Patent Application
20240377394
A Sequence Listing is provided herewith in a text file, (STAN-1819WO_SEQ_LIST_ST25), created on Apr. 5, 2022 and having a size of 420,000 bytes of file. The contents of the text file are incorporated herein by reference in its entirety.
There is a current lack of technologies that can be used to purify cells engineered with multiple genetic modifications. Current limitations in payload capacity require the use of multiple expression constructs for delivering transgenes. Serial sorting on multiple surface markers is expensive, time consuming, and results in massive cell losses. These purification problems impose limitations on the ability to engineer cells, e.g., for cell-based therapies. Improved approaches for purifying cells engineered with multiple genetic modifications are therefore needed.
Provided are methods of selecting for cells that comprise two or more separate expression constructs. In certain embodiments, the methods comprise contacting a population of cells with two or more separate expression constructs under conditions in which the two or more expression constructs are delivered to cells of the population of cells. The two or more separate expression constructs comprise a first expression construct that encodes a fusion protein comprising a selection marker, a protein localization tag, and a protease cleavage site disposed between the selection marker and the protein localization tag. The second expression construct encodes a protein required for cell surface expression of the selection marker. Such methods further comprise selecting for cells exhibiting cell surface expression of the selection marker. Related cells, compositions, kits, and therapeutic methods are also provided.
Before the methods and compositions of the present disclosure are described in greater detail, it is to be understood that the methods and compositions are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the methods and compositions will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the methods and compositions. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the methods and compositions, subject to any specifically excluded limit in the stated range.
Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and compositions.
Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions belong. Although any methods and compositions similar or equivalent to those described herein can also be used in the practice or testing of the methods and compositions, representative illustrative methods and compositions are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and/or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present methods and compositions are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.
It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
It is appreciated that certain features of the methods and compositions, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods and compositions, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and/or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present methods and compositions and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
Aspects of the present disclosure include methods of selecting for cells that comprise two or more separate expression constructs. The methods find use in a variety of applications including both research and clinical applications. By way of example, the methods find use in any application in which it is desirable to efficiently engineer and select for cells comprising multiple genetic modifications (e.g., transgenic modifications, gene knockouts, and/or the like), where such genetic modifications are difficult or not feasible using a single expression construct, e.g., due to the limitations in expression construct payload capacity. The methods of the present disclosure enable the selection of cells comprising the multiple desired genetic modification using a single selection marker. That is, cell surface expression of a single selection marker is the readout for cells comprising each of the desired multiple genetic modifications, obviating the need for serial sorting on multiple surface markers to obtain cells comprising the multiple modifications. Cells comprising the multiple desired genetic modifications can be readily selected (sometimes referred to herein as “purified” or “enriched”) based on the single selection marker using existing reagents and systems for magnetic-activated cell sorting (MACS), fluorescence-activated cell sorting (FACS), and the like.
According to some embodiments, the multiple genetic modifications find use in the context of cell-based therapies, such that the methods of the present disclosure find use in producing and selecting cells for such therapies. Non-limiting examples of genetic modifications that find use in cell-based therapies include transgenic modification to express a recombinant receptor (e.g., a chimeric antigen receptor (CAR), a T cell receptor (TCR), etc.) that targets undesirable cells (e.g., cancer cells) when administered to an individual, transgenic and/or knockout modifications that reduce immunogenicity of the engineered cells upon administration to an individual, transgenic and/or knockout modifications that confer upon the cells resistance to cell exhaustion upon administration to an individual, transgenic and/or knockout modifications that enhance the effectiveness of the cells in the tumor microenvironment (TME) for treatment of solid tumors, and/or the like. Any desired combination of such modifications may be made and selected for according to the methods of the present disclosure.
The selection approaches of the present disclosure are sometimes referred to herein as the “STASH selection system”, “STASH select”, etc. by virtue of the selection marker being “stashed” intracellularly in the absence of the desired combination of expression constructs being present in the cell. According to the selection system, one of the expression constructs encodes a fusion protein comprising the selection marker, a protein localization tag, and a protease cleavage site disposed between the selection marker and the protein localization tag. In the absence of one or more additional expression constructs which provide a protease capable of cleaving the protease cleavage site, the selection marker remains localized to (i.e., retained or “stashed” at) the intracellular location (e.g., organelle) determined by the particular protein localization tag employed. When the one or more additional expression constructs are present in the cell, thereby providing a protease capable of cleaving the protease cleavage site, the selection marker is cleaved from the protein localization tag and traffics to the surface of the cell, such that the cell comprising the desired multiple genetic modifications exhibits cell surface expression of the selection marker. The selection systems of the present disclosure are modular and include configurations such that the delivery to the cell of 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more separate expression constructs (each of which may provide a desired genetic modification, e.g., transgene, targeted gene knockout, and/or the like) is required to provide the protease activity necessary for cell surface expression of the selection marker.
Shown in
Select system. Cells which satisfy the two input requirements (expression of construct A and expression of construct B) result in the output surface expression of the selection marker.
In certain embodiments, the methods of the present disclosure comprise contacting a population of cells with two or more separate expression constructs under conditions in which the two or more expression constructs are delivered to cells of the population of cells. The two or more separate expression constructs comprise a first expression construct that encodes a fusion protein comprising a selection marker, a protein localization tag, and a protease cleavage site disposed between the selection marker and the protein localization tag. The two or more separate expression constructs further comprise a second expression construct that encodes a protein required for cell surface expression of the selection marker. The methods further comprise selecting for cells exhibiting cell surface expression of the selection marker.
The contacting step may comprise contacting the population of cells with the two or more separate expression constructs simultaneously, e.g., by combining the cells and each of the two or more separate expression constructs in a single mixture under conditions suitable for delivery (e.g., transfection, transduction, etc.) of each of the two or more separate expression constructs into cells of the population of cells. The contacting step may comprise contacting the population of cells with the two or more separate expression constructs sequentially, e.g., where the population of cells is first combined with less than each of the two or more separate expression constructs under expression construct delivery conditions, followed by combining the population of cells with the remaining two or more separate expression constructs in one or more further combining steps under suitable conditions.
A variety of suitable approaches and conditions for the delivery of expression constructs to cells are known. According to some embodiments, the two or more separate expression constructs are delivered to cells of the population of cells by microinjection, transfection, lipofection, heat-shock, electroporation, transduction, gene gun, DEAE-dextran-mediated transfer, and/or the like. In certain embodiments, the two or more separate expression constructs are introduced into cells of the population of cells by AAV transduction. The AAV vector may comprise ITRs from AAV2, and a serotype from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV 10. According to some embodiments, the AAV vector comprises ITRs from AAV2 and a serotype from AAV6. In certain embodiments, the nucleic acid (e.g., expression vector) encoding the CAR is introduced into the cell (e.g., a T cell) by lentiviral or retroviral transduction. The lentiviral vector backbone may be derived from HIV-1, HIV-2, visna-maedi virus (VMV) virus, caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immune deficiency virus (BIV), or simian immunodeficiency virus (SIV). The lentiviral vector may be integration competent or an integrase deficient lentiviral vector (TDLV). In one embodiment, IDLV vectors including an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) are employed. Non-limiting example approaches for the preparation of retroviral expression constructs and the transduction of cells with such constructs is provided in the Experimental section hereinbelow.
As used herein, an “expression construct” is a circular or linear polynucleotide (a polymer composed of naturally-occurring and/or non-naturally-occurring nucleotides) comprising a region that encodes a component of the cell selection system (e.g., a fusion protein comprising a selection marker, a protein localization tag, and a protease cleavage site; and/or a protein required for cell surface expression of the selection marker) operably linked to a suitable promoter, e.g., a constitutive or inducible promoter. In some embodiments, expression of the cell selection system component is under the control of one or more exogenous (including heterologous) regulatory elements, e.g., promoter, enhancer, etc., present in the expression construct, and operably linked to the region encoding the cell selection system component, prior to contacting with the population of cells. In some embodiments, expression of the cell selection system component may be controlled by one or more endogenous regulatory elements, e.g., promoter, enhancer, etc., at or near a genomic locus into which the expression construct is inserted.
One or more of the two or more separate expression constructs may further comprise one or more regions encoding one or more proteins of interest (e.g., any of the proteins of interest described elsewhere herein), each operably linked to a suitable promoter, where the promoter may be a single shared promoter among each of the protein-encoding regions of the expression construct (including the cell selection system component), or at least one of the protein-encoding regions may be operably linked to a promoter which is not shared with any other protein-encoding region of the expression construct. In certain embodiments, when an expression construct comprises one or more protein-encoding regions in addition to the region encoding the component of the cell selection system, the expression construct may be configured to allow for polycistronic expression of two or more (e.g., each) of the protein-encoding regions. That is, two or more (e.g., each) of the proteins encoded by the expression construct may be expressed as separate proteins from the same promoter. In certain embodiments, the expression construct includes a ribosome skipping site to allow for polycistronic expression of two or more (e.g., each) of the protein-encoding regions. A non-limiting example of a suitable ribosome skipping site which may be incorporated into expression constructs is the P2A ribosome skipping site from porcine teschovirus.
The expression constructs (e.g., vectors) can be suitable for replication and integration in prokaryotes, eukaryotes, or both. The expression constructs may contain functionally appropriately oriented transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid encoding the component of the cell selection system. The expression constructs optionally contain generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in both eukaryotes and prokaryotes, e.g., as found in shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.
To obtain high levels of expression of a cloned nucleic acid it is common to construct expression constructs which typically contain a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator, each in functional orientation to each other and to the protein-encoding sequence. Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator region of the E. coli tryptophan biosynthetic pathway, the leftward promoter of phage lambda (PL), and the L-arabinose (araBAD) operon. The inclusion of selection markers in DNA vectors transformed in E. coli is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol. Expression systems for expressing the selection system components are available using, for example, E. coli, Bacillus sp. and Salmonella. E. coli systems may also be used. Nucleic acids encoding the selection system components. Transducing cells with nucleic acids can involve, for example, incubating lipidic microparticles containing nucleic acids with cells or incubating viral vectors containing nucleic acids with cells within the host range of the vector.
The two or more expression constructs are “separate”, meaning that none of the two or more expression constructs are part of the same polynucleotide molecule.
In certain embodiments, upon delivery of the two or more separate expression constructs to cells of the population of cells, one or more of the expression constructs are episomal (e.g., extra-chromosomal), where by “episome” or “episomal” is meant a polynucleotide that replicates independently of the cell's chromosomal DNA. A non-limiting example of an episome that may be employed in the present methods is a plasmid.
According to some embodiments, upon delivery of the two or more separate expression constructs to cells of the population of cells, one or more of the expression constructs integrates into the genome of the cell. In certain embodiments, one or more of the expression constructs are adapted for site-specific integration into the genome. For example, an expression construct may be adapted for site-specific integration into the genome, where the site-specific integration inactivates a target gene within the genome of the cell. By way of example, the site-specific integration may knock-out the target gene by knock-in of the expression construct. Any suitable approach for site-specific gene editing and functional integration may be employed. Functional integration of an expression construct may be achieved through various means, including through the use of integrating vectors, including viral and non-viral vectors. In some instances, a retroviral vector, e.g., a lentiviral vector, may be employed. In some instances, a non-retroviral integrating vector may be employed. An integrating vector may be contacted with the cells in a suitable transduction medium, at a suitable concentration (or multiplicity of infection), and for a suitable time for the vector to infect the target cells, facilitating functional integration of the expression construct. Non-limiting examples of useful viral vectors include retroviral vectors, lentiviral vectors, adenoviral (Ad) vectors, adeno-associated virus (AAV) vectors, hybrid Ad-AAV vector systems, and the like.
Strategies for site-specific integration that find use in the methods of the present disclosure include those that employ homologous recombination, nonhomologous end-joining (NHEJ), and/or the like. Such strategies may employ a non-naturally occurring or engineered nuclease, including, but not limited to, zinc-ringer nucleases (ZNFs), meganucleases, transcription activator-like effector nucleases (TALENs)), or a CRISPR-Cas system. Eukaryotic cells utilize two distinct DNA repair mechanisms in response to DNA double strand breaks (DSBs): Homologous recombination (HR) and nonhomologous end-joining (NHEJ). Mechanistically, HR is an error-free DNA repair mechanism because it requires a homologous template to repair the damaged DNA strand. Because of its homology-based mechanism, HR has been used as a tool to site-specifically engineer the genome. Gene targeting by HR requires the use of two homology arms that flank the transgene/target site of interest. HR efficiency can be increased by the introduction of DSBs at the target site using specific rare-cutting endonucleases. The discovery of this phenomenon prompted the development of methods to create site-specific DSBs in the genome of different species. Various chimeric enzymes have been designed for this purpose over the last decade, namely ZFNs, meganucleases, and TALENs. ZFNs are modular chimeric proteins that contain a ZF-based DNA binding domain (DBD) and a FokI nuclease domain. DBD is usually composed of three ZF domains, each with 3-base pair specificity; the FokI nuclease domain provides a DNA nicking activity, which is targeted by two flanking ZFNs. Owing to the modular nature of the DBD, any site in a genome could be targeted. TALENs are similar to ZFNs except that the DBD is derived from transcription activator-like effectors (TALEs). The TALE DBD is modular, and it is composed of 34-residue repeats, and its DNA specificity is determined by the number and order of repeats. Each repeat binds a single nucleotide in the target sequence through only two residues.
The methods of the present disclosure may be performed on any cell populations of interest. In certain embodiments, the population of cells is a population of prokaryotic cells (e.g., bacteria), a population of yeast cells, a population of insect (e.g., drosophila) cells, a population of amphibian (e.g., frog, e.g., Xenopus) cells, a population of plant cells, etc. According to some embodiments, the population of cells is a population of mammalian cells. Mammalian cells of interest include human cells, rodent cells, and the like. According to some embodiments, the population of cells is a population of peripheral blood mononuclear cells (PBMCs). In certain embodiments, the population of cells is a population of immune cells. The population of immune cells may comprise one or any combination of T cells, B cells, natural killer (NK) cells, macrophages, monocytes, neutrophils, dendritic cells, mast cells, basophils, eosinophils. When the immune cells comprise T cells, the T cells may comprise one or any combination of naive T cells (TN), cytotoxic T cells (TCTL), memory T cells (TMEM), T memory stem cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM), tissue resident memory T cells (TRM), effector T cells (TEFF), regulatory T cells (TREGs), helper T cells, CD4+ T cells, CD8+ T cells, virus-specific T cells, alpha beta T cells (Tαβ), gamma delta T cells (Tγδ). According to some embodiments, the population of cells is a population of cells comprises stem cells, e.g., mammalian (e.g., human) stem cells. For example, the population of cells may comprise embryonic stem (ES) cells, adult stem cells, hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), neural stem cells (NSCs), or any combination thereof.
As used herein, the term “protein localization tag” refers to an amino acid sequence that directs the cellular localization of the fusion protein comprising the selection marker (and optionally, any other cell selection system components expressed by the two or more separate expression constructs) to a particular cellular compartment. In certain embodiments, the protein localization tag is selected from an endoplasmic reticulum (ER) localization tag, a Golgi apparatus (Golgi) localization tag, a lysosome localization tag, a plasma membrane localization tag, a mitochondria localization tag, a peroxisome localization tag, a cytosolic localization tag, and a nuclear localization tag.
The fusion protein comprising the selection marker (and optionally, any other cell selection system component(s) expressed by the two or more separate expression constructs) may include any suitable protein localization tag for directing localization to the desired cellular compartment. In some embodiments, when two or more cell selection system components comprise a protein localization tag, the protein localization tag of each component may direct each component to the same cellular compartment (e.g., organelle). For example, in certain embodiments, when two or more cell selection system components comprise a protein localization tag, the protein localization tags are identical or substantially identical to each other.
Suitable protein localization tags are known. In certain embodiments, a cell selection system component includes a protein localization tag in LocSigDB (a database of protein localization signals/tags available at genome.unmc.edu/LocSigDB/and described in Negi et al. (2015) Database, Volume 2015:1-7); DBSubLoc (a database of protein subcellular localization—available at bioinfo.tsinghua.edu.cn/dbsubloc.html); LOCATE (a mammalian protein subcellular localization database available at locate.imb.uq.edu.au); LocDB (a protein localization database available at rostlab.org/services/locDB); eSLDB (a eukaryotic subcellular localization database available at gpcr.biocomp.unibo.it/esldb); and/or any other convenient database of protein localization tags. According to some embodiments, the protein localization tag is located at the N-terminus of the cell selection system component. For example, there are naturally-occurring N-terminal protein localization tags for type II membrane proteins (see, e.g., Schutz et al. (1994) EMBO J. 13 (7): 1696-1705) and other proteins.
According to some embodiments, the protein localization tag is an ER localization tag. In certain embodiments, the ER localization tag comprises the amino acid sequence KKMP. A non-limiting example of an ER localization tag that may be included in a cell selection system component of the present disclosure is an ER localization tag comprising 85% or greater, 90% or greater, or 100% amino acid sequence identity to an ER localization tag comprising, consisting of, or present within, an amino acid sequence selected from LYKYKSRRSFIDEKKMP (SEQ ID NO: 1); AEKDEL (SEQ ID NO:2); EQKLISEEDLKDEL (SEQ ID NO:3); GGGGSGGGGSKDEL (SEQ ID NO:4); GGGGSGGGGSGGGGSGGGGSKDEL ID (SEQ NO: 5); GGGGSGGGGSGGGGSGGGGSAEKDEL (SEQ ID NO:6); KYKSRRSFIEEKKMP (SEQ ID NO: 7); LKYKSRRSFIEEKKMP (SEQ ID NO:8); LYKYKSRRSFIEEKKMP (SEQ ID NO:9); LYCKYKSRRSFIEEKKMP (SEQ ID NO:10); LYCNKYKSRRSFIEEKKMP (SEQ ID NO:11); LYCNKYKSRRSFIDEKKMP (SEQ ID NO:12); LYEQKLISEEDLKYKSRRSFIEEKKMP (SEQ ID NO: 13); LYCYPYDVPDYAKYKSRRSFIEEKKMP (SEQ ID NO:14); LYKKLETFKKTN (SEQ ID NO: 15); LYEQKLISEEDLKKLETFKKTN (SEQ ID NO:16); LYYQRL (SEQ ID NO:17); LYEQKLISEEDLYQRL (SEQ ID NO:18); LYKRKIIAFALEGKRSKVTRRPKASDYQRL (SEQ ID NO: 19); LYRNIKCD (SEQ ID NO:20); and LYEQKLISEEDLRNIKCD (SEQ ID NO:21). Another example of an ER localization tag that may be included in a cell selection system component of the present disclosure is an ER localization tag comprising 85% or greater, 90% or greater, 95% or greater, or 100% amino acid sequence identity to an ER localization tag comprising, consisting of, or present within, an amino acid sequence selected from:
In certain embodiments, the protein localization tag is a Golgi localization tag. A non-limiting example of a Golgi localization tag that may be included in a cell selection system component of the present disclosure is a Golgi localization tag comprising the amino acid sequence YQRL (SEQ ID NO:36).
According to some embodiments, the protein localization tag is a lysosome localization tag. A non-limiting example of a lysosome localization tag that may be included in a cell selection system component of the present disclosure is a lysosome localization tag comprising the amino acid sequence KFERQ (SEQ ID NO:37).
As described above, the first expression construct encodes a fusion protein comprising a protease cleavage site disposed between the selection marker and the protein localization tag. The term “cleavage site” refers to the bond (e.g., a scissile bond) cleaved by an agent, e.g., a protease. A cleavage site for a protease includes the specific amino acid sequence recognized by the protease during proteolytic cleavage and may include surrounding amino acids (e.g., from one to six amino acids) on either side of the scissile bond, which bind to the active site of the protease and are needed for recognition as a substrate. In some embodiments, the cleavage site is provided as a cleavable linker, where “cleavable linker” refers to a linker including the protease cleavage site. A cleavable linker is typically cleavable under physiological conditions.
