" refers to a range of -10% to +10% of the numerical value, unless the term is otherwise specifically defined in context.

[0091] As used herein, the singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0092] As used herein, "optional" or "optionally" means that the following circumstance or limitation of the scope does or does not occur, and means that the description includes instances where the circumstance or limitation of the scope occurs and instances where it does not occur. For example, a composition that optionally contains additional exogenous enzymes means that the enzymes may or may not be present in the composition.

[0093] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of exclusive terminology such as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0094] Still, it should be noted that, as used herein, the term "consisting essentially of" refers to a composition wherein the component(s) following the term, in the presence of other known component(s), is a total amount of less than 30% by weight of the total composition and does not affect or interfere with the action or activity of the component(s).

[0095] It is further noted that, as used herein, the term "comprising" is meant to include but is not limited to one or more components following the term "comprising". The one or more components following the term "comprising" are required or mandatory, but the composition comprising the one or more components may further include other non-mandatory or optional one or more components.

[0096] It is also noted that, as used herein, the term "consisting of" is meant to include and be limited to the component or components following the term "consisting of." Thus, the component or components following the term "consisting of" are required or mandatory, and one or more other components are not present in the composition.

[0097] Each maximum numerical limit given throughout this specification is intended to include each lower numerical limit, as if such lower numerical limits were expressly written herein. Each minimum numerical limit given throughout this specification will include each higher numerical limit, as if such higher numerical limits were expressly written herein. Each numerical range given throughout this specification will include each narrower numerical range falling within such wider numerical range, as if such narrower numerical ranges were all expressly written herein.

[0098] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0099] Additional definitions of terms may appear throughout this specification.

[0100] II. Composition

[0101] A. BASI

[0102] Barley α-amylase subtilisin inhibitor (BASI) polypeptide belongs to the Kunitz-type trypsin inhibitor family (Leah and Mundy, 1989, Plant Mol. Biol. 12, 673-682). BASI is a single-chain protein consisting of 181 amino acids and contains two disulfide bridges, which are conserved in the structure of Kunitz inhibitors. BASI shares 92% and 58% sequence identity with similar inhibitors from wheat (WASI) and rice (RASI), respectively (Mundy et al., 1984, FEBS Lett. 167, 210-214; Ohtsubo and Richardson, 1992, FEBS Lett. 309, 68-72) (Micheelsen et al., 2008, Journal of Biotechnology 133 (2008) 424-432).

[0103] The inventors of the present application have found that when used in a monoclonal antibody (or a functional fragment thereof) production process, BASI reduces protease activity, resulting in higher intact protein yields, lower degraded protein yields, and improved performance in downstream protease-sensitive applications. BASI can be used by recombinant co-expression with a monoclonal antibody or a functional fragment thereof or by adding exogenous BASI (or a fermentation broth containing BASI) to a fermentation broth, a recovered protein solution, or a formulated monoclonal antibody (or a functional fragment thereof) product.

[0104] In some embodiments, the BASI comprises the amino acid sequence of SEQ ID NO: 1. In other embodiments, the BASI may have at least about 50% identity to the mature peptide of SEQ ID NO: 1, or it may comprise a sequence that has at least about 50% identity to residues 67-96 of SEQ ID NO: 1. The identity may specifically be at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% identity to SEQ ID NO: 1 or to residues 67-96 of SEQ ID NO: 1.

[0105] B. Antibody

[0106] Provided herein are antibodies, fragments thereof, or variants thereof, whose co-expression or co-fermentation with BASI results in reduced cleavage (such as proteolytic cleavage) and / or increased titer or fermentation yield. The recombinant monoclonal antibodies disclosed herein can be produced by methods well known in the art.

[0107] Relative to antibodies or functional fragments thereof not produced according to the methods disclosed herein, the same antibodies or functional fragments thereof produced by the methods disclosed herein may also show one or more of the following: improved purification yield; reduced formulation issues; reduced immunogenicity and increased bioavailability. In some embodiments, the improvement in manufacturability may result from the reduction in proteolytically mediated aggregation tendency and the increase in productivity brought about by the reduction in protein cleavage and degradation.

[0108] The antibodies or functional fragments thereof produced according to the methods disclosed herein exhibit increased or improved or enhanced protease resistance compared to antibodies or functional fragments thereof not produced according to the methods disclosed herein. The term "protease resistance" refers to the ability of a molecule comprising peptide bonds to resist hydrolytic cleavage of one or more of its peptide bonds in the presence of a proteolytic enzyme. Resistance to proteolytic enzymes is a relative property and is compared to molecules (such as molecules not produced according to the methods disclosed herein (such as by co-expression or co-fermentation with a BASI polypeptide)) that are less able to withstand hydrolytic cleavage of one or more of their peptide bonds within a specific time period and under specified conditions (including pH and / or temperature when testing for cleavage resistance). One result of proteolysis, indicating that cleavage has occurred, is the generation of smaller fragments (lower molecular weight) compared to the molecular weight of the intact, non-cleaved parent molecule. At a given temperature (e.g., 37° C.), a given pH, a given antibody concentration and a given protease concentration (such as a ratio of about 1%-2% (w / w) to IgG), when the antibody is in a given buffer (e.g., Tris-buffered saline) and is reacted with a protease (such as, but not limited to, pepsin, matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-12 (MMP-12), pepsin, glutamyl endopeptidase V8 (GluV8) of Staphylococcus aureus, Streptococcus pyogenes, pyogenes) immunoglobulin degrading enzyme (IdeS)) or expression host protease digestion, when more than about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the full-length antibody remains intact within a given time period (such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours), the antibodies or functional fragments thereof disclosed herein comprising hinge, CH2 domain and CH3 domain have "protease resistance" or "resistance to proteolysis", or have "increased resistance to proteolysis". The amount of intact IgG can be assessed by SDS-PAGE.

[0109] C. Antibody modification

[0110] The antibodies or functional fragments thereof (such as antigen-binding fragments thereof) provided herein may be modified. Modification of the antibody or antigen-binding fragment may improve one or more properties of the antibody, including but not limited to reducing the immunogenicity of the antibody or antigen-binding fragment, increasing the half-life of the antibody or antigen-binding fragment, such as reducing sensitivity to proteolysis and / or reducing sensitivity to oxidation, and changing or improving the binding properties of the antibody or antigen-binding fragment thereof. Exemplary modifications include but are not limited to modifications to the primary amino acid sequence of the antibody or its functional fragment and / or BASI polypeptide and changes to the post-translational modifications of the antibody or its functional fragment and / or BASI polypeptide.

[0111] Exemplary post-translational modifications include, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins. Other exemplary modifications include attaching one or more heterologous peptides to the antibody or its functional fragment and / or BASI polypeptide to alter or improve one or more properties of the antibody or its antigen-binding fragment.

[0112] 1. Signal sequence

[0113] In some embodiments, the antibodies disclosed herein may include a signal sequence. The signal sequence may be any signal sequence that promotes secretion of a protein from a host cell (e.g., a filamentous fungal host cell). In specific embodiments, the antibody may comprise a signal sequence of a protein that is known to be highly secreted from a host cell that will produce the fusion protein. The signal sequence employed may be endogenous or non-endogenous to the host cell that will produce the antibody or its functional fragment and / or BASI polypeptide.

[0114] Suitable signal sequences are known in the art (see, e.g., Ward et al., Bio / Technology 1990 8:435-440; and Paloheimo et al., Applied and Environmental Microbiology 2003 69:7073-7082). Non-limiting examples of suitable signal sequences include those for cellobiohydrolase I, cellobiohydrolase II, endoglucanase I, II and III, α-amylase, aspartyl protease, glucoamylase, phytase, mannanase, α and β glucosidase, bovine chymosin, human interferon and human tissue plasminogen activator; and synthetic consensus eukaryotic signal sequences such as those described by Gwynne et al., (1987) Bio / Technology 5:713-719.

[0115] In some embodiments, if Trichoderma (e.g., Trichoderma reesei) is used as a host cell, the signal sequence or carrier of Trichoderma reesei mannanase I (Man5A, or MANI), Trichoderma reesei cellobiohydrolase II (Cel6A or CBHII), endoglucanase I (Cel7b or EGI), endoglucanase II (Cel5a or EGII), endoglucanase III (Cel12A or EGIII), xylanase I or II (XynIIa or XynIIb), or Trichoderma reesei cellobiohydrolase I (Cel7a or CBHI) can be used for antibodies or functional fragments thereof and / or BASI polypeptides.

[0116] In other embodiments, if Aspergillus (e.g., Aspergillus niger) is used as a host cell, the signal sequence or carrier of Aspergillus niger glucoamylase (GlaA) or alpha amylase can be used in the fusion polypeptide. Aspergillus niger and Aspergillus awamori glucoamylase have the same amino acid sequence. Two forms of enzymes are generally identified in the culture supernatant. GAI is a full-length form (amino acid residues 1-616), while GAII is a natural proteolytic fragment comprising amino acid residues 1-512. It is known that GAI folds into two separate domains connected by an extended linker region. The two domains are a catalytic domain of 471 residues (amino acids 1-471) and a starch binding domain of 108 residues (amino acids 509-616), and the linker region between the two domains is 36 residues (amino acids 472-508). GAII lacks a starch binding domain. With reference to Libby et al., (1994) Protein Engineering 7: 1109-1114. In some embodiments, the glucoamylase used as a carrier protein and including a signal sequence will have greater than 95%, 96%, 97%, 98%, and 99% sequence identity to the catalytic domain of an Aspergillus or Trichoderma glucoamylase. The term "catalytic domain" refers to a structural portion or region of a protein amino acid sequence that has the catalytic activity of the protein.

[0117] 2. Carrier

[0118] In specific embodiments, the signal sequence may be included in a "vector" containing the signal sequence at its N-terminus, wherein the vector is at least the N-terminal portion of a protein that is efficiently secreted by a cell. In certain embodiments, the signal sequence and the vector protein are obtained from the same gene. In some embodiments, the signal sequence and the vector protein are obtained from different genes. In additional embodiments, the vector protein can be cleaved from the antibody during secretion, thereby producing a mature antibody that does not contain the vector.

[0119] The carrier protein may include all or part of the mature sequence of the secreted polypeptide. In some embodiments, the full-length secreted polypeptide is used. However, a functional portion of the secreted polypeptide may be employed. As used herein, a "portion" or grammatical equivalent of a secreted polypeptide means a truncated secreted polypeptide that retains the ability to fold into a normal but truncated configuration.

[0120] In some cases, the truncation of the secreted polypeptide means that the functional protein retains biological function. In some embodiments, the catalytic domain of the secreted polypeptide is used, but other functional domains, such as substrate binding domains, may also be used. In one embodiment, when glucoamylase (e.g., glucoamylase from Aspergillus niger) is used as a carrier protein, the functional portion retains the catalytic domain of the enzyme and includes amino acids 1-471 (see, WO 03089614, e.g., Example 10, the disclosure of which is incorporated herein by reference). In another embodiment, when CBH I (i.e., CBHI from Trichoderma reesei) is used as a carrier protein, the functional portion retains the catalytic domain of the enzyme. Referring to WO 05093073 Figure 2 SEQ ID NO: 1 of WO 05093073, the disclosure of which is incorporated herein by reference, discloses sequences encoding the Trichoderma reesei CBH1 signal sequence, the Trichoderma reesei CBH1 catalytic domain (also referred to as the catalytic core or core domain), and the Trichoderma reesei CBH1 linker. In some embodiments, the CBH1 carrier protein and the signal sequence will be combined with the Figure 2 The disclosure of the patent is incorporated herein by reference.

[0121] Typically, if the carrier protein is a truncated protein, it is C-terminally truncated (i.e., containing an intact N-terminus). Alternatively, the carrier protein can be N-terminally truncated, or optionally truncated at both ends to leave a functional portion. Generally speaking, such a portion of the secreted protein comprising the carrier protein comprises greater than 50%, greater than 70%, greater than 80%, and greater than 90% of the secreted protein, and in some embodiments, comprises the N-terminal portion of the secreted protein. In some embodiments, the carrier protein will also include a linker region in addition to the catalytic domain. In some embodiments, a portion of the linker region of the CBHI protein can be used in the carrier protein.

[0122] In some embodiments, the first amino acid sequence comprising a signal sequence that functions as a secretory sequence is encoded by a first DNA molecule. The second amino acid sequence comprising a carrier protein is encoded by a second DNA sequence. However, as described above, the signal sequence and the carrier protein can be obtained from the same gene.

[0123] 3. Antibody conjugates and derivatives

[0124] Any antibody disclosed herein may include modified (i.e., by covalent attachment of any type of molecule to the antibody) derivatives. For example, but not by way of limitation, antibody derivatives include antibodies that have been modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization via known protecting groups / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, and the like. Any of a variety of chemical modifications may be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the derivatives may contain one or more non-classical amino acids.

