HK1125028A - Methods and compositions for improving recombinant protein production - Google Patents

Description

Methods and compositions for improved recombinant protein production

Cross Reference to Related Applications

This application claims priority from U.S. patent application No. 60/616,474, filed 10/5/2004, which is incorporated herein by reference in its entirety.

Background

Expression vectors for recombinant proteins have been prepared since at least the mid-1980 s. In general, vector-based strategies for recombinant protein expression are used extensively in basic research and small scale experiments where absolute purity of protein preparations is not important. In contrast, when recombinant proteins are used in therapeutic applications, even minor amounts of contamination, such as the presence of mis-spliced or intron read-through byproducts, can reduce the activity and yield of the resulting therapeutic protein. Administration of therapeutic proteins having mis-spliced or read-through protein sequences to patients increases the likelihood of undesirable side effects.

Such by-products can also be troublesome to produce. The presence of by-products can be detrimental to the purification process, since such by-products are generally very similar in size, affinity, or biological activity to the desired protein. Furthermore, it has been observed that scaling up protein expression using recombinant host cells compared to the desired product often leads to an increase in the amount of by-products, especially if the cells are cultured under slightly less than optimal cell culture conditions. Such non-optimal cell culture conditions often occur in large scale protein production, for example, at the later stages of the operation of a bio-fermentor, or, for other reasons, when the health of the large scale culture deteriorates.

Thus, there is a need for improved methods for recombinant protein production, particularly for large-scale therapeutic protein production.

Summary of The Invention

The present invention provides methods and compositions for improving recombinant protein or peptide expression and/or production. In one embodiment, nucleic acid molecules are provided that have been modified to reduce or eliminate mis-splicing and/or intron read-through byproducts, and/or to enhance expression of recombinant proteins. In certain embodiments, the nucleic acid encodes a recombinant antibody (also referred to herein as an immunoglobulin), or a fragment thereof.

The invention also includes vectors (e.g., expression vectors) that have been modified to reduce or eliminate mis-splicing and/or intron read-through byproducts and/or to enhance recombinant protein expression; host cells, e.g., mammalian cells, including such nucleic acid molecules and vectors; and methods of culturing such cells to produce, e.g., produce the recombinant protein or peptide on a large scale. Also disclosed are compositions, e.g., pharmaceutical compositions, of recombinant proteins or peptides, e.g., antibodies, that are substantially free of mis-spliced and/or intron read-through products. These compositions are suitable for therapeutic use, including, for example, neurodegenerative and malignant diseases.

In particular, the invention provides compositions and methods for expressing therapeutic antibodies for the treatment of neurodegenerative diseases. The integrity of these antibodies is particularly important so that, for example, the therapeutic activity of the molecule per unit dose is maintained and its purification is improved. There is a great need to improve the purity of these proteins, which are intended to have a specialized functionality, for delivery and use in the treatment of certain biological indications, such as neurodegenerative diseases, where therapeutic polypeptides are able to cross the Blood Brain Barrier (BBB) and bind to target antigens in the brain. Exemplary antibodies according to the invention are prepared in high purity and bind to a neurodegenerative disease target, e.g., an antibody to amyloid protein of Alzheimer's disease, i.e., amyloid-beta peptide (A β).

Thus, in one aspect, the invention features nucleic acid molecules (e.g., modified or recombinant nucleic acid molecules) that include nucleotide sequences having one or more intron and exon sequences, wherein at least one intron sequence is modified to enhance protein expression and/or reduce or eliminate mis-spliced or intron read-through (IRT) by-products, as compared to naturally occurring genomic sequences. In one embodiment, the nucleic acid molecule is intended to enhance expression of a desired protein or peptide, e.g., an antibody or fragment thereof (e.g., an immunoglobulin heavy chain) and/or reduce or eliminate an intron read-through (IRT) by-product thereof, relative to a naturally occurring sequence (e.g., a genomic sequence). The protein or peptide may be of mammalian, e.g. human or murine, typically human, origin. Nucleic acid molecules described in this specification are also understood to be modified forms derived from naturally occurring sequences. In certain embodiments, the nucleic acid molecule is isolated or purified. In other embodiments, the nucleic acid molecule is a recombinant molecule.

In one embodiment, the nucleic acid molecule has at least one, two, three introns or up to deletion of all but one intron compared to the naturally occurring sequence (e.g., genomic sequence). For example, an intron capable of facilitating intron read-through (IRT) may be deleted from the naturally occurring sequence. In other embodiments, rearrangement may be by intron/exon configuration (e.g., intron/exon 5 'to 3' order); a deletion of one or more intronic portions; or replacing an intron or portion thereof with a heterologous intron sequence: thereby resulting in enhanced protein expression and/or reduced or eliminated mis-spliced or intron read-through (IRT) by-products.

In related embodiments, the nucleic acid molecule comprises a nucleotide sequence (e.g., a human genomic sequence) encoding an antibody heavy chain or fragment thereof. For example, the nucleotide sequence may comprise a heavy chain variable region encoding an immunoglobulin subtype, such as an immunoglobulin G subtype (e.g., IgG1, IgG2, IgG3, or IgG4 antibody subtypes), a hinge region, and first, second, and third constant regions (e.g., C)_H1，C_H2，C_H3) One or more nucleotide (e.g., exon) sequences of (a). Typically, the immunoglobulin subtype is from a mammalian source, such as murine or human. In one embodiment, human IgG1 or IgG4, or mutated forms thereof, are selected. For example, the constant region of an immunoglobulin may be mutated to result in one or more of the following: increased stability, decreased effector function, or decreased complement fixation. In one embodiment, human IgG4 is mutated to increase stability, e.g., a serine to proline substitution at residue 241 increases the stability of the hinge region. In other embodiments, the constant region is mutated so as to reduce glycosylation.

In one embodiment, the nucleic acid molecule is modified to lack at least one intron that facilitates intron read-through of the sequence. For example, C in the constant region of an immunoglobulin heavy chain_H2 and C_HThe intron between 3 may be deleted. Examples of other heavy chain immunoglobulin introns which may be deleted alone or in combination include heavy chain variable region and immunoglobulin heavy chain constant region C_H1 intron, C_H1 and the hinge region, and the hinge region and the immunoglobulin heavy chain constant region C_H2. Any combination of the foregoing introns may be deleted, including combinations of two, three, or up to all but one of the foregoing introns. In some embodiments, the heavy chain constant region is within three ofIntrons, e.g. C_H1 and the intron between the hinge region, the hinge region and C_H2, and C_H2 and C_H3 in the sequence. Exemplary combinations of the following heavy chain immunoglobulin intron deletions are also within the scope of the invention: immunoglobulin heavy chain constant region C_H1 and the hinge region, and C_H2 and C_H3 or a variant thereof; c_H1 and the hinge region, and the hinge region and C_HAn intron between 2; hinge region and C_H2, and C_H2 and C_H3, or a pharmaceutically acceptable salt thereof.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence represented by the formula:

V_H-Int1-C_H1-Int 2-hinge-Int 3-C_H2-C_H3，

Wherein V_HIs a nucleotide sequence encoding a heavy chain variable region;

C_H1，C_H2 and C_H3 is a nucleotide sequence encoding a naturally occurring or mutated form of the corresponding heavy chain constant region, e.g., the human IgG1 or IgG4 heavy chain gene;

the hinge is a nucleotide sequence encoding the hinge region of a heavy chain constant region, e.g., the naturally occurring or mutated form of the human IgG1 or IgG4 heavy chain gene; and is

Int1, Int2, Int3, and Int4 are introns from heavy chain genomic sequences. In one embodiment, at C_H2 and C_H3, the intron represented by Int4 in this specification is deleted. In other embodiments, at C_H1 and the hinge region, between the hinge region and C_H2, and/or at C_H2 and C_H3, one, two or generally three of the introns represented by Int2, Int3 and Int4 in the present specification. Schematic intron/exon alignment of other heavy chain genomic sequences can be seen in fig. 1, fig. 5 and fig. 7.

Typically, at least one intron is present in the nucleic acid molecule, e.g., in the heavy chain variable region and C_H1, denoted in this specification as Int 1. Other examples of heavy chain immunoglobulin introns that may be present alone or in combination include immunoglobulin heavy chain constant region C_H1 and the hinge region; hinge region and C_HAn intron between 2; and C_H2 and C_H3, or a pharmaceutically acceptable salt thereof. It is often desirable to include at least one intron in the modified nucleic acid molecule. Without being bound by theory, it is believed that introns can affect numerous events in protein production including transcription rate, polyadenylation, mRNA export, translation efficiency and mRNA senescence.

In one embodiment, the nucleic acid molecule comprises a nucleotide sequence represented by the formula:

V_H-Int1-C_H1-Int 2-hinge-Int 3-C_H2-C_H3，

Wherein V_HIs a nucleotide sequence encoding a heavy chain variable region;

C_H1，C_H2 and C_H3 is a nucleotide sequence encoding a naturally occurring or mutated form of the corresponding heavy chain constant region, e.g., the human IgG1 or IgG4 heavy chain gene;

the hinge is a nucleotide sequence encoding a hinge region of a heavy chain constant region, e.g., a naturally occurring or mutated form of a human IgG1 or IgG4 heavy chain gene; and is

Int1, Int2, and Int3 are introns from heavy chain genomic sequences. In one embodiment, the nucleotide sequence consists essentially of the components described above, e.g., an insertion sequence that does not alter structure or function.

In other embodiments, the nucleic acid molecule comprises a nucleotide sequence represented by the formula:

V_H-Int1-C_H1-hinge-C_H2-C_H3，

Wherein V_HIs a nucleotide sequence encoding a heavy chain variable region;

C_H1，C_H2 and C_H3 is a nucleotide sequence encoding a naturally occurring or mutated form of the corresponding heavy chain constant region, e.g., the human IgG1 or IgG4 heavy chain gene;

the hinge is a nucleotide sequence encoding the hinge region of a heavy chain constant region, e.g., the naturally occurring or mutated form of the human IgG1 or IgG4 heavy chain gene; and

int1 is an intron from the heavy chain genomic sequence. In one embodiment, the nucleotide sequence consists essentially of the components described above, e.g., an insertion sequence that does not alter its structure or function.

The genomic nucleotide sequence and the corresponding amino acid sequence of human IgG1 are shown in FIG. 8(SEQ ID NOS: 1 and 2, respectively). Code C_H1, hinge region, C_H2 and C_HThe exons 3 are located at about nucleotides 231, 524, 916, 960, 1079, 1408, and 1506, 1829 of FIG. 8(SEQ ID NO: 1), respectively. The Int1, Int2, Int3 and Int4 corresponding to the intron from the human IgG1 heavy chain genomic sequence are located at nucleotides about 1-230, about 525-915, about 961-1078 and about 1409-1505, respectively, of FIG. 8(SEQ ID NO: 1).

The genomic nucleotide sequence and the corresponding amino acid sequence of the mutated human IgG4 are shown in FIG. 9(SEQ ID NOS: 3 and 4, respectively). Code C_H1, hinge region, C_H2 and C_HThe exons 3 are located at about nucleotides 231, 524, 916, 952, 1071, 1400 and 1498, 1820 of FIG. 9(SEQ ID NO: 3), respectively. The Int1, Int2, Int3 and Int4 corresponding to the intron from the human IgG4 heavy chain genomic sequence are located at nucleotides about 1-230, about 525-916, about 953-1070 and about 1401-1497 of FIG. 9(SEQ ID NO: 3), respectively.

Examples of modified nucleic acid molecules of the invention include deletion of the intron between CH2 and CH3 of human IgG1, corresponding to about 140 of FIG. 8(SEQ ID NO: 1)Nucleotides 9-1505; or a mutated human IgG4 corresponding to the human genomic heavy chain constant region sequence at nucleotide 1401-1497 of FIG. 9(SEQ ID NO: 3). Other examples of heavy chain immunoglobulin introns that may be deleted alone or in combination include the intron between the heavy chain variable region of human IgG1 and CH1, corresponding to nucleotides about 1-230 of FIG. 8(SEQ ID NO: 1), or mutated IgG4, corresponding to nucleotides about 1-230 of FIG. 9(SEQ ID NO: 3); the intron between CH1 and the hinge region of human IgG1, corresponding to nucleotide position 525 and 915 of FIG. 8(SEQ ID NO: 1); or a mutated IgG4 corresponding to nucleotide position 525 and 916 of FIG. 9(SEQ ID NO: 3); and an intron between the hinge region and CH2 of human IgG1 corresponding to nucleotides 961-1078 of FIG. 8(SEQ ID NO: 1); or a mutated IgG4 corresponding to nucleotide position 1070 about 953-fold as shown in FIG. 9(SEQ ID NO: 3). Any combination of the foregoing introns may be deleted, including combinations of two, three, or up to all but one of the foregoing introns. In some embodiments, three introns of the heavy chain constant region, e.g., C_H1 and the intron between the hinge region, the hinge region and C_H2, and C_H2 and C_H3 in the sequence. In some embodiments, the nucleic acid molecule comprises one or more exon nucleotide sequences and one or more (but not all) intron nucleotide sequences of human IgG1 or IgG4 disclosed in the present invention, or sequences substantially identical thereto. In related embodiments, the nucleic acid sequence has one or more (but not all) intron nucleotide sequence deletions of human IgG1 or IgG4, or sequences substantially identical thereto, as disclosed in the present invention.

In one embodiment, the modified nucleic acid molecule comprises a nucleotide sequence encoding human IgG1 as shown in FIG. 10(SEQ ID NO: 5) or a sequence substantially identical thereto (e.g., a sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 5, or a sequence having 1, 5, 10, 50 or more nucleotide changes as compared to the nucleotide sequence of SEQ ID NO: 5).

In another embodiment, the modified nucleic acid molecule comprises a nucleotide sequence encoding human IgG4 as shown in FIG. 11(SEQ ID NO: 6) or a sequence substantially identical thereto (e.g., a sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 6, or a sequence having 1, 5, 10, 50 or more nucleotide changes as compared to the nucleotide sequence of SEQ ID NO: 6).

