Patent Application
20160369258
The present invention relates generally to constructs and methods that restrict nuclear proteins and polypeptides to specific phases of the cell cycle. One application is in mutagenesis of target genes that enhances the natural mutagenic capabilities of adaptive immune cells by stimulating the process of diversification while protecting the cells from mutagenic factors that can kill cells as they progress through the cell cycle. The invention provides a method for safely initiating mutations and other types of diversification in expressed genes, such as antibody genes. This method can be coupled with selection to identify B cell clones that produce, for example, antibodies of high affinity or specificity. The diversification process can also be used to produce optimized T cells that express chimeric antigen receptors for use in therapeutic applications. The invention thus provides a means of developing a repertoire of variant immunoglobulins and other polypeptides.
Antibodies are molecules that provide a key defense against infection in humans. They are used as therapeutics in treatment of a variety of diseases, from infectious disease to cancer. They are also used as diagnostic reagents in a huge variety of tests carried out daily in clinical and research laboratories.
Antibody specificity and affinity are modified in vivo by processes of mutation, targeted to specific regions within the genes that encode antibodies. Variability in the V region primary sequence (and hence three-dimensional structure and antigen specificity) is the result of processes which alter V region sequence by causing irreversible genetic changes. These changes are programmed during B cell development, and can also be induced in the body in response to environmental signals that activate B cells. Several genetic mechanisms contribute to this variability. Two subpathways of the same mechanism lead to two different mutagenic outcomes, referred to as somatic hypermutation and gene conversion (reviewed (Maizels, 2005)). Somatic hypermutation inserts point mutations. Somatic hypermutation provides the advantage of enabling essentially any mutation to be produced, so a collection of mutated V regions has essentially sampled a large variety of possible mutations.
Activation-induced cytosine deaminase (AID) initiates immunoglobulin (Ig) gene diversification in activated B cells by deaminating C to U (1, 2). This triggers error-prone repair leading to somatic hypermutation (SHM), class switch recombination (CSR) and gene conversion (3-8), and to the chromosomal translocations characteristic of B cell malignancies (9, 10). AID also participates in erasing CpG methylation to reprogram the genome in early development (11-15), promotes B cell tolerance (16, 17) and limits autoimmunity (18, 19).
AID is tightly regulated. Increased AID levels stimulate Ig gene diversification, and also promote translocation (20-23). The AID active site is not optimized for catalysis, but mutations that increase catalytic activity not only accelerate Ig gene diversification but also stimulate translocation and compromise cell viability (24). AID deaminates single-stranded DNA, but not RNA (25-30). AID localizes predominately to the cytoplasm but requires access to the nucleus to function, and subcellular localization is regulated by other proteins (7). AID persistence in the nucleus is limited by proteosomal degradation (31, 32) and by CRM1-dependent nuclear export (33-35). Mutation or deletion of the C-terminal region that includes the nuclear export signal (NES) diminishes AID stability and the efficiency of CSR, and compromises cell viability (36-38). There remains a need for improved methods of stimulating gene diversification, and for methods that can exploit the diversification-enhancing capabilities of AID without compromising cell viability.
The invention meets these needs and others by providing materials and methods for restricting nuclear activity of a polypeptide to G1 or to S-G2/M phase of the cell cycle. In one embodiment, the method comprises restricting expression of an enzyme to G1 or to S-G2/M phase of the cell cycle in a host cell. In one embodiment, the enzyme whose expression or nuclear activity is restricted is an enzyme that modifies the sequence and/or structure of a nucleic acid. In one embodiment, the enzyme is AID. In another embodiment, the AID is a catalytically inactive derivative of AID. One example of a catalytically inactive variant of AID is AID H56A. Thus, a representative example of a fusion construct is one that encodes AIDH56A,F193A-CDT1. In another embodiment, the enzyme is CRISPR/Cas9 or CRISPR/Cas9D10A.
In one embodiment, the method comprises transfecting a host cell with a fusion construct comprising a nucleotide sequence that expresses the polypeptide fused to a nucleotide sequence that expresses CDT1 or geminin (GEM), wherein a fusion construct expressing CDT1 restricts expression of the enzyme to G1 and a fusion construct expressing GEM restricts expression of the enzyme to S/G2-M phase (Sakaue-Sawano et al. 2008. Cell 132:487).
Additional variations for restricting expression to particular phases of the cell cycle are contemplated. For example, fragments from RAG2 (Li et al. 1996. Immunity 5: 575) for G1 restriction; and Cyclins can be used for cell cycle restricted expression. In some embodiments, the nucleotide sequence that expresses CDT1 or GEM is positioned downstream of the nucleotide sequence that expresses the polypeptide whose nuclear activity is to be restricted.
The invention additionally provides a method of diversification of target sequences while protecting cell viability. The invention provides a cell, which in one embodiment is a lymphocyte, such as a B cell or T cell, modified to enhance diversification of a target gene. The cell comprises a construct as described herein and a target gene of interest. The B cell can be a chicken DT40 B cell or other vertebrate B cell, with a human B cell or a chicken DT40 B cell containing humanized immunoglobulin (Ig) genes (in which human IgH and IgL replace chicken IgH and IgL) preferred for some embodiments.
In one embodiment, the invention provides a nucleic acid construct that expresses a fusion of nuclear export deficient enzyme that initiates or enhances diversification and a polypeptide targeted for cell cycle-dependent nuclear destruction (a “fusion construct”). One representative example of an enzyme that initiates or enhances diversification is a deaminase. Deamination accelerates mutagenesis. In one embodiment, the construct comprises a first nucleotide sequence that expresses activation-induced cytosine deaminase (AID), wherein the AID is modified to prevent nuclear export; and a second nucleotide sequence that expresses chromatin licensing and DNA replication factor 1 (CDT1) or another polypeptide targeted for cell cycle-dependent nuclear destruction, wherein the second nucleotide sequence is operably linked to and downstream of the first nucleotide sequence. AID is a B cell-specific DNA deaminase that initiates Ig gene diversification.
Mutants that promote AID accumulation in the nucleus include, but are not limited to: AIDF198A (McBride et al. 2004. J Exp Med 199:1235); AID196X and other C-terminal deletion mutants that remove the nuclear export signal (see, e.g., Ito et al. 2004. PNAS 101: 1975); AIDF193A, F193E, F193H, L196A (Geisberger et al. 2009. PNAS 106:6736); and L198S (Patenaude et al. 2009, NSMB 16:17).
Fragments of other proteins that are targeted for nuclear destruction in specific phases of cell cycle can function analogously to the CDT1 tag (Sakaue-Sawano et al. 2008. Cell 132:487) that is exemplified herein to target proteolysis to a fusion protein. These include but are not limited to fragments from: Geminin (Sakaue-Sawano et al. 2008. Cell 132:487): S/G2-M restriction; RAG2 (Li et al. 1996. Immunity 5: 575): G1 restriction; and Cyclins.