According to some embodiments, the protease cleavage site is a viral protease cleavage site. Non-limiting examples of viral protease cleavage sites include cleavage sites for potyviral family proteases. Potyviral family proteases of interest include Tobacco Etch Virus (TEV) protease, plum pox virus protease (PPVp), soybean mosaic virus protease (SbMVp), sunflower mild mosaic virus protease (SuMMVp), tobacco vein mottling virus protease (TVMVp), and West Nile virus protease (WNVp). In certain embodiments, the viral protease cleavage site is a TEV protease cleavage site. The amino acid sequence of an example TEV protease cleavage site is ENLYFQS. The amino acid sequence of an example TEV protease is the following:
In some embodiments, the protease is a TEV protease comprising the amino acid sequence set forth above, or is a functional (proteolytic) variant thereof having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, or 99% or greater amino acid sequence identity to such a sequence, and/or a functional (proteolytic) fragment thereof such as a fragment having a length of from 100 to 125, 125 to 150, 150 to 175, 175 to 200, 200 to 225, or from 225 to 235 amino acids. Such a protease may be provided by two or more (e.g., two) complementary fragments of the protease, wherein the two or more (e.g., two) complementary fragments form an active protease complex.
According to some embodiments, the viral protease cleavage site is for an HCV protease. In certain embodiments, the viral protease cleavage site is for a viral protease derived from HCV nonstructural protein 3 (NS3). NS3 consists of an N-terminal serine protease domain and a C-terminal helicase domain. By “derived from HCV NS3” is meant the protease is the serine protease domain of HCV NS3 or a proteolytically active variant thereof capable of cleaving a cleavage site for the serine protease domain of HCV NS3. The protease domain of NS3 forms a heterodimer with the HCV nonstructural protein 4A (NS4A), which activates proteolytic activity. A protease derived from HCV NS3 may include the entire NS3 protein or a proteolytically active fragment thereof, and may further include a cofactor polypeptide, such as a cofactor polypeptide derived from HCV nonstructural protein 4A (NS4A), e.g., an activating NS4A region. NS3 protease is highly selective and can be inhibited by a number of non-toxic, cell-permeable drugs, which are currently available for use in humans. NS3 protease inhibitors that may be employed include, but are not limited to, simeprevir, danoprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, and any combination thereof. Non-limiting examples of proteases derived from HCV NS3 are provided below.
Example Proteases Derived from HCV NS3
In some embodiments, the protease comprises one of the sequences set forth above, or is a functional (proteolytic) variant thereof having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, or 99% or greater amino acid sequence identity to one of such sequences, and/or a functional (proteolytic) fragment thereof such as a fragment having a length of from 100 to 185, 120 to 185, 140 to 185, 160 to 185, 170 to 185, from 180 to 185, from 182 to 185, or from 184 to 185 amino acids.
In some embodiments, the protease cleavage site is a viral protease cleavage site. For example, when a protease derived from HCV NS3 is employed, the cleavage site should comprise an NS3 protease cleavage site. An NS3 protease cleavage site may include the four junctions between nonstructural (NS) proteins of the HCV polyprotein normally cleaved by the NS3 protease during HCV infection, including the NS3/NS4A, NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B junction cleavage sites. For a description of NS3 protease and representative sequences of its cleavage sites for various strains of HCV, see, e.g., Hepatitis C Viruses: Genomes and Molecular Biology (S. L. Tan ed., Taylor & Francis, 2006), Chapter 6, pp. 163-206; the disclosure of which is incorporated herein by reference in its entirety.
In some embodiments, the protease is derived from HCV NS3 and engineered to include one or more amino acid substitutions relative to an HCV NS3 protease amino acid sequence set forth above. For example, the protease may include a substitution at the position corresponding to position 54 of the amino acid sequence APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWAVYHGAGTRTIA SPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLL SPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTD (SEQ ID NO: 39). In some embodiments, such a substitution is a threonine to alanine substitution.
NS3 nucleic acid and protein sequences may be derived from HCV, including any isolate of HCV having any genotype (e.g., genotypes 1-7) or subtype. A number of NS3 nucleic acid and protein sequences are known and described, e.g., in U.S. Ser. No. 15/737,712, the disclosure of which is incorporated herein by reference in their entirety for all purposes. Additional representative NS3 sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. YP_001491553, YP_001469631, YP_001469632, NP_803144, NP_671491, YP_001469634, YP_001469630, YP_001469633, ADA68311, ADA68307, AFP99000, AFP98987, ADA68322, AFP99033, ADA68330, AFP99056, AFP99041, CBF60982, CBF60817, AHH29575, AIZ00747, AIZ00744, ABI36969, ABN05226, KF516075, KF516074, KF516056, AB826684, AB826683, JX171009, JX171008, JX171000, EU847455, EF154714, GU085487, JX171065, and JX171063; all of which sequences are herein incorporated by reference. Any of these sequences or functional variants thereof having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater amino acid sequence identity to any one of these sequences, or proteolytic fragments thereof, may be employed.
NS4A nucleic acid and protein sequences may be derived from HCV, including any isolate of HCV having any genotype (e.g., seven genotypes 1-7) or subtype. A number of NS4A nucleic acid and protein sequences are known. Representative NS4A sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. NP_751925, YP_001491554, GU945462, HQ822054, FJ932208, FJ932207, FJ932205, and FJ932199; all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or functional variants thereof having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, or 99% or greater amino acid sequence identity to any one of these sequences, or proteolytic fragments thereof, may be employed.
HCV polyprotein nucleic acid and protein sequences may be derived from HCV, including any isolate of HCV having any genotype (e.g., genotypes 1-7) or subtype. A number of HCV polyprotein nucleic acid and protein sequences are known. Representative HCV polyprotein sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries: Accession Nos. YP_001469631, NP_671491, YP_001469633, YP_001469630, YP_001469634, YP_001469632, NC_009824, NC_004102, NC_009825, NC_009827, NC_009823, NC_009826, and EF108306; all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or functional variants thereof having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, or 99% or greater amino acid sequence identity to any one of these sequences, or proteolytic fragments thereof, may be employed.
In some embodiments, the protease is derived from HCV NS3 and the cleavage site includes an NS3 protease cleavage site. An NS3 protease cleavage site may include the HCV polyprotein NS3/NS4A, NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B junction cleavage sites. Representative HCV NS4A/4B protease cleavage sites include DEMEECSQH and DEMEECSQH. Representative HCV NS5A/5B protease cleavage sites include EDVVPCSMG and EDVVPCSMGS. A representative NS4B/5A protease cleavage site is ECTTPCSGSWL. Additional NS3 protease cleavage sites that may be included in a recombinant polypeptide of the present disclosure include those described in Shiryaev et al. (2012) PLOS One 7 (4): e35759.
In certain embodiments, the protease cleavage site is a human protease cleavage site. Non-limiting examples of human protease cleavage sites include cleavages sites for a human kallikrein (KLK) protease, human enterokinase protease, human thrombin, a human matrix metalloprotease (MMP), human urokinase-type plasminogen activator receptor (uPAR), human plasmin, or human cathepsin. According to some embodiments, the protease cleavage site is a cleavage site for a human kallikrein (KLK) protease, non-limiting examples of which include human KLK3 (UniProtKB-Q546G3), human KLK4 (UniProtKB-Q9Y5K2), human KLK6 (UniProtKB-Q92876), human KLK8 (UniProtKB-060259), human KLK11 (UniProtKB-Q9UBX7), human KLK13 (UniProtKB-Q9UKR3), human KLK14 (UniProtKB-Q9P0G3), and human KLK15 (UniProtKB-Q9H2R5). Data demonstrating the utility of human KLK proteases and corresponding cleavage sites in the STASH Select system is provided in the Experimental section herein. Any of these sequences or functional variants thereof having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater amino acid sequence identity to any one of these sequences, or proteolytic fragments thereof, may be employed.
In certain embodiments, the protease cleavage site is a protease cleavage site for a human protease selected from acrosin (ACR), AGBL carboxypeptidase 1 (AGBL1), AGBL carboxypeptidase 2 (AGBL2), AGBL carboxypeptidase 3 (AGBL3), AGBL carboxypeptidase 4 (AGBL4), AGBL carboxypeptidase 5 (AGBL5), ATP/GTP binding carboxypeptidase 1 (AGTPBP1), asparaginase and isoaspartyl peptidase 1 (ASRGL1), astacin like metalloendopeptidase (ASTL), ATP23 metallopeptidase and ATP synthase assembly factor homolog (ATP23), ataxin 3 (ATXN3), ataxin 3 like (ATXN3L), azurocidin 1 (AZU1), beta-secretase 1 (BACE1), beta-secretase 2 (BACE2), bone morphogenetic protein 1 (BMP1), BRCA1/BRCA2-containing complex subunit 3 (BRCC3), calpain 14 (CAPN14), calpain 3 (CAPN3), caspase recruitment domain family member 8 (CARD8), caspase 4 (CASP4), chymotrypsin like elastase 1 (CELA1), chymotrypsin like elastase 2A (CELA2A), chymotrypsin like elastase 2B (CELA2B), chymotrypsin like elastase 3A (CELA3A), chymotrypsin like elastase 3B (CELA3B), CUGBP Elav-like family member 3 (CELF3), CUGBP Elav-like family member 4 (CELF4), CUGBP Elav-like family member 5 (CELF5), CUGBP Elav-like family member 6 (CELF6), cell growth regulator with EF-hand domain 1 (CGREF1), charged multivesicular body protein 3 (CHMP3), CLN5 intracellular trafficking protein (CLN5), chymase 1 (CMA1), collectin subfamily member 11 (COLEC11), COP9 signalosome subunit 5 (COPS5), corin, serine peptidase (CORIN), carboxypeptidase A4 (CPA4), carboxypeptidase vitellogenic like (CPVL), cystatin SN (CST1), cystatin 11 (CST11), cystatin C (CST3), cystatin S (CST4), cystatin D (CST5), cystatin E/M (CST6), cystatin 8 (CST8), cystatin 9 (CST9), cystatin like 1 (CSTL1), chymotrypsinogen B2 (CTRB2), chymotrypsin like (CTRL), cathepsin L (CTSL), DNA damage inducible 1 homolog 2 (DDI2), DAP3 binding cell death enhancer 1 (DELE1), adipsin (DF), dickkopf WNT signaling pathway inhibitor 2 (DKK2), dickkopf WNT signaling pathway inhibitor 4 (DKK4), dipeptidase 1 (DPEP1), dipeptidyl peptidase 3 (DPP3), dipeptidyl peptidase 9 (DPP9), FAM111 trypsin like peptidase A (FAM111A), ficolin 1 (FCN1), ficolin 2 (FCN2), ficolin 3 (FCN3), G3BP stress granule assembly factor 1 (G3BP1), hepsin (HPN), HtrA serine peptidase 1 (HTRA1), insulin degrading enzyme (IDE), inner mitochondrial membrane peptidase subunit 2 (IMMP2L), jumonji domain containing 7 (JMJD7), Josephin domain containing 2 (JOSD2), kallikrein 1 (KLK1), kallikrein related peptidase 10 (KLK10), kallikrein related peptidase 11 (KLK11), kallikrein related peptidase 12 (KLK12), kallikrein related peptidase 13 (KLK13), kallikrein related peptidase 14 (KLK14), kallikrein related peptidase 15 (KLK15), kallikrein related peptidase 2 (KLK2), kallikrein related peptidase 3 (KLK3), kallikrein related peptidase 4 (KLK4), kallikrein related peptidase 5 (KLK5), kallikrein related peptidase 6 (KLK6), kallikrein related peptidase 7 (KLK7), kallikrein related peptidase 8 (KLK8), kallikrein related peptidase 9 (KLK9), kallikrein pseudogene 1 (KLKP1), lipocalin 2 (LCN2), legumain (LGMN), leishmanolysin like peptidase (LMLN), MAS1 proto-oncogene like, G protein-coupled receptor (MAS1L), MBL associated serine protease 1 (MASP1), MBL associated serine protease 2 (MASP2), mannose binding lectin 2 (MBL2), matrix metallopeptidase 10 (MMP10), matrix metallopeptidase 11 (MMP11), matrix metallopeptidase 13 (MMP13), matrix metallopeptidase 16 (MMP16), matrix metallopeptidase 2 (MMP2), napsin A aspartic peptidase (NAPSA), neurolysin (NLN), NLR family CARD domain containing 4 (NLRC4), NLR family pyrin domain containing 1 (NLRP1), aminopeptidase puromycin sensitive (NPEPPS), opiorphin prepropeptide (OPRPN), OTU deubiquitinase, ubiquitin aldehyde binding 2 (OTUB2), poly (ADP-ribose) polymerase family member 9 (PARP9), proprotein convertase subtilisin/kexin type 1 (PCSK1), proprotein convertase subtilisin/kexin type 1 inhibitor (PCSK1N), proprotein convertase subtilisin/kexin type 2 (PCSK2), proprotein convertase subtilisin/kexin type 4 (PCSK4), proprotein convertase subtilisin/kexin type 5 (PCSK5), proprotein convertase subtilisin/kexin type 6 (PCSK6), proprotein convertase subtilisin/kexin type 7 (PCSK7), proprotein convertase subtilisin/kexin type 9 (PCSK9), platelet derived growth factor C (PDGFC), pepsinogen A3 (PGA3), pepsinogen A4 (PGA4), pepsinogen A5 (PGA5), pyroglutamyl-peptidase I like (PGPEP1L), PTEN induced kinase 1 (PINK1), prolyl endopeptidase like (PREPL), parkin RBR E3 ubiquitin protein ligase (PRKN), serine protease gene group (PRSS), serine protease 2 (PRSS2), serine protease 21 (PRSS21), serine protease 22 (PRSS22), serine protease 23 (PRSS23), serine protease 27 (PRSS27), serine protease 33 (PRSS33), serine protease 46, pseudogene (PRSS46P), serine protease 55 (PRSS55), serine protease 8 (PRSS8), proteinase 3 (PRTN3), presenilin 2 (PSEN2), PYD and CARD domain containing (PYCARD), retinoic acid receptor responder 1 (RARRES1), ring finger and FYVE like domain containing E3 ubiquitin protein ligase (RFFL), rhomboid like 2 (RHBDL2), SEC11 homolog A, signal peptidase complex subunit (SEC11A), SEC11 homolog B, signal peptidase complex subunit (SEC11B), SEC11 homolog C, signal peptidase complex subunit (SEC11BC), SUMO peptidase family member, NEDD8 specific (SENP8), SET nuclear proto-oncogene (SET), synaptosome associated protein 25 (SNAP25), secreted phosphoprotein 2 (SPP2), small proline rich protein 3 (SPRR3), spleen associated tyrosine kinase (SYK), transcription factor EB (TFEB), transglutaminase 2 (TGM2), toll like receptor adaptor molecule 1 (TICAM1), tubulointerstitial nephritis antigen like 1 (TINAGL1), transmembrane serine protease 11D (TMPRSS11D), transmembrane serine protease 11E (TMPRSS11E), transmembrane serine protease 4 (TMPRSS4), transmembrane serine protease 5 (TMPRSS5), transmembrane serine protease 6 (TMPRSS6), transmembrane serine protease 7 (TMPRSS7), TNF receptor superfamily member 10a (TNFRSF10A), tryptase alpha/beta 1 (TPSAB1), tryptase beta 2 (TPSB2), tryptase delta 1 (TPSD1), tryptase gamma 1 (TPSG1), tryptase pseudogene 2 (TPSP2), tyrosylprotein sulfotransferase 1 (TPST1), tyrosylprotein sulfotransferase 2 (TPST2), tyrosylprotein sulfotransferase 2 pseudogene 1 (TPST2P1), thyrotropin releasing hormone degrading enzyme (TRHDE), thyroid hormone receptor interactor 4 (TRIP4), ubiquitin C-terminal hydrolase L1 (UCHL1), ubiquitin specific peptidase 27 X-linked (USP27X), vasohibin 2 (VASH2), valosin containing protein (VCP), and WAP four-disulfide core domain 1 (WFDC1).
In some embodiments, the protease is highly selective for the cleavage site. Additionally, the protease activity may be capable of inhibition by known small molecule inhibitors that are cell-permeable and not toxic to the cell or individual under study or treatment. For a discussion of proteases, see, e.g., V. Y. H. Hook, Proteolytic and cellular mechanisms in prohormone and proprotein processing, RG Landes Company, Austin, Tex., USA (1998); N. M. Hooper et al., Biochem. J. 321:265-279 (1997); Z. Werb, Cell 91:439-442 (1997); T. G. Wolfsberg et al., J. Cell Biol. 131:275-278 (1995); T. Berg et al., Biochem. J. 307:313-326 (1995); M. J. Smyth and J. A. Trapani, Immunology Today 16:202-206 (1995); R. V. Talanian et al., J. Biol. Chem. 272: 9677-9682 (1997); and N. A. Thornberry et al., J. Biol. Chem. 272:17907-17911 (1997), the disclosures of which are incorporated herein by reference in their entireties for all purposes. In some embodiments, the protease employed is a sequence-specific non-human protease for which FDA-approved pharmacological inhibitors are available.
Any of the proteases employed according to the methods of the present disclosure, including any of the proteases described above, may be provided by two or more (e.g., two) complementary fragments of the protease, where the two or more (e.g., two) complementary fragments form an active protease complex. A protease may be provided by two or more (e.g., two) complementary fragments of the protease, e.g., in order to increase the number of separate expression constructs required for cell surface expression of the selection marker.
Any of the cell selection system components of the present disclosure may comprise a membrane association domain. Non-limiting examples of membrane association domains include transmembrane domains. A transmembrane (Tm) domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. In some embodiments, the Tm domain is derived from (e.g., includes at least the transmembrane region(s) or a functional portion thereof) of the alpha or beta chain of CD35, CD3ζ, CD3γ, CD3δ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, or PD-1. In certain embodiments, the transmembrane domain is a CD8α transmembrane domain. According to some embodiments, the transmembrane domain is a CD28 transmembrane domain. Non-limiting examples of transmembrane domains that may be included in one or more (e.g., each) of the cell selection system components are a transmembrane domain comprising 80% or greater, 85% or greater, 90% or greater, 95% or greater, or 100% amino acid sequence identity to a transmembrane domain comprising, consisting of, or present within, an amino acid sequence selected from WLRLLPFLGVLALLGYLAVRPFL (SEQ ID NO:42); VLWWSIAQTVILILTGIW (SEQ ID NO:43); LGPEWDLYLMTIIALLLGTVI (SEQ ID NO:44); YYASAFSMMLGLFIFSIVFL (SEQ ID NO:45); IAFLLACVATMIFMITKCCLF (SEQ ID NO:46); VIGFLLAVVLTVAFITF (SEQ ID NO:47); GLFLSAFLLLGLFKALGWAAV (SEQ ID NO:48); VGLVLAAILALLLAFYAFFYL (SEQ ID NO:49); TFCSTALLITALALVCTLLYL (SEQ ID NO:50); WYVWLAIFFAIIIFILILGWVLL (SEQ ID NO:51); WLWVVYILT VALPVFLVILFC (SEQ ID NO:52); IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:53); and FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:54).
Any of the cell selection system components of the present disclosure may comprise a hinge domain, e.g., a CD8α hinge domain, a CD28 hinge domain, or the like.
Exemplary amino acid sequences of transmembrane domains and hinge domains that may be included in one or more (e.g., each) of the cell selection system components are provided herein.
Non-limiting examples of membrane association domains also include post-translational modifications that tether the cell selection system component to a membrane. That is, the cell selection system component may comprise a post-translationally added membrane-tethering domain. By “membrane-tethering domain” is meant a domain (e.g., moiety) capable of stably associating with a membrane (e.g., ER membrane) of the cell. Suitable membrane-tethering domains include, but are not limited to, post-translational modifications such as palmitoylation, myristoylation, prenylation, a glycosylphosphatidylinositol (GPI) anchor, and the like.
In some embodiments, when two or more cell selection system components comprise a membrane association domain (e.g., transmembrane domain), the membrane association domain of each component may be identical or substantially identical to each other.
In some embodiments, in order to increase the number of separate expression constructs required for cell surface expression of the selection marker, two or more cell selection system components each comprise a dimerization domain, where dimerization of the cell selection system components is required for cell surface expression of the selection marker. Examples of cell selection system configurations that employ one or more pairs of dimerization domains are described elsewhere. Non-limiting examples of dimerization domains that may be employed include domains comprising a coiled coil structure. When the dimerization domain comprises a coiled coil structure, in some embodiments, the dimerization domain comprises a leucine zipper domain.