The modified nucleic acid molecule may comprise nucleotide sequences encoding light and heavy chain antibody or immunoglobulin sequences. These sequences may be present in the same nucleic acid molecule (e.g., the same expression vector) or may be expressed in separate nucleic acid molecules (e.g., separate expression vectors). Typically, the encoded antibody or immunoglobulin or fragment thereof can comprise at least one, and preferably two, full-length heavy chains, and at least one, and preferably two, light chains. Alternatively, the encoded immunoglobulin or fragment thereof can include only antigen binding fragments (e.g., Fab, F (ab')₂Fv or single chain Fv fragments). The antibody or fragment thereof may be monoclonal or a single specific antibody. The antibody or fragment thereof can also be a human antibody, a humanized antibody, a chimeric antibody, a CDR-grafted antibody, or an in vitro generated antibody. In yet another embodiment, the antibody has a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; more particularly, a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3 and IgG 4. In other embodiments, the antibody has a light chain selected from, for example, kappa or lambda.

In other embodiments, the nucleic acid molecule comprises a variable region, e.g., a humanized, chimeric, CDR-grafted or in vitro generated variable region. Typically, the variable region specifically binds to a predetermined antigen, e.g., an antigen associated with a disease, e.g., a neurodegenerative or malignant disease.

In one embodiment, the disease is a neurodegenerative disease and the antibody binds to an amyloid protein, such as an a β peptide (e.g., a human a β peptide). For example, the antibody may be a humanized antibody directed against an a β peptide comprising heavy and light chain regions from one or more Complementarity Determining Regions (CDRs) of a murine antibody, such as a murine anti-a β 3D6 antibody, or a murine anti-a β 12a11 antibody, or a murine anti-a β 10D5 antibody, or a murine anti-a β 12B4 antibody. The variable regions of humanized antibodies typically comprise human or substantially human framework regions. In one embodiment, the nucleic acid molecule comprises the heavy and light chain variable regions of a humanized anti-a β -peptide antibody.

In another aspect, the invention features a vector (e.g., an expression vector) that includes one or more of the foregoing modified nucleic acid molecules. The carrier may additionally comprise a compound capable of enhancing: a nucleotide sequence for one or more of replication, selection, transcription of mRNA, stability of mRNA, expression of a protein, or secretion of a protein in a host cell. For example, the vector may include a nucleotide sequence responsible for replication or enhancer expression, an enhancer promoter element, a nucleotide sequence encoding a leader sequence, a gene encoding a selectable marker (e.g., DHFR), an internal ribosome entry site sequence (IRES), and a polyadenylation sequence.

In another aspect, the invention provides a cell, e.g., a eukaryotic host cell, e.g., a mammalian cell (e.g., a Chinese Hamster Ovary (CHO) cell), comprising one of the aforementioned nucleic acid molecules and/or vectors, e.g., expression vectors.

In another aspect, the invention provides methods of enhancing the expression of a recombinant protein or peptide, e.g., an antibody, or expression of a recombinant protein or peptide, e.g., an antibody, having a reduced level (e.g., substantially no) of mis-splicing and/or intron read-through products as compared to a reference sequence, e.g., a naturally occurring genomic sequence. The method comprises introducing a nucleic acid molecule into a host cell, e.g., a mammalian cell (e.g., a CHO cell), as described herein; culturing the host cell under conditions that allow expression of the recombinant protein or peptide, thereby producing a culture of host cells; and, optionally, obtained, e.g., purified, from a culture of host cells (e.g., host cell supernatant).

The method further comprises the step of identifying (e.g., detecting and/or determining the level of) an IRT or IRT product in a nucleic acid sample, e.g., an mRNA sample from a host cell, contacting the nucleic acid sample with a nucleic acid probe that is complementary to an intron and adjacent exon sequences, or complementary to adjacent exon sequences, under conditions that allow hybridization of the nucleic acid sample and the probe; the resulting complex is detected, for example, by amplifying the probe sequence by PCR. In samples containing nucleic acid probes complementary to intron sequences, detection of a complex, e.g., a PCR amplification product, indicates the presence of IRT or an IRT product. The level of IRT product can be quantified as described, for example, in example 1.

In another aspect, the invention provides methods of making antibodies or fragments thereof having reduced (e.g., substantially no) intron read-through (IRT) heavy chain by-products as compared to a standard reference, e.g., a naturally occurring genomic sequence. The method comprises culturing a cell, e.g., a mammalian cell (e.g., a CHO cell), containing a nucleic acid molecule as described herein, and, optionally, encoding an antibody light chain nucleic acid, under conditions that allow expression of the heavy and light chains, and, optionally, operably linked. Optionally, the antibody or fragment thereof can be purified from the cell culture. Typically, the antibody, or fragment thereof, has reduced mis-spliced or intron read-through (IRT) heavy chain by-products.

The method further comprises the step of detecting and/or determining the level of IRT or IRT product in a sample, e.g., an mRNA sample from a host cell; contacting said nucleic acid sample with a nucleic acid probe complementary to intron and adjacent exon sequences, or complementary to adjacent exon sequences, under conditions permitting hybridization of said nucleic acid sample and probe; the resulting complex is detected, for example, by amplifying the probe sequence by PCR. In samples containing nucleic acid probes complementary to intron sequences, detection of a complex, e.g., a PCR amplification product, indicates the presence of IRT or an IRT product. The level of IRT product can be quantified as described, for example, in example 1.

In another aspect, the invention provides methods for reducing intron read-through (IRT) heavy chain by-products expressed from genomic heavy chain sequences by deleting an IRT-facilitating intron from the sequence.

In yet another aspect, the invention features methods of identifying (e.g., detecting and/or determining the level of) an IRT or IRT product in a sample, e.g., a nucleic acid sample. The method comprises the following steps: obtaining a nucleic acid sample, e.g., an mRNA sample, from a cell, e.g., a recombinant cell (e.g., a host cell as described herein); contacting said nucleic acid sample with a nucleic acid probe complementary to intron and adjacent exon sequences, or complementary to adjacent exon sequences, under conditions permitting hybridization of said nucleic acid sample and probe; the resulting complex is detected, for example, by amplifying the probe sequence by PCR. In samples containing nucleic acid probes complementary to intron sequences, detection of a complex, e.g., a PCR amplification product, indicates the presence of IRT or an IRT product. The level of IRT product can be quantified as described, for example, in example 1.

In yet another aspect, the invention features antibodies (e.g., recombinant antibodies) or fragments thereof prepared according to the disclosed methods having reduced (e.g., substantially free) of mis-splicing and/or intron read-through products as compared to a reference, e.g., naturally occurring genomic sequence. In one embodiment, the antibody or fragment thereof is chimeric, humanized, CDR-grafted or generated in vitro. Typically, the antibody or fragment thereof has a variable region that specifically binds to a predetermined antigen, e.g., an antigen associated with a disease, e.g., a neurodegenerative or malignant disease.

In yet another aspect, the invention provides compositions, e.g., pharmaceutical compositions, comprising a recombinant protein or peptide, e.g., an antibody, having reduced (e.g., substantially no) mis-splicing and/or intron read-through products, as compared to a reference, e.g., a naturally occurring genomic sequence; and a pharmaceutically acceptable carrier. These compositions are suitable for therapeutic use, including, for example, the treatment of neurodegenerative diseases.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Drawings

FIG. 1 shows the expected pre-mRNA transcribed from the expression vector containing the 3D6IgG gene (top) as well as the correctly spliced mRNA (middle) and intron read-through mRNA (bottom).

FIG. 2 shows the nucleic acid sequence spanning the intron between the constant regions of the 3D6 heavy chain expression vector CH2 and CH3 (referred to as the fourth intron) indicating the genomic 5 'and 3' splice junctions (SEQ ID NO: 7). Also shown are the partial amino acid sequences predicted from polypeptides of correctly (SEQ ID NO: 8) and incorrectly (SEQ ID NO: 9) spliced mRNAs. The RNA splice junction is shown by the filled double line.

FIG. 3 is a schematic of quantitative-polymerase chain reaction (Q-PCR) probes used to assess total levels of transcription of the 3D6 heavy chain gene (levels of CH2 containing mRNA transcripts) and intron 4 read-through transcription levels.

FIG. 4 is a bar graph illustrating the sustained accumulation of transcripts containing intron 4 as a function of culture time and induction of protein expression.

FIG. 5 shows the genomic arrangement of the intron and exon of 3D6 and the modified arrangement used in the development of a solution for intron read-through transcription expression vector.

FIG. 6 is a reverse phase high performance liquid chromatography (RP-HPLC) plot illustrating the lack of intron read-through heavy chain by-product in cell lines transformed with the modified expression vector.

FIG. 7 shows the arrangement of introns and exons in a heavy chain genomic construct, a construct lacking the last three intron sequences, and a cDNA construct without introns.

FIG. 8 shows the genomic nucleotides and pairs of human IgG1The corresponding amino acid sequences (SEQ ID NOS: 1 and 2, respectively). Code C_H1, hinge region, C_H2 and C_HThe exons of 3 are located at about nucleotides 231-, 524-, 916-, 960-, 1079-, 1408-, and 1506-, 1829- (SEQ ID NO: 1), respectively. The Int1, Int2, Int3 and Int4 corresponding to the intron from the human IgG1 heavy chain genomic sequence are located at nucleotides about 1-230, about 525-915, about 961-1078 and about 1409-1505, respectively (SEQ ID NO: 1).

FIG. 9 shows the genomic nucleotide sequence and the corresponding amino acid sequence of human IgG4 (SEQ ID NOS: 3 and 4, respectively). Code C_H1, hinge region, C_H2 and C_H3 are located at about nucleotides 231, 524, 916, 952, 1071, 1400 and 1498, 1820 of (SEQ ID NO: 3), respectively. The Int1, Int2, Int3 and Int4 corresponding to the intron from the human IgG4 heavy chain genomic sequence are located at nucleotides 1-230, 525-916, 953-1070 and 1401-1497, respectively (SEQ ID NO: 3).

FIG. 10 shows the genomic nucleotide sequence of human IgG 1(SEQ ID NO: 5) with deletion of the intron between constant regions CH2 and CH 3.

FIG. 11 shows the modified human IgG4 genomic nucleotide sequence (SEQ ID NO: 6) with the following intron deletions: an intron between CH1 and the hinge region, an intron between the hinge region and CH2, and an intron between CH2 and CH 3.

Figure 12-sets the heavy chain amino acid sequences for various other 3D6, 10D5, 12B4 and 266 antibodies.

Figure-sets the heavy chain amino acid sequences for various other 3D6, 10D5, 12B4 and 266 antibodies.

Detailed description of the invention

Expression vectors have been designed and constructed in a number of ways, and such procedures often require considerable trial and error before reasonable protein levels can be obtained. An important consideration in the design process is the use of intron sequences in the construction of the vector. In one approach, the entire gene sequence can be utilized as it naturally occurs-containing all intronic and exonic sequence elements. In such cases, it is expected that the post-transcriptional splicing machinery within the cell will cleave the intron sequence, thereby producing a mature mRNA containing only the exon sequences of the gene. Another approach is to use a sequence corresponding only to the cDNA of the gene. In this case, no splicing event is predicted to occur, and the pre-mRNA sequence is also substantially identical to the mRNA sequence in the encoded protein component. In a third case, vector construction involves selection and placement of introns that are not normally associated with the original gene sequence.

The effect of intron sequences on gene sequences expressed in vectors is not fully understood. Introns have been reported to affect a number of events during protein production, including transcription rate, polyadenylation, mRNA export, translation efficiency, and mRNA senescence (Nott et al (2003) RNA 9: 607-617). In the context of mRNA expression, there is currently no clear predictability in considering the results of intron pairs for protein production from vectors. For example, it has been previously reported that inclusion of various intron sequences can cause a large increase in expression, have no effect on mRNA expression, or can reduce mRNA expression (Berg et al (1988) MoI. cell. biol. 8: 4395-. Because most eukaryotic genes contain introns, the development of a system that expresses intron-containing genes at high levels and has a high degree of identity to the exon sequences in the absence of undesirable read-through by-products can significantly aid in the predictable development of protein expression systems.

Although the unpredictability associated with intron sequences prevents reliable expression vector design, significant design benefits can be achieved when the protein of interest has a model for acceptable genetic engineering techniques. Antibodies provide one such example, where intron sequences are included to assist in the design of the expression vector.

Certain terms are defined below for use in the specification and claims.

As used herein, the term "intron" includes a segment of DNA that has been transcribed, but has been removed from an RNA transcript by splicing together either end of the sequence (exon). Introns are considered interfering sequences within the protein coding region of a gene and generally do not contain the information represented by the protein produced by the gene.

The term "exon" includes any fragment present in the mature RNA product of a gene containing an interfering sequence. Exons contain information within the gene that is translated into protein.

The term "pre-mRNA" includes the initial RNA product obtained from a transcribed gene by RNA polymerase. RNA designed as pre-mRNA contains both intron and exon sequences and is therefore not processed by the splicing machinery of the cell.

The term "mRNA" includes RNA transcripts that have been treated to remove introns and that can be translated into polypeptides.

The term "splicing" includes cellular events that occur within the nucleus of eukaryotic cells in which introns are removed from pre-mRNA species. Generally, this process requires the formation of a splice body complex in which the 5 'splice donor site is brought into proximity with the 3' splice acceptor site, while interfering intron sequences are removed from the transcript.

The term "vector" includes nucleic acid constructs which typically contain a gene of interest and also contain the most essential elements necessary for the replication and/or transcription of the nucleic acid in a host cell. Such constructs may exist as extrachromosomal elements or may be integrated into the genome of the host cell.

The term "intron read-through" ("IRT") refers to a process in which an aberrantly spliced pre-mRNA transcript produces a protein or peptide that varies in size or amino acid composition. Different results may be produced in view of the final protein produced from this mis-spliced transcript. For example, a larger protein or a protein with an incorrect stop codon may be present than the predicted protein, in which case the protein may be longer or shorter than the predicted protein, respectively. In addition, the protein may also have incorrect or additional residues that may assist in protein modification for glycosylation, myristoylation, phosphorylation, ubiquitination, or other post-translational modifications.

