Patent Application
20120149880
The invention relates to the field of molecular biology and immunology, particularly the field of recombinant antibody production.
Antibodies have been used as research tools for decades. More recently, antibodies have found use as diagnostic and therapeutic tools. For example, trastuzumab (sold by Genentech under the trademark Herceptin), an antibody that binds selectively to the HER2 protein, is FDA approved for the treatment of patients with HER2-positive breast cancer. Similarly, several antibodies have received FDA approval for use as diagnostic tools, including CEA-Scan for colorectal cancer detection, Myoscint for detecting myocardial injury, and Verluma for advanced small cell lung cancer.
However, production of antibodies by classical means (e.g., from an immortalized hybridoma cell line according to the method of Kohler and Milstein) may be hampered by the secretion rates of the antibody-producing hybridoma. Thus, efforts have been made to produce antibodies using recombinant DNA technology.
Various methods for generating recombinant antibodies are known in the art (see, e.g., U.S. Patent Publication No. 20070065912; U.S. Pat. No. 5,969,108; U.S. Pat. No. 6,331,415; U.S. Pat. No. 7,498,024; and U.S. Pat. No. 7,485,291, all of which are herein incorporated by reference in their entirety). Each of these methods (and other known in the art) has its weaknesses. Thus, there is a need for a new system to generate recombinant antibodies.
The invention provides a genetic cassette that can be used, applying standard molecular biology and cell biology techniques, to produce a recombinant antibody.
Accordingly, in a first aspect, the invention provides a nucleic acid cassette comprising components in the following structure in a 5′ to 3′ direction on a sense strand: A-B-C, where “A” is a nucleic acid sequence encoding at least an antigen binding domain of a light chain of a first antibody, “B” is a nucleic acid sequence encoding a 2A peptide, “C” is a nucleic acid sequence encoding at least an antigen binding domain of a heavy chain of a second antibody, and “-” is a bond selected from the group consisting of a phosphodiester bond and a phosphorothioate bond. In some embodiments, the “-” is a phosphodiester bond.
In another aspect, the invention provides a nucleic acid cassette comprising components in the following structure in a 5′ to 3′ direction on a sense strand: A-B-C, wherein “A” is a nucleic acid sequence encoding a light chain of a first antibody, “B” is a nucleic acid sequence encoding a 2A peptide, “C” is a nucleic acid sequence encoding a heavy chain of a second antibody, and “-” is a bond selected from the group consisting of a phosphodiester bond and a phosphorothioate bond. In some embodiments, the “-” is a phosphodiester bond.
In some embodiments, the nucleic acid cassette further comprises components in the following structure: A!-A-B-C!-C, where “A!” is a nucleic acid sequence encoding a first leader peptide, and “C!” is a nucleic acid sequence encoding a second leader peptide. In some embodiments, the first leader peptide is a light chain leader peptide. In some embodiments, the second leader peptide is a heavy chain leader peptide.
In some embodiments, the nucleic acid cassette further comprises components in the following structure: A-B-C-D, wherein “D” is a nucleic acid sequence encoding a tag. In yet a further embodiment, the nucleic acid cassette further comprising components in the following structure: A-p-B-C; wherein “p” is a nucleic acid sequence encoding a protease recognition site. In some embodiments, the protease recognition site is recognized by a thrombin protease. In some embodiments, the protease recognition site comprises the arginine residue and at least four amino acid residues N-terminally adjacent to the arginine residue in the amino acid sequences set forth in SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54. In some embodiments, the protease recognition site comprises the arginine residue and at least nine amino acid residues N-terminally adjacent to the arginine residue in the amino acid sequences set forth in SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54. In some embodiments, the protease recognition site comprises the arginine residue and at least eleven amino acid residues N-terminally adjacent to the arginine residue in the amino acid sequences set forth in SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54. In some embodiments, the protease recognition site comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54. In some embodiments, the protease recognition site comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 72, and SEQ ID NO: 73.
In a further aspect, the invention provides a nucleic acid cassette comprising components in the following structure: A-a-B-C, wherein “A” is a nucleic acid sequence encoding an antigen binding domain of a light chain of a first antibody, “a” is a nucleic acid sequence encoding a stem of a light chain of a second antibody, “B” is a nucleic acid sequence encoding a 2A peptide, “C” is a nucleic acid sequence encoding an antigen binding domain of a heavy chain of a third antibody, and “-” is a bond selected from the group consisting of a phosphodiester bond and a phosphorothioate bond. In some embodiments, the “-” is a phosphodiester bond. In some embodiments, the nucleic acid cassette has the structure: A-a-B-C-c, where “c” is a nucleic acid sequence encoding a stem of a heavy chain of a fourth antibody,
In some embodiments, the nucleic acid cassette further comprises components in the following structure: A!-A-a-B-C!-C, wherein “A!” is a nucleic acid sequence encoding a first leader peptide, and “C!” is a nucleic acid sequence encoding a second leader peptide. In some embodiments, the first leader peptide is from a light chain of an antibody. In some embodiments, the second leader peptide is from a heavy chain of an antibody.
In further embodiments, the nucleic acid cassette further comprises components in the following structure: A-a-B-C-D, where “D” is a nucleic acid sequence encoding a tag.
In another aspect, the nucleic acid cassette further comprising components in the following structure: A-a-p-B-C, wherein “p” is a nucleic acid sequence encoding a protease recognition site. In some embodiments, the protease recognition site is recognized by a thrombin protease. In some embodiments, the protease recognition site comprises the arginine residue and at least five amino acid residues N-terminally adjacent to the arginine residue in the amino acid sequences set forth in SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, or SEQ ID NO: 54. In some embodiments, the protease recognition site comprises the arginine residue and at least ten amino acid residues N-terminally adjacent to the arginine residue in the amino acid sequences set forth in SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 72, or SEQ ID NO: 73. In some embodiments, the protease recognition site comprises the arginine residue and at least twelve amino acid residues N-terminally adjacent to the arginine residue in the amino acid sequences set forth in SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 72, or SEQ ID NO: 73. In some embodiments, the protease recognition site comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 72, or SEQ ID NO: 73. In some embodiments, the protease recognition site comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 72, and SEQ ID NO: 73.
In various embodiments of all of the aspects of the invention, the first antibody and the second antibody are the same. In some embodiments, the third antibody and the fourth antibody are the same. In various embodiments, the “-” is a phosphodiester bond. In some embodiments, the first antibody and the second antibody are from the same species of animal. In some embodiments, the animal is a human, a mouse, a rabbit, or a rat. In some embodiments, the first antibody and the second antibody are of an isotype selected from the group consisting of IgG, IgD, IgA, IgE, and IgM.
In some embodiments, the 2A peptide comprises an amino acid sequence selected from the group consisting of DVEXNPGP (SEQ ID NO: 1) and DIEXNPGP (SEQ ID NO: 2), where X is any amino acid residue. In some embodiments, the 2A comprises an amino acid sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 3).
In a further aspect, the invention provides a vector, such as an expression vector, comprising the nucleic acid cassette of the invention.
In another aspect, the invention provides a method for making a recombinant antibody comprising (a) introducing the nucleic acid cassette of the invention into a cell such that it is expressed by the cell; (b) maintaining the cell of step (a) in a culture media, and isolating the antibody from the cell or the culture media of step (b).
In another aspect, the invention provides a method for producing a recombinant antibody comprising (a) introducing the nucleic acid cassette of the invention into a cell such that the cell expresses the nucleic acid cassette; (b) maintaining the cell of step (a) in a culture media, (c) isolating the antibody from the cell or the culture media of step (b), and (d) incubating the antibody of step (c) with a protease that cleaves the protease recognition site. In some embodiments, step (d) is performed under conditions whereby the protease cleaves the protease recognition site. In some embodiments, the protease is thrombin. In some embodiments, the protease recognition site is recognized by a thrombin protease. In some embodiments, the protease recognition site comprises the arginine residue and at least five amino acid residues N-terminally adjacent to the arginine residue in the amino acid sequences set forth in SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 72, or SEQ ID NO: 73. In some embodiments, the protease recognition site comprises the arginine residue and at least ten amino acid residues N-terminally adjacent to the arginine residue in the amino acid sequences set forth in SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 72, or SEQ ID NO: 73. In some embodiments, the protease recognition site comprises the arginine residue and at least twelve amino acid residues N-terminally adjacent to the arginine residue in the amino acid sequences set forth in SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 72, or SEQ ID NO: 73. In some embodiments, the protease recognition site comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 72, or SEQ ID NO: 73. In some embodiments, the protease recognition site comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 72, and SEQ ID NO: 73.
In another aspect, the invention provides a cell introduced with a nucleic acid cassette of the invention. In some embodiments, the cell expresses the nucleic acid cassette.
In a further aspect, the invention provides a recombinant antibody produced by a cell expressing a nucleic acid cassette of the invention.
In another aspect, the invention provides a kit comprising a first primer comprising a 5′ portion comprising a recognition site of a first restriction endonuclease and a 3′ portion that hybridizes to an antisense strand of a nucleic acid sequence encoding a leader peptide of a light chain of a first antibody; a second primer comprising a 5′ portion comprising a nucleic acid sequence that is complementary to a first part of a 2A-peptide encoding nucleic acid sequence and a 3′ portion that hybridizes to a nucleic acid sequence encoding a constant region of a light chain of a second antibody; a third primer comprising a 5′ portion comprising a nucleic acid sequence that encodes a second part of a 2A peptide and a 3′ portion that hybridizes to an antisense strand of a nucleic acid sequence encoding a leader peptide of a heavy chain of an third antibody; a fourth primer comprising a 3′ portion that hybridizes to a nucleic acid sequence encoding a constant region of a heavy chain; and instructions for using the first, second, third, and fourth primers to generate a nucleic acid cassette from a sample comprising nucleic acid encoding the first antibody, the second antibody, the third antibody, and the fourth antibody. In some embodiments, the fourth primer further comprises a 5′ portion comprising a recognition site of a second restriction endonuclease (or the two sites may be recognized by the same endonuclease with interrupted palindromic recognition sites with degenerate sequences for directional cloning).
In further aspects, the invention provides a kit comprising a first primer comprising a 5′ portion comprising a recognition site of a first restriction endonuclease and a 3′ portion that hybridizes to an antisense strand of a nucleic acid sequence encoding a leader peptide of a light chain of a first antibody; a second primer comprising a 5′ portion comprising a nucleic acid sequence that hybridizes to a 2A-peptide encoding nucleic acid sequence, a middle portion that hybridizes to a nucleic acid sequence encoding a protease recognition site, and a 3′ portion that hybridizes to a nucleic acid sequence encoding a constant region of a light chain of a second antibody; a third primer comprising a 5′ portion comprising a nucleic acid sequence that encodes the protease recognition site, a middle portion that encodes a 2A peptide and a 3′ portion that hybridizes to an antisense strand of a nucleic acid sequence encoding a leader peptide of a heavy chain of a third antibody; a fourth primer comprising a 3′ portion that hybridizes to a nucleic acid sequence encoding a constant region of a heavy chain of a fourth antibody; and instructions for using the first, second, third, and fourth primers to generate a nucleic acid cassette from a sample comprising nucleic acid encoding the first antibody, the second antibody, the third antibody, and the fourth antibody. In some embodiments, the fourth primer further comprises a 5′ portion comprising a recognition site of a second restriction endonuclease (or the two sites may be recognized by the same endonuclease with interrupted palindromic recognition sites with degenerate sequences for directional cloning). In some embodiments, the kit further comprises a protease that cleaves the protease recognition site (e.g., thrombin) and buffer for the protease cleavage reaction.
In some embodiments, the first antibody and the second antibody are the same. In some embodiments, the third antibody and the fourth antibody are the same. In some embodiments, the first, second, third, and fourth antibodies are the same:
In some embodiments, the kit further comprises a thermostable DNA polymerase (e.g., Taq polymerase). In some embodiments, the kit further comprises a first restriction endonuclease and a second restriction endonuclease. In some embodiments, the first restriction endonuclease and the second restriction endonuclease are the same. In some embodiments, the kit further comprises a vector comprising a polylinker (also known as a multi-cloning site) comprising the first restriction endonuclease recognition site and the second restriction endonuclease recognition site. In some embodiments, the kit further comprises a vector fragment of a vector comprising a polylinker comprising the first restriction endonuclease recognition site and the second restriction endonuclease recognition site digested with the first restriction endonuclease and the second restriction endonuclease.
In various embodiments, the kit comprises the amount of the first primer and the fourth primer exceeds the amount of the second primer and the third primer.
In a further aspect, the invention provides a method for making a nucleic acid cassette. The method comprises (a) amplifying a nucleic acid molecule encoding a light chain comprising a leader peptide and a constant region of a first antibody with a first primer comprising a 5′ portion comprising a recognition site of a first restriction endonuclease and a 3′ portion that hybridizes to an antisense strand of a nucleic acid sequence encoding the leader peptide of the light chain and a second primer comprising a 5′ portion comprising a nucleic acid sequence that is complementary to a first part of a 2A-peptide encoding nucleic acid sequence and a 3′ portion that hybridizes to a nucleic acid sequence encoding the constant region of the light chain and (b) amplifying a nucleic acid molecule encoding a heavy chain comprising a leader peptide and a constant region of a second antibody with a third primer comprising a 5′ portion comprising a nucleic acid sequence that encodes a second part of a 2A peptide and a 3′ portion that hybridizes to an antisense strand of a nucleic acid sequence encoding the leader peptide of the heavy chain and a fourth primer comprising a 3′ portion that hybridizes to a nucleic acid sequence encoding the constant region of the heavy chain of a third antibody. In step (c), the products of steps (a) and (b) are mixed and amplified with the first primer and the fourth primer to generate a full-length cassette. In some embodiments, the amplifying step is by polymerase chain reaction (PCR) amplification. In some embodiments, the fourth primer further comprises a 5′ portion comprising a recognition site of a second restriction endonuclease (or the two sites may be recognized by the same endonuclease with interrupted palindromic recognition sites with degenerate sequences for directional cloning).
In various embodiments, the method of the reaction is carried out in a two-step PCR reaction. In various embodiments, the method of the reaction is carried out in a single-step PCR reaction.
In further aspects, the invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34. SEQ ID NO: 36, SEQ ID. NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 57, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 67.
In yet further aspects, the invention provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 35, SEQ ID NO: 43, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, and SEQ ID NO: 73.