The two or more separate expression constructs may each provide a genetic modification to the cells to which the two or more separate expression constructs are delivered. Non-limiting examples of genetic modifications include providing a region of the expression construct that encodes a protein of interest. Non-limiting examples of proteins of interest include a receptor, a ligand, a transcription factor, an antibody, a bispecific T-cell engager (BiTE), an enzyme, a cytokine, a chemokine, a toxin, a protein conferring resistance to cell exhaustion, and a suicide switch protein.
In some embodiments, a protein of interest further encoded by one or more expression constructs of the two or more separate expression constructs is a receptor. For example, one or more expression constructs of the two or more separate expression constructs may encode a receptor independently selected from a chimeric antigen receptor (CAR), a T cell receptor (TCR) such as a recombinant TCR, a chimeric cytokine receptor (CCR), a chimeric chemokine receptor, a synthetic notch receptor (synNotch), a Modular Extracellular Sensor Architecture (MESA) receptor, a Tango receptor, a ChaCha receptor, a generalized extracellular molecule sensor (GEMS) receptor, a growth factor receptor, a cytokine receptor, a chemokine receptor, a switch receptor, an adhesion molecule, an integrin, an inhibitory receptor, a stimulatory receptor, an immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor, an immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor, a hormone receptor, a receptor tyrosine kinase, an immune receptor such as CD28, CD80, ICOS, CTLA4, PD1, PD-L1, BTLA, HVEM, CD27, 4-1BB, 4-1BBL, OX40, OX40L, DR3, GITR, CD30, SLAM, CD2, 2B4, TIM1, TIM2, TIM3, TIGIT, CD226, CD160, LAG3, LAIR1, B7-1, B7-H1, and B7-H3, a type I cytokine receptor such as Interleukin-1 receptor, Interleukin-2 receptor, Interleukin-3 receptor, Interleukin-4 receptor, Interleukin-5 receptor, Interleukin-6 receptor, Interleukin-7 receptor, Interleukin-9 receptor, Interleukin-11 receptor, Interleukin-12 receptor, Interleukin-13 receptor, Interleukin-15 receptor, Interleukin-18 receptor, Interleukin-21 receptor, Interleukin-23 receptor, Interleukin-27 receptor, Erythropoietin receptor, GM-CSF receptor, G-CSF receptor, Growth hormone receptor, Prolactin receptor, Leptin receptor, Oncostatin M receptor, Leukemia inhibitory factor, a type II cytokine receptor such as interferon-alpha/beta receptor, interferon-gamma receptor, Interferon type Ill receptor, Interleukin-10 receptor, Interleukin-20 receptor, Interleukin-22 receptor, Interleukin-28 receptor, a receptor in the tumor necrosis factor receptor superfamily such as Tumor necrosis factor receptor 2 (1B), Tumor necrosis factor receptor 1, Lymphotoxin beta receptor, OX40, CD40, Fas receptor, Decoy receptor 3, CD27, CD30, 4-1BB, Decoy receptor 2, Decoy receptor 1, Death receptor 5, Death receptor 4, RANK, Osteoprotegerin, TWEAK receptor, TACI, BAFF receptor, Herpesvirus entry mediator, Nerve growth factor receptor, B-cell maturation antigen, Glucocorticoid-induced TNFR-related, TROY, Death receptor 6, Death receptor 3, Ectodysplasin A2 receptor, a chemokine receptor such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CX3CR1, XCR1, ACKR1, ACKR2, ACKR3, ACKR4, CCRL2, a receptor in the epidermal growth factor receptor (EGFR) family, a receptor in the fibroblast growth factor receptor (FGFR) family, a receptor in the vascular endothelial growth factor receptor (VEGFR) family, a receptor in the rearranged during transfection (RET) receptor family, a receptor in the Eph receptor family, a receptor that can induce cell differentiation (e.g., a Notch receptor), a cell adhesion molecule (CAM), an adhesion receptor such as integrin receptor, cadherin, selectin, and a receptor in the discoidin domain receptor (DDR) family, transforming growth factor beta receptor 1, and transforming growth factor beta receptor 2. In some embodiments, such a receptor is an immune cell receptor selected from a T cell receptor, a B cell receptor, a natural killer (NK) cell receptor, a macrophage receptor, a monocyte receptor, a neutrophil receptor, a dendritic cell receptor, a mast cell receptor, a basophil receptor, and an eosinophil receptor.
According to some embodiments, one or more expression constructs of the two or more separate expression constructs may encode a CAR. When two or more separate expression constructs encode a CAR, the CAR may be the same CAR, or the two or more separate expression constructs may encode two or more different CARs. In certain embodiments, when the protein of interest is a CAR, the extracellular binding domain of the CAR comprises a single chain antibody. The single-chain antibody may be a monoclonal single-chain antibody, a chimeric single-chain antibody, a humanized single-chain antibody, a fully human single-chain antibody, and/or the like. In one non-limiting example, the single chain antibody is a single chain variable fragment (scFv). Suitable CAR extracellular binding domains include those described in Labanieh et al. (2018) Nature Biomedical Engineering 2:377-391. In some embodiments, the extracellular binding domain of the CAR is a single-chain version (e.g., an scFv version) of an antibody approved by the United States Food and Drug Administration and/or the European Medicines Agency (EMA) for use as a therapeutic antibody, e.g., for inducing antibody-dependent cellular cytotoxicity (ADCC) of certain disease-associated cells in a patient, etc. Non-limiting examples of single-chain antibodies which may be employed when the protein of interest is a CAR include single-chain versions (e.g., scFv versions) of Adecatumumab, Ascrinvacumab, Cixutumumab, Conatumumab, Daratumumab, Drozitumab, Duligotumab, Durvalumab, Dusigitumab, Enfortumab, Enoticumab, Figitumumab, Ganitumab, Glembatumumab, Intetumumab, Ipilimumab, Iratumumab, Icrucumab, Lexatumumab, Lucatumumab, Mapatumumab, Narnatumab, Necitumumab, Nesvacumab, Ofatumumab, Olaratumab, Panitumumab, Patritumab, Pritumumab, Radretumab, Ramucirumab, Rilotumumab, Robatumumab, Seribantumab, Tarextumab, Teprotumumab, Tovetumab, Vantictumab, Vesencumab, Votumumab, Zalutumumab, Flanvotumab, Altumomab, Anatumomab, Arcitumomab, Bectumomab, Blinatumomab, Detumomab, Ibritumomab, Minretumomab, Mitumomab, Moxetumomab, Naptumomab, Nofetumomab, Pemtumomab, Pintumomab, Racotumomab, Satumomab, Solitomab, Taplitumomab, Tenatumomab, Tositumomab, Tremelimumab, Abagovomab, Igovomab, Oregovomab, Capromab, Edrecolomab, Nacolomab, Amatuximab, Bavituximab, Brentuximab, Cetuximab, Derlotuximab, Dinutuximab, Ensituximab, Futuximab, Girentuximab, Indatuximab, Isatuximab, Margetuximab, Rituximab, Siltuximab, Ublituximab, Ecromeximab, Abituzumab, Alemtuzumab, Bevacizumab, Bivatuzumab, Brontictuzumab, Cantuzumab, Cantuzumab, Citatuzumab, Clivatuzumab, Dacetuzumab, Demcizumab, Dalotuzumab, Denintuzumab, Elotuzumab, Emactuzumab, Emibetuzumab, Enoblituzumab, Etaracizumab, Farletuzumab, Ficlatuzumab, Gemtuzumab, Imgatuzumab, Inotuzumab, Labetuzumab, Lifastuzumab, Lintuzumab, Lorvotuzumab, Lumretuzumab, Matuzumab, Milatuzumab, Nimotuzumab, Obinutuzumab, Ocaratuzumab, Otlertuzumab, Onartuzumab, Oportuzumab, Parsatuzumab, Pertuzumab, Pinatuzumab, Polatuzumab, Sibrotuzumab, Simtuzumab, Tacatuzumab, Tigatuzumab, Trastuzumab, Tucotuzumab, Vandortuzumab, Vanucizumab, Veltuzumab, Vorsetuzumab, Sofituzumab, Catumaxomab, Ertumaxomab, Depatuxizumab, Ontuxizumab, Blontuvetmab, Tamtuvetmab, or an antigen-binding variant thereof. According to some embodiments, when the protein of interest is a CAR, the extracellular binding domain of the CAR specifically binds an antigen expressed on the surface of a cancer cell. For example, the extracellular binding domain may bind a cancer cell-surface antigen selected from B7-H3 (CD276), CD19, GD2, CD22, and HER2.
According to some embodiments, one or more expression constructs of the two or more separate expression constructs may encode an antibody. When two or more separate expression constructs encode an antibody, the antibody may be the same antibody, or the two or more separate expression constructs may encode two or more different antibodies. The term “antibody” (also used interchangeably with “immunoglobulin”) encompasses antibodies of any isotype (e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodies composed of a tetramer which in turn is composed of two dimers of a heavy and light chain polypeptide); single chain antibodies (e.g., scFv); fragments of antibodies (e.g., fragments of whole or single chain antibodies) which retain specific binding to the antigen, including, but not limited to single chain Fv (scFv), Fab, (Fab′)2, (scFv′)2, and diabodies; chimeric antibodies; monoclonal antibodies, humanized antibodies, human antibodies; and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein.
Immunoglobulin polypeptides include the kappa and lambda light chains and the alpha, gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu heavy chains or equivalents in other species. Full-length immunoglobulin “light chains” (usually of about 25 kDa or about 214 amino acids) comprise a variable region of about 110 amino acids at the NH2-terminus and a kappa or lambda constant region at the COOH-terminus. Full-length immunoglobulin “heavy chains” (of about 150 kDa or about 446 amino acids), similarly comprise a variable region (of about 116 amino acids) and one of the aforementioned heavy chain constant regions, e.g., gamma (of about 330 amino acids).
An immunoglobulin light or heavy chain variable region (VL and VH, respectively) is composed of a “framework” region (FR) interrupted by three hypervariable regions, also called “complementarity determining regions” or “CDRs”. The extent of the framework region and CDRs have been defined (see, E. Kabat et al., Sequences of proteins of immunological interest, 4th ed. U.S. Dept. Health and Human Services, Public Health Services, Bethesda, MD (1987); and Lefranc et al. IMGT, the international ImMunoGeneTics information System®. Nucl. Acids Res., 2005, 33, D593-D597)). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope of an antigen. All CDRs and framework provided by the present disclosure are defined according to Kabat, supra, unless otherwise indicated.
An “antibody” thus encompasses a protein having one or more polypeptides that can be genetically encodable, e.g., by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. In some embodiments, an antibody of the present disclosure is an IgG antibody, e.g., an IgG1 antibody, such as a human IgG1 antibody. In some embodiments, an antibody of the present disclosure comprises a human Fc domain.
A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
Antibodies encompass intact immunoglobulins as well as a number of well characterized fragments which may be genetically encoded or produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′2, a dimer of Fab which itself is a light chain joined to VH-CHI by a disulfide bond. The F(ab)′2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab′) 2 dimer into an Fab′ monomer. The Fab′ monomer is essentially a Fab with part of the hinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab′ fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. In certain embodiments, an antibody of the present disclosure is selected from an IgG, Fv, single chain antibody, scFv, Fab, F (ab′) 2, and Fab′.
One or more of the two or more separate expression constructs may encode a protein of interest that finds use in the context of cell therapy (e.g., a cell-based cancer therapy), non-limiting examples of which include a therapy comprising administration of therapeutic immune cells such as T cells (e.g., CAR T cells, T cells that express an engineered T cell receptor (TCR), and the like), NK cells (e.g., CAR NK cells), macrophages (e.g., CAR macrophages), etc. Examples of such proteins that may be expressed by one or more of the two or more separate expression constructs include those described in Rodriguez-Garcia et al. (2020) Front Immunol. 11:1109; Martinez & Moon (2019) Front. Immunol. 10:128; Knochelmann et al. (2018) Front Immunol. 9:1740; and the like, the disclosures of which are incorporated herein in their entireties for all purposes.
In some embodiments, one or more of the two or more separate expression constructs express a protein independently selected from a protein that reduces immunogenicity of the engineered cells upon administration to an individual, a protein that confers upon the cells resistance to cell exhaustion upon administration to an individual (e.g., cJun, etc.), a protein that enhances the effectiveness of the cells in the tumor microenvironment (TME) for treatment of solid tumors (e.g., a switch receptor, a dominant negative receptor), an HLA-E protein, a CD47 protein, a homing protein (e.g., a chemokine receptor), a persistence promoting protein (e.g., a cytokine receptor), an autonomous control unit protein (e.g., a gene circuit protein, an oscillator protein, etc.), a protein that rewires the metabolism of the cells, logic gating proteins (e.g., SynNotch, iCAR), a suicide switch protein (e.g., EGFRt, iCASP9, etc.), and any other proteins useful in the context of cell therapy.
Non-limiting examples of genetic modifications also include inactivating (e.g., knocking out) one or more genes in the genome of the cell. Accordingly, in some embodiments, one or more of the two or more separate expression constructs are configured to site-specifically integrate into the genome of the cell in a manner that inactivates one or more target genes.
In some embodiments, one or more of the two or more separate expression constructs are configured to site-specifically integrate into the genome of the cell in a manner that inactivates one or more target genes, where such gene inactivation finds use in the context of cell therapy (e.g., a cell-based cancer therapy), non-limiting examples of which include a therapy comprising administration of therapeutic immune cells such as T cells (e.g., CAR T cells, T cells that express an engineered T cell receptor (TCR), and the like), NK cells (e.g., CAR NK cells), macrophages (e.g., CAR macrophages), etc. Examples of such gene inactivations include those described in Rodriguez-Garcia et al. (2020) Front Immunol. 11:1109; Martinez & Moon (2019) Front. Immunol. 10:128; Knochelmann et al. (2018) Front Immunol. 9:1740; and the like, the disclosures of which are incorporated herein in their entireties for all purposes.
In some embodiments, one or more of the two or more separate expression constructs are configured to site-specifically integrate into the genome of the cell in a manner that inactivates one or more target genes, where such gene inactivation reduces immunogenicity of the engineered cells upon administration to an individual (e.g., knockout of one or more T cell receptor genes, e.g., a TRAC knockout), confers upon the cells resistance to cell exhaustion upon administration to an individual, enhances the effectiveness of the cells in the tumor microenvironment (TME) for treatment of solid tumors, promotes persistence of the cells, and any other gene inactivation useful in the context of cell therapy.
The first expression construct may encode a fusion protein comprising any convenient selection marker that enables selection of cells comprising the two or more separate expression constructs. In some embodiments, the selection marker is one that is already used for cell selection purposes for which there are existing reagents (e.g., antibodies, etc.) and devices for selecting cells exhibiting cell surface expression of the selection marker. For example, the selection marker may be one that is currently employed in magnetic-activated cell sorting (MACS) workflows, flow cytometry workflows (e.g., fluorescence-activated cell sorting (FACS) workflows), and the like.
The selection marker may be a protein tag. For example, the selection marker may be a Myc-tag, a His-tag, an HA-tag, a FLAG-tag, a Strep-tag, an NE-tag, an Xpress tag, an Avi-tag, a polyglutamate tag, a polyarginine tag, or the like.
In certain embodiments, the selection marker comprises a cluster of differentiation (CD) protein. A non-limiting example of a CD protein that finds use as a selection marker is CD34.
According to some embodiments, the selection marker comprises a truncated receptor comprising the extracellular domain of the receptor. Examples of the truncated receptors that find use as selection markers include a truncated epidermal growth factor receptor (EGFRt), a truncated nerve growth factor receptor (NGFRt), a truncated CD19 (CD19t), and a truncated CD20 (CD20t).
As will be appreciated with the benefit of the present disclosure, the selection marker may be chosen such that the selection marker provides a functionality in addition to facilitating selection of the cells comprising each of the two or more expression constructs. For example, the selection marker may further serve a useful function in the context of cell therapy, e.g., during a cell manufacturing process, or subsequent to administration of the cells to an individual in need thereof. In one non-limiting example, the selection marker may further serve as a suicide switch enabling ablation of the cells when the individual experiences excessive adverse side effects from the cell therapy. The use of a selection marker as a suicide switch is schematically illustrated in
Any convenient approach may be used to selecting for cells exhibiting cell surface expression of the selection marker. In certain embodiments, a magnetic-based cell selection approach is employed. By way of example, cells exhibiting cell surface expression of the selection marker may be selected (purified, enriched) by magnetic-activated cell sorting (MACS). MACS involves labeling cells exhibiting cell surface expression of a selection marker with magnetic beads, e.g., by combining the population of cells with magnetic beads coated with a moiety (antibody, lectin, enzyme, or the like) that binds the selection marker on the cell surface. The labeled cells may then be transferred to a column, where a magnetic field applied is applied and magnetizes the labeled cells to the walls of the column while non-labeled cells flow through the column. The magnetic field is then removed and the labeled cells (i.e., those exhibiting cell surface expression of the selection marker) may be retrieved from the column.
Also by way of example, cells exhibiting cell surface expression of the selection marker may be selected (purified, enriched) by flow cytometry, e.g., fluorescence-activated cell sorting (FACS). FACS involves labeling cells exhibiting cell surface expression of a selection marker with a fluorophore, e.g., by combining the population of cells with fluorophore-labeled antibodies that bind the selection marker on the cell surface. The fluorescently-labeled cells may then be separated from unlabeled cells using a fluorescence-activated cell sorter according to the manufacturer's instructions.
Further non-limiting examples of cell selection systems according to embodiments of the present disclosure will now be described.
An example three-way cell selection system is schematically illustrated in
An example five-way cell selection system is schematically illustrated in
An example cell selection system in which a truncated epidermal growth factor receptor (EGFRt) is used as a selectable surface marker and a suicide switch is schematically illustrated in
An example three-way cell selection system is schematically illustrated in
As used herein, a “degron” is a sequence of amino acids which provides a degradation signal that directs a polypeptide to intracellular pathways for proteolytic degradation. The degron may promote degradation of an attached polypeptide through either the proteasome or autophagy-lysosome pathways. In some embodiments, the degron induces rapid degradation of the polypeptide. For a discussion of degrons and their function in protein degradation, see, e.g., Kanemaki et al. (2013) Pflugers Arch. 465 (3): 419-425, Erales et al. (2014) Biochim Biophys Acta 1843 (1): 216-221, Schrader et al. (2009) Nat. Chem. Biol. 5 (11): 815-822, Ravid et al. (2008) Nat. Rev. Mol. Cell. Biol. 9 (9): 679-690, Tasaki et al. (2007) Trends Biochem Sci. 32 (1 I): 520-528, Meinnel et al. (2006) Biol. Chem. 387 (7): 839-851, Kim et al. (2013) Autophagy 9 (7): 1100-1103, Varshavsky (2012) Methods Mol. Biol. 832:1-11, and Fayadat et al. (2003) Mol Biol Cell. 14 (3): 1268-1278; the disclosures of which are incorporated herein by reference in their entireties for all purposes.
According to some embodiments, the degron is one found in p53, HIF1alpha, ubiquitin, or a functional variant thereof. In certain embodiments, the degron includes portions of the HCV nonstructural proteins NS3 and NS4A. According to some embodiments, the degron comprises or consists of amino the acid sequence PITKIDTKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLST (the amino acid sequence of a degron from HCV genotype 1a; SEQ ID NO:55), or a functional variant thereof having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, or 99% or greater amino acid sequence identity to such an amino acid sequence, or a fragment thereof, such as a fragment having a length of from 30 to 41 amino acids, 32 to 41 amino acids, 34 to 41 amino acids, 36 to 41 amino acids, or 38 to 41 amino acids, wherein a functional variant of the degron is capable of promoting degradation of the polypeptide.
An example four-way cell selection system is schematically illustrated in
An example five-way cell selection system is schematically illustrated in
Cut A, Cut B, and Cut C are cleavage sites for Prot A, Prot B, and Prot C, respectively. When all five components are present within the same cell, they associate at the ER membrane. Prot C on component 5 cleaves at Cut C, which removes the degron from component 4 and allows it to be expressed at high levels. Protease B is reconstituted by association of components 3 and 4, which cleaves at Cut B, removing the degron from component 2 and allowing component 2 to be expressed at high levels. Prot A on component 2 in turn cleaves at Cut A, which removes the ER Tag from the epitope marker and allows it to translocate to the cell surface. The surface-expressed selection tag can then be used as a selection handle to isolate cells expressing all five constructs.