The term "intron read-through byproduct" refers to a protein or peptide translated from an aberrantly spliced mRNA resulting from intron read-through, e.g., a protein having an unpredictable size or amino acid composition. The intron read-through by-product can be shorter or longer than a polypeptide predicted from a gene of known amino acid sequence and/or predicted from a cDNA of the gene. Intron read-through byproducts can also have molecular weights that are far different from those of proteins with accepted molecular weights that are produced from the correct splicing of mRNA by the gene. Moreover, the term "intron read-through byproduct" also includes proteins that result from proteolytic processing events that are not normally associated with the protein of interest, which can potentially occur from protein products that are shifted in frame due to intron-exon-linker reads-through.

The term "heavy chain by-product" refers to a polypeptide translated from an aberrantly spliced immunoglobulin heavy chain mRNA produced by intron read-through, e.g., a heavy chain protein having an unpredictable size or amino acid composition. The heavy chain by-product may be shorter or longer than the polypeptide predicted from the known amino acid sequence of the immunoglobulin and/or predicted from the cDNA of the gene. The heavy chain by-product may also have a molecular weight that is far different from the accepted molecular weight protein produced from the correct splicing of mRNA by the heavy chain gene. Moreover, the term "heavy chain by-product" also includes polypeptides produced from proteolytic processing events not normally associated with the protein.

The term "naturally occurring sequence" or "naturally occurring genomic sequence" refers to the intron and exon genetic constructs found in nature or in nature. Naturally occurring sequences can be found, for example, in their natural chromosomal location or in the location in which they are cloned into a vector, so long as the intron and exon organization of the sequence is retained.

The term "immunoglobulin" or "antibody" (used interchangeably herein) refers to a protein having a 4-polypeptide chain structure consisting of two heavy and two light chains, which chains can be stabilized by, for example, interchain disulfide bonds, wherein the immunoglobulin or antibody has the ability to selectively or specifically bind to an antigen.

The term a β -immunoglobulin or a β -antibody refers to an antibody that selectively or specifically binds to a β peptide.

The term "single chain immunoglobulin" or "single chain antibody" (used interchangeably herein) refers to a protein having a 2-polypeptide chain structure consisting of one heavy chain and one light chain, which chains may be stabilized by, for example, an interchain peptide linker, wherein the immunoglobulin or antibody has the ability to specifically bind to an antigen.

The term "immunoglobulin or antibody domain" refers to a globular region within a heavy or light chain polypeptide that includes peptide loops (e.g., including 3-4 peptide loops) stabilized by, for example, β -pleated sheets and/or intrachain disulfide bonds. In this application, domains are further referred to as "constant" or "variable", wherein the term "constant" refers to the relative lack of sequence variation within the domains of the various modules in the case of a "constant" domain, and wherein the term "variable" refers to the significant variation within the domains of the various modules in the case of a "variable" domain. In the art, an antibody or polypeptide "domain" can often be interchangeably referred to as an antibody or polypeptide "region". The "constant" domain of an antibody light chain may be alternatively referred to as a "light chain constant region", a "light chain constant domain", a "CL" region or a "CL" domain. The "constant" domain of an antibody heavy chain may be alternatively referred to as a "heavy chain constant region", a "heavy chain constant domain", a "CH" region or a "CH" domain. The "variable" domain of an antibody light chain may be alternatively referred to as a "light chain variable region", "light chain variable domain", "VL" region or "VL" domain. The "variable" domain of an antibody heavy chain may be alternatively referred to as a "heavy chain variable region", "heavy chain variable domain", "VH" region or "VH" domain.

The term "region" also refers to parts or portions of an antibody chain or antibody chain domain (e.g., parts or portions of a heavy or light chain, or parts or portions of a constant or variable domain, as defined in the specification), as well as more discrete parts or portions of the chain or domain. For example, as defined herein, light and heavy chains or light and heavy chain variable domains include "complementarity determining regions" or "CDRs" interspersed between "structural regions" or "FRs".

The immunoglobulin or antibody may be present in monomeric or multimeric form, e.g., an IgM antibody in pentameric form, and/or an IgA antibody in monomeric, dimeric or multimeric form. The term "fragment" refers to a part or portion of an antibody or antibody chain that includes fewer amino acid residues than the entire or complete antibody or antibody chain. Fragments may be obtained by chemical or enzymatic treatment of the whole or all antibodies or antibody chains. Fragments may also be obtained by recombinant means. Exemplary fragments include Fab, Fab ', F (ab') 2, Fabc, and/or Fv fragments. The term "antigen-binding fragment" refers to an immunoglobulin or polypeptide fragment of an antibody that binds to an antigen or competes with (i.e., specifically binds to) an intact antibody (i.e., is derived from the intact antibody).

The term "conformation" refers to the tertiary structure of a protein or polypeptide (e.g., an antibody chain, and domains or regions thereof). For example, the phrase "light (or heavy) chain conformation" refers to the tertiary structure of the light (or heavy) chain variable region, while the phrase "antibody conformation" or "antibody fragment conformation" refers to the tertiary structure of an antibody or fragment thereof. The term "conformation" may also refer to a quaternary structure resulting from the three-dimensional relationship of one or several protein or peptide chains. In contrast to antigenic determinants, the phrase "conformational epitope" is meant to include antigenic determinants arranged in a specific spatial arrangement of amino acids within one or several proteins that are present in close proximity. Given the multifunctional nature of antibodies (i.e., the ability of an IgG molecule to bind to several epitopes on more than one protein molecule simultaneously), antibodies are considered to have the intrinsic property of binding conformational epitopes consisting of several amino acid chains. For example, precipitation of plaque-forming a □ provides a conformational epitope in which one antibody can bind several adjacently placed a □ peptides.

Binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins. Binding fragments include Fab, Fab ', F (ab') 2, Fabc, Fv, single chain and single chain antibodies. In addition to "bispecific" or "bifunctional" immunoglobulins or antibodies, an immunoglobulin or antibody is understood to be identical for each binding site. A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody having two distinct pairs of heavy/light chains and two distinct binding sites. Bispecific antibodies can be prepared by a variety of methods including hybridoma fusion or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, clin. exp. immunol.79: 315- > 321 (1990); kostelny et al, J.Immunol.148, 1547-1553 (1992).

"specific binding" or "selective binding" of an antibody refers to the antibody exhibiting significant affinity for a particular antigen or epitope, and, in general, does not exhibit significant cross-reactivity. "significant" or preferred combinations include combinations of at least 10⁶，10⁷，10⁸，10⁹M^-1Or 10¹⁰M^-1Has an affinity binding of more than 10⁷M^-1Preferably greater than 10⁸M^-1Is more preferred. Intermediate values of the values set forth in this specification are also included within the scope of the invention, and preferred binding affinities may be expressed as ranges of affinities, e.g., 10⁶-10¹⁰M^-1Preferably 10⁷-10¹⁰M^-1More preferably 10⁸-10¹⁰M^-1. "does not show significant cross-reactivity"Is an antibody that does not significantly bind to an undesired entity (e.g., an undesired proteinaceous entity). For example, an antibody that specifically binds to a β can significantly bind to a β, but does not significantly react with non-a β proteins or peptides (e.g., non-a β proteins or peptides included within a plaque). Antibodies specific for a particular epitope will generally not, for example, significantly cross-react with a distal epitope on the same protein or peptide. In exemplary embodiments, the antibodies do not exhibit cross-reactivity (e.g., do not cross-react with non-a β peptides or with distal epitopes on a β). Specific binding can be determined according to any art-recognized manner of determining such binding. Preferably, specific binding can be determined according to Scatchard analysis and/or competitive binding analysis.

The term "significant identity" refers to two sequences, e.g., two polypeptide sequences, which share at least 50-60% sequence identity, preferably at least 60-70% sequence identity, more preferably at least 70-80% sequence identity, more preferably at least 80-90% identity, even more preferably at least 90-95% identity, and even more preferably at least 95% sequence identity or even greater identity (e.g., 99% sequence identity or greater) when optimally aligned, e.g., by the programs GAP or BESTFIT using default GAP weights. The term "substantial identity" or "substantially identical" refers to two sequences, e.g., two polypeptide sequences, which share at least 80-90% sequence identity, preferably at least 90-95% sequence identity, and more preferably at least 95% sequence identity or even greater identity (e.g., 99% sequence identity or greater) when optimally aligned, e.g., by the programs GAP or BESTFIT using default GAP weights. For sequence comparison, one sequence is usually used as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, secondary sequence coordinates are specified, if necessary, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence compared to the reference sequence based on the programmed parameters.

The homology can be determined by, for example, the local homology algorithm of Smith and Waterman, adv. 482(1981), homology alignment algorithm by Needleman and Wunsch, J moi. biol.48: 443(1970), methods for finding similarities to Pearson and Lipman, proc.nat' l.acad.sci.usa 85: 2444(1988), Computer applications of these algorithms (GAP in Wisconsingenetics Software Package, BESTFIT, FASTA and TFASTA, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al, Current Protocols in Molecular Biology). One example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which can be found in Altschul et al, J.MoI.biol.215: 403 (1990). Software for performing BLAST analysis is publicly available through the national center for bioengineering information (publicly available through the NCBI Internet Server of the national institutes of health). Typically, sequence comparisons can be performed using default program parameters, although custom parameters can also be used. For amino acid sequences, the BLASTP program uses default parameters for a word size (W) of 3, an expectation value (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff & Henikoff, proc. natl. acad. sci. usa 89: 10915 (1989)).

Preferably, residue positions that are not identical will differ by conservative amino acid substitutions. For the purpose of classifying amino acid substitutions as conservative or non-conservative, amino acids may be classified as follows: class I (hydrophobic side chains): leu, met, ala, val, leu, ile; class II (neutral hydrophilic side chain): cys, ser, thr; class III (acidic side chain): asp, glu; class IV (basic side chain): asn, gln, his, lys, arg; class V (residues affecting chain orientation): gly, pro; and class VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids of the same class. Non-conservative substitutions involve the exchange of an amino acid from one of these classes of amino acids for an amino acid from another class.

Antibodies

The method of the present invention is applicable to a variety of antibody production processes in which unwanted or undesirable by-products can be detected. In particular, the methods are applicable to the production of recombinant antibodies, such as chimeric or humanized monoclonal antibodies, wherein the sequence of the produced antibody is known.

The term "humanized immunoglobulin" or "humanized antibody" refers to an immunoglobulin or antibody that includes at least one humanized immunoglobulin or antibody chain (i.e., at least one humanized light or heavy chain). The term "humanized immunoglobulin chain" or "humanized antibody chain" (i.e., "humanized immunoglobulin light chain" or "humanized immunoglobulin heavy chain") refers to an immunoglobulin or antibody chain (i.e., light chain or heavy chain, respectively) having variable regions that include Complementarity Determining Regions (CDRs) substantially from a human immunoglobulin or antibody variable framework region, and substantially from a non-human immunoglobulin or antibody (e.g., at least one CDR, preferably two CDRs, more preferably three CDRs), and further includes constant regions (e.g., in the case of a light chain, at least one constant region or portion thereof, and in the case of a heavy chain, optionally three constant regions). The term "humanized variable region" (e.g., "humanized light chain variable region" or "humanized heavy chain variable region") refers to a variable region that includes variable framework regions that are substantially from a human immunoglobulin or antibody and Complementarity Determining Regions (CDRs) that are substantially from a non-human immunoglobulin or antibody.

The phrase "substantially from a human immunoglobulin or antibody" or "substantially human" means that the region shares at least 80-90%, 90-95%, or 95-99% identity (i.e., native sequence identity) with a human framework or constant region sequence when aligned with human immunoglobulin or antibody amino acids for comparison purposes, thereby allowing, for example, conservative substitutions, consensus sequence substitutions, germline substitutions, back mutations, and the like. Introduction of conservative substitutions, consensus sequence substitutions, germline substitutions, back mutations, etc., generally refers to "optimization" of the humanized antibody or chain. The phrase "substantially from a non-human immunoglobulin or antibody" or "substantially non-human" refers to an immunoglobulin or antibody sequence that shares at least 80-95%, preferably at least 90-95%, more preferably 96%, 97%, 98%, or 99% identity with an immunoglobulin or antibody sequence of a non-human organism, e.g., a non-human mammal.

Thus, all regions and residues of a humanized immunoglobulin or antibody, or humanized immunoglobulin or antibody chain, except perhaps the CDRs, are substantially identical to corresponding regions or residues of one or more human immunoglobulin sequences. The term "corresponding region" or "corresponding residue" refers to a region or residue on a second amino acid or nucleotide sequence that occupies the same (i.e., equivalent) position as a region or residue on the first amino acid or nucleotide sequence when the first and second sequences are optimally aligned for comparison purposes.

Preferably, the humanized immunoglobulin or antibody binds to the antibody with 3,4, or 5 fold affinity relative to the non-humanized antibody. For example, if the non-humanized antibody has 10⁹M^-1With an affinity of (3), the humanized antibody has at least 3x10⁹M^-1，4x10⁹M^-1Or 5x10⁹M^-1The affinity of (a). When describing the binding properties of an immunoglobulin or antibody, this can be described in terms of the ability of the chain to produce "direct antigen (e.g., a β or 5T4) binding". When an intact immunoglobulin or antibody (or antigen-binding fragment thereof) is provided with specific binding properties or binding avidity, the chain is said to be "antigen binding directed". A mutation (e.g., a back mutation) is considered to greatly affect the ability of the heavy or light chain to direct antigen binding if the mutation affects (e.g., reduces) the binding avidity of an intact immunoglobulin or antibody (or antigen-binding fragment thereof) containing the chain by at least one order of magnitude, as compared to the effect caused by an antibody (or antigen-binding fragment thereof) containing the equivalent chain lacking the mutation. If it affects (e.g., reduces) the integrity of the chain containing the equivalent chain lacking the mutation, as compared to the effect caused by an antibody (or antigen-binding fragment thereof) containing the chainAn immunoglobulin or antibody (or antigen-binding fragment thereof) has only two, three, or four times its binding avidity, and the mutation is considered to "not significantly affect (e.g., reduce) chain-directed antigen binding.

