Patent Application
20250154494
T lymphocytes comprise approximately 20% of all leukocytes in human peripheral blood and are a major component of the adaptive immune system. A unique feature of each newly formed T cell is its T-cell receptor (TCR), which is formed during T cell genesis in the bone marrow and thymus via somatic recombination of genomic elements. As such, each TCR has a unique ligand-binding site, forming a receptor repertoire with the potential to bind up to 1022 distinct ligands. In order to aid in the diagnosis, prevention, and treatment of diseases and disorders such as diseases associated with pathogen infection, autoimmunity disorders, and cancer, it is crucial to determine functional relationships between TCRs and their ligands.
In droplet-based microfluidics, cells can be physically isolated from each other in aqueous droplets emulsified in immiscible oil. This offers the major advantage of culture conditions with a controlled extracellular environment and the prevention of cross-contamination on a microscale level. Droplet-based microfluidics have been applied to a variety of biological experimental systems, including mammalian cells.
In high-throughput cellular interaction screening, microfluidic co-encapsulation can be used to bring together small numbers of cells and screen in parallel for millions of potential interactions, owing to the high frequency of droplet generation, enabling the formation of millions of droplets per hour. This is performed in a controlled fashion, e.g. one-to-one droplet encapsulation of T cells and antigen-presenting cells (APCs), such as B cells. For the generation of monodisperse droplets with two cell types in a two-phase system (aqueous phase droplets in an oil phase), microfluidic designs have been described in the literature (Mazutis L et al., Nature protocols. 2013 May; 8(5):870-91). These generally consist of two cell inlets containing filter regions and a fluid resistor area, both of which converge with a fluorinated oil-based detergent phase at a T-junction nozzle. This facilitates the formation of micelle-based, cell-containing droplets.
Being able to perform high-throughput screening for millions of potential cell interactions necessitates a microfluidic device that allows droplet encapsulation for several hours. Using existing designs, droplet encapsulation of T cells with APCs could only be maintained for up to 15 min, as the filters of the aqueous inlets clog due to the tendency of suspension immune cells to aggregate together and form clumps, even when in motion. Additionally, droplet encapsulation of cells is a random process; that is, the discrete cellular encapsulation frequencies are distributed according to the Poisson distribution. Even when cells are optimized for single cell conditions, working with an average cell density of one cell per droplet volume, this results in a single-cell encapsulation rate of approximately 30% (for each cell type, when applicable).
There is thus a need in the art for art for improved microfluidic devices that reliably co-encapsulate single cells in an evenly distributed manner for prolonged periods of time without clogging as well as for a platform to rapidly deconvolute antigen:TCR interactions. The present invention addresses these unmet needs in the art.
In one embodiment, the invention relates to a system for identifying an antigen as a ligand for binding by a TCR comprising:
In one embodiment, the device comprises a microfluidic co-culture device comprising two inlets.
In one embodiment, the APC library is in a solution comprising EDTA.
In one embodiment, the at least one cell is in a solution comprising Ca2+ In one embodiment, the at least one cell is a T cell.
In one embodiment, the invention relates to a method of identifying an antigen as a ligand for binding by a TCR, the method comprising:
In one embodiment, the nucleotide sequence encoding at least one antigenic polypeptide for presentation further comprises a unique nucleotide barcode sequence encoding the same amino acid sequence which is shared by each member of the minigene library.
In one embodiment, the method of co-culturing comprises applying the at least one cell comprising at least one TCR to a first inlet of a microfluidic co-culture device comprising two inlets and applying the APC library to a second inlet of the microfluidic co-culture device.
In one embodiment, the APC library is applied to the microfluidic co-culture device in a solution comprising EDTA.
In one embodiment, the at least one cell comprising at least one TCR is applied to the microfluidic co-culture device in a solution comprising Ca2+.
In one embodiment, the TCR responsive promoter is activated by NFAT.
In one embodiment, the TCR responsive promoter comprises a sequence selected from the group consisting of SEQ ID NO:50-SEQ ID NO:74. In one embodiment, the expression cassette is under the control of at least one, at least two, at least three, at least four, or more than four copies of the TCR responsive promoter comprising a sequence as set forth in SEQ ID NO:50 (NBV promoter).
In one embodiment, the expression cassette comprises a nucleotide sequence encoding at least one protein selected from the group consisting of a fluorescent marker and an antibody, or fragment thereof, specific for binding to an APC marker. In one embodiment, the APC is a B cell, and wherein the APC marker is selected from the group consisting of CD19, CD20, CD38, CD40, CD45R, CD79a or CD79b. In one embodiment, the sequence encoding the antibody or fragment thereof specific for binding to the APC marker further comprises at least one selected from the group consisting of a linker sequence, a leader sequence and a tag.
In one embodiment, the TCR responsive promoter is operably linked to a nucleic acid molecule encoding at least two tandem scFv molecules for binding to an APC marker, wherein each of the at least two tandem scFv molecules is separated by a linker sequence. In one embodiment, the cell comprising the TCR promoter further comprises a nucleic acid molecule comprising a nucleotide sequence encoding an NBV transcription factor for inducing transcription at an NBV promoter comprising the nucleotide sequence of SEQ ID NO:50, wherein the NBV transcription factor comprises a fusion of
In one embodiment, the NBV transcription factor comprises an amino acid sequence as set forth in SEQ ID NO:76. In one embodiment, the nucleotide sequence is set forth in SEQ ID NO:75.
In one embodiment, the sequence encoding the NBV transcription factor is operably linked to a TCR responsive promoter. In one embodiment, the sequence encoding the NBV transcription factor is further regulated by at least one insulator element, an enhancer, or a combination thereof.
In one embodiment, the cell comprising the TCR responsive promoter further comprises at least one nucleotide sequence encoding a T cell co-stimulatory molecule. In one embodiment, the T cell co-stimulatory molecule is selected from the group consisting of CD2, CD226, CD40L, ICOS, OX40 and 41BB.
In one embodiment, the nucleic acid molecule of the minigene library further comprises a sequence encoding at least one transmembrane domain, or fragment thereof. In one embodiment, the transmembrane domain is SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO: 128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, or SEQ ID NO:135.
In one embodiment, the nucleic acid molecule of the minigene library further comprises a nucleotide sequence encoding an 6X flexible Flag construct. In one embodiment, the nucleic acid molecule further comprises a nucleotide sequence of SEQ ID NO:42.
In one embodiment, the nucleic acid molecule of the minigene library further comprises a nucleotide sequence encoding at least one T cell co-stimulatory molecule. In one embodiment, the T cell co-stimulatory molecule is selected from the group consisting of CD40, CD58, CD80, CD83, CD86, OX40L, 4-1BBL, and a combination thereof.
In one embodiment, the invention relates to a nucleic acid molecule encoding an antibody or fragment thereof specific for binding to an antigen presenting cell (APC) marker. In some embodiments, the APC marker is CD19 or CD20.
In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain variable region of an anti-CD19 synthetic antibody. In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the anti-CD19 synthetic antibody is SEQ ID NO:4 or SEQ ID NO:12.