The invention provides a nucleic acid cassette that can be manipulated, using standard molecular biology and cell biology techniques, to produce a recombinant antibody. Schematic diagrams of two non-limiting representative nucleic acid cassettes of the invention are provided in
The further aspects, advantages, and embodiments of the invention are described in more detail below. The patents, published applications, and scientific literature referred to herein establish the knowledge of those with skill in the art and are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter. As used herein, the following terms have the meanings indicated. As used in this specification, the singular forms “a,” “an” and “the” specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise. The term “about” is used to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20%.
Technical and scientific terms used herein have the meaning commonly understood by one of skill in the art to which the present invention pertains, unless otherwise defined. Reference is made herein to various methodologies and materials known to those of skill in the art. Standard reference works setting forth the general principles of recombinant DNA technology and immunology include Ausubel et al., Current Protocols in Molecular Biology, Wiley InterScience, New York, N.Y., (2007, and updates up to and including 2009), Coligan et al., Current Protocols in Immunology Wiley InterScience, New York, N.Y., (2007, and updates up to and including 2009), Lo et al., Antibody Engineering: Methods and Protocols, Humana Press, 2003; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, New York (1989); Kaufman et al., Eds., Handbook of Molecular and Cellular Methods in Biology in Medicine, CRC Press, Boca Raton (1995); McPherson, Ed., Directed Mutagenesis: A Practical Approach, IRL Press, Oxford (1991). Standard reference works setting forth the general principles of pharmacology include Goodman and Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., McGraw Hill Companies Inc., New York (2006).
In a first aspect, the invention provides a nucleic acid cassette comprising components in the following structure in a 5′ to 3′ direction on a sense strand: A-B-C, where “A” is a nucleic acid sequence encoding at least an antigen binding domain of a light chain of a first antibody, “B” is a nucleic acid sequence encoding a 2A peptide, “C” is a nucleic acid sequence encoding at least an antigen binding domain of a heavy chain of a second antibody, and “-” is a bond selected from the group consisting of a phosphodiester bond and a phosphorothioate bond. In some embodiments, the “-” is a phosphodiester bond.
The invention stems from the unexpected discovery that the order of the nucleic acid encoding the light chain and the nucleic acid encoding the heavy chain in the nucleic acid cassette is important to the amount and quality of the encoded antibody secreted by a cell containing the nucleic acid cassette. For example, the order of heavy and light chain could be, instead, the heavy chain-encoding nucleic acid first, then the 2A peptide-encoding nucleic acid, followed by the light chain-encoding nucleic acid. Indeed, this H-2A-L cassette is described below in the Examples and compared to the L-2A-H cassette of the invention. The presence of the 2A peptide sequence adds a series of amino acids (17 amino acids in the case of T2A peptide) to the C-terminus of the first polypeptide and only a single proline to the N-terminus of the second polypeptide. Therefore, it is logical to place the heavy chain before and the light chain after the 2A peptide sequence (i.e., in an H-2A-L order) because the heavy chain constant region 3 (at the extreme C-terminus of the heavy chain) is the furthest away from the N-terminal variable region and the hydrophobic transmembrane domain lies in the membrane-bound forms of immunoglobulins (an isoform that is expressed due to alternate splicing that changes the site of polyadenylation (see Alt et al., Cell. 20(2): 293-301, 1980; Nelson et al., Mol Cell Biol. 3(7): 1317-1332, 1983). Thus, the addition of extra amino acids from the 2A peptide would seem to be least likely to affect binding of the antibody to its target antigen.
Accordingly, as described below in the Examples, nucleic acid cassettes encoding four antibodies were constructed and tested in both the H-2A-L and L-2A-H format to determine whether there was a difference in expression, secretion and activity of the antibody. Surprisingly, it was found that the light chain-2A-heavy chain (L-2A-H) configuration secreted a much higher level of functional antibody than the heavy chain-2A-light chain (H-2A-L) configuration. Thus, the order of the components of light chain-encoding and heavy chain-encoding components of the nucleic acid cassette of the invention is an unexpectedly important feature of the invention.
In another aspect, the invention provides a nucleic acid cassette comprising components in the following structure in a 5′ to 3′ direction on a sense strand: A-B-C, wherein “A” is a nucleic acid sequence encoding a light chain of a first antibody, “B” is a nucleic acid sequence encoding a 2A peptide, “C” is a nucleic acid sequence encoding a heavy chain of a second antibody, and “-” is a bond selected from the group consisting of a phosphodiester bond and a phosphorothioate bond. In some embodiments, the “-” is a phosphodiester bond.
In accordance with the invention, by a “nucleic acid cassette” is meant a structure into which one or more nucleic acid sequences can be inserted or from which one or more nucleic acid sequences can be removed, where the entire cassette itself can be inserted into or removed from a vector such as a plasmid, or the genome of a cell.
The terms “nucleic acid molecule,” and “nucleic acid sequence” are used interchangeably herein to refer to polymers of nucleotides of any length, and include, without limitation, DNA, RNA, DNA/RNA hybrids, and modifications thereof. Unless otherwise specified, where the nucleotide sequence is provided, the nucleotides are set forth in a 5′ to 3′ orientation. Thus, the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports. The 5′ and 3′ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. The nucleic acid molecules described herein may also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside.
As used herein, by “sense” strand of a double stranded nucleic acid molecule is meant the strand that encodes for a polypeptide. Thus, the orientation of the sense strand of a DNA molecule is the same as the orientation of an mRNA molecule (where all the T residues in the DNA are replaced by U in the mRNA molecule). Similarly, by “antisense” is meant the strand of a double stranded nucleic acid molecule that is complementary to the sense strand.
It shall be understood that when a structure of a cassette is provided (e.g., A-B-C), the “-” symbol may be a phosphodiester linkage, but may also be any type of linkage to join together two nucleotides (e.g., the 3′ nucleotide of the A molecule with the 5′ nucleotide of the B molecule). One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), “(O)NR.sub.2 (“amidate”), P(O)R, P(O)OR′, CO or CH.sub.2 (“formacetal”), in which each R or R′ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a nucleic acid cassette of the invention need be identical. The preceding description applies to all nucleic acid molecules referred to herein, including RNA and DNA.
Unless otherwise indicated, each nucleotide sequence (also called a nucleic acid sequence) set forth herein is presented as a sequence of deoxyribonucleotides (abbreviated A, G, C and T). However, by “sequence” of a nucleic acid molecule is intended, for a DNA nucleic acid molecule, a sequence of deoxyribonucleotides, and for an RNA nucleic acid molecule, the corresponding sequence of ribonucleotides (A, G, C and U), where each thymidine deoxyribonucleotide (T) in the specified deoxyribonucleotide sequence is replaced by the ribonucleotide uridine (U) in the corresponding ribonucleotide sequence. For instance, reference to an RNA molecule having the sequence set forth using deoxyribonucleotide abbreviations is intended to indicate an RNA molecule having a sequence in which each deoxyribonucleotide A, G or C of a DNA sequence has been replaced by the corresponding ribonucleotide A, G or C, and each deoxyribonucleotide T has been replaced by a ribonucleotide U.
The nucleic acid cassette of the invention includes a component encoding a 2A peptide. 2A peptides, which were identified in the Aphthovirus subgroup of picornaviruses, causes a ribosomal “skip” from one codon to the next without the formation of a peptide bond between the two amino acids encoded by the codons (see Donnelly et al., J. of General Virology 82: 1013-1025 (2001); Donnelly et al., J. of Gen. Virology 78: 13-21 (1997); Doronina et al., Mol. And Cell. Biology 28(13): 4227-4239 (2008); Atkins et al., RNA 13: 803-810 (2007)). By “codon” is meant three nucleotides on an mRNA (or on the sense strand of a DNA molecule) that are translated by a ribosome into one amino acid residue. Thus, two polypeptides can be synthesized from a single, contiguous open reading frame within an mRNA when the polypeptides are separated by a 2A oligopeptide sequence that is in frame. 2A peptides have been used to generate a recombinant, multi-chain T cell receptor (see, e.g., Szymczak et al., Nat. Biotechnol. 22: 589-594 (2004) and transgenic mice expressing a membrane-localized red fluorescent protein and nucleus-localized green fluorescent protein (see, e.g., Trichas et al., BMC Biology 6:40 (2008)).
In various embodiments, the 2A peptide comprises the amino acid sequence Asp-Val/Ile-Glu-X-Asn-Pro-Gly-Pro, where X is any amino acid residue and where translation can prematurely terminate after the glycine at the 7th position and restart from the proline at the 8th position. Note that in single letter code, this sequence is DVEXNPGP (SEQ ID NO: 1) or DIEXNPGP (SEQ ID NO: 2), where X is any amino acid residue and where translation can prematurely terminate after the glycine at the 7th position and restart from the proline at the 8th position. In some embodiments, the 2A peptide comprises the amino acid sequence QGWVPDLTVDGDVESNPGP (SEQ ID NO: 4), where translation can prematurely terminate after the glycine at the 18th position and restart from the proline at the 19th position. In some embodiments, the 2A peptide comprises the amino acid sequence GGGQKDLTQDGDIEPSNPGP (SEQ ID NO: 5), where translation can prematurely terminate after the glycine at the 19th position and restart from the proline at the 20th position. In some embodiments, the 2A peptide is from the Thosea asigna virus (and may be referred to as a T2A peptide) and comprises the amino acid sequence EGRGSLLTCGDVEENPGP (SEQ ID NO: 3), where translation can prematurely terminate after the glycine at the 17th position and restart from the proline at the 18th position.
The nucleic acid cassette of the invention may be flanked by sites (e.g., a restriction endonuclease recognition site) that facilitate its insertion or removal from a backbone nucleic acid molecule (e.g., a vector or the genome of a cell or animal). In some embodiments, where the cassette is flanked by restriction endonuclease sites, those sites do not occur within the cassette itself. For example, some relatively rare-cutting restriction endonucleases include AscI, Nod, SfiI, NruI, MluI, SacII, BssHII, PacI, BstEII, FseI, and BstXI.
In some embodiments, where the cassette is flanked by restriction endonuclease sites, the restriction endonuclease is able to cut a′ linearized nucleic acid molecule close to the end. For example, BamHI, EcoRI, HindIII, MluI, NcoI, NotI, XbaI, XhoI, and Pst1 are some non-limiting examples of restriction endonucleases that can cut at their recognition site when the recognition site is close to the end of a linearized nucleic acid molecule. Use of such restriction endonucleases facilitates cloning of a linearized nucleic acid molecule (e.g., a PCR product) into a vector or genome of a cell or animal.
In a further aspect, the invention provides a vector, such as an expression vector, comprising the nucleic acid cassette of the invention.
As described below in the examples (see, e.g, Example 1, and
Thus, used herein, by a “vector” is meant any construct capable of delivering one or more nucleic acid molecule(s) of interest to a host cell when the vector is introduced to the host cell or host animal. The host cell may be a eukaryotic or prokaryotic cell. In some embodiments, the cassette of the invention is constructed while components of the cassette are in a vector. In one non-limiting example, a vector comprising nucleic acid encoding the leader peptide sequence of a light chain of the antibody may be used as a backbone into which can be inserted a nucleic acid encoding the light chain of the antibody, the 2A peptide, the leader peptide of the heavy chain, and the heavy chain of the antibody.
An “expression vector” is capable of delivering and expressing the one or more nucleic acid molecule(s) of interest as encoded polypeptide in a host cell introduced with the expression vector. Thus, in an expression vector, a nucleic acid cassette is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, polyA signal/tail, etc., either within the vector or in the genome of the host cell at or near or flanking the integration site of the nucleic acid molecule(s) of interest such that the nucleic acid molecule(s) of interest will be translated in the host cell introduced with the expression vector.
Additional, non-limiting regulatory elements to which a nucleic acid cassette of the invention may be operably linked to facilitate its expression when introduced into a cell include promoters (e.g., the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs), sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by an expression vector comprising a nucleic acid cassette may include a translation initiation codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the nucleic acid cassette.
The nucleic acid cassette of the invention may be inserted into a vector containing a selectable marker for propagation in a host. In some embodiments, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells. The invention may be practiced with vectors comprising cis-acting control regions to the polynucleotide of interest. Appropriate trans-acting factors may be supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host. In certain embodiments in this regard, the vectors provide for specific expression, which may be inducible and/or cell type-specific (e.g., those inducible by environmental factors that are easy to manipulate, such as temperature and nutrient additives).
Thus, in another aspect, the invention provides a cell introduced with a nucleic acid cassette of the invention. In some embodiments, the cell expresses the nucleic acid cassette.
By “introduced” or “introducing” is meant that a nucleic acid molecule (e.g., a vector or a nucleic acid cassette) is inserted into the host cell by any means including, without limitation, electroporation, fusion with a vector-containing liposomes, chemical transfection (e.g., DEAE-dextran or calcium phosphate), transformation, cationic lipid-mediated transfection, transvection, and infection and/or transduction (e.g., with recombinant virus). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant virus), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents. Such methods are described in many standard laboratory manuals, such as Davis et al., B
Any cell of any species may be introduced with the nucleic acid cassette of the invention. Thus, mammalian cells (e.g., HeLa cells, COS cells, 293 cells (and variants thereof including 293T or 293EBNA), CV-1 cells, CHO cells), insect cells (e.g., S19 cells), yeast cells, and bacterial cells may be introduced with the nucleic acid cassette of the invention. In some embodiments, the introduced nucleic acid is positioned for expression in the cell such that the cell expresses the nucleic acid cassette (i.e., transcribes and/or translates the cassette) as antibody. To be expressed, the nucleic acid cassette may be on an expression vector, where the expression vector containing the nucleic acid cassette is introduced into a cell.
In some embodiments, the nucleic acid cassette of the invention may be introduced into a cell using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus, or may use a replication defective virus. In the latter case, viral propagation generally will occur only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. N.Y. Acad Sci. 569:86-103; Flexner et al., 1990, Vaccine 8:17-21; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner-Biotechniques 6:616-627, 1988; Rosenfeld et al., 1991, Science 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA 90:11498-11502; Guzman et al., 1993, Circulation 88:2838-2848; and Guzman et al., 1993, Cir. Res. 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art.
Of course, the inserted nucleic acid cassette of the invention need not be inserted into an expression vector prior to being introduced into a host cell. For example, a nucleic acid cassette may be introduced into a host cell by homologous recombination into the host cell's genome. If the inserted nucleic acid cassette is introduced into the genome such that it is operably linked to regulatory elements in that genome (e.g., the nucleic acid cassette is inserted downstream of a host cell's endogenous promoter), the nucleic acid cassette may be expressed (i.e., transcribed and translated) to allow the host cell to make recombinant antibody.