The invention provides an adaptive immune cell, such as a B cell or a T cell. A typical example of a B cell for use in the invention is a Ramos human B cell. The B cell can be a human B cell, or a chicken B cell such as DT40, or other vertebrate B cell, or a B cell that has been humanized by replacement of endogenous IgH and IgL genes with human IgH and IgL genes. A typical example of a T cell for use with the invention is a chimeric antigen receptor (CAR) T cell. Candidate lymphocytes for use in the invention are those which can benefit from modulation of the affinity and/or specificity of the cell for its target.
The lymphocyte can be from any vertebrate species. In a typical embodiment, the lymphocyte is from a mammalian or avian species, and in one embodiment, the lymphocyte is a human B cell or human T cell. Other (non-lymphocyte) host cells are suitable for use with the invention as well. In one embodiment, the invention provides a yeast or bacterial cell transfected with the nucleic acid construct.
Typically, the target gene comprises a promoter and a coding region. The coding region of the target gene in the lymphocyte of the invention can be one that encodes any protein or peptide of interest, and need not comprise a complete coding region. In some embodiments, a particular region or domain is targeted for diversification, and the coding region may optionally encode only a portion that includes the region or domain of interest.
In one embodiment, the target gene comprises an immunoglobulin (Ig) gene, wherein the Ig gene comprises an Ig gene enhancer and coding region. The Ig gene can be all or part of an IgL and/or IgH gene. The coding region can be native to the Ig gene, or a heterologous gene. In some embodiments, the target gene is or contains a non-Ig target domain for diversification, as well as domains permitting display of the gene product on the B cell surface, including a transmembrane domain and a cytoplasmic tail.
In one embodiment, the invention provides a method of producing a repertoire of polypeptides having variant sequences of a polypeptide of interest. In one embodiment, the method comprises culturing a lymphocyte transfected with a nucleic acid construct of the invention in conditions that allow expression of the nucleic acid construct. The lymphocyte contains the coding region of the polypeptide of interest, thereby permitting diversification of the coding region. The method further comprises maintaining the culture under conditions that permit proliferation of the lymphocyte until a plurality of lymphocytes and the desired repertoire is obtained. The method optionally further comprises selecting lymphocytes that express a polypeptide exhibiting desired characteristics. For example, a cell expressing an enzyme modified to metabolize an otherwise toxic compound can be selected by growth in a medium containing that compound. Alternatively, a cell that expresses a cytoplasmic fluorescent protein with enhanced fluorescence can be selected by flow for cells with higher mean fluorescent intensity than the starting population. As another example, a cell that expresses a steroid hormone receptor with higher affinity for the hormone can be selected by a fluorescence based assay for increased activity, and a cell that expresses a signaling molecule with higher affinity for a small molecule can be selected by a fluorescence-based signaling assay or other form of such assay that is not toxic to the cell. Likewise, a cell that expresses a DNA damage repair protein with increased activity can be selected for the ability to survive damage by that agent.
In another embodiment, the invention provides a method of producing lymphocytes that produce an optimized polypeptide of interest. In one embodiment, the method comprises culturing a lymphocyte transfected with a nucleic acid construct of the invention in conditions that allow expression of the nucleic acid construct, wherein the lymphocyte contains the coding region of the polypeptide of interest, and wherein and the lymphocyte expresses the polypeptide of interest on the surface of the lymphocyte. The method further comprises selecting cells from the culture that bind a ligand that specifically binds the polypeptide of interest expressed on the lymphocyte surface; and repeating these two steps until cells are selected that have a desired affinity and/or specificity for the ligand that specifically binds the polypeptide of interest. In one embodiment, the polypeptide of interest is an Ig. In a typical embodiment, the Ig is an IgL, IgH or both.
The invention provides a method of producing a repertoire of polypeptides having variant sequences of a polypeptide of interest via diversification of polynucleotide sequences that encode the polypeptide. The cell to be used in the method comprises both the nucleic acid construct of the invention and a nucleic acid encoding the polypeptide of interest. Typically, the method comprises culturing the cell of the invention in conditions that allow expression of the nucleic acids, wherein the target gene contains the coding region of the polypeptide of interest, thereby permitting diversification of the coding region. The method can further comprise maintaining the culture under conditions that permit proliferation of the cell until a plurality of variant polypeptides and the desired repertoire is obtained. The repertoire can then be used for selection of polypeptides having desired properties.
Also provided is a kit that can be used to carry out the methods of the invention. The kit comprises a lymphocyte or other cell of the invention and one or more fusion constructs described herein. The kit further comprises one or more containers, with one or more fusion constructs stored in the containers. Each fusion construct comprises a polynucleotide that can be expressed in the cell. The kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In addition, a label can be provided on the container to indicate that the composition is used for a specific therapeutic or non-therapeutic application, and can also indicate directions for use. Directions and or other information can also be included on an insert which is included with the kit.
The present invention is based on the unexpected discovery that an enzyme useful for genome engineering can be regulated by fusion of its encoding gene to a protein whose expression is restricted to selected phases of the cell cycle. This allows for an improved method of mutagenesis of target genes by stimulating the process of diversification while protecting the cells from mutagenic factors that can kill cells. The invention provides a method for safely initiating mutations and other types of diversification in expressed genes, such as antibody genes. This method can be coupled with selection to identify B cell clones that produce, for example, antibodies of high affinity or specificity. The diversification process can also be used to produce T cells bearing optimized chimeric antigen receptor for use in therapeutic applications. The invention thus provides a means of developing a repertoire of variant immunoglobulins and other polypeptides.
All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified.
As used herein, “polypeptide” includes proteins, fragments of proteins, and peptides, whether isolated from natural sources, produced by recombinant techniques or chemically synthesized. Peptides of the invention typically comprise at least about 6 amino acids.
As used herein, a “polypeptide targeted for cell cycle-dependent nuclear destruction” means a polypeptide that can target proteolysis to a fusion protein comprising this polypeptide during select phases of the cell cycle. Examples of such polypeptides include fragments of CDT1 (Sakaue-Sawano et al. 2008. Cell 132:487), Geminin (GEM; Sakaue-Sawano et al. 2008. Cell 132:487), RAG2 (Li et al. 1996. Immunity 5: 575), and Cyclins.
As used herein, “CDT1” refers to chromatin licensing and DNA replication factor 1, and includes fragments of CDT1 that can be fused to another polypeptide and that target this fusion protein for degradation in the nucleus during S-G2/M phase of cell cycle.