In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a light chain variable region of an anti-CD19 synthetic antibody. In some embodiments, the nucleotide sequence encoding the light chain variable region of the anti-CD19 synthetic antibody is SEQ ID NO:8 or SEQ ID NO:16.
In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain variable region of an anti-CD20 synthetic antibody. In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the anti-CD20 synthetic antibody is SEQ ID NO:20 or SEQ ID NO:28.
In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a light chain variable region of an anti-CD20 synthetic antibody. In some embodiments, the nucleotide sequence encoding the light chain variable region of the anti-CD20 synthetic antibody is SEQ ID NO:24 or SEQ ID NO:32.
In some embodiments, the nucleic acid molecule encodes an scFV antibody fragment. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain variable region of an anti-CD19 synthetic antibody comprising SEQ ID NO:4 and a light chain variable region of an anti-CD19 synthetic antibody comprising SEQ ID NO:8. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain variable region of an anti-CD19 synthetic antibody comprising SEQ ID NO:12 and a light chain variable region of an anti-CD19 synthetic antibody comprising SEQ ID NO:16. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain variable region of an anti-CD20 synthetic antibody comprising SEQ ID NO:20 and a light chain variable region of an anti-CD20 synthetic antibody comprising SEQ ID NO:24. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a heavy chain variable region of an anti-CD20 synthetic antibody comprising SEQ ID NO:28 and a light chain variable region of an anti-CD20 synthetic antibody comprising SEQ ID NO:32.
In some embodiments, the sequence encoding the antibody or fragment thereof specific for binding to an antigen presenting cell marker further comprises a linker sequence, a leader sequence, a tag, or a combination thereof.
In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding at least two tandem scFv molecules separated by a linker sequence.
In one embodiment, the invention relates to a nucleic acid molecule comprising an expression cassette for expression under the control of a T-cell receptor (TCR) responsive promoter that is activated by a molecule that is expressed upon binding of a TCR to its ligand. In some embodiments, the ligand is a polypeptide:major histocompatibility complex (MHC) complex. In some embodiments, the ligand is a polypeptide:human leukocyte antigen (HLA) complex.
In one embodiment, the TCR responsive promoter is activated by NFAT. In one embodiment, the TCR responsive promoter comprises a sequence of SEQ ID NO:50-SEQ ID NO:74. In one embodiment, the TCR responsive promoter comprises a promoter comprising the sequence of SEQ ID NO:50, which is referred to elsewhere hereinafter as the “NBV promoter”.
In one embodiment, the nucleic acid molecule comprising an expression cassette for expression under the control of a TCR responsive promoter that is activated by a molecule that is expressed, or activated, upon binding of a TCR to an antigenic polypeptide, comprises a TCR responsive promoter operably linked to a nucleic acid molecule encoding an antibody or fragment thereof specific for binding to an APC marker.
In one embodiment, the APC is a B cell, dendritic cell (DC), monocyte, macrophage, or engineered APC. In one embodiment, the APC is a B cell. In one embodiment, the APC marker is CD19, CD20, CD38, CD40, CD45R, CD79a or CD79b.
In one embodiment, the nucleic acid molecule comprises an NBV promoter comprising a sequence as set forth in SEQ ID NO:50 operably linked to a nucleotide sequence encoding a heavy chain variable region of an anti-CD19 synthetic antibody. In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the anti-CD19 synthetic antibody is SEQ ID NO:4 or SEQ ID NO:12.
In one embodiment, the nucleic acid molecule comprises an NBV promoter comprising a sequence as set forth in SEQ ID NO:50 operably linked to a nucleotide sequence encoding a light chain variable region of an anti-CD19 synthetic antibody. In some embodiments, the nucleotide sequence encoding the light chain variable region of the anti-CD19 synthetic antibody is SEQ ID NO:8 or SEQ ID NO:16.
In one embodiment, the nucleic acid molecule comprises an NBV promoter comprising a sequence as set forth in SEQ ID NO:50 operably linked to a nucleotide sequence encoding a heavy chain variable region of an anti-CD20 synthetic antibody. In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the anti-CD20 synthetic antibody is SEQ ID NO:20 or SEQ ID NO:28.
In one embodiment, the nucleic acid molecule comprises an NBV promoter comprising a sequence as set forth in SEQ ID NO:50 operably linked to a nucleotide sequence encoding a light chain variable region of an anti-CD20 synthetic antibody. In some embodiments, the nucleotide sequence encoding the light chain variable region of the anti-CD20 synthetic antibody is SEQ ID NO:24 or SEQ ID NO:32.
In one embodiment, the nucleic acid molecule comprises an NBV promoter comprising a sequence as set forth in SEQ ID NO:50 operably linked to a nucleotide sequence encoding a heavy chain variable region of an anti-CD19 synthetic antibody comprising SEQ ID NO:4 and a light chain variable region of an anti-CD19 synthetic antibody comprising SEQ ID NO:8.
In one embodiment, the nucleic acid molecule comprises an NBV promoter comprising a sequence as set forth in SEQ ID NO:50 operably linked to a nucleotide sequence encoding a heavy chain variable region of an anti-CD19 synthetic antibody comprising SEQ ID NO:12 and a light chain variable region of an anti-CD19 synthetic antibody comprising SEQ ID NO:16.
In one embodiment, the nucleic acid molecule comprises an NBV promoter comprising a sequence as set forth in SEQ ID NO:50 operably linked to a nucleotide sequence encoding a heavy chain variable region of an anti-CD20 synthetic antibody comprising SEQ ID NO:20 and a light chain variable region of an anti-CD20 synthetic antibody comprising SEQ ID NO:24.
In one embodiment, the nucleic acid molecule comprises an NBV promoter comprising a sequence as set forth in SEQ ID NO:50 operably linked to a nucleotide sequence encoding a heavy chain variable region of an anti-CD20 synthetic antibody comprising SEQ ID NO:28 and a light chain variable region of an anti-CD20 synthetic antibody comprising SEQ ID NO:32.
In one embodiment, the sequence encoding the antibody or fragment thereof specific for binding to the APC marker further comprises at least one of a linker sequence, a leader sequence, a tag, or a combination thereof.
In one embodiment, the nucleic acid molecule comprises a TCR responsive promoter operably linked to a nucleic acid molecule encoding at least two tandem scFv molecules for binding to an APC marker, wherein each of the at least two tandem scFv molecules is separated by a linker sequence.
In one embodiment, the invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding an NBV transcription factor for inducing transcription at an NBV promoter, wherein the NBV transcription factor comprises a fusion of a) the cytoplasmic retention and DNA-binding domains from the N′-terminus of the nuclear factor in activated T cells (NFAT), b) the octamer motif (‘ATGCAAAT’)-binding domain from the transcriptional co-activator Bob1, and c) the C′-terminal transactivation domain (TAD) from the herpesvirus VP16 protein.
In one embodiment, the NBV transcription factor comprises an amino acid sequence as set forth in SEQ ID NO:76.