The term "chimeric immunoglobulin" or antibody refers to an immunoglobulin or antibody whose variable regions are derived from a first species and whose constant regions are derived from a second species. Chimeric immunoglobulins or antibodies can be constructed, for example, by genetic engineering, from immunoglobulin gene fragments belonging to different species. The term "humanized immunoglobulin" or "humanized antibody" is not intended to include chimeric immunoglobulins or antibodies as defined herein. Although a humanized immunoglobulin or antibody is chimeric in its construction (i.e., contains regions from more than one protein), it also includes other features not found in a chimeric immunoglobulin or antibody as defined herein (i.e., a variable region containing donor CDR residues and acceptor framework residues).

May be prepared by recombinant DNA techniques well known in the art, for example, as described in International application No. PCT/US86/02269 to Robinson et al; akira et al, European patent application No. 184,187; taniguchi, M, european patent application No. 171,496; european patent application No. 173,494 to Morrison et al; PCT International publication WO 86/01533 to Neuberger et al; cabilly et al, U.S. Pat. No. 4,816,567; european patent application No. 125,023 to Cabilly et al; better et al (1988) Science 240: 1041-1043; liu et al (1987) Proc.Natl.Acad.Sci USA 84: 3439-; liu et al (1987) J.Immunol.139: 3521-3526; sun et al (1987) Proc. Natl. Acad. Sci. USA 84: 214-218; nishimura et al (1987) can. res.47: 999-; wood et al (1985) Nature 314: 446- > 449; and Shaw et al (1988) j. natl. cancer inst.80: 1553 1559); morrison, s.l. (1985) Science 229: 1202-1207; oi et al (1986) BioTechniques 4: 214; winter U.S. Pat. Nos. 5,225,539; jones et al (1986) Nature 321: 552-525; verhoeyan et al (1988) Science 239: 1534; and Beidler et al (1988) j.immunol.141: 4053-.

Monoclonal, chimeric and humanized antibodies obtained by, for example, deletion, addition, or substitution of other portions of the antibody, such as modification of the constant region, are also within the scope of the present invention. For example, the following modifications may be made to the antibody: (i) replacing the constant region with another constant region, e.g., one intended to increase the half-life, stability or affinity of the antibody, or one from another species or antibody type; or (ii) by modifying one or more amino acids within the constant region, thereby altering, for example, the number of glycosylation sites, effector cell function, Fc receptor (FcR) binding, complement binding, and the like. Methods for altering antibody constant regions are well known in the art. Antibodies with altered functions, e.g., affinity for effector ligands, such as FcR on cells, or the C1 component of complement, can be prepared by replacing at least one amino acid of the antibody constant region with a different residue (see, e.g., european patent No. 388,151 a1, U.S. patent No. 5,624,821, and U.S. patent No. 5,648,260, which are incorporated herein by reference in their entirety). Similar types of alterations can also be described which, if applied to murine, or other species, immunoglobulins, reduce or eliminate these functions.

For example, it is possible to alter the Fc region of an antibody (e.g., IgG, such as human IgG), affinity for FcR (e.g., Fc γ R1), or for Clq binding (see, e.g., U.S. patent No. 5,624,821), by replacing a particular residue with a residue having a suitable functional group on its side chain, or by introducing a charged functional group, such as glutamic acid or aspartic acid, or possibly an aromatic nonpolar residue, such as phenylalanine, tyrosine, tryptophan, or alanine.

Human antibodies from transgenic animals and phage display

Alternatively, it is now possible to produce transgenic animals (e.g., mice) that, following immunization, produce the full repertoire of human antibodies without the production of endogenous immunoglobulins(reporter). For example, it has been described that antibody heavy chain junction regions (J) are thought to occur in chimeric and germline mutant mice_H) Homozygous deletion of the gene results in complete inhibition of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays in such germline mutant mice results in the production of human antibodies following antigen stimulation. See, for example, U.S. patent nos. 6,150,584; 6,114,598, and 5,770,429.

All human antibodies can also be obtained from phage display libraries (Hoogenboom et al, J.MoI.biol., 227: 381 (1991); Marks et al, J.MoI.biol., 222: 581: 597 (1991)).

Bispecific antibodies, antibody fusion polypeptides, and single chain antibodies

Bispecific antibodies (BsAbs) are antibodies that have binding specificity for at least two different epitopes. Such antibodies can be derived from full-length antibodies or antibody fragments (e.g., F (ab)'₂Bispecific antibody). Methods for making bispecific antibodies are well known in the art. Conventional preparation of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al, Nature, 305: 537-539 (1983)). These hybridomas (cell hybridomas) produce a potential mixture of different antibody molecules due to the random coordination of the immunoglobulin heavy and light chains (see, WO 93/08829 and Traunecker et al, EMBO J., 10: 3655-3659 (1991)).

Bispecific antibodies also include cross-linked or "heteroconjugate" antibodies. For example, one of the heteroconjugate antibodies may bind avidin, while the other binds biotin or other payload (payload). Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with a number of crosslinking techniques.

In another embodiment, the antibody may be chemically or genetically fused to a payload domain, e.g., an immunotoxin, to produce an antibody fusion polypeptide. Such payloads include, for example, immunotoxins, chemotherapeutic agents, and radioisotopes, all of which are well known in the art.

Single chain antibodies, as described herein, are also suitable for stabilization. The fragment includes a heavy chain variable region (VH) linked to a light chain variable region (VL) by a linker, which allows each variable region to contact each other and reconstruct the antigen binding pocket of the parent antibody from which the VL and VH regions can be derived. See Gruber et al, j.immunol., 152: 5368(1994).

anti-Abeta antibodies

In general, the antibodies of the invention include antibodies useful for treating amyloidosis, particularly alzheimer's disease, by targeting the a β peptide.

The term "amyloidogenic disease" includes any disease associated with (or caused by) the formation or precipitation of insoluble amyloid fibrils. Exemplary amyloidosis disorders include, but are not limited to, systemic amyloidosis, Alzheimer's disease, mature diabetic onset, Parkinson's disease, Huntington's chorea, frontotemporal dementia, and prion-related transmissible spongiform encephalopathies (Kuru and Kluyveromyces in humans and pruritus and BSE in sheep and cattle, respectively). Different amyloidosis disorders are defined and characterized according to the nature of the deposited fibrous polypeptide component. For example, in a subject or patient with alzheimer's disease, beta-amyloid (e.g., wild-type, variant, or truncated beta-amyloid) is a characteristic polypeptide component of amyloid deposits. Thus, alzheimer's disease is an example of a "disease characterized by a β precipitation" or a "disease associated with a β precipitation" in, for example, the brain of a subject or patient. The terms "beta-amyloid", "beta-amyloid peptide", "beta-amyloid body", "a β" and "a β peptide" are used interchangeably in this specification. When administered to a mammal, an "immunogenic agent" or "immunogen" is capable of inducing an immune response in the animal, optionally in combination with an adjuvant.

In the present specification, the terms "a □ antibody," "anti-a □ antibody," and "anti-a □" are used interchangeably and refer to an antibody that binds to one or more epitopes or epitopes of APP, a □ protein, or both. Exemplary epitopes or antigenic determinants can be found in human Amyloid Precursor Protein (APP), but are preferably found in the a β peptide of APP. APP exists in several isomeric forms, e.g. APP⁶⁹⁵，APP⁷⁵¹And APP⁷⁷⁰. Can be based on APP⁷⁷⁰The sequences of the isoforms assign numerical numbers to amino acids within APP (see, e.g., genbank accession No. p 05067). The A β peptide (which may also be referred to herein as β amyloid peptide and A β 0) is a 39-43 amino acid fragment of APP (A β 139, A β 240, A β 341, A β 442 and A β 543) within about 4 kDa. A β 640, for example, consists of residues 672-711 of APP, while A β 742 consists of residues 672-713 of APP. As a result of proteolytic processing of APP by different secretases in vivo or in situ, both "short form", 40 amino acids in length, and "long form", 42-43 amino acids in length, A β 8 peptides can be obtained. The epitope or antigenic determinant may be located within the N-terminus of the a β 9 peptide and comprises 1-10 amino acid residues of a β, preferably 1-3, 1-4, 1-5, 1-6, 1-7, 2-7, 3-6 or 3-7 from a β 042, or within 2-4, 5,6, 7 or 8 residues of a β, 3-5, 6, 7, 8 or 9 residues of a β, or 4-7, 8, 9 or 10 residues of a β 42. An "intermediate" epitope or antigenic determinant may be located in the middle or middle portion of an A.beta.peptide and includes amino acids 16-24, 16-23, 16-22, 16-21, 19-21, 19-22, 19-23 or 19-24 of A.beta.. A "C-terminal" epitope or antigenic determinant is located within the C-terminus of the A.beta.peptide and includes amino acid residues 33-40, 33-41 or 33-42 of A.beta..

In various embodiments, the a β antibody is end-specific. In the present specification, the term "terminal specificity" refers to antibodies which are capable of specifically binding to the N-terminal or C-terminal residues of the a β peptide, but which are not capable of recognizing the same residues when present in the longer a β class containing said residues or when present in APP.

In various embodiments, the a β antibody has "C-terminal specificity". In the present specification, the term "C-terminal specificity" refers to an antibody that specifically recognizes the free C-terminus of the A.beta.peptide. Examples of C-terminal specific A.beta.antibodies include the following: a β peptide terminating at residue 40 but not at residue 41, 42 and/or 43; a β peptides that recognize residues 42 at the terminus, but not 40, 41 and/or 43 at the terminus, and so on.

In one embodiment, the antibody may be A3D 6 antibody or variant thereof, or a 10D5 antibody or variant thereof, both of which are found in U.S. patent publication No. 2003/0165496a1, U.S. patent publication No. 2004/0087777a1, and international patent publication No. WO02/46237 A3. The descriptions of 3D6 and 10D5 can also be found in International patent publication Nos. WO02/088306A2 and WO02/088307A 2. 3D6 is a monoclonal antibody (mAb) capable of specifically binding to the N-terminal epitope located within human β -amyloid peptide, specifically residues 1-5. In contrast, 10D5 is also a monoclonal antibody (mAb) that is capable of specifically binding to the N-terminal epitope located within human β -amyloid peptide, specifically residues 3-6. In yet another embodiment, the antibody may be the 12B4 antibody or a variant thereof, as described in U.S. patent publication No. 20040082762a1 and international patent publication No. WO03/077858a 2. 12B4 is a monoclonal antibody (mAb) capable of specifically binding to the N-terminal epitope located within human β -amyloid peptide, specifically residues 3-7. In yet another embodiment, the antibody may be the 12a11 antibody or a variant thereof, as described in U.S. patent application No. 10/858,855 and international patent application No. PCT/US 04/17514. 12a11 is a monoclonal antibody (mAb) capable of specifically binding to the N-terminal epitope located within human β -amyloid peptide, specifically residues 3-7. In yet another embodiment, the antibody may be a 266 antibody, as described in U.S. patent application No. 10/789,273 and international patent application No. WO01/62801a 2. Antibodies suitable for use in the present invention that are designed to specifically bind to a C-terminal epitope located within human β -amyloid peptide include, but are not limited to 369.2B, as described in U.S. Pat. No. 5,786,160.

In exemplary embodiments, the antibody may be selected from the group consisting of a humanized anti-a β peptide 3D6 antibody, a humanized anti-a β peptide 12a11 antibody, a humanized anti-a β peptide 10D5 antibody, a humanized anti-a β peptide 12B4 antibody, and a humanized anti-a β peptide 266 antibody capable of selectively binding to a β peptide. More specifically, humanized anti-A β peptide 3D6 antibodies are designed to specifically bind NH located within the human β -amyloid 1-40 or 1-42 peptide₂-terminal epitopes, said amyloid peptides being found in plaque deposits in the brain (e.g. in patients suffering from alzheimer's disease).

Fc fusion protein

In certain embodiments, the nucleic acid molecules of the invention encode fusion or chimeric proteins. Fusion proteins can include targeting moieties, e.g., soluble receptor fragments or ligands, as well as immunoglobulin chains, Fc fragments, heavy chain constant regions including various subtypes: IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE. For example, the fusion protein can include the extracellular domain of the receptor, and, for example, fused to a human immunoglobulin Fc chain (e.g., a human IgG, e.g., human IgG1 or human IgG4, or mutated forms thereof). In one embodiment, the human Fc sequence may be mutated at one or more amino acids, for example at residues 254 and 257 of the wild-type sequence, to reduce Fc receptor binding. The fusion protein may additionally comprise a linker sequence linking the first portion to a second portion, such as an immunoglobulin fragment. For example, the fusion protein can include a peptide linker, e.g., a peptide linker of about 4-20, more preferably 5-10 amino acids in length; the peptide linker is 8 amino acids in length. For example, the fusion protein can include a peptide linker having the general formula (Ser-Gly-Gly-Gly-Gly) y, where y is 1, 2, 3,4, 5,6, 7, or 8. In other embodiments, additional amino acid sequences may be added to the N-or C-terminus of the fusion protein to facilitate expression, spatial structure flexibility, assay and/or isolation or purification.

The chimeric or fusion proteins of the invention can be prepared by standard DNA recombination techniques. For example, DNA fragments encoding different polypeptide sequences may be ligated in frame by conventional techniques, e.g., blunt-ended or sticky-ended ligation, restriction enzyme digestion to provide appropriate ends, sticky end filling if desired, alkaline phosphatase treatment to avoid unwanted ligation, and enzymatic ligation. In other embodiments, the fusion gene may be synthesized by conventional techniques including automated DNA synthesis. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that are capable of increasing the complementary overhang between two consecutive gene fragments, which can then be annealed and re-amplified to produce a chimeric gene sequence (see, e.g., Ausubel et al (eds.) Current Protocols in Molecular Biology, John Wiley & Sons, 1992). In addition, there are many expression vectors that encode fusion moieties (e.g., the Fc region of an immunoglobulin heavy chain) that are commercially available. Immunoglobulin fusion polypeptides are well known in the art and can be found, for example, in U.S. patent nos. 5,516,964; 5,225,538 No; U.S. Pat. No. 5,428,130; 5,514,582 No; 5,714,147, 5,455,165.