An example six-way cell selection system is schematically illustrated in
The fifth component (5) is a transmembrane domain fused to the N-terminal fragment of Protease C (nC), and ER retention tag. The sixth component (6) is a transmembrane domain fused to the C-terminal fragment of Protease C (cC), and ER retention tag. Cut A, Cut B, and Cut C are cleavage sites for Prot A, Prot B, and Prot C respectively. When all six components are present within the same cell, they associate at the ER membrane. Component 5 and 6 associate and reconstitute Prot C, which cleaves at Cut C, removing the degron from component 4 and allows it to be expressed at high levels. Protease B is reconstituted by association of components 3 and 4, which cleaves at Cut B, removing the degron from component 2 and allowing component 2 to be expressed at high levels. Prot A on component 2 in turn cleaves at Cut A, which removes the ER Tag from the epitope marker and allows it to translocate to the cell surface. The surface-expressed selection tag can then be used as a selection handle to isolate cells expressing all six constructs.
An example seven-way cell selection system is schematically illustrated in
The fifth component (5) is a transmembrane domain fused to the N-terminal fragment of Protease C (nC), and ER retention tag. The sixth component (6) is comprised of an N-terminal ER retention Tag, a transmembrane domain fused to the C-terminal fragment of Protease C (cC), cleavage site D (Cut D), a degron which induces degradation of the protein, and an ER retention tag. The seventh component (7) is comprised of a transmembrane domain fused to Protease D (Prot D), and an ER retention tag. Cut A, Cut B, Cut C, and Cut D are cleavage sites for Prot A, Prot B, Prot C, and Prot D, respectively. When all seven components are present within the same cell, they associate at the ER membrane. Prot D on component 7 cleaves at Cut D, which removes the degron from component 6 and allows component 6 to be expressed at high levels. Component 5 and 6 associate and reconstitute Prot C, which cleaves at Cut C, removing the degron from component 4 and allows it to be expressed at high levels. Protease B is reconstituted by association of components 3 and 4, which cleaves at Cut B, removing the degron from component 2 and allowing component 2 to be expressed at high levels. Prot A on component 2 in turn cleaves at Cut A, which removes the ER Tag from the epitope marker and allows it to translocate to the cell surface. The surface-expressed selection tag can then be used as a selection handle to isolate cells expressing all seven constructs.
An example eight-way cell selection system is schematically illustrated in
The fifth component (5) is a transmembrane domain fused to the N-terminal fragment of Protease C (nC), and ER retention tag. The sixth component (6) is comprised of an N-terminal ER retention Tag, a transmembrane domain fused to the C-terminal fragment of Protease C (cC), cleavage site D (Cut D), a degron which induces degradation of the protein, and an ER retention tag.
The seventh component (7) is a transmembrane domain fused to the N-terminal fragment of Protease D (nD), and ER retention tag. The eighth component (8) is a transmembrane domain fused to the C-terminal fragment of Protease D (cD), and ER retention tag. Cut A, Cut B, Cut C, and Cut D are cleavage sites for Prot A, Prot B, Prot C, and Prot D, respectively. When all eight components are present within the same cell, they associate at the ER membrane. Prot D, which is reconstituted by association of components 7 and 8, cleaves at Cut D, which removes the degron from component 6 and allows component 6 to be expressed at high levels. Component 5 and 6 associate and reconstitute Prot C, which cleaves at Cut C, removing the degron from component 4 and allows it to be expressed at high levels. Protease B is reconstituted by association of components 3 and 4, which cleaves at Cut B, removing the degron from component 2 and allowing component 2 to be expressed at high levels. Prot A on component 2 in turn cleaves at Cut A, which removes the ER Tag from the epitope marker and allows it to translocate to the cell surface. The surface-expressed selection tag can then be used as a selection handle to isolate cells expressing all eight constructs.
An example nine-way cell selection system is schematically illustrated in
The fifth component (5) is a transmembrane domain fused to the N-terminal fragment of Protease C (nC), and ER retention tag. The sixth component (6) is comprised of an N-terminal ER retention Tag, a transmembrane domain fused to the C-terminal fragment of Protease C (cC), cleavage site D (Cut D), a degron which induces degradation of the protein, and an ER retention tag. The seventh component (7) is a transmembrane domain fused to the N-terminal fragment of Protease D (nD), and ER retention tag. The eighth component (8) is comprised of an N-terminal ER retention Tag, a transmembrane domain fused to the C-terminal fragment of Protease D (CD), cleavage site E (Cut E), a degron which induces degradation of the protein, and an ER retention tag. The ninth component (9) is comprised of a transmembrane domain fused to Protease E (Prot E), and an ER retention tag.
Cut A, Cut B, Cut C, Cut D, and Cut E are cleavage sites for Prot A, Prot B, Prot C, Prot D, and Prot E, respectively. When all nine components are present within the same cell, they associate at the ER membrane. Protease E on component 9 cleaves at Cut E, which removes the degron from component 8 and allows component 8 to be expressed at high levels. Prot D, which is reconstituted by association of components 7 and 8, cleaves at Cut D, which removes the degron from component 6 and allows component 6 to be expressed at high levels. Component 5 and 6 associate and reconstitute Prot C, which cleaves at Cut C, removing the degron from component 4 and allows it to be expressed at high levels. Protease B is reconstituted by association of components 3 and 4, which cleaves at Cut B, removing the degron from component 2 and allowing component 2 to be expressed at high levels. Prot A on component 2 in turn cleaves at Cut A, which removes the ER Tag from the epitope marker and allows it to translocate to the cell surface. The surface-expressed selection tag can then be used as a selection handle to isolate cells expressing all nine constructs.
An example ten-way cell selection system is schematically illustrated in
The fifth component (5) is a transmembrane domain fused to the N-terminal fragment of Protease C (nC), and ER retention tag. The sixth component (6) is comprised of an N-terminal ER retention Tag, a transmembrane domain fused to the C-terminal fragment of Protease C (cC), cleavage site D (Cut D), a degron which induces degradation of the protein, and an ER retention tag. The seventh component (7) is a transmembrane domain fused to the N-terminal fragment of Protease D (nD), and ER retention tag. The eighth component (8) is comprised of an N-terminal ER retention Tag, a transmembrane domain fused to the C-terminal fragment of Protease D (cD), cleavage site E (Cut E), a degron which induces degradation of the protein, and an ER retention tag.
The ninth component (9) is a transmembrane domain fused to the N-terminal fragment of Protease E (nE), and ER retention tag. The tenth component (10) is a transmembrane domain fused to the C-terminal fragment of Protease E (cE), and ER retention tag.
Cut A, Cut B, Cut C, Cut D, and Cut E are cleavage sites for Prot A, Prot B, Prot C, Prot D, and Prot E, respectively. When all ten components are present within the same cell, they associate at the ER membrane. Protease E, which is reconstituted by association of components 9 and 10, cleaves at Cut E, which removes the degron from component 8 and allows component 8 to be expressed at high levels. Prot D, which is reconstituted by association of components 7 and 8, cleaves at Cut D, which removes the degron from component 6 and allows component 6 to be expressed at high levels. Component 5 and 6 associate and reconstitute Prot C, which cleaves at Cut C, removing the degron from component 4 and allows it to be expressed at high levels. Protease B is reconstituted by association of components 3 and 4, which cleaves at Cut B, removing the degron from component 2 and allowing component 2 to be expressed at high levels. Prot A on component 2 in turn cleaves at Cut A, which removes the ER Tag from the epitope marker and allows it to translocate to the cell surface. The surface-expressed selection tag can then be used as a selection handle to isolate cells expressing all ten constructs.
An example five-way cell selection system is schematically illustrated in
Binding events between Zip2+Zip4, Zip3+Zip5, and the transmembrane domains result in reconstitution of the Proteolytic complex A and localization at the ER in close proximity to component 1. The Proteolytic complex A cleaves at Cut A, which removes the ER Tag from the epitope marker and allows it to translocate to the cell surface. The surface-expressed selection tag can then be used as a selection handle to isolate cells expressing all five constructs.
An example nine-way cell selection system is schematically illustrated in
Binding events between Zip6+Zip8, Zip7+Zip9, and the transmembrane domains result in reconstitution of the Proteolytic complex B and localization at the ER in close proximity to component Proteolytic complex A. Protease Complex B cleaves at Cut B, which removes the degron from component 5, allowing component 5 to be expressed at high levels.
Binding events between Zip2+Zip4, Zip3+Zip5, and the transmembrane domains result in reconstitution of the Proteolytic complex A and localization at the ER in close proximity to component 1. The Proteolytic complex A cleaves at Cut A, which removes the ER Tag from the epitope marker and allows it to translocate to the cell surface. The surface-expressed selection tag can then be used as a selection handle to isolate cells expressing all nine constructs.
An example thirteen-way cell selection system is schematically illustrated in
Binding events between Zip10+Zip12, Zip11+Zip13, and the transmembrane domains result in reconstitution of the Proteolytic complex C and localization at the ER in close proximity to Proteolytic complex B. Protease Complex C cleaves at Cut C, which removes the degron from component 9, allowing component 9 to be expressed at high levels.
Binding events between Zip6+Zip8, Zip7+Zip9, and the transmembrane domains result in reconstitution of the Proteolytic complex B and localization at the ER in close proximity to Proteolytic complex A. Protease Complex B cleaves at Cut B, which removes the degron from component 5, allowing component 5 to be expressed at high levels.
Binding events between Zip2+Zip4, Zip3+Zip5, and the transmembrane domains result in reconstitution of the Proteolytic complex A and localization at the ER in close proximity to component 1. The Proteolytic complex A cleaves at Cut A, which removes the ER Tag from the epitope marker and allows it to translocate to the cell surface. The surface-expressed selection tag can then be used as a selection handle to isolate cells expressing all thirteen constructs.
Also provided by the present disclosure are fusion proteins. In some embodiments, provided are any of the fusion proteins employed in the cell selection methods described above, e.g., any of the fusion proteins encoded by the first, second, etc. expression constructs described elsewhere herein, including any of the fusion proteins or equivalents thereof for which the amino acid sequences are provided herein, e.g., in the sequence table(s) herein. Also provided are nucleic acids encoding such fusion proteins and expression vectors comprising such nucleic acids. Cells comprising such fusion proteins, nucleic acids and/or expression vectors are also provided.
In certain embodiments, provided are fusion proteins comprising a protein fused to an ER localization tag, wherein the ER localization tag comprises 80% or greater, 85% or greater, 90% or greater, 95% or greater, or 100% amino acid sequence identity to an ER localization tag comprising, consisting of, or present within, an amino acid sequence selected from
According to some embodiments, provided are fusion proteins comprising a protein fused to an ER localization tag, where the ER localization tag comprises a transmembrane (Tm) domain, an intracellular domain (ICD), or both, of an ER localization tag of a polypeptide set forth in Table 1, or a variant Tm and/or ICD thereof which retains the ability to localize a polypeptide to the ER. By a “variant” Tm and/or ICD is meant a variant that comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater amino acid sequence identity to the parental/reference sequence, or a fragment thereof, where the variant retains the ability of the ER localization tag to localize a polypeptide to the ER.
In certain embodiments, provided are fusion proteins comprising a protein fused to an ER localization tag, wherein the ER localization tag comprises a Tm domain, an ICD, or both, of an ER localization tag of a human ER-resident protein, or a variant Tm and/or ICD thereof which retains the ability to localize a polypeptide to the ER. For example, as demonstrated in the Experimental section herein, aspects of the present disclosure include novel human ER localization tags that find use in localizing proteins to the ER. According to some embodiments, the human ER-resident protein is CDGSH iron sulfur domain 2 (CISD2). In certain embodiments, such an ER localization tag comprises the Tm domain, the ICD, or both, of the polypeptide set forth in SEQ ID NO:91, or a variant Tm and/or ICD thereof which retains the ability to localize a polypeptide to the ER. According to some embodiments, the human ER-resident protein is UDP glucuronosyltransferase family 2 member B17 (UGT2B17). In certain embodiments, such an ER localization tag comprises the Tm domain, the ICD, or both, of the polypeptide set forth in SEQ ID NO:95, or a variant Tm and/or ICD thereof which retains the ability to localize a polypeptide to the ER.
The fusion protein may be fused directly to the ER localization tag, or indirectly via one or more domains, e.g., other protein-encoding domain(s), linker(s), and/or the like. The fusion protein may further comprise a protease cleavage site, e.g., disposed between the protein and the ER localization tag. The fusion protein may further comprise a membrane association domain, e.g., any of the transmembrane domains described elsewhere herein. The fusion protein may further comprise a protein localization tag, e.g., any of the protein localization tags described elsewhere herein. Also provided are nucleic acids encoding such fusion proteins and expression vectors comprising such nucleic acids. Cells comprising such fusion proteins, nucleic acids and/or expression vectors are also provided. Methods of producing such fusion proteins are also provided. In some embodiments, such methods comprise culturing a cell comprising an expression vector encoding the fusion protein under conditions suitable for the cell to express the fusion protein, wherein the fusion protein is produced.
Also provided are fusion proteins comprising a protein fused to a transmembrane domain, wherein the transmembrane domain comprises 80% or greater, 85% or greater, 90% or greater, 95% or greater, or 100% amino acid sequence identity to a transmembrane domain comprising, consisting of, or present within, an amino acid sequence selected from
The fusion protein may be fused directly to the transmembrane domain, or indirectly via one or more domains, e.g., other protein-encoding domain(s), linker(s), and/or the like. The fusion protein may further comprise a protease cleavage site. The fusion protein may further comprise a membrane association domain, e.g., any of the transmembrane domains described elsewhere herein. The fusion protein may further comprise a protein localization tag, e.g., any of the protein localization tags described elsewhere herein. Also provided are nucleic acids encoding such fusion proteins and expression vectors comprising such nucleic acids. Cells comprising such fusion proteins, nucleic acids and/or expression vectors are also provided. Methods of producing such fusion proteins are also provided. In some embodiments, such methods comprise culturing a cell comprising an expression vector encoding the fusion protein under conditions suitable for the cell to express the fusion protein, wherein the fusion protein is produced.
The amino acid sequences of exemplary cell selection system components are provided in Table 1 below. For each sequence, the domains as ordered from N- to C-terminus are listed in the left column. The sequence in the right column indicates the domains by alternating underlining. The present disclosure provides each of the proteins provided in Table 1, and each of the individual domains therein, as well as nucleic acids that encode such proteins and individual domains. Cells comprising such proteins and nucleic acids are also provided. As will be appreciated, the present disclosure also provides variants of any of the proteins and individual domains therein, where in some instances a variant protein or domain thereof comprises an amino acid sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater amino acid sequence identity to the parental/reference sequence, or a functional fragment thereof, where the variant retains the functionality (e.g., protease activity, cleavability by the protease, localization/retention (e.g., at the ER), selectability by a cell selection system, and/or the like) of the parental/reference sequence.
MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGE
GKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKT
FINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWE
AFTETLYPADGGLEGRNDMALKLVGGSHLIANAKTTY
RSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQH
EVAVARYCDLPSKLGHKLNNASATNFSLLKQAGDVE
ENPGPMGTSLLCWMALCLLGADHADACPYSNPSLC
SGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNP
SLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPA
RIVLSGSGTSAPITAYAQQTRGLLGCIITSLTGRDKNQ
VEGEVQIMSTATQTFLATCINGVCWAVYHGAGTRTIA
SPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCG
SSDLYLVTRHADVIPVRRRGDGRGSLLSPRPISYLKG
SSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVE
NLETTMRSPVFTDNSSPPAVTLTHAAASTGSSGGGG
GSGGLYKYKSRRSFIDEKKMP
MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGE
GKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKT
FINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWE
AFTETLYPADGGLEGRNDMALKLVGGSHLIANAKTTY
RSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQH
EVAVARYCDLPSKLGHKLNNASATNFSLLKQAGDVE
ENPGPMGTSLLCWMALCLLGADHADACPYSNPSLC
SGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNP
SLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPA
DGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGV
FKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFR
EPQREERICLVTTNFQTKSMSSMVSDTSCTFPSSDGI
FWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTN
TNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSV
LWGGHKVFMVKPEEPFQPVKEATQLMNAAASTGSS
MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGE
GKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKT
FINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWE
AFTETLYPADGGLEGRNDMALKLVGGSHLIANAKTTY
RSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQH
EVAVARYCDLPSKLGHKLNNASATNFSLLKQAGDVE
ENPGPMGTSLLCWMALCLLGADHADACPYSNPSLC
SGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNP
SLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPA
DGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGV
FKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFR
EPQREERICLVTTNFQTAAASTGSSGGGGGSGGLYK
YKSRRSFIDEKKMP
MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGE
GRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSK
AYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGLVT
VTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTM
GWEASTERLYPRDGVLKGEIHQALKLKDGGHYLVEF
KTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
RSEGRHHLFLGHGTGSTGSGSSGTASSEDNNMAVIK
EFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPD
YKKLSFPEGFKWERVMNFEDGGLVTVTQDSSLQDG
TLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYP
RDGVLKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQL
PGYYYVDTKLDITSHNEDYTIVEQYERSEGRHHLFLY
GMDELYKNAGNSSIGATNFSLLKQAGDVEENPGPM
ELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGG
GGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH
GQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKN
FMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMV
KPEEPFQPVKEATQLMNAAASTGSSGGGGGSGGLY
KYKSRRSFIDEKKMP
MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGE
GKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKT
FINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWE
AFTETLYPADGGLEGRNDMALKLVGGSHLIANAKTTY
RSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQH
EVAVARYCDLPSKLGHKLNNASATNFSLLKQAGDVE
ENPGPMGTSLLCWMALCLLGADHADACPYSNPSLC
SGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNP
SLCSGGGGSIEVMYPPPYLDNEKSNGTIIHVKGKHLC
VGSSGGSSGSSGGGSSGSGSSGNSSGGSGSTGSS
SDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHG
VFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKE
REPQREERICLVTTNFQTAAASTGSSGGGGGSGGLY
KYKSRRSFIDEKKMP
MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGE
GRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSK
AYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGLVT
VTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTM
GWEASTERLYPRDGVLKGEIHQALKLKDGGHYLVEF
KTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
RSEGRHHLFLGHGTGSTGSGSSGTASSEDNNMAVIK
EFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPD
YKKLSFPEGFKWERVMNFEDGGLVTVTQDSSLQDG
TLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYP
RDGVLKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQL
PGYYYVDTKLDITSHNEDYTIVEQYERSEGRHHLFLY
GMDELYKNAGNSSIGATNFSLLKQAGDVEENPGPM
ELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGG
GGSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFP
DGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPK
NFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFM
VKPEEPFQPVKEATQLMNAAASTGSSGGGGGSGGL
YKYKSRRSFIDEKKMP
MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLG
LSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGC
NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE
GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ
GLSTATKDTYDALHMQALPPRNASATNFSLLKQAGD
VEENPGPMGTSLLCWMALCLLGADHADACPYSNPS
LCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYS
NPSLCSGGGGSIEVMYPPPYLDNEKSNGTIIHVKGKH
WVGSSGGSSGSSGGGSSGSGSSGNSSGGSGSTGS
ESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLH
GVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLK
FREPQREERICLVTTNFQTAAASTGSSGGGGGSGGL
YKYKSRRSFIDEKKMP
MARSVTLVFLVLVSLTGLYAADIQMTQSPSSLSASVG
SLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCS
ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG
LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG
LSTATKDTYDALHMQALPPRSIGATNESLLKQAGDVE
ENPGPMGTSLLCWMALCLLGADHADACPYSNPSLC
SGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNP
SLCSGGGGSIEVMYPPPYLDNEKSNGTIIHVKGKHLC
VGSSGGSSGSSGGGSSGSGSSGNSSGGSGSTGSS
HWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNY
FTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWG
GHKVFMVKPEEPFQPVKEATQLMNAAASTGSSGGG
MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGE
GKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKT
FINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWE
AFTETLYPADGGLEGRNDMALKLVGGSHLIANAKTTY
RSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQH
EVAVARYCDLPSKLGHKLNNASATNFSLLKQAGDVE
ENPGPSIGMGTSLLCWMALCLLGADHADGNSACPY
SNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTA
CPYSNPSLCSGGGGSGGSWLRLLPFLGVLALLGYLA
GSGESLFKGPRDYNPISSTICHLTNESDGHTTSLYGI
MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGE
GKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKT
FINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWE
AFTETLYPADGGLEGRNDMALKLVGGSHLIANAKTTY
RSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQH
EVAVARYCDLPSKLGHKLNNASATNFSLLKQAGDVE
ENPGPMGTSLLCWMALCLLGADHADACPYSNPSLC
SGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNP
SLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPA
LLCGASLVSSDWLVSAAHCVYGRNLEPSKWTAILGL
HMKSNLTSPQTVPRLIDEIVINPHYNRRRKDNDIAMM
HLEFKVNYTDYIQPICLPEENQVFPPGRNCSIAGWGT
VVYQGTTANILQEADVPLLSNERCQQQMPEYNITEN
MICAGYEEGGIDSCQGDSGGPLMCQENNRWFLAGV
TSFGYKCALPNRPGVYARVSRFTEWIQSFLHAAAST
MLLLVTSLLLCELPHPAFLLIPGGSEQKLISEEDLTTTP
EEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPN
DGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYP
DGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNY
RYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAAD
WCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTART
TYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFK
EWQKAFTDVMGMDELYKSIGGGSGGSDEMEECSQ
HGSTGGSGGSLYKYKSRRSFIDEKKMP
MLLLVTSLLLCELPHPAFLLIPGGSEQKLISEEDLTTTP
EEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPN
DGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYP
DGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNY
RYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAAD
WCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTART
TYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNEK
EWQKAFTDVMGMDELYKSIGGGSGGSTENLYFQSG
MGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGL
YTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSV
TFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVC
RCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQ
NTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLR
ECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQE
PEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDN
LIPVYCSILAAVVVGLVAYIAFKRAAAGSVSKGEEDNM
ASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEE
LNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSP
FQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEG
SHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKT
YPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPM
AANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTD
VMGMDELYKSIGGGSGGSTENLYFQSGSTGGSGGS
MGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGL
YTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSV
TFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVC
RCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQ
NTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLR
ECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQE
PEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDN
LIPVYCSILAAVVVGLVAYIAFKRAAAGSVSKGEEDNM
ASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEE
LNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSP
FQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEG
SHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKT
YPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPM
AANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTD
VMGMDELYKSIGGGSGGSDEMEECSQHGSTGGSG
MLLLVTSLLLCELPHPAFLLIPGGSACPYSNPSLCSG
GGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSL
CSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAG
GQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGY
GFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFE
DGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVM
TNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKR
YRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHS
KTELNFKEWQKAFTDVMGMDELYKSIGGGSGGSTE
NLYFQSGSTGGSGGSLYKYKSRRSFIDEKKMP
MGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGL
YTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSV
TFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVC
RCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQ
NTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLR
ECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQE
PEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDN
LVITLYCAAAGSVSKGEEDNMASLPATHELHIFGSING
VDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWIL
VPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHR
TMQFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFPA
DGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTT
GNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKT
ELKHSKTELNFKEWQKAFTDVMGMDELYKSIGGGSG
MLLLVTSLLLCELPHPAFLLIPGGSEQKLISEEDLIEVM
VLVVVGGVLACYSLLVTVAFIIFWVAAAGSVSKGEED
NMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGY
EELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGM
SPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTY
EGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRS
KKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFA
KPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKA
FTDVMGMDELYKSIGGGSGGSTENLYFQSGSTGGS
MLLLVTSLLLCELPHPAFLLIPGGSACPYSNPSLCSG
GGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSL
CSGGGGSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPS
GQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGY
GFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFE
DGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVM
TNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKR
YRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHS
KTELNFKEWQKAFTDVMGMDELYKSIGGGSGGSTE
NLYFQSGSTGGSGGSLYKYKSRRSFIDEKKMP
MGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGL
YTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSV
TFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVC
RCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQ
NTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLR
ECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQE
PEAPPEQDLIASTVAGVVTTVMGSSQPVVTRGTTDN
FWVAAAGSVSKGEEDNMASLPATHELHIFGSINGVD
FDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVP
HIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTM
QFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFPADG
PVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGN
GKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTEL
KHSKTELNFKEWQKAFTDVMGMDELYKSIGGGSGG
MLLLVTSLLLCELPHPAFLLIPGGSDYKDDDDKGGTT
TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR
GLDFACDIYIWAPLAGTCGVLLLSLVITLYCKGGGGS
GGAAAGSNLVAQLENEVASLENENETLKKKNLHKKD
MLLLVTSLLLCELPHPAFLLIPGGSYPYDVPDYAGGTT
TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR
GLDFACDIYIWAPLAGTCGVLLLSLVITLYCKGGGGS
GGAAAGSNEVTTLENDAAFIENENAYLEKEIARLRKE
MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGE
GKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKT
FINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWE
AFTETLYPADGGLEGRNDMALKLVGGSHLIANAKTTY
RSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQH
EVAVARYCDLPSKLGHKLNNASATNFSLLKQAGDVE
ENPGPGGSARNAYLRKKIARLKKDNLQLERDEQNLE
KIIANLRDEIARLENEVASHEQGSTGSSGGSGGSGSS
IGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTL
QQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERIC
LVTTNFQTAAA
MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGE
GRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSK
AYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGLVT
VTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTM
GWEASTERLYPRDGVLKGEIHQALKLKDGGHYLVEF
KTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
RSEGRHHLFLGHGTGSTGSGSSGTASSEDNNMAVIK
EFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPD
YKKLSFPEGFKWERVMNFEDGGLVTVTQDSSLQDG
TLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYP
RDGVLKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQL
PGYYYVDTKLDITSHNEDYTIVEQYERSEGRHHLFLY
GMDELYKNAGNSGGGATNFSLLKQAGDVEENPGPG
KNELATLENEVARLENDVAEGSTGSSGGSGGSGSS
CGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFM
ELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKP
MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLS
MASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMS
PFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYE
GSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSK
KTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAK
PMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAF
TDVMGMDELYKSIGGGSGGSTENLYFQSGSTGGSG
MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLS
EACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLL
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVERKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVERKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVERKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVERKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVERKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI
SLYKYKSRRSFIDEKKMP
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVERKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVERKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
GGTENLYFQSGSTQMRHLKSFFEAKKLV
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVERKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
AAAGGTENLYFQSGSTAYRQRQHQDMPAPRPPGPR
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVERKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
AAGGTENLYFQSGSTHMKEKEKSD
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
AAAGGTENLYFQSGSTCFRKLAKTGKKKKRD
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
GTENLYFQSGSTKCCAYGYRKCLGKKGRVKKAHKSKTH
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
AAAGGTENLYFQSGSTRLTTDVDPDLDQDED
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
AAAGGTENLYFQSGSTKYKSRRSFIDEKKMP
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
AAAGGTENLYFQSGSTNRSPRNRKPRRE
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
VTVAFIIFWVAAAGGTENLYFQSGSTPITKIDTKYIMTC
MSADLEVVTSTWVLVGGVLAALAAYCLSTLYKYKSR
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
VTVAFIIFWVAAAGGTENLYFQSGSTPKKKQQKDSLI
NLKIQKENPKVVNEINIEDLCLTKAAYCRCWRSKTFP
ACDGSHNKHNELTGDNVGPLILKKKEV
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
VTVAFIIFWVAAAGGTENLYFQSGSTCFRKLAKTGKK
KKRD
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
VTVAFIIFWVAAAGGTENLYFQSGSTMTGCCGCCCG
CFGIIPLMSKCGKKSSYYTTFDNDVVIEQYRPKKSV
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI
KSRRSFIEEKKMP
MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQG
TGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFH
QYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGA
SLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNS
LTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYR
STARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKT
ELNFKEWQKAFTDVMGMDELYKNASGATNFSLLKQ
AGDVEENPGPIASMLLLVTSLLLCELPHPAFLLIPRKV
PAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI
DPDRHIERVTELQELFLTRVGLDIGKVWVADDGAAVA
VWTTPESVEAGAVFAEIGPRMAELSGSRLAAQQQM
EGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVL
PGVEAAERAGVPAFLETSAPRNLPFYERLGFTVTAD
VEVPEGPRTWCMTRKPGAAAAGGTENLYFQSGSTPI
TKIDTKYIMTCMSADLEVVTSTWVLVGGVLAALAAYC
LSTLYKYKSRRSFIEEKKMP
MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPS
RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE
GGCELRVKFSRSADAPAYKQGQNQLYNELNLGRRE
ASMLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDS
LSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPL
DPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEII
RGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIIS
GNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKA
TGQVCHALCSPEGCWGPEPRDCVSCRNVSRGREC
VDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCT
GRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLV
WKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPK
IPSGGSWLRLLPFLGVLALLGYLAVRPFLAAAGGTEN
LYFQSGSTPKKKQQKDSLINLKIQKENPKVVNEINIED
LCLTKAAYCRCWRSKTFPACDGSHNKHNELTGDNV
GPLILKKKEV
MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPS
RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE
GGCELRVKFSRSADAPAYKQGQNQLYNELNLGRRE
HFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDIL
KTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQH
GQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCY
ANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHA
LCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLE
GEPREFVENSECIQCHPECLPQAMNITCTGRGPDNC
IQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGH
VCHLCHPNCTYGCTGPGLEGCPTNGPKIPSGGSWL
RLLPFLGVLALLGYLAVRPFLAAAGGTENLYFQSGST
PKKKQQKDSLINLKIQKENPKVVNEINIEDLCLTKAAY
CRCWRSKTFPACDGSHNKHNELTGDNVGPLILKKKE
V
LLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIK
MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGE
GKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKT
FINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWE
AFTETLYPADGGLEGRNDMALKLVGGSHLIANAKTTY
RSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQH
EVAVARYCDLPSKLGHKLNNASATNFSLLKQAGDVE
ENPGPMGTSLLCWMALCLLGADHADGGSIAFLLACV
MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGE
GKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKT
FINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWE
AFTETLYPADGGLEGRNDMALKLVGGSHLIANAKTTY
RSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQH
EVAVARYCDLPSKLGHKLNNASATNFSLLKQAGDVE
ENPGPMGTSLLCWMALCLLGADHADACPYSNPSLC
SGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNP
SLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPA
AVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPE
DTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHD
LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASG
WGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVT
KFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITS
WGSEPCALPERPSLYTKVVHYRKWIKDTIVANPAAAS
MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGE
GKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKT
FINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWE
AFTETLYPADGGLEGRNDMALKLVGGSHLIANAKTTY
RSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQH
EVAVARYCDLPSKLGHKLNNASATNFSLLKQAGDVE
ENPGPMGTSLLCWMALCLLGADHADACPYSNPSLC
SGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNP
SLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPA
FCSGVLVHPQWVLSAAHCFQNSYTIGLGLHSLEADQ
EPGSQMVEASLSVRHPEYNRPLLANDLMLIKLDESVS
ESDTIRSISIASQCPTAGNSCLVSGWGLLANGRMPTV
LQCVNVSVVSEEVCSKLYDPLYHPSMFCAGGGHDQ
KDSCNGDSGGPLICNGYLQGLVSFGKAPCGQVGVP
GVYTNLCKFTEWIEKTVQASAAASTGSSGGGGGSG
MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGE
GKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKT
FINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWE
AFTETLYPADGGLEGRNDMALKLVGGSHLIANAKTTY
RSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQH
EVAVARYCDLPSKLGHKLNNASATNFSLLKQAGDVE
ENPGPMGTSLLCWMALCLLGADHADACPYSNPSLC
SGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNP
SLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPA
LCGGVLIHPLWVLTAAHCKKPNLQVFLGKHNLRQRE
SSQEQSSVVRAVIHPDYDAASHDQDIMLLRLARPAKL
SELIQPLPLERDCSANTTSCHILGWGKTADGDFPDTI
QCAYIHLVSREECEHAYPGQITQNMLCAGDEKYGKD
SCQGDSGGPLVCGDHLRGLVSWGNIPCGSKEKPGV
YTNVCRYTNWIQKTIQAKAAASTGSSGGGGGSGGLY
KYKSRRSFIDEKKMP
MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGE
GKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKT
FINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWE
AFTETLYPADGGLEGRNDMALKLVGGSHLIANAKTTY
RSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQH
EVAVARYCDLPSKLGHKLNNASATNFSLLKQAGDVE
ENPGPMGTSLLCWMALCLLGADHADACPYSNPSLC
SGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNP
SLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPA
LLCGGVLVGGNWVLTAAHCKKPKYTVRLGDHSLQN
KDGPEQEIPVVQSIPHPCYNSSDVEDHNHDLMLLQL
RDQASLGSKVKPISLADHCTQPGQKCTVSGWGTVTS
PRENFPDTLNCAEVKIFPQKKCEDAYPGQITDGMVC
AGSSKGADTCQGDSGGPLVCDGALQGITSWGSDPC
GRSDKPGVYTNICRYLDWIKKIIGSKGAAASTGSSGG
MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGE
GKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKT
FINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWE
AFTETLYPADGGLEGRNDMALKLVGGSHLIANAKTTY
RSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQH
EVAVARYCDLPSKLGHKLNNASATNFSLLKQAGDVE
ENPGPMGTSLLCWMALCLLGADHADACPYSNPSLC
SGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNP
SLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPA
CGATLIAPRWLLTAAHCLKPRYIVHLGQHNLQKEEGC
EQTRTATESFPHPGFNNSLPNKDHRNDIMLVKMASP
VSITWAVRPLTLSSRCVTAGTSCLISGWGSTSSPQLR
LPHTLRCANITIIEHQKCENAYPGNITDTMVCASVQEG
GKDSCQGDSGGPLVCNQSLQGIISWGQDPCAITRKP
GVYTKVCKYVDWIQETMKNNAAASTGSSGGGGGSG
MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGE
GKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKT
FINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWE
AFTETLYPADGGLEGRNDMALKLVGGSHLIANAKTTY
RSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQH
EVAVARYCDLPSKLGHKLNNASATNFSLLKQAGDVE
ENPGPMGTSLLCWMALCLLGADHADACPYSNPSLC
SGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNP
SLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPA
TCFPHSQPWQAALLVQGRLLCGGVLVHPKWVLTAA
HCLKEGLKVYLGKHALGRVEAGEQVREVVHSIPHPE
YRRSPTHLNHDHDIMLLELQSPVQLTGYIQTLPLSHN
NRLTPGTTCRVSGWGTTTSPQVNYPKTLQCANIQLR
SDEECRQVYPGKITDNMLCAGTKEGGKDSCEGDSG
GPLVCNRTLYGIVSWGDFPCGQPDRPGVYTRVSRY
VLWIRETIRKYETQQQKWLKGPQAAASTGSSGGGG
MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGE
GKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKT
FINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWE
AFTETLYPADGGLEGRNDMALKLVGGSHLIANAKTTY
RSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQH
EVAVARYCDLPSKLGHKLNNASATNFSLLKQAGDVE
ENPGPMGTSLLCWMALCLLGADHADACPYSNPSLC
SGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNP
SLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPA
RFLCGGALLSGQWVITAAHCGRPILQVALGKHNLRR
WEATQQVLRVVRQVTHPNYNSRTHDNDLMLLQLQQ
PARIGRAVRPIEVTQACASPGTSCRVSGWGTISSPIA
RYPASLQCVNINISPDEVCQKAYPRTITPGMVCAGVP
QGGKDSCQGDSGGPLVCRGQLQGLVSWGMERCAL
PGYPGVYTNLCKYRSWIEETMRDKAAASTGSSGGG
MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGE
GKPYEGTQTMRIKVVEGGPLPFAFDILATSFLYGSKT
FINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWE
AFTETLYPADGGLEGRNDMALKLVGGSHLIANAKTTY
RSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQH
EVAVARYCDLPSKLGHKLNNASATNFSLLKQAGDVE
ENPGPMGTSLLCWMALCLLGADHADACPYSNPSLC
SGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNP
SLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPA
FNCGASLISPHWVLSAAHCQSRFMRVRLGEHNLRKR
DGPEQLRTTSRVIPHPRYEARSHRNDIMLLRLVQPAR
LNPQVRPAVLPTRCPHPGEACVVSGWGLVSHNEPG
TAGSPRSQVSLPDTLHCANISIISDTSCDKSYPGRLTN
TMVCAGAEGRGAESCEGDSGGPLVCGGILQGIVSW
GDVPCDNTTKPGVYTKVCHYLEWIRETMKRNAAAST
CCTATTAAATAAAAGAATAAGCAGTATTATTAAGTA
GCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCA
GGCCTGGCCGTGAACGTTCACTGAAATCATGGCCT
CTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGA
GTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGAT
GCTATTTCCCGTATAAAGCATGAGACCGTGACTTG
CCAGCCCCACAGAGCCCCGCCCTTGTCCATCACT
GGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGA
GGGAAATGAGATCATGTCCTAACCCTGATCCTCTT
GTCCCACAGATATCCAGAACCCTGACCCTGCCGTG
TACCAGCTGAGAGACTCTAAATCCAGTGACAAGTC
TGTCTGCCTATTCACCAACGCGTCTTAGAAGGATC
TGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGC
GCACATCGCCCACAGTCCCCGAGAAGTTGGGGGG
AGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGG
TGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGT
ACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGA
ACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTC
TTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTG
AAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCG
CCCGCCGCCCTACCTGAGGCCGCCATCCACGCCG
GTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGT
GCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTT
AAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCG
CTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTC
CACGCTTTGCCTGACCCTGCTTGCTCAACTCTACG
TCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGAT
CCAAGCTGTGACCGGCGCCTAatcgcgagcGCCGCC
GAACTTCCCCATCCGGCCTTCCTTTTGATACCCCG
CAAGGTGTGTAACGGAATTGGGATTGGTGAATTCA
AGGATTCACTTAGTATAAATGCCACAAACATTAAGC
ATTTTAAGAACTGTACGTCCATTTCAGGAGACCTG
CATATTCTTCCCGTAGCTTTCCGGGGCGATTCATT
CACCCACACACCGCCACTTGATCCACAAGAACTTG
ACATCCTGAAAACGGTTAAGGAGATAACAGGATTC
CTCCTGATACAGGCCTGGCCCGAGAATAGAACCG
ACTTGCACGCCTTTGAAAATTTGGAGATAATTCGG
GGTCGGACTAAGCAACATGGACAATTTTCACTGGC
GGTAGTTTCTTTGAATATTACGAGCCTCGGCCTTA
GATCTCTCAAGGAGATCTCAGACGGCGACGTTATA
ATATCTGGGAACAAGAACCTGTGCTACGCTAACAC
AATCAATTGGAAAAAGCTGTTCGGCACGTCTGGAC
AAAAGACAAAGATAATTTCAAATCGAGGCGAAAATA
GCTGCAAGGCTACGGGACAGGTTTGTCACGCCCT
CTGTAGCCCAGAGGGCTGTTGGGGACCCGAGCCA
AGAGATTGCGTCTCATGTCGGAATGTGTCCCGAGG
CCGAGAATGTGTCGATAAATGCAATCTTCTGGAGG
GAGAACCACGGGAATTCGTTGAAAACAGTGAGTGC
ATTCAATGTCACCCGGAGTGCCTTCCGCAAGCGAT
GAATATTACATGTACAGGCCGGGGTCCCGATAATT
GCATCCAGTGTGCTCATTATATTGACGGACCACAC
TGTGTAAAGACATGCCCTGCCGGCGTTATGGGTGA
AAACAATACGCTGGTCTGGAAGTATGCAGACGCAG
GACATGTTTGTCACCTGTGCCATCCTAACTGCACG
TATGGCTGTACAGGACCGGGTCTGGAAGGCTGCC
CTACGAATGGTCCCAAAATACCATCAggaggatccATA
GCCTTCCTCCTCGCaTGCGTCGCTACCATGATCTT
CATGATAACTAAATGCTGTCTCTTCGCGGCCGCAg
gtGGTACAgagaaccTCTATTTTCAGTCAgggtcgacaTG
TTTCAGGAAACTGGCGAAGACAGGTAAGAAAAAAA
AAAGAGACtaatgatgagtataccctgcaggTtaattagct
CAAACAAATGTGTCACAAAGTAAGGATTCTGATGT
GTATATCACAGACAAAACTGTGCTAGACATGAGGT
CTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGG
AGCAACAAATCTGACTTTGCATGTGCAAACGCCTT
CAACAACAGCATTATTCCAGAAGACACCTTCTTCC
CCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGC
AGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCT
GCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCT
CTGATTGGTGGTCTCGGCCTTATCCATTGCCACCA
AAACCCTCTTTTTACTAAGAAACAGTGAGCCTTGTT
CTGGCAGTCCAGAGAATGACACGGGAAAAAAGCA
GATG
RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHIL
PVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAW
PENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSL
GLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQ
KTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRD
RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHIL
PVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAW
PENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSL
GLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQ
KTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRD
RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHIL
PVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAW
PENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSL
GLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQ
KTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRD
RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHIL
PVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAW
PENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSL
GLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQ
KTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRD
RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHIL
PVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAW
PENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSL
GLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQ
KTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRD
Also provided by the present disclosure are cells. According to some embodiments, provided is a cell comprising two or more separate expression constructs, wherein the two or more separate expression constructs comprise a first expression construct that encodes a fusion protein comprising a selection marker, a protein localization tag, and a protease cleavage site disposed between the selection marker and the protein localization tag. The two or more separate expression constructs further comprise a second expression construct that encodes a protein required for cell surface expression of the selection marker. The first and/or second expression construct may further encode a protein of interest, e.g., any of the proteins of interest described elsewhere herein. In certain embodiments, the first and/or second expression construct is site-specifically integrated into the genome of the cell. The site-specific integration may result in the inactivation of one or more target genes in the genome of the cell.
