Engineered antibodies

Abstract

Disclosed herein, inter alia, are cleavage-resistant antibodies and antibody-producing host cells, as well as methods for their production, use, and improved secretion.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 825,448, filed March 28, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Provided herein are, inter alia, antibodies having a modified hinge region that are more resistant to cleavage and / or have increased stability compared to the same antibody with an unmodified hinge region. [Background technology]

[0003] Antibodies are immunological proteins that bind to specific antigens. In most mammals, including humans and mice, antibodies are composed of paired heavy and light polypeptide chains. Each chain consists of two distinct regions called the variable (Fv) and constant (Fc) regions. The light and heavy chain Fv regions contain the antigen-binding determinants of the molecule and are responsible for binding the target antigen. The Fc region defines the class (or isotype) of the antibody (e.g., IgG) and is responsible for binding several natural proteins to trigger important biochemical events. The constant region of the heavy chain can be further divided into four smaller domains called CH1, hinge region, CH2, and CH3. The Fc region, which is part of the constant region, is responsible for several important cellular functions. Generally, the Fc region is defined as including only CH2 and CH3, and may also include part of the hinge region.

[0004] Antibodies of the IgG isotype are extremely flexible molecules. Indeed, the biological functions of IgG require a very specific and controlled way of deformation. The structure mainly responsible for the internal flexibility of the IgG molecule is located between the first domain (CH1) and the second domain (CH2) of the constant region and is called the hinge region. The hinge can be divided into three peptide regions, the upper, middle and lower hinge, respectively (Brekke et al., 1995, Immunol Today 16:85-90). Biochemical and structural studies point to the hinge region of antibodies as a key structural element that controls flexibility and modulates effector functions. The crystal structure of IgG1b12 (Saphire et al., 2001, Science 293:1155-1159 and Saphire et al., 2002, Journal of Molecular Biology, 319(1):9-18) revealed extreme asymmetry indicative of extreme interdomain flexibility within the antibody. The b12 structure revealed another feature of the hinge: its fragility; the b12 structure shows extensive damage in the hinge region, consistent with prior knowledge of antibody hinge properties. Summary of the Invention [Problem to be solved by the invention]

[0005] It is therefore not surprising that significant proteolytic cleavage (i.e., clipping) events are often observed in the antibody hinge region when recombinant antibodies are expressed in host cells. Such cleavage reduces the efficiency of the antibody production process, resulting in reduced titers of functional immunoglobulin. Thus, what is needed are improved compositions and methods for reducing the amount of proteolytic clipping in the hinge region of host cell-expressed antibodies during the production and purification process. The subject matter disclosed herein addresses these needs and also provides additional benefits. [Means for solving the problem]

[0006] Provided herein are, inter alia, non-naturally occurring modified monoclonal antibodies with altered amino acid sequences within the antibody hinge region that result in reduced or absent protease-mediated cleavage (i.e., clipping) during production and purification, and methods of producing the same. The disclosed methods, modified antibodies, and recombinant host cells result in increased antibody production and / or purification compared to antibodies that do not contain the disclosed altered hinge region amino acid sequences and / or are not used in accordance with the methods disclosed herein.

[0007] Thus, in some aspects, provided herein is a monoclonal IgG1 antibody heavy chain polypeptide comprising a hinge region comprising one or more amino acid modifications that reduce proteolysis of the polypeptide. In some embodiments, the polypeptide further comprises an IgG1 antibody light chain polypeptide. In some embodiments of any of the embodiments disclosed herein, the modification comprises a modification at one or more of amino acid positions 216, 217, 222, 226, and / or 234, where the amino acid positions are numbered according to the numbering in SEQ ID NO:1. In some embodiments, the modification comprises one or more of 216T or V; 217T or S; 222C, D, or E; 226N or P; and / or 234R. In some embodiments of any of the embodiments disclosed herein, the modification further comprises a modification at position 227. In some embodiments, the modification comprises 227P. In some embodiments of any of the embodiments disclosed herein, the modification comprises a modification at position 216 and one or more modifications at amino acid positions 222, 226, 227, and / or 234. In some embodiments, the modifications include 216T and one or more of 222C, D, or E; 226N or P; 227P; and / or 234R. In some embodiments of any of the embodiments disclosed herein, the modifications include a modification at position 217 and one or more modifications at amino acid positions 222, 226, 227, and / or 234. In some embodiments, the modifications include 217T and one or more of 222C, D, or E; 226N or P; 227P; and / or 234R. In some embodiments of any of the embodiments disclosed herein, the modifications are: (a) R217S-T226N; (b) R217S-S222C; (c) T216V-R217S-T226N; (d) S222C-H227P; (e) R217S-S222E-H227P; (f) T216V-R217S-S222C-T226P; (g) T226P-H227P; (h) R217S-S222C-T226P; (i) T216V-S222D-T226P; (j) T216V-S222C-T226P-H227P; (k) T216V-S222E-T226N;(l)T216V-S222C-H227P;(m)R217S-S222C-T226N;(n)S222C-T226P-H227P;(o)T216V-S222C-T226N-H227P;(p)R217S-S222D-T226P-H227P;(q)T216V-H227P;(r)S222E-T226P-H227P;(s)R217S-S222D-T226N-H227P;(t)R217S-S222E-T226P-H227P;(u)T216V-S222C;(v)R217S-S222D;(w)S222E-T226P;(x)R217S-S222C-T226P-H227P;(y)T216V-T226P-H227P;(Z)T216V-S222D-H227P;(aa)T226N-H227P;(bb)S222D-T226N-H227P;(cc)R217S-S222E-T226N;(dd)R217S-T226N-H227P;(ee)R217S-S222E-T226N-H227P;(ff)T216V-R217S-H227P;(gg)T216V-S222E-T226P-H227P;(hh)T216V-S222C-T226N;(ii)R217S-T226P-H227P;(jj)T216V-T226P;(kk)S222E-T226N-H227P;(ll)S222E-H227P;(mm)R217S-S222E-T226P;(nn)R217S-T226P;(oo)T216V-S222D;(pp)R217S-S222C-H227P;(qq)T216V-S222E-T226N-H227P;(rr)S222C-T226N-H227P;(ss)T216V-R217S-S222C-T226N-H227P;(tt)R217S-S222D-T226P;and (uu)S222D-T226N(vv). In some embodiments of any of the embodiments disclosed herein, the polypeptide exhibits at least about 50% less proteolysis compared to a monoclonal IgG1 antibody heavy chain polypeptide that does not comprise the one or more amino acid modifications. In some embodiments of any of the embodiments disclosed herein, the polypeptide exhibits no detectable proteolysis. In some embodiments of any of the embodiments disclosed herein, the polypeptide further comprises a polypeptide encoding a signal sequence. In some embodiments of any of the embodiments disclosed herein, the polypeptide further comprises a polypeptide encoding a carrier protein. In some embodiments of any of the embodiments disclosed herein, the polypeptide encoding the carrier protein is adjacent to a polypeptide encoding a signal sequence. In some embodiments of any of the embodiments disclosed herein, the carrier protein comprises CBH1 or a fragment thereof. In some embodiments of any of the embodiments disclosed herein, the antibody is an anti-respiratory syncytial virus (RSV) antibody, an anti-Ebola virus antibody, an anti-aggregating beta amyloid (Aβ) antibody, an anti-human immunodeficiency virus (HIV) antibody, an anti-herpes simplex virus (HSV) antibody, an anti-sperm antibody (e.g., an anti-human contraceptive antigen (HCA) antibody), or an anti-HER2 / neu antibody. In some embodiments of any of the embodiments disclosed herein, the polypeptide exhibits increased stability compared to a monoclonal IgG1 antibody heavy chain polypeptide that does not comprise said one or more amino acid modifications;

[0008] In other aspects, provided herein are nucleic acids encoding any of the polypeptides disclosed herein. In yet a further aspect, provided herein are vectors encoding any of the nucleic acids disclosed herein. In some embodiments, the vector further comprises a nucleic acid sequence encoding a promoter.

[0009] In another aspect, provided herein is a host cell comprising any of the polypeptides disclosed herein, any of the nucleic acids disclosed herein, or any of the vectors disclosed herein. In some embodiments, the host cell is selected from the group consisting of a mammalian host cell, a bacterial host cell, and a fungal host cell. In some embodiments, the mammalian cell is a Chinese Hamster Ovary (CHO) cell. In some embodiments, the bacterial cell is an E. coli cell. In some embodiments, the fungal cell is a yeast cell or a filamentous fungal cell. In some embodiments, the yeast cell is a species of Saccharomyces. In some embodiments of any of the embodiments disclosed herein, the fungal cell is selected from the group consisting of Trichoderma species, Penicillium species, Humicola species, Chrysosporium species, Gliocladium species, Aspergillus species, Fusarium species, Mucor species, Neurospora species, Hypocrea species; Myceliophthora species, and Emericella species.In some embodiments, the fungal cell is selected from the group consisting of Trichoderma reesei, Trichoderma viride, Trichoderma koningii, Trichoderma harzianum, Humicola insolens, Humicola grisea, Chrysosporium lucknowense, Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans, Aspergillus kawachi, Aspergillus aculeatus, and the like. aculeatus, Aspergillus japonicus, Aspergillus sojae, Myceliophthora thermophila, and Aspergillus awamori.

[0010] In a further aspect, provided herein is a method for producing any of the polypeptides disclosed herein, comprising culturing any of the host cells disclosed herein under conditions suitable for producing the polypeptide. In some embodiments, the method further comprises isolating the polypeptide. In some embodiments of any of the embodiments disclosed herein, the polypeptide exhibits at least about 50% less proteolysis compared to a monoclonal IgG1 antibody heavy chain polypeptide that does not comprise the one or more amino acid modifications. In some embodiments of any of the embodiments disclosed herein, the polypeptide exhibits no detectable proteolysis.

[0011] In some aspects, provided herein are methods of modifying a monoclonal IgG1 antibody heavy chain polypeptide to increase its resistance to proteolysis, comprising modifying one or more amino acid residues in the hinge region of the polypeptide. In some embodiments, the modification comprises a modification at one or more of amino acid positions 216, 217, 222, 226, and / or 233, the amino acid positions being numbered according to the numbering in SEQ ID NO: 1. In some embodiments, the modification comprises one or more of 216T or V; 217S; 222C, D, or E; 226N or P; and / or 234R. In some embodiments, the modification further comprises 227P. In some embodiments of any of the embodiments disclosed herein, the modifications are: (a) R217S-T226N; (b) R217S-S222C; (c) T216V-R217S-T226N; (d) S222C-H227P; (e) R217S-S222E-H227P; (f) T216V-R217S-S222C-T226P; (g) T226P-H227P ;(h)R217S-S222C-T226P;(i)T216V-S222D-T226P;(j)T216V-S222C-T226P-H227P;(k)T216V-S22 2E-T226N;(l)T216V-S222C-H227P;(m)R217S-S222C-T226N;(n)S222C-T226P-H227P;(o)T216V-S2 22C-T226N-H227P;(p)R217S-S222D-T226P-H227P;(q)T216V-H227P;(r)S222E-T226P-H227P;(s) R217S-S222D-T226N-H227P;(t)R217S-S222E-T226P-H227P;(u)T216V-S222C;(v)R217S-S222D;( w)S222E-T226P;(x)R217S-S222C-T226P-H227P;(y)T216V-T226P-H227P;(Z)T216V-S222D-H227P ;(aa)T226N-H227P;(bb)S222D-T226N-H227P;(cc)R217S-S222E-T226N;(dd)R217S-T226N-H227P;(ee)R217S-S222E-T226N-H227P;(ff)T216V-R217S-H227P;(gg)T216V-S222E-T226P-H227P;(hh)T216V-S222C-T226N; (ii)R217S-T226P-H227P;(jj)T216V-T226P;(kk)S222E-T226N-H227P;(ll)S222E-H227P;(mm)R217S-S222E-T226P;(nn (uu)S222D-T226N(vv). In some embodiments of any of the embodiments disclosed herein, the polypeptide exhibits at least about 50% less proteolysis compared to a monoclonal IgG1 antibody heavy chain polypeptide that does not comprise said one or more amino acid modifications. In some embodiments of any of the embodiments disclosed herein, the polypeptide does not exhibit detectable proteolysis. In some embodiments of any of the embodiments disclosed herein, the polypeptide exhibits increased stability compared to a monoclonal IgG1 antibody heavy chain polypeptide that does not contain the one or more amino acid modifications.

[0012] In a further aspect, provided herein is a monoclonal IgG1 antibody heavy chain polypeptide produced by any of the methods disclosed herein.

[0013] In still further aspects, provided herein are a) written instructions for producing any of the polypeptides disclosed herein, and b) a kit comprising one or more of: 1) any of the nucleic acids disclosed herein; 2) any of the vectors disclosed herein; and / or 3) any of the host cells disclosed herein.

[0014] In another aspect, provided herein is a syringe, cannula, or catheter comprising any of the polypeptides disclosed herein.

[0015] Each of the aspects and embodiments described herein can be used together unless expressly or specifically excluded by the context of that embodiment or aspect.

[0016] Throughout this specification, various patents, patent applications, and other types of publications are cited, such as, for example, journal articles, electronic database entries, etc. The disclosures of all patents, patent applications, and other publications cited herein are incorporated by reference in their entirety for all purposes. [Brief description of the drawings]

[0017] [Figure 1] 1 shows the P entry clones used for Synagis HC heavy chain SEL library construction.

[0018] [Diagram 2] The expression vector pTTTpyr2-ISceI-Synagis HC_Geneart_SEL heavy chain is shown.

[0019] [Diagram 3] 1 shows the P entry clones used for c2G4_HC3 SEL library construction.

[0020] [Figure 4] 1 shows an expression vector for producing the c2G4_HC3 heavy chain.

[0021] [Diagram 5] The expression cassette for the c2G4_LC2 light chain is shown.

[0022] [Figure 6]Graph showing reduction in hinge clipping for modified C2G4 variants versus wild type C2G4 control samples. The x-axis shows the final sum of bands, which is used to ensure that the calculated clipping range is based on significant bands and not on noise. The solid line depicted is the average delta clipping of WT samples, and the dashed lines are the average WT plus and minus one standard deviation.

[0023] [Figure 7] Graphs are shown highlighting the reduction in hinge clipping for modified variants versus wild-type control samples. The x-axis shows the final sum of bands, which is used to ensure that the calculated clipping range is based on significant bands and not noise. The square data points are for pooled WT-hinge samples. Each data point represents a biological replicate that was pooled after harvest, but then purified and assayed independently. The "+" data point is for HC:W105F, a known variant that has a stronger binding interaction with the antigen. This variant has a wild-type hinge sequence. The triangle data points are biological WT-hinge replicates that were not pooled before being purified and assayed. The circle data points represent modified variants as described in Table 4.

[0024] [Figure 8] Graph showing the reduction in hinge clipping for modified variants versus wild-type hinge control samples. The x-axis shows the final sum of bands, which is used to ensure that the calculated clipping range is based on significant bands and not noise.

[0025] [Figure 9] 1 shows a sequence alignment for the hinge region of anti-RSV and C2G4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The invention disclosed herein is based, in part, on the inventors' discovery that undesired antibody cleavage is eliminated or reduced when a wild-type (i.e., naturally occurring) antibody hinge region domain is modified to include one or more alternatively substituted amino acids.

[0027] Thus, provided herein are DNA constructs, vectors, antibodies, host cells expressing DNA constructs and / or cleavage-resistant antibodies, and methods for enhancing secretion and / or preventing or reducing unwanted antibody cleavage. More specifically, and in some non-limiting embodiments, modified antibody hinge sequences are included in the antibodies to prevent or reduce antibody cleavage (i.e., clipping) during host cell production. The antibodies disclosed herein exhibit better secretion, stability, and / or purification compared to antibodies that do not include the modified hinge sequences disclosed herein. Thus, the present disclosure provides an alternative and improved method for antibody production, particularly for the production of therapeutic proteins, which results in high levels of purified antibody with limited risk of contamination with unwanted cleavage products.