Nucleic acid molecules, constructs and vectors

Exemplary embodiments of the invention feature engineered constructs designed to eliminate unwanted or undesirable by-products, particularly unwanted or undesirable antibody (or immunoglobulin) by-products. In certain aspects, the constructs include a component of a naturally occurring antibody gene sequence, where the component can be genetically altered, modified, or engineered (e.g., genetically engineered) such that the resulting construct is capable of expressing a desired protein of interest (e.g., an antibody) without the need or undesired by-products. Constructs may be made using techniques well known in the art for making recombinant nucleic acid molecules (e.g., comprising components of immunoglobulin chain genes), as described in detail below.

The antibody gene sequences may encode multiple subtypes of antibodies, including: IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, IgD, or IgE. Preferably, the antibody encoded by the antibody gene sequence is of the IgG subtype. The encoded immunoglobulin or antibody molecule may comprise a full length (e.g., an IgG1 or IgG4 immunoglobulin) or may comprise only fragments (e.g., an Fc fragment).

It will be appreciated by those skilled in the art that the nucleotide sequence encoding the antibody of the present invention may be derived from the nucleotide and amino acid sequences described herein, or from immunoglobulin gene sequences from other sources known in the art, using the genetic code and standard molecular biology techniques. The nucleic acid composition of the present invention can be obtained from a known immunoglobulin DNA (e.g., cDNA sequence). In particular, the nucleotide sequence may be substantially identical to or derived from a native V, D, J, or constant region cDNA sequence. The sequences of the heavy and light chain constant region genes are well known in the art. Preferably, the constant region is human, but constant regions from other species, e.g., rodents (e.g., mice or rats), primates (macaques), camelids, or rabbits, may also be used. Constant regions from these species are well known in the art (see, e.g., Kabat, E.A. et al (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. department of Health and Human Services, NIH Publication No.91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be a constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD. As to the heavy chain constant region sequence is known in the art, and can be found, for example, in NCBI NG _ 001019. In some embodiments, the constant region is an IgG1 or IgG4 constant region. For the heavy chain gene of an Fc fragment, the DNA encoding Fc can be operably linked to a heavy chain leader sequence (e.g., a heavy chain variable region leader sequence) for direct expression.

Other aspects of the invention include spliced immunoglobulin DNA cassette sequences. The spliced immunoglobulin cassette sequences include the nucleic acid sequence encoded by the immunoglobulin DNA cassette nucleotide sequence as well as the amino acid sequence.

Provided herein are exemplary human IgG1 constant region genomic sequences:

GTGAGTCCTGTCGACTCTAGAGCTTTCTGGGGCAGGCCAGGC

CTGACTTTGGCTGGGGGCAGGGAGGGGGCTAAGGTGACGCA

GGTGGCGCCAGCCAGGCGCACACCCAATGCCCATGAGCCCA

GACACTGGACGCTGAACCTCGCGGACAGTTAAGAACCCAGG

GGCCTCTGCGCCCTGGGCCCAGCTCTGTCCCACACCGCGGTC

ACATGGCACCACCTCTCTTGCAGCCTCCACCAAGGGCCCATC

GGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGG

CACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGA

ACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG

CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTAC

TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGC

ACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAAC

ACCAAGGTGGACAAGAAAGTTGGTGAGAGGCCAGCACAGGG

AGGGAGGGTGTCTGCTGGAAGCCAGGCTCAGCGCTCCTGCCT

GGACGCATCCCGGCTATGCAGTCCCAGTCCAGGGCAGCAAG

GCAGGCCCCGTCTGCCTCTTCACCCGGAGGCCTCTGCCCGCC

CCACTCATGCTCAGGGAGAGGGTCTTCTGGCTTTTTCCCCAG

GCTCTGGGCAGGCACAGGCTAGGTGCCCCTAACCCAGGCCCT

GCACACAAAGGGGCAGGTGCTGGGCTCAGACCTGCCAAGAG

CCATATCCGGGAGGACCCTGCCCCTGACCTAAGCCCACCCCA

AAGGCCAAACTCTCCACTCCCTCAGCTCGGACACCTTCTCTCC

TCCCAGATTCCAGTAACTCCCAATCTTCTCTCTGCAGAGCCCA

AATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGGTA

AGCCAGCCCAGGCCTCGCCCTCCAGCTCAAGGCGGGACAGGT

GCCCTAGAGTAGCCTGCATCCAGGGACAGGCCCCAGCCGGGT

GCTGACACGTCCACCTCCATCTCTTCCTCAGCACCTGAACTCC

TGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGG

ACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGG

TGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT

GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAG

CCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAG

CGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGA

GTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCAT

CGAGAAAACCATCTCCAAAGCCAAAGGTGGGACCCGTGGGG

TGCGAGGGCCACATGGACAGAGGCCGGCTCGGCCCACCCTCT

GCCCTGAGAGTGACCGCTGTACCAACCTCTGTCCCTACAGGG

CAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGG

GAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGT

CAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAG

CAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCG

TGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCAC

CGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCAT

GCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGA

AGAGCCTCTCCCTGTCCCCGGGTAAATGA(SEQ ID NO：1)

also provided herein are exemplary IgG4 constant region genomic sequences:

GTGAGTCCTGTCGACTCTAGAGCTTTCTGGGGCAGGCCAGGC

CTGACTTTGGCTGGGGGCAGGGAGGGGGCTAAGGTGACGCA

GGTGGCGCCAGCCAGGCGCACACCCAATGCCCATGAGCCCA

GACACTGGACGCTGAACCTCGCGGACAGTTAAGAACCCAGG

GGCCTCTGCGCCCTGGGCCCAGCTCTGTCCCACACCGCGGTC

ACATGGCACCACCTCTCTTGCAGCCTCCACCAAGGGCCCATC

GGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAG

CACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGA

ACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCG

GCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCT

ACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGG

GCACGAAGACCTACACCTGCAATGTAGATCACAAGCCCAGC

AACACCAAGGTGGACAAGAGAGTTGGTGAGAGGCCAGCAC

AGGGAGGGAGGGTGTCTGCTGGAAGCCAGGCTCAGCCCTCC

TGCCTGGACGCACCCCGGCTGTGCAGCCCCAGCCCAGGGCA

GCAAGGCAGGCCCCATCTGTCTCCTCACCTGGAGGCCTCTGA

CCACCCCACTCATGCTCAGGGAGAGGGTCTTCTGGATTTTTC

CACCAGGCTCCGGGCAGCCACAGGCTGGATGCCCCTACCCC

AGGCCCTGCGCATACAGGGGCAGGTGCTGCGCTCAGACCTG

CCAAGAGCCATATCCGGGAGGACCCTGCCCCTGACCTAAGC

CCACCCCAAAGGCCAAACTCTCCACTCCCTCAGCTCAGACAC

CTTCTCTCCTCCCAGATCTGAGTAACTCCCAATCTTCTCTCTG

CAGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGGTA

AGCCAACCCAGGCCTCGCCCTCCAGCTCAAGGCGGGACAGG

TGCCCTAGAGTAGCCTGCATCCAGGGACAGGCCCCAGCCGG

GTGCTGACGCATCCACCTCCATCTCTTCCTCAGCACCTGAGT

TCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCA

AGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCG

TGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTC

AACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGAC

AAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGG

TCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCA

AGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCC

TCCATCGAGAAAACCATCTCCAAAGCCAAAGGTGGGACCCA

CGGGGTGCGAGGGCCACATGGACAGAGGTCAGCTCGGCCCA

CCCTCTGCCCTGGGAGTGACCGCTGTGCCAACCTCTGTCCCT

ACAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCC

ATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCT

GCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGT

GGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCAC

GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAG

CAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAAT

GTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC

TACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGA

(SEQ ID NO：3)

antibody preparation

The antibodies of the invention are typically prepared by recombinant expression. The nucleic acids encoding the light and heavy chains may be inserted into an expression vector. The light and heavy chains may be cloned into the same or different expression vectors. The DNA segment encoding the immunoglobulin chain may be operably linked to control sequences in an expression vector that ensure expression of the immunoglobulin polypeptide. Expression control sequences include, but are not limited to, promoters (e.g., naturally associated or heterologous promoters), signal sequences, enhancing elements, and transcription termination sequences. Preferably, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell (e.g., a COS or CHO cell).

After manipulation of the isolated genetic material to provide the polypeptides of the invention as described above, the genes are typically inserted into expression vectors for introduction into host cells, which vectors can be used to produce the desired amount of modified antibodies, which in turn provide the polypeptides of the invention. The term "vector" encompasses nucleic acid constructs, which often include nucleic acids, such as genes, and also include the minimal elements necessary for nucleic acid replication, transcription, stability, and/or protein expression or secretion from a host cell. Such constructs may exist as extrachromosomal elements, or may be integrated into the genome of the host cell.

The term "expression vector" includes a specific type of vector in which the nucleic acid construct is optimized for high level expression of the desired protein product. Expression vectors often contain transcriptional regulators such as promoter and enhancer elements that are optimized to achieve high levels of transcription in a particular cell type and/or optimized such that the expression is constitutive when a particular inducer is used. The expression vector also has sequences that provide for proper and/or enhanced translation of the protein. Such vectors may conveniently be selected from plasmids, bacteriophages, viruses and retroviruses, as is well known to those skilled in the art. The term "expression cassette" includes a vector containing a gene and elements other than the gene that allow for the proper and or enhanced expression of the gene in a host cell.

The term "operably linked" includes contiguous (e.g., functionally linked) wherein the components are linked in a relationship that allows them to function in their intended manner. For example, a promoter/enhancer operably linked to a polynucleotide of interest can be linked to the polynucleotide such that expression of the polynucleotide of interest is obtained under conditions that activate expression directed by the promoter/enhancer. As to the invention described in this specification, operably linked also includes the relationship of splice donor and splice acceptor sites found in the original transcript (pre-mRNA) of the gene of interest. Typically, the splice acceptor and donor sites are operably linked, in that two sequences are required and act together to produce the resulting mature messenger RNA.

The phrase "naturally operably linked" refers to the structure of an intron and an exon of a gene found in its natural or native state (organization). One method of cloning a gene of interest involves isolating nucleic acids, including exons and introns, from the gene and inserting the nucleic acid sequences into a vector to amplify the nucleic acid sequences. The entire gene sequence may also be inserted into an expression vector for expressing the protein in the same or a different species. Introns and exons are considered to retain their natural operative linkage when the gene containing them is found to be cloned in its naturally occurring form.

Expression vectors are usually replicable in host organisms, as are either episomes (episomes) or integrated parts of the host chromosomal DNA. Typically, expression vectors contain a selectable marker (e.g., ampicillin, hygromycin, tetracycline, kanamycin, or neomycin resistance) to allow detection of those transformed cells with the desired DNA sequence (see, e.g., U.S. patent No. 4,704,362 to Itakura et al). In addition to immunoglobulin cassette sequences, insertion sequences and control sequences, the recombinant expression vectors of the invention may also carry other sequences, such as sequences capable of regulating replication of the vector in a host cell (e.g., an origin of replication) and selectable marker gene sequences. Selectable marker genes can assist in the selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017 to Axel et al). For example, generally, a selectable marker gene confers drug resistance, such as G418 resistance, hygromycin resistance or methotrexate resistance, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (suitable for methotrexate selection/amplification in a DHFR-host) and the neo gene (for G418 selection).

Once the vector is incorporated into an appropriate host, the host may be maintained under conditions suitable for high level expression of the nucleotide sequence, and collection and purification of the desired antibody. Mammalian cells preferred for expression and production of the antibodies of the invention. See, e.g., Winnacker, From Genes to Clones, VCH Publishers, n.y., n.y. (1987). Eukaryotic cells are preferred because a variety of suitable host cell lines capable of secreting heterologous proteins (e.g., intact immunoglobulins) have been developed in the art, including CHO cell lines, various COS cell lines, HeLa cells, preferably, myeloma cell lines, or transplantsChemotransformed B-cells or hybridomas. Preferably, the cell is non-human. Preferred mammalian cells suitable for expression of the antibodies of the invention include Chinese hamster ovary (CHO cells) (including dhfr cells)^-CHO cells, see Urlaub and Chasin (1980) proc.natl.acad.sci.usa 77: 4216-: 601-621), lymphocyte cell lines, e.g., NSO myeloma cells and SP2 cells, COS cells, and cells derived from transgenic animals, e.g., mammary epithelial cells. Other suitable host cells are well known to those skilled in the art.

Expression vectors suitable for use in these cells can include expression control sequences such as origins of replication, promoters, and enhancers (Queen et al, Immunol. Rev.89: 49(1986)), as well as necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Preferred expression control sequences are promoters from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus and the like. See, e.g., Co et al (1992) j.immunol.148: 1149. preferred regulatory sequences suitable for expression in mammalian host cells include viral elements which direct the expression of high levels of proteins in mammals, such as promoters and/or enhancers from the FF-1a promoter and BGH polya, Cytomegalovirus (CMV) (e.g., CMV promoter/enhancer), simian nephrovirus 40(SV40) (e.g., SV40 promoter/enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and polyoma virus. For further description of viral regulatory elements, and sequences thereof, see, e.g., U.S. Pat. No. 5,168,062 to Stinski et al, U.S. Pat. No. 4,510,245 to Bell et al, and U.S. Pat. No. 4,968,615 to Schaffher et al. In exemplary embodiments, antibody heavy and light chain genes may be operably linked to enhancer/promoter regulatory elements (e.g., from SV40, CMV, adenovirus, etc., such as CMV enhancer/AdMLP promoter regulatory element or SV40 enhancer/AdMLP promoter regulatory element) to drive high levels of transcription of the genes. In an exemplary embodiment of the invention, the construct includes an Internal Ribosome Entry Site (IRES) to provide relatively high levels of the polypeptide of the invention in a eukaryotic host cell. U.S. Pat. No. 6,193,980, which is incorporated herein by reference in its entirety, discloses compatible IRES sequences.