[0125] Antibodies or fragments thereof with increased half-life in vivo can be produced by attaching a polymer molecule, such as a high molecular weight polyethylene glycol (PEG), to the antibody or antibody fragment. PEG can be attached to the antibody or antibody fragment via site-specific conjugation of PEG to the N-terminal or C-terminal of the antibody or antibody fragment or via the ε-amino group present on the lysine residue with or without the use of a multifunctional joint. Linear or branched polymer derivatization resulting in minimal loss of biological activity will be used. The degree of conjugation will be closely monitored by SDS-PAGE and mass spectrometry to ensure the correct conjugation of the PEG molecule to the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate by, for example, size exclusion chromatography or ion exchange chromatography. Further, antibodies can be conjugated to albumin to make the antibody or antibody fragment more stable in vivo or have a longer half-life in vivo. These technologies are well known in the art, see, for example, International Publication Nos. WO 93 / 15199, WO 93 / 15200 and WO 01 / 77137 and European Patent No. EP 413,622. The present invention encompasses the use of antibodies or fragments thereof conjugated or fused to one or more moieties including, but not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetics, synthetic drugs, inorganic molecules, and organic molecules.

[0126] The present invention encompasses the use of antibodies or fragments thereof recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to heterologous proteins or polypeptides (or fragments thereof, e.g., to polypeptides of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids) to produce fusion proteins. The fusion need not necessarily be direct, but may occur via a linker sequence. For example, the antibodies can be used to target heterologous polypeptides to specific cell types in vitro or in vivo by fusing or conjugating the antibodies to antibodies that are specific for specific cell surface receptors. Antibodies fused or conjugated to heterologous polypeptides can also be used in in vitro immunoassays and purification methods using methods known in the art. See, e.g., International Publication No. WO 93 / 21232; European Patent No. EP 439,095; Naramura et al., 1994, Immunol. Lett. 39:91-99; U.S. Pat. No. 5,474,981; Gillies et al., 1992, PNAS 89:1428-1432; and Fell et al., 1991, J. Immunol. 146:2446-2452.

[0127] The present invention further includes compositions comprising heterologous proteins, peptides or polypeptides fused or conjugated to antibody fragments. For example, the heterologous polypeptide can be fused or conjugated to a Fab fragment, a Fd fragment, a Fv fragment, a F(ab)2 fragment, a VH domain, a VL domain, a VH CDR, a VL CDR or a fragment thereof. Methods for fusing or conjugating polypeptides to antibody portions are well known in the art. See, e.g., U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patent Nos. EP 307,434 and EP 367,166; International Publication Nos. WO 96 / 04388 and WO 91 / 06570; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88:10535-10539; Zheng et al., 1995, J. Immunol. 154:5590-5600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337-11341.

[0128] Additional fusion proteins (e.g., antibodies that specifically bind to an antigen (e.g., supra)) can be generated by techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to alter the activity of an antibody or a functional fragment thereof disclosed herein (e.g., an antibody or fragment thereof with higher affinity and lower dissociation rate). See generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252 and 5,837,458; and Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-313. The antibody or fragment thereof or the encoded antibody or fragment thereof may be altered by random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. One or more portions of a polynucleotide encoding an antibody or antibody fragment that specifically binds to an antigen may be recombined with one or more components, motifs, segments, parts, domains, fragments, etc., of one or more heterologous molecules.

[0129] In addition, the antibody or its fragment can be fused with a marker sequence (such as a peptide that promotes purification). In certain embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as a tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, California, 91311), etc., many of which are commercially available. As described in Gentz ​​et al., 1989, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 86: 821-824, for example, hexa-histidine provides convenience for purifying fusion proteins. Other peptide tags that can be used for purification include, but are not limited to, hemagglutinin ("HA") tags corresponding to epitopes derived from influenza hemagglutinin proteins (Wilson et al., 1984, Cell [Cell] 37: 767) and "flag" tags.

[0130] In other embodiments, the antibodies disclosed herein or their analogs or derivatives can be conjugated with diagnostic agents or detectable agents. Such antibodies can be used to monitor or prognose the development or progression of cancer as part of a clinical testing procedure (such as determining the efficacy of a particular therapy). Such diagnosis and detection can be achieved by coupling the antibody to a detectable substance, which includes but is not limited to various enzymes, such as but not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; prosthetic groups, such as but not limited to streptavidin / biotin and avidin / biotin; fluorescent materials, such as but not limited to umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as but not limited to luminol; bioluminescent materials, such as but not limited to luciferase, luciferin and aequorin; radioactive materials, such as but not limited to iodine ( 131 I. 125 I. 123 I. 121 I), carbon ( 14 C) Sulfur ( 35 S), tritium ( 3 H), Indium ( 115 In, 113 In, 112 In, 111 In), and technetium ( 99 Tc), thallium ( 201 Ti、Ga( 68 Ga, 67 Ga), Palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe、Fluorine( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y. 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 Such as 68 Ge, 57 Co. 65 Zn, 85 Sr. 32 P. 153 Gd, 169 Yb, 51 Cr, 54 Mn,75 Se, 113 Sn and 117 Tin; positron emitting metals, non-radioactive paramagnetic metal ions and molecules radiolabeled or conjugated to specific radioisotopes using various positron emission tomography.

[0131] It is also contemplated that the antibody or fragment thereof disclosed herein is conjugated to a therapeutic agent. Antibodies or fragments thereof can be conjugated to a therapeutic moiety, such as a cytotoxin (e.g., a cell growth inhibitor or a cytocidal agent), a therapeutic agent, or a radioactive metal ion (e.g., an α-emitter). Cytotoxins or cytotoxic agents include any agent that is harmful to cells. Examples include ribonucleases, monomethylauristatin E and F, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and their analogs or homologues. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., nitrogen mustard, thioepa chlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). A more extensive list of therapeutic moieties can be found in PCT Publication WO 03 / 075957 (incorporated herein by reference).

[0132] Further, the antibody or its fragment can be conjugated to a therapeutic agent or drug moiety that changes a given biological response. The therapeutic agent or drug moiety should not be construed as being limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide with a desired biological activity. Such proteins can include, for example, toxins such as abrin, ricin A, onconase (or another cytotoxic RNase), Pseudomonas exotoxin, cholera toxin or diphtheria toxin; proteins such as tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, apoptotic agents (e.g., TNF-α, TNF-β, AIM I (see, International Publication No. WO 97 / 33899), AIM II (see, International Publication No. WO 97 / 34911), Fas ligand (Takahashi et al., 1994, J. Immunol. [Journal of Immunology], 6: 1567) and VEGI (see, International Publication No. WO 99 / 23105)), thrombotic agents or anti-angiogenic agents (e.g., angiostatin or endostatin); or biological response modifiers, such as, for example, lymphokines (e.g., interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), and granulocyte colony stimulating factor ("G-CSF")) or growth factors (e.g., growth hormone ("GH")).

[0133] In addition, the antibody can be conjugated to a therapeutic moiety, such as a radioactive material or a macrocyclic chelator that can be used to conjugate a radioactive metal ion (see above for examples of radioactive materials). In certain embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″-tetraacetic acid (DOTA), which can be attached to the antibody via a linker molecule. Such linker molecules are well known in the art and are described in Denardo et al., 1998, Clin Cancer Res. 4:2483; Peterson et al., 1999, Bioconjug. Chem. 10:553; and Zimmerman et al., 1999, Nucl. Med. Biol. 26:943.

[0134] The techniques for conjugating therapeutic moieties to antibodies and related molecules are well known. Moieties can be conjugated to antibodies by any method known in the art, including but not limited to aldehyde / Schiff linkages, sulfhydryl linkages, acid labile linkages, cis-aconityl linkages, hydrazone linkages, enzymatically degradable linkages (see generally, Garnett, 2002, Adv Drug Deliv Rev [Advanced Drug Delivery Review] 53: 171).Techniques for conjugating therapeutic moieties to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," In Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (Eds.), pp. 243-56 (Alan R. Liss, Inc., 1985); Hellstrom et al., "Antibodies For Drug Delivery," In Controlled Drug Delivery (2nd ed.), Robinson et al. (Eds.), pp. 623-53 (Marcel Dekker, Inc., 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press, 1985); and Thorpe et al., 1982, Immunol. Rev. 62:119.

[0135] Methods for fusing or conjugating antibodies and related molecules to polypeptide moieties are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851 and 5,112,946; EP 307,434; EP 367,166; PCT Publications WO 96 / 04388 and WO 91 / 06570; Ashkenazi et al., 1991, PNAS USA [Proceedings of the National Academy of Sciences of the United States of America] 88:10535; Zheng et al., 1995, J Immunol [Journal of Immunology] 154:5590; and Vil et al., 1992, PNAS USA [Proceedings of the National Academy of Sciences of the United States of America] 89:11337. Fusion of the antibody to the moiety need not necessarily be direct, but may occur via a linker sequence. Such linker molecules are well known in the art and are described in Denardo et al., 1998, Clin Cancer Res 4:2483; Peterson et al., 1999, Bioconjug Chem 10:553; Zimmerman et al., 1999, Nucl Med Biol 26:943; Garnett, 2002, Adv Drug Deliv Rev 53:171.

[0136] Antibodies can also be attached to solid supports, which are particularly useful for immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

[0137] The therapeutic moiety or drug conjugated to an antibody (such as any of those disclosed herein) should be selected to achieve one or more desired prophylactic or therapeutic effects for a particular condition in a subject. In deciding which therapeutic moiety or drug to conjugate to an antibody, a clinician or other medical personnel should consider the following factors: the nature of the disease, the severity of the disease, and the condition of the subject.

[0138] D. Polynucleotide

[0139] Another aspect of the compositions and methods disclosed herein is a polynucleotide or nucleic acid sequence encoding an antibody or functional fragment thereof or encoding a BASI polypeptide for use in any of the methods disclosed herein.

[0140] Provided herein is a fusion DNA construct encoding an antibody or a functional fragment thereof and / or a BASI polypeptide, comprising: an operably linked promoter; a first DNA molecule encoding a signal sequence; a second DNA molecule encoding a carrier protein; a third DNA molecule encoding an antibody (e.g., a heavy chain and / or a light chain) or a functional fragment thereof; and a fourth DNA molecule encoding a BASI polypeptide. The components of the fusion DNA construct may appear in any order. Since the genetic code is known, the design and production of these nucleic acids are well within the skill of ordinary technicians, given the description of the antibodies or functional fragments thereof and / or BASI polypeptides disclosed herein. In certain embodiments, the nucleic acids may be codon-optimized to express the antibodies or functional fragments thereof and / or BASI polypeptides in specific host cells. Since codon usage tables for many species (e.g., mammalian cells and filamentous fungi) are available, the design and production of codon-optimized nucleic acids encoding the subject antibodies and / or BASI polypeptides will be well within the skill of those skilled in the art.

[0141] E. Promoter

[0142] Examples of suitable promoters for directing transcription of nucleic acids in a host cell (e.g., a filamentous fungal host cell) are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-amylase (Korman et al. (1990) Curr. Genet [Current Genetics] 17:203-212; Gines et al., (1989) Gene [Gene] 79:107-117), Aspergillus niger or Aspergillus awamori glucoamylase (glaA) (Nunberg et al., (1984) Mol. Cell Biol. [Molecular and Cellular Biology] 4:2306-2315; Boel E. et al., (1984) EMBO J. [Journal of the European Molecular Biology Association] 3:1581-1585), Rhizomucor miehei lipase, Aspergillus oryzae (Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-amylase (Korman et al., (1990) Curr. Genet [Current Genetics] 17:203-212; Gines et al., (1989) Gene [Gene] 79:107-117), Aspergillus niger or Aspergillus awamori glucoamylase (glaA) (Nunberg et al., (1984) Mol. Cell Biol. [Molecular and Cellular Biology] 4:2306-2315; Boel oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase (Hyner et al., (1983) Mol. Cell. Biol. 3:1430-1439), Fusarium venenatum amyloglucosidase, Fusarium oxysporum trypsin-like protease (WO 96 / 00787), Trichoderma reesei cellobiohydrolase I (Shoemaker et al. (1984) EPA EPO 0137280), Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei β-xylosidase, and the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral α-amylase and Aspergillus oryzae triose phosphate isomerase); and mutants, truncated and hybrid promoters thereof. See also Yelton et al., (1984) Proc. Natl. Acad. Sci. USA 81:1470-1474; Mullaney et al., (1985) Mol. Gen. Genet. 199:37-45; Lockington et al., (1986) Gene 33:137-149; Macknight et al., (1986) Cell 46:143-147; Hynes et al., (1983) Mol. Cell Biol. 3:1430-1439.Higher eukaryotic promoters such as the SV40 early promoter (Barclay et al. (1983) Molecular and Cellular Biology 3:2117-2130) may also be useful. The promoter may be a constitutive promoter or an inducible promoter. Exemplary promoters include Trichoderma reesei cellobiohydrolase I or II, Trichoderma reesei endoglucanase I, II or III, and Trichoderma reesei xylanase II.