The present disclosure also provides cells or progeny thereof selected according to the cell selection methods of the present disclosure.
Cells of the present disclosure may be autologous/autogeneic (“self”) or non-autologous (“non-self,” e.g., allogeneic, syngeneic or xenogeneic). “Autologous” as used herein, refers to cells derived from the same individual to which they are subsequently administered. “Allogeneic” as used herein refers to cells of the same species that differ genetically from the cell in comparison. “Syngeneic,” as used herein, refers to cells of a different individual that are genetically identical to the cell in comparison.
In some embodiments, the cells are T cells obtained from a mammal. In some embodiments, the mammal is a primate. In some embodiments, the primate is a human.
T cells may be obtained from a number of sources including, but not limited to, peripheral blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from an individual using any number of known techniques such as sedimentation, e.g., FICOLL™ separation.
In some embodiments, an isolated or purified population of T cells is used. In some embodiments, TCTL and TH lymphocytes are purified from PBMCs. In some embodiments, the TCTL and TH lymphocytes are sorted into naïve (TN), memory (TMEM), stem cell memory (TSCM), central memory (TCM), effector memory (TEM), and effector (TEFF) T cell subpopulations either before or after activation, expansion, and/or genetic modification. Suitable approaches for such sorting are known and include, e.g., magnetic-activated cell sorting (MACS), where TN are CD45RA+ CD62L+ CD95−; TSCM are CD45RA+ CD62L+ CD95+; TCM are CD45RO+ CD62L+ CD95+; and TEM are CD45RO+ CD62L− CD95+. An exemplary approach for such sorting is described in Wang et al. (2016) Blood 127 (24): 2980-90.
A specific subpopulation of T cells expressing one or more of the following markers: CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62, CD127, and HLA-DR can be further isolated by positive or negative selection techniques. In some embodiments, a specific subpopulation of T cells, expressing one or more of the markers selected from the group consisting of CD62L, CCR7, CD28, CD27, CD122, CD127, CD197; or CD38 or CD62L, CD127, CD197, and CD38, is further isolated by positive or negative selection techniques. In some embodiments, the manufactured T cell compositions do not express one or more of the following markers: CD57, CD244, CD 160, PD-1, CTLA4, TIM3, and LAG3. In some embodiments, the manufactured T cell compositions do not substantially express one or more of the following markers: CD57, CD244, CD 160, PD-1, CTLA4, TIM3, and LAG3.
In order to achieve therapeutically effective doses of T cell compositions, the T cells may be subjected to one or more rounds of stimulation, activation and/or expansion. T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041, each of which is incorporated herein by reference in its entirety for all purposes. In some embodiments, T cells are activated and expanded for about 1 to 21 days, e.g., about 5 to 21 days. In some embodiments, T cells are activated and expanded for about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 3 days, about 2 days to about 4 days, about 3 days to about 4 days, or about 1 day, about 2 days, about 3 days, or about 4 days prior to introduction of a nucleic acid (e.g., expression vector) encoding the polypeptide into the T cells.
In some embodiments, T cells are activated and expanded for about 6 hours, about 12 hours, about 18 hours or about 24 hours prior to introduction of a nucleic acid (e.g., expression vector) encoding the cell surface receptor the into the T cells. In some embodiments, T cells are activated at the same time that a nucleic acid (e.g., an expression vector) encoding the cell surface receptor is introduced into the T cells.
In some embodiments, conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) and one or more factors necessary for proliferation and viability including, but not limited to serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, IL-21, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α or any other additives suitable for the growth of cells known to the skilled artisan. Further illustrative examples of cell culture media include, but are not limited to RPMI 1640, Clicks, AEVI-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells.
Also provided by the present disclosure are compositions. According to some embodiments, provided are compositions comprising any of the cells of the present disclosure or progeny thereof, e.g., cells selected according to the methods of the present disclosure, etc.
Such compositions may comprise the cells present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like. One or more additives such as a salt (e.g., NaCl, MgCl2, KCl, MgSO4), a buffering agent (a Tris buffer, N-(2-Hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino) ethanesulfonic acid (MES), 2-(N-Morpholino) ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino) propanesulfonic acid (MOPS), N-tris [Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), a solubilizing agent, a detergent (e.g., a non-ionic detergent such as Tween-20, etc.), a nuclease inhibitor, glycerol, a chelating agent, and the like may be present in such compositions. In certain embodiments, the liquid medium is a cell culture medium. Non-limiting examples of cell culture media include Minimal Essential Media, DMEM, a-MEM, RPMI Media, Clicks, F-12, X-Vivo 15, X-Vivo 20, Optimizer, and the like.
In certain embodiments, provided are pharmaceutical compositions comprising cells or progeny thereof selected according to the methods of the present disclosure. The pharmaceutical compositions may comprise such cells and a pharmaceutically acceptable carrier. The pharmaceutical compositions generally include a therapeutically effective amount of the cells. By “therapeutically effective amount” is meant a number of cells sufficient to produce a desired result, e.g., an amount sufficient to effect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a disease (e.g., cancer) or disorder associated, e.g., with the target cell or a population thereof (e.g., cancer cells), as compared to a control. An effective amount can be administered in one or more administrations. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the cells are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” an individual, e.g., a patient. When a therapeutic amount is indicated, the precise amount of the compositions contemplated in particular embodiments, to be administered, can be determined by a physician in view of the specification and with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (individual). In some embodiments, a pharmaceutical composition of the present disclosure includes from 1×106 to 5×1010 of the cells of the present disclosure.
The cells of the present disclosure can be incorporated into a variety of formulations for therapeutic administration. More particularly, the cells of the present disclosure can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable excipients or diluents.
Formulations of the cells suitable for administration to a patient (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration.
The cells may be formulated for parenteral (e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration, or any other suitable route of administration.
Pharmaceutical compositions that include the cells of the present disclosure may be prepared by mixing the cells having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and/or tonicity agents. Acceptable carriers, excipients and/or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and/or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).
An aqueous formulation of the cells may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.
A tonicity agent may be included in the formulation to modulate the tonicity of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.
A surfactant may also be added to the formulation to reduce aggregation and/or minimize the formation of particulates in the formulation and/or reduce adsorption. Example surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20TM) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable Polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Example concentrations of surfactant may range from about 0.001% to about 1% w/v.
In some embodiments, the pharmaceutical composition comprises cells of the present disclosure, and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w/v).
Also provided by the present disclosure are methods of using the cells and compositions of the present disclosure. In certain embodiments, the methods comprise administering a therapeutically effective amount of any of the pharmaceutical compositions of the present disclosure to an individual in need thereof.
In some embodiments, the individual in need thereof has cancer, and one or more of the two or more separate expression constructs encode a receptor (e.g., a CAR, a TCR, and/or the like) that binds to a molecule on the surface of the cancer cells. The pharmaceutical composition typically includes a therapeutically effective amount of such cells as described above. The cells may be any cells capable of effecting the desired therapy. In some embodiments, the cells are immune cells. Non-limiting examples of immune cells which may be administered include T cells, B cells, natural killer (NK) cells, macrophages, monocytes, neutrophils, dendritic cells, mast cells, basophils, and eosinophils. In certain embodiments, the cells are T cells. According to some embodiments, the cells are T cells and a protein of interest expressed by one or more of the two or more expression constructs is a CAR, such that the cells are CAR T cells. In certain embodiments, the cells are stem cells, e.g., embryonic stem cells or adult stem cells. In some embodiments, the pharmaceutical composition is an autologous composition produced by a method including removing cells from the individual and introducing into the removed cells or progeny thereof the desired two or more expression constructs, followed by selection of such cells based on cell surface expression of the selection marker.
In certain embodiments, the individual in need thereof has a cell proliferative disorder. By “cell proliferative disorder” is meant a disorder wherein unwanted cell proliferation of one or more subset(s) of cells in a multicellular organism occurs, resulting in harm, for example, pain or decreased life expectancy to the organism. Cell proliferative disorders include, but are not limited to, cancer, pre-cancer, benign tumors, blood vessel proliferative disorders (e.g., arthritis, restenosis, and the like), fibrotic disorders (e.g., hepatic cirrhosis, atherosclerosis, and the like), psoriasis, epidermic and dermoid cysts, lipomas, adenomas, capillary and cutaneous hemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas, myofibromatosis, osteoplastic tumors, dysplastic masses, mesangial cell proliferative disorders, and the like.
In some embodiments, the individual has cancer. The subject methods may be employed for the treatment of a large variety of cancers. “Tumor”, as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancers that may be treated according to the methods of the present disclosure include, but are not limited to, carcinoma, lymphoma, blastoma, and sarcoma. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bile duct cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, various types of head and neck cancer, and the like. In certain embodiments, the individual has a cancer selected from a solid tumor, recurrent glioblastoma multiforme (GBM), non-small cell lung cancer, metastatic melanoma, melanoma, peritoneal cancer, epithelial ovarian cancer, glioblastoma multiforme (GBM), metastatic colorectal cancer, colorectal cancer, pancreatic ductal adenocarcinoma, squamous cell carcinoma, esophageal cancer, gastric cancer, neuroblastoma, fallopian tube cancer, bladder cancer, metastatic breast cancer, pancreatic cancer, soft tissue sarcoma, recurrent head and neck cancer squamous cell carcinoma, head and neck cancer, anaplastic astrocytoma, malignant pleural mesothelioma, breast cancer, squamous non-small cell lung cancer, rhabdomyosarcoma, metastatic renal cell carcinoma, basal cell carcinoma (basal cell epithelioma), and gliosarcoma. In certain aspects, the individual has a cancer selected from melanoma, Hodgkin lymphoma, renal cell carcinoma (RCC), bladder cancer, non-small cell lung cancer (NSCLC), and head and neck squamous cell carcinoma (HNSCC).
Also provided by the present disclosure are kits. In certain embodiments, provided are kits that include any reagents that find use in practicing the methods of the present disclosure. By way of example, in certain embodiments, provided are kits that comprise a first expression construct that encodes a fusion protein comprising a selection marker, a protein localization tag, and a protease cleavage site disposed between the selection marker and the protein localization tag, and a second expression construct that encodes a protein required for cell surface expression of the selection marker. The first and/or second expression constructs may further comprise a cloning site for a nucleic acid encoding a protein of interest. In certain embodiments, the first and/or second expression constructs further encode one or more proteins of interest, e.g., any of the proteins of interest described elsewhere herein.
The kits of the present disclosure may further include any other reagents useful for practicing the methods of the present disclosure, such as transfection/transduction reagents useful for introducing the expression constructs into cells of interest, e.g., immune cells (e.g., T cells) or other cells of interest.
Components of the kits may be present in separate containers, or multiple components may be present in a single container. For example, the first and second expression constructs may be provided in separate containers or the same container. A suitable container includes a single tube (e.g., vial), one or more wells of a plate (e.g., a 96-well plate, a 384-well plate, etc.), or the like.
The kits of the present disclosure may further comprise instructions for contacting a population of cells with the two or more expression constructs under conditions in which the two or more expression constructs are delivered to cells of the population of cells. The kits of the present disclosure may further comprise instructions for selecting for cells exhibiting cell surface expression of the selection marker.
The instructions of the kits may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging), etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., portable flash drive, DVD, CD-ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, the means for obtaining the instructions is recorded on a suitable substrate.
Notwithstanding the appended claims, the present disclosure is also defined by the following embodiments:
1. A method of selecting for cells that comprise two or more separate expression constructs, the method comprising:
2. The method according to embodiment 1, wherein the first expression construct further encodes a protein of interest.
3. The method according to embodiment 1 or embodiment 2, wherein the first expression construct site-specifically integrates into the genome of the cell.
4. The method according to embodiment 3, wherein site-specific integration of the first expression construct into the genome of the cell inactivates a target gene within the genome of the cell.
5. The method according to any one of embodiments 1 to 4, wherein the second expression construct further encodes a protein of interest.
6. The method according to any one of embodiments 1 to 5, wherein the second expression construct site-specifically integrates into the genome of the cell.
7. The method according to embodiment 6, wherein site-specific integration of the second expression construct into the genome of the cell inactivates a target gene within the genome of the cell.
8. The method according to any one of embodiments 1 to 7, wherein the protein localization tag is selected from the group consisting of: an endoplasmic reticulum (ER) localization tag, a Golgi apparatus (Golgi) localization tag, a lysosome localization tag, a plasma membrane localization tag, a mitochondria localization tag, a peroxisome localization tag, a cytosolic localization tag, and a nuclear localization tag.
9. The method according to any one of embodiments 1 to 7, wherein the protein localization tag is an ER localization tag.
10. The method according to embodiment 9, wherein the ER localization tag comprises the amino acid sequence KKMP.
11. The method according to embodiment 9, wherein the ER localization tag comprises 85% or greater, 90% or greater, or 100% amino acid sequence identity to an ER localization tag comprising, consisting of, or present within, an amino acid sequence selected from the group consisting of: LYKYKSRRSFIDEKKMP (SEQ ID NO:1); AEKDEL (SEQ ID NO:2); EQKLISEEDLKDEL (SEQ ID NO:3); GGGGSGGGGSKDEL (SEQ ID NO:4); GGGGSGGGGSGGGGSGGGGSKDEL (SEQ ID NO:5); GGGGSGGGGSGGGGSGGGGSAEKDEL (SEQ ID NO:6); KYKSRRSFIEEKKMP (SEQ ID NO: 7); LKYKSRRSFIEEKKMP (SEQ ID NO:8); LYKYKSRRSFIEEKKMP (SEQ ID NO:9); LYCKYKSRRSFIEEKKMP (SEQ ID NO:10); LYCNKYKSRRSFIEEKKMP (SEQ ID NO:11); LYCNKYKSRRSFIDEKKMP (SEQ ID NO:12); LYEQKLISEEDLKYKSRRSFIEEKKMP (SEQ ID NO: 13); LYCYPYDVPDYAKYKSRRSFIEEKKMP (SEQ ID NO:14); LYKKLETFKKTN (SEQ ID NO: 15); LYEQKLISEEDLKKLETFKKTN (SEQ ID NO:16); LYYQRL (SEQ ID NO:17); LYEQKLISEEDLYQRL (SEQ ID NO:18); LYKRKIIAFALEGKRSKVTRRPKASDYQRL (SEQ ID NO: 19); LYRNIKCD (SEQ ID NO:20); and LYEQKLISEEDLRNIKCD (SEQ ID NO:21).
12. The method according to embodiment 9, wherein the ER localization tag comprises 85% or greater, 90% or greater, 95% or greater, or 100% amino acid sequence identity to an ER localization tag comprising, consisting of, or present within, an amino acid sequence selected from the group consisting of:
13. The method according to embodiment 11 or embodiment 12, wherein the C-terminus of the ER localization tag comprises the four C-terminal residues of one of the sequences recited in embodiment 11 or embodiment 12.
14. The method according to embodiment 9, wherein the ER localization tag comprises a transmembrane (Tm) domain, an intracellular domain (ICD), or both, of an ER localization tag of a polypeptide set forth in Table 1, or a variant Tm and/or ICD thereof which retains the ability to localize a polypeptide to the ER.
15. The method according to embodiment 9, wherein the ER localization tag comprises a Tm domain, an ICD, or both, of an ER localization tag of a human ER-resident protein, or a variant Tm and/or ICD thereof which retains the ability to localize a polypeptide to the ER.
16. The method according to embodiment 15, wherein the human ER-resident protein is CDGSH iron sulfur domain 2 (CISD2).
17. The method according to embodiment 16, wherein the ER localization tag comprises the Tm domain, the ICD, or both, of the polypeptide set forth in SEQ ID NO:91, or a variant Tm and/or ICD thereof which retains the ability to localize a polypeptide to the ER.
18. The method according to embodiment 15, wherein the human ER-resident protein is UDP glucuronosyltransferase family 2 member B17 (UGT2B17).
19. The method according to embodiment 18, wherein the ER localization tag comprises the Tm domain, the ICD, or both, of the polypeptide set forth in SEQ ID NO:95, or a variant Tm and/or ICD thereof which retains the ability to localize a polypeptide to the ER.
20. The method according to any one of embodiments 1 to 7, wherein the protein localization tag is a Golgi localization tag.
21. The method according to embodiment 20, wherein the wherein the Golgi localization tag comprises the amino acid sequence YQRL (SEQ ID NO:36).
22. The method according to any one of embodiments 1 to 7, wherein the protein localization tag is a lysosome localization tag.
23. The method according to embodiment 22, wherein the lysosome localization tag comprises the amino acid sequence KFERQ (SEQ ID NO:37). 24. The method according to any one of embodiments 1 to 23, wherein the protease cleavage site is a viral protease cleavage site.
25. The method according to embodiment 24, wherein the viral protease cleavage site is a cleavage site for a potyviral family protease.
26. The method according to embodiment 25, wherein the potyviral family protease is Tobacco Etch Virus (TEV) protease, plum pox virus protease (PPVp), soybean mosaic virus protease (SbMVp), sunflower mild mosaic virus protease (SuMMVp), tobacco vein mottling virus protease (TVMVp), or West Nile virus protease (WNVp).
27. The method according to embodiment 25, wherein the viral protease cleavage site is a TEV protease cleavage site.
28. The method according to embodiment 24, wherein the viral protease cleavage site is for a viral protease derived from hepatitis C virus (HCV) nonstructural protein 3 (NS3).
29. The method according to embodiment 28, wherein the viral protease cleavage site is for a viral protease that further comprises a cofactor polypeptide derived from HCV nonstructural protein 4A (NS4A).
30. The method according to embodiment 28 or embodiment 29, wherein the viral protease cleavage site is selected from the group consisting of: an NS4A/4B junction cleavage site, an NS3/NS4A junction cleavage site, an NS4A/NS4B junction cleavage site, an NS4B/NS5A junction cleavage site, an NS5A/NS5B junction cleavage site, and variants thereof cleavable by the viral protease.
31. The method according to any one of embodiments 1 to 23, wherein the protease cleavage site is a human protease cleavage site.
32. The method according to embodiment 31, wherein the human protease cleavage site is a cleavage site for a human protease selected from the group consisting of: a human kallikrein (KLK) protease, human enterokinase protease, human thrombin, a human matrix metalloprotease (MMP), human urokinase-type plasminogen activator receptor (uPAR), human plasmin, and human cathepsin.