[0028] I. Definition The term "polypeptide" or "protein" refers to any polymer containing any of the 20 naturally occurring amino acids, regardless of its size. The term "protein" is often used in reference to relatively large proteins, and "peptide" is often used in reference to small polypeptides, although the use of these terms in the field often overlaps. Thus, the term "polypeptide" generally refers to proteins, polypeptides, and peptides, unless otherwise noted. Conventional one-letter or three-letter abbreviations for amino acid residues are used herein.

[0029] The term "nucleic acid" or "polynucleotide" includes DNA, RNA, single-stranded or double-stranded, and chemical modifications thereof. The terms "nucleic acid" and "polynucleotide" can be used interchangeably herein. Because the genetic code is degenerate, more than one codon may be used to code for a particular amino acid, and the subject matter of the present invention includes polynucleotides that code for a particular amino acid sequence.

[0030] As used herein, the terms "antibody" and "antibodies" refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, single chain Fvs (scFvs), disulfide-linked Fvs (sdFvs), Fab fragments, F(ab') fragments, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to the engineered antibodies disclosed herein), as well as epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site, which may or may not be fused to another immunoglobulin domain, including, but not limited to, an Fc region or fragment thereof. As outlined herein, the terms "antibody" and "antibodies" specifically include hinge region variants described herein, full length antibodies, and hinge variant-fusions comprising modified hinges described herein fused to immunologically active fragments of immunoglobulins or to other proteins. Such Fc variant-fusions include, but are not limited to, scFv-Fc fusions, variable region (e.g., VL and VH)-Fc fusions, scFv-scFv-Fc fusions. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0031] As used herein, the "hinge region" is generally defined as the interval 212-238 (EU numbering) or 222-251 (Kabat numbering) of human IgG1. The hinge can be further divided into three distinct regions: the upper, middle and lower hinge. In some embodiments, the hinge region is defined as the interval from amino acids 216 to 238 of the sequence shown in SEQ ID NO: 1. Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence or the sequence shown in SEQ ID NO: 1 using a number of publicly available sequence alignment programs.

[0032] The terms "proteolysis", "proteolytic degradation", "cleavage", and "clipping", as used interchangeably herein in the context of antibody production, refer to the unwanted degradation of antibody polypeptide components (e.g., antibody heavy chain polypeptides) into smaller polypeptides that are generally considered to be undesirable by-products of antibody expression and production in recombinant host cells (e.g., filamentous fungal host cells). In some embodiments, degradation can occur by cleavage, by enzymatic or chemical mechanisms, of peptide bonds located in the antibody heavy chain hinge region. In alternative embodiments, degradation can occur by cleavage of cross-links between homologous or heterologous proteins. In some embodiments, proteolysis occurs during antibody expression in a host cell (e.g., a eukaryotic, e.g., mammalian or fungal host cell). In other embodiments, proteolysis occurs during or after isolation and / or purification of the antibody.

[0033] "Stability" and "stable" refer to the resistance of the modified antibody in the formulation to aggregation, degradation or fragmentation under given manufacturing, preparation, shipping and storage conditions. Modified antibodies with improved stability will retain biological activity under given manufacturing, preparation, shipping and storage conditions. The stability of the modified 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 techniques, intrinsic tryptophan fluorescence, differential scanning calorimetry, and / or ANS binding techniques. The stability of the modified antibody can be compared to comparable molecules under the same conditions. The overall stability of the modified antibody can also be assessed by various immunological assays, including, for example, ELISA and radioimmunoassays, using isolated antigen molecules or cells expressing them.

[0034] The terms "wild-type," "wild-type," "parent," or "reference" with respect to a polypeptide refer to a naturally occurring polypeptide that does not contain an artificial substitution, insertion, or deletion at one or more amino acid positions. Similarly, the terms "wild-type," "wild-type," "parent," or "reference" with respect to a polynucleotide refer to a naturally occurring polynucleotide that does not contain an artificial nucleoside change. However, a polynucleotide that encodes a wild-type, parent, or reference polypeptide is not limited to naturally occurring polynucleotides, but includes any polynucleotide that encodes a wild-type, parent, or reference polypeptide.

[0035] As used herein, the term "non-naturally occurring" refers to anything that is not found in nature (e.g., recombinant nucleic acid and protein sequences produced in a laboratory), such as modifications of wild-type nucleic acid and / or amino acid sequences. In some embodiments, a non-naturally occurring polypeptide contains amino acid substitutions (i.e., mutations) that are not found in the corresponding wild-type or naturally occurring amino acid sequence.

[0036] As used herein, a "derivative" or "variant" of a polypeptide means a polypeptide that is derived from a precursor polypeptide (e.g., a naturally occurring polypeptide) by the addition of one or more amino acids to either or both of the C-terminus and N-terminus, the substitution of one or more amino acids at one or several different sites in the amino acid sequence, the deletion of one or more amino acids at one or both termini of the polypeptide or at one or more sites in the amino acid sequence, or the insertion of one or more amino acids at one or more sites in the amino acid sequence.

[0037] As used herein, a "variant polynucleotide" encodes a variant polypeptide or has a specified degree of homology / identity with a parent polynucleotide or hybridizes under stringent conditions with the parent polynucleotide or its complement. Suitably, the variant polynucleotide has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% nucleotide sequence identity with the parent polynucleotide or the complement of the parent polynucleotide. Methods for determining percent identity are known in the art.

[0038] The term "derived from" encompasses the terms "originating from," "obtained from," "obtainable from," "isolated from," and "made from," and generally indicates that one specified material has characteristics that find their origin in, or that can be described with respect to, another specified material.

[0039] "Control sequences" are defined herein to include all components necessary or advantageous for the expression of a polynucleotide or polypeptide of interest. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters and transcriptional and translational stop signals. Control sequences may provide linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding the polypeptide.

[0040] "Operably linked" is defined herein as an arrangement in which a control sequence is suitably placed in a functional relationship (i.e., in a relative position) with a polynucleotide, such as a coding sequence in a DNA sequence, or a polypeptide of interest, such that the control sequence induces or regulates expression of the polynucleotide and / or polypeptide.

[0041] The term "DNA construct" refers to a DNA sequence that is operably linked to a suitable control sequence capable of causing the expression of a protein in a suitable host. Such control sequences may include a promoter for causing transcription, an optional operator sequence for controlling transcription, a sequence encoding a suitable ribosome binding site on the mRNA, an enhancer, and sequences that control the termination of transcription and translation.

[0042] The term "fusion DNA construct" or "fusion nucleic acid" refers to a nucleic acid that includes several 5' to 3' polynucleotide sequences (e.g., but not limited to, a DNA molecule encoding a signal sequence, a DNA molecule encoding a carrier protein, a DNA molecule encoding a KEX2 site, and a DNA molecule encoding a polypeptide of interest) that are operably linked together and encode a fusion polypeptide.

[0043] "Vector" refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes, etc.

[0044] "Expression vector" refers to a vector capable of incorporating and expressing heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are commercially available.

[0045] A "promoter" or "promoter sequence" is a nucleic acid sequence recognized by a host cell for expression of a polynucleotide of interest, such as a coding region. Generally, a promoter sequence contains transcriptional control sequences that mediate expression of a polynucleotide of interest. The promoter can be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell, including mutated, truncated, and hybrid promoters, and can be derived from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0046] The term "signal sequence" refers to a sequence of amino acids at the amino terminus of a protein that directs the protein to the secretory system for secretion from a cell. The signal sequence is cleaved from the protein prior to secretion of the protein. In some cases, the signal sequence can be referred to as a "signal peptide" or a "leader peptide." The definition of a signal sequence is a functional one. The mature form of an extracellular protein lacks the signal sequence, which is cleaved off during the secretion process.

[0047] As used herein, the term "carrier protein" refers to a protein that functions in or facilitates the folding and secretion of a polypeptide from a host cell. Exemplary carrier proteins are discussed in more detail below.

[0048] The term "recombinant" when used with respect to a subject cell, nucleic acid, polypeptide / enzyme, or vector indicates that the subject has been modified from its native state. Thus, for example, a recombinant cell expresses a gene not found in the native (non-recombinant) form of the cell, or expresses a native gene at a different level or under different conditions than found in nature. A recombinant nucleic acid may differ from the native sequence by one or more nucleotides and / or is operably linked to a heterologous sequence in an expression vector, such as a heterologous promoter, a signal sequence allowing secretion, etc. A recombinant polypeptide / enzyme may differ from the native sequence by one or more amino acids and / or is fused to a heterologous sequence. A vector comprising a nucleic acid encoding an antibody heavy chain is, for example, a recombinant vector.

[0049] As used herein, "microorganism" refers to bacteria, fungi, viruses, protozoa, and other microorganisms or microscopic microorganisms.

[0050] "Host strain" or "host cell" refers to a suitable host for an expression vector or DNA construct comprising a polynucleotide encoding a polypeptide, particularly a recombinant fusion polypeptide encompassed by the present disclosure. In specific embodiments, the host strain may be a filamentous fungal cell or a mammalian cell. The term "host cell" includes both cells and protoplasts.

[0051] The term "filamentous fungi" refers to all filamentous forms of the subdivision Eumycotina (see Alexopoulos, CJ (1962), INTRODUCTORY MYCOLOGY, Wiley, New York). These fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, glucan, and other complex polysaccharides. The filamentous fungi disclosed herein are morphologically, physiologically, and genetically distinct from yeasts. Vegetative growth by filamentous fungi is by hyphal elongation and carbon catabolism is obligately aerobic.

[0052] The term "culturing" refers to growing a population of microbial cells in a liquid or solid medium under suitable conditions.

[0053] The term "heterologous" with respect to a polynucleotide or polypeptide refers to a polynucleotide or polypeptide that does not naturally occur in a host cell. In some embodiments, the protein is a commercially important industrial protein, and in some embodiments, the heterologous protein is a therapeutic protein. The term is intended to encompass proteins encoded by naturally occurring genes, mutated genes, and / or synthetic genes.

[0054] The term "homologous" with respect to a polynucleotide or protein refers to a polynucleotide or protein that is naturally present in a host cell.

[0055] As used herein, the terms "recovered," "isolated," and "separated" refer to a protein (e.g., a polypeptide of interest), cell, nucleic acid, or amino acid that has been removed from at least one component with which it is associated.

[0056] As used herein, the terms "transformed," "stably transformed," and "transgenic" as used with respect to a cell mean that the cell has additional copies of a non-native (e.g., heterologous) or native (e.g., homologous) nucleic acid sequence integrated into its genome or has an episomal plasmid that is maintained through multiple generations.

[0057] As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene, which process includes both transcription and translation.

[0058] The term "secreted protein" refers to the region of a polypeptide that is released from the cell during protein secretion.

[0059] The term "secretion" refers to the selective movement of a protein across a membrane in a host cell into the extracellular space and surrounding medium.

[0060] Certain ranges are provided herein with numerical values ​​preceded by the term "about". The term "about" is used herein to provide a textual aid for the exact number it precedes, as well as numbers that are close to or approximately the same as the number it precedes. In determining whether a number is close to or approximately the same as a specifically recited number, the close or approximately same number that is not recited may also be a number that provides a substantial equivalent to the specifically recited number in the context in which it is present. For example, with respect to a numerical value, the term "about" refers to a range of -10% to +10% of that numerical value, unless the term is otherwise specifically defined in the context.

[0061] As used herein, the singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0062] It is further noted that the claims may be drafted to exclude any optional element, and thus, this statement shall serve as a predicate for use of exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements or the use of a "negative" limitation.

[0063] It should also be noted that the term "consisting essentially of," as used herein, refers to a composition in which the component following this term is less than 30% by weight of the total composition and in the presence of other known components in a total amount that does not contribute to or interfere with the action or activity of the component.

[0064] It should be further noted that the term "comprising," as used herein, means including, but not limited to, the component following the term "comprising." The component following the term "comprising" is required or essential, but the composition that includes the component can further include other non-essential or optional components.

[0065] It should also be noted that the term "consisting of," as used herein, is meant to include and be limited to the component following the term "consisting of." Thus, the component following the term "consisting of" is required or essential, and no other component is present in the composition.

[0066] Every maximum numerical limitation given throughout this specification will include every lower numerical limitation, as if such lower numerical limitation were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitation were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0067] 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.

[0068] Other definitions of terms may appear throughout the specification.

[0069] II. Composition A. Engineered Antibodies Provided herein are non-naturally occurring antibodies, fragments thereof, or variants thereof, having modified hinge regions with improved expression and / or cleavage characteristics. The modified hinge regions may exhibit alterations in one or more of the hinge characteristics, including but not limited to, stability, flexibility, length, conformation, charge, resistance to cleavage (e.g., proteolytic cleavage), and hydrophobicity, as compared to wild-type antibody hinges. The modified hinge regions disclosed herein may be generated by methods well known in the art, such as, for example, introducing modifications into wild-type hinges. Modifications that may be utilized to generate modified hinge regions include, but are not limited to, amino acid insertions, deletions, substitutions, and rearrangements. The hinge modifications and the disclosed modified hinge regions together are referred to herein as "hinge modifications" or simply "modified hinges." The modified hinge regions disclosed herein may be incorporated into a molecule of choice, including, but not limited to, antibodies and fragments thereof.

[0070] As demonstrated herein, molecules comprising a modified hinge may exhibit reduced or eliminated proteolysis during host cell production and / or purification as compared to molecules having the same amino acid sequence except for the modified hinge, such as, for example, a molecule having the same amino acid sequence but for including a wild-type hinge. As demonstrated herein, molecules comprising a modified hinge may have improved stability and / or storage (i.e., increased shelf life) and resistance to cleavage as compared to molecules having the same amino acid sequence except for the modified hinge, such as, for example, a molecule having the same amino acid sequence but for including a wild-type hinge.

[0071] In one embodiment, the modified antibody can have at least a modified hinge (e.g., a hinge region comprising one or more amino acid insertions, deletions, substitutions, or rearrangements), wherein the modified antibody has improved stability and / or resistance to cleavage compared to a comparable molecule.

[0072] The modified antibodies disclosed herein include antibody variants that include hinge modifications that alter one or more characteristics of the hinge, including but not limited to stability, resistance to cleavage (e.g., proteolytic cleavage), flexibility, length, conformation, charge, and hydrophobicity. The modified antibodies disclosed herein also include variants that include modified hinges that exhibit one or more altered characteristics compared to the wild-type hinge, including but not limited to stability, flexibility, length, conformation, charge, and hydrophobicity. The disclosed modified hinges can be generated by methods well known in the art, such as, for example, introducing modifications into a wild-type hinge. Hinge modifications that can be utilized in generating the modified hinges include, but are not limited to, insertion, deletion, inversion, and substitution of one or more amino acid residues. It will be appreciated by those skilled in the art that a combination of insertion and / or deletion and / or substitution can also be used to generate the modified hinge.

[0073] In certain embodiments, the engineered antibody includes a hinge modification that is a substitution of at least one amino acid residue in the hinge. In one embodiment, at least one, or at least two, or at least three, or at least four, or at least five, or at least ten, or at least fifteen amino acid residues are substituted in the hinge. In one embodiment, the substitution is made in the upper hinge. In another embodiment, the substitution is made in the middle hinge. In another embodiment, the substitution is made in the lower hinge. In yet another embodiment, the substitution is made in two or more positions, including but not limited to the upper hinge, middle hinge, and lower hinge.