Alternatively, the antibody-coding sequence may be integrated into a transgene, introduced into the genome of a transgenic animal, and subsequently expressed in the milk of the transgenic animal (see, e.g., U.S. Pat. No. 5,741,957 to Deboer et al, U.S. Pat. No. 5,304,489 to et al, and U.S. Pat. No. 5,849,992 to Meade et al). Suitable transgenes include coding sequences for light and/or heavy chains operably linked to promoters and enhancers from mammary gland-specific genes, such as casein or beta lactoglobulin.

Prokaryotic host cells may also be suitable for use in the production of antibodies of the invention. Coli is a prokaryotic host particularly suitable for cloning the polynucleotides (e.g., DNA sequences) of the present invention. Other microbial hosts suitable for this use include Bacillus species, such as Bacillus subtilis, Enterobacter species, such as E.coli, Salmonella and Serratia species, and various Pseudomonas species. In these prokaryotic hosts, one skilled in the art can also prepare expression vectors, which can typically contain expression control sequences (e.g., origins of replication) compatible with the host. In addition, any number of various well-known promoters may also be present, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or a promoter system from bacteriophage lambda. The promoter generally controls expression, optionally through an operator, and has ribosome binding site sequences and the like to initiate and complete transcription and translation.

Protein expression in prokaryotes is generally performed in e.coli using vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors can add multiple amino acids to the constant region of an antibody, typically a recombinant antibody, encoded therein without affecting the specificity or antigen recognition of the antibody. The addition of fusion peptide amino acids can add additional functionality to the antibody, for example, as a tag (e.g., an epitope tag, such as myc or flag).

Other microorganisms, such as yeast, may also be used for expression. Saccharomyces (Saccharomyces) is a preferred yeast host, with suitable vectors possessing desired expression control sequences (e.g., promoters), origins of replication, termination sequences, and the like. Common promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, inter alia, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.

Alternatively, antibodies of the invention can be made in transgenic plants (e.g., tobacco, corn, soybean, and alfalfa). Improved 'plant antibody' vectors (Hendy et al (1999) J. immunological. methods 231: 137-146) and purification strategies and the increase of transformable crop varieties make such methods a practical and efficient method for the preparation of recombinant immunoglobulins not only for human and animal therapy, but also for industrial applications (e.g., catalytic antibodies). Moreover, plant-produced antibodies also appear to be safe and effective and avoid the use of animal-derived materials, and thus avoid the risk of contamination with infectious spongiform encephalopathy (TSE) preparations. In addition, differences in the glycosylation pattern of plant and mammalian cells producing antibodies have little or no effect on antibody binding or specificity. Furthermore, no evidence of toxicity or HAMA was observed in patients receiving topical oral application of secretory dimeric IgA antibodies derived from plants (see, e.g., Larrick et al (1998) Res. immunological.149: 603-608).

Recombinant antibodies can be expressed in transgenic plants using a variety of methods. For example, antibody heavy and light chains can be cloned independently into an expression vector (e.g., an agrobacterium tumefaciens (agrobacterium tumefaciens) vector), and then transformed in vitro using recombinant bacteria or direct transformation methods, e.g., using particles coated with a vector that is subsequently physically introduced into plant tissue by bombardment. Subsequently, whole plants expressing the individual chains are reconstructed and then sexually crossed, ultimately producing fully assembled and functional antibodies. Similar protocols have been used to express functional antibodies in tobacco plants (see, e.g., Hiatt et al (1989) Nature 342: 76-87). In various embodiments, a signal sequence may be used to initiate the expression by directing the chains into the appropriate plant environment (e.g., the aqueous environment of apoplams or other specific plant tissues, including tubers, fruits, or seeds) for binding and folding of unassembled antibody chains (see Fiedler et al (1995) Bio/Technology 13: 1090-. 1093). Plant bioreactors can also be used to increase antibody production and significantly reduce costs.

Suitable host cells are described in Goeddel (1990) Gene Expression Technology: methods in Enzymology 185, Academic Press, San Diego, Calif., are discussed in more detail. Alternatively, recombinant expression vectors can be transcribed and translated in vitro using, for example, T7 promoter regulatory sequences and T7 polymerase.

Vectors containing the polynucleotide sequences of interest (e.g., heavy and light chain coding sequences and expression control sequences) can be transferred into host cells by well-known methods, depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biolistic (biolistic) or virus-based transfection may be used for other cellular hosts (see generally, Sambrook et al, Molecular Cloning: organism Manual (Cold Spring Harbor Press, 2nd ed., 1989, incorporated herein by reference in its entirety for all purposes.) other methods for transforming mammalian cells include polybrene (polybrene), protoplast fusion, liposomes, electroporation, and microinjection (see generally, Sambrook et al, supra.) for the preparation of transgenic animals, the transgene may be microinjected into fertilized eggs, or into the genome of embryonic stem cells, and the nucleus of such cells may be transferred into enucleated egg cells.

When the heavy and light chains are cloned into separate expression vectors, the vectors can be co-transfected to obtain expression and assembly of the intact immunoglobulin. Once expressed, the intact antibodies of the invention, dimers thereof, light and heavy chains alone, or other immunoglobulin forms (see generally Scopes, Protein Purification (Springer-Verlag, N.Y., (1982)). Once expressed, substantially pure immunoglobulins that are at least about 90-95% homogeneous are preferred, and 98-99% or greater homogeneity are more preferred, using procedures standard in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, HPLC Purification, gel electrophoresis, and the like.

The immunoglobulins or antibodies prepared according to the present invention may be derivatized or linked to other functional molecules (e.g., other peptides or proteins). Thus, the antibodies or antibody portions of the invention or modified forms of said antibodies of the invention in the present specification can be further derivatized for use in research, diagnostic and/or therapeutic areas. For example, an antibody or antibody portion of the invention can be functionally linked (by chemical coupling, genetic fusion, non-covalent linkage, or otherwise) to one or more other molecular entities, such as other antibodies (e.g., bispecific antibodies or diabodies), detectable agents, cytotoxic agents, pharmaceutical agents, and/or proteins or peptides, which can mediate the linking of the antibody or antibody portion to other molecules (e.g., streptavidin core region or polyhistidine tag).

One class of derivatized antibodies can be prepared by cross-linking two or more antibodies (of the same type or of different types, e.g., to make a bispecific antibody). Suitable crosslinkers include those heterobifunctional crosslinkers having groups of different reactivity separated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such cross-linking agents are available from Pierce chemical company, Rockford, IL.

Exemplary fluorescently detectable agents include fluorescenceBiotin, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and the like. Antibodies can also be derivatized with detectable enzymes such as alkaline phosphatase, horseradish peroxidase, P-galactosidase, acetylcholinesterase, glucose oxidase, and the like. When the antibody is derivatized with a detectable enzyme, the assay may be performed by adding additional reagents for the enzyme to produce a detectable reaction product. For example, when horseradish peroxidase detection reagent is present, the addition of hydrogen peroxide and diaminobenzidine can result in a colored reaction product that is detectable. Antibodies can also be derivatized using prosthetic groups (e.g., streptavidin/biotin and avidin/biotin). For example, antibodies can be derivatized with biotin and detected by indirect determination of avidin or streptavidin binding. Examples of suitable fluorescent materials include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of the light emitting material include: luminol; examples of bioluminescent materials include luciferase, luciferin and aequorin, and examples of suitable radioactive materials include¹²⁵I，¹³¹I，³⁵S or³H. The antibody (or fragment thereof) may also be conjugated to a therapeutic moiety, such as a cytotoxin or other therapeutic protein. Alternatively, the antibody can be conjugated to a second antibody to form an antibody heteroconjugate, as described in U.S. Pat. No. 4,676,980 to Segal et al.

Expression vectors for reducing or eliminating undesirable polypeptide by-products

In the development of protein expression systems suitable for therapeutic proteins, HPLC analysis of purified target products identified unexpected Low Molecular Weight (LMW) types of peptides. More specifically, unexpected polypeptide by-products were observed in the development of CHO (chinese hamster ovary) cell lines for expression of the 3D6 antibody. The antibody is also described elsewhere and is the result of many efforts to develop immunotherapeutic agents useful for the treatment of alzheimer's disease. It has specificity for A-beta peptide and has been shown to be effective in removing A-beta plaques. In addition to the genes used for selective culture of cells containing the expression cassette, CHO cell lines can be developed using methods acceptable in the art and containing copies of the heavy and light chains of the 3D6 antibody.

The tests carried out on numerous clonal isolates of the cells indicated that the production of the LMW type was not a clone-specific phenomenon used, i.e.only a small fraction of the total protein produced in all the tested cell lines was of the unexpected LMW type. It was further observed that when protein expression was induced, the proportion of LMW types relative to total protein increased. Further determination of the polypeptide using mass spectrometry demonstrated that the LMW type contains amino acids that were not predicted by the exon sequence of the gene.

The top panel of fig. 1 schematically shows the 3D6 heavy chain expression cassette with an intron and exon relationship and the positions of an Internal Ribosome Entry Site (IRES) and the di-light folate reductase (DHFR) selectable marker gene. The exons shown are the variable region heavy chain (V)_H1) Hinge and constant region heavy chains 1, 2, and 3 (C)_H1，C_H2，C_H3). The introns of the expression cassettes are denoted Int1, Int2, Int3 and Int 4. FIG. 1 also shows the correct splicing events predicted from mRNA obtained from the expression cassettes. The middle panel shows correctly spliced mRNA containing only the bicistronic transcript intron sequence.

Close examination of intron and exon sequences in the expression vectors and mass spectral data indicated RNA polymerase intron read-through (IRT) to specific splice site linkers. Since the organization structure between introns and exons and splice site donor and acceptor sites in the expression vector is essentially identical to that in the original genomic structure of the gene, the wrong indirect event is difficult to predict.

The bottom panel of fig. 1 shows the predicted product resulting from an intron read-through of the fourth intron. Figure 2 provides sequence information for the sense and antisense strands of the DNA sequence in the fourth intron region of the genomic sequence of the 3D6 antibody expression vector. The splice junctions (splice donor and acceptor sites) are indicated by vertical lines perpendicular to the nucleic acid sequence. DNA corresponding to intron sequences is underlined and in italics. The predicted amino acids of the desired product and of the read-through byproduct polypeptide are shown below the antisense strand of genomic DNA. The amino acid sequence of a polypeptide derived from a correctly spliced RNA is indicated in bold uppercase letters; polypeptide by-products from mis-spliced RNA are shown in lower case.

The present invention describes materials and methods for designing protein expression cassettes and vectors such that intron read-through (IRT) and undesirable polypeptide by-products can be greatly reduced or completely eliminated. In part, the present invention provides novel vector designs in which the natural operative linkage of introns and exons in an isolated nucleic acid encoding a protein of interest is altered, thereby reducing or eliminating IRT, and thereby reducing or eliminating undesirable types of IRT polypeptides. The unique changes are particularly suitable for IgG1 or IgG4 antibodies, but can be used for any gene of interest. In addition, vectors of the invention having altered naturally operably linked introns and exons have been shown to not only reduce or eliminate IRT by-products, but also to increase protein expression levels, in relation to vectors designed using standard techniques recognized in the art.

Examples

Materials and methods

In all examples, unless otherwise indicated, the materials and methods illustrated below were used:

in general, the practice of the present invention employs techniques well known in the art of molecular biology, recombinant DNA technology, and immunology, particularly, for example, antibody technology. See, e.g., Sambrook, Fritsch and manitis, Molecular Cloning: cold spring Harbor laboratory Press (1989); antibody Engineering Protocols (Methods in Molecular Biology), 510, Paul, s., Human Press (1996); antibody Engineering: a Practical Approach (Practical Approach) (Practical approaches Series, 169), McCafferty, Ed IRL Press (1996); antibodies: a Laboratory Manual (antibodies: Laboratory Manual), Harlow et al, Cold Spring Harbor Press, (1999); and Current Protocols in Molecular Biology (latest protocol in Molecular Biology) eds. Ausubel et al, John Wiley & Sons (1992).

Example 1 quantification of intron read-through transcription

In order to quantify the relative amount of aberrant transcripts formed by intron read-through, a quantitative PCR assay was designed. The method for evaluating IRT transcription is shown in FIG. 3. Specifically, quantitative PCR analysis was designed using the TaqMan3 system, where PCR amplification was used to quantify the nucleic acid type of interest. Three sets of probe primers were designed to determine the portion of intron read-through mRNA produced. The first set of probe primers is designed to quantify the level of transcription of an exon sequence naturally operably linked to the intron of interest. In the case of the 3D6 heavy chain expression cassette, the mRNA type containing the 3D6 second constant region heavy chain (CH2) exon is the target site. This provides a measure for the total product of 3D6 mRNA. The second set of probe primers bridged the operably linked intron and exon where the interface CH2 exon-fourth intron of the 3D6 expression cassette was ligated. Amplification of the primer set from this probe indicated the presence of an intron read-through transcript containing the 5' splice donor sequence, and the presence of a sequence bridging the CH2 exon and intron 4. The third set of probe primers targets the sequence of the fourth intron. This set of probes provides a quantitative analysis of the portion of mis-spliced RNA containing the internal intron 4 sequence.

FIG. 4 shows the results of Q-PCR analysis using the probe primer set. Briefly, CHO cells containing stably integrated expression vectors were inoculated and maintained in culture for two weeks. The cultures were induced at day 7 to increase expression. During the course of the experiment, samples of cell cultures were lysed and RNA content was assessed using a probe and primer set specific for the CH2 exon or intron as described in the previous paragraph. The graph shows low levels of mis-spliced RNA products prior to induction, and the percentage of RNA containing intron 4 after induction increased over time. The Q-PCR methods described herein predict the likelihood that a particular expression cassette containing introns and exons linked in a naturally operable manner will be able to produce intron read-through byproducts.