As used herein, “lymphocyte” refers to adaptive immune cells, including B cells and T cells. A typical example of a B cell for use in the invention is a Ramos human B cell. A typical example of a T cell for use with the invention is a T cell bearing a chimeric antigen receptor (CAR). Candidate lymphocytes for use in the invention are those which can benefit from modulation of the affinity and/or specificity of a cell surface receptor for its target.
As used herein, “nuclear export deficient activation-induced cytosine deaminase (AID)”, means a derivative of the AID protein deficient in nuclear export, such as an AID that lacks a functional nuclear export signal due to one or more mutations at the C terminus or deletion of a portion of the C terminus, including, for example, mutation or deletion of one or more amino acids within the C-terminal residues 183-198, or mutation of another region necessary to enable nuclear export. Examples of nuclear export deficient AIDs include, but are not limited to, AIDF193A, AIDF193E, AIDF193H, AIDL196A, AIDF198A, AIDF198S, AID193X or AID196X. Additional information about AID variants that are deficient in nuclear export can be found in Ito, et al., PNAS 101 (7):1975-1980, 2004; and in Patenaude et al., Nat. Struct. Mol. Biol. 16(5):517-27, 2009.
As used herein, “diversification” of a target gene means a change or mutation in sequence or structure of the target gene. Diversification includes the biological processes of somatic hypermutation, gene conversion, and class switch recombination, which can result in point mutation, templated mutation, DNA deletion and DNA insertion. The diversification factors of the invention can induce, enhance or regulate any of these methods of diversification.
A “mutation” is an alteration of a polynucleotide sequence, characterized either by an alteration in one or more nucleotide bases, or by an insertion of one or more nucleotides into the sequence, or by a deletion of one or more nucleotides from the sequence, or a combination of these.
As used herein, “promoter” means a region of DNA, generally upstream (5′) of a coding region, which controls at least in part the initiation and level of transcription. Reference herein to a “promoter” is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including a TATA box or a non-TATA box promoter, as well as additional regulatory elements (i.e., activating sequences, enhancers and silencers) that alter gene expression in response to developmental and/or environmental stimuli, or in a tissue-specific or cell-type-specific manner. A promoter is usually, but not necessarily, positioned upstream or 5′, of a structural gene, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene, although they may also be many kb away. Promoters may contain additional specific regulatory elements, located more distal to the start site to further enhance expression in a cell, and/or to alter the timing or inducibility of expression of a structural gene to which it is operably connected.
As used herein, “operably connected” or “operably linked” and the like means that the polynucleotide elements are linked in a functional relationship. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the relevant nucleic acid sequences are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. “Operably linking” a promoter to a transcribable polynucleotide means placing the transcribable polynucleotide (e.g., protein encoding polynucleotide or other transcript) under the regulatory control of a promoter, which then controls the transcription and optionally translation of that polynucleotide.
The term “nucleic acid” or “polynucleotide” refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally-occurring nucleotides.
As used herein, “prevent” means to reduce, hinder, or otherwise minimize the occurrence of an event.
As used herein, “a” or “an” means at least one, unless clearly indicated otherwise.
The invention provides a nucleic acid construct that expresses a fusion of an enzyme that modifies the sequence or structure of DNA or RNA when localized to the nucleus, and a polypeptide targeted for cell cycle-dependent nuclear destruction (a “fusion construct”). In one embodiment, the enzyme is a nuclear export deficient enzyme that initiates or enhances diversification. One representative example of an enzyme that initiates or enhances diversification is a deaminase. Deamination accelerates mutagenesis. In one embodiment, the construct comprises a first nucleotide sequence that expresses activation-induced cytosine deaminase (AID), wherein the AID is modified to prevent nuclear export; and a second nucleotide sequence that expresses chromatin licensing and DNA replication factor 1 (CDT1) or another polypeptide targeted for cell cycle-dependent nuclear destruction, wherein the second nucleotide sequence is operably linked to and downstream of the first nucleotide sequence. AID is a B cell-specific DNA deaminase that initiates Ig gene diversification.
Mutants that prevent AID nuclear export include, but are not limited to: AIDF198A(McBride et al. 2004. J Exp Med 199:1235); AID196X and other C-terminal deletion mutants that remove the nuclear export signal (see, e.g., Ito et al. 2004. PNAS 101: 1975); AIDF193A, F193E, F193H, L196A (Geisberger et al. 2009. PNAS 106:6736); and L198S (Patenaude et al. 2009, NSMB 16:17).
Fragments of other proteins that are targeted for nuclear destruction in specific phases of cell cycle can function analogously to the CDT1 tag (Sakaue-Sawano et al. 2008. Cell 132:487) that is exemplified herein to target proteolysis to a fusion protein. These include but are not limited to fragments from: Geminin (Sakaue-Sawano et al. 2008. Cell 132:487): S/G2-M restriction; RAG2 (Li et al. 1996. Immunity 5: 575): G1 restriction; and Cyclins.
AID has been fused to a variety of tags to regulate its stability or to visualize it by flow, microscopy, and western blotting. Representative examples of such tags, or fusion partners, include CDT1, GEM, mK02, mAG, GFP, mCherry and T7 tags. Fusion constructs of the invention may optionally include a tag to facilitate visualization, detection, or tracking.
Fusion constructs may generally be prepared using standard techniques. For example, DNA sequences encoding the peptide components may be assembled separately, and ligated into an appropriate expression vector. The ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements. The 3′ end of the DNA sequence encoding one peptide component is ligated, with or without a linker, to the 5′ end of a DNA sequence encoding the second peptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion protein that retains the biological activity of both component peptides. Additional fusion partners, or visualization tags, may be joined in a similar manner. Thus, a fusion construct of the invention optionally further comprises a detectable marker. In one embodiment, the detectable marker is a fluorescent protein.
A peptide linker sequence may be employed to separate the first and the second peptide components by a distance sufficient to ensure that each peptide folds into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional regions on the first and second peptides; and (3) the lack of hydrophobic or charged residues that might react with the peptide functional regions. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence.
The invention provides an adaptive immune cell, such as a B cell or a T cell. A typical example of a B cell for use in the invention is a Ramos human B cell. The B cell can be a human B cell, or a chicken B cell such as DT40, or other vertebrate B cell, or a B cell that has been humanized by replacement of endogenous IgH and IgL genes with human IgH and IgL genes. A typical example of a T cell for use with the invention is a chimeric antigen receptor (CAR) T cell. Candidate lymphocytes for use in the invention are those which can benefit from modulation of the affinity and/or specificity of the cell for its target.
The lymphocyte can be from any vertebrate species. In a typical embodiment, the lymphocyte is from a mammalian or avian species, and in one embodiment, the lymphocyte is a human B cell or human T cell. Other (non-lymphocyte) host cells are suitable for use with the invention as well. In one embodiment, the invention provides a yeast or bacterial cell transfected with the nucleic acid construct.