[0074] In yet another embodiment, the engineered antibody comprises at least one substitution in the hinge region, the substitution being located at an amino acid position selected from the group consisting of 216, 217, 222, 226, 227, and / or 234 (wherein the amino acid positions are numbered according to the numbering in SEQ ID NO:1), or at positions 213, 214, 221, 223, 224, and / or 231 (wherein the numbering system is that of the EU index as described in Kabat), or at positions 223, 224, 232, 236, 237, and / or 244 (wherein the numbering system is that of the Kabat index as described in Kabat). Any combination of hinge modifications are described in Table 1 and are specifically contemplated as embodiments. [Table 1]

[0075] In one embodiment, the modified antibodies disclosed herein include modified hinges that have altered (e.g., increased or decreased) hinge flexibility compared to a wild-type hinge. Modified hinges with altered hinge flexibility can be generated by incorporating specific modifications into a wild-type hinge. Hinge modifications that increase hinge flexibility include, but are not limited to, substitution of one or more amino acid residues with one or more amino acid residues that increase flexibility (e.g., glycine), substitution of cysteines involved in disulfide bond formation with amino acid residues that cannot form disulfide bonds (e.g., serine, alanine, glycine), insertion of one or more amino acid residues that allow a high degree of local flexibility (e.g., glycine), and deletion of one or more amino acid residues that increase the rigidity of the polypeptide (e.g., proline). Hinge modifications that decrease the flexibility of the hinge include, but are not limited to, substitution of one or more amino acid residues with one or more amino acid residues that increase the rigidity of the polypeptide (e.g., proline), substitution of an amino acid residue that cannot form disulfide bonds (e.g., serine, alanine, glycine) with an amino acid residue that can form disulfide bonds (e.g., cysteine), insertion of one or more amino acid residues that increase the rigidity of the polypeptide (e.g., proline), and deletion of one or more amino acid residues that increase flexibility (e.g., glycine).

[0076] In specific embodiments, the modified antibodies disclosed herein include modified hinges that have an altered hinge conformation compared to a wild-type hinge. Modified hinges with altered hinge conformation can be generated by incorporating specific modifications into a wild-type hinge. Hinge modifications that alter the conformation of the hinge include, but are not limited to, substitution of one or more amino acid residues with a small side chain (e.g., alanine, glycine) for an amino acid residue with a larger, bulkier side chain (e.g., tryptophan, proline), substitution of one or more amino acid residues with a larger, bulkier side chain (e.g., tryptophan, proline) for an amino acid residue with a small side chain (e.g., alanine, glycine), inversion of two or more amino acid residues within the hinge, insertion or deletion of one or more amino acid residues with a large or bulky side chain (e.g., tryptophan, proline). In addition, hinge modifications that alter the length and / or flexibility of the hinge can also result in altered conformation.

[0077] In a specific embodiment, the modified antibodies disclosed herein comprise a modified hinge with an IgG2a camelid-like modification. A modified hinge with a camelid-like modification may be generated by replacing a portion of a wild-type hinge with a portion of a camelid IgG2a hinge. Alternatively, or optionally, a modified hinge with a camelid-like modification may be generated by replacing one or more amino acid residues in the hinge with the corresponding amino acid residue found in a camelid IgG2a hinge. A modified hinge with a camelid-like modification may also incorporate additional amino acid substitutions and / or insertions and / or deletions. In addition, the camelid-like modification of the hinge may alter the characteristics of the hinge, including, but not limited to, length, flexibility, conformation, charge, and hydrophobicity.

[0078] In another embodiment, the engineered antibodies disclosed herein comprise a modified hinge that has an altered charge relative to the wild-type hinge. The modified charge hinge can be generated by incorporating specific modifications into the wild-type hinge. Hinge modifications that alter the charge of the hinge include, but are not limited to, substitution of one or more neutrally charged amino acid residues (e.g., valine, threonine) for a charged amino acid residue (e.g., aspartic acid, glutamic acid, lysine, arginine), substitution of one or more positively charged amino acid residues (e.g., lysine, arginine) for a neutrally charged (e.g., valine, threonine) or negatively charged (e.g., aspartic acid, glutamic acid) amino acid residue, substitution of one or more negatively charged amino acid residues (e.g., aspartic acid, glutamic acid) for a neutrally charged (e.g., valine, threonine) or positively charged (e.g., lysine, arginine) amino acid residue, and insertion or deletion of one or more charged amino acid residues (e.g., aspartic acid, glutamic acid, lysine, arginine).

[0079] In another specific embodiment, the modified antibodies disclosed herein include modified hinges that have altered (e.g., increased or decreased) hydrophobicity compared to the wild-type hinge. Modified hinges with altered hydrophobicity can be generated by incorporating specific modifications into the wild-type hinge. Hinge modifications that alter the hydrophobicity of the hinge include, but are not limited to, substitution of one or more hydrophobic amino acid residues (e.g., valine, leucine) for hydrophilic amino acid residues (e.g., serine, threonine, tyrosine), substitution of one or more hydrophilic amino acids (e.g., serine, threonine, tyrosine) for hydrophobic amino acid residues (e.g., valine, leucine), and insertion or deletion of one or more hydrophobic or hydrophilic amino acid residues (e.g., valine, leucine, serine, threonine, tyrosine).

[0080] It will be appreciated by those skilled in the art that any given hinge modification can change more than one characteristic of the hinge. For example, the addition of one or more proline residues to the hinge results in a hinge modification that increases the length of the hinge and potentially reduces its flexibility at the same time. Similarly, the replacement of glycine residues with aspartic acid can change both the charge and hydrophobicity of the hinge. Other combinations are described above, and still other combinations will be apparent to those skilled in the art.

[0081] In particular, it is contemplated that one may choose to analyze the properties of the amino acid residues present in the hinge before making any hinge modifications. Those skilled in the art will recognize that in some cases, the antibody of interest already has a suitable amino acid sequence in the hinge, such that one or more characteristics of the hinge (e.g., flexibility, length, conformation, charge and hydrophobicity) are altered compared to the wild-type hinge. In this situation, additional hinge modifications will only be introduced if further modifications are desired. In addition to the specific amino acid residues described above and in Table 1, those skilled in the art will understand that some additional amino acid residues can be inserted, deleted and / or substituted in the hinge to change the characteristics of the hinge. Families of amino acid residues with similar properties have been defined in the art, and some examples are shown in Table 2. [Table 2]

[0082] In particular, it is contemplated that conservative amino acid substitutions may be made for the above-mentioned modification of the hinge. It is well known in the art that "conservative amino acid substitution" refers to an amino acid substitution that replaces a functionally equivalent amino acid. A conservative amino acid change results in a silent change in the amino acid sequence of the resulting peptide. For example, one or more amino acids with similar polarity act as functional equivalents, resulting in a silent change in the amino acid sequence of the peptide. A substitution that replaces a residue with a charge-neutral and smaller residue can also be considered as a "conservative substitution" even if the residue is from a different group (e.g., the replacement of phenylalanine with the smaller isoleucine). Families of amino acid residues with similar side chains have been defined in the art. Some families of conservative amino acid substitutions are shown in Table 2 (above).

[0083] The term "conservative amino acid substitution" also refers to the use of amino acid analogs or variants. Guidance on how to make phenotypically silent amino acid substitutions is provided in Bowie et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," (1990, Science 247:1306-1310).

[0084] The stability of the modified antibodies disclosed herein can be examined by measuring a variety of different characteristics of the polypeptide that can alter its biological function and / or activity. Non-limiting examples of such characteristics include aggregation, fragmentation, the presence or absence of protein modifications (e.g., acetylation, glycosylation, methylation, phosphorylation), biological activity, and dissociation of multi-subunit complexes. Typically, one or more of these characteristics are monitored (i.e., measured) over a period of time under a set of defined conditions. The stability of the disclosed modified antibodies can be characterized using in vitro stability assays known in the art to determine the stability of a polypeptide. Such assays include, but are not limited to, monitoring the integrity of the polypeptide over time using assays that monitor the size and / or activity of the polypeptide. The stability of the modified antibodies can be examined in solution or as a solid. In addition, stability can be monitored in the presence of components known to affect antibody stability, such as, but not limited to, metal ions and proteases.

[0085] In one embodiment, the modified antibodies disclosed herein are antibodies or Fc fusion proteins that contain a modified hinge that has improved stability or is resistant or less prone to cleavage compared to a comparable molecule lacking the modified hinge. Such antibodies include IgG molecules that contain a hinge that can be modified to generate a modified hinge. Thus, the modified antibodies disclosed herein can include any antibody molecule incorporating a modified hinge that preferably binds specifically to an antigen (i.e., competes out non-specific binding as determined by immunoassays well known in the art to assay for specific antigen-antibody binding). Such antibodies include, but are not limited to, polyclonal, monoclonal, bispecific, multispecific, human, humanized, chimeric antibodies, single chain antibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv, and fragments modified to contain or fused to a hinge region-containing polypeptide, in certain cases, containing either a VL or VH domain, or further containing a complementarity determining region (CDR) that specifically binds to an antigen.

[0086] Also disclosed herein are modified antibodies with improved stability. In certain embodiments, antibodies comprising a modified hinge region are more resistant to cleavage than comparable molecules. In specific embodiments, the modified antibodies are more resistant to metal ion-mediated cleavage, particularly cation ion-mediated cleavage, at the hinge. In one embodiment, the modified antibodies are at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 50-fold, or at least 100-fold more resistant to cleavage than comparable molecules lacking the modified hinge region. In another embodiment, the modified antibodies disclosed herein are at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200% more resistant to cleavage than comparable molecules lacking the modified hinge region.

[0087] It is contemplated that the modified antibodies disclosed herein may have other altered characteristics, including increased in vivo half-life (e.g., serum half-life) in mammals (particularly humans), increased stability in vivo (e.g., serum half-life) and / or in vitro (e.g., shelf life), and / or increased melting temperature (Tm) compared to comparable molecules. In one embodiment, the modified antibodies disclosed herein have an in vivo half-life of greater than 15 days, greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. In another embodiment, the modified antibodies have an in vitro half-life (e.g., liquid or powder formulation) of greater than 15 days, greater than 30 days, greater than 2 months, greater than 3 months, greater than 6 months, or greater than 12 months, or greater than 24 months, or greater than 36 months, or greater than 60 months. In yet another embodiment, the modified antibodies have a Tm value greater than about 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, or 95°C.

[0088] It is also specifically contemplated that the altered antibodies disclosed herein may contain one or more additional amino acid residue substitutions, mutations and / or modifications that result in the antibody having preferred characteristics including, but not limited to, increased serum half-life, increased binding affinity, decreased immunogenicity, increased production, enhanced or decreased ADCC or CDC activity, altered glycosylation and / or disulfide bonds, and modified binding specificity.

[0089] The modified antibodies disclosed herein can be combined with other modifications, including, but not limited to, modifications that alter effector function. For example, the modified antibodies disclosed herein can be combined with other modifications to provide additive, synergistic, or novel properties in an antibody or fusion. Such modifications can be in the CH1, CH2, or CH3 domains, or combinations thereof. It is contemplated that modified antibodies enhance the properties of the modifications with which they are combined. For example, when a modified antibody (e.g., any of the modified antibodies with modified hinge regions disclosed herein) is combined with a variant known to bind FcγRIIIA with higher affinity than a comparable molecule containing a wild-type Fc region, the combination with the variant results in a greater fold enhancement in FcγRIIIA affinity.

[0090] In some embodiments, the modified antibodies disclosed herein comprise one or more modified glycoforms, i.e., carbohydrate compositions covalently attached to the molecule comprising the modified hinge region. Modified glycoforms can be useful for a variety of purposes, including, but not limited to, enhancing or reducing effector function. Modified glycoforms can be generated by any method known to one of skill in the art, for example, by using modified or variant expression strains, by co-expression with one or more enzymes, such as β(1,4)-N-acetylglucosaminyltransferase III (GnTI11), by expressing the molecule comprising the modified hinge region in various organisms or cell lines derived from various organisms, or by modifying the carbohydrate after the molecule comprising the modified hinge region is expressed. Methods for producing modified glycoforms are known in the art and include, but are not limited to, those described in Umana et al, 1999, Nat. Biotechnol 17:176-180; Davies et al., 20017 Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473) U.S. Patent No. 6,602,684; U.S. Patent Application No. 10 / 277,370; U.S. Patent Application No. 10 / 113,929; PCT Publication No. WO 00 / 61739 A1; PCT Publication No. WO 01 / 292246 A1; PCT Publication No. WO 02 / 311140 A1; PCT Publication No. WO 02 / 30954 A1; Potillegent TM technology(Biowa,Inc.Princeton,NJ);GlycoMAb TMGlycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland) includes those described in WO 00061739; EA 01229125; U.S. Patent Publication No. 20030115614; Okazaki et al., 2004, JMB, 336:1239-49.

[0091] The modified antibodies disclosed herein are intended to include antibodies comprising a variable region, an Fc region, and a modified hinge (e.g., any of the modified hinge regions disclosed herein). Modified antibodies may be produced "de novo" by combining a variable domain or fragment thereof that specifically binds to at least one antigen with an Fc region that incorporates a modified hinge. Alternatively, modified antibodies may be produced by modifying the hinge of an Fc region-containing antibody that binds an antigen.

[0092] Antibody types contemplated for use with the modified hinge regions disclosed herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies, bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, single chain FvFc (scFvFc), single chain Fv (scFv), and anti-idiotypic (anti-Id) antibodies. In particular, antibodies used in the methods disclosed herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule.

[0093] The engineered antibodies disclosed herein can be from any animal origin, including birds and mammals (e.g., human, murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken). In specific embodiments, the antibodies are human or humanized monoclonal antibodies. As used herein, "human" antibodies include antibodies having a human immunoglobulin amino acid sequence, as well as antibodies isolated from human immunoglobulin libraries or from mice expressing antibodies derived from human genes.

[0094] The antibodies disclosed herein can be monospecific, bispecific, trispecific, or of greater multispecificity. Multispecific antibodies may specifically bind to different epitopes of a desired target molecule, or may specifically bind to both the target molecule and to a heterologous epitope, such as a heterologous polypeptide or solid support material. See, for example, WO 94 / 04690; WO 93 / 17715; WO 92 / 08802; WO 91 / 00360; and WO 92 / 05793; Tutt, et al., 1991, J.Immunol.147:60-69; U.S. Patent Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, and 5,601,819; and Kostelny et al., 1992, J.Immunol.148:1547). Antibodies with three or more valencies incorporating the modified hinges disclosed herein are contemplated. For example, trispecific antibodies can be made. See, e.g., Tutt et al. J. Immunol. 147:60 (1991).

[0095] Modified antibodies contemplated herein also include single domain antibodies, including camelized single domain antibodies (see, e.g., Muyldermans et al., 2001, Trends Biochem. Sci. 26:230; Nuttall et al., 2000, Cur. Pharm. Biotech. 1:253; Reichmann and Muyldermans, 1999, J. Immunol. Meth. 231:25; WO 94 / 04678 and WO 94 / 25591; U.S. Pat. No. 6,005,079).

[0096] The modified antibodies disclosed herein can further include antibody-like and antibody-domain fusion proteins. An antibody-like molecule is any molecule generated with desired binding properties, see, for example, PCT Publications WO 04 / 044011; WO 04 / 058821; WO 04 / 003019 and WO 03 / 002609. An antibody-domain fusion protein can incorporate one or more antibody domains, such as an Fc domain or a variable domain. For example, the heterologous polypeptide can be a Fab fragment, an Fd fragment, an Fv fragment, an F(ab) fragment, an Fc fragment, an Fv fragment, an F(ab) ... 2 The antibody may be fused or conjugated to a fragment, VH domain, VL domain, VH CDR, VL CDR, or fragment thereof. Numerous antibody-domain molecules are known in the art, including but not limited to diabodies (dsFv). 2(Bera et al., 1998, J. Mol. Biol. 281:475-83); minibodies (homodimers of scFv-CH3 fusion proteins) (Pessi et al., 1993, Nature 362:367-9), tetravalent di-diabodies (Lu et al., 2003 J. Immunol. Methods 279:219-32), and tetravalent bispecific antibodies called Bs(scFv)4-IgG (Zuo et al., 2000, Protein Eng. 13:361-367. Fc domain fusions combine the Fc region of an immunoglobulin, particularly an Fc region containing a modified hinge, with a fusion partner, which may generally be a protein, including but not limited to a ligand, an enzyme, a ligand portion of a receptor, an adhesion protein, or any other protein or domain. See, e.g., Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200; Heidaran et al., 1995, FASEB J. 9:140-5. 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. 307,434 and 367,166; PCT Publication Nos. WO 96 / 04388 and WO 91 / 06570; Ashkenazi et al. See, e.g., Zheng et al., 1995, J. Immunol. 154:5590-5600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337-11341.