In one embodiment, the nucleic acid molecule encoding an NBV transcription factor for inducing transcription at an NBV promoter comprises a nucleotide sequence as set forth in SEQ ID NO:75. In one embodiment, the sequence encoding the NBV transcription factor is operably linked to a TCR responsive promoter. In one embodiment, the sequence encoding the NBV transcription factor is further regulated by at least one insulator element, an enhancer, or a combination thereof.
In one embodiment, the invention relates to a cell comprising at least one nucleic acid molecule comprising an expression cassette for expression under the control of a T cell receptor (TCR) responsive promoter that is activated by a molecule that is expressed upon binding of a TCR to an antigenic polypeptide. In one embodiment, the cell further comprises at least one nucleic acid molecule comprising a nucleotide sequence encoding an NBV transcription factor for inducing transcription at an NBV promoter, wherein the NBV transcription factor comprises a fusion of a) the cytoplasmic retention and DNA-binding domains from the N′-terminus of the nuclear factor in activated T cells (NFAT), b) the octamer motif (‘ATGCAAAT’)-binding domain from the transcriptional co-activator Bob1, and c) the C′-terminal transactivation domain (TAD) from the herpesvirus VP16 protein.
In one embodiment, the cell further comprises at least one nucleotide sequence encoding a co-stimulatory molecule. In one embodiment, the co-stimulatory molecule is CD2, CD226, CD40L, ICOS, OX40 or 41BB, or any combination thereof.
In one embodiment, the invention relates to a minigene library comprising a plurality of nucleic acid molecules wherein each nucleic acid molecule comprises a nucleotide sequence encoding at least one antigenic polypeptide for presentation, wherein the sequence encoding the at least one antigenic polypeptide is of a predetermined length.
In one embodiment, each nucleic acid molecule further comprises a unique nucleotide barcode sequence encoding the same amino acid sequence which is shared by each member of the minigene library.
In some embodiments, the nucleic acid molecule encoding the minigene antigen expression cassette further comprises a sequence encoding at least one transmembrane domain for class II antigen presentation, or fragment thereof. In some embodiments, the nucleic acid molecule encoding the minigene antigen expression cassette further comprises SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, or SEQ ID NO:135.
In one embodiment, the nucleic acid molecule further comprises a nucleotide sequence encoding a 6× flexible Flag construct. In one embodiment, each nucleic acid molecule further comprises a nucleotide sequence of SEQ ID NO:42.
In one embodiment, each nucleic acid molecule further comprises a nucleotide sequence encoding at least one co-stimulatory molecule. In one embodiment, the co-stimulatory molecule is CD40, CD58, CD80, CD83, CD86, OX40L, 4-1BBL, or a combination thereof. In one embodiment, each nucleic acid molecule comprises a nucleotide sequence of SEQ ID NO:43-SEQ ID NO:49.
In one embodiment, the invention relates to an APC library comprising a plurality of APCs, wherein each APC comprises a nucleic acid molecule of a minigene library comprising a plurality of nucleic acid molecules wherein each nucleic acid molecule comprises a nucleotide sequence encoding at least one antigenic polypeptide for presentation, wherein the sequence encoding the at least one antigenic polypeptide is of a predetermined length. In one embodiment, each nucleic acid molecule further comprises a unique nucleotide barcode sequence encoding the same amino acid sequence which is shared by each member of the minigene library.
In one embodiment, the APC is a B cell, dendritic cell (DC), monocyte or macrophage. In one embodiment, the APCs are B cells. In one embodiment, the APCs are immortalized patient-derived B cells.
In one aspect, the present invention relates to a microfluidic device for co-encapsulation of individual cells, comprising: two or more proximal inlets fluidly connected to one or more distal outlets; a filter positioned downstream from each proximal inlet; a series of asymmetrical focusing loops positioned downstream from each filter; a nozzle positioned upstream from the one or more distal outlets; and one or more isolation fluid inlets connected to the nozzle; wherein each of the series of asymmetrical focusing loops comprise arcuate channel lengths curving in alternating directions about a central axis, such that arcuate channel lengths on a first side of the central axis are larger than arcuate channel lengths on an opposing second side of the central axis; and wherein each of the series of asymmetrical focusing loops converge into a single microchannel fluidly connected to the nozzle.
In one embodiment, a channel fluidly connecting each proximal inlet to a filter has a width that gradually expands to a width between about 10 μm and 10000 μm. In one embodiment, a channel fluidly connecting each proximal inlet to a filter comprises one or more stream separators, wherein each stream separator is an elongated barrier substantially in parallel alignment with the channel and is configured to evenly distribute fluid flow from each proximal inlet.
In one embodiment, the filter comprises a plurality of pillars spaced apart by a distance between about 50 μm and 5000 μm. In one embodiment, increasing spacing between pillars is configured to filter particles while minimizing clogging occurrences. In one embodiment, each pillar has a cross-sectional shape selected from the group consisting of: a circle, an oval, a triangle, a square, a rectangle, a diamond, a polygon, a V-shape, and a U-shape. In one embodiment, each pillar has a width between about 1 μm and 100 μm.
In one embodiment, each of the series of asymmetrical focusing loops has an overall wave-like shape. In one embodiment, each of the series of asymmetrical focusing loops is configured to encourage a flow of particles to evenly space apart both laterally and longitudinally within a flow of fluid.
In one embodiment, the arcuate channel lengths on the first side and the second side have equal widths. In one embodiment, the arcuate channel lengths on the first side have a width between about 50 μm and 10000 μm. In one embodiment, the arcuate channel lengths on the second side have a width between about 20 μm and 5000 μm. In one embodiment, the arcuate channel lengths on the first side and the second side comprise a curvature defined by a degree of a substantially circular path. In one embodiment, the degree is between about 5° and 355°. In one embodiment, the degree is about 180°. In one embodiment, the arcuate channel lengths on the first side and the second side comprise a curvature defined by a diameter of a substantially circular path. In one embodiment, the diameter is between about 20 μm and 1000 μm. In one embodiment, the diameter is about 110 μm for the arcuate channel lengths on the first side. In one embodiment, the diameter is about 50 μm for the arcuate channel lengths on the second side.
In one embodiment, a channel fluidly connects each of the series of asymmetrical focusing loops to the single microchannel fluidly connected to the nozzle. In one embodiment, the channel has a tapered width. In one embodiment, a channel fluidly connects the nozzle with each of the one or more outlets. In one embodiment, the channel has an expanded width. In one embodiment, the channel is aligned in-line with the single microchannel. In one embodiment, two channels fluidly connect each of the isolation fluid inlets to the nozzle. In one embodiment, the two channels fluidly connect on opposing sides of the nozzle.
In one embodiment, the device further comprises one or more additional inlets fluidly connected directly to or to a position between one or more of the proximal inlets, filters, focusing loops, nozzle, and outlet. In one embodiment, the device further comprises one or more flow modulators positioned between one or more of the proximal inlets, filters, focusing loops, nozzle, and outlet, wherein the one or more flow modulators is selected from the group consisting of: valves, fluid resistors, expandable elements, contractible elements, pumps, and membranes.