[0143] F. Carrier

[0144] The polynucleotide encoding any antibody or functional fragment thereof and / or BASI polypeptide disclosed herein may be present in a vector, for example, a phage, plasmid, virus, or retroviral vector. In certain embodiments, the vector may be an expression vector for expressing a subject fusion polypeptide in a filamentous fungal cell.

[0145] Vectors for expressing recombinant proteins are well known in the art (Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., (1989) Cold Spring Harbor, NY).

[0146] Fusion DNA constructs can be constructed using well-known techniques, as generally described in, for example, European Patent Application Publication No. 0 215 594, the disclosure of which is incorporated herein by reference.

[0147] Natural or synthetic polynucleotide fragments encoding a polypeptide of interest (eg, an immunoglobulin) can be incorporated into a heterologous nucleic acid construct or vector capable of introduction into a host cell (eg, a filamentous fungal cell) and replication in the host cell.

[0148] Once a DNA construct or more specifically a fusion DNA construct is prepared, it can be incorporated into a number of vectors known in the art. While in some embodiments, the DNA construct will include a promoter sequence, in other embodiments, the vector will include other regulatory sequences that function in the host to be transformed, such as ribosome binding sites, transcription start and stop sequences, terminator sequences, polyadenylation signals, enhancers and or activators. In some embodiments, polynucleotides encoding antibodies or functional fragments thereof and / or BASI polypeptides are inserted into a vector comprising a promoter, signal sequence and carrier protein at appropriate restriction endonuclease sites by standard procedures. Such procedures and related subcloning procedures are considered to be within the knowledge of those skilled in the art.

[0149] The terminator sequence recognized by the expression host to terminate transcription can be operably linked to the 3' end of the fusion DNA construct encoding the antibody to be expressed or its functional fragment and / or BASI polypeptide. Those of ordinary skill in the art are familiar with various terminator sequences that can be used with host cells (such as filamentous fungi). Non-limiting examples include the terminator from the Aspergillus nidulans trpC gene (Yelton M. et al., (1984) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 81: 1470-1474) or the terminator from the Aspergillus niger glucoamylase gene (Nunberg et al. (1984) Mol. Cell. Biol. [Molecular and Cellular Biology] 4: 2306-2353) or the terminator from the Trichoderma reesei cellobiohydrolase I gene.

[0150] The polyadenylation sequence is a DNA sequence that, when transcribed, is recognized by the expression host to add polyadenosine residues to the transcribed mRNA. Examples include the polyadenylation sequence from the following genes: Aspergillus nidulans trpC gene (Yelton et al. (1984) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 81; 1470-1474); Aspergillus niger glucoamylase gene (Nunberg et al. (1984) Mol. Cell. Biol. [Molecular and Cell Biology] 4: 2306-2315); Aspergillus oryzae or Aspergillus niger alpha amylase gene and Rhizomucor miehei carboxyproteinase gene.

[0151] In a further embodiment, the fusion DNA construct or a vector comprising the fusion DNA construct will contain a selectable marker gene to allow selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used. Examples of selectable markers include, but are not limited to, selectable markers that confer antimicrobial resistance (e.g., hygromycin, bleomycin, chloramphenicol, and phleomycin). Genes that confer metabolic advantages, such as nutritional selective markers, can also be used. Some of these markers include amdS. In addition, sequences encoding genes that complement auxotrophic defects can be used as selectable markers (e.g., pyr4 complements of pyr4-deficient Aspergillus nidulans, Aspergillus awamori, or Trichoderma reesei and argB complements of argB-deficient strains). See Kelley et al., (1985) EMBO J. 4:475-479; Penttila et al., (1987) Gene 61:155-164 and Kinghorn et al. (1992) Applied Molecular Genetics of Filamentous Fungi, Blackie Academic and Professional, Chapman and Hall, London, the disclosures of each of which are incorporated herein by reference.

[0152] G. Host cells

[0153] Provided herein are recombinant host cells comprising a heterologously expressed barley alpha-amylase subtilisin inhibitor (BASI) polypeptide and a heterologously expressed monoclonal antibody or a functional fragment thereof.

[0154] An expression cassette or vector expressing a polynucleotide encoding the above-mentioned BASI polypeptide and / or monoclonal antibody or a functional fragment thereof can be introduced into a suitable expression host cell, and then the expression host cell expresses the corresponding polynucleotide encoding the antibody or a functional fragment thereof and / or BASI polypeptide.

[0155] Suitable host cells include cells of any microorganism (e.g., bacteria, protists, algae, fungi (e.g., yeast or filamentous fungi) or other microorganisms), and can be cells of bacteria, yeast, plants or filamentous fungi. Fungal expression hosts can be, for example, yeast. Also suitable are mammalian expression hosts, such as mouse (e.g., NS0), Chinese hamster ovary (CHO), human embryonic kidney (HEK) or baby hamster kidney (BHK) cell lines. Other eukaryotic hosts such as insect cells (such as Drosophila S2 cells) or viral expression systems (e.g., phages such as M13, T7 phage or lambda, or viruses such as baculovirus) are also suitable for producing polypeptides.

[0156] Suitable host cells of the bacterial genera include, but are not limited to, cells of the genera Escherichia, Proteus, Bacillus, Ralstonia, Lactobacillus, Lactococcus, Pseudomonas, Staphylococcus, and Streptomyces. Suitable cells of bacterial species include, but are not limited to, cells of the following species: Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Lactobacillus brevis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas stutzerei, Staphylococcus carnosus, Lactococcus lactis, Ralstonia eutropha, Proteus mirabilis, and Streptomyces lividans.

[0157] Suitable host cells of the genus Saccharomyces include, but are not limited to, cells of the genus Saccharomyces, Schizosaccharomyces, Candida, Hansenula, Pichia, Kluyveromyces, Yarrowia, and Phaffia. Suitable cells of yeast species include, but are not limited to, cells of the species Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Hansenula polymorpha, Yarrowia lipolytica, Pichia pastoris, P. canadensis, Kluyveromyces marxianus, and Phaffia rhodozyma.

[0158] Suitable host cells for filamentous fungi include all filamentous forms of the subdivision Eumycotina. Suitable cells for filamentous fungi include, but are not limited to, cells of the following genera: Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Corynascus, Chaertomium, Cryptococcus, Filobasidium, Fusarium, Gibberella, Humicola, Magnaporthe, Mucor, Myceliophthora, Mucor and fungi.

[0159] Suitable cells of filamentous fungal species include, but are not limited to, cells of the following species: Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium lukenowensis, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium spicata, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium pseudosporum, Fusarium spp. sporotrichioides), Fusarium sulphureum, Fusarium torulosum, Fusariumtrichothecioides, Fusarium veneer, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Coprinus cinereus, Coriolushirsutus, Humicola insolens, Humicola velutipes lanuginosa), Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Neurospora intermedia, Penicilliumpurpurogenum), Penicillium canescens, Penicillium solitum, Penicillium funiculosum, Phanerochaete chrysosporium, Phlebia radiate, Pleurotus eryngii, Talaromyces flavus, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride.

[0160] Promoters and / or signal sequences associated with secreted proteins in a particular host of interest are candidates for heterologous production and secretion of antibodies or functional fragments thereof and / or BASI polypeptides in that or other hosts. As a non-limiting example, in filamentous fungal systems, promoters driving genes for cellobiohydrolase I (cbhl), glucoamylase A (glaA), TAKA-amylase (amyA), xylanase (exlA), gpdA-promoter cbhl, cbhlll, endoglucanase genes egl-eg5, Cel61B, Cel74A, gpd promoter, Pgkl, pkil, EF-1alpha, tefl, cDNAl and hexl are suitable and can be derived from many different organisms (e.g., Aspergillus niger, Trichoderma reesei, Aspergillus oryzae, Aspergillus awamori, Aspergillus nidulans).

[0161] In some embodiments, the polynucleotide encoding the antibody or its functional fragment and / or BASI polypeptide is recombinantly associated with a polynucleotide encoding a suitable homologous or heterologous signal sequence, which results in secretion of the recombinant polypeptide into the extracellular (or periplasmic) space, thereby allowing direct detection in the cell supernatant (or periplasmic space or lysate). Suitable signal sequences for E. coli, other Gram-negative bacteria, and other organisms known in the art include those that drive the expression of HlyA, DsbA, Pbp, PhoA, PelB, OmpA, OmpT, or the M13 phage Gill gene. For Bacillus subtilis, Gram-positive organisms, and other organisms known in the art, suitable signal sequences further include those that drive the expression of AprE, NprB, Mpr, AmyA, AmyE, Blac, SacB, and for Saccharomyces cerevisiae or other yeasts, include killer toxins, Bar1, Suc2, mating factor alpha, Inu1A, or Ggplp signal sequences. The signal sequence can be cleaved by a number of signal peptidases, thereby removing it from the remainder of the expressed protein.

[0162] In some embodiments, the antibody or its functional fragment and / or BASI polypeptide is expressed alone or as a fusion with an additional peptide, tag or protein (e.g., 6XHis, HA or FLAG tag) located at the N-terminus or C-terminus. Suitable fusions include tags, peptides or proteins (e.g., 6XHis, HA, chitin binding protein, thioredoxin or FLAG tags) that facilitate affinity purification or detection and those that promote expression, secretion or processing of the target β-glucosidase. In addition to KEX2, further suitable processing sites include enterokinase, STE13 or other protease cleavage sites known in the art for in vivo or in vitro cleavage.

[0163] The polynucleotide encoding the antibody or functional fragment thereof and / or BASI polypeptide can be introduced into the expression host cell by a number of transformation methods, including but not limited to electroporation, lipid-assisted transformation or transfection ("lipofection"), chemical-mediated transfection (e.g., CaCl and / or CaP), lithium acetate-mediated transformation (e.g., of host cell protoplasts), biolistic "gene gun" transformation, PEG-mediated transformation (e.g., of host cell protoplasts), protoplast fusion (e.g., using bacterial or eukaryotic protoplasts), liposome-mediated transformation, Agrobacterium tumefaciens, adenovirus or other viral or bacteriophage transformation or transduction.

[0164] III. Methods

[0165] A. Methods for producing antibodies

[0166] The antibodies used in the methods disclosed herein can be produced by any method known in the art for the synthesis of antibodies, in particular by chemical synthesis or by recombinant expression techniques.

[0167] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridomas, recombinant and phage display techniques or combinations thereof. For example, monoclonal antibodies can be produced using hybridoma technology, including hybridoma technology known in the art and taught in the following documents: for example, Harlow et al., Antibodies: A Laboratory Manual [antibodies: laboratory manual], (Cold Spring Harbor Laboratory Press [Cold Spring Harbor Laboratory Press], 2nd Edition 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas [monoclonal antibodies and T cell hybridomas] 563-681 (Elsevier, NY [New York Elsevier Press], 1981). The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic or phage clone) rather than a method for producing it.

[0168] The method for producing and screening specific antibodies using hybridoma technology is conventional and well-known in the art. In short, mice can be immunized with antigens or their immunogenic fragments, and once an immune response is detected, for example, antibodies specific to the applied antigen are detected in mouse serum, mouse spleens are harvested and splenocytes are separated. Then splenocytes are fused with any suitable myeloma cells (e.g., cells from the SP20 cell line available from ATCC) by well-known technology. In addition, RIMMS (repeated immunity, multiple sites) technology can be used for immune animals (Kilpatrick et al., 1997, Hybridoma [hybridoma] 16: 381-9). Hybridomas are selected and cloned by limiting dilution. Then hybridoma clones are determined for cells secreting antibodies capable of binding polypeptides of the present invention by methods known in the art. Ascites (which generally contain high levels of antibodies) can be produced by cloning immune mice with positive hybridomas.

[0169] Thus, monoclonal antibodies can be produced by culturing antibody-secreting hybridoma cells, wherein hybridomas can be produced by fusing spleen cells isolated from mice immunized with the antigen or an immunogenic fragment thereof with myeloma cells and then screening the hybridomas produced by the fusion for hybridoma clones that secrete antibodies capable of binding the administered antigen.

[0170] The antibody used in the methods disclosed herein may contain novel amino acid residues in its hinge region in addition. Engineered antibodies can be produced by a variety of methods well known to those skilled in the art. Non-limiting examples include separating antibody coding regions (e.g., from hybridomas) and introducing one or more hinge modifications of the present invention into separated antibody coding regions. Alternatively, the variable region can be subcloned into a vector encoding a modified hinge region (such as any of those disclosed herein). Additional methods and details are provided hereinafter.