Although details of quantifying IRT for the 3D6 antibody expression system are clearly provided, the technique can be implemented in any protein expression system where there is a potential IRT. The novel methods of the invention are therefore particularly useful for assessing whether the vectors of the invention (described in detail below) can be tailored to a particular protein of interest, thereby avoiding the production of undesirable IRT polypeptide by-products. When the abundance of IRT transcripts is greater than about 0.1% -1%, vectors with altered native operable linkage can be used to express the desired protein. One of skill in the art will readily appreciate methods for determining intron read-through mRNA, and, thus, methods for predicting whether an intron read-through polypeptide can be applied to any protein expression system in which a splicing event occurs. For example, the system can be used in any eukaryotic cell system, e.g., yeast, drosophila, mouse, monkey, rabbit, rat, or human cell-based systems.

Example 2 vectors with modified naturally operably linked introns and exons

Expression vectors can be designed in which the natural operable linkage of introns and exons is modified. Two exemplary vector sequences can be found in FIG. 5. The figure shows the development of expression constructs to solve the problem of intron read-through by-products. The top panel is shown as containing variable regions (V)_H) Three constant regions (C)_H1，C_H2，C_H3) And the genome, intron-exon organization of the hinge region exon for general antibody heavy chains. Middle and bottom panels describe the integration of expression vectorsThe genomic sequence of (a), which is capable of eliminating intron read-through heavy chain by-products.

CHO cells expressing a 3D6 light chain may be transformed with a modified 3D6 heavy chain expression vector having the complete genomic heavy chain sequence of the 3D6 antibody or in which the natural operable linkage of introns and exons is modified. The cells can be cultured using standard techniques for protein expression purposes as described in the materials and methods. The antibody was purified from the treated supernatant and then fractionated by denaturing Reverse Phase (RP) HPLC (figure 6). The heavy and light chain components of the antibody are separated by passing through the column.

The peaks of the heavy and light chains were evident in the top trace representing the protein preparation component of the 3D6 genomic clone. In addition, a small peak corresponding to a heavy chain intron read-through product can be identified between the heavy and light chain components.

The bottom trace is an example of an expression system that reduces the problem of intron read-through. As with the top trace, both light and heavy chain peaks are clearly visible, however, IRT levels are reduced below the detection limit. This finding can be extended to other vectors in which the natural operative linkage of introns and exons is altered. For example, the HC Δ intron 4 sequence shown in figure 5 similarly reduces IRT to undetectable levels.

Table 1 shows details of the HC.DELTA.intron 4 sequence shown in FIG. 5.

Table 1: hu3D6v2HC- Δ 4

ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTG

TCCAGTGTGAGGTGCAGCTGCTGGAGTCCGGCGGCGGCCTGGTGCAGCC

CGGCGGCTCCCTGCGCCTGTCCTGCGCCGCCTCCGGCTTCACCTTCTCC

AACTACGGCATGTCCTGGGTGCGCCAGGCCCCCGGCAAGGGCCTGGAGT

GGGTGGCCTCCATCCGCTCCGGCGGCGGCCGCACCTACTACTCCGACAA

CGTGAAGGGCCGCTTCACCATCTCCCGCGACAACTCCAAGAACACCCTG

TACCTGCAGATGAACTCCCTGCGCGCCGAGGACACCGCCGTGTACTACT

GCGTGCGCTACGACCACTACTCCGGCTCCTCCGACTACTGGGGCCAGGG

CACCCTGGTGACCGTGTCCTCCGGTGAGTCCTGTCGACTCTAGAGCTTT

CTGGGGCAGGCCAGGCCTGACTTTGGCTGGGGGCAGGGAGGGGGCTAAG

GTGACGCAGGTGGCGCCAGCCAGGCGCACACCCAATGCCCATGAGCCCA

GACCTGGACGCTGAACCTCGCGGACAGTTAAGAACCCAGGGGCCTCTGC

GCCCTGGGCCCAGCTCTGTCCCACACCGCGGTCACATGGCACCACCTCT

CTTGCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCTGGCACCCTCCTC

CAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC

TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCA

GCGGCGTGCACACTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCC

CTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCT

ACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGACAAGAAA

GTTGGTGAGAGGCCAGCACAGGGAGGGAGGGTGTCTGCTGGAAGCCAGG

CTCAGCGCTCCTGCCTGGACGCATCCCGGCTATGCAGTCCCAGTCCAGG

GCAGCAAGGCAGGCCCCGTCTCCTCTTCACCCGGAGGCCTCTGCCCGCC

CCACTCATGCTCAGGGAGAGGGTCTTCTGGCTTTTTCCCCAGGCTCTGG

GCAGGCACAGGCTAGGTGCCCCTAACCCAGGCCCTGCACACAAAGGGGC

AGTGCTGGGCTCAGACCTGCCAAGAGCCATATCCGGGAGGACCCTGCCC

CTGACCTAAGCCCACCCCAAAGGCCAAACTCTCCACTCCCTCAGCTCGG

ACACCTTCTCTCCTCCCAGATTCCAGTAACCCCAATCTTCTCTCTGCAG

AGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGGTAA

GCCAGCCCAGGCCTCGCCCTCCAGCTCAAGGCGGGACAGGTGCCCTAGA

GTAGCCTGCACCAGGGACAGGCCCCAGCCGGGTGCTGACACGTCCACCT

CCATCTCTTCCTCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCT

CTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGACCCCTGAGG

TCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT

CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCG

CGGGAGGAGCAGTACAACACACGTACCGTGTGGTCAGCGTCCTCACCGT

CCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC

AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAG

GGCAGCCCCGAGAACCACAGGTGTACACCCGCCCCCATCCCGGGAGGAG

ATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATC

CCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAA

CTACAAGACCCGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCT

ATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTT

CTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTAACGCAGAAGA

GCCTCTCCCTGTCCCCGGGTAAATGA

Example 3 removal of introns enhances protein expression

To determine the effect of intron removal on antibody expression, expression constructs of antibodies constructed using different numbers of introns may be used. The 12a11v3.1 variable region is stably expressed in CHO cells, while three constant region expression constructs having genomic sequences, cDNA sequences, and genomic sequences deleted for three introns (i.e., the intron between CH1 and the hinge region, the intron between the hinge region and CH2, and the intron between CH2 and CH3) are also stably expressed in CHO cells. For 12A11v3.1, the removal of the intron resulted in a significant increase in antibody expression. More specifically, the expression determined for the construct lacking the three introns of 12a11v3.1 was increased by about 5-fold compared to the genomic sequence. The 12a11v3.1 construct with the cDNA sequence showed an approximately 6-fold increase in expression compared to the genomic sequence. However, well-expressed antibodies generally do not show significant variation between intron-deleted sequences and genomic sequences when expressed in CHO-cells

Although the foregoing invention has been described in some detail for purposes of clarity of understanding, it will be apparent that certain modifications may be practiced within the scope of the appended claims. All publications and patent documents cited in this specification, and all words appearing in the figures and sequence listing, are herein incorporated by reference in their entirety for all purposes to the same extent as if each were individually indicated.

The specification is fully understood in light of the teachings of the references within the scope of the specification to which reference is made. The embodiments within the scope of the description provide illustration of embodiments in the present disclosure and should not be construed as limiting the scope of the invention. Those skilled in the art will readily appreciate that many other embodiments are encompassed by the present disclosure. All publications and patents cited in this disclosure, as well as sequences identified by accession numbers or database reference numbers, are incorporated by reference in their entirety. To the extent that the reference and the specification conflict or disagree, the specification can substitute any such material. Any reference cited herein is not meant to be an admission that such reference is prior art to the present disclosure.

Unless otherwise indicated, all numbers expressing quantities of ingredients, cell cultures, processing conditions, and so forth used in the specification, including the claims, are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters are values which are approximate and may vary depending upon the circumstances in which the desired properties of the invention are to be obtained. Unless otherwise indicated, the term "at least" preceding a list of elements is to be understood to refer to each element of the list. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the scope of the following claims.

Sequence listing

<110> Neurolab, Inc. (NEURALAB LIMITED) Huishi corporation (WYETH)

<120> METHODS AND COMPOSITIONS FOR improving recombinant protein PRODUCTION (METHODS AND COMPOSITIONS FOR improved recombinant protein PRODUCTION IN protein PRODUCTION)

<130>SCT071450-66

<150>60/616,474

<151>2005-10-04

<160>3

<170>FastSEQ for Windows Version 4.0

<210>1

<211>138

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized 3D6 v2 heavy chain variable region

<221>SIGNAL

<222>(1)...(19)

<400>1

Met Asn Phe Gly Leu Ser Leu Ile Phe Leu Val Leu Val Leu Lys Gly

-15 -10 -5

Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln

1 5 10

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

15 20 25

Ser Asn Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

30 35 40 45

Glu Trp Val Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser

50 55 60

Asp Asn Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

65 70 75

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

80 85 90

Tyr Tyr Cys Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp

95 100 105

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

110 115

<210>2

<211>414

<212>DNA

<213>Artificial Sequence

<220>

<223>Humanized 3D6 v2 heavy chain variable region

<400>2

atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggtgt ccagtgtgag 60

gtgcagctgc tggagtccgg cggcggcctg gtgcagcccg gcggctccct gcgcctgtcc 120

tgcgccgcct ccggcttcac cttctccaac tacggcatgt cctgggtgcg ccaggccccc 180

ggcaagggcc tggagtgggt ggcctccatc cgctccggcg gcggccgcac ctactactcc 240

gacaacgtga agggccgctt caccatctcc cgcgacaact ccaagaacac cctgtacctg 300

cagatgaact ccctgcgcgc cgaggacacc gccgtgtact actgcgtgcg ctacgaccac 360

tactccggct cctccgacta ctggggccag ggcaccctgg tgaccgtgtc ctcc 414

<210>3

<211>139

<212>PRT

<213>Artificial Sequence

<220>

<223>Humanized 12A11 v3.1heavy chain variable region

<221>SIGNAL

<222>(1)...(19)

<400>1

Met Glu Phe Gly Leu Ser TrpValPheLeuValAlaLeuLeuArgGlyValGlnCys

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly Phe Thr Leu Ser Thr Ser

20 25 30

Gly Met Ser Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Arg Thr Thr Thr Ala Asp Tyr Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210>4

<211>417

<212>DNA

<213>Artificial Sequence

<220>

<223>humanized 12A11 v3.1 VH sequence

<220>

<221>sig_peptide

<222>(1)...(57)

<400>4

atggagtttg ggctgagctg ggttttcctc gttgctcttc tgagaggtgt ccagtgtcaa 60

gttcagctgg tggagtctgg cggcggggtg gtgcagcccg gacggtccct caggctgtct 120

tgtgctttct ctgggttCAC actgagcact tctggtatga gtgtgggctg gattcgtcag 180

gctccaggga agggtctgga gtggctggca cacatttggt gggatgatga taagtactat 240

aacccatccc tgaagagccg ATTcacaatc tcCAGggaCA Actccaaaaa cacGCTgtac 300

ctccagatga acagtctgcg ggctgaagat actgccgtgt actactgtgc tcgaagaact 360

actaccgctg actactttgc ctactggggc caaggcacca ctgtcacagt ctcctca 417

SEQ ID NO：5

>nucleotide sequence for human IgG1

GTGAGTCCTGTCGACTCTAGAGCTTTCTGGGGCAGGCCAGGCCTGACTTTGGCTGGGGGCAGGGAGGGGGCTAAGGT

GACGCAGGTGGCGCCAGCCAGGCGCACACCCAATGCCCATGAGCCCAGACCTGGACGCTGAACCTCGCGGACAGTTA

AGAACCCAGGGGCCTCTGCGCCCTGGGCCCAGCTCTGTCCCACACCGCGGTCACATGGCACCACCTCTCTTGCAGCC

TCCACCAAGGGCCCATCGGTCTTCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGC

CTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACTTC

CCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCA

GACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGACAAGAAAGTTGGTGAGAGGCCAGCACAGGG

AGGGAGGGTGTCTGCTGGAAGCCAGGCTCAGCGCTCCTGCCTGGACGCATCCCGGCTATGCAGTCCCAGTCCAGGGC

AGCAAGGCAGGCCCCGTCTCCTCTTCACCCGGAGGCCTCTGCCCGCCCCACTCATGCTCAGGGAGAGGGTCTTCTGG

CTTTTTCCCCAGGCTCTGGGCAGGCACAGGCTAGGTGCCCCTAACCCAGGCCCTGCACACAAAGGGGCAGTGCTGGG

CTCAGACCTGCCAAGAGCCATATCCGGGAGGACCCTGCCCCTGACCTAAGCCCACCCCAAAGGCCAAACTCTCCACT

CCCTCAGCTCGGACACCTTCTCTCCTCCCAGATTCCAGTAACCCCAATCTTCTCTCTGCAGAGCCCAAATCTTGTGA

CAAAACTCACACATGCCCACCGTGCCCAGGTAAGCCAGCCCAGGCCTCGCCCTCCAGCTCAAGGCGGGACAGGTGCC

CTAGAGTAGCCTGCACCAGGGACAGGCCCCAGCCGGGTGCTGACACGTCCACCTCCATCTCTTCCTCAGCACCTGAA

CTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGACCCCTGAGGTC

ACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCA

TAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACACACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA

GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCT

CCAAAGCCAAAGTGGGACCCGTGGGGTGCGAGGGCCACATGGACAGAGGCCGGCTCGGCCCACCCTCTGCCCTGAGA

GTGACCGCTGTACCAACCTCTGTCCCTACAGGGCAGCCCCGAGAACCACAGGTGTACACCCGCCCCCATCCCGGGAG

GAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGA

GAGCAATGGGCAGCCGGAGAACAACTACAAGACCCGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAG

CAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACA

ACCACTAACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA

Claims (81)

1. A nucleic acid molecule encoding an a β -antibody chain, said nucleic acid molecule comprising a nucleotide sequence having one or more intron and exon sequences, wherein at least one intron sequence is deleted compared to the naturally occurring genomic sequence, thereby reducing mis-spliced or intron read-through (IRT) by-products.

2. A nucleic acid molecule encoding an a β -antibody chain, said nucleic acid molecule comprising a nucleotide sequence having one or more intron and exon sequences, wherein at least one intron sequence is deleted compared to the naturally occurring genomic sequence, thereby enhancing protein expression.