B cells are natural producers of antibodies, making them an attractive cell for production of both improved antibodies and improved non-immunoglobulin proteins and polypeptides. DT40 B cells are an effective starting point for evolving specific and high affinity antibodies by iterative cycles of hypermutation and selection (Cumbers et al., 2002; Seo et al., 2005). DT40 cells have several advantages over other vehicles tested for this purpose. DT40 constitutively diversifies its Ig genes in culture, and proliferates more rapidly than human B cell lines (10-12 hr generation time, compared to 24 hr); clonal populations can be readily isolated because cells are easily cloned by limiting dilution, without addition of special factors or feeder layers; and DT40 carries out efficient homologous gene targeting (Sale, 2004), so specific loci can be replaced at will allowing one to manipulate factors that regulate hypermutation.
The invention provides a novel platform for generating high affinity antibodies and other optimized polypeptides. In one embodiment, the vehicle for antibody evolution is a B cell line, DT40, which naturally produces antibodies, and which has been engineered to facilitate mutagenesis. Like other B cells, DT40 expresses antibodies on the cell surface, allowing convenient clonal selection for high affinity and optimized specificity, by fluorescence or magnetic-activated cell sorting. In the DT40 cell line, hypermutation is carried out by the same pathway that has been perfected over millions of years of vertebrate evolution to Ig gene hypermutation in a physiological context. This highly conserved pathway targets mutations preferentially (though not exclusively) to the complementarity-determining regions (CDRs), the subdomains of the variable (V) regions that make contact with antigen.
Thus far, the use of DT40 (and other cultured B cell lines) for antibody selection has been limited because the rate of hypermutation is very slow, about 0.1%-1% that of physiological hypermutation. To accelerate hypermutation, key regulatory sites and factors have been manipulated, taking advantage of our current sophisticated understanding of the molecular mechanisms of hypermutation.
Although chicken DT40 B cells offer many advantages, in some embodiments it may be desired to use human B cells. Alternatively, one can employ humanized Ig genes with the chicken DT40 B cells. By humanizing the DT40 immunoglobulin genes, the utility of this platform for therapeutics can be broadened, as the antibodies generated in the DT40 platform could be used directly for treatment.
There is ample documentation of the utility of humanized antibody genes, and a number of validated approaches for humanization, as reviewed recently (Waldmann and Morris, 2006; Almagro and Fransson, 2008). Humanization is effected by substitution of human Ig genes for the chicken Ig genes, and this is readily done in DT40 by taking advantage of the high efficiency of homologous gene targeting. The substitutions are designed to modify distinct parts of the heavy and light chain loci. Substitution could produce DT40 derivatives that generate entirely humanized antibodies, by swapping V(D)J and C regions; or chimeric antibodies (humanized C regions but not V regions). These replacements will not alter the adjacent cis-regulatory elements or affect their ability to accelerate hypermutation. The conserved mechanisms that promote hypermutation will target mutagenesis to the CDRs of humanized sequences. The humanized line can thus be used for accelerated development of human monoclonals in cell culture, providing a dual platform for rapid production of useful antibodies for either therapeutic or diagnostic purposes.
In addition, one can optimize antibody effector function by C region replacement. Antibody-based immunotherapy is a powerful approach for therapy, but this approach thus far been limited in part by availability of specific antibodies with useful effector properties (Hung et al., 2008; Liu et al., 2008). The constant (C) region of an antibody determines effector function. Substitutions of either native or engineered human C regions can be made by homologous gene targeting in the DT40 vehicle to generate antibodies with desired effector function.
Typically, the target gene comprises a promoter and a coding region. The coding region of the target gene in the lymphocyte of the invention can be one that encodes any protein or peptide of interest, and need not comprise a complete coding region. In some embodiments, a particular region or domain is targeted for diversification, and the coding region may optionally encode only a portion that includes the region or domain of interest.
In one embodiment, the target gene comprises an immunoglobulin (Ig) gene, wherein the Ig gene comprises an Ig gene enhancer and coding region. The Ig gene can be all or part of an IgL and/or IgH gene. The coding region can be native to the Ig gene, or a heterologous gene. In some embodiments, the target gene is or contains a non-Ig target domain for diversification, as well as domains permitting display of the gene product on the B cell surface, including a transmembrane domain and a cytoplasmic tail.
The invention provides a method of producing a repertoire of polypeptides having variant sequences of a polypeptide of interest. In one embodiment, the method comprises culturing a lymphocyte transfected with a nucleic acid construct of the invention in conditions that allow expression of the nucleic acid construct. The lymphocyte contains the coding region of the polypeptide of interest, thereby permitting diversification of the coding region. The method further comprises maintaining the culture under conditions that permit proliferation of the lymphocyte until a plurality of lymphocytes and the desired repertoire is obtained. In another embodiment, the invention provides a method of producing lymphocytes that produce an optimized polypeptide of interest.
In one embodiment, the method comprises culturing a lymphocyte transfected with a nucleic acid construct of the invention in conditions that allow expression of the nucleic acid construct, wherein the lymphocyte contains the coding region of the polypeptide of interest, and wherein and the lymphocyte expresses the polypeptide of interest on the surface of the lymphocyte. The method further comprises selecting cells from the culture that bind a ligand that specifically binds the polypeptide of interest expressed on the lymphocyte surface; and repeating these two steps until cells are selected that have a desired affinity and/or specificity for the ligand that specifically binds the polypeptide of interest. In one embodiment, the polypeptide of interest is an Ig. In a typical embodiment, the Ig is an IgL, IgH or both.
The invention provides a method of producing a repertoire of polypeptides having variant sequences of a polypeptide of interest via diversification of polynucleotide sequences that encode the polypeptide. The cell to be used in the method comprises both the nucleic acid construct of the invention and a nucleic acid encoding the polypeptide of interest. Typically, the method comprises culturing the cell of the invention in conditions that allow expression of the nucleic acids, wherein the target gene contains the coding region of the polypeptide of interest, thereby permitting diversification of the coding region. The method can further comprise maintaining the culture under conditions that permit proliferation of the cell until a plurality of variant polypeptides and the desired repertoire is obtained. The repertoire can then be used for selection of polypeptides having desired properties.
In embodiments in which the polypeptide of interest is an Ig, such as an IgL, IgH or both, the ligand may be a polypeptide, produced by recombinant or other means, that represents an antigen. The ligand can be bound to or linked to a solid support to facilitate selection, for example, by magnetic-activated cell selection (MACS). In another example, the ligand can be bound to or linked to a fluorescent tag, to allow for or fluorescence-activated cell sorting (FACS). Those skilled in the art appreciate that other methods of labeling and selecting cells are known and can be used in this method.