33. The method according to embodiment 32, wherein the human kallikrein protease is selected from the group consisting of: human KLK3, human KLK4, human KLK6, human KLK8, human KLK11, human KLK13, human KLK14, and human KLK15.
34. The method according to any one of embodiments 1 to 33, wherein the protein required for cell surface expression of the selection marker is a protease, wherein the protease cleavage site is a cleavage site for the protease.
35. The method according to embodiment 34, wherein the protease is fused to a protein localization tag that localizes the protease to the same cellular compartment as the fusion protein comprising the selection marker.
36. The method according to embodiment 35, wherein the protease is fused to a protein localization tag having the same amino acid sequence as that of the protein localization tag of the fusion protein comprising the selection marker.
37. The method according to embodiment 35 or embodiment 36, wherein the protease is fused to a membrane association domain.
38. The method according to embodiment 37, wherein the membrane association domain is a transmembrane domain.
39. The method according to embodiment 38, wherein the transmembrane domain is a CD8α transmembrane domain.
40. The method according to embodiment 38, wherein the transmembrane domain is a CD28 transmembrane domain.
41. The method according to embodiment 38, wherein the transmembrane domain comprises 80% or greater, 85% or greater, 90% or greater, 95% or greater, or 100% amino acid sequence identity to a transmembrane domain comprising, consisting of, or present within, an amino acid sequence selected from the group consisting of:
42. The method according to embodiment 38 or embodiment 41, wherein the protease is fused to a hinge domain.
43. The method according to embodiment 42, wherein the hinge domain is a CD8α hinge domain.
44. The method according to embodiment 34, wherein the protease is fused to a dimerization domain.
45. The method according to embodiment 44, wherein the method comprises contacting the population of cells with a third expression construct that encodes a fusion protein comprising a membrane association domain, a dimerization domain that dimerizes with the dimerization domain fused to the protease, and a protein localization tag that localizes the dimerization domain to the same cellular compartment as the fusion protein comprising the selection marker.
46. The method according to embodiment 45, wherein the third expression construct further encodes a protein of interest.
47. The method according to embodiment 45 or embodiment 46, wherein the first expression construct site-specifically integrates into the genome of the cell.
48. The method according to embodiment 47, wherein site-specific integration of the first expression construct into the genome of the cell inactivates a target gene within the genome of the cell.
49. The method according to any one of embodiments 1 to 33, wherein the protein required for cell surface expression of the selection marker is a first complementary fragment of a protease, wherein the protease cleavage site is a cleavage site for the protease.
50. The method according to embodiment 49, wherein the two or more expression constructs comprise a third expression construct that encodes a second complementary fragment of the protease, wherein the first and second complementary fragments form an active protease complex.
51. The method according to embodiment 50, wherein the third expression construct further encodes a protein of interest.
52. The method according to embodiment 50 or embodiment 51, wherein the first expression construct site-specifically integrates into the genome of the cell.
53. The method according to embodiment 52, wherein site-specific integration of the first expression construct into the genome of the cell inactivates a target gene within the genome of the cell.
54. The method according to any one of embodiments 50 to 53, wherein the first and second complementary fragments are each fused to a protein localization tag that localizes the first and second complementary fragments to the same cellular compartment as the fusion protein comprising the selection marker.
55. The method according to embodiment 54, wherein the first and second complementary fragments are each fused to a protein localization tag having the same amino acid sequence as that of the protein localization tag of the fusion protein comprising the selection marker.
56. The method according to any one of embodiments 50 to 55, wherein the first and second complementary fragments are each fused to a membrane association domain. 57. The method according to embodiment 56, wherein the membrane association domain is transmembrane domain.
58. The method according to embodiment 57, wherein the transmembrane domain is as defined in any one of embodiments 39 to 41.
59. The method according to embodiment 50, wherein the first and second complementary fragments are each fused to a dimerization domain.
60. The method according to embodiment 59, wherein the two or more expression constructs comprise:
61. The method according to embodiment 60, wherein the fourth expression construct further encodes a protein of interest.
62. The method according to embodiment 60 or embodiment 61, wherein the fourth expression construct site-specifically integrates into the genome of the cell.
63. The method according to embodiment 62, wherein site-specific integration of the fourth expression construct into the genome of the cell inactivates a target gene within the genome of the cell.
64. The method according to any one of embodiments 60 to 63, wherein the fifth expression construct further encodes a protein of interest.
65. The method according to any one of embodiments 60 to 64, wherein the fifth expression construct site-specifically integrates into the genome of the cell.
66. The method according to embodiment 65, wherein site-specific integration of the fourth expression construct into the genome of the cell inactivates a target gene within the genome of the cell.
67. The method according to any one of embodiments 60 to 66, wherein the membrane association domain of the fusion protein encoded by each of the fourth and fifth expression constructs is, independently, a transmembrane domain as defined in any one of embodiments 39 to 41.
68. The method according to any one of embodiments 44, 45, or 59 to 67, wherein the dimerization domain comprises a coiled coil structure.
69. The method according to embodiment 68, wherein the dimerization domain comprises a leucine zipper domain.
70. The method according to any one of embodiments 2 to 69, wherein a protein of interest further encoded by one or more expression constructs of the two or more separate expression constructs is independently selected from the group consisting of: a receptor, a ligand, a transcription factor, an antibody, a bispecific T-cell engager (BiTE), an enzyme, a cytokine, a chemokine, a toxin, a protein conferring resistance to cell exhaustion, and a suicide switch protein.
71. The method according to embodiment 70, wherein a protein of interest further encoded by one or more expression constructs of the two or more separate expression constructs is a receptor.
72. The method according to embodiment 71, wherein the receptor is a chimeric antigen receptor (CAR), a T cell receptor (TCR), a synthetic Notch (SynNotch) receptor, a Modular Extracellular Sensor Architecture (MESA) receptor, a Tango receptor, a ChaCha receptor, a generalized extracellular molecule sensor (GEMS) receptor, a cytokine receptor, a chemokine receptor, a switch receptor, an adhesion molecule, an integrin, an inhibitory receptor, a stimulatory receptor, an immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor, or an immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor.
73. The method according to embodiment 72, wherein the receptor is a CAR.
74. The method according to any one of embodiments 1 to 73, wherein the selection marker comprises a protein tag.
75. The method according to embodiment 74, wherein the protein tag is selected from the group consisting of: a Myc-tag, a His-tag, an HA-tag, a FLAG-tag, a Strep-tag, an NE-tag, an Xpress tag, an Avi-tag, a polyglutamate tag, and a polyarginine tag.
76. The method according to any one of embodiments 1 to 75, wherein the selection marker comprises a cluster of differentiation (CD) protein.
77. The method according to embodiment 76, wherein the CD protein is CD34.
78. The method according to any one of embodiments 1 to 75, wherein the selection marker comprises a truncated receptor comprising the extracellular domain of the receptor.
79. The method according to embodiment 78, wherein the truncated receptor is truncated epidermal growth factor receptor (EGFRt), a truncated nerve growth factor receptor (NGFRt), a truncated CD19 (CD19t), or a truncated CD20 (CD20t).
80. The method according to any one of embodiments 1 to 79, wherein the selection marker is fused to a membrane association domain.
81. The method according to embodiment 80, wherein the membrane association domain is a transmembrane domain as defined in any one of embodiments 39 to 41.
82. The method according to any one of embodiments 1 to 81, wherein the fusion protein encoded by the first expression construct further comprises a degron, wherein the protease cleavage site disposed between the selection marker and the degron.
83. The method according to any one of embodiments 1 to 82, wherein the fusion protein encoded by the first expression construct further comprises a domain that confers antibiotic resistance.
84. The method according to embodiment 83, wherein the domain that confers antibiotic resistance is disposed between the selection marker and the protease cleavage site.
85. The method according to embodiment 83 or embodiment 84, wherein the domain that confers antibiotic resistance confers puromycin resistance.
86. The method according to embodiment 85, wherein the domain that confers puromycin resistance comprises a puromycin-N-acetyltransferase (PuroR).
87. The method according to any one of embodiments 1 to 86, wherein the selecting comprises magnetic-activated cell sorting (MACS).
88. The method according to any one of embodiments 1 to 86, wherein the selecting comprises flow cytometry.
89. The method according to embodiment 88, wherein the flow cytometry comprises fluorescence-activated cell sorting (FACS).
90. The method according to any one of embodiments 1 to 89, wherein the population of cells is a population of mammalian cells.
91. The method according to embodiment 90, wherein the mammalian cells comprise immune cells.
92. The method according to embodiment 91, wherein the immune cells comprise T cells, B cells, natural killer (NK) cells, macrophages, monocytes, neutrophils, dendritic cells, mast cells, basophils, eosinophils, and any combination thereof.
93. The method according to embodiment 91, wherein the immune cells comprise T cells.
94. The method according to embodiment 93, wherein the T cells comprise naive T cells (TN), cytotoxic T cells (TCTL), memory T cells (TMEM), T memory stem cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM), tissue resident memory T cells (TRM), effector T cells (TEFF), regulatory T cells (TREGs), helper T cells, CD4+ T cells, CD8+ T cells, virus-specific T cells, alpha beta T cells (Tαβ), gamma delta T cells (Tγδ), and any combination thereof.
95. The method according to embodiment 90, wherein the mammalian cells comprise stem cells.
6. The method according to embodiment 95, wherein the stem cells comprise embryonic stem (ES) cells, adult stem cells, hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), neural stem cells (NSCs), or any combination thereof.
97. A cell comprising two or more separate expression constructs, wherein the two or more separate expression constructs comprise:
98. The cell of embodiment 97, wherein the first expression construct further encodes a protein of interest.
99. The cell of embodiment 97 or embodiment 98, wherein the first expression construct is site-specifically integrated into the genome of the cell.
100. The cell of embodiment 99, wherein a target gene within the genome of the cell is inactivated as a result of the site-specific integration of the first expression construct.
101. The cell of any one of embodiments 97 to 100, wherein the second expression construct further encodes a protein of interest.
102. The cell of any one of embodiments 97 to 101, wherein the second expression construct is site-specifically integrated into the genome of the cell.
103. The cell of embodiment 102, wherein a target gene within the genome of the cell is inactivated as a result of the site-specific integration of the second expression construct.
104. The cell of any one of embodiments 97 to 103, wherein the cell is a mammalian cell.
105. The cell of embodiment 104, wherein the mammalian cell is a human cell.
106. The cell of embodiment 104 or embodiment 105, wherein the cell is an immune cell.
107. The cell of embodiment 106, wherein the immune cell is a T cell, a B cell, a natural killer (NK) cell, a macrophage, a monocyte, a neutrophil, a dendritic cell, a mast cell, a basophil, or an eosinophil.
108. The cell of embodiment 106, wherein the immune cell is a T cell.
109. The cell of embodiment 108, wherein the T cell is a naive T cell (TN), a cytotoxic T cell (TCTL), a memory T cell (TMEM), a T memory stem cell (TSCM), a central memory T cell (TCM), an effector memory T cell (TEM), a tissue resident memory T cell (TRM), an effector T cell (TEFF), a regulatory T cell (TREGs), a helper T cell, a CD4+ T cell, a CD8+ T cell, a virus-specific T cell, an alpha beta T cell (Tαβ), or a gamma delta T cell (Tγδ).
110. The cell of embodiment 104 or embodiment 105, wherein the cell is a stem cell.
111. The cell of embodiment 110, wherein the stem cell is an embryonic stem (ES) cell, an adult stem cell, a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPSC), a mesenchymal stem cell (MSC), or a neural stem cell (NSC).
112. A kit comprising two or more separate expression constructs, wherein the two or more separate expression constructs comprise:
113. The kit of embodiment 112, wherein the first expression construct further encodes a protein of interest.
114. The kit of embodiment 112, wherein the first expression construct comprises a cloning site for a nucleic acid encoding a protein of interest.
115. The kit of any one of embodiments 112 to 114, wherein the second expression construct further encodes a protein of interest.
116. The kit of any one of embodiments 112 to 114, wherein the second expression construct comprises a cloning site for a nucleic acid encoding a protein of interest.
117. The kit of any one of embodiments 112 to 116, further comprising instructions for contacting a population of cells with the two or more expression constructs under conditions in which the two or more expression constructs are delivered to cells of the population of cells.
118. The kit of any one of embodiments 112 to 117, further comprising instructions for selecting for cells exhibiting cell surface expression of the selection marker.
119. The cell of any one of embodiments 97 to 118, wherein the protein localization tag is as defined in any one of embodiments 8 to 23.
120. The cell or kit of any one of embodiments 97 to 119, wherein the protease cleavage site is as defined in any one of embodiments 24 to 33.
121. The cell or kit of any one of embodiments 97 to 120, wherein the protein required for cell surface expression of the selection marker is a protease, wherein the protease cleavage site is a cleavage site for the protease.
122. The cell or kit of embodiment 121, wherein the protease is fused to a protein localization tag that localizes the protease to the same cellular compartment as the fusion protein comprising the selection marker.
123. The cell or kit of embodiment 122, wherein the protease is fused to a protein localization tag having the same amino acid sequence as that of the protein localization tag of the fusion protein comprising the selection marker.
124. The cell or kit of embodiment 122 or embodiment 123, wherein the protease is fused to a membrane association domain.
125. The cell or kit of embodiment 124, wherein the membrane association domain is a transmembrane domain as defined in any one of embodiments 39 to 41.
126. The cell or kit of embodiment 121, wherein the protease is fused to a dimerization domain.
127. The cell or kit of embodiment 126, comprising a third expression construct that encodes a fusion protein comprising a transmembrane domain, a dimerization domain that dimerizes with the dimerization domain fused to the protease, and a protein localization tag that localizes the dimerization domain to the same cellular compartment as the fusion protein comprising the selection marker.
128. The cell or kit of any one of embodiments 97 to 120, wherein the protein required for cell surface expression of the selection marker is a first complementary fragment of a protease, wherein the protease cleavage site is a cleavage site for the protease.
129. The cell or kit of embodiment 128, comprising a third expression construct that encodes a second complementary fragment of the protease, wherein the first and second complementary fragments form an active protease complex.
130. The cell or kit of embodiment 129, wherein the third expression construct further encodes a protein of interest.
131. The cell or kit of embodiment 129 or embodiment 130, wherein the first and second complementary fragments are each fused to a protein localization tag that localizes the protease to the same cellular compartment as the fusion protein comprising the selection marker.
132. The cell or kit of embodiment 131, wherein the first and second complementary fragments are each fused to a protein localization tag having the same amino acid sequence as that of the protein localization tag of the fusion protein comprising the selection marker.
133. The cell or kit of any one of embodiments 129 to 132, wherein the first and second complementary fragments are each fused to a membrane association domain.
134. The cell or kit of embodiment 133, wherein the membrane association domain is a transmembrane domain.
135. The cell or kit of embodiment 134, wherein the transmembrane domain is as defined in any one of embodiments 39 to 41.
136. The cell or kit of any one of embodiments 129 to 132, wherein the first and second complementary fragments are each fused to a dimerization domain.
137. The cell or kit of embodiment 136, comprising:
138. The cell or kit of embodiment 137, wherein the fourth expression construct further encodes a protein of interest.
139. The cell or kit of embodiment 137 or embodiment 138, wherein the fifth expression construct further encodes a protein of interest.
140. The cell or kit of any one of embodiments 137 to 139, wherein the membrane association domain of the fusion protein encoded by each of the fourth and fifth expression constructs is, independently, a transmembrane domain as defined in any one of embodiments 39 to 41.
141. The cell or kit of embodiment 126, 127, or 136 to 140, wherein the dimerization domain comprises a coiled coil structure.
142. The cell or kit of embodiment 141, wherein the dimerization domain comprises a leucine zipper domain.
143. The cell or kit of any one of embodiments 97 to 142, wherein a protein of interest further encoded by one or more expression constructs of the two or more separate expression constructs is independently selected from the group consisting of: a receptor, a ligand, a transcription factor, an antibody, a bispecific T-cell engager (BiTE), an enzyme, a cytokine, a chemokine, a toxin, a protein conferring resistance to cell exhaustion, and a suicide switch protein.
144. The cell or kit of embodiment 143, wherein a protein of interest further encoded by one or more expression constructs of the two or more separate expression constructs is a receptor.
145. The cell or kit of embodiment 144, wherein the receptor is a chimeric antigen receptor (CAR), a T cell receptor (TCR), a synthetic Notch (SynNotch) receptor, a Modular Extracellular Sensor Architecture (MESA) receptor, a Tango receptor, a ChaCha receptor, a generalized extracellular molecule sensor (GEMS) receptor, a cytokine receptor, a chemokine receptor, a switch receptor, an adhesion molecule, an integrin, an inhibitory receptor, a stimulatory receptor, an immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor, or an immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor.
146. The cell or kit of embodiment 144, wherein the receptor is a CAR.
147. The cell or kit of any one of embodiments 97 to 146, wherein the selection marker is as defined in any one of embodiments 74 to 81.
148. The cell or kit of any one of embodiments 97 to 147, wherein the fusion protein encoded by the first expression construct further comprises a degron, wherein the protease cleavage site disposed between the selection marker and the degron.
149. The cell or kit of any one of embodiments 97 to 148, wherein the fusion protein encoded by the first expression construct further comprises a domain that confers antibiotic resistance.
150. The cell or kit of embodiment 149, wherein the domain that confers antibiotic resistance is disposed between the selection marker and the protease cleavage site.
151. The cell or kit of embodiment 149 or embodiment 150, wherein the domain that confers antibiotic resistance confers puromycin resistance.
152. The cell or kit of embodiment 151, wherein the domain that confers puromycin resistance comprises a puromycin-N-acetyltransferase (PuroR).
153. A composition comprising cells or progeny thereof selected according to the method of any one of embodiments 1 to 96 present in a liquid medium.
154. A composition comprising the cell of any one of embodiments 97 to 111 or 119 to 152 present in a liquid medium.
155. The composition of embodiment 153 or embodiment 154, wherein the liquid medium is a cell culture medium.
156. The composition of embodiment 153 or embodiment 154, wherein the liquid medium is suitable for administration of the composition to an individual in need thereof.
157. The composition of embodiment 156 formulated for parenteral administration to the individual.
158. A method comprising administering a therapeutically effective amount of the composition of embodiment 156 or embodiment 157 to an individual in need thereof.
159. A fusion protein comprising a protein fused to an ER localization tag, wherein the ER localization tag comprises 80% or greater, 85% or greater, 90% or greater, 95% or greater, or 100% amino acid sequence identity to an ER localization tag comprising, consisting of, or present within, an amino acid sequence selected from the group consisting of:
160. The fusion protein of embodiment 159, wherein the C-terminus of the ER localization tag comprises the four C-terminal residues of one of the sequences recited in embodiment 159.
161. A fusion protein comprising a protein fused to an ER localization tag, wherein the ER localization tag comprises a Tm domain, an ICD, or both, of an ER localization tag of a polypeptide set forth in Table 1, or a variant Tm and/or ICD thereof which retains the ability to localize a polypeptide to the ER.
162. A fusion protein comprising a protein fused to an ER localization tag, wherein the ER localization tag comprises a Tm domain, an ICD, or both, of an ER localization tag of a human ER-resident protein, or a variant Tm and/or ICD thereof which retains the ability to localize a polypeptide to the ER.
163. The fusion protein of embodiment 162, wherein the human ER-resident protein is CISD2.
164. The fusion protein of embodiment 163, wherein the ER localization tag comprises the Tm domain, the ICD, or both, of the polypeptide set forth in SEQ ID NO:91, or a variant Tm and/or ICD thereof which retains the ability to localize a polypeptide to the ER.
165. The fusion protein of embodiment 162, wherein the human ER-resident protein is UGT2B17.
166. The fusion protein of embodiment 165, wherein the ER localization tag comprises the Tm domain, the ICD, or both, of the polypeptide set forth in SEQ ID NO:95, or a variant Tm and/or ICD thereof which retains the ability to localize a polypeptide to the ER.
167. The fusion protein of embodiment 159, wherein the protein is fused directly to the ER localization tag.
168. The fusion protein of embodiment 159, wherein the protein is fused indirectly to the ER localization tag.
169. The fusion protein of any one of embodiments 159 to 168, further comprising a protease cleavage site.
170. The fusion protein of embodiment 169, wherein the protease cleavage site is disposed between the protein and the ER localization tag.