In one aspect, the present invention relates to a method of forming co-encapsulated particles, comprising the steps of: providing a microfluidic device of the present invention; providing a first suspension of a particle providing at least one second suspension of a particle; flowing each of the suspensions through a proximal inlet of the device; and flowing an isolation fluid through an isolation fluid inlet of the device.
In one embodiment, the particle of the first suspension and the at least one second suspension is selected from the group consisting of: cells, viruses, bacteria, amoeba, protozoa, paramecium, microparticles, nanoparticles, beads, microorganisms, vesicles, nucleic acid oligonucleotides, proteins, polypeptides, carbohydrates, and fragments thereof.
In one embodiment, the first suspension and the at least one second suspension comprises a suspension fluid selected from the group consisting of: water, cell growth media, serum, plasma, and oil. In one embodiment, the isolation fluid is immiscible with the suspension fluid.
In one embodiment, the particle of the first suspension is a B cell. In one embodiment, the first suspension comprises one or more additives. In one embodiment, the one or more additives comprises a chelating agent. In one embodiment, the chelating agent is configured to inhibit B-cell clumping in a B cell suspension. In one embodiment, the chelating agent is selected from EDTA and EGTA.
In one embodiment, the particle of the at least one second suspension is a T cell. In one embodiment, the at least one second suspension comprises one or more additives. In one embodiment, the one or more additives comprises an ion additive. In one embodiment, the ion additive is configured to restore ion concentration balance in a B-cell and T-cell co-encapsulate, such that T-cell activation is rescued within the B-cell and T-cell co-encapsulate. In one embodiment, the ion additive is calcium.
In one embodiment, the invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding at least one antigenic polypeptide for presentation at a cell surface, and further comprising at least one transmembrane sequence for enhanced presentation, or fragment thereof. In one embodiment, the transmembrane domain is SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO: 125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, or SEQ ID NO:135.
In one embodiment, the antigenic polypeptide is a pathogenic peptide. In one embodiment, the antigenic polypeptide is a viral antigen.
In one embodiment, the invention relates to an immunogenic composition comprising at least one nucleic acid molecule comprising a nucleotide sequence encoding at least one antigenic polypeptide for presentation at a cell surface, and further comprising at least one transmembrane sequence for enhanced presentation. In one embodiment, the transmembrane domain is SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO: 129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, or SEQ ID NO:135.
In one embodiment, the antigenic polypeptide is a pathogenic peptide. In one embodiment, the antigenic polypeptide is a viral antigen.
In one embodiment, the composition comprises a vaccine or a vaccine adjuvant.
In one embodiment, the invention relates to a method of inducing an immune response, the method comprising administering an immunogenic composition comprising at least one nucleic acid molecule comprising a nucleotide sequence encoding at least one antigenic polypeptide for presentation at a cell surface, and further comprising at least one transmembrane sequence for enhanced presentation to a subject in need thereof. In one embodiment, the transmembrane domain is SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO: 123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, or SEQ ID NO:135.
In one embodiment, the invention relates to a method of enhancing a vaccine response, the method comprising administering an immunogenic composition comprising at least one nucleic acid molecule comprising a nucleotide sequence encoding at least one antigenic polypeptide for presentation at a cell surface, and further comprising at least one transmembrane sequence for enhanced presentation to a subject in need thereof. In one embodiment, the transmembrane domain is SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO: 123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO: 128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, or SEQ ID NO:135.
The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
Representative flow cytometry histograms of V5 (left) and HA (right) signals from both scFvs and quantified median fluorescence intensity (MFI).
The present invention relates generally to a system for the identification of target antigens for T cell receptors (TCRs), compositions for use in the system of the invention and methods of use of the system.
In one embodiment, the system comprises a modified T cell expressing a marker for tagging an antigen presenting cell (APC) upon binding of a TCR expressed by the T cell to an antigen expressed by the APC. In one embodiment, the marker for tagging an APC comprises an antibody or binding fragment thereof specific for binding to a receptor expressed on the surface of the APC. In one embodiment, the marker for tagging an APC comprises an scFv binding molecule specific for binding to a surface molecule of the APC. In some embodiments, the APC is a B cell. In some embodiments, the surface molecule of the APC (e.g., a B cell) is CD19 or CD20.
Therefore, in one embodiment, the invention provides compositions comprising an antibody or binding fragment thereof specific for binding to a receptor expressed on the surface of the APC, or a nucleic acid molecule encoding the same. In one embodiment, the invention relates to an scFv binding molecule specific for binding to a surface molecule of an APC, or a nucleic acid molecule encoding the same. In some embodiments, the APC is a B cell. In some embodiments, the surface molecule of the APC (e.g., a B cell) is CD19 or CD20.
In one embodiment, the invention provides T cells modified to express at least one antibody or binding fragment thereof specific for binding to a receptor expressed on the surface of the APC, or a T cell comprising a nucleic acid molecule encoding at least one antibody or binding fragment thereof specific for binding to a receptor expressed on the surface of the APC. In one embodiment, the invention relates to an scFv binding molecule specific for binding to a surface molecule of an APC, or a nucleic acid molecule encoding the same. In some embodiments, the APC is a B cell. In some embodiments, the surface molecule of the APC (e.g., a B cell) is CD19 or CD20.
In one embodiment, the marker for tagging an APC upon binding of a TCR expressed by the T cell to an antigen expressed by the APC is under the control of one or more regulatory elements that induce expression of the marker upon binding of a TCR expressed by the T cell to an antigen expressed by the APC. In such an embodiment, the expression of the marker for tagging the APC is restricted spatially and temporally so that only the interacting APC is tagged by the marker, which is expressed upon binding of the antigen expressed by the APC (e.g., a B cell) to the TCR expressed by the T cell.
Therefore, in some embodiments, the invention provides nucleic acid molecules comprising an expression cassette under the control of one or more inducible regulatory element(s), wherein the one or more inducible regulatory element(s) is/are induced upon binding of a TCR to an antigen. In some embodiments, one or more regulatory element(s) is/are an inducible promoter that is activated upon binding of a TCR to an antigen (referred to herein as a TCR responsive promoter). In one embodiment, one or more TCR responsive promoter(s) is/are inducible by NFAT. In one embodiment, the TCR responsive promoter is SEQ ID NO:50.
In some embodiments, the invention provides T cells comprising a nucleic acid molecule encoding a marker for tagging an APC upon binding of a TCR expressed by the T cell to an antigen expressed by the APC, wherein the sequence encoding the marker is under the control of one or more inducible regulatory element(s), wherein the one or more inducible regulatory element(s) is/are induced upon binding of a TCR to an antigen. In some embodiments, one or more regulatory element(s) is a TCR responsive promoter. In one embodiment, one or more TCR responsive promoter(s) is/are inducible by NFAT. In one embodiment, the TCR responsive promoter is SEQ ID NO:50.