As indicated, when an expression vector is used, the expression vector may include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture media and conditions for the above-described host cells are known in the art.
Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
Non-limiting bacterial promoters suitable for use in the present invention include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV), the EF1α promoter, and metallothionein promoters, such as the mouse metallothionein-I promoter.
In the yeast, Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al. C
Transcription of DNA encoding an antibody of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually from about 10 to 300 by that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at basepairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
In some embodiments, the nucleic acid cassette of the invention (or an encoded antibody) may be purified. By “purified” (or “isolated”) refers to a molecule such as a nucleic acid sequence (e.g., a polynucleotide) or polypeptide (e.g., an antibody) that is removed or separated from other components present in its natural environment. For example, an isolated antibody is one that is separated from other components of a eukaryotic cell (e.g., the endoplasmic reticulum or cytoplasmic proteins and RNA). An antibody produced (e.g., expressed) by a nucleic acid cassette of the invention may also be purified from the 2A tail after cleavage of the tail from the light chain by a protease (e.g., thrombin). An isolated antibody-encoding nucleic acid sequence (e.g., a sequence that is a component of a nucleic acid cassette of the invention) is one that is separated from other nuclear components (e.g., histones) and/or from upstream or downstream nucleic acid sequences (e.g., an isolated antibody-encoding polynucleotide may be separated from the endogenous heavy chain or light chain promoter). An isolated nucleic acid molecule or polypeptide of the invention may be at least 60% free, or at least 75% free, or at least 90% free, or at least 95% free from other components present in natural environment of the indicated nucleic acid molecule or polypeptide.
As used herein, the terms “peptide”, “polypeptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, and it may comprise modified amino acids. Where the amino acid sequence is provided, unless otherwise specified, the sequence is in an N′ terminal (amino-terminal) to C′ terminal (carboxy terminal) orientation (e.g., a PPL sequence is N′ proline-proline-leucine-C′). In some embodiments, the polymer may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.); as well as other modifications known in the art. It is understood that, because the polypeptides of this invention are based upon an antibody, the polypeptides can occur as single chains or associated chains.
In a further aspect, the invention provides a recombinant antibody produced by a cell expressing a nucleic acid cassette of the invention.
Naturally occurring antibodies are made up of two classes of polypeptide chains, light chains and heavy chains. A non-limiting antibody of the invention can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains. The heavy chain of the antibody can be of any isotype including IgM, IgG, IgE, IgA or IgD or sub-isotype including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain.
As used herein, the term “antibody” is meant to include an immunoglobulin molecule of any isotype IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA) from any species (e.g., human, camelids (e.g., camels and llamas), chickens, goats, horse, cows, donkey, rabbits, and rodents (e.g., rats, mice, and hamsters).
In some embodiments, an antibody encoded by a nucleic acid cassette of the invention specifically binds to a target molecule. As used herein, by “specifically binding” or “specifically binds” means that an antibody of the invention interacts with its target molecule, where the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the target molecule; in other words, the reagent is recognizing and binding to a specific structure rather than to all molecules in general. An antibody that specifically binds to a target may be referred to a target-specific antibody (e.g., a HBsAg-specific antibody, which specifically binds the hepatitis H surface antigen). In some embodiments of the invention, an antibody that specifically binds to a target molecule provide a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay (e.g., (Western blotting, immunohistochemistry (IHC), flow cytometry, ELISA, Immunofluorescence, etc.). In some embodiments, antibodies that specifically bind to a target molecule do not detect other proteins in immunochemical assays and can immunoprecipitate the target molecule from solution.
In some embodiments, an antibody encoded by a nucleic acid cassette of the invention has a KD for its target molecule of 1×10−6M or less. In some embodiments, a binding agent of the invention binds to its target molecule with a KD of 1×10−7 M or less, or a KD of 1×10−8 M or less, or a KD of 1×10−9M or less, or a KD of 1×10−1° M or less, or a KD of 1×10−11M or less, or a KD of 1×10−12M or less. In certain embodiments, the KD of a binding agent of the invention for its target molecule is 1 pM to 500 pM, or between 500 pM to 1 μM, or between 1 μM to 100 nM, or between 100 mM to 10 nM. As used herein, by the term “KD”, is intended to refer to the dissociation constant of an interaction between two molecules (e.g., the dissociation constant between a binding agent (e.g., an antibody) and its specific target molecule).
A single naturally-occurring antibody comprises two identical copies of a light chain and two identical copies of a heavy chain. The heavy chains, which each contain one variable domain (VH) and multiple constant region domains (CHI, hinge, CH2, and CH3), bind to one another via disulfide bonding within their constant domains to form the stem of the antibody. The light chains, which each contain one variable domain (VL) and one constant region domain (CL), each bind to one heavy chain via disulfide bonding. The variable domain of each light chain is aligned with the variable domain of the heavy chain to which it is bound. The variable regions of both the light chains and heavy chains contain three hypervariable regions known as the complementary determining regions (CDRs), sandwiched between four more conserved framework regions (FR) for a structure FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
As used herein, an “antigen binding domain” is any portion of an antibody that is capable of specifically binding to a target molecule and includes, without limitation, some or all of one or more of the following elements, CDR1, CDR2, CDR3, from either the heavy chain or the light chain.
Methods for identifying the CDR and FR regions of an antibody by analyzing the amino acid sequence of the antibody are well known (see, e.g., Wu, T. T. and Kabat, E. A. (1970) J. Exp. Med. 132: 211-250; Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987)); Martin et al., Methods Enzymol. 203:121-53 (1991); Morea et al., Biophys Chem. 68(1-3):9-16 (October 1997); Morea et al., J Mol Biol. 275(2):269-94 (January 1998); Chothia et al., Nature 342(6252):877-83 (December 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007).
As one non-limiting example, the following method can be used to identify the CDRs of an antibody in most species.
For the CDR-L1, the CDR-L1 is approximately 10-17 amino acid residues in length. Generally, the start is at approximately residue 24 (the residue before the 24th residue is typically a cysteine. The CDR-L1 ends on the residue before a tryptophan residue. Typically, the sequence containing the tryptophan is either Trp-Tyr-Gln, Trp-Leu-Gln Trp-Phe-Gln, or Trp-Tyr-Leu, where the last residue within the CDR-L1 domain is the residue before the TRP in all of these sequences.
For the CDR-L2, the CDR-L2 is typically seven residues in length. Generally, the start of the CDR-L2 is approximately sixteen residues after the end of CDR-L1 and typically begins on the on the residue after the sequences of Ile-Tyr, Val-Tyr, Ile-Lys, or Ile-Phe.
For the CDR-L3, the CDR-L3 is typically 7-11 amino acid residues in length. Generally, the domain starts approximately 33 residues after the end of the CDR-L2 domain. The residue before the start of the domain is often a cysteine and the domain ends on the residue before Phe in the sequence Phe-Gly-XXX-Gly (where XXX is the three letter code of any single amino acid).
For the CDR-H1, the CDR-H1 domain is typically 10-12 amino acid residues in length and often starts on approximately residue 26. The domain typically starts four or five residues after a cysteine residue, and typically ends on the residue before a Trp (the Trp is often found in one of the following sequences: Trp-Val, Trp-Ile, or Trp-Ala.
For the CDR-H2, the CDR-H2 domain is typically 16 to 19 residues in length and typically starts 15 residues after the final residue of the CDR-H1 domain. The domain typically ends on the amino acid residue before the sequence Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala (which includes, for example, the sequences Lys-Leu-Thr and Arg-Ala-Ala).
For the CDR-H3, the CDR-H3 domain is typically 3-25 amino acids in length and typically starts 33 amino acid residues after the final residues of the CDR-H2 domain (which is frequently two amino acid residues after a cysteine residue, e.g., a cysteine in the sequence Cys-Ala-Arg). The domain ends on the amino acid immediately before the Trp in the sequence Trp-Gly-XXX-Gly (where XXX is the three letter code of any single amino acid; SEQ ID NO: 6).
In a further aspect, the invention provides a nucleic acid cassette comprising components in the following structure: A-a-B-C-c, wherein “A” is a nucleic acid sequence encoding an antigen binding domain of a light chain of a first antibody, “a” is a nucleic acid sequence encoding a stem of a light chain of a second antibody, “B” is a nucleic acid sequence encoding a 2A peptide, “C” is a nucleic acid sequence encoding an antigen binding domain of a heavy chain of a third antibody, “c” is a nucleic acid sequence encoding a stem of a heavy chain of a fourth antibody, and “-” is a bond selected from the group consisting of a phosphodiester bond and a phosphorothioate bond. In some embodiments, the “-” is a phosphodiester bond.
As used herein, a “stem” is any portion of an antibody that is located, in the naturally occurring antibody, carboxy-terminally to the variable domain of the antibody and includes, without limitation, some or all of one or more of the following elements: a CH1 region, a hinge region, a CH2 region, a CH3 region, and a light chain constant region (CL region). An antibody encoded by a nucleic acid cassette of the invention may comprise a light chain constant region that comprises some or all of a CL region.
Antibodies encoded by the nucleic acid cassettes of the invention include but are not limited to polyclonal, monoclonal, monospecific, polyspecific antibodies and fragments thereof and chimeric antibodies comprising an immunoglobulin binding domain fused to another polypeptide. Similarly, antibodies encoded by the nucleic acid cassettes of the invention can be derived from any species of animal, including mammals (e.g., rabbit, mouse, human, rat). Non-limiting exemplary natural antibodies include antibodies derived from human, camelids (e.g., camels and llamas), horse, cow, donkey, chicken, goats, and rodents (e.g., rats, mice, hamsters and rabbits), including transgenic rodents genetically engineered to produce human antibodies (see, e.g., Lonberg et al., WO93/12227; U.S. Pat. No. 5,545,806; and Kucherlapati, et al., WO91/10741; U.S. Pat. No. 6,150,584, which are herein incorporated by reference in their entirety). Natural antibodies are the antibodies produced by a host animal. Genetically altered antibodies refer to antibodies wherein the amino acid sequence has been varied from that of a native antibody. Because of the relevance of recombinant DNA techniques to this application, one need not be confined to the sequences of amino acids found in natural antibodies; antibodies can be redesigned to obtain desired characteristics. The possible variations are many and range from the changing of just one or a few amino acids to the complete redesign of, for example, the variable or constant region. Changes in the constant region will, in general, be made in order to improve or alter characteristics, such as complement fixation, interaction with membranes and other effector functions. Changes in the variable region will be made in order to improve the antigen binding characteristics.
The antibody encoded by the nucleic acid cassette of the invention may be expressed in a modified form, such as a fusion protein (e.g., a GST-fusion), and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
In one non-limiting example, an antibody encoded by the nucleic acid cassette of the invention may comprise a heterologous region from an immunoglobulin that is useful to solubilize proteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262). On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified in the advantageous manner described. This is the case when Fc portion proves to be a hindrance to use in therapy and diagnosis, for example when the fusion protein is to be used as antigen for immunizations. In drug discovery, for example, human proteins, such as, hIL-5 has been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. See Bennett et al., Journal of Molecular Recognition 8: 52-58 (1995) and Johanson et al., Journal of Biological Chemistry 270(16): 9459-9471 (1995).
In another aspect, the invention provides a method for making a recombinant antibody comprising introducing the nucleic acid cassette into a cell such that the nucleic acid cassette is expressed by the cell; maintaining the cell in a culture medium, and isolating the antibody from the cell or the culture medium.
The antibodies generated by using the nucleic acid cassettes of the invention can be recovered and purified from recombinant cell cultures by well-known methods including, without limitation, ammonium sulfate or ethanol precipitation, protein A-binding acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Where the antibody is not secreted, the host cell may first be lysed, and the antibody purified from the cell lysate. In some embodiments, high performance liquid chromatography (“HPLC”) is employed for purification. Depending upon the host employed in a recombinant production procedure (e.g., a eukaryotic or prokaryotic host cell), the antibodies generated using the nucleic acid cassettes of the present invention may be glycosylated or may be non-glycosylated. In addition, the antibodies may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
In some embodiments, the antibody generated as described herein are secreted by the host cell into which the nucleic acid cassette has been introduced. For secretion of the translated antibody into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate leader peptides may be incorporated into the expressed polypeptide (or nucleic acid sequence encoding the same in the nucleic acid cassette).
Thus, in some embodiments, the nucleic acid cassette of the invention further comprises leader peptide sequences upstream (i.e., 5′) of the nucleic acid sequence encoding the light chain (or antigen binding domain thereof) and upstream of the nucleic acid sequence encoding the heavy chain (or antigen binding domain thereof). In some embodiments, the leader peptide sequence located 5′ to the nucleic acid sequence encoding the light chain (or antigen binding domain thereof) is a light chain leader peptide. In some embodiments, the leader peptide sequence located 5′ to the nucleic acid sequence encoding the heavy chain (or antigen binding domain thereof) is a heavy chain leader peptide.
As used herein, by “leader peptide” or a “secretory signal peptide” is meant a peptide sequence comprising a sequence that enables the polypeptide positioned C′ to the leader peptide to be secreted from a cell expressing (e.g., transcribing and/or translating) the polypeptide. In some embodiments, the leader peptide is attached to the polypeptide by a peptide bond. In some embodiments, the leader peptide may be cleaved from the polypeptide. In some embodiments, the cleavage of the leader peptide from the polypeptide occurs prior to the secretion of the polypeptide from the cell. The leader peptide may be endogenous to the polypeptide or it may be heterologous (i.e., the leader peptide naturally occurs N-terminally to a different molecule). Thus, leader peptide from a secreted hormone (e.g., cholecystokinin) is heterologous to light chain polypeptide.
Leader peptides (i.e., secretory signals) are well known in the art, since all secreted proteins (including antibodies) comprise them. For example, Barash et al., Biochemical and Biophysical Research Communications 294 (4): 835-842 (2002) describe a hidden Markov model (HMM) has been used to describe, predict, identify, and generate secretory signal peptide sequences. Similarly, U.S. Pat. No. 6,733,997 describes a universal secretory signal peptide sequence. A nucleic acid sequence encoding secretory signal peptide can be ligated, in frame, to a nucleic acid sequence encoding an antibody chain according to standard methods.