[0097] Other molecules specifically contemplated are small engineered protein domains such as, for example, immune domains and / or monomer domains (see, for example, US Patent Publication Nos. 2003082630 and 2003157561). The immune domains contain at least one complementarity determining region (CDR) of an antibody, and the monomer domains are based on known naturally occurring non-antibody domain families, in particular protein extracellular domains, which contain a conserved scaffold and a variable binding site, an example being the LDL receptor A domain, which is involved in ligand binding. Such protein domains can fold correctly, for example, independent of the presence or with limited assistance of chaperonins or metal ions. This ability avoids misfolding of the domain when inserted into a new protein environment, thereby preserving the binding affinity of the protein domain for a particular target. The variable binding sites of the protein domains are randomized using various diversity generating methods, such as, for example, random mutagenesis, site-directed mutagenesis, and by directed evolution methods, such as, for example, recursive error-prone PCR, recursive recombination, etc. For details of various diversity generation methods, see U.S. Patent Nos. 5,811,238; 5,830,721; 5,834,252; PCT Publication Nos. WO 95 / 22625; WO 96 / 33207; WO 97 / 20078; WO 97 / 35966; WO 99 / 41368; WO 99 / 23107; WO 00 / 00632; WO 00 / 42561; and WO 01 / 23401. The mutagenized protein domains are then expressed using a display system, such as phage display, which can generate a library of at least 10 variants and facilitate the isolation of protein domains with improved affinity and potency for the intended target by subsequent panning and screening.Such methods are described in PCT Publications WO 91 / 17271; WO 91 / 18980; WO 91 / 19818; WO 93 / 08278. Additional examples of display systems are described in U.S. Patent Nos. 6,281,344; 6,194,550; 6,207,446; 6,214,553 and 6,258,558. These methods can be utilized to rapidly generate highly diverse engineered protein domains with sub-nM binding affinities (Kd) and blocking functions (IC50). Once identified, 2-10 such engineered protein domains can be linked together to form binding proteins using natural protein linkers of about 4-15 amino acids in length. Individual domains can target a single type of protein or several, depending on the use / disease indication. The variant protein domain can then be linked to a modified hinge, e.g., an Fc region in an antibody containing any of the modified hinge regions disclosed herein.

[0098] It is also contemplated that a modified hinge (eg, any of the modified hinges disclosed herein) can be introduced into an antibody already described in the art. Antibodies into which modified hinges have been introduced include, but are not limited to, KS1 / 4 pan-carcinoma antigen (Perez and Walker, 1990, J. Immunol. 142:3662-3667; Bumal, 1988, Hybridoma 7(4):407-415), ovarian cancer antigen (CAl25) (Yu et al., 1991, Cancer Res. 51(2):468-475), prostatic acid phosphate (Tailor et al., 1990, Nucl. Acids Res. 18(16):4928), prostate specific antigen (Henttu and Vihko, 1989, Biochem. Biophys. Res. Comm. 160(2):903-910; Israeli et al., 1993, Cancer Res. 53:227-230), melanoma-associated antigen p97 (Estin et al., 1989, J. Natl. Cancer Instil. 81(6):445-446), melanoma antigen gp75 (Vijayasardahl et al., 1990, J. Exp. Med. 171(4):1375-1380), high molecular weight melanoma antigen (HMW-MAA) (Natali et al., 1987, Cancer 59:55-63; Mittelman et al., 1990, J. Clin. Invest. 86:2136-2144), prostate-specific membrane antigen, carcinoembryonic antigen (CEA) (Foon et al. al., 1994, Proc. Am. Soc. Clin. Oncol. 13:294), polymorphic epithelial mucin antigen, human milk fat globule antigen, colorectal tumor associated antigens such as CEA, TAG-72 (Yokata et al., 1992, Cancer Res. 52:3402-3408), CO17-1A (Ragnhammar et al., 1993, Int. J. Cancer 53:751-758); GICA 19-9 (Herlyn et al., 1982, J. Clin. Immunol. 2:135), CTA-1 and LEA, Burkitt's lymphoma antigen-38.13, CD19 (Ghetie et al., 1994, Blood 83:1329-1336), human B lymphoma antigen-CD20 (Reff et al., 1994, Blood 83:435-445), CD33 (Sgouros et al., 1993, J. Nucl. Med. 34:422-430), melanoma-specific antigens such as ganglioside GD2 (Saleh et al., 1993, J. Immunol., 151, 3390-3398), ganglioside GD3 (Shitara et al., 1993, Cancer Immunol. Immunother. 36:373-380), ganglioside GM2 (Livingston et al., 1993, J. Immunol. Immunother. 36:373-380), al., 1994, J. Clin. Oncol. 12:1036-1044), ganglioside GM3 (Hoon et al., 1993, Cancer Res. 53:5244-5250), tumor-specific transplantation-type cell surface antigens (TSTA), such as virus-induced tumor antigens (including T antigen DNA tumor viruses and envelope antigens of RNA tumor viruses), carcinoembryonic antigen-alpha-fetoprotein, such as colon CEA, bladder tumor carcinoembryonic antigen (Hellstrom et al., 1985, Cancer. Res. 45:2210-2188), differentiation antigens, such as human lung cancer antigens L6, L20 (Hellstrom et al., 1986, Cancer Res. 46:3917-3923), fibrosarcoma antigens, human leukemia T cell antigen-Gp37 (Bhattacharya-Chatterjee et al., 1999, Cancer Res. 53:5244-5250), tumor specific ... al., 1988, J. of Immun. 141:1398-1403), neoglycoproteins, sphingolipids, breast cancer antigens, e.g., EGFR (epidermal growth factor receptor), HER2 antigen (p185. HER2), polymorphic epithelial mucin (PEM) (Hilkens et al., 1992, Trends in Bio. Chem. Sci. 17:359), malignant human lymphocyte antigen-APO-1 (Bernhard et al., 1989, Science 245:301-304), differentiation antigens (Feizi, 1985, Nature 314:53-57), such as I antigen found in fetal erythrocytes, early endoderm I antigen found in adult erythrocytes, preimplantation embryos, I(Ma) found in gastric adenocarcinoma, M18, M39 found in breast epithelium, SSEA-1, VEP8, VEP9, Myl, VIM-D5 found in bone marrow cells, D found in colorectal cancer 1 56-22, TRA-1-85 (blood type H), C14 found in colon adenocarcinoma, F3 found in lung adenocarcinoma, AH6 found in gastric cancer, Y hapten, Lc found in embryonal carcinoma cells y , TL5 (blood type A), EGF receptor found in A431 cells, E found in pancreatic cancer 1 series (blood type B), FC10.2 found in embryonal carcinoma cells, gastric adenocarcinoma antigen, CO-514 found in adenocarcinoma (blood type Le a ), NS-10, CO-43 (blood group Le b ), G49 found in the EGF receptor of A431 cells, MH2 (blood type ALe) found in colon adenocarcinoma b / Le v ), 19.9 found in colon cancer, gastric cancer mucin, T found in bone marrow cells 5 A 7 , R found in melanoma 24 , 4.2, found in embryonal carcinoma cells, G D3 , D1.1, OFA-1, G M2 , OFA-2, G D2and M1:22:25:8, as well as SSEA-3 and SSEA-4 found in 4-8 cell stage embryos. In one embodiment, the antigen is a T cell receptor-derived peptide from cutaneous T cell lymphoma (see Edelson, 1998, The Cancer Journal 4:62).

[0099] In some embodiments, the modified hinge can be introduced into the anti-fluorescein monoclonal antibody, 4-4-20 (Kranz et al., 1982 J. Biol. Chem. 257(12):6987-6995). In other embodiments, the modified hinge is introduced into the mouse-human chimeric anti-CD20 monoclonal antibody 2H7, which recognizes the CD20 cell surface phosphoprotein on B cells (Liu et al., 1987, Journal of Immunology, 139:3521-6). In yet other embodiments, the modified hinge is introduced into a humanized antibody (Ab4D5) against human epidermal growth factor receptor 2 (p185HER2), as described by Carter et al. (1992, Proc. Natl. Acad. Sci. USA 89:4285-9). In yet another embodiment, the modified hinge is incorporated into a humanized anti-TAG72 antibody (CC49) (Sha et al., 1994 Cancer Biother. 9(4):341-9). In another embodiment, the modified hinge is incorporated into Rituxan, which is used to treat lymphoma.

[0100] In some embodiments, the modified hinge (e.g., any of the modified hinges disclosed herein that confer resistance to proteolytic cleavage) can be, but is not limited to, Erbitux®, a chimerized monoclonal antibody against EGFR. TM(also known as IMC-C225) (ImClone Systems Inc.); HERCEPTIN® (trastuzumab), a humanized anti-HER2 monoclonal antibody for the treatment of metastatic breast cancer patients (Genentech, Calif.); REOPRO® (abciximab) (Centocor), an anti-glycoprotein IIb / IIIa receptor on platelets for the prevention of blood clot formation; ZENAPAX® (daclizumab), an immunosuppressant humanized anti-CD25 monoclonal antibody for the prevention of acute kidney allograft rejection (Roche Pharmaceuticals, Switzerland). Another example is the humanized anti-CD18 F(ab') 2 (Genentech); humanized anti-CD18 F(ab') 2 CDP860 (Celltech, UK); PRO542 (Progenics / Genzyme Transgenics), an anti-HIV gp120 antibody fused to CD4; C14 (ICOS Pharm), an anti-CD14 antibody; humanized anti-VEGF IgG1 antibody (Genentech); and OVAREX, a murine anti-CA125 antibody. TM (Altarex); PANOREX, a mouse anti-17-IA cell surface antigen IgG2a antibody TM (Glaxo Wellcome / Centocor); IMC-C225 (ImClone Systems), a chimeric anti-EGFR IgG antibody; and VITAXIN, a humanized anti-αVβ3 integrin antibody. TM (Applied Molecular Evolution / MedImmune); Campath 1H / LDP-03 (Leukosite), a humanized anti-CD52 IgG1 antibody; Smart M195 (Protein Design Lab / Kanebo), a humanized anti-CD33 IgG antibody; and RITUXAN, a chimeric anti-CD20 IgG1 antibody. TM (IDEC Pharm / Genentech, Roche / Zettyaku); LYMPHOCIDE, a humanized anti-CD22 IgG antibody TM(Immunomedics); Smart ID10 (Protein Design Lab), a humanized anti-HLA antibody; ONCOLYM TM (Lym-1) is a radiolabeled mouse anti-HLA DR antibody (Techniclone); anti-CD11a is a humanized IgG1 antibody (Genetech / Xoma); ICM3 is a humanized anti-ICAM3 antibody (ICOS Pharm); IDEC-114 is a primatized anti-CD80 antibody (IDEC Pharm / Mitsubishi); ZEVALIN TM is a radiolabeled mouse anti-CD20 antibody (IDEC / Schering AG); IDEC-131 is a humanized anti-CD40L antibody (IDEC / Eisai); IDEC-151 is a primatized anti-CD4 antibody (IDEC); IDEC-152 is a primatized anti-CD23 antibody (IDEC / Seikagaku); SMART anti-CD3 is a humanized anti-CD3 IgG (Protein Design Lab); 5G1.1 is a humanized anti-complement factor 5 (C5) antibody (Alexion Pharm); IDEC-151 is a primatized anti-CD4 IgG1 antibody (IDEC Pharm / SmithKline Beecham; MDX-CD4 is a human anti-CD4 IgG antibody (Medarex / Eisai / Genmab); CDP571 is a humanized anti-TNF-α IgG4 antibody (Celltech); LDP-02 is a humanized anti-α4β7 antibody (LeukoSite / Genentech); OrthoClone OKT4A is a humanized anti-CD4 IgG antibody (Ortho Biotech); ANTOVA TM is a humanized anti-CD40L IgG antibody (Biogen); ANTEGREN TMis a humanized anti-VLA-4 IgG antibody (Elan); MDX-33 is a human anti-CD64 (FcγR) antibody (Medarex / Centeon); rhuMab-E25 is a humanized anti-IgE IgG1 antibody (Genentech / Norvartis / Tanox Biosystems); IDEC-152 is a primatized anti-CD23 antibody (IDEC Pharm); ABX-CBL is a mouse anti-CD-147 IgM antibody (Abgenix); BTI-322 is a rat anti-CD2 IgG antibody (Medimmune / Bio Transplant); Orthoclone / OKT3 is a mouse anti-CD3 IgG2a antibody (ortho Biotech); SIMULECT TM is a chimeric anti-CD25 IgG1 antibody (Novartis Pharm); LDP-01 is a humanized anti-β 2 -integrin IgG antibody (LeukoSite); anti-LFA-1 is a mouse anti-CD18 F(ab') 2 (Pasteur-Merieux / Immunotech); CAT-152 is a human anti-TGF-β 2 antibody (Cambridge Ab Tech); and Corsevin M is a chimeric anti-Factor VII antibody (Centocor).

[0101] In some embodiments, the engineered antibody or functional fragment thereof is an anti-respiratory syncytial virus (RSV) antibody, an anti-Ebola virus antibody, an anti-aggregating beta amyloid (Aβ) antibody, an anti-human immunodeficiency virus (HIV) antibody, an anti-herpes simplex virus (HSV) antibody, an anti-sperm antibody (e.g., an anti-human contraceptive antigen (HCA) antibody), and an anti-HER2 / neu antibody.

[0102] In yet another embodiment, the modified antibodies disclosed herein can specifically bind to the same antigen as a known therapeutic antibody, including but not limited to those described above, although in some embodiments the variable region of the modified antibody is not that of said therapeutic antibody, while in other embodiments the variable region of the modified antibody is the same as that of the therapeutic antibody.

[0103] 1. Signal sequence In some embodiments, the modified antibodies disclosed herein can include a signal sequence. The signal sequence can be any signal sequence that facilitates secretion of the protein from a host cell (e.g., a filamentous fungal host cell). In certain embodiments, the modified antibodies can include a signal sequence for a protein that is known to be highly secreted from the host cell in which the fusion protein is to be produced. The signal sequence used can be endogenous or non-endogenous to the host cell in which the modified antibody is to be produced.

[0104] 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 of 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.

[0105] In some embodiments, if a Trichoderma (e.g., T. reesei) is used as a host cell, the signal sequence or carrier of T. reesei mannanase I (Man5A, or MANI), T. 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 T. reesei cellobiohydrolase I (Cel7a or CBHI) can be used in the engineered antibody.

[0106] In other embodiments, if Aspergillus (e.g., A. niger) is used as the host cell, the A. niger glucoamylase (GlaA) or alpha amylase signal sequence or carrier can be used in the fusion polypeptide. The A. niger and A. awamori glucoamylases have identical amino acid sequences. Two forms of the enzyme are generally recognized in the culture supernatant. GAI is the full-length form (amino acid residues 1-616) and GAII is a naturally occurring proteolytic fragment containing amino acid residues 1-512. GAI is known to fold as two separate domains linked by an extended linker region. These two domains are a 471 residue catalytic domain (amino acids 1-471) and a 108 residue starch binding domain (amino acids 509-616), with a linker region between the two domains of 36 residues (amino acids 472-508). GAII lacks a starch binding domain. See Libby et al., (1994) Protein Engineering 7:1109-1114. In some embodiments, glucoamylases used as carrier proteins and including signal sequences will have greater than 95%, 96%, 97%, 98%, and 99% sequence identity with the catalytic domain of Aspergillus or Trichoderma glucoamylases. The term "catalytic domain" refers to a structural portion or region of a protein's amino acid sequence that has the catalytic activity of the protein.

[0107] 2. Carrier In certain embodiments, the signal sequence can include a "carrier" that contains a signal sequence at its N-terminus, where the carrier is at least the N-terminal portion of a protein that is endogenous to the cell and efficiently secreted by the cell. In certain embodiments, the signal sequence and the carrier protein are obtained from the same gene. In some embodiments, the signal sequence and the carrier protein are obtained from different genes.