The invention also provides a vehicle for selection of T cell receptors. T cell-based immunotherapy has great potential (Blattman and Greenberg, 2004). T cell receptor specificity and affinity is governed by CDR contacts (Chlewicki et al., 2005). Selection for specificity or high affinity T cell receptors can be carried out in a DT40 vehicle, which has been modified by substitution of T cell receptors (V regions or entire genes) for the Ig loci; or directly in human T cells.
Production of catalytic Igs is another aspect of the invention. The Ig-related methods of the invention are not simply limited to the production of Igs for binding and recognition, as the target Ig could also be used for catalysis. After development of a stable molecule that mimics the transition state of an enzymatic reaction, DT40 cells can be used to evolve an antibody that binds and stabilizes the actual chemical transition state. After identifying clones that produce an Ig capable of binding the intermediate, the system can be used again to screen for catalytic activity of Igs on the real substrate in culture. Once some activity has been demonstrated in this system, optimization of activity can proceed by further evolution of the Ig loci through mutagenesis. Thus, invention does not require animal immunization (a slow step), immortalization by hybridoma technology, and the inefficiency of later having to screen hybridomas for antibodies that demonstrate catalytic activity.
The genomic structure at the Ig loci has evolved to promote mutagenesis of 1-1.5 kb downstream of the promoter. This system can be harnessed to mutate short regions of genes. Clonal selection based on surface protein expression can be incorporated by fusion of the region of interest to a portion of a gene expressing elements that mediate surface expression. Exemplary elements for surface expression include a signal peptide, transmembrane domain and cytoplasmic tail from a protein expressed on the B cell surface (Chou et al., 1999; Liao et al., 2001).
The invention can also be used for the production of recognition arrays. The ability to evolve cells harboring receptors with affinities for a large spectrum of antigens allows the development of recognition arrays. Combining this technology with intracellular responses/signaling from receptor stimulation in DT40 (such as measurement of Ca2+ by aequorin (Rider et al., 2003) or use of reporter gene transcription) would create a useful biosensor. Diversified clones would be spotted into arrays or 96 well plates, and exposed to samples. Each sample would yield a “fingerprint” of stimulation. The arrays would permit qualitative comparisons of biological/medical, environmental, and chemical samples. Analysis need not be limited to the analysis of proteins, as is the case for comparative techniques like 2D gels, since all forms of compounds could have antigenic properties. Furthermore, the arrays would lead to the identification of components without knowledge of their presence beforehand.
The invention additionally provides a method of restricting nuclear activity of a polypeptide to G1 or to S-G2/M phase of the cell cycle. In one embodiment, the method comprises restricting expression of an enzyme to G1 or to S-G2/M phase of the cell cycle in a host cell. In one embodiment, the enzyme whose expression or nuclear activity is restricted is AID. In one embodiment, the AID is a catalytically inactive derivative of AID. One example of a catalytically inactive variant of AID is AID H56A. Thus, a representative example of a fusion construct is one that encodes AIDH56A,F193A-CDT1. In another embodiment, the enzyme is CRISPR/Cas9 or CRISPR/Cas9D10A.
In one embodiment, the method comprises transfecting a host cell with a fusion construct comprising a nucleotide sequence that expresses the polypeptide fused to a nucleotide sequence that expresses CDT1 or geminin (GEM), wherein a fusion construct expressing CDT1 restricts expression of the enzyme to G1 and a fusion construct expressing GEM restricts expression of the enzyme to S/G2-M phase (Sakaue-Sawano et al. 2008. Cell 132:487).
Additional variations for restricting expression to particular phases of the cell cycle are contemplated. For example, fragments from RAG2 (Li et al. 1996. Immunity 5: 575) for G1 restriction; and Cyclins can be used for cell cycle restricted expression. In some embodiments, the nucleotide sequence that expresses CDT1 or GEM is positioned downstream of the nucleotide sequence that expresses the polypeptide whose nuclear activity is to be restricted.
For use in the methods described herein, kits are also within the scope of the invention. Such kits can comprise a carrier, package or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements (e.g., cells, constructs) to be used in the method.
Typically, the kit comprises a lymphocyte or other cell of the invention and one or more fusion constructs described herein. The kit further comprises one or more containers, with one or more fusion constructs stored in the containers. Each fusion construct comprises a polynucleotide that can be expressed in the cell. The kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In addition, a label can be provided on the container to indicate that the composition is used for a specific therapeutic or non-therapeutic application, and can also indicate directions for use. Directions and or other information can also be included on an insert which is included with the kit.
The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention.
This example illustrates features that support the invention, namely the means by which a diversification factor like AID can be modified to persist in the nucleus and also coupled with a nuclear destruction signal to protect cell viability. AID (Activation Induced Deaminase) deaminates cytosines in DNA to initiate immunoglobulin gene diversification and to reprogram the genome in early development. This example demonstrates how the cell cycle regulates AID and the cellular response to AID. Using high content screening microscopy to quantify subcellular localization, we show that AID undergoes nuclear degradation more slowly in G1 phase than in S or G2-M phase. Using CDT1 and GEM tags to promote degradation of nuclear AID in specific phases of cell cycle, we show that elevated nuclear AID accelerates somatic hypermutation and class switch recombination. Strikingly, nuclear AID is tolerated in G1 phase but compromises cell viability in other phases of cell cycle. These results establish that cell cycle regulates subcellular localization and nuclear stability of AID, and identify an unexpected connection between spatiotemporal regulation of AID and cell viability
AID levels are constant during cell cycle (31, 36), but several observations suggested that cell cycle may regulate AID. In DT40 chicken B cells, brief treatment with leptomycin B (LMB), an inhibitor of the CRM1-dependent nuclear export, increases nuclear AID signal in G1 phase cells (39); Polη, which copies donor DNA in AID-initiated gene conversion, co-localizes with the diversifying IgλR allele predominately in G1 phase (40); UNG2 removes uracils produced upon deamination by AID predominately in G1 phase (41); and RPA initially accumulates at Ig switch regions in G1 phase (42).
We have now asked if cell cycle regulates subcellular localization, stability or physiological activity of AID. We demonstrate that nuclear degradation occurs more slowly in G1 phase than in S-G2/M phase cells, and that the presence of AID in the nucleus in G1 phase accelerates SHM and CSR. Strikingly, elevated nuclear AID is tolerated in G1 phase, but it compromises fitness in other stages of cell cycle. These results establish that cell cycle regulates both nuclear AID and the ability of cells to respond to AID.
Nuclear AID is More Stable in G1 Phase than in S or G21M Phases.