[0171] Antibody fragments that recognize specific antigens can be produced by any technique known to those skilled in the art. For example, Fab and F(ab')2 fragments of the present invention can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab')2 fragments contain variable regions, light chain constant regions, and the CH1 domain of heavy chains. Further, antibodies disclosed herein or their functional fragments can also be generated using various phage display methods known in the art.

[0172] In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying polynucleotide sequences encoding them. Specifically, the DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or mouse cDNA libraries of lymphoid tissue). The DNA encoding VH and VL domains is recombined together with scFv joints by PCR, and cloned into phagemid vectors (e.g., p CANTAB 6 or pComb 3HSS). The vector is electroporated into Escherichia coli, and Escherichia coli is infected with helper phage. The phage used in these methods is typically a filamentous phage, including fd and M13, and VH and VL domains are usually recombinantly fused with phage gene III or gene VIII. The phage expressing the antigen binding domains combined with the target antigen epitope can be selected or identified using, for example, labeled antigens or antigens combined or captured on solid surfaces or beads. Examples of phage display methods that can be used to prepare the antibodies or functional fragments thereof disclosed herein include those disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186; Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology 57:191-280; PCT Publication Nos. WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, WO 95 / 15982, WO 95 / 20401 and WO 97 / 13844; and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108.

[0173] As described in the above references, after phage selection, the antibody coding region can be isolated from the phage and used to produce complete antibodies (including human antibodies) or any other desired antigen-binding fragments and expressed in any desired host (including mammalian cells, insect cells, plant cells, yeast and bacteria), for example as described below. Techniques for recombinant production of Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art, such as those disclosed in International Publication No. WO 92 / 22324; Mullinax et al., 1992, BioTechniques [Biotechnology] 12 (6): 864-869; Sawai et al., 1995, AJRI [American Journal of Reproductive Immunology] 34: 26-34; and Better et al., 1988, Science [Science] 240: 1041-1043.

[0174] In order to produce complete antibodies, PCR primers (including VH or VL nucleotide sequences), restriction sites and flanking sequences protecting restriction sites can be used to amplify the VH or VL sequences in scFv clones. Using cloning techniques known to those skilled in the art, the VH domain amplified by PCR can be cloned into a vector expressing a VH constant region (e.g., a human γ constant region), and the VL domain amplified by PCR can be cloned into a vector expressing a VL constant region (e.g., a human κ or λ constant region). It is contemplated that the constant region comprises a modified hinge (such as any modified hinge disclosed herein). In certain embodiments, the vector for expressing the VH or VL domain comprises a promoter, a secretion signal, a cloning site for both the variable domain and the constant domain, and a selection marker (such as neomycin). The VH and VL domains can also be cloned into a vector expressing the desired constant region. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into a cell line using techniques known to those skilled in the art to produce a stable or transient cell line expressing a full-length antibody (e.g., IgG).

[0175] Chimeric antibodies are molecules in which different portions of the antibody are derived from different immunoglobulin molecules. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567, 4,816397, and 6,311,415.

[0176] For some uses, including in vivo uses of antibodies in humans and in vitro detection assays, it may be preferred to use human antibodies or chimeric antibodies. Fully human antibodies are particularly desirable for therapeutic treatments of human subjects. Human antibodies can be prepared by a variety of methods known in the art, including the above-mentioned phage display methods using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT Publication Nos. WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741.

[0177] Humanized antibody is such antibody or its variant or fragment, it can be combined with predetermined antigen and comprises framework region with the amino acid sequence of human immunoglobulin substantially and CDR with the amino acid sequence of non-human immunoglobulin substantially.Humanized antibody comprises at least one, typically all of two variable domains (Fab, Fab', F(ab')2, Fabc, Fv), wherein all or substantially all CDR regions correspond to the CDR regions of non-human immunoglobulin (i.e., donor antibody), and all or substantially all framework regions are framework regions of human immunoglobulin consensus sequence.In a specific embodiment, humanized antibody also comprises at least a portion of immunoglobulin constant region (Fc) (typically the constant region of human immunoglobulin).Usually, antibody will contain at least variable domains of light chain and heavy chain.Antibody can also include CH1, hinge, CH2, CH3 and CH4 regions of heavy chain. Humanized antibodies can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE; and any isotype, including IgG1, IgG2, IgG3 and IgG4. Typically, in the case where the humanized antibody is required to exhibit cytotoxic activity, the constant domain is a complement fixation constant domain, and the class is typically IgG.sub.1. In the case where this cytotoxic activity is not required, the constant domain may belong to the IgG.sub.2 class. Humanized antibodies may include sequences from more than one class or isotype, and selecting a specific constant domain to optimize the desired effector function is within the ordinary skill of the art. The framework region and CDR region of the humanized antibody do not need to correspond exactly to the parent sequence, for example, the donor CDR or the consensus framework may be mutated by substitution, insertion or deletion of at least one residue so that the CDR or framework residue at the site does not correspond to the consensus sequence or the input antibody. However, such mutations will not be extensive. Usually, at least 75%, and more often 90% or greater than 95% of the humanized antibody residues will correspond to those of the parental framework region (FR) and CDR sequences.Humanized antibodies can be produced using a variety of techniques known in the art, including, but not limited to, CDR grafting (European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089); veneering or resurfacing (European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering [Protein Engineering] 7(6):805-814; and Roguska et al., 1994, PNAS [Proceedings of the National Academy of Sciences of the United States of America] 91:969-973); chain shuffling (U.S. Pat. No. 5,565,332) and techniques disclosed in, for example, U.S. Pat. No. 6,407,213; U.S. Pat. No. 5,766,886; WO 9317105; Tan et al., J. Immunol. [Journal of Immunology] 169:1119-25 (2002); Caldas et al., Protein Eng. 13(5):353-60 (2000); Morea et al., Methods 20(3):267-79 (2000); Baca et al., J. Biol. Chem. 272(16):10678-84 (1997); Roguska et al., Protein Eng. 9(10):895-904 (1996); Couto et al., Cancer Res. 55(23 Suppl):5973s-5977s (1995); Couto et al., Cancer Res. 55(8):1717-22 (1995); Sandhu JS, Gene 150 (2): 409-10 (1994) and Pedersen et al., J. Mol. Biol. 235 (3): 959-73 (1994). Often, framework residues in the framework region will be substituted with corresponding residues from the CDR donor antibody to alter (preferably improve) antigen binding. These framework substitutions are identified by methods well known in the art, such as by modeling the interactions of the CDR and framework residues to identify framework residues that are important for antigen binding and sequence comparison to identify unusual framework residues at specific positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature 332: 323).

[0178] Transgenic mice can also be used to produce human antibodies, which cannot express functional endogenous immunoglobulins, but can express human immunoglobulin genes. For example, human heavy chain and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, in addition to human heavy chain and light chain genes, human variable regions, constant regions and diversity regions can also be introduced into mouse embryonic stem cells. Mouse heavy chain and light chain immunoglobulin genes can be made non-functional separately or simultaneously by introducing human immunoglobulin loci via homologous recombination. Specifically, homozygous deletions in the JH region prevent endogenous antibody production. Modified embryonic stem cells are amplified and microinjected into blastocysts to produce chimeric mice. Chimeric mice are then bred to produce homozygous offspring expressing human antibodies. Transgenic mice are immunized in a normal manner with selected antigens or their immunogenic fragments. Conventional hybridoma technology can be used to obtain monoclonal antibodies against antigens from immunized transgenic mice. The human immunoglobulin transgenes possessed by transgenic mice are rearranged during B cell differentiation, and then undergo class switching and somatic mutations. Therefore, using this technology, IgG, IgA, IgM and IgE antibodies useful in treatment can be produced. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. [International Immunology Review] 13: 65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and the scheme for preparing such antibodies, see, for example, International Publication Nos. WO 98 / 24893, WO 96 / 34096 and WO 96 / 33735; and U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318 and 5,939,598.

[0179] Further, the antibodies disclosed herein or their functional fragments can be used to generate anti-idiotype antibodies that "mimic" receptors using techniques well known to those skilled in the art. (See, for example, Greenspan and Bona, 1989, FASEBJ. [Journal of the Federation of American Societies for Experimental Biology] 7 (5): 437-444; and Nissinoff, 1991, J. Immunol. [Journal of Immunology] 147 (8): 2429-2438). For example, antibodies of the present invention that bind to a receptor and competitively inhibit the binding of a receptor (as determined by assays well known in the art and disclosed below) to its ligand can be used to generate anti-idiotypes that "mimic" ligands and thus bind to and neutralize receptors and / or their ligands. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize ligands and / or their receptors. Provided herein are methods of using polynucleotides comprising nucleotide sequences encoding antibodies or fragments thereof.

[0180] In one embodiment, the nucleotide sequence of the antibody encoding specific binding antigen is obtained, and it is used to produce the antibody disclosed herein or its functional fragment. The nucleotide sequence can be obtained by sequencing the hybridoma clone DNA. If there is no clone containing the nucleic acid encoding a specific antibody or its epitope binding fragment, but the sequence of the antibody molecule or its epitope binding fragment is known, the nucleic acid encoding the immunoglobulin can be chemically synthesized, or by using synthetic primers hybridized with the 3' end and 5' end of the sequence to carry out PCR amplification or by using oligonucleotide probes specific to a specific gene sequence to clone to identify, for example, cDNA clones from a cDNA library encoding the antibody and obtain from a suitable source (e.g., an antibody cDNA library, or a cDNA library produced from any tissue or cell expressing the antibody (such as a hybridoma cell selected to express the antibody) or a nucleic acid separated therefrom, preferably poly A+RNA). The amplified nucleic acid produced by PCR can then be cloned into a reproducible cloning vector using any method well known in the art.

[0181] Once the nucleotide sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for manipulating nucleotide sequences, such as recombinant DNA techniques, site-directed mutagenesis, PCR, and the like (see, for example, the techniques described in Current Protocols in Molecular Biology, FM Ausubel et al., eds., John Wiley & Sons (Chichester, England, 1998); Molecular Cloning: A Laboratory Manual, 3rd ed., J. Sambrook et al., eds., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, New York, 2001); Antibodies: A Laboratory Manual, E. Harlow and D. Lane et al., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, New York, 1988); and Using Antibodies: A Laboratory Manual, E. Harlow and D. Lane, eds., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, New York, 1989). Laboratory [Cold Spring Harbor Laboratory] (Cold Spring Harbor, NY, 1999)) to produce antibodies with different amino acid sequences by, for example, introducing deletions and / or insertions into desired regions of the antibody.

[0182] In a specific embodiment, one or more CDRs are inserted into the framework region using conventional recombinant DNA techniques. The framework region may be a naturally occurring or shared framework region, including but not limited to a human framework region (for a list of human framework regions, see, for example, Chothia et al., 1998, J. Mol. Biol. [Journal of Molecular Biology] 278: 457-479). It is contemplated that the polynucleotide encoding the antibody specifically bound to the antigen is generated by the combination of the framework region and the CDR. In one embodiment, as discussed above, one or more amino acid substitutions may be made in the framework region, and in certain embodiments, the amino acid substitution improves the binding of the antibody to its antigen. In addition, such methods may be used to carry out amino acid substitutions or deletions of one or more variable region cysteine ​​residues that participate in intrachain disulfide bonds to produce antibody molecules lacking one or more intrachain disulfide bonds. Other changes to the polynucleotide are encompassed by the present invention and are within the technical scope of the art.

[0183] The hinge of the antibody identified from such screening methods can be modified as described above to produce an antibody incorporating a modified hinge (such as any of those disclosed above). It is further contemplated that the antibodies disclosed herein can be used to prevent, manage and treat diseases, disorders, infections, including but not limited to inflammatory diseases, autoimmune diseases, bone metabolism-related disorders, angiogenesis-related disorders, infections and cancers. Such antibodies can be used in the methods and compositions disclosed herein.

[0184] B. Recombinant expression of antibodies and BASI peptides

[0185] Also provided herein are methods for reducing proteolysis of heterologously expressed monoclonal antibodies or functional fragments thereof, which methods comprise culturing recombinant cells comprising a heterologously expressed BASI polypeptide and a heterologously expressed monoclonal antibody or functional fragment thereof under suitable conditions for producing the heterologously expressed antibody or functional fragment thereof and a barley alpha-amylase subtilisin inhibitor (BASI) polypeptide.

[0186] The recombinant expression of antibodies or functional fragments thereof (as well as derivatives, analogs or fragments thereof) and BASI polypeptides requires the construction of expression vectors containing polynucleotides encoding antibodies or functional fragments thereof and BASI polypeptides. Once one or more polynucleotides encoding antibodies and / or BASI polypeptides have been obtained, vectors for producing antibodies and / or BASI polypeptides can be generated by recombinant DNA technology using techniques well known in the art. Therefore, methods for preparing proteins by expressing polynucleotides containing nucleotide sequences encoding antibodies and / or nucleotide sequences encoding BASI are described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody and BASI coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

[0187] The expression vector is transferred into a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce antibodies and / or BASI polypeptides (such as any of those disclosed herein). In specific embodiments for expressing antibodies comprising double-chain antibodies, vectors encoding both the heavy and light chains can be co-expressed in the host cell to express the entire immunoglobulin molecule.