[0108] A carrier protein can comprise all or a portion of the mature sequence of a secreted polypeptide. In some embodiments, a full-length secreted polypeptide is used. However, a functional portion of a secreted polypeptide can also be used. As used herein, a "portion" of a secreted polypeptide or grammatical equivalent means a truncated secreted polypeptide that retains its ability to fold into the normal configuration even when truncated.

[0109] In some cases, truncation of a secreted polypeptide means that the functional protein retains a biological function. In some embodiments, the catalytic domain of the secreted polypeptide is used, although 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 the carrier protein, the functional portion retains the catalytic domain of the enzyme, including amino acids 1-471 (see WO 03089614, the disclosure of which is incorporated herein by reference, e.g., Example 10). In another embodiment, when CBH I (i.e., CBH I from Trichoderma reesei) is used as the carrier protein, the functional portion retains the catalytic domain of the enzyme. Reference is made to SEQ ID NO:1 of Figure 2 of WO 05093073, the disclosure of which is incorporated herein by reference, which discloses sequences encoding the Trichoderma reesei CBH1 signal sequence, the T. reesei CBH1 catalytic domain (also referred to as the catalytic core or core domain) and the T. reesei CBH1 linker. In some embodiments, the CBH1 carrier protein comprising a signal sequence will have greater than 95%, 96%, 97%, 98% and 99% sequence identity to SEQ ID NO:1 of Figure 2 of WO 05093073, the disclosure of which is incorporated herein by reference.

[0110] Generally, if the carrier protein is a truncated protein, it is truncated at the C-terminus (i.e., contains an intact N-terminus). Alternatively, the carrier protein may be truncated at the N-terminus, or in some cases, truncated at both ends to leave a functional portion. Generally, such portions of the secreted protein that comprise the carrier protein include more than 50%, more than 70%, more than 80%, and more than 90% of the secreted protein, in some embodiments, the N-terminal portion of the secreted protein. In some embodiments, the carrier protein will 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 for the carrier protein.

[0111] In some embodiments, a first amino acid sequence comprising a signal sequence functional as a secretory sequence is encoded by a first DNA molecule. A 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.

[0112] 3. Antibody Conjugates and Derivatives Any of the altered antibodies disclosed herein can include derivatives that have been modified (i.e., by the covalent attachment of any type of molecule to the antibody). For example, and not by way of limitation, antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, and the like. Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.

[0113] Antibodies or fragments thereof with increased in vivo half-life can be generated by conjugating polymer molecules such as high molecular weight polyethylene glycol (PEG) to the antibody or antibody fragment. PEG can be attached to the antibody or antibody fragment by site-specific conjugation of PEG to the N- or C-terminus of the antibody or antibody fragment, with or without a multifunctional linker, or by using epsilon-amino groups present on lysine residues. Linear or branched polymer derivatization that results in minimal loss of biological activity will be used. The degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate, for example, by size exclusion or ion exchange chromatography.

[0114] Furthermore, the antibody or antibody fragment can be conjugated to albumin to make it more stable in vivo or have a longer half-life in vivo.The technique is well known in the art, see, for example, WO 93 / 15199, WO 93 / 15200, and WO 01 / 77137; and 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, mimetic drugs, synthetic drugs, inorganic molecules, and organic molecules.

[0115] The invention encompasses the use of antibodies or fragments thereof recombinantly fused or chemically conjugated (including covalently and non-covalently) to heterologous proteins or polypeptides (or fragments thereof, e.g., 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 generate fusion proteins. The fusion need not necessarily be direct, but may occur through a linker sequence. For example, antibodies may be used to target heterologous polypeptides to particular cell types, either in vitro or in vivo, by fusing or conjugating the antibody to an antibody specific for a particular cell surface receptor. Antibodies fused or conjugated to heterologous polypeptides may also be used in in vitro immunoassays and purification methods, using methods known in the art. See, e.g., WO 93 / 21232; 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.

[0116] 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 a Fab fragment, Fd fragment, Fv fragment, F(ab) fragment, or a combination thereof. 2The polypeptides may be fused or conjugated to antibody portions, VH domains, VL domains, VHCDRs, VLCDRs, or fragments 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. 307,434 and 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 88:10535-10539. Please see 89:11337-11341.

[0117] For example, additional fusion proteins of antibodies that specifically bind to an antigen (e.g., as described above) can be generated by the 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 modified antibodies or fragments thereof disclosed herein (e.g., antibodies or fragments thereof with higher affinities and lower off-rates). See generally U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, as well as 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. Antibodies or fragments thereof, or the encoded antibodies or fragments thereof, may be altered by being subjected to 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, which portion specifically binds to an antigen, can be recombinant with one or more components, motifs, sections, parts, domains, fragments, etc., of one or more heterologous molecules.

[0118] Additionally, the antibody or fragment thereof can be fused to a marker sequence, e.g., a peptide, to facilitate purification. In certain embodiments, the marker amino acid sequence is a hexa-histidine peptide, e.g., a tag provided in, among others, pQE vectors (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), many of which are commercially available. For example, hexa-histidine provides for convenient purification of the fusion protein, as described in Gentz ​​et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767), and the "flag" tag.

[0119] In other embodiments, the modified antibodies disclosed herein, or analogs or derivatives thereof, are conjugated to a diagnostic or detectable agent. Such antibodies may be useful for monitoring or prognosing the onset or progression of cancer, as part of a clinical testing procedure, such as determining the efficacy of a particular treatment. Such diagnosis and detection can be accomplished using a variety of enzymes, including, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, including, but not limited to, streptavidin / biotin and avidin / biotin; fluorescent materials, including, but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, including, but not limited to, luminol; bioluminescent materials, including, but not limited to, luciferase, luciferin, and aequorin; 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), Gallium ( 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 Ru, 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 113 Sn, and 117 This can be achieved by coupling the antibody to a detectable substance, including radioactive materials such as (but not limited to) tin; various positron emitting metals using positron emission tomography, noradioactive paramagnetic metal ions, as well as molecules that are radiolabeled or conjugated to specific radioisotopes.

[0120] The use of modified antibodies or fragments thereof disclosed herein conjugated to a therapeutic agent is also contemplated. The antibody or fragment thereof may be conjugated to a therapeutic moiety, such as a cytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agent, or a radioactive metal ion (e.g., an alpha-emitter). A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include ribonuclease, monomethyl auristatin 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, as well as analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil), chlorambucil), melphalan, carmustine (BCNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotics (e.g., vincristine and vinblastine). A more extensive list of therapeutic moieties can be found in PCT Publication WO 03 / 075957, which is incorporated herein by reference.

[0121] Additionally, the antibody or fragment thereof may be conjugated to a therapeutic agent or drug moiety that modifies a given biological response. The therapeutic agent or drug moiety should not be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide having a desired biological activity. Such proteins include, for example, toxins such as abrin, ricin A, onconase (or another cytotoxic RNase), Pseudomonas exotoxin, cholera toxin, or diphtheria toxin; tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, apoptotic agents such as TNF-alpha, TNF-beta, AIM I (see WO 97 / 33899), AIM II (see WO 97 / 34911), Fas Ligand (Takahashi et al., J. Immunol. 1999, 144:1311-1323), and the like. al., 1994, J. Immunol., 6:1567), and VEGI (see WO 99 / 23105), thrombotic or anti-angiogenic agents, such as proteins such as angiostatin or endostatin; or biological response modifiers, such as 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")).

[0122] Additionally, the antibodies can be conjugated to therapeutic moieties, such as radioactive materials or macrocyclic chelators useful for conjugating radioactive metal ions (see, e.g., radioactive materials, above). 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 generally known in the art and have been 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.

[0123] Techniques for conjugating therapeutic moieties to antibodies and related molecules are well known. The moieties can be conjugated to the antibodies by any method known in the art, including, but not limited to, aldehyde / Schiff linkage, sulphydryl linkage, acid labile linkage, cis-acotinyl linkage, hydrazone linkage, enzymatically degradable linkage (see generally, Garnett, 2002, Adv Drug Deliv Rev 53:171).Techniques for conjugating therapeutic moieties to antibodies are well known and are described, for example, in 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 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 Therapeutic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); See Therapeutic Use Of Radiolabeled Antibodies 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.

[0124] Methods for fusing or conjugating antibodies and related molecules to polypeptide moieties are known in the art. See, for example, U.S. Patent 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 88:10535; Zheng et al., 1995, J Immunol 154:5590; and Vil et al., 1992, PNAS USA 89:11337. The fusion of the antibody to the moiety does not necessarily have to be direct, but can be via a linker sequence. Such linker molecules are generally 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.

[0125] The antibodies may also be attached to a solid support, which is particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

[0126] The therapeutic moiety or drug to be conjugated to the modified antibody (e.g., any of those disclosed herein) should be selected to achieve the desired prophylactic or therapeutic effect for a particular disorder in a subject. In determining which therapeutic moiety or drug to conjugate to the modified antibody, the clinician or other medical practitioner should consider the following: the nature of the disease, the severity of the disease, and the condition of the subject.

[0127] B. Polynucleotides Another aspect of the compositions and methods disclosed herein is a polynucleotide or nucleic acid sequence encoding a modified antibody, such as any of the modified antibodies disclosed herein.

[0128] Provided herein is a fusion DNA construct encoding the modified antibody disclosed above, comprising in operable linkage: a promoter; a first DNA molecule encoding a signal sequence; a second DNA molecule encoding a carrier protein; and a third DNA molecule encoding an antibody (e.g., heavy and / or light chain) or a functional fragment thereof. The components of the fusion DNA construct can be present in any order. Since the genetic code is known, the design and production of these nucleic acids is well within the skill of the art, given the description of the modified antibodies disclosed herein. In certain embodiments, the nucleic acid can be codon-optimized for expression of the modified antibody in a particular host cell. Since codon usage tables are available for many species of, for example, mammalian cells and filamentous fungi, the design and production of a codon-optimized nucleic acid encoding a subject modified antibody will be well within the skill of the art.

[0129] C. Promoter Examples of suitable promoters for directing transcription of a nucleic acid in a host cell (e.g., a filamentous fungal host cell) include promoters encoding 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 17:203-212; Gines et al., (1989) Gene 79:107-117), Aspergillus niger or Aspergillus awamori glucoamylase (glaA) (Nunberg et al., (1984) Mol. Cell 10:111-112). Biol.4:2306-2315;Boel E.et al.,(1984)EMBO J.3:1581-1585), Rhizomucor miehei lipase, Aspergillus 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 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 reesei beta-xylosidase, and the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase); as well as mutant, truncated, and hybrid promoters thereof. 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 may also be useful, such as the SV40 early promoter (Barclay et al (1983) Molecular and Cellular Biology 3:2117-2130). The promoter may be a constitutive or inducible promoter. Exemplary promoters include Trichoderma reesei cellobiohydrolase I or II, Trichoderma reesei endoglucanase I, II or III, and Trichoderma reesei xylanase II.

[0130] D. Vector A polynucleotide encoding any of the modified antibodies disclosed herein can be present in a vector, such as a phage, plasmid, viral, or retroviral vector, hi certain embodiments, the vector can be an expression vector for expressing a subject fusion polypeptide in a filamentous fungal cell.

[0131] Vectors for expression of 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, Second Edition, (1989) Cold Spring Harbor, NY).

[0132] Fusion DNA constructs can be constructed using well known techniques, for example as generally described in EP 0215594, the disclosure of which is incorporated herein by reference.

[0133] Natural or synthetic polynucleotide fragments encoding a polypeptide of interest (e.g., an immunoglobulin) can be incorporated into a heterologous nucleic acid construct or vector that allows for introduction into and replication in a host cell (e.g., a filamentous fungal host cell).

[0134] Once the DNA construct, more particularly the fusion DNA construct, has been generated, it can be incorporated into any number of vectors as known in the art. The DNA construct will in some embodiments contain a promoter sequence, while in other embodiments the vector will contain other regulatory sequences functional in the host to be transformed, such as ribosome binding sites, transcriptional start and stop sequences, terminator sequences, polyadenylation signals, enhancers, and / or activators. In some embodiments, the polynucleotide encoding the modified antibody is inserted into the appropriate restriction endonuclease site of the vector containing the promoter, signal sequence, and carrier protein by standard procedures. These procedures and associated subcloning procedures are considered to be within the knowledge of one of ordinary skill in the art.

[0135] A terminator sequence recognized by the expression host for terminating transcription can be operably linked to the 3' end of the fusion DNA construct encoding the modified antibody to be expressed. Those skilled 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 81:1470-1474), or the Aspergillus niger glucoamylase gene (Nunberg et al. (1984) Mol. Cell. Biol. 4:2306-2353), or the Trichoderma reesei cellobiohydrolase I gene.

[0136] A polyadenylation sequence is a DNA sequence that, when recognized by an expression host, adds polyadenosine residues to the transcribed mRNA. Examples include the polyadenylation sequences from the A. nidulans trpC gene (Yelton et al (1984) Proc. Natl. Acad. Sci. USA 81;1470-1474), the A. niger glucoamylase gene (Nunberg et al. (1984) Mol. Cell. Biol. 4:2306-2315), the A. oryzae or A. niger alpha-amylase genes, and the Rhizomucor miehei carboxyl protease gene.

[0137] In further embodiments, the fusion DNA construct, or a vector containing the fusion DNA construct, will contain a selectable marker gene that allows for the 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, those that confer antibiotic resistance (e.g., hygromycin, bleomycin, chloramphenicol, and phleomycin). Genes that confer metabolic advantages, such as nutritional selection markers, can also be used. Some of these markers include amdS. Sequences encoding genes that complement auxotrophic defects can also be used as selection markers (e.g., pyr4 complementation of pyr4-deficient A. nidulans, A. awamori, or Trichoderma reesei, and argB complementation 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.

[0138] E. host cell The expression cassette or vector can be introduced into a suitable expression host cell, which then expresses the corresponding polynucleotide encoding the altered antibody.

[0139] Suitable host cells include any microbial cell (e.g., bacteria, protists, algae, fungi (e.g., yeast or filamentous fungi), or other microbial cells), and suitable host cells may be bacterial, yeast, or filamentous fungal cells. Fungal expression hosts may be, for example, yeast, which may also serve as an ethanologen. Also suitable are mammalian expression hosts, such as mouse (e.g., NS0), Chinese hamster ovary (CHO), or baby hamster kidney (BHK) cell lines. Other eukaryotic hosts, such as insect cells or viral expression systems (e.g., bacteriophages, such as M13, T7 phage, or lambda, or viruses, such as baculovirus), are also suitable for producing polypeptides.

[0140] Suitable host cells of bacterial genera include, but are not limited to, cells of the genera Escherichia, Proteus, Bacillus, Ralstonia, Lactobacillus, Lactococcus, Pseudomonas, Staphylococcus, and Streptomyces. Suitable bacterial species cells include, but are not limited to, 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 the like. mirabilis, and Streptomyces lividans cells.

[0141] Suitable host cells of the yeast genus include, but are not limited to, cells of the genera Saccharomyces, Schizosaccharomyces, Candida, Hansenula, Pichia, Kluyveromyces, Yarrowia, and Phaffia. Suitable yeast species cells include, but are not limited to, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Hansenula polymorpha, Yarrowia lipolytica, Pichia pastoris, P. canadensis, Kluyveromyces marxianus, and Phaffia rhodozyma cells.

[0142] Suitable filamentous fungal host cells include all filamentous fungal subdivision Eumycotina. Suitable filamentous fungal genera include, but are not limited to, cells of the genera Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysoporium, Coprinus, Coriolus, and the like. ), Corynascus, Chaertomium, Cryptococcus, Filobasidium, Fusarium, Gibberella, Humicola, Magnaporthe, Mucor, Myceliophthora hthora, Mucor, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Scytaldium, Schizophyllum, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma.

[0143] Suitable cells of filamentous fungal species include, but are not limited to, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium lucknowense, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, and the like. crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Bjerkandera adusta, Ceriporiopsis aneirinaaneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis-subvermispora, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Neurospora intermedia, Penicillium purpurogenum, Penicillium canescens, Penicillium solitum, Penicillium funiculosum, white rot fungus, Phlebia radiate, Pleurotus eryngii, Talaromyces flavus, Thielavia terrestris terrestris), Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningikoningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride cells.