We analyzed subcellular distribution of AID in the human B cell line, Ramos, transduced with a lentiviral construct expressing human AID fused to the mCherry fluorescent protein at the C-terminus. Ramos B cells express endogenous AID and actively diversify their Ig genes, so the pathways that regulate and respond to damage by AID are intact. Cells were analyzed by high content screening (HCS) microscopy (43), a flow-based approach that automatically quantifies signals per unit area (pixels) in each compartment of each cell (
Statistical tests were performed using two-tailed, unpaired Student's t-test, assuming unequal variances, for comparison of nuclear and cytoplasmic AID-mCherry signal and the N/C ratio between different treatment groups and between different times post treatment and untreated control in each treatment group.
We used HCS to quantify AID-mCherry subcellular distribution in Ramos B cells in each phase of cell cycle (
Statistical tests were performed using two-tailed, unpaired Student's t-test, assuming unequal variances, for comparison of nuclear and cytoplasmic AID-mCherry signal and the N/C ratio between G1 and S; G1 and G2/M; and S and G2/M at different times post-treatment in each treatment group.
Comparison of the slopes of the LMB response curves between the 1 and 2 hr time points (
Elevated Nuclear AID Compromises Viability of AID-mCherry-CDT1 Transductants.
With the goal of restricting the presence of AID-mCherry in the nucleus to G1 or S/G2-M phases, we fused AID-mCherry to tags derived from the CDT1 and GEM cell cycle regulators, which target a fused protein for destruction in the nucleus in S-G2/M phase (CDT1) or G1/early S phase (GEM) (44). Control experiments confirmed that, in Ramos B cells, these tags fused to monomeric Kusabira Orange 2 (mKO2) or monomeric Azami-Green (mAG) promoted nuclear localization and conferred the predicted cell cycle regulation: signals from mKO2-CDT1 or mAG-GEM were restricted to G1 phase or late G1/S-G2/M phase, respectively (
The number of cells (N) and the mean total, cytoplasmic, and nuclear mCherry signals are tabulated for Ramos AID-mCherry, AID-mCherry-CDT1, AID-mCherry-GEM, AIDF193A-mCherry, AIDF193A-mCherry-CDT1 and AIDF193A-mCherry-GEM transductants. Nuclear signals as determined by HCS were corrected for cytoplasmic baseline (see Materials and Methods). Statistical tests were performed using two-tailed, unpaired Student's t-test, assuming unequal variances for comparisons among transductant populations.
The nuclear localization of the AID-mCherry-CDT1 derivative could reflect more rapid nuclear import. However, while the nuclear signal and the ratio of nuclear to cytoplasmic signal (N/C) peaked more quickly in AID-mCherry-CDT1 than in AID-mCherry or AID-mCherry-GEM transductants following treatment with LMB (
HCS analysis also showed that AID-mCherry and AID-mCherry-GEM signals were exclusively cytoplasmic, independent of cell cycle (
The number of cells (N) and the mean total, cytoplasmic, and nuclear mCherry signals are tabulated for G1, S and G2/M cells in Ramos AID-mCherry, AID-mCherry-CDT1 and AID-mCherry-GEM, AIDF193A-mCherry, AIDF193A-mCherry-CDT1, AIDF193A-mCherry-GEM transductant populations. Nuclear signals as determined by HCS were corrected for cytoplasmic baseline (see Materials and Methods). Statistical tests were performed using two-tailed, unpaired Student's t-test, assuming unequal variances for comparisons among G1, S and G2/M phase cells in transductant populations.
AID-mCherry-CDT1 Reduced Viablity and Accelerated Ig Gene Diversification.
The distinctive spatiotemporal regulation of AID-mCherry, AID-mCherry-CDT1 and AID-mCherry-GEM allowed us to analyze the physiological consequences of nuclear AID at different stages of cell cycle. Strikingly, AID-mCherry-CDT1 transductants exhibited diminished cell viability relative to AID-mCherry or AID-mCherry-GEM transductants (
sIgM loss frequency was 7.9% in AID-mCherry transductants, 41.1% (p=0.003) in AID-mCherry-CDT1 transductants, and 6.5% in AID-mCherry-GEM transductants (
We assayed the effects of the tagged AID derivatives in a more physiological context by transducing primary murine B cells with AID-mCherry, AID-mCherry-CDT1 or AID-mCherry-GEM, and culturing in vitro with IL-4 and anti-CD40 to stimulate CSR. The mCherry signal in transduced primary B cells was too low for HCS analysis (
We sequenced IgVH regions amplified from single cells (
Elevated Nuclear AID is Tolerated in G1 Phase but not in S-G2/M Phase Cells.
The presence of a nuclear AID-mCherry-CDT1 signal in both G1 and S phase cells (
HCS analysis showed that total and cytoplasmic mCherry signals were significantly lower in AIDF193A-mCherry-CDT1 and AIDF193A-mCherry-GEM transductants than in AIDF193A-mCherry transductants, as predicted for tags that target the protein for nuclear degradation during a portion of cell cycle (
HCS documented persistent nuclear localization of AIDF193A-mCherry and AIDF193A-mCherry-GEM in all phases of cell cycle, while nuclear localization of AIDF193A-mCherry-CDT1 occurred exclusively in G1 phase (
The Ramos AIDF193A-mCherry, AIDF193A-mCherry-CDT1 and AIDF193A-mCherry-GEM transductants all exhibited greatly elevated sIgM loss rates (
AIDF193A-mCherry-CDT1 was distinguished by its ability to accelerate SHM without vastly compromising cell viability. This will make AIDF193A-mCherry-CDT1 a useful tool for accelerating mutagenesis in platforms designed to optimize evolution of antibodies and other targets.
We have shown that cell cycle regulates AID nuclear stability and the cellular response to AID. The role of cell cycle regulation of AID-initiated mutagenesis has previously been elusive. Although total AID levels had been found to remain constant during cell cycle (31, 36), evidence that AID-initiated DNA damage occurred in G1 phase (39-42) had suggested that temporal regulation might be important. We have distinguished nuclear from total AID levels, to demonstrate that AID is degraded in the nucleus more slowly in G1 than S-G2/M phases, and that G1 phase nuclear AID accelerates SHM and CSR, without compromising cell viability. Thus, G1 phase is the sweet spot for AID-initiated mutagenesis.