[0188] In some embodiments, the recombinant co-expression of a BASI polypeptide and a monoclonal antibody or a functional fragment thereof results in reduced proteolysis of the monoclonal antibody or a functional fragment thereof, compared to recombinant expression of the monoclonal antibody or a functional fragment thereof alone (i.e., without co-expression with a BASI polypeptide). In some embodiments, the recombinant co-expression of a BASI polypeptide and a monoclonal antibody or a functional fragment thereof results in reduced proteolysis of the monoclonal antibody or a functional fragment thereof by about 5%-100%, 10-90%, 20%-80%, 30%-70%, 40%-60%, 50%-100%, 50%-90%, 50%-75%, 60%-100%, 60%- 90%, 60%-80%, 75%-100%, 75%-95%, 80%-100%, 80%-90%, such as about any of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (including all values ​​falling between these percentages).

[0189] Further, the present invention provides a method for reducing proteolysis of a heterologously expressed monoclonal antibody or a functional fragment thereof, the method comprising isolating or purifying the antibody or a functional fragment thereof in the presence of an exogenously added barley alpha-amylase subtilisin inhibitor (BASI) polypeptide. In some embodiments, isolating or purifying the antibody or a functional fragment thereof in the presence of an exogenously added BASI polypeptide results in reduced proteolysis of the monoclonal antibody or a functional fragment thereof compared to isolating or purifying the monoclonal antibody or a functional fragment thereof in the absence of an exogenously added BASI polypeptide. In some embodiments, isolating or purifying the antibody or a functional fragment thereof in the presence of an exogenously added BASI polypeptide results in reduced proteolysis of the monoclonal antibody or a functional fragment thereof by about 5%-100%, 10-90%, 20%-80%, 30%-70%, 40%-60%, 50%-100%, 50%-90%, 50%-75%, 60%-80%, 80%-90%, 80%-10 ... %-100%, 60%-90%, 60%-80%, 75%-100%, 75%-95%, 80%-100%, 80%-90%, such as about any of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (including all values ​​falling between these percentages).

[0190] C. Antibody purification

[0191] The method for purifying polypeptide (comprising antibody or its Fab provided herein) from host cell will depend on selected host cell and expression system.For secreted molecules, generally after removing cells, protein is purified from culture medium.For intracellular expression, cell lysis and protein purification from extract can be performed.In one example, by centrifugation and cell lysis (for example, by repeated freezing and thawing in dry ice / ethanol bath), centrifugation is subsequently performed, and the supernatant containing polypeptide is retained to separate polypeptide from host cell.When transgenic organisms such as transgenic plants and animals are used for expression, tissues or organs can be used as starting materials for preparing lysed cell extracts.In addition, the production of transgenic animals can include producing polypeptide in milk or eggs, milk or eggs can be collected, and if necessary, protein can be further extracted, and further purified using standard methods in this area.

[0192] Proteins such as antibodies or their antigen-binding fragments provided herein can be purified from cracked cell extracts using, for example, standard protein purification techniques known in the art, including but not limited to SDS-PAGE, size fractionation and size exclusion chromatography, ammonium sulfate precipitation, and ion exchange chromatography (such as anion exchange). Affinity purification techniques can also be used to improve the efficiency and purity of preparation. For example, antibodies, receptors and other molecules in conjunction with proteases can be used for affinity purification. Expression constructs can also be engineered to add affinity tags to proteins (such as myc epitopes, GST fusions or His6), and affinity purification is performed using myc antibodies, glutathione resins, and Ni resins, respectively. Purity can be assessed by any method known in the art, including gel electrophoresis and staining and spectrophotometric techniques.

[0193] Typically, the antibody and its part are purified by any program known to those skilled in the art. Standard protein purification techniques known in the art can be used to purify the antibody to a fairly high purity, including but not limited to SDS-PAGE, size fractionation and size exclusion chromatography, ammonium sulfate precipitation, chelate chromatography, ion exchange chromatography or column chromatography. For example, the antibody can be purified by column chromatography. An example of a method for purifying the antibody is by using column chromatography, wherein a solid support column material is connected to protein G, which is a cell surface-associated protein from streptococci that binds immunoglobulins with high affinity. The antibody can be purified to 60%, 70%, 80% purity, and typically at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% purity. Purity can be assessed by standard methods, such as by SDS-PAGE and Coomassie staining.

[0194] The gel can then be separated, for example, on a gel (e.g., an SDS-Page gel), size fractionated (e.g., on a Sephacryl gel). TMS-200HiPrep TM The isolated polypeptides are analyzed by separation on a 16×60 size exclusion column (Amersham, GE Healthcare Life Sciences, Piscataway, NJ). The isolated polypeptides can also be analyzed in binding assays, typically using binding partners bound to a solid support (e.g., bound to a plate (e.g., ELISA-based binding assay) or beads) to determine their ability to bind to a desired binding partner. The binding assays described in the following sections for evaluating the binding of precipitated phage displaying the polypeptides can also be used to evaluate polypeptides isolated directly from host cell lysates. For example, a binding assay can be performed to determine whether an antibody polypeptide binds to one or more antigens, for example, by coating the antigen on a solid support (such as the wells of an assay plate) and incubating the isolated polypeptide on the solid support, followed by washing and detection with a secondary reagent (e.g., an enzyme-labeled antibody and substrate).

[0195] The invention may be further understood by reference to the following examples, which are provided by way of illustration and not limitation.

[0196] Examples

[0197] Example 1: Construction of Trichoderma reesei strains for co-expression of BASI and antibodies

[0198] pIT and pT1A vectors: Two plasmids, pI1 and pT1A, which are pUC18-based E. coli vectors, were used for subcloning of fungal expression cassettes. Each plasmid contains two approximately 1 kb long Trichoderma reesei QM6a DNA sequences (upstream and downstream), allowing homologous recombination at the respective genomic loci. The pI1 vector is used for targeted integration at chr3 and has the native pyr2 as a selectable marker, while the pT1A vector is used for targeted integration at chr2 and encodes amdS for selection with acetamide as the sole nitrogen source. Plasmid pI1 was constructed using Gibson assembly using the following four PCR products: (1) UHR (upstream homology region), amplified from QM6a genomic DNA (from chr3); (2) pyr2 gene (Trichoderma reesei orotate phosphoribosyltransferase), as (2014) from the vector pTrex8gM; (3) DHR (downstream homology region), amplified from QM6a genomic DNA (chr3); (4) pUC18 backbone (pUC18 replication origin and E. coli bla, encoding β-lactamase), amplified from the vector pUC18. PCR reactions were performed using Q5 High-Fidelity DNA Polymerase (NEB - Ipswich, US) according to standard protocols. The assembled products were added to 50 μL TOP10 chemically competent E. coli (Thermofisher, Waltham US) cells and transformed according to standard protocols. Plasmid DNA was isolated from E. coli colonies using the NucleoSpin Plasmid Miniprep Kit (Macherey-Nagel, Düren, Germany) according to standard protocols. The construction of pT1A plasmid was similar, but with two differences, namely, the upstream and downstream homologous sequences targeted the genomic location of chr2, and the selection marker was the Aspergillus nidulans amdS gene (GenBank: BN001303.1).

[0199] pI1_X_antibody C LC construct: using GeneArt TM The plasmid pI1_X_antibody C LC plasmid was constructed by assembling the following two PCR products using the Seamless Cloning and Assembly Kit (Thermo Fisher Scientific, Waltham, USA): (1) a vector backbone amplified from the pI1 vector containing homology regions for targeted integration at chr3 and native pyr2 as a selectable marker; (2) the antibody C light chain (LC) fused to the Aspergillus niger glucoamylase core and linker (GenBank: HQ537427.1) and flanked by the Aspergillus tubingensis xlnA promoter and native cbhl terminator amplified from synthetic DNA synthesized by Twist Bioscience (San Francisco, US). PCR reactions were performed using Q5 High-Fidelity DNA Polymerase (NEB - Ipswich, USA) according to standard protocols. The assembled product was added to 50 μL TOP10 chemically competent E. coli (Thermo Fisher Scientific, Waltham, USA) and transformed according to standard protocols. Plasmid DNA was isolated from E. coli colonies using the Qiaquick Spin DNA Miniprep Kit (Hilden, Germany) according to standard protocols.

[0200] pI1_C_antibody C HC construct: using GeneArt TMThe plasmid pI1_C_antibody C HC plasmid was constructed by assembling the following two PCR products using the Seamless Cloning and Assembly Kit (Thermo Fisher Scientific, Waltham, USA): (1) a vector backbone amplified from the pI1 vector containing homology regions for targeted integration at chr3 and native pyr2 as a selectable marker; (2) the antibody C heavy chain (HC) fused to the Trichoderma reesei CBH1 core and linker (XM_006969162.1) and flanked by the native cbh1 promoter and terminator amplified from synthetic DNA synthesized by Tovisit Biotech (San Francisco, USA). PCR reactions were performed using Q5 High-Fidelity DNA Polymerase (NEB - Ipswich, USA) according to standard protocols. The assembled products were added to 50 μL TOP10 chemically competent Escherichia coli (Thermo Fisher Scientific, Waltham, USA) and transformed according to standard protocols. Plasmid DNA was isolated from E. coli colonies using the Qiaquick Spin DNA Miniprep Kit (Hilden, Germany) according to standard protocols.

[0201] pT1A_C_antibody C HC construct: using GeneArt TM The pT1A_X_antibody C HC plasmid was constructed using the Seamless Cloning and Assembly Kit (Thermo Fisher Scientific, Waltham, USA) to assemble the following three PCR products: (1) a vector backbone PCR-amplified from the pT1A vector containing homology regions for targeted integration at ch2; (2) an Aspergillus nidulans amdS marker (GenBank: BN001303.1); (3) an antibody C heavy chain (HC) fused to the Trichoderma reesei CBH1 core and linker (XM_006969162.1) and flanked by the native cbh1 promoter and terminator amplified from synthetic DNA synthesized by Tovisit Biotech (San Francisco, USA). PCR reactions were performed using Q5 High-Fidelity DNA Polymerase (NEB - Ipswich, USA) according to standard protocols. The assembled products were added to 50 μL TOP10 chemically competent Escherichia coli (Thermo Fisher Scientific, Waltham, USA) and transformed according to standard protocols. Plasmid DNA was isolated from E. coli colonies using the Qiaquick Spin DNA Miniprep Kit (Hilden, Germany) according to standard protocols.

[0202] pT1A_Accessory_X_C_Antibody C HC construct: GeneArt TMPlasmid pT1A_Auxiliary_X_X_Antibody C HC construct was constructed using the Seamless Cloning and Assembly Kit (Thermo Fisher Scientific, Waltham, USA) to assemble the following four PCR products: (1) a vector backbone amplified from the pT1A vector containing homology regions for targeted integration at ch2; (2) an Aspergillus nidulans amdS marker (GenBank: BN001303.1); (3) an overexpression cassette for native T. reesei proteins; (4) an Antibody C heavy chain (HC) fused to the T. reesei CBH1 core and linker (XM_006969162.1) and flanked by the native cbh1 promoter and terminator amplified from synthetic DNA synthesized by Tovisit Biotech (San Francisco, USA). PCR reactions were performed using Q5 High-Fidelity DNA Polymerase (NEB - Ipswich, USA) according to standard protocols. The assembled product was added to 50 μL TOP10 chemically competent E. coli (Thermo Fisher Scientific, Waltham, USA) and transformed according to standard protocols. Plasmid DNA was isolated from E. coli colonies using the Qiaquick Spin DNA Miniprep Kit (Hilden, Germany) according to standard protocols.

[0203] Transformation of Antibody C: The donor DNA amplified from the pI1_cHC expression vector used for the transformation reaction contained approximately 1.0 kb of upstream homology region for targeting at chr3, expression cassette, and partial fragment of pyr2 tag. The expression cassette consisted of the heavy chain (HC) fused to the native cbhl core and linker (XM_006969162.1) and flanked by the native cbhl promoter and terminator. The donor DNA amplified from the pI1_X_xLC expression vector used for the transformation reaction contained approximately 1.0 kb of downstream homology region, expression cassette, and partial fragment of pyr2 tag. The expression cassette consisted of the light chain (LC) fused to the Aspergillus niger glucoamylase core and linker (GenBank: HQ537427.1) and flanked by the Aspergillus tubingensis xlnA promoter and native cbhl terminator.