[0144] Promoters and / or signal sequences associated with secreted proteins in a particular host of interest are candidates for use in heterologous production and secretion of modified antibodies in that host or other hosts. As non-limiting examples, in filamentous fungal systems, promoters driving genes for cellobiohydrolase I (cbh1), glucoamylase A (glaA), TAKA-amylase (amyA), xylanase (exlA), gpdA-promoter cbh1, cbhll, endoglucanase genes eg1-eg5, Cel61B, Cel74A, gpd promoter, Pgk1, pki1, EF-1 alpha, tef1, cDNA1, and hex1 are suitable and can be obtained from many different microorganisms (e.g., A. niger, T. reesei, A. oryzae, A. awamori, A. nidulans).

[0145] In some embodiments, the polynucleotide encoding the modified antibody is recombinantly linked to a polynucleotide encoding a suitable homologous or heterologous signal sequence that provides for 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 Escherichia coli, other Gram-negative bacteria, and other microorganisms known in the art include those that drive expression of HlyA, DsbA, Pbp, PhoA, PelB, OmpA, OmpT, or the M13 phage Gill genes. For Bacillus subtilis, gram-positive microorganisms, and other microorganisms known in the art, suitable signal sequences further include those driving expression of AprE, NprB, Mpr, AmyA, AmyE, Blac, SacB, and for S. cerevisiae or other yeasts, the killer toxin, Bar1, Suc2, mating factor alpha, Inu1A, or Ggplp signal sequences. Signal sequences are cleaved by several signal peptidases and can therefore be removed from the remainder of the expressed protein.

[0146] In some embodiments, the modified antibodies are expressed alone or as fusions with additional peptides, tags, or proteins (e.g., 6XHis, HA, or FLAG tags) located at the N- or C-terminus. Suitable fusions include tags, peptides, or proteins that facilitate affinity purification or detection (e.g., 6XHis, HA, chitin-binding protein, thioredoxin, or FLAG tags), as well as those that facilitate expression, secretion, or processing of the target beta-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.

[0147] Polynucleotides encoding modified antibodies can be introduced into expression host cells by several transformation methods, including, but not limited to, electroporation, lipid-mediated transformation or gene transfer ("lipofection"), chemically-mediated gene transfer (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 virus, or phage transformation or transduction.

[0148] III. Method A. Methods for generating antibodies The altered antibodies disclosed herein (i.e., antibodies incorporating a modified hinge as described above) may be produced by any method known in the art for the synthesis of antibodies, in particular by chemical synthesis or recombinant expression techniques.

[0149] Polyclonal antibodies that recognize a particular antigen can be produced by a variety of procedures well known in the art. For example, various host animals, including but not limited to rabbits, mice, rats, etc., can be administered an antigen or an immunogenic fragment thereof to induce the production of serum containing polyclonal antibodies specific to the antigen. Depending on the host species, various adjuvants may be used to increase the immunological response, including but not limited to Freund's (complete and incomplete), mineral gels, such as aluminum hydroxide, surfactants, such as lysolecithin, Pluronic® polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants, such as BCG (bacilli Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.

[0150] Monoclonal antibodies can be produced using a variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technology, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques, including those known in the art, and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981). The term "monoclonal antibody" as used herein is not limited to antibodies produced via hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

[0151] Methods for producing and screening specific antibodies using hybridoma technology are routine and well known in the art. Briefly, mice can be immunized with an antigen or an immunogenic fragment thereof, and once an immune response is detected, e.g., antibodies specific to the administered antigen are detected in the mouse serum, the mouse spleen is removed and splenocytes are isolated. The splenocytes are then fused to any suitable myeloma cells, e.g., cells from cell line SP20 available from ATCC, by well-known techniques. Additionally, animals can be immunized using RIMMS (repeated immunization, multiple sites) techniques (Kilpatrick et al., 1997, Hybridoma 16:381-9). Hybridomas are selected and cloned by limiting dilution. Hybridoma clones are then assayed for cells secreting antibodies capable of binding to the polypeptide of the present invention by methods known in the art. Ascites fluid, which usually contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.

[0152] Thus, monoclonal antibodies can be produced by culturing antibody-secreting hybridoma cells, where hybridomas can be produced by fusing spleen cells isolated from mice immunized with an antigen or an immunogenic fragment thereof with myeloma cells, and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete antibodies capable of binding to the administered antigen.

[0153] The modified antibodies disclosed herein may further contain novel amino acid residues in their hinge region. Modified antibodies may be generated by a number of methods well known to those skilled in the art. Non-limiting examples include isolating an antibody coding region (e.g., from a hybridoma) and introducing one or more hinge modifications of the invention into the isolated antibody coding region. Alternatively, the variable region may be subcloned into a vector encoding a modified hinge region (e.g., any of those disclosed herein). Additional methods and details are provided below.

[0154] Antibody fragments that recognize specific antigens can be generated by any technique known to those of skill 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 (yielding Fab fragments) or pepsin (yielding F(ab')2 fragments). F(ab')2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain. Additionally, modified antibodies disclosed herein can also be generated using various phage display methods known in the art.

[0155] In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from an animal cDNA library (e.g., a human or mouse cDNA library of lymphoid tissues). The DNA encoding the VH and VL domains are recombined with an scFv linker by PCR and cloned into a phagemid vector (e.g., pCANTAB6 or pComb3HSS). The vector is electroporated into E. coli, which is infected with helper phage. The phages used in these methods are usually filamentous phages, including fd and M13, and the VH and VL domains are generally recombinantly fused to either the phage gene III or gene VIII. Phage expressing an antigen-binding domain that binds to an antigen epitope of interest can be selected or identified by antigen, for example, using labeled antigen, or antigen bound or captured to a solid surface or bead.Examples of phage display methods that can be used to make the modified antibodies disclosed herein include those described in, for example, 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 publications 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. Pat. 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.

[0156] After phage selection, as described in the above references, the antibody coding region from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, for example, as described below. Techniques for recombinantly producing Fab, Fab', and F(ab')2 fragments can also be used, using methods known in the art, such as those disclosed in WO 92 / 22324; Mullinax et al., 1992, BioTechniques 12(6):864-869; Sawai et al., 1995, AJRI 34:26-34; and Better et al., 1988, Science 240:1041-1043.

[0157] To generate a full antibody, the VH or VL sequence can be amplified in the scFv clone using PCR primers that contain the VH or VL nucleotide sequence, a restriction site, and flanking sequences to protect the restriction site. Using cloning techniques known to those of skill in the art, the PCR amplified VH domain can be cloned into a vector expressing a VH constant region, e.g., a human gamma constant region, and the PCR amplified VL domain can be cloned into a vector expressing a VL constant region, e.g., a human kappa or lambda constant region. It is contemplated that the constant region comprises a modified hinge (e.g., any of the modified hinges 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 and constant domains, and a selection marker such as neomycin. The VH and VL domains may also be cloned into one vector expressing the desired constant region. The heavy chain conversion vector and the light chain conversion vector are then co-introduced into a cell line to generate a stable or transient cell line expressing a full-length antibody, e.g., an IgG, using techniques known to those of skill in the art.

[0158] Chimeric antibodies are molecules in which different parts 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. Patent Nos. 5,807,715, 4,816,567, 4,816397, and 6,311,415.

[0159] For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use human or chimeric antibodies. Fully human antibodies are particularly desirable for therapeutic treatment of human subjects. Human antibodies can be produced by a variety of methods known in the art, including the above-mentioned phage display method using antibody libraries derived from human immunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741.

[0160] A humanized antibody is an antibody or variant thereof or fragment thereof that is capable of binding to a predetermined antigen and comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin. A humanized antibody comprises substantially all of at least one, and usually two, variable domains (Fab, Fab', F(ab')2, Fabc, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are of a human immunoglobulin consensus sequence. In specific embodiments, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Typically, the antibody will contain both a light chain and at least the variable domain of a heavy chain. The antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. The humanized antibody can be selected from any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4. Usually, the constant domain is a complement fixing constant domain if it is desired that the humanized antibody exhibit cytotoxic activity, which class is usually IgG.sub.1. If such cytotoxic activity is not desired, the constant domain can be of IgG.sub.2 class. A humanized antibody can contain sequences from more than one class or isotype, and it is within the ordinary skill in the art to select a particular constant domain to optimize the desired effector function. The framework and CDR regions of the humanized antibody, which may not correspond exactly to the parental sequence, for example the donor CDR or consensus framework, can be mutagenized by substitution, insertion, or deletion of at least one residue so that the CDR or framework residue at that site does not correspond to either the consensus or the import antibody. However, such mutations will not be extensive. Usually, at least 75%, and more often 90% or more 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 by techniques including, but not limited to, CDR grafting (EP 239,400; WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska et al., 1994, PNAS 91:969-973), chain shuffling, and the like. shuffling) (U.S. Patent No. 5,565,332), as well as, for example, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, WO 9317105 pamphlet, Tan et al., J. Immunol. 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 Supp):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 regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.These framework substitutions are identified by methods well known in the art, for example, by modeling the interactions of CDR and framework residues to identify important framework residues for antigen binding and sequence comparison and to identify unusual framework residues at particular positions (see, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature 332:323).

[0161] Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but that can express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable, constant, and diversity regions can also be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be rendered non-functional separately or simultaneously by the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then bred to give rise to homozygous progeny that express human antibodies. The transgenic mice are immunized with a selected antigen or an immunogenic fragment thereof in the usual manner. Monoclonal antibodies against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technique for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735; and U.S. Pat. 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.

[0162] Furthermore, the modified antibodies disclosed herein can then be utilized to generate anti-idiotypic antibodies that "mimic" the receptor, using techniques well known to those of skill in the art (see, e.g., Greenspan & Bona, 1989, FASEB J. 7(5):437-444; and Nissinoff, 1991, J. Immunol. 147(8):2429-2438). For example, an antibody of the invention that binds to its ligand and competitively inhibits receptor binding (as determined by assays well known in the art and discussed below) can be used to generate anti-idiotypes that "mimic" the ligand and thus bind and neutralize the receptor and / or its ligand. Such neutralizing anti-idiotypes, or Fab fragments of such anti-idiotypes, can be used in therapeutic regimens to neutralize the ligand and / or its receptor. Methods are provided herein that employ the use of polynucleotides that include nucleotide sequences encoding the modified antibodies or fragments thereof.

[0163] In one embodiment, a nucleotide sequence encoding an antibody that specifically binds to an antigen is obtained and used to generate the modified antibodies disclosed herein. The nucleotide sequence can be obtained from sequencing hybridoma clone DNA. If a clone containing a nucleic acid encoding a particular antibody or epitope-binding fragment thereof is not available, but the sequence of the antibody molecule or epitope-binding fragment thereof is known, the nucleic acid encoding the immunoglobulin can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library made from any tissue or cell that expresses the antibody, such as a hybridoma cell selected to express the antibody, or nucleic acid isolated from that tissue or cell, preferably polyA+ RNA) by PCR amplification using synthetic primers hybridizing to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for a particular gene sequence to identify, for example, a cDNA clone from a cDNA library that encodes the antibody. The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method well known in the art.

[0164] Once the nucleotide sequence of the antibody is determined, the antibody nucleotide sequence can be modified using methods well known in the art for the manipulation of nucleotide sequences, such as recombinant DNA techniques, site-directed mutagenesis, PCR, and the like (e.g., Current Protocols in Molecular Biology, FMAusubel et al., ed., John Wiley & Sons (Chichester, England, 1998); Molecular Cloning: A Laboratory Manual, 3rd Edition, J. Sambrook et al., ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY, 2001); Antibodies: A Laboratory Manual, E. Harlow and D. Lane, ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY, 1988); and Using Antibodies: A Laboratory Manual, E. Harlow and D. Lane, ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY, 2002)). Antibodies can be engineered to generate antibodies with different amino acid sequences, for example, by introducing deletions and / or insertions into desired regions of the antibody, using techniques described in methods such as those described in U.S. Pat. No. 5,992,341 (Harbor, NY, 1999).

[0165] In specific embodiments, one or more of the CDRs are inserted into the framework regions using routine recombinant DNA techniques. The framework regions may be naturally occurring or consensus framework regions, including but not limited to human framework regions (see, e.g., Chothia et al., 1998, J. Mol. Biol. 278:457-479 for a list of human framework regions). It is contemplated that the polynucleotide generated by the combination of the framework regions and the CDRs encodes an antibody that specifically binds to an antigen. In one embodiment, one or more amino acid substitutions can be made in the framework regions, as discussed above, and in certain embodiments, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods can be used to make amino acid substitutions or deletions of one or more variable region cysteine ​​residues involved in an intrachain disulfide bond to generate an antibody molecule lacking one or more intrachain disulfide bonds. Other modifications to the polynucleotide are encompassed by the present invention and are within the skill of the art.

[0166] The hinge of an antibody identified from such screening methods can be modified as described above to generate an antibody incorporating a modified hinge, such as any of those disclosed above. Furthermore, it is contemplated that the modified antibodies disclosed herein are useful in the prevention, management and treatment of 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.

[0167] B. Recombinant Expression Recombinant expression of any of the modified antibodies disclosed herein, as well as derivatives, analogs or fragments thereof (e.g., antibodies or fusion proteins of the invention), requires the construction of an expression vector containing a polynucleotide encoding the modified antibody. Once a polynucleotide encoding the modified antibody is obtained, a vector for the production of the modified antibody can be generated by recombinant DNA technology using techniques well known in the art. Thus, methods for producing a protein by expressing a polynucleotide containing a nucleotide sequence encoding the modified antibody are described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing the modified antibody coding sequence and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

[0168] The expression vector is introduced into a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce the modified antibody (e.g., any of those disclosed herein). In certain embodiments for expression of modified antibodies, including double-chain antibodies, vectors encoding both the heavy and light chains can be co-expressed in a host cell for expression of the entire immunoglobulin molecule.

[0169] C. Administration of Pharmaceutical Compositions Also provided herein are methods and pharmaceutical compositions comprising any of the modified antibodies disclosed herein (e.g., any antibody comprising a modified hinge region disclosed herein). Additionally provided herein are methods of treating, preventing, and ameliorating one or more symptoms associated with a disease, disorder, or infection by administering to a subject an effective amount of at least one modified antibody disclosed herein, or a pharmaceutical composition comprising at least one modified antibody disclosed herein. In one aspect, the modified antibody is substantially purified (i.e., substantially free of substances that limit its effect or produce undesirable side effects). In specific embodiments, the subject is an animal, such as a mammal, including non-primates (e.g., cows, pigs, chickens or other poultry, horses, cats, dogs, rats, etc.) and primates (e.g., monkeys, such as cynomolgus monkeys, and humans). In specific embodiments, the subject is a human. In yet another specific embodiment, the antibody of the invention is derived from the same species as the subject.

[0170] The route of administration of the composition depends on the condition to be treated. For example, intravenous injection may be preferred for the treatment of systemic disorders, such as metastatic lymphatic cancer or tumors. The dosage of the composition to be administered can be determined by those skilled in the art without undue experimentation, together with standard dose-response studies. Relevant circumstances to be considered when making these decisions include the condition(s) to be treated, the choice of the composition to be administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms. Depending on the condition, the composition can be administered to the patient orally, parenterally, intranasally, vaginally, rectally, lingually, sublingually, buccally, intrabuccally, and / or transdermally.

[0171] Thus, compositions designed for oral, lingual, sublingual, buccal and buccal administration can be prepared by means well known in the art without undue experimentation, for example, by using inert diluents or edible carriers.The composition can be enclosed in gelatin capsules or compressed into tablets.For therapeutic oral administration, the pharmaceutical composition of the present invention can be combined with excipients and used in the form of tablets, pellets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums, etc.In a further embodiment, the composition can be included in animal feed.