The unanticipated resilience of G1 phase cells to AID-initiated damage was especially evident in the contrast between the high viability of AIDF193A-mCherry-CDT1 transductants, in which AID is in the nucleus only in G1 phase, and the poor viability of AIDF193A-mCherry and AIDF193A-mCherry-GEM transductants, in which AID is in the nucleus outside G1 phase (
The GEM tag was predicted to restrict nuclear protein to S-G2/M phase, but there was no nuclear AID-mCherry-GEM signal in any stage of cell cycle. Nuclear AID is degraded more slowly in G1 than S or G2/M phase (
AIDF193A-mCherry-GEM accumulated in the nucleus during S-G2/M phase, while AID-mCherry-GEM did not (compare
The CDT1 tag destabilizes nuclear protein outside G1 phase (44) and would not be predicted to increase nuclear levels at any stage of cell cycle. Nonetheless, AID-mCherry-CDT1 nuclear signal exceeded that of AID-mCherry (
The CDT1 and GEM tags somewhat altered the spectrum of SHM. A reduced frequency of mutations at A and T was evident in AID-mCherry-CDT1 (6.8%) and AID-mCherry-GEM (8.4%) relative to AID-mCherry transductants (17.9%;
The use of CDT1 and GEM tags to destabilize nuclear protein outside specific windows of cell cycle (44) proved unexpected insights into regulation of AID and the response to AID-initiated DNA damage. These tags can be readily applied to study repair in other contexts, and they should also prove useful for optimizing the nucleases (CRISPR/Cas9, TALENs, etc.) that target nicks and double-strand breaks for genome engineering and gene correction applications. The utility of these tags is especially evident in the AIDF193A-mCherry-CDT1 derivative. AIDF193A-mCherry-CDT1 expression greatly accelerates hypermutation, but without the negative impact on cell proliferation associated with other AID derivatives that increase the frequency of SHM but compromise cell viability, including AID mutants selected for increased deamination activity (24); NES mutants (36, 37); and the naturally occurring human AIDΔE5 dominant negative mutant, which exhibits increased hypermutation activity coupled with diminished cell viability (38). AIDF193A-mCherry-CDT1 should prove to be useful for defining the mechanisms that protect the genome from AID-initiated DNA damage in G1 phase, and in very practical applications directed toward evolving or optimizing antibodies and other proteins.
Expression Constructs.
The pEGFP-N3 construct for expression of AID-GFP was a gift from Dr. Javier Di Noia (Department of Microbiology and Immunology, University of Montreal, Montreal, Quebec, Canada). We substituted mCherry for a region of GFP flanked by ApaI and BsrGI restriction sites in the pEGFP-N3 construct to generate an AID-mCherry expression construct, pAID-mCh. Cell cycle reporter constructs p-mKO2-CDT1 CSII and p-mAG-GEM CSII, in a lentiviral vector, were a gift from Dr. Atsushi Miyawaki (Brain Science Institute, RIKEN, Hirosawa, Wako-city, Saitama 351-0198, Japan).
pAID-mCh CSII: We amplified AID-mCherry from pAID-mCh with primers PQL31, 5′-ATATCAATTGAGATCCCAAATGGACAGCC-3′ (SEQ ID NO: 7) and PQL32, 5′-ATATTCTAGATTACTTGTACAGCTCGTCCATGC-3′, (SEQ ID NO: 8) and inserted it between EcoRI and XbaI sites in p-mAG-GEM CSII, thereby replacing mAG-GEM with AID-mCherry.
pAID-mCh-CDT1 and pAID-mCh-GEM: We amplified CDT1 with primers PQL44 5′-TATATGTACAAGGGATATCCATCACACTGGCGGCC-3′ (SEQ ID NO: 9) and PQL45 5′-TATATGTACATCTAGATTAGATGGTGTCCTGGTCC-3′ (SEQ ID NO: 10) from p-mKO2-CDT1 CSII, and GEM with primers PQL44 5′-TATATGTACAAGGGATATCCATCACACTGGCGGCC-3′ (SEQ ID NO: 9) and PQL46 5′-TATATGTACATCTAGATTACAGCGCCTTTCTCCG-3′ (SEQ ID NO: 11) from p-mAG-GEM CSII, and inserted the resulting fragments between BsrGI and XbaI restriction sites of pAID-mCh CSII.
pAID-mKO2-CDT1 and pAID-mKO2-GEM: We amplified mKO2 with primers mKO2 FOR 5′-ATATGGATCCATCGCCACCATGGTGAGTGTG-3′ (SEQ ID NO: 12) and mKO2 REV 5′-ATATGCGGCCGCCAGTGTGATGGATATCCGC-3′ (SEQ ID NO: 13), and inserted the resulting fragment between BamHI and NotI restriction sites in pAID-mCh-CDT1 or pAID-mCh-GEM CSII, respectively.
pAIDF193A-mCh-CDT1, pAIDF193A-mCh-CDT1 and pAIDF193A-mCh-GEM: F193A mutants were generated using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent) with primer set, F193A FOR 5′-CTTACGAGACGCAGCTCGTACTTTGGGAC-3′ (SEQ ID NO: 14) and F193A REV 5′-GTCCCAAAGTACGAGCTGCGTCTCGTAAG-3′(SEQ ID NO: 15).
Cell Culture and Transduction.
The human Burkitt lymphoma cell line, Ramos, was cultured in supplemented RPMI 1640 (Gibco), which contained 10% FBS, 2 mM L-glutamine, penicillin/streptomycin, 1× non-essential amino acids (Gibco), 1 mM sodium pyruvate, and 10 mM HEPES. Lentiviral transductions used 2×105 cells cultured in medium containing 8 μg/ml of polybrene. Following transduction, cells were cultured for 3-4 days and these recent transductants then sorted for mCherry+ to enrich for transduced cells, typically constituting 0.1-10% of the population. Cells were treated with leptomycin B (LMB; LC Laboratories) at 50 ng/ml and MG132 (Z-Leu-Leu-Leu-aldehyde; Sigma-Aldrich) at 50 μM. Viable cells were counted after trypan blue staining. Cell viability was confirmed by CellTiter-Glo® Luminescent Cell Viability Assay (Promega).
Assays of Cell Cycle.
To determine cell cycle distribution, cells were fixed, permeabilized with 0.5% Triton X-100, stained with DAPI (2 μg/ml) and analyzed by FACS.
High Content Screening (HCS) Microscopy and Analysis.
Cells were fixed in 3.7% formaldehyde at a density of 2×106 cells/ml and stained with whole cell stain (HCS CellMask, Invitrogen) and DAPI (0.2 μg/ml). Fixed cells were then washed, resuspended in PBS and spun down in a 96-well μclear microplate (Greiner Bio One) for imaging. Cells were imaged by Thermo Scientific ArrayScan VTI HCS reader, analyzing 3000-6000 cells in each treatment group. Cells with very low or very high mCherry signals were eliminated, gating based on the mock transduction control (low) and eliminating cells with signals more than 5 SD from the mean (high). The HCS Colocalization BioApplication protocol was used to determine nuclear and whole cell boundaries in individual cells as defined by DAPI and HCS CellMask, respectively, thereby defining the cytoplasmic region as the region between nuclear and whole cell boundaries. The average signal in the nuclear and cytoplasmic compartments was determined in individual cells by measuring the total intensity of mCherry signal divided by area within each compartment. The ratio of nuclear to cytoplasmic signal (N/C) was calculated as the ratio of the average signals of nuclear and cytoplasmic mCherry.