[0204] Antibody A and B constructs: Four different constructs were constructed based on the "pI1" and "pT1A" backbones, with expression cassettes for both antibody A and antibody B sequences for expression in Trichoderma reesei. This enables one copy of each antibody chain to be targeted to two respective natural loci for integration at the loci on chr2 and chr3. For the p1A construct, the Aspergillus nidulans amdS marker was used for selection. DNA encoding the antibody peptides was synthesized by Tovisit Biotech (San Francisco, USA). These synthetic heavy and light chain fragments were cloned into pI1 and p1A vectors to obtain: pI1_cHC_Antibody A, pI1_cHC_Antibody B, pI1_X_xLC_Antibody A, pI1_X_xLC_Antibody B, p1A_cHC_Antibody A, p1A_cHC_Antibody B, p1A_X_xLC_Antibody A and p1A_X_xLC_Antibody B. The donor DNA amplified from the pI1_cHC expression vector used for the transformation reaction with primer pair RAS210 and JC831 contained approximately 1.0 kb of upstream homology region, expression cassette, and partial fragment of pyr2 tag. The expression cassette consisted of a heavy chain (HC) fused to the native cbhl core and linker (XM_006969162.1) and flanked by the native cbhl promoter and terminator. The donor DNA amplified from the pI1_X_xLC expression vectors RAS500 and RAS213 used for the transformation reaction contained approximately 1.0 kb of downstream homology region, expression cassette, and partial fragment of pyr2 tag. The expression cassette consisted of a light chain (LC) fused to the Aspergillus niger glucoamylase core and linker (GenBank: HQ537427.1) and flanked by the Aspergillus fusiformis xlnA promoter and native cbhl terminator ( Figure 1 ).

[0205] Donor DNA amplified from the p1A_cHC expression vector for transformation reactions with primer pairs SK4242 and SK4244 contained approximately 1.0 kb of upstream homology region for targeting at chr3, expression cassette, and partial fragment of amdS tag. The expression cassette consisted of antibody A or antibody B heavy chain (HC) fused to the native cbhl core and linker (XM_006969162.1) and flanked by the native cbhl promoter and terminator. Donor DNA amplified from the p1A_X_xLC expression vector for transformation reactions with primer pairs RAS680 and RAS693 contained approximately 1.0 kb of downstream homology region, expression cassette, and partial fragment of amdS tag. The expression cassette contained a light chain (LC) fused to the Aspergillus niger glucoamylase core and linker (GenBank: HQ537427.1) and flanked by the Aspergillus fusiformis xlnA promoter and native cbhl terminator. PCR reactions were performed using Q5 high-fidelity DNA polymerase (NEB - Ipswich, USA) according to standard protocols. This split-tag strategy increases the probability of single-copy integration at the target locus.

[0206] LC-BASI constructs: To test the effect of BASI co-expression on antibody proteolysis, a fusion BASI expression cassette was incorporated into the pI1_X_xLC_antibody A and pI1_X_xLC_antibody B vectors. The BASI expression cassette contains the native Trichoderma reesei cbh2 (GenBank: M55080.1) promoter and the Aspergillus nidulans trpC (GenBank: Z32524.1) terminator that regulates BASI expression. The synthetic BASI mature sequence was synthesized by Tovisit Biotechnologies (San Francisco, USA). The mature sequence was fused to the native Cbh1 core and linker (XM_006969162.1) to obtain a fusion chimera, which is similar to the heavy and light chain fusion constructs. This Cbh1_BASI fusion expression cassette containing the cbh2 promoter, cbh1 core and linker, BASI mature sequence and trpC terminator was cloned into the pI1_X_xLC_antibody A and pI1_X_xLC_antibody B plasmids. Using standard methods known to those skilled in the art, High-fidelity DNA polymerase (NEB-Ipswich, USA), GeneArt seamless cloning and assembly kit (Thermo Fisher Scientific, Waltham, USA), Qiagen DNA purification kit (Hilden, Germany), primers from Integrated DNA Technologies (Coralville, US) make these BASI constructs. The resulting constructs are called pI1_X_xLC_antibody A_BASI fusions and pI1_X_xLC_antibody B_BASI fusions. Donor DNA for conversion is generated by PCR. The donor DNA fragment contains a partial fragment of pyr2 marker, a light chain expression cassette as described above, a BASI fusion expression cassette, and a homologous region downstream of chromosome 3 of about 1.0 kb. The donor DNA fragments of the heavy chain expression cassette and the light chain expression cassette (with and without additional BASI expression cassettes) are introduced into Trichoderma reesei strain T4_X.

[0207] pAS0025: Using GeneArt TM Plasmid pAS0025 was constructed using the Seamless Cloning and Assembly Kit (Thermo Fisher Scientific, Waltham, MD) to assemble the following two products: (1) a vector backbone containing the T. reesei CBH1 core flanked by the CBH1 promoter, CBH1 terminator, and T. reesei pyr2 marker, cut with restriction enzymes BamHI and XmaI according to standard methods; and (2) synthetic DNA (Integrated DNA Technologies, Coralville, IA) of the ccdB gene and chloramphenicol resistance marker (Invitrogen). The assembled products were added to 50 μL of Ne Shot TM ccdB Survival Cells chemically competent E. coli (Thermo Fisher Scientific, Waltham, USA) and transformed according to standard protocols. Plasmid DNA was isolated from E. coli colonies using the Qiaquick Spin DNA Miniprep Kit (Hilden, Germany) according to standard protocols.

[0208] pJC110: Using GeneArt TMPlasmid pJC110 was constructed using the Seamless Cloning and Assembly Kit (Thermo Fisher Scientific, Waltham, MA, USA) to assemble the following two products: (1) a vector backbone containing the Aspergillus nidulans amdS marker (GenBank: BN001303.1) and cut with the restriction enzyme AvrII directly downstream of the marker according to standard protocols; (2) an expression cassette with the Trichoderma reesei CBH1 promoter and core, PaeI restriction site, ccdB gene and chloramphenicol resistance marker (Invitrogen) and the Trichoderma reesei terminator amplified from the vector pAS0025. The ccdB gene and chloramphenicol resistance marker require specific E. coli strains for survival and reduce the amount of background during screening, as described by Invitrogen. PCR reactions were performed using Q5 High-Fidelity DNA Polymerase (NEB - Ipswich, TN, USA) according to standard protocols. The assembled products were added to 50 μL One Shot TM ccdB Survival Cells chemically competent E. coli (Thermo Fisher Scientific, Waltham, USA) and transformed according to standard protocols. Plasmid DNA was isolated from E. coli colonies using the Qiaquick Spin DNA Miniprep Kit (Hilden, Germany) according to standard protocols.

[0209] Antibody D LC construct (pJC160): GeneArt TM Plasmid pJC160 was constructed using the Seamless Cloning and Assembly Kit (Thermo Fisher Scientific, Waltham, USA) to assemble the following two products: (1) vector backbone pJC110 with the T. reesei CBH1 promoter, core, terminator and Aspergillus nidulans amdS marker (GenBank: BN001303.1) containing the expression cassette, which was cut with restriction enzymes PaeI and AvrII according to standard protocols; (2) antibody D light chain, which was flanked by a portion of the T. reesei CBH1 core and the T. reesei CBH1 terminator amplified from synthetic DNA synthesized by Tovisit Biotech (San Francisco, USA). PCR reactions were performed using Q5 High-Fidelity DNA Polymerase (NEB - Ipswich, USA) according to standard protocols. The assembled products were added to 50 μL TOP10 chemically competent E. coli (Thermo Fisher Scientific, Waltham, USA) and transformed according to standard protocols. Plasmid DNA was isolated from E. coli colonies using the Qiaquick Spin DNA Miniprep Kit (Hilden, Germany) according to standard protocols.

[0210] Pegl1-auxiliary protein_X-antibody D_LC construct (pSK736): GeneArt TMPlasmid pSK736 was constructed using the Seamless Cloning and Assembly Kit (Thermo Fisher Scientific, Waltham, USA) to assemble the following three PCR products: (1) a vector backbone PCR amplified from pCR TOPO Blunt II (Invitrogen); (2) an accessory protein_X expression cassette containing the Trichoderma reesei promoter egl1 amplified from Trichoderma strain RL-P37 and the Trichoderma reesei accessory protein_X CDS and terminator amplified from Trichoderma strain RL-P37 and fused together by fusion PCR; (3) an antibody D LC expression cassette containing the antibody D LC fused to the Trichoderma reesei CBH1 core and linker (XM_006969162.1) and flanked by the native cbh1 promoter and terminator from Trichoderma strain RL-P37 and the native Aspergillus nidulans amdS marker (GenBank: BN001303.1) amplified from vector pJC160. PCR reactions were performed using Q5 high-fidelity DNA polymerase (NEB-Ipswich, USA) according to standard protocols. The assembled products were added to 50 μL TOP10 chemically competent E. coli (Thermo Fisher Scientific, Waltham, USA) and transformed according to standard protocols. Plasmid DNA was isolated from E. coli colonies using Qiaquick Spin DNA Miniprep Kit (Hilden, Germany) according to standard protocols.

[0211] Pcbh2-auxiliary protein_X-antibody D LC construct (pJC173): GeneArt TM Plasmid pJC173 was constructed by assembling the following two PCR products using the Seamless Cloning and Assembly Kit (Thermo Fisher Scientific, Waltham, USA): (1) a vector backbone amplified from pSK736 containing the T. reesei accessory protein_X and antibody D LC expression cassettes fused to the T. reesei CBH1 core and linker (XM_006969162.1) and flanked by the native cbh1 promoter and terminator and the Aspergillus nidulans amdS marker (GenBank: BN001303.1); (2) the T. reesei CBH2 promoter amplified from genomic DNA from T. reesei strain RL-P37. PCR reactions were performed using Q5 High-Fidelity DNA Polymerase (NEB - Ipswich, USA) according to standard protocols. The assembled products were added to 50 μL TOP10 chemically competent E. coli (Thermo Fisher Scientific, Waltham, USA) and transformed according to standard protocols. Plasmid DNA was isolated from E. coli colonies using the Qiaquick Spin DNA Miniprep Kit (Hilden, Germany) according to standard protocols.

[0212] Antibody D_HC construct (pJC158): GeneArt TM Plasmid pJC158 was constructed using the Seamless Cloning and Assembly Kit (Thermo Fisher Scientific, Waltham, USA) to assemble the following two products: (1) vector backbone pAS0025 containing the expression cassette with the T. reesei CBH1 promoter, core, terminator and pyr2 marker, which was cut with restriction enzymes PaeI and AvrII according to standard protocols; (2) antibody D HC, which is flanked by a portion of the T. reesei CBH1 core and the T. reesei CBH1 terminator amplified from synthetic DNA synthesized by Tovisit Biotech (San Francisco, USA). PCR reactions were performed using Q5 High-Fidelity DNA Polymerase (NEB - Ipswich, USA) according to standard protocols. The assembled products were added to 50 μL TOP10 chemically competent E. coli (Thermo Fisher Scientific, Waltham, USA) and transformed according to standard protocols. Plasmid DNA was isolated from E. coli colonies using the Qiaquick Spin DNA Miniprep Kit (Hilden, Germany) according to standard protocols.

[0213] [001303.1] Generation of donor DNA for transformation of Antibody D: Donor DNA was generated from vector pJC173 by PCR amplification. The PCR fragment contained the T. reesei accessory protein_X driven by the T. reesei cbh2 promoter, and the antibody D LC expression cassette containing the antibody D LC fused to the T. reesei Cbh1 core and linker (XM_006969162.1) and flanked by the native cbh1 promoter and terminator sequences. The selection marker for the pJC173 donor fragment was the Aspergillus nidulans amdS gene (GenBank: BN001303.1). The donor DNA for vector pJC158 contained the antibody D HC fused to the T. reesei CBH1 core and linker (XM_006969162.1) flanked by the native cbh1 promoter and terminator. The selection marker for the expression cassette was the T. reesei pyr2 gene. PCR reactions were performed using Q5 High-Fidelity DNA Polymerase (NEB - Ipswich, USA) according to standard protocols.

[0214] Transformation of Antibody D: The host Trichoderma reesei strain used for transformation was deficient in major cellulases and xylanases. The strain was transformed using a standard PEG-protoplast transformation method. A protoplast containing approximately 5 μg of each DNA and 5×10 6The transformation mixture of 10 protoplasts (total volume of 250 μl) was diluted with 2 volumes of 1.2M sorbitol / 10mM Tris, pH 7.5 / 10mM CaCl2 solution and mixed with 26mL of 2% low melting point agarose in minimal medium containing 1M sorbitol and acetamide as the sole nitrogen source and distributed on four 10cm culture dishes, which were previously poured with minimal medium containing 1.5% agarose, 1M sorbitol and acetamide. Single colonies were picked on fresh 10cm culture dishes containing 1.5% agar, 1g / L uridine and acetamide. Once the stable transformants were fully sporulated, the spores were harvested and used to inoculate liquid cultures.