In further embodiments, the invention provides a method for producing a recombinant antibody by maintaining host cell comprising a nucleic acid cassette of the invention under conditions suitable for the expression of antibody and recovering the antibody, either from lysates made from the cells or, where the nucleic acid cassette included a secretory signal, from the conditioned media of the host cells. Culture conditions suitable for the maintenance and/or growth of host cells and the expression of recombinant polypeptides (such as antibodies) from such cells are well known to those of skill in the art. See, e.g., C
The invention also provides cell lines that produce an antibody encoded by the nucleic acid cassettes of the invention. For example, the invention includes recombinant host cells producing an antibody of the invention, where such recombinant host cells may be constructed by introducing into them the nucleic acid cassette of the invention. Host cells may be eukaryotic or prokaryotic cells (see, e.g., A
In some embodiments, it may be desirable to insert a nucleic acid sequence encoding a protease recognition site after the light chain-encoding nucleic acid sequence in the cassette. The addition of such a protease recognition site would enable cleavage of the 2A peptide from the light chain encoded by the nucleic acid cassette of the invention. Accordingly, in another aspect, the invention provides a nucleic acid cassette comprising components in the following structure:
A-p-B-C,
where “A” is a nucleic acid sequence encoding at least an antigen binding domain of a light chain of a first antibody, “B” is a nucleic acid sequence encoding a 2A peptide, “C” is a nucleic acid sequence encoding at least an antigen binding domain of a heavy chain of a second antibody, “-” is a phosphodiester bond or a phosphorothioate bond, and “p” “p” is nucleic acid sequence encoding a protease recognition site.
As used herein, by “protease recognition site” is meant a specific amino acid sequence within a polypeptide (e.g., a protein) at which or after which (i.e., within which) a protease will cleave the polypeptide. Such proteases and their recognition sites include, without limitation, the furin protease (which cleaves after the final arginine residue in the sequences Arg-X-X-Arg (SEQ ID NO: 7); Arg-X-Lys-Arg (SEQ ID NO: 8); or Arg-X-Arg-Arg (SEQ ID NO: 9), where X is any amino acid residue), the enterokinase protease (which cleaves after the final lysine residue in the sequence Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 10)), the thrombin protease (which cleaves after the arginine residue in the sequence Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO: 55) and cleaves its natural ligand, fibrinogen, after the arginine residues), and the Factor Xa protease (which cleaves after the final arginine residues in the sequences Ile-Glu-Gly-Arg (SEQ ID NO: 11) or Ile-Asp-Gly-Arg (SEQ ID NO: 12)). All of the former proteases are commercially available (e.g., from New England Biolabs, Inc., Ipswich, Mass., or Sigma-Aldrich, Inc., St. Louis, Mo.).
In some embodiments, the protease of the invention is thrombin, and the protease recognition site is a sequence from a fibrinogen molecule. Fibrinogen (also called factor I) is a soluble plasma glycoprotein which, during blood coagulation, is converted into insoluble fibrin following cleavage by thrombin. Fibrinogen comprises two sets of three different chains (α, β, and γ). During blood clotting, thrombin cleaves the N-termini of the α and β chains of fibrinogen to form fibrinopeptide A and fibrinopeptide B, respectively. The amino acid sequences of human fibrinopeptide A and fibrinopeptide B, are thrombin protease recognition sites, are set forth below:
Note that in the human fibrinogen a chain, there is a second arginine residue following (i.e., C′ terminal to) the first arginine. In some embodiments, it may be desirable to include both arginine residues, to ensure cleavage after at least one of the arginines. Thus, another thrombin protease recognition site may have the following amino acid sequence:
Additional thrombin protease recognition sites include the following sequences:
Thrombin also cleaves at a consensus sequence, namely after the final Arg residue in the sequence Leu-Val-Pro-Arg (SEQ ID NO: 54).
In some embodiments, the “p” is nucleic acid sequence encoding a protease recognition site comprising the arginine residue (e.g., an arginine residue within the amino acid sequences set forth in SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 72, or SEQ ID NO: 73). In some embodiments, the “p” is nucleic acid sequence encoding a protease recognition site comprising the arginine residue and at least a few amino acid residues N-terminally adjacent to the arginine residue. By “N-terminally adjacent” means that the referenced amino acid residues are directly covalently linked to the N′ terminus of the arginine residue via a peptide bond. For example, the sequence Gly-Val-Arg-Gly-Pro-Arg (SEQ ID NO: 56) contains five amino acids that are N-terminally adjacent to the arginine residue in SEQ ID NO: 53.
In some embodiments, the “p” is nucleic acid sequence encoding a protease recognition site comprising the arginine residue and at least four amino acid residues N-terminally adjacent to the arginine residue in the amino acid sequences set forth in SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 72, or SEQ ID NO: 73. In some embodiments, the “p” is nucleic acid sequence encoding a protease recognition site comprising the arginine residue and at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten, or at least eleven, or at least twelve, or at least thirteen, or at least fourteen amino acid residues N-terminally adjacent to the arginine residue in the amino acid sequences set forth in SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 72, or SEQ ID NO: 73.
In particular embodiments, the “p” encodes a protease recognition site comprising an amino acid sequence set forth in SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 72, or SEQ ID NO: 73.
In various embodiments, where the “p” encodes an amino acid sequence from a chain of a fibrinogen molecule, the fibrinogen molecule is the fibrinogen that naturally occurs within the species from which the antibody sequences are derived. For example, SEQ ID NOs: 50, 51, 52, 72, and 73, are from human fibrinogen, and nucleic acid sequences encoding, e.g., SEQ ID NO: 50 (or a portion thereof) may be used for a genetic cassette encoding an antibody with constant regions from human antibodies (e.g., human Heavy constant and human Light constant regions):
The antibodies generated in accordance with the present invention may be employed in various methods. For example, the antibodies of the invention may be used in any known assay method, such competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987). For use in in vitro assays, the antibodies may be detectably labeled (e.g., with a fluorophore such as FITC or phycoerythrin or with an enzyme substrate, such as a substrate for horse radish peroxidase) for easy detection. The antibodies may also be generated such that one or both of the heavy and light chain is tagged.
Accordingly, in another aspect, the invention provides a nucleic acid cassette comprising components in the following structure:
A-B-C-D or A-D-B-C,
where “A” is a nucleic acid sequence encoding at least an antigen binding domain of a light chain of a first antibody, “B” is a nucleic acid sequence encoding a 2A peptide, “C” is a nucleic acid sequence encoding at least an antigen binding domain of a heavy chain of a second antibody, “-” is a phosphodiester bond and a phosphorothioate bond, and “D” is a nucleic acid sequence encoding a tag,
As used herein, a “tag” means a peptide structure that can be detected. Thus, a tag includes, without limitation, an epitope that can be recognized by an antibody, the ligand of a receptor, one partner of a binding partner pair (e.g., the streptavidin-biotin binding partner pair), a mass tag that produces an identifiable spectrum by mass spectrometry, a fluorescent label, a chromophoric label, and a marker protein. By “marker protein” is meant a polypeptide whose expression or activity indicates the amount of the polypeptide in the sample (e.g., in a cell). Non-limiting marker proteins include green fluorescent protein and horseradish peroxidase.
Non-limiting examples of tags (and their amino acid sequences) include the c-myc tag (EQKLISEEDL; SEQ ID NO: 13), the His tag (HHHHHH; SEQ ID NO: 14); the HA tag (YPYDVPDYA; SEQ ID NO: 15), the VSV-G tag (YTDIEMNRLGK; SEQ ID NO: 16), the HSV tag (QPELAPEDPED; SEQ ID NO: 17), the V5 tag (GKPIPNPLLGLDST; SEQ ID NO: 18), and Flag tag (DYKDDDDK; SEQ ID NO: 19).
In some embodiments, the tag identifies the source of the antibody encoded by the nucleic acid cassette.
In some embodiments, the tag is cleavable from the chain of the antibody. For example, as described below, many proteases (e.g., furin, Factor Xa, etc. . . . ) cleave at specific sites. Thus, a nucleic acid sequence encoding a protease recognition site (e.g., the Factor Xa protease recognition site Ile-Glu-Gly-Arg (SEQ ID NO: 11) or Ile-Asp-Gly-Arg (SEQ ID NO: 12)), can be inserted between the nucleic acid sequence encoding the heavy chain of an antibody and the nucleic acid sequence encoding the tag. This configuration would result in a nucleic acid cassette comprising components in the following structure:
A-B-C-p-D or A-p-D-B-C,
where “A” is a nucleic acid sequence encoding at least an antigen binding domain of a light chain of a first antibody, “B” is a nucleic acid sequence encoding a 2A peptide, “C” is a nucleic acid sequence encoding at least an antigen binding domain of a heavy chain of a second antibody, “-” is a phosphodiester bond and a phosphorothioate bond, “D” is a nucleic acid sequence encoding a tag, and “p” is a nucleic acid sequence encoding a protease recognition site.
It may also be desirable to introduce two protease recognition sites (which may or may not be cleavable by the same protease. For example, the invention provides the cassette:
A-p1-B-C-p2-D
where “A” is a nucleic acid sequence encoding at least an antigen binding domain of a light chain of a first antibody, “B” is a nucleic acid sequence encoding a 2A peptide, “C” is a nucleic acid sequence encoding at least an antigen binding domain of a heavy chain of a second antibody, “-” is a phosphodiester bond and a phosphorothioate bond, “D” is a nucleic acid sequence encoding a tag, “p1” is a nucleic acid sequence encoding a first protease recognition site and “p2” is a nucleic acid sequence encoding a second protease recognition site. In some embodiments, the first and second proteases are the same.
The ability to cleave the tag off of a recombinant antibody produced by a nucleic acid cassette of the invention is particularly useful, for example, where the antibody is a therapeutic antibody and it is being validated for its binding activity prior to introduction into the patient. For example, if the tag is detectable (e.g., a fluorophore), it may be desirable to validate its ability to bind its target (e.g., via a Western blot, ELISA, or by immunohistochemistry (IHC) blot). Once the antibody is found to have the required specificity, it can simply be incubated with the protease that cleaves the protease recognition site under conditions where the protease will cleave its site (e.g., where the protease is thrombin, conditions may include incubation with thrombin in thrombin cleavage buffer at 37° C. for at least an hour). After purification of the antibody to separate the antibody from its removed tag and 2A tail (e.g., by binding to and elution from a protein A sepharose column). The purified antibody may then be used to inject into a patient (e.g., a human or other mammal).
Note that additional elements can be inserted in component D in the above-described nucleic acid cassette. Thus, instead of a tag, the D can be nucleic acid sequence encoding any polypeptide. Some non-limiting examples include the J chain of an antibody (e.g., where an IgM isotype antibody is encoded by components A and C) or a selectable marker. In the latter example, where D encodes a selectable marker (e.g., neomycin resistance), cells expressing the nucleic acid cassette will be able to survive and/or grow in the presence of the drug (e.g., G418).
In further embodiments the antibodies generated in accordance with the invention may be used for in vivo diagnostic assays, such as in vivo imaging. In some embodiments, the antibody is labeled with a radionucleotide (such as 3H, 111In, 14C, 32P, or 123I) so that the cells or tissue of interest can be localized using immunoscintigraphy.
Methods of conjugating labels or tags to antibodies are known in the art. In other embodiments of the invention, the antibodies generated in accordance with the invention are not labeled, and the presence thereof is detected using a labeled secondary antibody, which binds to the first, unlabeled antibody.
The antibody may also be used as staining reagent in pathology, following techniques well known in the art.
In additional aspects, the invention provides numerous kits for generating the nucleic acid cassette of the invention. For example, the kits may comprise a first primer comprising a 5′ portion comprising a recognition site of a first restriction endonuclease and a 3′ portion that hybridizes to an antisense strand of a nucleic acid sequence encoding a leader peptide of a light chain of a first antibody; a second primer comprising a 5′ portion comprising a nucleic acid sequence that is complementary to a nucleic acid sequence that encodes a first part of a 2A peptide and a 3′ portion that hybridizes to a nucleic acid sequence encoding a constant region of a light chain of a second antibody; a third primer comprising a 5′ portion comprising a nucleic acid sequence that encodes a second part of a 2A peptide and a 3′ portion that hybridizes to an antisense strand of a nucleic acid sequence encoding a leader peptide of a heavy chain of a third antibody; a fourth primer comprising a 3′ portion that hybridizes to a nucleic acid sequence encoding a constant region of a heavy chain of a fourth antibody; and instructions for using the first, second, third, and fourth primers to generate a nucleic acid cassette from a sample comprising nucleic acid encoding the first antibody, the second antibody, the third antibody, and the fourth antibody. In some embodiments, the fourth primer may also include a 5′ portion comprising a recognition site of a second restriction endonuclease.
The invention also provides kits for generating a nucleic acid cassette with instructions, and a first and a fourth primer as described above, but with a second primer comprising a 5′ portion comprising a nucleic acid sequence that hybridizes to a 2A-peptide encoding nucleic acid sequence, a middle portion that hybridizes to a nucleic acid sequence encoding a protease recognition site, and a 3′ portion that hybridizes to a nucleic acid sequence encoding a constant region of a light chain of a second antibody and a third primer comprising a 5′ portion comprising a nucleic acid sequence that encodes the protease recognition site, a middle portion that encodes a 2A peptide and a 3′ portion that hybridizes to an antisense strand of a nucleic acid sequence encoding a leader peptide of a heavy chain of a third antibody. In some embodiments, the kit further comprises a protease that will cleave the protease recognition site. For example, if the protease recognition site encoded by the nucleic acid cassette is a thrombin recognition site, the kit further comprises thrombin protease. The kit may also comprise instructions for cleaving the antibody encoded by the nucleic acid cassette with the protease and a buffer to create conditions under which the protease will cleave the antibody.
As used herein, by “hybridize” is meant that a primer (e.g., a PCR primer) anneals to another single stranded nucleic acid molecule to form a double stranded nucleic acid molecule. In general, hybridization (i.e., annealing) should occur under stringent conditions (see, e.g., Ausubel et al., supra).
A typical PCR program consists of:
Step 1—95 C for 2-5 minutes
Step 2—95 C for 30 seconds
Step 3—55-72 C for 30 seconds
Step 4—72 C for 1 minute/kb of product
Step 5—go back to Step 2, 29 times (for 30 cycle reaction)—can be adjusted as needed
Step 6—72 C for 5-10 minutes
The annealing temperature at Step 3 can be raised to increase specificity of priming (i.e., increasing stringency). Typically, it is set at 2-5 degrees lower than the predicted Tm of the oligos (which should have similar Tm to each others'), but it can be raised to as high as the extension temperature (Step 4). Another method for increasing the specificity of priming (i.e., increasing stringency) is by decreasing Mg2+ concentration to below 2 mM. To determine Tm of a primer, any of a variety of methods may be used.