3. The nucleic acid molecule of claim 1 or 2, wherein at least 3 intron sequences are deleted.

4. The nucleic acid molecule of any one of claims 1-3, wherein the antibody chain is a heavy chain or a fragment thereof.

5. The nucleic acid molecule of claim 4, wherein the antibody heavy chain or fragment thereof comprises a heavy chain variable region, a hinge region, a first constant region (CH1), a second constant region (CH2) and a third constant region (CH3) of a human immunoglobulin G subtype.

6. The nucleic acid molecule of claim 5, wherein the immunoglobulin G subtype is human IgG1 or human IgG 4.

7. The nucleic acid molecule of claim 6, wherein the human IgG1 or human IgG4 is mutated.

8. The nucleic acid molecule of claim 5, wherein the intron between the CH2 region and the CH3 region of the immunoglobulin heavy chain constant region is deleted.

9. The nucleic acid molecule of claim 8, further comprising a deletion of an intron between the CH1 region and the hinge region.

10. The nucleic acid molecule of claim 8, further comprising a deletion of an intron between the hinge region and the CH2 region.

11. The nucleic acid molecule of claim 5 having a heavy chain comprising an intron between the variable region of the heavy chain and the CH1 region.

12. The nucleic acid molecule of claim 5, wherein the nucleotide sequence encoding the heavy chain hinge region, and the first, second and third constant regions comprises a sequence that is at least 95% identical to the nucleotide sequence set forth in FIG. 8(SEQ ID NO: 1).

13. The nucleic acid molecule of claim 5, wherein the nucleotide sequence encoding the heavy chain hinge region, and the first, second and third constant regions comprises a sequence that is at least 95% identical to the nucleotide sequence set forth in FIG. 9(SEQ ID NO: 3).

14. The nucleic acid molecule as claimed in claim 8, wherein the deletion of the intron between CH2 and CH3 corresponds to nucleotide positions approximately 1409-1505 of human IgG1 as shown in figure 8(SEQ ID NO: 1).

15. The nucleic acid molecule according to claim 8, wherein the deletion of the intron between CH2 and CH3 corresponds to nucleotide position 1401-1497 of human IgG4 as depicted in FIG. 9(SEQ ID NO: 3).

16. The nucleic acid molecule as claimed in claim 9, wherein the deletion of the intron between CH1 and the hinge region corresponds to nucleotide position 525 and 915 of human IgG1 as shown in figure 8(SEQ ID NO: 1).

17. The nucleic acid molecule as claimed in claim 9, wherein the deletion of the intron between CH1 and the hinge region corresponds to nucleotide position 525-916 of human IgG4 as shown in figure 9(SEQ ID NO: 3).

18. The nucleic acid molecule of claim 10 wherein the deletion of the intron between the hinge region and CH2 corresponds to nucleotides 961-1078 of human IgG1 as depicted in figure 8(SEQ ID NO: 1).

19. The nucleic acid molecule of claim 10 wherein the deletion of the intron between the hinge region and CH2 corresponds to nucleotide position 953-1070 of human IgG4 as depicted in figure 9(SEQ ID NO: 3).

20. A nucleic acid molecule comprising a nucleotide sequence encoding human IgG1, wherein said nucleotide sequence has at least 90% identity to the sequence set forth in FIG. 10(SEQ ID NO: 5).

21. A nucleic acid molecule comprising a nucleotide sequence encoding human IgG4, wherein said nucleotide sequence has at least 90% identity to the sequence set forth in FIG. 11(SEQ ID NO: 6).

22. A genomic nucleotide sequence encoding a human heavy chain constant region, or a mutated form thereof, wherein the nucleotide sequence lacks at least one intron that is present in the naturally-occurring genomic sequence, and wherein the intron facilitates intron read-through.

23. A genomic nucleotide sequence encoding human IgG1, or a mutated form thereof, wherein the nucleotide sequence lacks at least one intron that is present in the naturally occurring genomic sequence, and wherein the intron facilitates intron read-through.

24. The nucleotide sequence of claim 22 or 23, wherein the at least one intron is an intron located between constant regions CH2 and CH 3.

25. A genomic nucleotide sequence encoding human IgG4, or a mutated form thereof, wherein the genomic sequence lacks three introns that are present in the naturally occurring genomic sequence.

26. The nucleotide sequence of claim 25, wherein the intron is an intron between CH1 and the hinge region, an intron between the hinge region and CH2, and an intron between CH2 and CH 3.

27. A nucleic acid molecule encoding an antibody that selectively binds to a β peptide, said nucleic acid molecule comprising a nucleotide sequence represented by the formula:

V_H-Int1-C_H1-Int 2-hinge-Int 3-C_H2-C_H3，

Wherein V_HIs a nucleotide sequence encoding a heavy chain variable region;

C_H1，C_H2 and C_H3 is a nucleotide sequence encoding the corresponding heavy chain constant region;

the hinge is a nucleotide sequence encoding the hinge region of the heavy chain constant region; and is

Int1, Int2, and Int3 are introns from heavy chain genomic sequences.

28. The nucleotide sequence of claim 27, wherein the nucleotide sequence encodes the heavy chain of human immunoglobulin G.

29. A nucleic acid molecule encoding an antibody that selectively binds to a β peptide, said nucleic acid molecule comprising a nucleotide sequence represented by the formula:

V_H-Int1-C_H1-hinge-C_H2-C_H3，

Wherein V_HIs a nucleotide sequence encoding a heavy chain variable region;

C_H1，C_H2 and C_H3 is a nucleotide sequence encoding the corresponding heavy chain constant region;

the hinge is a nucleotide sequence encoding the hinge region of the heavy chain constant region; and

int1 is an intron from the heavy chain genomic sequence.

30. The nucleotide sequence of claim 29, wherein the nucleotide sequence encodes the heavy chain of human immunoglobulin G.

31. An expression cassette comprising the nucleic acid molecule of claim 5.

32. An expression vector comprising the nucleic acid molecule of claim 5.

33. The expression vector of claim 32, further comprising one or more nucleotide sequences that enhance replication, selection, mRNA transcription, mRNA stability, protein expression, or protein secretion in a host cell.

34. A host cell comprising the nucleic acid molecule of claim 5.

35. A host cell comprising the expression cassette of claim 31.

36. A host cell comprising the expression vector of claim 32.

37. The host cell of claim 36, which is a Chinese Hamster Ovary (CHO) cell.

38. A method of expressing a recombinant antibody or fragment thereof that selectively binds to an a β peptide and is substantially free of an intron read-through (IRT) product, the method comprising:

introducing into a mammalian host cell the nucleic acid molecule of claim 5;

culturing the host cell under conditions that allow for expression of the recombinant antibody or fragment thereof, thereby producing a culture of host cells; and

obtaining the recombinant antibody or fragment thereof from a culture of the host cell.

39. The method of claim 38, further comprising the step of identifying an IRT product in a nucleic acid sample from said host cell.

40. The method of claim 39, wherein said identifying step comprises:

obtaining a nucleic acid sample from a culture of host cells;

contacting the nucleic acid sample with a nucleic acid probe complementary to intron and adjacent exon sequences under conditions that allow hybridization of the nucleic acid sample and probe;

detecting the resulting complex, wherein detection in a sample of the complex using a nucleic acid probe complementary to an intron sequence indicates the presence of an IRT product.

41. The method of claim 38, wherein the host cell comprises a nucleotide sequence encoding a light chain variable and constant region.

42. A method of enhancing expression of a recombinant antibody or fragment thereof that selectively binds to an a β peptide, comprising:

introducing into a mammalian host cell the nucleic acid molecule of claim 5;

culturing the host cell under conditions that allow expression of the recombinant antibody, thereby producing a culture of host cells; and

recombinant antibodies are obtained from a culture of host cells.

43. The method of claim 42, wherein the host cell comprises a nucleotide sequence encoding a light chain variable region and a constant region.

44. A method of making a recombinant antibody or fragment thereof that selectively binds to an Α β peptide and is substantially free of intron read-through (IRT) heavy chain by-products, the method comprising:

culturing a mammalian host cell containing the nucleic acid molecule of claim 5 and nucleic acid encoding an antibody light chain of an antibody that selectively binds to an a β peptide under conditions such that the heavy and light chains are expressed.

45. The method of claim 44, further comprising purifying the heavy and light chains from the culture.

46. A method of enhancing expression of a recombinant antibody or fragment thereof that selectively binds to a β peptide, comprising:

culturing a mammalian host cell containing the nucleic acid molecule of claim 5 and nucleic acid encoding an antibody light chain of an antibody that selectively binds to an a β peptide under conditions such that the heavy and light chains are expressed.

47. The method of claim 46, further comprising purifying the heavy and light chains from the culture.

48. A method of detecting IRT product in a sample comprising:

obtaining a nucleic acid sample from the recombinant cell;

contacting the nucleic acid sample with a nucleic acid probe complementary to intron and adjacent exon sequences under conditions that allow hybridization of the nucleic acid sample and probe;

detecting the resulting complex, wherein detection in a sample of said complex using a nucleic acid probe complementary to the intron sequence indicates the presence of an IRT product.

49. An antibody or antigen-binding fragment thereof that selectively binds to an a β peptide, prepared by a method comprising the steps of claim 40, under suitable conditions that allow expression and assembly of the antibody or fragment.

50. The antibody of claim 49, which is a chimeric antibody, a humanized antibody, a CDR-grafted antibody or an in vitro generated antibody.

51. The antibody of claim 50 which is a humanized antibody.

52. The antibody of claim 51 which binds to human 5T 4.

53. A pharmaceutical composition comprising the antibody of claim 49 and a pharmaceutically acceptable carrier.

54. An isolated nucleic acid molecule encoding an antibody heavy chain that selectively binds to an a β peptide, said nucleic acid molecule comprising modified operably linked human genomic intron and exon sequences, wherein the modification of the natural operable linkage of the intron and exon sequences reduces or eliminates expression of an intron read-through heavy chain byproduct.

55. The isolated nucleic acid molecule of claim 54, wherein expression of said heavy chain is enhanced as compared to a polypeptide comprising naturally operably linked human genomic intron and exon sequences.

56. The nucleic acid molecule of claim 55 wherein the genomic exon sequences encode a variable region, a hinge region, and first, second and third constant regions.

57. The nucleic acid molecule of claim 56, wherein the first, second and third constant regions are IgG1 constant regions.

58. The nucleic acid molecule of claim 56, wherein the first, second and third constant regions are IgG4 constant regions

59. The nucleic acid molecule of claim 56 wherein the variable region is a humanized variable region.

60. The nucleic acid molecule of claims 56-59 wherein the variable region specifically binds to the A β peptide.

61. A nucleic acid molecule according to claims 56-59 wherein the variable region comprises Complementarity Determining Regions (CDRs) from a mouse 3D6 antibody.

62. The nucleic acid molecule of claim 54 comprising an intron sequence.

63. The nucleic acid molecule of claim 54 comprising two intron sequences.

64. The nucleic acid molecule of claim 54 comprising three intron sequences.

65. A nucleic acid molecule encoding an antibody heavy chain that binds to an a β peptide, said nucleic acid molecule comprising an exon encoding a variable region operably linked to exons encoding a first, second and third constant region, said nucleic acid molecule further comprising at least one intron sequence, wherein said intron sequence enhances expression of said heavy chain from said nucleic acid molecule compared to a nucleic acid molecule encoding an antibody heavy chain that binds to an a β peptide without said intron, and wherein said nucleic acid molecule does not result in translation of an intron read-through (IRT) heavy chain byproduct.

66. An expression cassette comprising a nucleic acid molecule according to any one of the preceding claims.

67. An expression vector comprising the nucleic acid molecule of any preceding claim.

68. The expression vector of claim 67, further comprising a gene encoding a selectable marker and an internal ribosome entry site sequence (IRES).

69. A cell comprising the nucleic acid molecule of any one of claims 54-68.

70. A mammalian cell containing the cassette of claim 66.

71. A mammalian cell containing the vector of claim 67 or 68.

72. The cell of claim 70 or 71, which is a Chinese Hamster Ovary (CHO) cell.

73. A nucleotide sequence encoding the heavy chain of IgG1, said sequence having the general formula:

V_H-Int1-C_H1-Int 2-hinge-Int 3-C_H2-C_H3

Wherein V_HIs any exon encoding the variable region of the heavy chain;

C_H1，C_H2 and C_H3 is an exon encoding the human heavy chain constant region obtained from the naturally occurring IgG1 heavy chain gene;

int1, Int2, and Int3 are the corresponding introns from the gene, and Int2 and Int3 may optionally be present; and is

The hinge is the exon encoding the hinge obtained from the gene.

74. The sequence of claim 73, wherein Int2 and Int3 are present.

75. A method of making an antibody formulation substantially free of intron read-through (IRT) heavy chain by-products comprising:

culturing the cell of any one of claims 70-72, further comprising a nucleic acid molecule encoding an antibody light chain that binds to A β peptide, under conditions such that the heavy and light chains are expressed and the heavy and light chains are operably linked in the absence of an intron read-through (IRT) heavy chain byproduct.

76. The nucleic acid molecule of claim 1 or 2, wherein the antibody chain has the amino acid sequence of SEQ ID NO: 12, or a sequence shown in figure 12.

76. The nucleic acid molecule of claim 1 or 2, wherein the antibody chain has the amino acid sequence of SEQ ID NO: 13, or a sequence shown in figure 13.

77. An a β antibody comprising a heavy chain comprising an amino acid sequence consisting of SEQ ID NO: 6-11 and the constant region encoded by any one of the nucleic acids of SEQ ID NOs: 2, or a variable region encoded by the nucleic acid of (3).

78. An a β antibody comprising a heavy chain comprising an amino acid sequence consisting of SEQ ID NO: 6-11 and the constant region encoded by any one of the nucleic acids of SEQ ID NOs: 4, or a variable region encoded by the nucleic acid of (4).

79. An a β antibody comprising a heavy chain comprising an amino acid sequence consisting of SEQ ID NO: 6-11 and a variable region having an amino acid sequence as set forth in any of figures 12-14 or 16C-D.

80. The antibody of any one of claims 77-80, further comprising a corresponding light chain as set forth in one of figures 15 or 16A-B.