[0215] As described by Sheir-Neiss and Montenecourt (1984, incorporated herein by reference), the parent strain T4_X of Trichoderma reesei described in this example is derived from Trichoderma reesei strain RL-P37 (NRRL deposit number 15709). The strain designated herein as "T4_X" is a mutagenic derivative of RL-P37, which has a significant mutation in the pyr2 gene, so that the strain requires uridine for growth, and has a nik1 (M743T) mutation that increases total protein production (e.g., see, U.S. Patent Application Publication No. 2018 / 0037919, which is incorporated herein by reference).

[0216] Example 2: Production of BASI in Bacillus subtilis

[0217] This example demonstrates the production of BASI protein in liquid fermentation of Bacillus subtilis. The inoculum was grown in a seed flask containing LB medium.

[0218] The production medium for producing BASI protein contains minerals, one or more carbon sources and a complex nitrogen source. BASI protein accumulates in the broth / cells.

[0219] Bacillus subtilis of 14L scale in liquid culture: Growth in 14L fermentor consists of the following 2 steps: generating seed culture, and generating production culture while producing protein. Seed culture was started by inoculating 30mL LB medium into 350mL flask. Seed culture was incubated for about 2h at 180rpm and 37°C until it was turbid. 30mL volume of seed culture was inoculated into each tank so that final volume reached the appropriate production medium of 7kg. During the experiment, production culture had unilateral pH control (only adding alkali), and NH4OH was used to carry out alkali addition and pH was controlled at 7.1. Feed was triggered when OUR reached 25mmol / L / h, and rose to 1.65g / min from 0.28g / min through 10 hours.

[0220] Various parameters were monitored during the run, including but not limited to: CER (carbon dioxide evolution rate), OUR (oxygen uptake rate), pH, DO (dissolved oxygen), OD (optical density), etc.

[0221] Example 3: Production of monoclonal antibodies in Trichoderma reesei

[0222] This example demonstrates the production of a monoclonal antibody in liquid fermentation. Sufficient biomass is first generated and then the biomass is induced to produce a monoclonal antibody. The inoculum is grown in a seed flask containing citrate minimal medium and trace elements.

[0223] The production medium for the production of monoclonal antibodies contains minerals, one or more carbon sources and optionally a complex nitrogen source.

[0224] The type and amount of complex nitrogen are shown in Table 1. If a complex nitrogen source was used, cottonseed meal was used in all runs.

[0225]

[0226]

[0227] *CSF = cottonseed meal

[0228] Growth of Trichoderma reesei in fed-batch culture: Growth in a fermenter consists of two steps: generating a seed culture, and generating a production culture. The seed culture is necessary to accumulate biomass for the production culture. The seed culture is started by inoculating 50 mL of citrate minimal medium with the appropriate strain. The seed culture is incubated at 100-300 rpm and 28°C for 2 days. A 50 mL volume of the seed culture is inoculated into each tank containing the production medium. The production culture is grown at 34°C, and the temperature is lowered to T at the start of the feed. f . Various parameters were monitored during the run, including, but not limited to: CER (carbon dioxide evolution rate), OUR (oxygen uptake rate), pH, DO (dissolved oxygen), DCW (cell dry weight), antibody yield, and total protein (TP) yield. Runs 20204180 and 20204182 had BASI added during the run. BASI was added at the beginning of the run, and BASI was added again at the beginning of production (pH increased, temperature decreased, and feeds started). A sample was taken before the second addition, and analysis showed that BASI was not present.

[0229] Figure 2It is shown that co-expression of BASI in this strain has a positive effect on the titer of the 150 kDa antibody species (size exclusion chromatography (SEC), right axis). Protein A-based titers and SEC-based titers of BASI-negative strains appear to reach a maximum early on and then decline as the run progresses.

[0230] Example 4: Fermentation of strains co-expressing antibody B and BASI

[0231] Strains t-BBD55 expressing antibody B and BASI in run 20204157 and t-BBH66 expressing antibody B in run 20204164 were fermented under the same conditions. Antibody titers were determined using two methods. Protein A HPLC reports total antibody-related species, including degraded species. Size exclusion chromatography (SEC) reports the concentration of proteins separated by size. In this case, the peak eluting at a time consistent with a mass of approximately 150 kDa is quantified. This will only report intact antibodies, excluding antibody fragments and host proteins with masses significantly greater or less than 150 kDa.

[0232] The method of separating the various species from the fermentation (including fusion protein, free CBH1, light and heavy chains, minor degradation products and full-length, mature, 150 kd antibody molecule) was accomplished by size exclusion chromatography. This mode of chromatography separates molecules by size rather than molar mass.

[0233] The method used an SRT-SEC column, 7.8 mm x 300 mm long, 300A (Sepax, Inc). The running buffer (150 mM sodium phosphate and 150 mM sodium chloride, pH 6.8) was run under isocratic conditions. It was run at 1 ml / min at 25°C for 20 minutes, and the protein was detected by UV absorption at 222 nm compared to a CHO expressed antibody standard.

[0234] Total antibody species concentration was determined using a MacPac Protein A column (4×35 mm, Thermo Fisher Scientific) on an Agilent HPLC system. Sample supernatants were diluted 16-fold or 64-fold with eluent A-PBS at pH 7.5. pH 7 in an Agilent HPLC plate. 20 μl of this solution was loaded onto the column using the following method: 100% eluent A (50 mM sodium phosphate, 150 mM NaCl, pH 7.5) was run for 0.2 minutes, followed by 100% eluent B (50 mM sodium phosphate, 150 mM NaCl, pH 2.5) for 0.8 minutes, followed by 100% eluent A for 1.2 minutes, with detection at 220 nm. Antibody peak areas were recorded. Standard curves for gene-purified antibodies were used to convert peak areas to antibody concentrations in mg / ml. Supernatant concentration was corrected for cell mass by adjusting "DCW Correction = Supernatant Concentration * (1 - DCW * 4 / 1000)", where "DCW" is the dry weight of cells measured in g / kg.

[0235] Example 5: Stabilization of Antibodies in Trichoderma Broth by Supplementation with Exogenously Produced BASI

[0236] This example demonstrates the stabilizing effect of exogenous BASI on a monoclonal antibody produced in Trichoderma reesei.

[0237] Stabilization of Antibody D in Harvest Broth Using BASI Filtrate

[0238] Four 50 mL tubes were filled with the fermentation broth (broth) expressing Antibody D (defined medium), BASI filtrate, 1 M Bis-Tris buffer and water as shown in Table 2.

[0239] Table 2: Experimental conditions

[0240]

[0241] The tubes were mixed thoroughly by inversion. 5 mL of each treated broth was transferred to a 15 mL tube and subsequently stored at 5° C. SDS-PAGE analysis (Coomassie blue staining) was performed on days 7 and 28.

[0242] like Figure 3 As shown, the untreated broth (experimental condition 1) lost about 60% of the antibody band by 7 days, and only trace amounts remained after 28 days. For all levels (experimental conditions 2-4), the antibody bands treated with BASI remained after 28 days of storage at 5°C, with no significant loss compared to the reference stored at -20°C.

[0243] Stabilization of Antibody D in Clarified Broth Using BASI Filtrate

[0244] Each treated broth used in the above experiments was centrifuged at 30,000 rpm for 20 min. The supernatant was collected from each tube and filtered using a Steriflip filter device. Each supernatant was then divided into two separate tubes, one set of tubes was stored at -20°C (as a reference) and one set of tubes was stored at 22°C.

[0245] like Figure 4 As shown, after 7 days at 22° C., the antibody completely disappeared in the untreated clarified broth, while it was retained in the BASI treated clarified broth. After 28 days of storage at 22° C., no loss of antibody bands was observed at 10 g BASI filtrate / g broth.

[0246] Stabilization of Antibody C in Clarified Broth Using BASI Filtrate

[0247] Fill four 14 mL tubes with fermentation broth (broth) expressing Antibody C in composite (20200482: B2 2 copies Antibody C without endoT t-BAV65) defined (20200483: B2 2 copies Antibody C without endoT; t-BAV65) medium, BASI filtrate, 1 M Bis-Tris buffer and water as shown in Table 3.

[0248] Table 3: Experimental conditions

[0249]

[0250] Each treated broth was centrifuged at 30,000 rpm for 20 min. The supernatant was collected and sterile filtered using a Steriflip filter unit. Each sample was divided into three tubes. One set of tubes was stored at -20°C, one set was stored at 5°C, and one set was stored at 22°C. SDS-PAGE analysis (Coomassie blue staining) was performed on the 4th day.

[0251] like Figure 5 As shown, after 4 days, the untreated clarified broth showed complete disappearance of the antibody band when stored at 22°C. In contrast, in the case of the addition of BASI, the antibody band remained at a level comparable to the -20°C reference sample.

[0252] Stabilization of Antibody D in UF Concentrate Using BASI Filtrate

[0253] Ultrafiltration (UF) concentrates derived from fermentation broth (defined medium) expressing Antibody D as described above were prepared for storage stability studies as shown in Table 4.

[0254] Table 4: Experimental conditions

[0255]

[0256] Each solution was mixed by inversion and sterile filtered using a Steriflip filter unit. Each sample was divided into three tubes, one group was stored at -20°C, one group was stored at 5°C, and one group was stored at 22°C. SDS-PAGE analysis (Coomassie blue staining) was performed on day 5.

[0257] like Figure 6 As shown, after 5 days, the UF concentrate without BASI stored at 5°C lost most of the antibody bands. However, with the addition of BASI, the antibody bands for both doses were similar to the corresponding -20°C reference. When stored at 22°C, the UF concentrate without BASI treatment showed a complete disappearance of the antibodies, while the antibody bands were still visible in the UF concentrate treated with BASI.

[0258] Stabilization of Antibody D in UF Concentrates Using Purified BASI

[0259] Prepare the ultrafiltration (UF) concentrate derived from the fermentation broth (defined medium) expressing antibody D as described above, and the purified BASI solution prepared by the BASI shake flask filtrate as described above. Specifically, the purified BASI solution was prepared using a HiTrap SP XL 5mL pre-packed column. The column was balanced using a 50mM sodium acetate equilibration buffer at pH 5. Elution was performed from salt-free to 0.5M NaCl over 10CV using the same buffer. Solutions for storage stability studies were prepared as shown in Table 5.

[0260] Table 5: Experimental conditions

[0261]

[0262] Each solution was mixed by inversion and sterile filtered using a Steriflip filter unit. Each sample was divided into three tubes, one group was stored at -20°C, one group was stored at 5°C, and one group was stored at 22°C. SDS-PAGE analysis (Coomassie blue staining) was performed on days 2, 4, and 10.

[0263] like Figure 7 As shown, without the addition of BASI, complete disappearance of the antibody band was observed by day 2 at 22°C and between days 4 and 10 at 5°C. The addition of BASI stabilized the antibody, and all samples retained the antibody band.

[0264] Stabilization of Antibody A during Fermentation Using BASI UFC

[0265] A composite fermentation medium (containing cottonseed meal) expressing Antibody A and sterile filtered BASI centrate were prepared as described above. As shown in Table 6, sterile filtered BASI UF centrate (UFC) was added to each fermentor.

[0266] Table 6: Fermentation conditions

[0267]

[0268] Samples were collected at 40, 70, 91, 116 and 140 hours during the fermentation and stored at -20°C until SDS-PAGE analysis (Coomassie blue staining).

[0269] like Figure 8 As shown, higher antibody band intensity was observed in fermentations with BASI addition compared to fermentations lacking BASI addition.

[0270] Stabilize Antibody A during recovery and storage

[0271] BASI-free broth was prepared using 250 g of fermentation broth containing antibody A (generated as described above) and 125 g of sodium citrate buffer (pH 5.5). BASI-containing broth was prepared using 250 g of fermentation broth containing antibody A (generated as described above), 2.5 g of BASI UF concentrate (generated as described above) and 122.5 bis-tris buffer (pH 6.5). Each broth was centrifuged at 30,000 rpm for 30 min and then sterile filtered using a 0.2 μm Nalgene filter. Each broth was concentrated using an ultrafilter 10K MWCO. Each broth concentrate was then divided into two portions, one stored at 20° C. and one stored at 10° C. SDS-PAGE analysis (Coomassie blue staining) was performed on the 1st and 15th days.

[0272] No differences in antibody band intensity were observed on day 1 (data not shown). However, on day 15, UF concentrates from broth lacking BASI addition showed a significant loss of antibody bands. In the case of BASI addition, the antibody bands remained comparable to the -20°C reference sample (see Fig. 9 ).