One non-limiting formula is:
Tm=81.5° C.+16.6(log [M+])+0.41(% G+C)−(500/N), where [M+] is the concentration of monovalent cation in the PCR reaction (e.g., KCl) in moles/liter, N is the number of nucleotides in the primer, and % G+C is the percentage of G and C residues in the primer (e.g., a 14 nucleotide long primer with 4 G residues and 3 C residues has a % G+C of 50%).
Another non-limiting method for determining Tm of a primer is:
Tm=64.9° C.+41° C.×(number of G's and C's in the primer−16.4)/N, where N is the number of nucleotides in the primer.
Thus, as used herein, by “stringent conditions” is meant that the primer will hybridize (i.e., anneal) to the single stranded target molecule (a) within a range from about Tm minus 2° C. (2° C. below the melting temperature (Tm) of the probe or sequence) to about 20° C. to 25° C. above Tm or (b) in a solution containing a concentration of Mg2+ that is equal to or less concentrated than 2 mM. It will be understood by those of skill in the art that the stringency of hybridization may be determined on a PCR.
As used herein, by “portion” is meant any subset of the whole sequence of the primer. Thus, a portion of a primer of 20 nucleotides in length is meant any sequence that is 19 nucleotides in length or fewer. Of course, if there are two portions in the same primer (e.g., a 5′ portion and a 3′ portion), one of the two portions is at least 25% as long as the other portion. For example, in the 20 nucleotide long primer, the 5′ portion may comprise 5 nucleotides or more and the 3′ portion may comprise 15 nucleotides or fewer. In some embodiments, where there are two portions in the same primer, one of the two portions is at least 30% or at least 35% or at least 40% as long as the other portion.
In some embodiments of the kit of the invention, the first antibody and the second antibody are the same. In some embodiments, the third antibody and the fourth antibody are the same. In some embodiments, the first antibody, second antibody, third antibody, and fourth antibody are the same.
In some embodiments of the above kits of the invention, the amount of the first primer is approximately the same as the amount of fourth primer, and the amount of second primer is approximately the same as the amount of the third primer. In some embodiments, the amount of the first and fourth primers exceeds the amount of the second and third primers by twice as much, or five times as much, or ten times as much, or twenty times as much, or forty times as much, or one hundred times as much.
It should be noted that when referring to amounts of primers, it is within the skill of the ordinarily skilled artisan to determine what amount is appropriate to achieve the best results (in this case, generation of a nucleic acid cassette). For example, for primers, either the absolute concentration of the primer can be referred to (e.g., μg/ml), or the actual number of primers can be the same (if, for example, the length of one of the primers exceeds the other. For example, if the first primer is twice as long as the fourth primer, the same concentration of both the first and the fourth primers would result in there being twice as many fourth primer molecules as first primer molecules. In this example, to achieve approximately the same amount of the first and the fourth primers, the skilled artisan may choose to rely upon the number of molecules of each of the first and the fourth primers.
In some embodiments, the kit further comprises a thermostable DNA polymerase (e.g., Taq polymerase). In some embodiments, the kit further comprises a first restriction endonuclease and a second restriction endonuclease. In some embodiments, the first and the second restriction endonuclease are the same. For example, where a restriction endonuclease recognizes a site that has a degenerate (i.e., variable) sequences, the same restriction endonuclease can recognize two different recognition sites. For example, the restriction endonuclease SfiI recognizes the site: 5′GGCCNNNN*NGGCC3′ (SEQ ID NO: 20) cutting at the * to create a 3′ overhang of -NNNN 3′, where “N” can be any nucleotide. Those “N” sequences in the first and second endonuclease recognition sites can differ, but both the first and the second endonuclease sites would still be recognized and cut by SfiI.
In some embodiments, the kit further comprises a vector comprising a polylinker comprising the first restriction endonuclease recognition site and the second restriction endonuclease recognition site. In some embodiments, the kit further comprises a vector fragment of a vector comprising a polylinker comprising the first restriction endonuclease recognition site and the second restriction endonuclease recognition site digested with the first restriction endonuclease and the second restriction endonuclease.
As used herein, by “polylinker” is meant a portion of a vector (e.g., an expression vector) that contains many unique restriction endonuclease recognition sites (i.e., the site does not occur elsewhere in the vector). Typically, in an expression cloning vector, the polylinker occurs downstream of a promoter sequence and upstream of a polyA signal.
In a further aspect, the invention provides a method for making a nucleic acid cassette. The method comprises (a) amplifying a nucleic acid molecule encoding a light chain comprising a leader peptide and a constant region of a first antibody with a first primer comprising a 5′ portion comprising a recognition site of a first restriction endonuclease and a 3′ portion that hybridizes to an antisense strand of a nucleic acid sequence encoding the leader peptide of the light chain and a second primer comprising a 5′ portion comprising a nucleic acid sequence that is complementary to a first part of a 2A-peptide encoding nucleic acid sequence and a 3′ portion that hybridizes to a nucleic acid sequence encoding the constant region of the light chain and (b) amplifying a nucleic acid molecule encoding a heavy chain comprising a leader peptide and a constant region of a second antibody with a third primer comprising a 5′ portion comprising a nucleic acid sequence that encodes a second part of a 2A peptide and a 3′ portion that hybridizes to an antisense strand of a nucleic acid sequence encoding the leader peptide of the heavy chain and a fourth primer comprising a 3′ portion that hybridizes to a nucleic acid sequence encoding the constant region of the heavy chain. In some embodiments, the fourth primer also includes a 5′ portion comprising a recognition site of a second restriction endonuclease. In step (c), the products of step (a) and step (b) are allowed to anneal (i.e., hybridize) and in step (d), the product of step (c) is amplified with the first primer and the fourth primer. In some embodiments, the amplifying step is by polymerase chain reaction (PCR) amplification.
As described below, the steps (a) through (d) of the method of the invention may be performed in a two-step PCR method, or may be performed in a one-step PCR method (i.e., in the same PCR reaction). In various embodiments, the first and second antibody are the same.
The following examples are provided to illustrate, but not to limit, the invention.
The light chain-2A-heavy chain (L-2A-H) cassette encoding an anti-MRPL11 rabbit IgG antibody was assembled as a single molecule of DNA using two steps of polymerase chain reaction (PCR). A schematic representation of the resulting cassette is shown in
CCCCTATTGAAGCT 3′
ACTGGGCTGCGCT 3′
Primer A (sense) was designed to hybridize to the 5′ end of the rabbit kappa chain leader sequence and contains additional 5′ sequence that includes a HindIII restriction site. Primer B (antisense) was designed to hybridize to the 3′ end of the rabbit kappa chain constant region and also contains 39 nucleotides encoding the N-terminal half of the T2A peptide sequence in its 5′ end. Primer C (sense) was designed to hybridize to the 5′ end of the rabbit heavy chain leader sequence and also contains 40 nucleotides encoding the C-terminal half of the T2A peptide sequence in its 5′ end. Note that the number of nucleotides encoding the T2A peptide (or any other 2A peptide used) in Primers B and C may be varied as long as they contain enough complementary nucleotides (about 20 nucleotides) to each other for efficient assembly in the second step of PCR.
Primer D (antisense) was designed to hybridize to the 3′ end of sequence encoding the rabbit heavy chain constant region 3 and contained additional 5′ sequence that includes a Not I restriction site.
In this example, Primer B and Primer C are complementary in their 5′ portions, allowing hybridization to generate a template for the full length cassette in the second PCR step, when Primer A and Primer D are used to assemble the light chain and heavy chain sequences flanking the T2A peptide sequence. This method of using 4 primers to generate the final full length cassette is a modification of a method called an overlap PCR (or overlap extension PCR) (see Higuchi et al., Nucleic Acids Res. 16(15): 7351-67 (1988). The sequence of the T2A peptide (i.e., the 2A peptide from Thosea asigna) comprises the amino acid sequence N-terminus-EGRGSLLTCGDVEENPGP-C-terminus (SEQ ID NO: 3), where the ribosomal skip (i.e., no peptide bond is formed) between the C-terminal GP (underlined and bolded in the above sequence). These four primers may not be restricted to rabbit IgG sequence but can be designed to amplify IgG of any species including but not limited to human, mouse and chicken. Note that in the case of human or mouse IgG (or any other species where a diverse repertoire of IgG sequences exist), the primer pairs used for the amplification and assembly of the light and heavy chains may contain a set of multiple primers that hybridize to a range of sequences. In addition, the recognition sites in Primer A and Primer D are not limited to HindIII and NotI restriction enzymes for ligation. As mentioned above, a single restriction endonuclease with an interrupted palindromic recognition site with degenerate sequence (such as SfiI, AleI, BstAPI, DraIII etc.) may also be used instead of two distinct enzymes.
In an alternate method, a nucleic acid cassette of the invention can be generated using a one-step PCR method. This process is shown schematically in
In both cases (i.e., the one-step PCR method and the two-step PCR method), 3′ end sequences of Primers B and C contain complementary 2A peptide-encoding sequences that are part of the nucleic acid cassette of the invention. The 2A peptide sequence in Primers B and C may not be restricted to T2A (from Thosea asigna virus) but may be designed from 2A peptides of any of the Aphthoviruses.
For the two-step PCR method, the following PCR protocol was used for the first step independent amplification of heavy and light chains
Step 1—95 C for 5 minutes
Step 2—95 C for 30 seconds
Step 3—55 C for 30 seconds
Step 4—72 C for 1 minute
Step 5—go back to Step 2, 29 times
Step 6—72 C for 5 minutes
Amplification of the light chain with Primer A and Primer B generated a DNA fragment of approximately 750 bp, and amplification of the heavy chain with Primer C and Primer D generated a DNA fragment of approximately 1.4 kbp. DNA fragments generated in each reaction in the first PCR step was visualized in an ethidium bromide-stained 0.7% TAE agarose gel and gel-purified. 1/30th of each purified DNA fragment eluate after gel purification was mixed in a subsequent reaction for the second PCR step with Primer A and Primer D to assemble and amplify the full-length L-2A-H cassette.
The second PCR step (in the two-step PCR method) used the following PCR protocol:
Step 1—95 C for 5 minutes
Step 2—95 C for 30 seconds
Step 3—55 C for 30 seconds
Step 4—72 C for 2 minutes
Step 5—go back to Step 2, 29 times
Step 6—72 C for 5 minutes
As any routinely skilled scientist is aware, the temperatures used for each of the two steps in the two step PCR method described above may be varied to suit the polymerase being used in the reaction, buffer conditions, brand or model of the thermocycler.
In this manner, the following distinct L-2A-H cassette (nucleic acid sequence in a 5′ to 3′ orientation and amino acid sequence in a N-terminus to C-terminus orientation) were generated. (The cassette was sequenced according to standard methods.) Note that the below sequence includes only the coding region, and therefore does not show the HindIII restriction endonuclease recognition site at the 5′ end or the NotI restriction endonuclease recognition site at the 3′ end.
MRPL11 IgG L-2A-H cassette (nucleotide sequence is provided in SEQ ID NO: 25)
V R D N N Y L S W Y Q Q K P G Q P P K L L I Y R A
S T L E S G V P S R F K G N G S G T Q F T L T I S
N P G_P M E T G L R W L L L V A V L K G V Q C{circumflex over ( )}Q S
S G A T Y Y S T W A K G R F T I S K S S T T V D L
F S G P G T L V T V S L(G Q P K A P S V F P L A P
Note that in the above amino acid sequence, the predicted leader cleavage sites are indicated with a “̂” symbol, the CDRs are all underlined, the constant region is placed in parentheses, and the T2A sequence is bolded (where the “_” symbol indicates the translational skip within the T2A sequence).
For the one-step PCR method, briefly, amplification of light and full-length heavy chain occurs in a single reaction with Primer A, Primer B, Primer C and Primer D from above. For the one-step PCR method, amplification of light chain and heavy chain variable domain occurs in a single reaction with Primer A, Primer B, Primer C, and the following Primer D (HV) sequence, where the Primer D (HV was phosphorylated on its 5′ end:
The design of the first Primer D (here called FL Primer D) is as described above for Primer D. Thus, the first Primer D can be used to amplify nucleic acid encoding the entire heavy chain including the constant regions and creates a NotI recognition site at the 5′ terminal end of the nucleic acid cassette.
The Primer D (Hv) is an antisense primer that was designed to hybridize to the sense sequence of a highly conserved region in the 5′ end of rabbit heavy chain constant region 1. The 5′ end of the primer was phosphorylated so that it could be ligated to a blunt end on the expression vector encoding an in-frame Stul recognition site (that generates a blunt end) and constant regions 1, hinge, constant regions 2 and 3 of rabbit heavy chain IgG, without the need for restriction enzyme digestion of the PCR product.
Amplification of the light chain-2A-heavy chain variable domain (L-2A-Hv) cassette was accomplished in a single PCR process as a single tube reaction as follows. The reaction was carried out using New England Biolab's (produced by Finnzymes Oy) Phusion High-Fidelity DNA polymerase PCR master mix following the manufacturer's recommendation. Other PCR-compatible polymerases, commercially available or not, may be used to accomplish the same amplification. A mixture of both rabbit IgG heavy and light chain single-stranded or double-stranded DNA was used as template, either in the form of cDNA or plasmids. All four primers (A, B, C and D(Hv)) were added to the reaction, however, the concentration of the outer primers A and D(Hv) and inner primers B and C were varied to favor amplification of the full-length cassette. Primers A and D(Hv) were added at 0.3 μM final concentration, and primers B and C were added at 1/20th (for cDNA) or 1/50th (for plasmids) of the concentration of primers A and D(Hv). A 30-cycle PCR reaction as described below was sufficient to generate the 1.2-1.3 kb nucleic acid cassette encoding L-2A-Hv (as shown in
Step 1—98° C. for 30 seconds
Step 2—98° C. for 15 seconds
Step 3—55° C. for 15 seconds
Step 4—72° C. for 1 minute
Step 5—go back to Step 2, 29 times
Step 6—72° C. for 5 minutes
In this manner, the following distinct L-2A-Hv cassette (nucleic acid sequence in a 5′ to 3′ orientation and amino acid sequence in a N-terminus to C-terminus orientation) were generated. (The cassette was sequenced according to standard methods.) Note that the below sequence includes only the coding region, and therefore does not show the HindIII restriction endonuclease recognition site at the 5′ end.