In some embodiments, the nucleic acid molecules or cells of the invention comprise one or more additional nucleic acid sequences to further regulate the expression of the marker for tagging an APC. In some embodiments, one or more additional nucleic acid sequences include an NBV transcription factor for promoting transcription from an NBV TCR responsive promoter, and surface receptors known to be involved in T cell activation including, but not limited to, CD2, CD226, CD40L, ICOS, OX40, and 41BB. In some embodiments, the nucleic acid molecules comprise one or more insulator elements flanking the expression cassette to avoid spreading of activated chromatin from adjacent genetic elements or from a constitutively expressed downstream selection cassette. In some embodiments, the nucleic acid molecule comprises a 3′-untranslated region (UTR), derived from the human IgG1 heavy chain locus, to increase transcript stability and provide a dedicated polyadenylation sequence to the inducible cassette. In some embodiments, the nucleic acid molecules comprise one or more unique restriction enzyme sites for the incorporation of signal-responsive intron elements, which also serve to increase transcript stability, and downstream cis-regulatory elements (e.g., enhancers).
In one embodiment, the system of the invention employs APC cells expressing a library of antigens to identify new TCR:antigen interactions. In some embodiments, therefore, the invention relates to compositions comprising an APC cell library expressing a plurality of antigens and methods of screening the APC library for antigens that interact with a TCR.
In some embodiments, the antigen presenting cell library has been developed as a library of APCs wherein each APC comprises a nucleic acid molecule that is a member of a minigene library. In one embodiment, each nucleic acid molecule of the minigene library comprises a nucleotide sequence for expression of one or more antigen sequence(s) and further comprises a unique nucleotide sequence encoding the same amino acid tag sequence wherein the sequence encoding the amino acid tag serves as a barcode for identification of the expressed antigen(s). In one embodiment, each nucleotide sequence for expression of one or more antigen sequence(s) is designed to be a pre-determined length. In one embodiment, each nucleotide sequence for expression of one or more antigen sequence(s) comprises a single antigen sequence, or multiple antigen sequences separated by a linker sequence. In some embodiments, the nucleotide sequence for expression of one or more antigen sequence(s) is codon optimized for high-yield protein expression.
Therefore, in some embodiments, the invention relates to a plurality of nucleic acid molecules encoding an optimized minigene library, wherein each nucleic acid molecule comprises an antigen expression cassette (or minigene) comprising nucleotide sequence encoding one or more antigen sequence(s), or fragment thereof, and a unique nucleotide sequence encoding an amino acid tag. In some embodiments, each minigene sequence encodes a single antigen sequence, or multiple antigen sequences separated by linker sequences. In some embodiments, the minigene sequence is codon optimized for high-yield protein expression.
In some embodiments, the nucleic acid molecule encoding the minigene antigen expression cassette further encodes a flag-based surface receptor, a co-stimulatory molecule, or a combination thereof. In some embodiments, the co-stimulatory molecule is CD40, CD58, CD80, CD83, CD86, OX40L, 4-1BBL, or a combination thereof. In one embodiment the nucleotide sequence encoding the flag-based surface receptor comprises a sequence as set forth in SEQ ID NO:78, or a fragment or variant thereof. In one embodiment the nucleotide sequence encoding the co-stimulatory molecule is SEQ ID NO:79, which is a codon optimized sequence encoding CD40, or a fragment or variant thereof. In one embodiment the nucleotide sequence encoding the co-stimulatory molecule is SEQ ID NO:80, which is a codon optimized sequence encoding CD58, or a fragment or variant thereof. In one embodiment the nucleotide sequence encoding the co-stimulatory molecule is SEQ ID NO:81, which is a codon optimized sequence encoding CD80, or a fragment or variant thereof. In one embodiment the nucleotide sequence encoding the co-stimulatory molecule is SEQ ID NO:82, which is a codon optimized sequence encoding CD83, or a fragment or variant thereof. In one embodiment the nucleotide sequence encoding the co-stimulatory molecule is SEQ ID NO:83, which is a codon optimized sequence encoding CD86, or a fragment or variant thereof. In one embodiment the nucleotide sequence encoding the co-stimulatory molecule is SEQ ID NO:84, which is a codon optimized sequence encoding OX40L, or a fragment or variant thereof. In one embodiment the nucleotide sequence encoding the co-stimulatory molecule is SEQ ID NO:85, which is a codon optimized sequence encoding 41BBL, or a fragment or variant thereof. In various embodiments, the fragment of SEQ ID NO:79-85 comprises a sequence comprising at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the full-length sequence of SEQ ID NO:79-85. Specific fragments included in the invention may be a fragment lacking one or more codon at the 5′ or 3′ end of the sequence (e.g., a fragment lacking a start or stop codon.)
In some embodiments, the invention provides methods of generating a minigene library. In some embodiments, the invention provides method of optimizing a minigene library for high-yield, efficient expression of one or more antigen sequence(s).
In some embodiments, the invention relates to methods of co-culturing at least one APC of the invention and at least one T cell of the invention using a microfluidic co-culture system.
The present invention also provides microfluidic co-encapsulation devices configured to generate longitudinal flows of individual particles (such as individual cells) from fluid particle suspensions, combining two or more individual particle flows into a single stream of individual particles, and segmenting the single stream using an isolation fluid, resulting in a suspension of co-encapsulated individual particles from each fluid particle suspension. The present invention also provides methods of using the microfluidic co-encapsulation devices and methods of modifying fluid particle suspensions to promote individual particle separation.
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 this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.
As used herein, each of the following terms has the meaning associated with it in this section.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
“Activation”, as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cytokine production, effector functions, and detectable signals associated with an activated state, such as transcription factors and surface proteins.
The term “antibody,” as used herein, refers to an immunoglobulin molecule which is able to specifically bind to a specific epitope of an antigen. Antibodies can be intact immunoglobulins derived from natural sources, or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies (“intrabodies”), Fv, Fab, Fab′, F(ab)2 and F(ab′)2, as well as single chain antibodies (scFv), heavy chain antibodies, such as camelid antibodies, and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).
The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.
By the term “synthetic antibody” as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.
A “humanized antibody” refers to a type of engineered antibody having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one (or more) human immunoglobulin(s). In addition, framework support residues may be altered to preserve binding affinity (see, e.g., 1989, Queen et al., Proc. Natl. Acad Sci USA, 86:10029-10032; 1991, Hodgson et al., Bio/Technology, 9:421). A suitable human acceptor antibody may be one selected from a conventional database, e.g., the KABAT database, Los Alamos database, and Swiss Protein database, by homology to the nucleotide and amino acid sequences of the donor antibody. A human antibody characterized by a homology to the framework regions of the donor antibody (on an amino acid basis) may be suitable to provide a heavy chain constant region and/or a heavy chain variable framework region for insertion of the donor CDRs. A suitable acceptor antibody capable of donating light chain constant or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains are not required to originate from the same acceptor antibody. Methods of producing humanized antibodies are known in the art.
By the term “recombinant antibody” as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast cell expression system. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.