171. The fusion protein of embodiment 169 or embodiment 170, wherein the protease cleavage site is as defined in any one of embodiments 24 to 33.
172. The fusion protein of any one of embodiments 159 to 171, further comprising a transmembrane domain.
173. The fusion protein of embodiment 172, wherein the transmembrane domain is as defined in any one of embodiments 39 to 41.
174. A fusion protein comprising a protein fused to a transmembrane domain, wherein the transmembrane domain comprises 80% or greater, 85% or greater, 90% or greater, 95% or greater, or 100% amino acid sequence identity to a transmembrane domain comprising, consisting of, or present within, an amino acid sequence selected from the group consisting of:
175. The fusion protein of embodiment 174, wherein the protein is fused directly to the transmembrane domain.
176. The fusion protein of embodiment 174, wherein the protein is fused indirectly to the transmembrane domain.
177. The fusion protein of any one of embodiments 174 to 176, further comprising a protease cleavage site.
178. The fusion protein of embodiment 177, wherein the protease cleavage site is as defined in any one of embodiments 24 to 33.
179. The fusion protein of any one of embodiments 174 to 177, further comprising a protein localization tag.
180. The fusion protein of embodiment 179, wherein the protein localization tag is as defined in any one of embodiments 8 to 23.
181. The fusion protein of any one of embodiments 159 to 180, wherein the protein is a receptor, a ligand, a transcription factor, an antibody, a bispecific T-cell engager (BiTE), an enzyme, a cytokine, a chemokine, a toxin, a protein conferring resistance to cell exhaustion, and a suicide switch protein.
182. The fusion protein of any one of embodiments 159 to 180, wherein the protein is a receptor selected from the group consisting of: a chimeric antigen receptor (CAR), a T cell receptor (TCR), a synthetic Notch (SynNotch) receptor, a Modular Extracellular Sensor Architecture (MESA) receptor, a Tango receptor, a ChaCha receptor, a generalized extracellular molecule sensor (GEMS) receptor, a cytokine receptor, a chemokine receptor, a switch receptor, an adhesion molecule, an integrin, an inhibitory receptor, a stimulatory receptor, an immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor, and an immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor.
183. The fusion protein of embodiment 182, wherein the receptor is a CAR.
184. The fusion protein of any one of embodiments 159 to 176, wherein the protein is a selection marker.
185. A nucleic acid that encodes the fusion protein of any one of embodiments 159 to 184.
186. An expression construct comprising the nucleic acid of embodiment 185.
187. A cell comprising the nucleic acid of embodiment 185 or the expression construct of embodiment 186.
188. A method of producing the fusion protein of any one of embodiments 159 to 184, comprising culturing the cell of embodiment 186 or embodiment 187 under conditions suitable for the cell to express the fusion protein, wherein the fusion protein is produced.
The following examples are offered by way of illustration and not by way of limitation.
Selection of Cells with Multiple Genetic Modifications Using a Single Selection Marker
Described herein are cell selection systems according to embodiments of the present disclosure. The systems are sometimes referred to herein as “STASH selection systems”, “STASH select”, etc. by virtue of the selection marker being “stashed” intracellularly in the absence of the desired combination of expression constructs being present in the cell. According to the selection systems, one of the expression constructs encodes a fusion protein comprising the selection marker, a protein localization tag, and a protease cleavage site disposed between the selection marker and the protein localization tag. In the absence of one or more additional expression constructs which provide a protease capable of cleaving the protease cleavage site, the selection marker remains localized to (i.e., retained or “stashed” at) the intracellular location (e.g., organelle) determined by the particular protein localization tag employed. When the one or more additional expression constructs are present in the cell, thereby providing a protease capable of cleaving the protease cleavage site, the selection marker is cleaved from the protein localization tag and traffics to the surface of the cell, such that the cell comprising the desired multiple genetic modifications exhibits cell surface expression of the selection marker. Non-limiting examples and data providing proof-of-concept of cell selection systems according to embodiments of the present disclosure will now be described.
High Surface Expression of a Myc Tag Selection Marker in Cells that are Double Positive for Separate Expression Constructs A and B—NS3 Protease
Shown in
High Surface Expression of a Myc Tag Selection Marker in Cells that are Double Positive for Separate Expression Constructs A and B-TEV Protease Variant
High Surface Expression of a Myc Tag Selection Marker in Cells that are Triple Positive for Separate Expression Constructs A, B and C
High Surface Expression of a Myc Tag Selection Marker in Cells that are Quintuple Positive for Separate Expression Constructs A, B, C, D and E
EGFRt fusion proteins with various ER tags (set 1) were then produced.
Additional EGFRt fusion proteins with various ER tags (set 2) were then produced.
Two-Way STASH Select Using EGFRt with Novel ER Tags
Two-way STASH Select using EGFRt-STASH variant 493 at low initial double positive cell fractions
STASH Select Variant 493 with a Degron Domain Allows for Antibiotic Selection
Two-Way STASH Select with CD34 as the Epitope Marker
Two-Way STASH Select with TEV Protease Bearing a CISD2 ER Retention Tag
Three-Way STASH Select with Various Tm Combinations for EGFRt-STASH Variants with CISD2 Retention Signals
Three-Way STASH Select with Various Tm Combinations for EGFRt-STASH Variants with IBV S Protein ER Tag
Three-Way STASH Select with Various Tm Combinations for EGFRt-STASH Variants with a Degron Fused ER Tag
Three-Way STASH Select of cJun, CD19, and HER2 CAR-T Cells Using EGFRt-STASH Variant 507
Surface EGFR Expression with Protease 797, a Minimized Protease Construct with a 501 ER Retention Tag
Identification of a Human Protease for Use with STASH Select
Two-Way STASH Select with Various EGFR Truncations
The protease module was expressed as a bicistronic fusion construct according to the following from N to C terminus: Payload protein (BFP, CAR, cJun, etc.), ribosome skip sequence (P2A), leader sequence (TCR-β Leader), protease detection domain (RQR8), transmembrane domain (CD8α hinge and Tm), linker, NS4A cofactor domain, linker, HCV NS3 protease, NS3 helicase fragment, linker, and ER retention tag (adenovirus E3-19K tag).
The protease module was expressed as a bicistronic fusion construct according to the following from N to C terminus: Payload protein (BFP, tdTomato, CAR, cJun, etc.), ribosome skip sequence (P2A), leader sequence (TCR-Leader), protease detection domain (RQR8), transmembrane domain (CD8α hinge and Tm, CD28 hinge and Tm, or CISD2 Tm), linker, TEV protease, linker, and ER retention tag (adenovirus E3-19K tag or CISD2 intracellular domain).
The protease module was expressed as a bicistronic fusion construct according to the following from N to C terminus: Payload protein (BFP, tdTomato, CAR, cJun, etc.), ribosome skip sequence (P2A), leader sequence (TCR-β Leader), protease detection domain (RQR8), transmembrane domain (CD8α hinge and Tm, CD28 hinge and Tm, or CISD2 Tm), linker, human protease (such as Kallikrein-15 or enterokinase light chain), linker, and ER retention tag (adenovirus E3-19K tag or CISD2 intracellular domain).
nTEV Protease Expression Constructs for Three-Way STASH Selection
The protease module was expressed as a bicistronic fusion construct according to the following from N to C terminus: Payload protein (BFP, tdTomato, CAR, cJun, etc.), ribosome skip sequence (P2A), leader sequence (TCR-β Leader), protease detection domain (RQR8), transmembrane domain (CD8α hinge and Tm, CD28 hinge and Tm, or CISD2 Tm), linker, nTEV protease (N-terminal domain of split TEV protease comprising 118 N-terminal amino acids of the protease), linker, and ER retention tag (adenovirus E3-19K tag).
cTEV Protease Expression Constructs for Three-Way STASH Selection
The protease module was expressed as a bicistronic fusion construct according to the following from N to C terminus: Payload protein (BFP, tdTomato, CAR, cJun, etc.), ribosome skip sequence (P2A), leader sequence (TCR-β Leader), protease detection domain (RQR8), transmembrane domain (CD8α hinge and Tm, CD28 hinge and Tm, or CISD2 Tm), linker, cTEV protease (C-terminal domain of split TEV protease comprising 118 C-terminal amino acids of the protease), linker, and ER retention tag (adenovirus E3-19K tag).
nTEV Protease Expression Constructs for Five-Way STASH Selection
The protease module was expressed as a bicistronic fusion construct according to the following from N to C terminus: Payload protein (BFP, tdTomato, CAR, cJun, etc.), ribosome skip sequence (P2A), leucine zipper (SYNZIP1, SYNZIP2, SYNZIP1, or SYNZIP4), linker, and nTEV protease (N-terminal domain of split TEV protease comprising 118 N-terminal amino acids of the protease).
cTEV Protease Expression Constructs for Five-Way STASH Selection
The protease module was expressed as a bicistronic fusion construct according to the following from N to C terminus: Payload protein (BFP, tdTomato, CAR, cJun, etc.), ribosome skip sequence (P2A), leucine zipper (SYNZIP1, SYNZIP2, SYNZIP1, or SYNZIP4), linker, and cTEV protease (C-terminal domain of split TEV protease comprising 118 C-terminal amino acids of the protease).
The protease-recruiting transmembrane protein module was expressed as a bicistronic fusion construct according to the following from N to C terminus: Payload protein (BFP, tdTomato, CAR, cJun, etc.), ribosome skip sequence (P2A), leader sequence (GM-CSFR Leader), detection tag (FLAG Tag or Myc Tag), transmembrane domain (CD8α hinge and Tm or CD28 hinge and Tm), linker, leucine zipper (SYNZIP1, SYNZIP2, SYNZIP1, or SYNZIP4), linker, and ER retention tag (adenovirus E3-19K tag).
The protease-recruiting transmembrane protein module was expressed as a bicistronic fusion construct according to the following from N to C terminus: Payload protein (BFP, tdTomato, CAR, cJun, etc.), ribosome skip sequence (P2A), leader sequence (GM-CSFR Leader), detection tag (HA Tag), transmembrane domain (CD8α hinge and Tm or CD28 hinge and Tm), linker, leucine zipper (SYNZIP1, SYNZIP2, SYNZIP1, or SYNZIP4), linker, and ER retention tag (adenovirus E3-19K tag).
The STASH selection module was expressed as a bicistronic fusion construct according to the following from N to C terminus: Payload protein (CAR, cJun, GFP, BFP, tdTomato, etc.), ribosome skip sequence (P2A), leader sequence (GM-CSFR leader), extracellular domain of epitope marker (EGFRt, CD34, Myc Tag, NGFRt), linker, transmembrane domain (CD8α hinge and Tm, CD28 hinge and Tm, CISD2 Tm, TMED4 Tm, Sel1L Tm, DDOST Tm, UGT2B17 Tm, UGT1A1 Tm, TAPBP Tm, TMED4 Tm, TRIQK Tm, mastadenovirus C E3 19K Tm, IBV S Tm, or Calnexin Tm), linker, protease cleavage site (TEV cleavage site, HCV NS3 cleavage site, or human enterokinase light chain cleavage site), linker, and ER retention Tag (adenovirus E3-19K tag, adenovirus E3-19K variant 1 tag, adenovirus E3-19K variant 2 tag, adenovirus E3-19K variant 3 tag, KDELR2 ICD, carboxypeptidase D ICD, Coronavirus infectious bronchitis virus (IBV) S protein ER retention motif, HCV NS3 helix, HCV helicase domain, CISD2 ICD, TMED4 ICD, Sel1L ICD, DDOST ICD, UGT2B17 ICD, UGT1A1 ICD, TAPBP ICD, TRIQK ICD, mastadenovirus C E3 19K ICD, IBV S ICD, or Calnexin ICD).
STASH Selection Marker with Degron Domain Expression Constructs
The STASH selection module was expressed as a bicistronic fusion construct according to the following from N to C terminus: Payload protein (CAR, cJun, GFP, BFP, tdTomato, etc.), ribosome skip sequence (P2A), leader sequence (GM-CSFR leader), extracellular domain of epitope marker (EGFRt, CD34, Myc Tag, NGFRt), linker, transmembrane domain (CD8α hinge and Tm, CD28 hinge and Tm), linker, protease cleavage site (TEV cleavage site), linker, degron domain (HCV NS4A degron domain), and ER retention Tag (adenovirus E3-19K tag).
STASH Selection Marker with Puromycin Resistance and Degron Domains Expression Constructs
The STASH selection module was expressed as a bicistronic fusion construct according to the following from N to C terminus: Payload protein (CAR, cJun, GFP, BFP, tdTomato, etc.), ribosome skip sequence (P2A), leader sequence (GM-CSFR leader), extracellular domain of epitope marker (EGFRt), linker, transmembrane domain (CD8α hinge and Tm), linker, puromycin-N-acetyltransferase (PuroR), linker, protease cleavage site (TEV cleavage site), linker, degron domain (HCV NS4A degron domain), and ER retention Tag (adenovirus E3-19K tag).
STASH Selection Marker with Puromycin Resistance, Degron Domains, and Dual Cleavage Sites Expression Constructs
The STASH selection module was expressed as a bicistronic fusion construct according to the following from N to C terminus: Payload protein (CAR, cJun, GFP, BFP, tdTomato, etc.), ribosome skip sequence (P2A), leader sequence (GM-CSFR leader), extracellular domain of epitope marker (EGFRt), linker, transmembrane domain (CD8α hinge and Tm), linker, protease cleavage site (TEV cleavage site), puromycin-N-acetyltransferase (PuroR), linker, protease cleavage site (TEV cleavage site), linker, degron domain (HCV NS4A degron domain), and ER retention Tag (adenovirus E3-19K tag).
D. Isolation of Primary Human T Cells from Blood Donors
Primary human T cells were extracted from buffy coats by negative selection using the RosetteSep Human T cell Enrichment kit (Stem Cell Technologies) and SepMate-50 tubes. T cells were cryopreserved at CryoStor CS10 cryopreservation media (Stem Cell Technologies) until use.
DNA sequences were synthesized as gBlocks or oligonucleotides (Integrated DNA Technologies) and cloned into the MSGV1 retroviral expression construct by In-Fusion cloning. In-Fusion reaction products were transformed into chemically competent cells (Stellar Cell, Takara Bio) by heat shock method. Transformants were sequence verified by Sanger sequencing. Bacteria cultures from sequence verified clones were grown for 16 hours at 37C with shaking. Subsequently, the bacteria cells were harvested and DNA was extracted using a miniprep kit (QIAprep Spin Miniprep Kit, Qiagen).
Retroviral supernatant was prepared using 293GP cells and the RD114 envelope plasmid. In brief, 22 ug of the corresponding MSGV1 transfer plasmid and 11 ug of RD114 and were delivered to 293GP cells, grown to about 80% confluency on poly-D-lysine dishes (Corning), by transient transfection using the Lipofectamine 2000 reagent (Thermo Fisher). 293GP cells were cultured in media (DMEM, 10% FBS, 10 mM HEPES, 2 mM L-glutamine, 100 U/mL penicillin, and 100 ug/mL streptomycin, Gibco) at 37° C. in a 5% CO2 environment. Media was replenished every 24 hours. Retroviral supernatant was harvested 48 and 72-hour post transfection, centrifuged to deplete dead cells and debris, and stored at −80 C until further use.
The EGFR-STASH TRAC knock-in template was cloned into an AAV plasmid backbone in the following configuration ITR, TRAC left homology arm, EF1a promoter, EGFR STASH variant 501, bGH poly (A) signal, TRAC right homology arm, and ITR. AAV was produced by transfecting five 150 mm plates of 293T cells with 30 ug template plasmid and 110 ug AAV6 helper plasmid (pDGM6). 293T cells were cultured in media (DMEM, 10% FBS, 10 mM HEPES, 2 mM L-glutamine, 100 U/mL penicillin, and 100 ug/mL streptomycin, Gibco) at 37° C. in a 5% CO2 environment. Media was replenished every 24 hours. After 72 hours, AAV6 particles were extracted using the AAVpro® Purification Kit Maxi kit (Takara, catalog #6666), according to the manufacturer's instructions. See Wiebking et al. Nat. Biotechnology 2020 for related methods.
Primary human T cells were thawed at Day 0 and activated with anti-CD3/CD28 Human T-Expander Dynabeads (Thermo Fisher) at a bead to cell ratio of 3:1. On Day 2 virus coated culture plates were prepared on non TC-treated 12-well or 24 well plates that had been pre-coated with RetroNectin (Takara Bio) according to the manufacturer's instructions, by incubating with 0.1-1 mL of each retroviral component diluted in DMEM and centrifugation at 3200 RPM, 32° C. for about two hours. Subsequently, the supernatant was aspirated off of the wells and 0.25-0.5×106 T cells were added in 1 mL of T cell media comprised of: AIM V (Thermo Fisher), 5% fetal bovine serum (FBS), 100 U/mL penicillin (Gibco), 2 mM L-glutamine (Gibco), 100 mg/ml streptomycin (Gibco), 10 mM HEPES (Gibco), and 100 U/mL rhIL-2 (Peprotech). After addition of the T cells, the plates were gently spun down at 1200 RPM for 2 min then incubated for 24 hrs at 37° C. 5% CO2. This transduction process was repeated at Day 3. For some experiments with greater than 3 expression constructs, this process was repeated on Day 4. Dynabeads were removed on Day 4 by magnetic separation. Cells were maintained between 0.4-2×106 cells/mL and expanded until Day 7-21. T cells were transduced with 1-5 different viruses per transduction day.
CRISPR guides were synthesized by Synthego and resuspended according to the manufacturer's instructions. Alt-R® S.p. Cas9 Nuclease V3 was purchased from IDT. To generate Cas9 ribonucleoproteins, 0.5 uL of sgRNA was added to 0.4 uL of Cas9, allowed to complex at room temperature, then placed on ice until electroporation. Primary human T cells were thawed at Day 0 and activated with anti-CD3/CD28 Human T-Expander Dynabeads (Thermo Fisher) at a bead to cell ratio of 3:1. On Day 2, beads were removed from T cells by magnetic separation. 1×106 T cells were resuspended in 20 uL P3 buffer (Lonza), added to cas9 ribonucleoprotein complex, transferred to electroporation strips, then electroporated using the Lonza nucleofector 4D system using program EH-115. Immediately after electroporation, cells were transferred to 96 well plates containing T cell culture media and AAV6 viral particles.
Recombinant CD19 idiotype antibody, HER2-Fc, and CD22-Fc, fluorescently labeled with the DyLight 650 Microscale Antibody Labeling Kit (Thermo Fisher), were used for CAR detection. The following antibodies were used for staining: anti-human EGFR Antibody (clone AY13, BioLegend), Myc-Tag (71D10) Rabbit mAb (Cell Signaling Technology), anti-human CD271 (NGFR) Antibody (clone ME20.4, BioLegend), CD34 Monoclonal Antibody (QBEND/10, Thermo Fisher Scientific), DYKDDDDK (SEQ ID NO:131) Tag (9A3) Mouse mAb (Cell Signaling Technology), anti-HA. 11 Epitope Tag Antibody (BioLegend), EGF Receptor Antibody anti-human Biotin (Miltenyi Biotec), and c-myc Antibody Biotin (Miltenyi Biotec). Flow cytometry was performed on a BD Fortessa instrument and analyzed by FlowJo software (Tree Star).
Cell were stained with the indicated biotinylated antibody according to the manufacturer's instructions. Subsequently, cells were labeled with magnetic microbeads (Streptavidin MicroBeads or Anti-Biotin MicroBeads UltraPure, Miltenyi Biotec) according to the manufacturer's instruction. Cells were loaded onto LS columns, washed with MACS buffer, and magnetically separated using the QuadroMACS separator (Miltenyi Biotec) according to the manufacturer's instructions.
Primary human T cells transduced with the indicated expression constructs were grown to Day 7-Day 10 post activation, as described above, then cultured in T cell media containing the indicated concentration or puromycin dihydrochloride (Thermo Fisher Scientific) for 48-96 hours.
Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.
This application claims the benefit of U.S. Provisional Patent Application No. 63/171,841, filed Apr. 7, 2021, which application is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/023725 | 4/6/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63171841 | Apr 2021 | US |