MRPL11 IgG L-2A-Hv cassette (nucleic acid sequence provided in SEQ ID NO: 58)
V R D N N Y L S W Y Q Q K P G Q P P K L L I Y R A
S T L E S G V P S R F K G N G S G T Q F T L T I S
N P G_P M E T G L R W L L L V A V L K G V Q C Q S
S G A T Y Y S T W A K G R F T I S K S S T T V D L
F S G P G T L V T V S L(G Q P K A P S V F
Note that in the above amino acid sequence, the predicted leader cleavage sites are indicated with a “̂” symbol, the CDRs are all underlined, the beginning of the Heavy chain constant region is placed in an open parentheses, and the T2A sequence is bolded (where the “_” symbol indicates the translational skip within the T2A sequence).
Using the same two-step PCR method and same primers set forth in Example 1, an additional three cassettes were generated, namely a ERK2p IgG L-2A-H cassette (i.e., encoding an anti-ERK2p rabbit IgG antibody), a SUZ12 IgG L-2A-H cassette (i.e., encoding an anti-SUZ12 rabbit IgG antibody), and HER2 Ig G L-2A-H cassette (i.e., encoding an anti-HER2 rabbit IgG antibody).
To subclone the L-2A-H nucleic acid cassettes into plasmid vectors, the approximately 2 kb products from the second step PCR were gel purified and digested with HindIII and NotI (both from New England Biolabs) to generate directionally ligatable 5′ and 3′ ends. These fragments with “sticky ends” were then ligated using T4 DNA ligase (New England Biolabs, Ipswich, Mass.) into the vector fragment of either the pTT5 mammalian expression vector (from the National Research Council Canada) or the pcDNA3 expression vector (from Invitrogen, Carlsbad, Calif.) digested with HindIII and NotI. Note that expression vector need not be limited to this vector and any other vector (e.g., a eukaryotic expression vector such as pCI-Neo or simply a cloning vector such as puc9) may be applicable.
Competent E. coli were transformed with the ligation reactions and selected on LB ampicillin agar plates, and single colonies were inoculated into LB ampicillin broth for overnight growth. Plasmid DNA was isolated from the liquid cultures using a commercially available kit (Zymo Research), and the presence of the L-2A-H cassette insert in the plasmids was verified by visualization of a 2 kbp fragment on a 0.7% TAE gel following a HindIII/NotI digest.
To subclone the L-2A-Hv nucleic acid cassettes into plasmid vectors, the approximately 1.2-1.3 kb products from the single-step PCR were gel purified and digested with HindIII (from New England Biolabs) to generate directionally ligatable 5′ and 3′ ends. These fragments with a 5′ Hind III “sticky end” and a 3′ blunt end were then ligated using T4 DNA ligase (New England Biolabs, Ipswich, Mass.) into the vector fragment of the pTT5 mammalian expression vector (from the National Research Council Canada), containing rabbit constant regions 1 through 3 (including hinge), digested with HindIII and StuI. Note that expression vector need not be limited to this vector and any other vector (e.g., a eukaryotic expression vector such as pCI-Neo or simply a cloning vector such as puc9) may be applicable.
Competent E. coli were transformed with the ligation reactions and selected on LB ampicillin agar plates, and single colonies were inoculated into LB ampicillin broth for overnight growth. Plasmid DNA was isolated from the liquid cultures using a commercially available kit (Zymo Research), and the presence of the L-2A-H cassette insert in the plasmids was verified by visualization of a 2 kbp fragment on a 0.7% TAE gel following a HindIII/NotI digest. Note that ligation destroyed the StuI recognition site at the junction between the Hv chain-encoding nucleic acid from the cassette and the rabbit IgG1 constant regions 1-3-encoding nucleic acid from the vector.
Note that the StuI recognition sequence at the 5′ end of the constant region (the lowercase agg at the 5′ end of the above sequence compatible for ligation with the 3′ end of the LT2A-Hv cassette) has been engineered in so that a smooth junction with no amino acid change occurs upon ligation. Also, two internal StuI sites were eliminated by modifying the codon in each case without changing the amino acid sequence (residue changes shown as underlined lower-case letters in the above sequence). The lowercase tag sequence at the 3′ end of the above sequence is the stop codon. The sequence of the above rabbit IgG constant regions may not be limited to this exact sequence and, for example, may be substituted with other IgG isotype, IgM, IgA, IgE or IgD isotypes or IgG subclasses or allotypes or constant regions of immunoglobulins from other species.
The L-2A-H cassettes inserted into either the pTT5 or the pcDNA3 expression vectors were next transfected into mammalian cells. In addition, a heavy chain-2A-light chain (H-2A-L cassette) inserted into pTT5 or pcDNA3 mammalian expression vector was also transfected into mammalian cells. As a negative control, pTT5 vector or pcDNA3 vector with no insert was transfected into mammalian cells, while as a positive control, mammalian cells were transfected with two separate vectors (either pTT5 or pcDNA3, depending upon which vector backbone was used for the nucleic acid cassette), one encoding the light chain and one encoding the heavy chain.
To do this, expression vector (or pair of expression vectors for H+L) were transiently transfected into HEK293T cells (commercially available from the American Type Culture Collection, Manassas, Va.) plated at approximately 80% confluency on 12-well plates in 1 ml/well of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine and incubated at 37° C. with 5% CO2. Note that the cells used for transfection need not be limited to HEK293T cells, but can be any other transfectable cell line (e.g., 293 cells, COS cells, etc. . . . ), and can be transfected for transient or stable expression. For the transfection, the DNA to be transfected was complexed with a transfection carrier as follows. 1 μg of DNA was diluted in 25 μl of serum-free DMEM, 100 μl of serum-free DMEM containing 74 μg/ml of polyethylenimine was added to the diluted DNA and mixed gently, incubated at room temperature for 30 minutes, then gently added onto HEK293T cells that were seeded 24 hours prior. 5 days later, the supernatant was harvested for characterization of secreted IgG, and the cells were lysed in 100 μl of 1× Laemmli buffer (with 42 mM DTT) for Western blot analyses. As controls for each antibody, 1 μg of a 1:1 mixture of heavy chain- and light chain-encoding pTT5 plasmids or pcDNA3 plasmids were transfected in the same exact manner. Expression of IgG was driven by a CMV immediate early promoter in the vectors tested (i.e., pTT5 or pcDNA3), but other eukaryotic promoters (e.g., the spleen focus-forming virus (SFFV) promoter or the EF1alpha promoter) may also be used. Although transfection here was by chemical means, physical transfection (e.g., electroporation) may also be used. In fact, any method for inserting the expression vectors containing the nucleic acid cassette into a cell line may be used (e.g., transduction, infection, etc. . . . ).
The culture supernatant from the transfected cells harvested 5 days post-transfection was characterized for secretion of IgG with specific binding activity to target antigens by enzyme-linked immunosorbent assay (ELISA). High-binding 96-well polystyrene plates (Costar) were coated with 0.1 μg of antigen (immunizing peptides for HER2, ERK2p, MRPL11 or SUZ12 in the case of target antigens, or anti-rabbit IgG antibody for detection of total IgG) and blocked with 5% bovine serum albumin in tris-buffered saline (TBS). Each supernatant sample was tested at undiluted and diluted 10-fold in TBS when tested against peptides and diluted 100- and 1000-fold when tested for total IgG on plates coated with anti-rabbit IgG antibody. 50 μl of each supernatant was added per well and plates were incubated at 37° C. for 2 hours, after which the plates were washed 3 times with TBS-Tween (0.1%) (TBS-T), 50 μl of detection antibody (anti-rabbit HRP, Cell Signaling Technology, Inc., Danvers, Mass., product #7074) diluted 2000-fold in TBS-T was added to each well and plates were incubated at 37° C. for 1 hour then washed 3 times, and finally developed with 50 μl of TMB solution (BioFX labs), neutralized with 50 μl of stop solution (BioFX labs), and OD450 nm was read on a plate reader (Titertek). Tables 1-4 show the absorbance values of ELISAs for anti-ERK2p, anti-MRPL11, anti-SUZ12, and anti-HER2 antibodies, respectively. Each IgG was constructed and tested in both the L-2A-H and H-2A-L configurations. All samples tested were generated from a single transfection experiment. Total IgG concentration in each sample was quantified in a separate ELISA by comparing the signal to a standard curve ranging from 2 ng/ml to 0.05 ng/ml.
In each of Tables 1-4, “sup dilution” means the factor by which the culture supernatant was diluted in the ELISA assay, “vector only” means supernatant taken from cells transfected with empty pTT5 vector or empty pcDNA3, and “H+L+ve” means supernatant taken from cells transfected with two vectors, one containing the H chain and one containing the L chain. For each configuration (L-2A-H and H-2A-L) of the antibody, with the exception of the anti-ERK2p L-2A-H configuration, supernatants from two independent clones (indicated in Table 1 as H2AL#10 and #19, for example) were tested to demonstrate reproducibility.
Tables 1, 2, 3, and 4 respectively, show the binding specificity and quantification of anti-ERK2p, anti-MRPL11, anti-SUZ12, and anti-HER2 rabbit IgG. Supernatants of HEK293T cells transfected with each antibody cassette vector was tested for binding to all three antigens by ELISA at undiluted and 10-fold dilution. Total IgG secretion (bottom row, [IgG] μg/ml) was tested qualitatively (at 1/100 and 1/1000 dilutions) and quantitatively by quantitative ELISA-determined concentration. For samples where the IgG concentration is not indicated, the concentrations were not measured. The numbers in the table are absorbance values of optical density measured at 450 nm and are averages of duplicate measurements.
For each antibody, as shown in Tables 1-4, the secreted IgG displayed specific binding to its cognate target antigen without any non-specific binding to any other antigen tested, regardless of the order of the light and heavy chains flanking the 2A peptide. The antigen-specific signal levels for the undiluted and 10-fold supernatant dilution were saturated, and thus a ten-fold dilution did not reduce the obtained value. The total IgG ELISA, which tested the supernatants at 100-fold and 1000-fold dilution due to its high sensitivity, shows a qualitative difference between the H-2A-L and L-2A-H configurations. In all cases, L-2A-H configuration expressed as well as the positive control (i.e., transfection with two plasmids—one encoding the L chain and the other encoding the H chain), and the levels of secretion (into supernatant) of antibody encoded by the L-2A-H constructs were 3 to 5-fold higher than those encoded by the H-2A-L constructs. This indicates that translation of each chain from the L-2A-H expression cassette message is as efficient as each chain being expressed as two independent transcripts from two separate vectors (and thus each H and L chain driven by its own promoter).
IgG in total cell lysate and supernatant of transfected HEK293 cells was detected by Western blotting. 10% volume of total cell lysate and 0.5% volume of total supernatant were resuspended in 1× Laemmli buffer (i.e., 2% SDS, 10% glycerol, 0.002% bromophenol blue and 62.5 mM tris-HCl at pH 6.8) with 42 mM DTT and boiled for 5 minutes. The chromosomal DNA was shredded in the lysate using Qiashredder (Qiagen) prior to boiling. The samples were loaded and run on a 4-20% gradient Tris-glycine polyacrylamide gel (Invitrogen), then transferred onto a nitrocellulose membrane (Whatman). The nitrocellulose membrane was blocked with 5% milk in phosphate-buffered saline (PBS) for 30 minutes, then incubated with either HRP-conjugated anti-rabbit IgG antibody (Cell Signaling Technology #7074) diluted 1000-fold in 5% milk in TBS for 1 hour at room temperature, or with anti-rabbit kappa chain antibody (Cell Signaling Technology, Inc., product #3677) diluted 1000-fold in 5% BSA in TBS overnight at 4° C. followed by an incubation with HRP-conjugated anti-mouse IgG antibody (Cell Signaling Technology, Inc., product #7076) diluted 1000-fold in 5% milk in TBS for 1 hour at room temperature. The blots were washed 4 times in TBS-T, then immersed in chemiluminescence peroxidase substrate (Cell Signaling Technology, Inc.) and exposed to film (Kodak) for detection of signal.
Similar levels of heavy and light chains were detected intracellularly for both H-2A-L and L-2A-H configurations (see
In this example, a nucleic acid encoding proteolytic cleavage site is introduced into the nucleic acid cassette of the invention. In this example, a site recognized by the furin protease is inserted into the nucleic acid cassette of the invention.
Furin is a ubiquitous subtilisin-related serine protease that is expressed in almost all cell types and which cleaves after the last amino acid in the following sequence: Arg-X-X-Arg (where X can be any amino acid), such as Arg-X-Arg-Arg or Arg-X-Lys-Arg.
Using standard molecular biology methods (see, e.g., Ausubel et al., supra), the MRPL11 IgG-encoding nucleic acid cassette described in Example 1 is modified to encode a furin recognition site after the constant region of the L chain and before the 2A sequence. The resulting amino acid sequence encoded by the nucleic acid sequence of this example is set forth below:
V R D N N Y L S W Y Q Q K P G Q P P K L L I Y R A
S T L E S G V P S R F K G N G S G T Q F T L T I S
D Y E E N P G_P M E T G L R W L L L V A V L K G V
F I G G S G A T Y Y S T W A K G R F T I S K S S T
G S F D F S G P G T L V T V S L(G Q P K A P S V F
Note that in the above amino acid sequence, the predicted leader cleavage sites are indicated with a “̂” symbol, the CDRs are all underlined, the constant region is placed in parentheses, the T2A sequence is bolded (where the “_” symbol indicates the translational skip within the T2A sequence), and the furin protease recognition site is underlined and bolded, where X is any amino acid.
The nucleic acid cassette encoding the above MRPL11 IgG L-furin recognition site-2A-H cassette amino acid sequence is inserted into an expression cloning vector (e.g., pcDNA3) which used to transfect HEK293T cells according to the methods described in the above examples. Since furin is expressed in HEK293T cells, the 2A peptide sequences are cleaved off the C terminus of the light chain portion of the encoded antibody.
In this example, the components of the nucleic acid cassette are designed in a working vector prior to transferring the finished L2AH cassette, in its entirety, from the working vector to an expression vector.
The working vector is the puc9 vector (commercially available from the American Type Culture Collection, Manassas, Va.). The cloning sites on the puc9 vector used to insert components of the cassette are HindIII and BamHI.
In this example, the components of the cassette are A′-A-B-C′-C, where A′ encodes the light chain leader peptide sequence, A encodes the light chain, B encodes a 2A peptide, C′ encodes the heavy chain leader peptide sequence; and C encodes the heavy chain. The antibody described in this example is encoded by a hybridoma cell line. This example describes the process by which the nucleotide sequences encoding the chains of the antibody secreted by the hybridoma cell are isolated and used to make a nucleic acid cassette of the invention.
The light chain leader peptide and the expressed light chain (i.e., components A′-A) in this example has the nucleotide sequence set forth in SEQ ID NO: 28.