[0172] Tablets, pills, capsules, troches, etc. may also contain binders, recipients, disintegrants, lubricants, sweeteners, and / or flavoring agents. Some examples of binders include microcrystalline cellulose, tragacanth gum, and gelatin. Non-limiting examples of excipients include starch and lactose. Some examples of disintegrants include alginic acid, corn starch, etc. Examples of lubricants include magnesium stearate and potassium stearate. An example of a flow agent is colloidal silicon dioxide. Some non-limiting examples of sweeteners include sucrose, saccharin, etc. Examples of flavoring agents include peppermint, methyl salicylate, orange flavor, etc. The materials used in the preparation of these various compositions must be pharma- ceutical pure and non-toxic in the amounts used.

[0173] The pharmaceutical compositions disclosed herein can be administered parenterally, for example, by intravenous, intramuscular, intrathecal and / or subcutaneous injection. Parenteral administration can be accomplished by incorporating the compositions disclosed herein into a solution or suspension. Such solutions or suspensions can also contain sterile diluents, for example, water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol and / or other synthetic solvents. Parenteral formulations can also contain antibacterial agents, for example, benzyl alcohol and / or methyl parabens, antioxidants, for example, ascorbic acid and / or sodium bisulfite, and chelating agents, such as EDTA. Buffers, such as acetate, citrate and phosphate, and osmolality adjusters, such as sodium chloride and dextrose, can also be added. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes and / or multiple dose vials. Rectal administration includes administering the composition into the rectum and / or large intestine. This can be accomplished using suppositories and / or enemas. Suppository formulations can be made by methods known in the art. Transdermal administration includes percutaneous absorption of the composition through the skin. Transdermal formulations include patches, ointments, creams, gels, salves, and the like. The modified antibody-containing composition disclosed herein can be administered intranasally to a patient. As used herein, administering intranasally or intranasally includes administering the composition to the mucous membranes of the patient's nasal passages and / or nasal cavity.

[0174] The modified antibody-containing compositions disclosed herein can be used in accordance with the methods of the invention to prevent, treat, or ameliorate one or more symptoms associated with a disease, disorder, or infection. It is contemplated that the pharmaceutical compositions of the invention are sterile and in a form suitable for administration to a subject.

[0175] In one embodiment, the modified antibody-containing compositions disclosed herein are pyrogen-free formulations that are substantially free of endotoxins and / or related pyrogens. Endotoxins include toxins that are localized within microorganisms and released when the microorganism degrades or dies. Pyrogens also include pyrogenic, heat-stable substances (glycoproteins) derived from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension, and shock when administered to humans. Due to potential adverse effects, it is advantageous to remove even small amounts of endotoxins from pharmaceutical solutions administered intravenously. The Food and Drug Administration ("FDA") has set an upper limit of 5 endotoxin units (EU) per dose per kilogram of body weight for a single period of one hour for intravenous drug administration (The United States Pharmacopeial Convention, Pharmacopeial Forum 26(1):223 (2000)). When therapeutic proteins are administered in amounts of hundreds or thousands of milligrams per kilogram of body weight, as is the case with monoclonal antibodies, it is advantageous to remove even trace amounts of endotoxins. In specific embodiments, the endotoxin and pyrogen levels in the composition are less than 10 EU / mg, or less than 5 EU / mg, or less than 1 EU / mg, or less than 0.1 EU / mg, or less than 0.01 EU / mg, or less than 0.001 EU / mg.

[0176] Further provided herein are methods for preventing, treating, or ameliorating one or more symptoms associated with a disease, disorder, or infection, comprising (a) administering to a subject in need thereof a prophylactically or therapeutically effective amount of a composition comprising one or more of the modified antibodies disclosed herein; and (b) administering one or more subsequent doses of the modified antibody to maintain a plasma concentration of the modified antibody at a desired level (e.g., about 0.1 to about 100 μg / ml) that continuously binds to an antigen. In specific embodiments, the plasma concentration of the modified antibody is maintained at 10 μg / ml, 15 μg / ml, 20 μg / ml, 25 μg / ml, 30 μg / ml, 35 μg / ml, 40 μg / ml, 45 μg / ml, or 50 μg / ml. In specific embodiments, the effective amount of the modified antibody to be administered is at least 1 mg / kg to 8 mg / kg per dose. In another specific embodiment, the effective amount of the modified antibody to be administered is at least 4 mg / kg to 8 mg / kg per dose. In yet another specific embodiment, the effective amount of the modified antibody to be administered is 50 mg to 250 mg per dose. In yet another specific embodiment, the effective amount of the modified antibody to be administered is 100 mg to 200 mg per dose.

[0177] Also provided herein are protocols for preventing, treating, or ameliorating one or more symptoms associated with a disease, disorder, or infection, in which any of the modified antibodies disclosed herein are used in combination with a therapy (e.g., a prophylactic or therapeutic agent). The modified antibodies disclosed herein can augment and synergize, enhance the efficacy, improve the resistance, and / or reduce the side effects caused by other cancer therapies, including currently standard and experimental chemotherapies. The combination therapies of the present invention have additive efficacy, additive therapeutic effect, or synergistic effect. The combination therapies of the present invention allow for lower dosages of the therapies (e.g., prophylactic or therapeutic agents) utilized with the modified antibodies disclosed herein to prevent, treat, or ameliorate one or more symptoms associated with a disease, disorder, or infection, and / or improve the quality of life of a subject having a disease, disorder, or infection, and / or allow for less frequent administration of such prophylactic or therapeutic agents to said subject to achieve a prophylactic or therapeutic effect. Furthermore, the combination therapies of the present invention reduce or avoid unnecessary or adverse side effects associated with the implementation of current monotherapy and / or existing combination therapies, which in turn improves patient compliance with the treatment protocol. Numerous molecules that can be utilized in combination with the modified antibodies disclosed herein are known in the art, see, e.g., PCT Publication Nos. WO 02 / 070007; WO 03 / 075957, and U.S. Patent Application Publication No. 2005 / 064514.

[0178] IV. Kit Further provided herein are kits comprising one or more of the modified antibodies disclosed herein that specifically bind to an antigen conjugated or fused to a detectable agent, therapeutic agent or drug, with altered (e.g., improved) stability, and / or reduced likelihood of proteolytic cleavage, in one or more containers, for use in monitoring, diagnosing, preventing, treating, or ameliorating one or more symptoms associated with a disease, disorder, or infection.

[0179] The present invention can be further understood by reference to the following examples, which are provided by way of illustration and not by way of limitation. EXAMPLES

[0180] Example 1: Generation and evaluation of a site evaluation library A. Plasmid and site validation constructs for anti-RSV HC heavy chain The sequence for the heavy chain of a monoclonal antibody against respiratory syncytial virus (palivizumab or Synagis) was codon-optimized and synthesized by GeneArt GmH (Germany). Lysine at position 216 in the heavy chain was mutated to threonine (K216T) to prevent possible degradation by Kex2 furin-like protease during expression in fungal cells. First, the synthetic sequences of c2G4 and anti-RSV HC together with their linker region (1-479 aa) were cloned behind the catalytic core of Trichoderma reesei native cellobiohydrolase I (CBH1), respectively. A Kex2 cleavage site was introduced between the linker and the HC to release the mature antibody chain from the carrier partner.

[0181] The fusion construct of cbhI-HC_Synagis was then amplified by PCR using gene-specific primers extended at attB1 and attB2 sites to allow Gateway® BP recombination cloning into the pDonor221 vector (Invitrogen, USA). As shown in Figure 1, the plasmid pEntry-SynagisHC_Geneart_SEL was used by the supplier BaseClear (Netherlands) as a template for the construction of a site evaluation (SEL) library at aa positions 466-725 (counting from CbhI Met). The average number of mutation variants per aa position was approximately 17. The mutated sequence was further cloned into the pTTTpyr2-ISceI destination vector via Gateway® LR recombination technology, resulting in the final expression plasmid pTTTpyr2-ISceI-SynagisHC_Geneart_SEL (Figure 1).

[0182] This expression vector contains the T. reesei cbhI promoter and terminator regions, allowing strong inducible expression of the gene of interest, and the T. reesei pyr2 selection marker, which confers growth of transformants on minimal medium without added uridine. The plasmid is autonomously maintained in fungal cells by the telomeric regions derived from T. reesei. Plasmids were propagated in commercially available Escherichia coli TOP10 cells (Invitrogen, US), purified, sequenced, arrayed in 96-well MTPs, and used for fungal transformation as described below.

[0183] The pEntry-Synagis_LC_Geneart plasmid was constructed via Gateway® BP recombinational cloning and further recombined with the pTrex6g destination vector in a similar manner as described above to yield the expression vector pTrex6g-Synagis_LC, which served as the template to generate the PCR fragment expressing the light chain (Figure 4).

[0184] Anti-RSV antibodies were generated as described in International Patent Application No. PCT / US2020 / 021685, the disclosure of which is incorporated herein by reference in its entirety. Anti-herpes simplex virus antibodies (HSV08), anti-HIV antibodies (VRC01), and anti-HER2 / neu antibodies (trastuzumab) were generated in a similar manner.

[0185] B. Fungal host strain construction and transformation The expression cassette consists of the CBH1 promoter, CBH1 core, antibody HC and LC linked by the CBH1 linker, and kex2 sequences for processing of CBH1, the CBH1 terminator, and the alS marker that confers resistance to chlorimuron ethyl to fungal cells. The alS marker was used to create screening strains so that the pyr2 marker was available for the SEL variants. The complete expression cassette was amplified by PCR. The PCR product was purified and concentrated to 500-1000 ng / μL. The expression cassette was randomly integrated in multiple copies into the host T. reesei genome as described below.

[0186] The host T. reesei strain used for transformation was deleted for major cellulases and xylanases. The strain was transformed using standard PEG-protoplast transformation methods. Approximately 10 μg of DNA and 5×10 6The transformation mixture containing the protoplasts was treated with 2 mL of 25% PEG solution, diluted with 2 volumes of 1.2 M sorbitol / 10 mM Tris, pH 7.5 / 10 mM CaCl2 solution, mixed with 26 mL of 2% low melting point agarose containing 1 M sorbitol, 1 g / L uridine, 75 mg / L chlorimuron ethyl (in minimal medium), and distributed into four 10 cm Petri dishes containing pre-poured 1.5% agarose, 1 M sorbitol (in minimal medium). After sufficient growth, each dish was observed for transformants, and individual colonies were picked, four per dish, onto new 10 cm Petri dishes containing 1.5% agar, 1 g / L uridine, 75 mg / L chlorimuron ethyl to provide room to assess stability. The phenotype of stable colonies is concentric growth with smooth edges. Once stable transformants were observed and fully sporulated, spores were harvested and used to inoculate liquid cultures to assess the expression level of LC by Western blot analysis using a light chain specific antibody peroxidase conjugate from Sigma-Aldrich (USA). One transformant with high expression level of LC, #LC6, served as a screening host for further expression of the heavy chain SEL library.

[0187] All high-throughput transformations with Synagis HC variants were performed robotically in a 24-well MTP format using a Biomek robot (Beckman Coulter, USA). Plasmids carrying the variants were received from the supplier in a 96-well format arranged according to a pre-determined layout. Approximately 1 μg of DNA and 5x10 6 The transformation mixture containing the protoplasts of screening host #LC6 was treated with 200 μl of 25% PEG solution and diluted with 1 volume of 1.2 M sorbitol / 10 mM Tris, pH 7.5 / 10 mM CaCl 2The transformants were diluted in solution, robotically rearranged into 24-well MTPs, and poured into 1 ml of 3% low melting point agarose containing 1 M sorbitol (in minimal medium). After sufficient growth, transformants from each well were pooled together and plated onto a new 24-well agar dish with minimal medium. Once sporulated, spores were harvested and used to inoculate liquid cultures.

[0188] Fungal host strains for anti-RSV antibodies were constructed and transformed as described in International Patent Application No. PCT / US2020 / 021685, the disclosure of which is incorporated herein by reference in its entirety. Host strains for anti-herpes simplex virus antibodies (HSV08), anti-HIV antibodies (VRC01), and anti-HER2 / neu antibodies (trastuzumab) were constructed and transformed in a similar manner.

[0189] C. Fungal fermentation in slow-release 24-well MTPs To generate sufficiently high antibody titers, 5 ~10 6 T. reesei spores were inoculated into customer made 24-well MTPs composed of Sylgard 170 elastomer (from Dow Corning, USA) premixed with lactose, which was slowly released into the medium during fermentation to ensure continuous production. Cultures were grown in 1.25 ml of medium containing 16 g / L glucose, 9 g / L casamino acids, 10 g / L (NH4)2SO4, 4.5 g / L KH2PO4, 1 g / L MgSO4*7H2O, 1 g / L CaCl2*2H2O, 33 g / L PIPPS buffer [pH 5.5], 0.25% T. reesei trace elements (100%: 175 g / L citric acid (anhydrous), 200 g / L FeSO4*7H2O, 16 g / L ZnSO4*7H2O, 3.2 g / L CuSO4*5H2O, 1.4 g / L MnSO4*H2O, 0.8 g / L H3BO3).

[0190] Plates were incubated in an Infors shaker at 200 rpm and 28 C, 80% humidity, with a shaking amplitude of 50 mm. After 5-6 days of growth, cultures were reformatted into 96-well deep-well MTPs and filtered using 96-well microtiter filter plates (0.2 μm hydrophilic PVDF membrane, Corning, Tewksbury MA). Plates were frozen in Axygen half deep-well plates (P-DW-11-C).

[0191] D.miniSEL Screen Plasmid and library construction: The sequence of the humanized version of the Zmap c2G4 monoclonal antibody against Ebola virus was codon-optimized and synthesized by GeneArt GmH (Germany). All KR sites were removed from both the heavy chain (HC) and light chain (LC) to prevent possible degradation by Kex2 furin-like protease during expression in fungal cells. First, the synthetic sequences of c2G4 HC and LC, together with their linker regions (1-479aa), were cloned behind the catalytically inactive core of Trichoderma reesei native cellobiohydrolase I, respectively. To release the mature antibody chain from the carrier partner, a Kex2 cleavage site SVAVEKR was introduced between the linker and either the HC or LC.

[0192] Fusion constructs of cbhI-c2G4_HC3 and cbhI-c2G4_LC2 were then amplified by PCR using gene-specific primers extended at attB1 and attB2 sites to allow Gateway® BP recombination cloning into pDonor221 vector (Invitrogen, USA). As shown in Figure 3, plasmid pEntry-CbhIx-c2G4_HC3 was used by the supplier BaseClear (Netherlands) as template for the construction of a site evaluation (SEL) library at 217-236 aa positions (counts in mature HC). The number of mutation variants per each aa position varied between 13 and 19, with the average number being above 16. The mutation variants were further cloned into pTTTpyr2 destination vector via Gateway® LR recombination technology, resulting in the final expression plasmid pTTTpyr2-CBHI-c2G4_HC3 (Figure 3).

[0193] This expression vector contains the T. reesei cbhI promoter and terminator regions, allowing strong inducible expression of the gene of interest, the Aspergillus nidulans amdS and the T. reesei pyr2 selection markers, which allow growth of transformants on minimal medium containing acetamide in the absence of uridine. The plasmid is autonomously maintained in fungal cells by the telomeric regions derived from T. reesei. The use of replicating plasmids results in increased transformation frequency and avoids the problem of locus-dependent expression observed with integrative fungal transformation. Plasmids were propagated in commercially available Escherichia coli TOP10 cells (Invitrogen, US), purified, sequenced, arrayed in 96-well MTPs, and used for fungal transformation as described below.

[0194] The pEntry-CbhIx-c2G4_LC2 plasmid was recombined with the pTrex6g destination vector in a similar manner as described above to yield the expression vector pTrex6g-CbhIx-c2G4_LC2, which served as a template to generate a PCR fragment expressing c2G4_LC2, driven by the cbhI promoter and linked to the alS marker, which confers resistance to chlorimuron ethyl in fungal cells (Figure 4).