Confocal microscopy showed that AID-mCherry was mostly absent from the nucleus when out-of-focus signal was eliminated, regardless of the level of cytoplasmic signal (
G1, S, and G2/M phase cells were distinguished by ranking DNA content as determined by total DAPI signal, and specific fractions of the population assigned to G1, S and G2/M phases (
Assays of sIgM Loss Frequency in Ramos B Cell Transductants.
sIgM loss frequency provides a convenient surrogate assay for SHM (45, 46). To determine fractions of sIgM− cells, 2-5×105 cells were fixed in 3.7% formaldehyde and stained with anti-human IgM (1:500, Souther Biotech), and sIgM− variants quantified by FACS as described (47). To establish that selective pressure was not sufficient to affect the frequency of sIgM loss, we assayed loss of mCherry signal posttransduction (
Assay of CSR in Primary Splenic B Cells.
B cells were isolated from spleens of C57BL/6 mice and enriched through a negative selection in AUTOMACs with biotinylated anti-CD43 antibody (BD Pharmigen, Cat #5532269) and streptavidin magnetic microbeads (Miltenyi Biotech, Cat #130-048-102). Purified B cells were transduced for 24 hr in X-vivo medium (Lonza) containing 2 mM L-glutamine, 50 μM β-mercaptoethanol, 5 ng/mL IL-4 (R&D Systems, cat#404-ML-010) and 1 μg/mL anti-CD40 antibody (BioLegend, Cat#102802) in 100 μL total volume in a round bottom 96-well plate, then transferred at 24 hr to supplemented RPMI (see above) containing 5 ng/mL IL-4 and 1 μg/mL anti-CD40 antibody. Cells were cultured for 4-5 days, stained with anti-IgG1 (FITC anti-mouse IgG1; BioLegend, Cat#406605), and surface IgG1 quantified by flow-cytometry.
Single-Cell PCR and Sequencing of VH Regions.
At day 7 post sorting recent transductants for mCherry+ cells, single cells from AID-mCherry, AID-mCherry-CDT1 or AID-mCherry-GEM transductant populations were aliquoted, one cell per well, into 96-well plates containing 20 μl of Pfu reaction buffer (Agilent). Samples were frozen, thawed, and treated with 250 μg/ml proteinase K for 1 hr at 50° C. then 5 min at 95° C., the primers and high-fidelity Pfu Turbo DNA polymerase (Agilent) were added and the rearranged VH region amplified by nested PCR with first round primers, RVHFOR QL 5′-TCCCAGGTGCAGCTACAGCAG-3′ (SEQ ID NO: 16) and JOL48 QL 5′-GTACCTGAGGAGACGGTGACC-3′ (SEQ ID NO: 17) (52); followed by 1:30 dilution and second round amplification with primers 5′-AGGTGCAGCTACAGCAGTG-3′ (SEQ ID NO: 18) and 5′-GCCCCAGACGTCCATACC-3′ (SEQ ID NO: 19). Predicted sizes of PCR products were confirmed by gel electrophoresis and fragments purified and sequenced.
Cell Culture and Transduction.
Ramos B cells were transduced in medium containing polybrene, cultured for 3-4 days, then sorted for mCherry+ to enrich for transduced cells, typically constituting 0.1-10% of the population. Primary murine B cells were transduced in supplemented X-vivo medium, then cultured 4-5 days with IL-4 and anti-CD40, and the fraction of IgG1+ cells quantified.
High Content Screening (HCS) Microscopy.
Cells were fixed and stained with whole cell stain (HCS CellMask, Invitrogen) and DAPI, washed, and imaged by Thermo Scientific ArrayScan VTI HCS reader, analyzing 3000-6000 cells in each treatment group. To enable accurate comparisons among different transductant populations, nuclear signal for each cell was corrected by subtraction of the corresponding baseline value, as established by linear regression analysis. HCS results were expressed in terms of average signal, to ensure independence of cell size.
This example illustrates an embodiment of the invention that implements the principles described above for use with B cells to T cells. More specifically, one can use the invention described herein to modulate and optimize chimeric antigen receptor (CAR) T cells for use in therapeutic treatments. One can modulate and improve the affinity or specificity of a CAR T cell by transfecting a host T cell with a fusion construct of the invention. The fusion construct would couple a fragment of a protein targeted for nuclear destruction during a relevant portion of the cell cycle (e.g., CDT1 for destruction upon entry into S phase; GEM for G1 phase destruction) with AID modified to promote accumulation of AID in the nucleus. This construct stimulates diversification of the target gene to be optimized for immunotherapeutic use.
This example illustrates an embodiment of the invention, whereby cell cycle tags derived from CDT1 or GEM (or other proteins involved in cell cycle control) can confer cell cycle restriction to enzymes that function in the nucleus. This modulation of nuclear protein activity can be of use, for example, in genome engineering. The nuclease activities of enzymes used to target DNA and the pathways of downstream repair can reflect the stage of cell cycle in which the DSB or nick occurs. For example, the frequency of a desired outcome (e.g. homology-directed repair) would be higher if DNA is cleaved in G1 phase, by an enzyme bearing a CDT1 tag; or the frequency of an undesired outcome (mutagenic end-joining) would be lower if DNA is cleaved in S phase, by an enzyme bearing a GEM tag.
Two enzymes widely used for genome engineering are CRISPR/Cas9, which creates targeted double-strand breaks (DSBs); and the CRISPR/Cas9D10A nickase, which creates targeted single-strand breaks (nicks). This can be implemented by using standard cloning approaches to generate constructs that express Cas9-CDT1 and Cas9-GEM or Cas9D10A-CDT1 and Cas9D10A-GEM fusion proteins. These fusion proteins will be expressed upon transfection of cultured cells, and predicted cell cycle regulation confirmed by flow cytometry. Frequencies of homology-directed repair, targeted deletions and mutagenic end-joining can be measured, using standard published approaches (e.g. Davis and Maizels, PNAS, 111(10):E924-32, 2014). Comparison of these frequencies can be used to identify optimum stages of cell cycle (and corresponding fusion proteins) for genome engineering.
Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention pertains.
From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.
This application claims benefit of U.S. provisional patent application Nos. 61/951,312, filed Mar. 11, 2014, and 62/094,260, filed Dec. 19, 2014, the entire contents of each of which are incorporated by reference into this application.
This invention was made with U.S. government support under R01 GM041712, awarded by the National Institutes of Health. The U.S. government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US15/19990 | 3/11/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62094260 | Dec 2014 | US | |
| 61951312 | Mar 2014 | US |