The heavy chain leader peptide and the expressed heavy chain (i.e., components C′-C in the nucleic acid cassette) in this example has the nucleotide sequence set forth in SEQ ID NO: 29
The 2A peptide (i.e., component B) in this example will have the following nucleotide sequence: gacgtggaggagaatcccggccct (SEQ ID NO: 30).
To generate the cassette, the B-C components are generated first and are inserted into the puc9 vector.
To do this, the following PCR primers are generated. 5′ agtggatccgacgtggaggagaatcccggccctATGGAGACTGG3′ (SEQ ID NO: 31; where the BamHI recognition site is underlined and the nucleotide sequence encoding the 2A peptide is italicized and bolded, and the nucleotide sequence encoding the heavy chain is capitalized). 5′ taggacgcgtTCATTTACCCGGAGA3′ (SEQ ID NO: 32; where the Mlul recognition site is underlined and the antisense of the nucleotide sequence encoding the heavy chain is capitalized)
mRNA is isolated from the hybridoma cell line, reverse transcribed using-standard methods, and subjected to PCR using the above two PCR primers. The resulting PCR product is electrophoretically resolved on a low-melting point agarose gel, purified from the gel, and incubated at 37° C. with MluI and BamHI restriction endonucleases in buffer supplied by the manufacturer (New England Biolabs, Ipswich, Mass.).
A puc9 vector is digested with MluI and BamHI. The digested DNAs are electrophoretically resolved in low melting point agarose, and the vector fragment (i.e., the larger fragment) from the digested puc9 is ligated to the digested PCR product. The ligation is used to transform competent E. coli, and the resulting cells are plated onto LB agar plates containing ampicillin. Positive clones are picked and expanded, minipreps are performed, and ligated vector is isolated. The vector's insert is then sequenced (e.g., using a sequencing primer that hybridizes upstream of the BamHI site in puc9).
The A′-A components of the cassette is next constructed. A forward PCR primer is generated that adds a HindIII recognition site at the 5′ end of the above mentioned A′ sequence. For example, the PCR primer may have the following sequence: 5′ gggaagcttATGGACATGAGGG 3′ (SEQ ID NO: 33; where the HindIII recognition site is underlined and the nucleotide sequence encoding the light chain is capitalized.)
The reverse primer is constructed to add a MluI recognition site at the 3′ end of the A sequence, but removing the stop codon (in this case TGA) from the sequence. As an example, the reverse primer will have the sequence: 5′ ggacgcgtACAGTCACCCCTAT 3′ (SEQ ID NO: 34; where the MluI recognition site is underlined and the nucleotide sequence encoding the light chain is capitalized).
The heavy chain is PCR amplified from the cDNA generated from the hybridoma mRNA, and digested with HindIII and MluI. The B-C containing puc9 vector (i.e., the vector containing the nucleotide sequence encoding the 2A peptide and the light chain) is similarly digested with HindIII and MluI, and the digested vector fragment is ligated with the digested PCR fragment. The ligation mixture is used to transform E. coli, and positive clones (i.e., ampicillin resistant) are screened by digesting miniprep DNA with restriction endonucleases chosen to identify those vectors having the heavy chain inserted in the correct orientation.
After a positive clone is identified and sequenced, the entire cassette (which now runs from HindIII at the 5′ end to BamHI at the 3′ end) is excised by digesting the puc9 vector containing the cassette with HindIII and BamHI and isolating the insert fragment from the vector fragment.
The pcDNA3.1 vector is purchased from Invitrogen (Carlsbad, Calif.) and is digested with HindIII and BamHI. The vector fragment is ligated to the insert fragment (containing the nucleic acid cassette), and at least one positive clone is isolated, expanded, and the supercoiled plasmid DNA is purified.
COS cells are transfected using DEAE-dextran with the cassette/pcDNA3 vector linearized by digesting with a restriction endonuclease that does not recognize a site either in the cassette, in the neomycin resistance gene, or in either the promoter for the cassette or the promoter for the neo gene. (For example, PvuI is used to linearize the cassette/pcDNA3.1 construct. Transfected cells are selected in G418-containing media and the cells are cloned by limiting dilution. Expanded clones are then screened for high secretion rates of the antibody, and the ability of the secreted antibody to bind to its target molecule (using, for example, an ELISA assay as described in Example 3 above).
In this example, an episomally replicable expression vector containing components of the nucleic acid cassette of the invention is provided.
The expression plasmid pCEP4 is purchased from Invitrogen (Carlsbad, Calif.) and is digested with HindIII and BamHI. The digested plasmid is electrophoretically resolved and the vector band purified. The HindIII to BamHI nucleic acid cassette described in Example 8 is excised from puc9 and ligated into the digested pCEP4 vector. Transformed E. coli are selected on ampicillin-containing agar plates and positive clones sequenced. Plasmid DNA is purified from the selected positive clones and used to transfect Jurkat T cells. Since pCEP4 contains both EBNA1 and oriP (the origin of replication for EBV), it is capable of episomally replicating in the cell, and so does not require stable integration. Jurkat T cells are transfected using electroporation.
To generate stable cells, the transfected cells are selected in hygromycin-containing media. The hygromycin resistant cells are cloned by limiting dilution.
Note that if cells transiently expressing the antibody encoded by the nucleic acid cassette are desired, the cells are not selected or cloned.
Conditioned media from the cells is collected and antibody in the media is enriched using, for example, the ability of the Fc portion of antibody to bind protein A sepharose.
In this example, a cloning vector is generated containing the nucleic acid encoding the 2A peptide component of the cassette within the polylinker of the plasmid. This vector, together with instructions for inserting nucleic acid sequences encoding the heavy chain and the light chain of an antibody of interest and, optionally, with PCR primers for facilitating cloning in the H and L chain sequences, may be sold as a kit.
For example, the puc9 plasmid is used as the cloning vector. The 2A peptide in this example has the amino acid sequence DVEENPGP (SEQ ID NO: 35). Given the degeneracy of the genetic code, numerous different nucleotide sequences encode this 2A peptide sequence. For example, the following nucleotide sequence 5′ Gacgtcgaagagaacccagggccc3′ (SEQ ID NO: 36) is used. The 5′ end of this sequence is an AatII recognition site, while the 3′ end of this sequence is an ApaI site.
Thus, the cloning sites of the cassette within the polylinker of the puc9 plasmid may be: (puc9 backbone sequences)-AAGCTT (HindIII site)-(random sequences)-ggatcc(BamHI site)-gaagagaaccca-acgcgt(MluI site)-(random sequences)-gcggccgc (NotI site)-(puc9 sequences).
A map of this cloning vector will be provided with the kit (full length sequence and map of puc9 is available from the American Type Culture Collection), and instructions such that the practitioner will be able to design PCR primers which add the appropriate restriction endonuclease recognition site to the amplified sequence to facilitate insertion of nucleic acids encoding the H chain and L chain of the antibody into the vector. In the above-example, the H chain-encoding nucleic acid sequence will be inserted into the MluI to NotI insertion site, and the L chain-encoding nucleic acid sequence will be inserted into the HindIII to BamHI site. Care is taken to ensure that the inserted sequences are in frame with the 2A peptide, such that entire nucleic acid sequence contained within the HindIII and the NotI sites are in a single open reading frame and, but for the translation “stop” signal by the 2A peptide, would be translated as a single polypeptide. Note that the puc9 vector backbone can also be substituted with an expression cloning plasmid (e.g., the pcDNA3.1 vector described above).
Where the practitioner desires to obtain a cassette encoding a secreted antibody, the cloning vector can also include nucleic acid sequences encoding a leader peptide upstream of the HindIII site for the first chain and immediately after the MluI site for the second chain (a new restriction endonuclease site may be engineered at the 3′ end of the leader peptide to facilitate insertion of the second chain).
The kit may further include PCR primers designed to facilitate insertion of the nucleotide sequences encoding the antibody of interest into the cloning vector. In some embodiments, because of the huge diversity in the antigen binding domain portion of an antibody chain among different species (e.g., difference between mice and humans), however, provision of such primers with the kit may limit the number of species from which antibodies of interest are derived that the practitioner is able to insert into the cloning vector.
The light chain-2A-heavy chain (L-2A-H) cassette encoding a mouse antibody will be assembled as a single molecule of DNA using two steps of polymerase chain reaction (PCR).
Briefly, the first step will consist of two independent reactions, one that will amplify the light chain (or 5′ piece of the cassette) with Primer A and Primer B from a template encoding the full length light chain sequence including the light chain leader sequence, and the other that will amplify the heavy chain (or 3′ end of the cassette) with Primer C and Primer D from a template encoding the full length heavy chain sequence including the heavy chain leader sequence. The sequences of Primers A-D will be as follows (where the sequences that will be derived from murine sequences are underlined):
TTCCTGTTGAAGCT 3′
TGGGTGTGGACCTTG 3′
The template DNA to be used to construct the nucleic acid cassette will be derived from the following sources: heavy chain variable region from GenBank AB016619.1, IgG1 isotype heavy chain constant region from GenBank AK144480.1, light chain variable region from GenBank AB016620.1 and kappa chain isotype constant region from GenBank BC091750.1. The variable region of the expressed recombinant antibody is derived from FU-MK1 (see Arakawa F et al., “cDNA sequence analysis of monoclonal antibody FU-MK-1 specific for a transmembrane carcinoma-associated antigen, and construction of a mouse/human chimeric antibody”. Hybridoma. 18(2):131-138 (April 1999)), and is specific to a human gastric adenocarcinoma transmembrane antigen, GA733-2*. The methodology for construction of the mouse L-2A-H IgG cassette will be conducted as described for the rabbit L-2A-H IgG cassette described in Example 1.
The nucleotide sequence of the resulting mouse light chain 2A heavy chain cassette will be that set forth in SEQ ID NO: 42.
The amino acid sequence of the resulting mouse light chain 2A heavy chain cassette will be:
ISGYLSWLQQKPDGTVKRLIYAASTLHSGVPKRFSGSRSGSDYSLTISS
CGDVEENPGPMAWVWTLLFLMAAAQSIQA{circumflex over ( )}QIQLVQSGPELKKPGETVK
Note that in the above amino acid sequence, the predicted leader cleavage sites are indicated with a “̂” symbol, the CDRs are all underlined, the constant region is placed in parentheses, and the T2A sequence is bolded (where the “_” symbol indicates the translational skip within the T2A sequence).
The light chain-2A-heavy chain (L-2A-H) cassette encoding a human antibody can be assembled as a single molecule of DNA using two steps of polymerase chain reaction (PCR).
Briefly, the first step can consist of two independent reactions, one that will amplify the light chain (or 5′ piece of the cassette) with Primer A and Primer B from a template encoding the full length light chain sequence including the light chain leader sequence, and the other that will amplify the heavy chain (or 3′ end of the cassette) with Primer C and Primer D from a template encoding the full length heavy chain sequence including the heavy chain leader sequence. The sequences of Primers A-D will be as follows (where the sequences that will be derived from human sequences are underlined):
CCCTGTTGCTCTT 3′
GCACCTGGAGGAT 3′
The template DNA to be used to construct the nucleic acid cassette is cDNA generated from RNA isolated from an Epstein Barr Virus-immortalized human B-cell culture that secretes IgG with polyreactivity. The methodology for construction of the human L-2A-H IgG cassette will be conducted as described for the rabbit L-2A-H IgG cassette described in Example 1 (the two-step PCR method).
The nucleotide sequence of the resulting human light chain 2A heavy chain cassette will be that set forth in SEQ ID NO: 48.
The amino acid sequence of the resulting human light chain 2A heavy chain cassette will be:
SSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL
GDVEENPGPMDCTWRILLLVAAATGTHA{circumflex over ( )}EVQVQLVESGGGLVQPGGSL
YMDVWGKGTTVTVSS(ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF
Note that in the above amino acid sequence, the predicted leader cleavage sites are indicated with a “̂” symbol, the CDRs are all underlined, the constant region is placed in parentheses, and the T2A sequence is bolded (where the “_” symbol indicates the translational skip within the T2A sequence).
The light chain-T2A-heavy chain (L-2A-HV) cassette encoding a human antibody was constructed for an antibody with specificity to a protein antigen from human cytomegalovirus (called CMV antigen or CMV Ag). The template DNA used to construct the nucleic acid cassette was plasmids encoding either the heavy or light chain, each cloned originally, from human B-cells secreting antigen specific IgG. The 2-step PCR methodology for construction of the human L-2A-H IgG cassette was as described for the rabbit L-2A-H IgG cassette described in Example 1 and depicted schematically in
Briefly, the first step of the PCR consisted of two independent reactions, one that amplifies the light chain (or 5′ piece of the cassette) with Primer A and Primer B from a template encoding the full length light chain sequence including the light chain leader sequence, and the other that amplifies the heavy chain variable domain (or 3′ end of the cassette) with Primer C and Primer D(HV) from a template encoding the full length heavy chain sequence including the heavy chain leader sequence. The sequences of Primers A-D were as follows (where the sequences that are derived from human sequences are underlined):
The second step of the PCR was as described in Example 1. Briefly, the products of the two independent PCR reactions from the first step were gel-purified and mixed in a subsequent (second step) PCR reaction with Primer A and Primer D to assemble and amplify the final human L-2A-HV genetic cassette.
The nucleotide sequence of the open-reading-frame of the resulting anti-CMV Ag human light chain 2A heavy chain variable region (human L-2A-HV) cassette is set forth in SEQ ID NO: 65.
The amino acid sequence of the resulting human light chain 2A heavy chain variable region (L-2A-HV) cassette is:
ISTYLAWYQQKPGKAPNLLMYAASTLQSGVPSRFSGSGSGTDFTLTISR
CGDVEENPG_PMDWTWRFLFVVAAATGVQS{circumflex over ( )}QVQLVQSGAEVKKPGSSV
Note that in the above amino acid sequence, the predicted leader cleavage sites are indicated with a “̂” symbol, the CDRs are all underlined, the constant regions are placed in parentheses (note that only the beginning of the heavy chain constant region is shown, hence the open parenthesis), and the T2A sequence is bolded (where the “_” symbol indicates the translational skip within the T2A sequence).
Using the method described for Example 14, an additional human IgG L-2A-HV cassette was built using the sequences of an anti-HBsAg antibody isolated from a human B-cell. (HBsAg is short for Hepatitis B surface antigen). This nucleic acid cassette encodes the light chain and the variable region of the heavy chain of the antibody, where the light and the heavy chain components are separated by the 2A peptide.