An “antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.
An “antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.
As used herein, “antigen-binding domain” means that part of the antibody, recombinant molecule, the fusion protein, or the immunoconjugate of the invention which recognizes the target or portions thereof.
The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an adaptive immune response. This immune response may involve either antibody production, or the activation of specific immunogenically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA or RNA. A skilled artisan will understand that any DNA or RNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an adaptive immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, tumor sample, cell, biological fluid, body fluid, blood, serum, plasma, tissue, or any combination thereof.
As used herein, an “instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of a compound, composition, vector, or delivery system of the invention in the kit for effecting alleviation of the various diseases or disorders recited herein. Optionally, or alternately, the instructional material can describe one or more methods of alleviating the diseases or disorders in a cell or a tissue of a mammal. The instructional material of the kit of the invention can, for example, be affixed to a container which contains the identified compound, composition, vector, or delivery system of the invention or be shipped together with a container which contains the identified compound, composition, vector, or delivery system. Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.
“Operably linked,” “operatively linked,” or “under transcriptional control” as used herein may mean that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter may be positioned 5′ (upstream) or 3′ (downstream) of a gene under its control. The distance between the promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function.
The phrase “biological sample”, “sample” or “specimen” as used herein, is intended to include any sample comprising a cell, a tissue, or a bodily fluid in which expression of a nucleic acid or polypeptide can be detected. The biological sample may contain any biological material suitable for detecting the desired biomarkers, and may comprise cellular and/or non-cellular material obtained from the individual. Examples of such biological samples include but are not limited to blood, lymph, bone marrow, biopsies, and smears. Samples that are liquid in nature are referred to herein as “bodily fluids.” Biological samples may be obtained from a patient by a variety of techniques including, for example, by scraping or swabbing an area or by using a needle to obtain bodily fluids. Methods for collecting various body samples are well known in the art.
“CDRs” are defined as the complementarity determining region amino acid sequences of a TCR or TCR chain.
As used herein, an “immunoassay” refers to any binding assay that uses an antibody capable of binding specifically to a target molecule to detect and quantify the target molecule.
By the term “specifically binds,” as used herein with respect to a binding molecule (e.g., an antibody or TCR), is meant a binding molecule which recognizes a specific target molecule (e.g., an antigen or epitope), but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally.
A “coding region” of a gene consists of the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene.
A “coding region” of a mRNA molecule also consists of the nucleotide residues of the mRNA molecule which are matched with an anti-codon region of a transfer RNA molecule during translation of the mRNA molecule or which encode a stop codon. The coding region may thus include nucleotide residues comprising codons for amino acid residues which are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).
“Complementary” as used herein to refer to a nucleic acid, refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. In some embodiments, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and or at least about 75%, or at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. In some embodiments, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
The term “DNA” as used herein is defined as deoxyribonucleic acid.
“Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting there from. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
“Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in its normal context in a living subject is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural context is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
An “isolated nucleic acid” refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, i.e., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.
The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.
A “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.
As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
The term “RNA” as used herein is defined as ribonucleic acid.
The term “recombinant DNA” as used herein is defined as DNA produced by joining pieces of DNA from different sources.
The term “recombinant polypeptide” as used herein is defined as a polypeptide produced by using recombinant DNA methods.
As used herein, “conjugated” refers to covalent attachment of one molecule to a second molecule.
“Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ×100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.
“Variant” as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence, or peptide sequence respectively, but retains essential biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions, and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A variant of a nucleic acid or peptide can be a naturally occurring, such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis. In various embodiments, the variant sequence is at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85% identical to the reference sequence.
The term “regulating” as used herein can mean any method of altering the level or activity of a substrate. Non-limiting examples of regulating with regard to a protein include affecting expression (including transcription and/or translation), affecting folding, affecting degradation or protein turnover, and affecting localization of a protein. Non-limiting examples of regulating with regard to an enzyme further include affecting the enzymatic activity. “Regulator” refers to a molecule whose activity includes affecting the level or activity of a substrate. A regulator can be direct or indirect. A regulator can function to activate or inhibit or otherwise modulate its substrate.
“Variant” with respect to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Variant may also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of 2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101, incorporated fully herein by reference. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions may be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.
“Vector” as used herein may mean a nucleic acid sequence containing an origin of replication. A vector may be a plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be either a self-replicating extrachromosomal vector or a vector which integrates into a host genome.
As used herein, a “substantially purified” cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.
Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
In one embodiment, the invention relates to compositions comprising a cell expressing a T cell receptor (TCR) molecule and further expressing a secreted molecule specific for binding to a marker expressed on a surface of an antigen-presenting cell (APC), referred to herein as an APC tagging molecule.
In some embodiments, the cell is an immune cell. Exemplary immune cells that may comprise or express one or more TCRs include, but are not limited to, T cells (including killer T cells, helper T cells, regulatory T cells, gamma delta T cells, and laboratory-isolated or -generated T cell lines), engineered natural killer (NK) cells, NK T cells, or cells engineered to express a TCR. Therefore, in some embodiments, the invention provides a T cell expressing an APC tagging molecule. In some embodiments, the invention provides a T cell comprising a nucleic acid molecule comprising a nucleotide sequence encoding an APC tagging molecule.
In some embodiments, the TCR is an engineered TCR. In some embodiments, the nucleotide sequence encoding the TCR comprises a nucleotide sequence generated from a nucleotide sequence of a TCR isolated from a subject (i.e., a subject or patient derived TCR). In such an embodiment, the T cell of the invention comprises a deletion of the native TCR, and further comprises an exogenous nucleic acid sequence encoding a non-native TCR. In one embodiment, the non-native TCR is a subject or patient derived TCR. In one embodiment, the T cell further comprises a second exogenous nucleic acid molecule encoding an APC tagging molecule.
An APC is any cell expressing one or more major histocompatibility complex (MHC), or as they are specifically named in human cells, human leukocyte antigen (HLA), proteins on its surface. Exemplary APCs include, but are not limited to, dendritic cells (DCs), macrophages, Langerhans cells, B cells, and the like. In some embodiments, the APC is a cell engineered to express major histocompatibility complex (MHC), or human leukocyte antigen (HLA).
In one embodiment, the APC is a B cell and the APC tagging molecule is a molecule specific for binding to a B cell marker. Exemplary B cell markers include, but are not limited to CD19, CD20, CD38, CD40, CD45R, CD79a, and CD79b. Therefore, in some embodiments, the APC tagging molecule comprises a molecule specific for binding to CD19, CD20, CD38, CD40, CD45R, CD79a, or CD79b.
In one embodiment, the APC is a DC and the APC tagging molecule is a molecule specific for binding to a DC marker. Exemplary DC markers include, but are not limited to, CD11c and CD1a. Therefore, in some embodiments, the APC tagging molecule comprises a molecule specific for binding to CD11c or CD1a.