[0195] Fungal strains and transformation: Prior to screening of the c2G4_HC3 heavy chain SEL library in T. reesei, an intermediate strain #C1 expressing sufficient levels of the c2G4_LC2 light chain was constructed. For this purpose, the light chain expression fragment was randomly integrated into a T. reesei strain deleted for major cellulases and xylanases using standard PEG-protoplast transformation methods. Transformants resistant to 50-80 mg / L chlorimuron ethyl due to the presence of the alS marker linked to c2G4_LC2 were screened for LC expression by a combination of ELISA and Western blot assays using a light chain specific antibody peroxidase conjugate from Sigma-Aldrich (USA). One transformant, designated #C1, with a strong signal of LC in Western blot, served as a host for further expression of the c2G4_HC3 SEL library.

[0196] All high-throughput transformations with c2G4_HC3 variants were performed robotically in a 24-well MTP format using a Biomek robot (Beckman Coulter, USA). Plasmids carrying the variants were received from the supplier in a 96-well format arranged according to a pre-determined layout. Approximately 1 μg of DNA and 5x10 6The transformation mixture containing 100 μl of the screening strain #C1 protoplasts was treated with 200 μl of 25% PEG solution, diluted with 1 volume of 1.2 M sorbitol / 10 mM Tris, pH 7.5 / 10 mM CaCl2 solution, rearranged robotically into 24-well MTPs, and poured into 1 ml of 3% low melting point agarose containing 1 M sorbitol (in minimal medium). After sufficient growth, transformants from each well were pooled together and plated onto a new 24-well agar dish with minimal medium containing 10 mM acetamide as the sole nitrogen source. Upon sporulation, spores were harvested and used to inoculate liquid cultures.

[0197] Fungal fermentation in slow-release 24-well MTPs: 10 5 ~10 6 T. reesei spores were inoculated into customer made 24-well MTPs composed of Sylgard 170 elastomer (from Dow Corning, USA) premixed with lactose, which was slowly released into the medium during fermentation to ensure continuous production. Cultures were grown in 1.25 ml of medium containing 16 g / L glucose, 9 g / L casamino acids, 10 g / L (NH4)2SO4, 4.5 g / L KH2PO4, 1 g / L MgSO4*7H2O, 1 g / L CaCl2*2H2O, 33 g / L PIPPS buffer [pH 5.5], 0.25% T. reesei trace elements (100%: 175 g / L citric acid (anhydrous), 200 g / L FeSO4*7H2O, 16 g / L ZnSO4*7H2O, 3.2 g / L CuSO4*5H2O, 1.4 g / L MnSO4*H2O, 0.8 g / L H3BO3).

[0198] Plates were incubated in an Infors shaker at 200 rpm and 28 C, 80% humidity, with a shaking amplitude of 50 mm. After 5-6 days of growth, cultures were reformatted into 96-well deep-well MTPs and filtered using 96-well microtiter filter plates (0.2 μm hydrophilic PVDF membrane, Corning, Tewksbury Mass.). Clarified samples were analyzed for antibody expression.

[0199] Example 2: Evaluation of antibody hinge variants for resistance to cleavage Clipping assay: This method measures the amount of antibody proteolysis after expression and purification from host cells. The concentration of purified antibody variants produced in Example 1 was determined by either a BCA assay for total protein (C2G4 sample) or a FRET assay (anti-RSV, HSV08, VRC01, and trastuzumab samples). The BCA assay was performed as described by the manufacturer (Thermo Fisher Scientific 23225). The C2G4 antibody was then normalized to 60 ppm and the FRET quantification antibodies were normalized to 120 ppm. Some of the antibodies were not normalized because their purified concentration was lower than the concentration to which the other antibodies were normalized. The dilution buffer used for normalization was a glycine-Tris buffer with the same pH and concentration as the solution of the purified antibody.

[0200] Empty Cellulighter host strain was run through the Dasgip under the same conditions where hinge clipping was observed. The supernatant was isolated and concentrated approximately 10-fold.

[0201] For the C2G4 antibody, 100 μL of 60 ppm protein standardized sample was added to the Corning 3605 plate. For the FRET quantification antibodies, the antibody samples were diluted 4-fold in Mili-Q water before adding to the Corning 3605 plate. Then, 10 μL of concentrated cellulighter broth was added to each well to initiate the hinge clipping reaction. The plate was sealed with BioRad Microseal B and placed in an Eppendorf thermomixer at 25°C. The plate was incubated for 100 minutes with shaking. The reaction was quenched by mixing 50 μL of reaction mixture and 50 μL of protease inhibitor cocktail in the 3605 plate. The protease inhibitor cocktail was added in Halt Buffer with EDTA added to a final concentration of 10X the recommended dilution. TM The stressed samples were then stored on ice until processed by Western analysis. Aliquots of antibody samples at the same dilution as in the reaction but without enrichment broth were mixed with protease inhibitor cocktail in the same ratio as the stressed samples to obtain T 0 A time point (unstressed) was generated.

[0202] Unstressed and stressed samples were analyzed for hinge clipping by Western blot. Samples were run on Invitrogen E-PAGE 48-well 8% gels, transferred to nitrocellulose membranes with Invitrogen iBlot 2, and probed with Invitrogen iBind Flex. For more accurate comparison, unstressed and stressed samples for a given variant were run next to each other in the same row of a 48-well gel. SuperSignal TMSamples were probed using anti-human Fc-HRP (Sigma A0170) with West (Thermo 34076). Blots were visualized and quantified with a BioRad ChemiDoc MP and software. Percent clipped was calculated by dividing the volume of the bottom band (Fc fragment from clipped HC) by the sum of the three fragments (CBH1-HC, mature HC, Fc fragment). The reported delta of hinge clipping is the difference in percent hinge clipping before and after incubation of samples with concentrated Cellulighter broth.

[0203] D. Purification: Plates were moved from the freezer to the refrigerator and samples were allowed to thaw slowly overnight at 4°C. Prior to purification, grown WT samples were removed from plates and pooled. 1 mL per well of pooled WT, pooled low binding control, pooled high binding control, and pooled vector only (vector expressing CBH1 in the same strain) samples were added to designated wells. These library plates were grown in duplicate and the controls were added to both plates. The plates were gently rocked for 2 minutes to homogenize the fluid in the wells, followed by centrifugation for 1 minute to pellet any precipitates.

[0204] The robot processed four library plates at a time. The robot added 50 μL of 1 M KPi (pH 7) to increase the pH of the supernatant to improve antibody binding to the Protein A resin. The robot then transferred the raw material (up to 880 μL / well) from the four plates to a 2 mL filter plate (Pall 8275) pre-filled with 220 μL of Protein A resin (in PBS). The filter plates were then rocked on a shaker for 5 minutes. The plates were then filtered by centrifugation at 1000 g for 2 minutes and the flow-through was collected in an empty collection plate from which the samples were transferred. This material was stored until after quantification. The filter plate was returned to the robot platform and duplicate growth plates were added to the same filter plate. The plates were incubated and centrifuged as before. The resin was then washed with 880 μL of PBS buffer. The plates were rocked for 1 minute and then centrifuged at 1000 g for 2 minutes. The flow-through was discarded and the plate was returned to the robot for a second PBS wash. After the second wash, the plate was transferred to the robot which ran the elution program.

[0205] The elution program processed four plates at a time. The program added 11 μL of neutralization buffer (1M Tris pH 9) to the clean half-deep well plate where the samples were to be eluted. The program then added 440 μL of elution buffer (100 mM glycine pH 2.7) to the filter plate. The plate was then rocked for 1 minute at setting 7 and then filtered by centrifugation (1000 g, 2 minutes) into a freshly prepared collection plate. After centrifugation, the sample plate was rocked for 1 minute to ensure proper mixing of the neutralization buffer.

[0206] Results: For the C2G4 antibody, several hinge sequence variants were identified that resulted in significantly less antibody clipping compared to the mature antibody without modifications in the hinge region, as shown in Figure 6. A complete list of these variants and the reduction in hinge clipping that was assayed is shown in Table 3. [Table 3]

[0207] When the substitutions found to reduce clipping in the C2G4 antibody shown in Table 3 were introduced into the hinge of an anti-RSV antibody as shown in Figures 7 and 8, these mutations also resulted in significantly less antibody clipping compared to mature antibodies that were not modified in the hinge region, demonstrating that these hinge substitutions reduce clipping across multiple antibodies. A complete list of these C2G4 variants and the reduction in hinge clipping assayed is shown in Table 4. [Table 4-1] [Table 4-2]

[0208] When select substitutions found to reduce clipping in the C2G4 antibody were introduced into the hinges of an anti-herpes simplex virus antibody (HSV08), an anti-HIV antibody (VRC01), and an anti-HER2 / neu antibody (trastuzumab), these mutations also resulted in significantly less antibody clipping compared to mature antibodies without modifications in the hinge region, further demonstrating that these hinge substitutions reduce clipping across multiple antibodies (Table 5). [Table 5]

[0209] Example 3: Evaluation of CHO-expressed antibody hinge variants for resistance to cleavage CHO-Produced Anti-RSV Variant: Chinese Hamster Ovary (CHO) cell expressed variant was obtained from Bionova Scientific (Fremont, CA). This variant was purified and delivered in PBS buffer. The concentration was measured by FRET assay using Synagis, and the variant was diluted to 120 ppm with the same Tris-Gly as before.

[0210] FRET quantification assay and normalization: Protein A (Thermo Fisher Scientific 77674) was labeled with Alexa Fluor 546 NHS ester (Thermo Fisher Scientific A20102). Protein L (Thermo Fisher Scientific 77680) was labeled with Alexa Fluor 488 NHS ester (Thermo Fisher Scientific A20100). Labeled Protein A and Protein L were diluted with 107 mM KPi pH 7 in a ratio that resulted in a FRET signal for our standard curve with an appropriate dynamic range. The standard curve was commercially available Synagis from AbbVie. 40 μL of Protein A and L solution was mixed with 10 μL of purified antibody sample in a Corning 3605 plate. The FRET signal on the plate was read (ex: 485 nm em: 590 nm cutoff: 590 nm) and the concentration of unknowns was determined from the Synagis standard curve. Samples were run in duplicate.

[0211] After analyzing the data, the plate was normalized to 120 ppm. The dilution buffer was Tris-Gly buffer with the same pH and concentration as in the purified sample. Wells below 120 ppm were used without dilution. The results are shown in Table 6. As shown, the CHO-expressed anti-RSV variants showed significantly less clipping compared to the wild-type antibody hinge. [Table 6] array [ka] Nucleotide sequence of Synagis HC: [ka] Synagis HC amino acid sequence: [ka] Nucleotide sequence of Synagis LC: [ka] Synagis LC amino acid sequence: [ka] Nucleotide sequence of c2G4_HC3: [ka] Amino acid sequence of c2G4_LC2: [ka] Nucleotide sequence of c2G4_LC2: [ka] Amino acid sequence of c2G4_HC3 LC: [ka]

Claims

1. 1. A monoclonal IgG1 antibody heavy chain polypeptide comprising a hinge region comprising one or more amino acid modifications that reduce proteolysis of the polypeptide, The polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1, and the amino acid modifications are K216T; K216V; R217S; S222C; S222D; S222E; T226N; T226P; H227P; A234R; (a) R217S-T226N; (b) R217S-S222C; (c) T216V-R217S-T226N; (d) S222C-H227P; (e) R217S-S222E-H227P; (f) T216V-R217S-S222C-T226P; (g) T226P-H227P; (h) R 217S-S222C-T226P; (i) T216V-S222D-T226P; (j) T216V-S222C-T226P-H2 27P; (k) T216V-S222E-T226N; (l) T216V-S222C-H227P; (m) R217S-S222C-T 226N; (n) S222C-T226P-H227P; (o) T216V-S222C-T226N-H227P; (p) R217S- S222D-T226P-H227P; (q) T216V-H227P; (r) S222E-T226P-H227P; (s) R217S -S222D-T226N-H227P; (t) R217S-S222E-T226P-H227P; (u) T216V-S222C; (v) R217S-S222D; (w) S222E-T226P; (x) R217S-S222C-T226P-H227P; (y) T2 16V-T226P-H227P; (Z) T216V-S222D-H227P; (aa) T226N-H227P; (bb) S222D -T226N-H227P; (cc) R217S-S222E-T226N; (dd) R217S-T226N-H227P; (ee) R 217S-S222E-T226N-H227P; (ff) T216V-R217S-H227P; (gg) T216V-S222E- T226P-H227P; (hh) T216V-S222C-T226N; (ii) R217S-T226P-H227P; (jj) T2 16V-T226P; (kk) S222E-T226N-H227P; (ll) S222E-H227P; (mm) R217S-S222 E-T226P; (nn) R217S-T226P; (oo) T216V-S222D; (pp) R217S-S222C-H227P;(qq) T216V-S222E-T226N-H227P; (rr) S222C-T226N-H227P; (ss) T216V-R217S-S222C-T226N-H227P; (tt) R217S-S222D-T226P; and (uu) S222D-T226N, wherein said amino acid positions are numbered according to the numbering in SEQ ID NO:

1.

2. The polypeptide of claim 1, further comprising an IgG1 antibody light chain polypeptide.

3. 3. The polypeptide of claim 1 or 2, wherein the polypeptide exhibits at least about 50% less proteolysis compared to a monoclonal IgG1 antibody heavy chain polypeptide not comprising the one or more amino acid modifications.

4. The polypeptide of any one of claims 1 to 3, wherein the polypeptide does not exhibit detectable proteolysis.

5. The polypeptide of any one of claims 1 to 4, further comprising a polypeptide encoding a signal sequence.

6. The polypeptide of any one of claims 1 to 5, further comprising a polypeptide encoding a carrier protein.

7. The polypeptide of claim 6 , wherein the polypeptide encoding the carrier protein is adjacent to a polypeptide encoding a signal sequence.

8. 8. The polypeptide of claim 6 or 7, wherein the carrier protein comprises CBH1 or a fragment thereof.

9. 9. The polypeptide of any one of claims 1 to 8, wherein the antibody is an anti-respiratory syncytial virus (RSV) antibody, an anti-Ebola virus antibody, an anti-aggregating beta amyloid (Aβ) antibody, an anti-human immunodeficiency virus (HIV) antibody, an anti-herpes simplex virus (HSV) antibody, an anti-sperm antibody (e.g., an anti-human contraceptive antigen (HCA) antibody), or an anti-HER2 / neu antibody.

10. 10. The polypeptide of any one of claims 1 to 9, wherein the polypeptide exhibits increased stability compared to a monoclonal IgG1 antibody heavy chain polypeptide not comprising the one or more amino acid modifications.

11. A nucleic acid encoding a fusion polypeptide according to any one of claims 1 to 10.

12. A vector encoding the nucleic acid of claim 11.

13. The vector of claim 12 , further comprising a nucleic acid sequence encoding a promoter.

14. A host cell comprising a polypeptide according to any one of claims 1 to 10, a nucleic acid according to claim 11, or a vector according to claim 12 or claim 13.

15. The host cell of claim 14 , wherein the host cell is selected from the group consisting of a mammalian host cell, a bacterial host cell, and a fungal host cell.

16. 16. The host cell of claim 15, wherein the mammalian host cell is a Chinese Hamster Ovary (CHO) cell.

17. The host cell of claim 15 , wherein the bacterial host cell is an E. coli cell.

18. The host cell of claim 15 , wherein the fungal host cell is a yeast cell or a filamentous fungal cell.

19. 20. The host cell of claim 18, wherein the yeast cell is a species of Saccharomyces.

20. 19. The host cell of claim 15 or claim 18, wherein the fungal host cell is selected from the group consisting of Trichoderma species, Penicillium species, Humicola species, Chrysosporium species, Gliocladium species, Aspergillus species, Fusarium species, Mucor species, Neurospora species, Hypocrea species, Myceliophthora species, and Emericella species.

21. The fungal host cell is selected from the group consisting of Trichoderma reesei, Trichoderma viride, Trichoderma koningii, Trichoderma harzianum, Humicola insolens, Humicola grisea, Chrysosporium lucknowense, Aspergillus oryzae, Aspergillus niger, and the like. niger, Aspergillus nidulans, Aspergillus kawachi, Aspergillus aculeatus, Aspergillus japonicus, Aspergillus sojae, Myceliophthora thermophila, and Aspergillus awamori.

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