To subclone the human IgG L-2A-H full-length nucleic acid cassettes (amplified with primers A, B, C and D(FL) from Example 13) into plasmid vectors, the approximately 2 kb products from the second step PCR may be gel purified and digested with HindIII and NotI (both available from New England Biolabs) to generate directionally ligatable 5′ and 3′ ends. These fragments with “sticky ends” may then be ligated using T4 DNA ligase (New England Biolabs, Ipswich, Mass.) into the vector fragment of either the pTT5 mammalian expression vector (from the National Research Council Canada) or the pcDNA3 expression vector (from Invitrogen, Carlsbad, Calif.) digested with HindIII and NotI. Note that expression vector need not be limited to this vector and any other vector (e.g., a eukaryotic expression vector such as pCI-Neo or simply a cloning vector such as puc9) may be applicable.
For subcloning of the human IgG L-T2A-HV fragment from Examples 14 and 15, into a vector, the 1.2-1.3 kb L-2A-Hv fragment was isolated by agarose gel electrophoresis (1.5% agarose) and gel purified using a commercial kit (Qiagen or Marligen). The purified fragment was digested with HindIII (New England Biolabs), gel purified again, then ligated to a vector containing a HindIII site on the upstream (5′) end and a StuI site on the downstream (3′) end that is in-frame with the sequence encoding human IgG1 constant regions as shown below.
aggCCtCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCC
Note that the StuI restriction site used to generate a blunt end that is ligatable to the 5′ phosphorylated end of the L-2A-Hv that is generated by primer D(Hv) is underlined. The sequence of the above human IgG1 constant regions does not need be limited to IgG1 (e.g., the constant regions may be substituted with IgM, IgA, IgE or IgD isotype, or with other allotypes of IgG1 or with constant regions of immunoglobulins from other species).
Competent E. coli were transformed with the ligation reactions and selected on LB ampicillin agar plates, and single colonies were inoculated into LB ampicillin broth for overnight growth. Plasmid DNA was isolated from the liquid cultures using a commercially available kit (Zymo Research), and the presence of the L-2A-H cassette insert in the plasmids was verified by visualization of a 2 kbp fragment on a 0.7% TAE gel following a HindIII/NotI digest (a NotI site exists downstream of the stop codon of the IgG1 sequence in the vectors used, therefore a subcloned L-2A-Hv cassette will release the same size band as the full-length L-2A-H when digested with HindIII/NotI.
The L-2A-HV cassettes inserted into the pTT5 expression vector were next transfected into mammalian cells. In addition, a heavy chain-2A-light chain (H-2A-L cassette) inserted into pTT5 mammalian expression vector was also transfected into mammalian cells. As a negative control, pTT5 vector or pcDNA3 vector with no insert was transfected into mammalian cells, while as a positive control, mammalian cells were transfected with two separate vectors, one encoding the light chain and one encoding the heavy chain.
To do this, each expression vector (or pair of expression vectors for heavy and light chains) was transiently transfected into a derivative of HEK293 cells plated at approximately 80% confluency on 24-well plates in 1 ml/well of appropriate medium and incubated at 37° C. with 5% CO2. Note that the cells used for transfection need not be limited to HEK293 cells, but can be any other transfectable cell line, and can be transfected for transient or stable expression. For the transfection, the DNA to be transfected was complexed with a transfection carrier as follows. 1 μg of DNA was diluted in 25 μl of serum-free medium, 100 μl of serum-free medium containing 40 μg/ml of polyethylenimine was added to the diluted DNA and mixed gently, incubated at room temperature for 30 minutes, then gently added onto cells that were seeded 24 hours prior. 5 days later, the supernatant was harvested for characterization of secreted IgG, and the cells were lysed in 100 μl of 1× Laemmli buffer (with 42 mM DTT) for Western blot analyses. As controls for each antibody, 1 μg of a 1:1 mixture of heavy chain- and light chain-encoding pTT5 plasmids were transfected in the same exact manner. Expression of IgG was driven by a CMV immediate early promoter in the vectors tested (i.e., pTT5), but other eukaryotic promoters (e.g., the spleen focus-forming virus (SFFV) promoter or the EF1 alpha promoter) or expression vectors may also be used. Although transfection here was by chemical means, physical transfection (e.g., electroporation) may also be used. In fact, any method for inserting the expression vectors containing the nucleic acid cassette into a cell line may be used (e.g., transduction, infection, etc. . . . ).
The culture supernatant from the transfected cells harvested 5 days post-transfection was characterized for secretion of IgG with specific binding activity to target antigens by enzyme-linked immunosorbent assay (ELISA). To do this, high-binding 96-well polystyrene plates (Costar) were coated with 0.1 μg of antigen (CMV grade 2 antigen—lysate of MRC-5 cells infected by CMV strain AD169 (available from Microbix, Toronto, Canada) HBsAg adw subtype (available from Prospec, Rehovot, Israel) or anti-human IgG antibody for detection of total IgG) and blocked with 5% milk in phosphate-buffered saline (5% MPBS). Each supernatant sample was tested at undiluted and diluted 10-fold in PBS when tested against peptides and diluted 2-, 20-, 200- and 2000-fold when tested for total IgG on plates coated with either antigen or goat anti-human IgG antibody (Southern Biotech, Birmingham, Ala.). 50 μl of each supernatant dilution was added per well and plates were incubated at 37° C. for 2 hours, after which the plates were washed 3 times with PBS-Tween (0.1%) (PBS-T), 50 μl of detection antibody (goat anti-human HRP, Southern Biotech) diluted 5000-fold in PBS-T was added to each well and plates were incubated at 37° C. for 1 hour then washed 3 times, and finally developed with 50 μl of TMB solution (BioFX labs), neutralized with 50 μl of stop solution (BioFX labs), and OD450 nm was read on a plate reader (Titertek).
As non-limiting representative examples of the amount of specific antibody produced using the nucleic acid cassette of the invention, Tables 5-7 (and
To generate the data shown below in Tables 5 through 7 and shown schematically in
Tables 5, 6 and 7 shows the absorbance values of ELISAs for each antibody. For graphical representation of the data, the average value of duplicate wells from one representative clone for each construct was plotted on a bar graph, which is shown in
Note that the graphs shown in
Each IgG was constructed and tested in both the L-2A-H and H-2A-L configurations.
Note that in each of Tables 5-7, “sup dilution” means the factor by which the culture supernatant was diluted in the ELISA assay. In
For each antibody, the secreted IgG displayed specific binding to its cognate target antigen without any non-specific binding to the other (irrelevant) antigen tested, regardless of the order of the light and heavy chains flanking the 2A peptide. Both the antigen-specific ELISA as well as the total human IgG ELISA show that the reactivity of the L-2A-H orientation is stronger than that of the H-2A-L orientation, thus L-2A-H orientation produces a higher level of antibody than does H-2A-L.
Human IgG in total cell lysate and supernatant of transfected HEK293 cells was detected by Western blotting. 10% volume of total cell lysate and 0.5% volume of total supernatant were resuspended in 1× Laemmli buffer with 42 mM DTT and boiled for 5 minutes. The chromosomal DNA was shredded in the lysate using Qiashredder (Qiagen) prior to boiling. The samples were loaded and run on a 4-20% gradient Tris-glycine polyacrylamide gel (Invitrogen), then transferred onto a nitrocellulose membrane (Whatman). The nitrocellulose membrane was blocked with 5% milk in phosphate-buffered saline (PBS) for 30 minutes, then incubated with either HRP-conjugated anti-human IgG antibody (Southern Biotech) diluted 1000-fold in 5% milk in PBS for 1 hour at room temperature. The blots were washed 4 times in PBS-T, then immersed in chemiluminescence peroxidase substrate (Cell Signaling Technology, Inc.) and exposed to film (Kodak) for detection of signal.
Comparable levels of heavy chains were detected intracellularly for both H-2A-L and L-2A-H configurations (see
The L-2A-H cassette-mediated expression of IgG resulted in efficient production of functional IgG (see above examples). However, the resulting IgG contains light chains that have additional T2A sequence (17 amino acids) on their C-terminus. With the goal to eliminate the additional sequence or to minimize immunogenicity due to the T2A peptide, specific protease cleavage recognition sequences were engineered into the region between the C-terminus of the kappa chain of the described human IgG cassettes. Due to its robust yet highly sequence-specific activity, thrombin was used to achieve this goal. Other site-specific proteases such as, but not limited to, furin, factor Xa, TEV protease, other viral proteases, may also be used.
The most commonly used thrombin cleavage recognition site is LVPR_GS (SEQ ID NO: 68), where cleavage occurs between the arginine and glycine (as indicated by _). Such a sequence would leave four amino acids (LVPR) at the kappa chain C-terminus upon cleavage.
Another thrombin recognition site is within its natural ligand, fibrinogen. Accordingly, the N-terminal amino acid sequence from human fibrinogen a, thrombin's natural substrate that exists in great abundance in the blood, was chosen as the thrombin recognition sequence for these studies. Due to possible accessibility constraints that may exist at the C-terminus due to the tertiary or quaternary structure of kappa chains, different lengths of the N-terminus of fibrinogen, including the two thrombin cleavage sites, were introduced at the junction between the C-terminus of kappa chain and the T2A sequence to test the efficiency of cleavage by thrombin in the context of the anti-CMV human IgG (described above) expressed from the L-2A-H cassette. The amino acid sequences of the kappa chain-fibrinogen-T2A junction of the three constructs tested are shown below in Table 8, where the kappa light chain C-terminus (ending in a C) sequence is underlined, the fibrinogen sequences are bold and italicized and the T2A sequence is in normal upper case letters.
. . . FNRGEC
*
*
EGRGSL
. . . FNRGEC
*
*
EGRGSLLTCG
. . . FNRGEC
*
*
EGRGSLLTCGDVEEN
All L-fibrinogen-T2A-H cassette constructs were built using the identical overlap-PCR method described in Examples 13-15 but with modified primers B and C to incorporate fibrinogen sequences. The resulting cassettes were cloned into mammalian expression plasmids in a manner identical to that described in Example 16.
The various human IgG L-fibrinogen-T2A-H cassettes described in the previous example, inserted into the pTT5 expression vector, were next transfected into mammalian cells, as described in Example 17. As a control, the vector encoding the same anti-CMV IgG in the L-2A-H (without any fibrinogen sequence at the light chain-T2A junction, described in Example 14) was transfected into cells as well. IgG expressed from this construct was used as a control to monitor binding specificity and expression levels of the fibrinogen-containing constructs, as well as any non-specific degradation or cleavage occurring during thrombin treatment.
To do this, each expression vector (or pair of expression vectors for heavy and light chains) was transiently transfected into a derivative of HEK293 cells plated at approximately 80% confluency on 6-well plates in 2 ml/well of appropriate medium and incubated at 37° C. with 5% CO2. Note that the cells used for transfection need not be limited to HEK293 cells, but can be any other transfectable cell line, and can be transfected for transient or stable expression. For the transfection, the DNA to be transfected was complexed with a transfection carrier as follows. 2 μg of DNA was diluted in 50 μl of serum-free medium, 200 μl of serum-free medium containing 40 μg/ml of polyethylenimine was added to the diluted DNA and mixed gently, incubated at room temperature for 30 minutes, then gently added onto cells that were seeded 24 hours prior. Supernatant was harvested 5 days later for analysis of IgG expression, binding specificity and thrombin cleavage.
To test the effects of thrombin on the removal of T2A sequence via site-specific cleavage at the fibrinogen sequence engineered between the C-terminus of the kappa chain and the T2A peptide sequence, the supernatants from the transfections in the previous example were digested with thrombin as follows. Restriction-Grade Thrombin Kit (Novagen, La Jolla, Calif.) containing a 10× buffer and restriction grade thrombin was used for the assay. For each sample, 30 μl of 10× thrombin cleavage buffer was added to 270 μl of supernatant containing antibody, then the sample was divided into two separate tubes of 150 μl each, 1 μl of restriction grade thrombin (1U/μl) was added to one tube and in the other tube, no thrombin was added as the no-thrombin control. All tubes were incubated at 37° C., and after 1, 2, 4 and 24 hours, 20 μl were removed and added immediately to 20 μl of 2× Laemlli buffer supplemented with 42 mM DTT and boiled at 95° C. for 5 minutes. Specific removal of the light chain C-terminal T2A sequence was assessed by Western blot (see Example 23 and
In order to detect removal of the T2A peptide sequence from the kappa chain C-terminus of the human anti-CMV after thrombin digestion as described in the previous example, the tissue culture supernatant of transfection samples for different fibrinogen length sequence constructs that were harvested at different time-points after thrombin treatment (+ lanes on
10 μl of each sample boiled with Laemmli buffer (as described in the previous example) was loaded and run on a 4-20% gradient Tris-glycine polyacrylamide gel (Invitrogen), then transferred onto a nitrocellulose membrane (Whatman). The nitrocellulose membrane was blocked with 5% milk in phosphate-buffered saline (PBS) for 30 minutes, then incubated with either HRP-conjugated goat anti-human IgG antibody (Southern Biotech) diluted 1000-fold in 5% MPBS for 1 hour at room temperature, or with HRP-conjugated goat anti-human kappa chain F(ab′)2 (AbD Serotec, Oxford, UK) diluted 1000-fold in 5% MPBS1 hour at room temperature. The blots were washed 4 times in PBS-T, then immersed in chemiluminescence peroxidase substrate (Cell Signaling Technology, Inc.) and exposed to film (Kodak) for detection of signal.
As expected, the heavy chain in all cases was unaffected after thrombin treatment (upper blot,
The anti-kappa chain blot (bottom blot,
The effects of thrombin digestion on the binding activity of human anti-CMV IgG was tested and analyzed by ELISA. After 24 hours of thrombin digestion as described in Example 22, antibodies encoded by the L-2A-H (no fibrinogen linker, fibrinogen5aa, 10aa and 15aa linker nucleic acid cassette constructs were tested for binding to CMV-grade 2 antigen using a protocol identical to that described above in Example 18. Each supernatant sample, with or without the addition of thrombin, was five-fold serially diluted (1/10, 1/50; 1/250, 1/1250, 1/6250, and 1/31250) in PBS, then applied to a 96-well plate coated with the CMV antigen. As shown in the graphical representation of the OD450 nm absorbance of the ELISA in
Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 12624329 | Nov 2009 | US | national |
This application claims priority to U.S. Provisional Patent Application Ser. No. 61/274,723, filed Aug. 20, 2009, and U.S. patent application Ser. No. 12/624,329 filed Nov. 23, 2009, the entire contents of both of which are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2010/002309 | 8/20/2010 | WO | 00 | 2/17/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61274723 | Aug 2009 | US |