In some embodiments, the APC tagging molecule comprises an antibody, or fragment thereof, specific for binding to an APC cell marker. In some embodiments, the APC tagging molecule comprises an antibody, or fragment thereof, specific for binding to CD19, CD20, CD38, CD40, CD45R, CD79a, CD79b, CD11c or CD1a.
In various embodiments, the APC tagging molecule is an antibody, an antibody fragment, a peptide sequence, aptamer, a ligand, a gene component, or any combination thereof. Examples of APC tagging molecule include, but are not limited to antibodies, peptidomimetics, synthetic ligands, and the like which specifically bind desired target APCs. APC tagging molecules of particular interest include peptidomimetics, peptides, antibodies (e.g., monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, etc.) and antibody fragments (e.g., the Fab′ fragment).
The invention encompasses monoclonal, synthetic antibodies, and the like. One skilled in the art would understand, based upon the disclosure provided herein, that the crucial feature of the antibody of the invention is that the antibody binds specifically with an APC marker of interest. That is, the antibody of the invention recognizes a target of interest or a fragment thereof.
The skilled artisan would appreciate, based upon the disclosure provided herein, that present invention includes use of a single antibody recognizing a single antibody target but that the invention is not limited to use of a single antibody, or fragment thereof. Instead, the invention encompasses use of at least one antibody, or fragment thereof, where the antibodies can be directed to the same or different antibody targets.
Nucleic acid molecules encoding the binding molecule described herein may be cloned and sequenced using technology, which is available in the art and is described, for example, in Wright et al. (1992, Critical Rev. Immunol. 12:125-168), and the references cited therein. Further, the antibody of the invention may be “humanized” using the technology described in, for example, Wright et al., and in the references cited therein, and in Gu et al. (1997, Thrombosis and Hematocyst 77:755-759), and other methods of humanizing antibodies well-known in the art or to be developed.
In some embodiments, a non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in a human or fragment thereof. A humanized antibody can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (see, e.g., European Patent Nos. EP 592, 106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 91:969-973), chain shuffling (see, e.g., U.S. Pat. No. 5,565,332), and techniques disclosed in, e.g., U.S. Patent Application Publication No. US2005/0042664, U.S. Patent Application Publication No. US2005/0048617, U.S. Pat. Nos. 6,407,213, 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994). Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, for example improve, antigen binding. These framework substitutions are identified by methods well-known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323.)
In one embodiment, the antibody fragment provided herein is a single chain variable fragment (scFv). In various embodiments, the antibodies of the invention may exist in a variety of other forms including, for example, Fv and Fab, as well as bi-functional (i.e. bi-specific) hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In some embodiments, the antibodies and fragments thereof of the invention bind an APC. In some embodiments, the antibodies and fragments thereof of the invention bind a B cell. In some embodiments, the antibodies and fragments thereof of the invention bind a DC.
In one embodiment, the APC tagging molecule is specific for binding to CD19. In one embodiment, the APC tagging molecule specific for binding to a CD19B cell marker comprises an scFv antibody fragment comprising a heavy chain variable region comprising an amino acid sequence as set forth in SEQ NO:4, or a fragment or variant thereof, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:8, or a fragment or variant thereof. A fragment or variant of SEQ ID NO:4 may comprise a fragment or variant of SEQ ID NO:4 in which the CDR regions of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 are retained, but non-CDR sequences are modified or varied. A fragment or variant of SEQ ID NO:8 may comprise a fragment or variant of SEQ ID NO:8 in which the CDR regions of SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 are retained, but non-CDR sequences are modified or varied.
In one embodiment, the APC tagging molecule is an scFv antibody fragment specific for binding to CD19. In one embodiment, the APC tagging molecule specific for binding to a CD19 B cell marker comprises an scFv antibody fragment comprising a heavy chain variable region comprising an amino acid sequence as set forth in SEQ NO: 12, or a fragment or variant thereof, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:16, or a fragment or variant thereof. A fragment or variant of SEQ ID NO:12 may comprise a fragment or variant of SEQ ID NO:12 in which the CDR regions of SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11 are retained, but non-CDR sequences are modified or varied. A fragment or variant of SEQ ID NO:16 may comprise a fragment or variant of SEQ ID NO:16 in which the CDR regions of SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15 are retained, but non-CDR sequences are modified or varied.
In one embodiment, the APC tagging molecule is specific for binding to CD20. In one embodiment, the APC tagging molecule specific for binding to a CD20B cell marker comprises an scFv antibody fragment comprising a heavy chain variable region comprising an amino acid sequence as set forth in SEQ NO:20, or a fragment or variant thereof, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:24, or a fragment or variant thereof. A fragment or variant of SEQ ID NO:20 may comprise a fragment or variant of SEQ ID NO:20 in which the CDR regions of SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19 are retained, but non-CDR sequences are modified or varied. A fragment or variant of SEQ ID NO:24 may comprise a fragment or variant of SEQ ID NO:24 in which the CDR regions of SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23 are retained, but non-CDR sequences are modified or varied.
In one embodiment, the APC tagging molecule is an scFv antibody fragment specific for binding to CD20. In one embodiment, the APC tagging molecule specific for binding to a CD20 B cell marker comprises an scFv antibody fragment comprising a heavy chain variable region comprising an amino acid sequence as set forth in SEQ NO:28, or a fragment or variant thereof, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:32, or a fragment or variant thereof. A fragment or variant of SEQ ID NO:28 may comprise a fragment or variant of SEQ ID NO:28 in which the CDR regions of SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27 are retained, but non-CDR sequences are modified or varied. A fragment or variant of SEQ ID NO:32 may comprise a fragment or variant of SEQ ID NO:32 in which the CDR regions of SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31 are retained, but non-CDR sequences are modified or varied.
A variant of an amino acid sequence may be an amino acid sequence that is substantially identical over the full-length of the amino acid sequence or fragment thereof to a parental amino acid sequence. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full-length of the amino acid sequence or a fragment thereof to a parental amino acid sequence. In one embodiment, the variant of SEQ ID NO:4 comprises a sequence 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full-length of the amino acid sequence to SEQ ID NO:4, but in which the sequence of the CDR sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 are not varied (i.e. the CDR sequences are 100% identical to the CDR sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.) In one embodiment, the variant of SEQ ID NO:4 comprises a sequence 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full-length of the amino acid sequence to SEQ ID NO:8, but in which the sequence of the CDR sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7 are not varied (i.e. the CDR sequences are 100% identical to the CDR sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.) In one embodiment, the variant of SEQ ID NO:4 comprises a sequence 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full-length of the amino acid sequence to SEQ ID NO:12, but in which the sequence of the CDR sequences of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11 are not varied (i.e. the CDR sequences are 100% identical to the CDR sequences of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.) In one embodiment, the variant of SEQ ID NO:16 comprises a sequence 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full-length of the amino acid sequence to SEQ ID NO:16, but in which the sequence of the CDR sequences of SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15 are not varied (i.e. the CDR sequences are 100% identical to the CDR sequences of SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15.) In one embodiment, the variant of SEQ ID NO:20 comprises a sequence 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full-length of the amino acid sequence to SEQ ID NO:20, but in which the sequence of the CDR sequences of SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19 are not varied (i.e. the CDR sequences are 100% identical to the CDR sequences of SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.) In one embodiment, the variant of SEQ ID NO:24 comprises a sequence 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full-length of the amino acid sequence to SEQ ID NO:24, but in which the sequence of the CDR sequences of SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23 are not varied (i.e. the CDR sequences are 100% identical to the CDR sequences of SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23.) In one embodiment, the variant of SEQ ID NO:28 comprises a sequence 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full-length of the amino acid sequence to SEQ ID NO:28, but in which the sequence of the CDR sequences of SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27 are not varied (i.e. the CDR sequences are 100% identical to the CDR sequences of SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.) In one embodiment, the variant of SEQ ID NO:32 comprises a sequence 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full-length of the amino acid sequence to SEQ ID NO:32, but in which the sequence of the CDR sequences of SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31 are not varied (i.e. the CDR sequences are 100% identical to the CDR sequences of SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31.)