[0273] Recovery and stability profile of antibody B expressed in the BASI strain

[0274] By comparing -20 ℃ and 10 ℃ storage samples, the stability of UF concentrate was monitored by SDS-PAGE at 1 month. At 120h, the fermentation expressing antibody B in the BASI strain was stopped, cooled to 15 ℃ and transferred to a harvest tank. The broth was diluted with 1.5 parts of 50mM sodium citrate at pH 5.5. The diluted broth was treated with 0.033% C581 flocculant, 6% diatomaceous earth FW12, and clarified using a rotary vacuum filter pre-coated with FW12 diatomaceous earth. The collected filtrate was concentrated using a 10K MWCO UF membrane. Samples collected during each step were analyzed using SDS-PAGE to monitor antibody stability. The UF concentrate obtained was divided into two fractions for monitoring antibody stability during storage at 10 ℃ and -20 ℃.

[0275] like Fig. 10A As shown, the antibody bands were very similar throughout the recovery process, with no apparent loss or degradation. Fig. 10B As shown, the UF concentrate derived from the BASI expression strain has good stability. The antibody bands of the samples stored at 10°C for 1 month were similar to those of the control samples stored at -20°C.

[0276] Example 6: Expression of BASI in Bacillus subtilis

[0277] The yhfN region and rrnI (mutated) promoter (SEQ ID NO: 3) from Bacillus subtilis and the aprE signal sequence (SEQ ID NO: 4) from Bacillus subtilis were amplified from a Bacillus subtilis expression strain using primers CF 17-79 and CF 19-20 (Table 7). Primer CF 17-79 also contains an overhang at the end of the alrA box that allows assembly. The 20 bp overhang at the end of the aprE signal sequence, the barley amylase subtilisin inhibitor (BASI) gene (codon optimized by Geneious software; SEQ ID NO: 5) plus 20 base pairs at the start of the BPN' terminator from B. amyliquefaciens (SEQ ID NO: 6) were synthesized by an external supplier (Eurofins Genomics). The synthetic DNA fragment was amplified using primers CF 19-19 and CF 19-22 (Table 7). The two fragments were fused together using PCR with primers CF 17-79 and CF 19-22 (Table 7) to form PCR Fusion 1 using techniques known in the art.

[0278] Table 7: PCR primers

[0279]

[0280] A DNA fragment containing the BPN' terminator and the alrA expression cassette was amplified from a Bacillus subtilis expression strain using primers CF 19-21 and CF 17-80 (Table 7; CF 17-80 has an overhang in the upstream region of yhfN for future Gibson assembly. The PCR fragment and PCR fusion 1 were assembled using Gibson assembly (New England Biolabs) using techniques known in the art to generate a circular DNA cassette (SEQ ID NO: 7). The assembly was subjected to rolling circle amplification (Evomics), which was used to transform 200 μl of competent cells of a suitable Bacillus subtilis strain. The transformed cells were incubated at 37°C for 1 hour while shaking at 250 rpm. This method is based on the observation that the alrA gene encoding alanine racemase is essential in Bacillus subtilis (Ferrari et al., Bio / Technol. [Biotechnology], 3: 1003-1007, 1987), and can therefore be used as a selectable marker. Alanine racemase converts natural L-alanine into D-alanine required for cell wall synthesis. The alanine racemase inhibitor β-chloro-D-alanine (CDA) is used for selection / amplification of the alrA gene (Heaton et al., Biochem. Biophys. Res. Comm. [Biochemical and Biophysical Research Communications], 149: 576-579, 1987). The alrA gene cassette is integrated with the BASI cassette into a host lacking the natural alrA gene and selected on a plate without supplementation of D-alanine. Cells from the transformation mixture are plated on agar plates. Single colonies were selected and grown in Luria broth containing β-chloro-D-alanine (CDA) to an optical density of 1.0 at 600 nm. Samples of the strains were then frozen at -80°C with 20% glycerol.

[0281] The bacterial strain was grown on a Luria agar plate with a chromosome-integrated expression cassette encoding a barley amylase subtilisin inhibitor and inoculated into 50 mL of Luria broth in a 250 mL super-production shake flask (Thomson Instrument Company). The shake flask was incubated overnight at 37° C. while rotating at 250 rpm. The entire volume of the flask was transferred to 500 mL of the following bacillus culture medium (pH 7.3) in a 2.5 L super-production shake flask (Thomson Instrument Company). The culture was grown in a shaking incubator at 37° C., 150 rpm for 68 hours.

[0282] Bacillus medium is an enriched semi-defined medium based on MOPs buffer, with urea as the main nitrogen source, glucose as the main carbon source, and supplemented with 1% soy peptone for robust cell growth. After growth, cells were removed by centrifugation (10,000 rpm, 45 min), and the supernatant was sterilized using a Nalgene 0.2uM filter device.

[0283] Example 7: Use of BASI to Stabilize Antibodies in Concentrated, Formulated Cell-Free Broth

[0284] BASI produced in Bacillus subtilis was used as a formulation ingredient to stabilize antibodies produced by Trichoderma reesei and then recovered by cell separation and concentrated by ultrafiltration. The ultrafiltration concentrate (UFC) was then formulated with or without the addition of BASI.

[0285] Antibody stability was assessed by SDS-PAGE followed by densitometry of the 150 kDa mature antibody band. Prepared samples were diluted to a target 0.025 μg / μL initial concentration of antibody. HCl was added 2:1 to denature the protein and inactivate background proteases. 20 μL of 4X LDS sample buffer was added to 60 μL of HCl-treated sample. 10 μL of each sample was loaded into a well of a 10-well 4%-12% Bis-Tris gel, and the gel was run at 175 volts for 1 hour in MOPS buffer. The gel was stained with the eStain L1 protein stain system and imaged using the Bio-Rad ChemiDoc MP imaging system. Densitometry was performed using Image Lab 5.0 software.

[0286] The residual stability of the antibodies was calculated by dividing the intensity of samples stored at 4°C or 22°C by the intensity of samples frozen immediately after formulation (storage day 0).

[0287] Samples were prepared with buffer (A and B) or buffer and arginine-HCl (C and D) as shown in Table 8.

[0288] Table 8: Sample composition

[0289]

[0290] Stability analysis of antibodies produced in three separate fermentations is shown in Fig.11 , Fig.12 and Fig.13 middle.

[0291] like Fig.11As shown, antibody B fermented at pH 5.5 (UFC 20200577) at 14 L scale and stored at 4°C for 56 days showed that the addition of BASI increased the residual band density from 7.1% to 45.2% when using only Bis-Tris buffer. When stored in the presence of 2.3% arginine-HCl, the addition of BASI increased the residual band density from 27.9% to 79.8%. Fig.12 As shown, antibody B fermented at pH 7 (UFC 20208053) at 500L scale and stored at 22°C for 25 days showed that the addition of BASI increased the residual band density from 0.7% to 7.6% when using only buffer Bis-Tris. When buffered in the presence of 2.3% arginine-HCl, the addition of BASI increased the residual band density from 4.6% to 22.6%. Fig.13 As shown, antibody A fermented at pH 6.5 (UFC 20200874) at 14L scale and stored at 22°C for 25 days showed that when only buffer Bis-Tris was used, the addition of BASI increased the residual band density from 0% to 1.6%. When buffered in the presence of 2.3% arginine-HCl, the addition of BASI increased the residual band density from 0% to 14.6%. Table 9 summarizes the stability changes observed after adding BASI to antibody UFC under different conditions.

[0292] Table 9: Summary of stability changes after adding BASI

[0293]

[0294] References

[0295] Buckland,B.,Brix,T.,Fastert,H.,Gbewonyo,K.,Hunt,G.,&Jain,D.(1985).Fermentation Exhaust Gas Analysis Using Mass Spectrometry.Bio / Technology(3),982-988.

[0296] MS, Skovlund, DA, Johannesen, PF et al. A novel platform for heterologous gene expression in Trichoderma reesei (Teleomorph Hypocreajecorina). Microb Cell Fact 13, 33 (2014).

[0297] Sheir-Neiss,G.,Montenecourt,B.S.Characterization of the secretedcellulases of Trichoderma reesei wild type and mutants during controlledfermentations.Appl Microbiol Biotechnol 20,46-53(1984).

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[0304]

Claims

1. A recombinant cell comprising a) a heterologously expressed barley alpha-amylase subtilisin inhibitor (BASI) polypeptide; and b) a heterologously expressed monoclonal antibody or a functional fragment thereof.

2. The cell according to claim 1, wherein the antibody or a functional fragment thereof is a therapeutic antibody or a functional fragment thereof.

3. The cell of claim 1 or claim 2, wherein the cell is a bacterial, fungal, yeast, plant or mammalian cell.

4. The cell of claim 3, wherein the cell is a Trichoderma reesei cell or an Aspergillus niger cell.

5. The cell of claim 3, wherein the cell is a Bacillus subtilis cell.

6. The cell of claim 3, wherein the cell is a Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cell.

7. The cell of any one of claims 1-6, wherein the BASI polypeptide comprises the amino acid sequence of SEQ ID NO:

1.

8. The cell according to any one of claims 1 to 7, wherein the antibody or functional fragment thereof exhibits less proteolytic degradation compared to the antibody or functional fragment thereof not heterologously co-expressed with the BASI polypeptide.

9. The cell of any one of claims 1-8, wherein the functional fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a disulfide-linked Fv (dsFv), a Fd fragment, a Fd' fragment, a single-chain Fv (scFv), a single-chain Fab (scFab), a diabody, an anti-idiotypic (anti-Id) antibody, or an antigen-binding fragment of any one thereof.

10. A fermentation broth comprising the cells of any one of claims 1-9.

11. A method for reducing proteolysis of a heterologously expressed monoclonal antibody or a functional fragment thereof, the method comprising culturing a recombinant cell under suitable conditions for producing the heterologously expressed antibody or a functional fragment thereof and a barley alpha-amylase subtilisin inhibitor (BASI) polypeptide, the recombinant cell comprising a) a heterologously expressed BASI polypeptide; and b) the heterologously expressed monoclonal antibody or a functional fragment thereof.

12. The method of claim 11, further comprising isolating the antibody or a functional fragment thereof.

13. The method according to claim 11 or claim 12, wherein the antibody or functional fragment thereof is a therapeutic antibody or functional fragment thereof.

14. The method of any one of claims 11-13, wherein the cell is a bacterial, fungal, yeast, mammalian or plant cell.

15. The method of claim 14, wherein the cell is a Trichoderma reesei cell or an Aspergillus niger cell.

16. The method of claim 14, wherein the cell is a Bacillus subtilis cell.

17. The method of claim 14, wherein the cells are Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells.

18. The method of any one of claims 11-17, wherein the BASI polypeptide comprises the amino acid sequence of SEQ ID NO:

1.

19. The method of any one of claims 11 to 18, wherein the antibody or functional fragment thereof exhibits less proteolytic degradation compared to the antibody or functional fragment thereof not heterologously co-expressed with the BASI polypeptide.

20. The method of any one of claims 11-19, wherein the functional fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a disulfide-linked Fv (dsFv), a Fd fragment, a Fd' fragment, a single-chain Fv (scFv), a single-chain Fab (scFab), a diabody, an anti-idiotypic (anti-Id) antibody, or an antigen-binding fragment of any one thereof.

21. A method for reducing proteolysis of a recombinantly expressed monoclonal antibody or a functional fragment thereof, the method comprising isolating the recombinantly expressed antibody or a functional fragment thereof in the presence of an exogenously added barley alpha-amylase subtilisin inhibitor (BASI) polypeptide.

22. The method according to claim 21, wherein the antibody or functional fragment thereof is a therapeutic antibody or functional fragment thereof.

23. The method of claim 21 or claim 22, wherein the BASI polypeptide is recombinantly expressed in a bacterial, fungal, yeast, mammalian or plant cell.

24. The method of any one of claims 21-23, wherein the BASI polypeptide is recombinantly expressed in a Bacillus subtilis cell or an Aspergillus niger cell.

25. The method of any one of claims 21-24, wherein the BASI polypeptide comprises the amino acid sequence of SEQ ID NO:

1.

26. The method of any one of claims 21-25, wherein the monoclonal antibody or a functional fragment thereof is recombinantly expressed in bacteria, fungi, yeast, mammalian or plant cells.

27. The method of claim 26, wherein the monoclonal antibody or a functional fragment thereof is recombinantly expressed in a Trichoderma reesei cell or an Aspergillus niger cell.

28. The method of claim 26, wherein the monoclonal antibody or a functional fragment thereof is recombinantly expressed in Bacillus subtilis cells.

29. The method of claim 26, wherein the monoclonal antibody or a functional fragment thereof is recombinantly expressed in Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells.

30. The method of any one of claims 21-29, wherein the antibody or functional fragment thereof exhibits less proteolytic degradation and / or increased intact protein yield compared to the antibody or functional fragment thereof isolated in the absence of exogenously added BASI polypeptide.

31. The method of any one of claims 21-30, wherein the functional fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a disulfide-linked Fv (dsFv), a Fd fragment, a Fd' fragment, a single-chain Fv (scFv), a single-chain Fab (scFab), a diabody, an anti-idiotypic (anti-Id) antibody, or an antigen-binding fragment of any one thereof.

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