ScFvs can be prepared according to method known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be produced by linking VH and VL regions together using flexible polypeptide linkers. The scFv molecules comprise flexible polypeptide linker (e.g., a Ser-Gly linker) with an optimized length and/or amino acid composition. The flexible polypeptide linker length can greatly affect how the variable regions of an scFv fold and interact. In fact, if a short polypeptide linker is employed (e.g., between 5-10 amino acids, intrachain folding is prevented. Interchain folding is also required to bring the two variable regions together to form a functional epitope binding site. For examples of linker orientation and size see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCT publication Nos. WO2006/020258 and WO2007/024715.
The scFv can comprise a polypeptide linker sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues between its VL and VH regions. The flexible polypeptide linker sequence may comprise any naturally occurring amino acid. In some embodiments, the flexible polypeptide linker sequence comprises amino acids glycine and serine. In another embodiment, the flexible polypeptide linker sequence comprises sets of glycine and serine repeats such as (Gly4Ser)n, where n is a positive integer equal to or greater than 1. In one embodiment, the flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4, (Gly4Ser)3, and SEQ ID NO:42. Variation in the flexible polypeptide linker length may retain or enhance activity, giving rise to superior efficacy.
In some embodiments, the scFv molecule comprises a polypeptide leader sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues. In one embodiment, the leader sequence comprises a sequence as set forth in SEQ ID NO:43.
In some embodiments, the scFv molecule comprises a capture tag sequence which allows for isolation of the scFv and an associated “tagged” APC. Exemplary capture tags that can be included on an scFv of the invention include, but are not limited to, a 4×V5 tag (SEQ ID NO:44), a 3×HA tag (SEQ ID NO:45), a 6×HA tag (SEQ ID NO:46), a 6× FLAG tag (SEQ ID NO:47), or a 3× FLAG tag (the FLAG sequence is DYKDDDDK (SEQ ID NO:48).
In one embodiment, the APC tagging molecule comprises two or more tandem APC tagging molecules, separated by a linker sequence. In some embodiments, the linker sequence comprises SEQ ID NO:49. In some embodiments, the tandem APC tagging molecule comprises at least two, at least three, at least four, or more than four tandem APC molecules. In some embodiments, the tandem APC tagging molecule comprises at least two, at least three, at least four, or more than four tandem scFv molecules. In some embodiments, two or more tandem APC tagging molecules target the same APC marker. In some embodiments, two or more tandem APC tagging molecules target two or more different APC markers. An exemplary diabody comprising two tandem APC tagging molecules comprises the sequence as set forth in SEQ ID NO:33.
As described above, the composition can comprise a recombinant nucleic acid sequence. The recombinant nucleic acid sequence can encode the antibody, a fragment thereof, a variant thereof, or a combination thereof. The recombinant nucleic acid sequence can be a heterologous nucleic acid sequence. The recombinant nucleic acid sequence can include one or more heterologous nucleic acid sequences.
The recombinant nucleic acid sequence can be an optimized nucleic acid sequence. Such optimization can increase or alter the immunogenicity of the antibody. Optimization can also improve transcription and/or translation. Optimization can include one or more of the following: low GC content leader sequence to increase transcription; mRNA stability and codon optimization; addition of a Kozak sequence for increased translation; addition of an immunoglobulin (Ig) leader sequence encoding a signal peptide; addition of an internal IRES sequence and eliminating to the extent possible cis-acting sequence motifs (i.e., internal TATA boxes).
The recombinant nucleic acid sequence can include one or more recombinant nucleic acid sequence constructs. The recombinant nucleic acid sequence construct can include one or more components, which are described in more detail below.
The recombinant nucleic acid sequence construct can include a heterologous nucleic acid sequence that encodes a heavy chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The recombinant nucleic acid sequence construct can include a heterologous nucleic acid sequence that encodes a light chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The recombinant nucleic acid sequence construct can also include a heterologous nucleic acid sequence that encodes a protease or peptidase cleavage site. The recombinant nucleic acid sequence construct can also include a heterologous nucleic acid sequence that encodes an internal ribosome entry site (IRES). An IRES may be either a viral IRES or a eukaryotic IRES. The recombinant nucleic acid sequence construct can include one or more leader sequences, in which each leader sequence encodes a signal peptide. The recombinant nucleic acid sequence construct can include one or more promoters, one or more introns, one or more transcription termination regions, one or more initiation codons, one or more termination or stop codons, and/or one or more polyadenylation signals. The recombinant nucleic acid sequence construct can also include one or more linker or tag sequences, as described above.
The recombinant nucleic acid molecule construct can include the heterologous nucleic acid sequence encoding the heavy chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The heavy chain polypeptide can include a variable heavy chain (VH) region and/or at least one constant heavy chain (CH) region. The at least one constant heavy chain region can include a constant heavy chain region 1 (CH1), a constant heavy chain region 2 (CH2), and a constant heavy chain region 3 (CH3), and/or a hinge region.
In some embodiments, the heavy chain polypeptide can include a VH region and a CH1 region. In other embodiments, the heavy chain polypeptide can include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region.
The heavy chain polypeptide can include a complementarity determining region (“CDR”) set. The CDR set can contain three hypervariable regions of the VH region. Proceeding from N-terminus of the heavy chain polypeptide, these CDRs are denoted “CDR1,” “CDR2,” and “CDR3,” respectively. CDR1, CDR2, and CDR3 of the heavy chain polypeptide can contribute to binding or recognition of the antigen.
The recombinant nucleic acid sequence construct can include the heterologous nucleic acid sequence encoding the light chain polypeptide, a fragment thereof, a variant thereof, or a combination thereof. The light chain polypeptide can include a variable light chain (VL) region and/or a constant light chain (CL) region.
The light chain polypeptide can include a complementarity determining region (“CDR”) set. The CDR set can contain three hypervariable regions of the VL region. Proceeding from N-terminus of the light chain polypeptide, these CDRs are denoted “CDR1,” “CDR2,” and “CDR3,” respectively. CDR1, CDR2, and CDR3 of the light chain polypeptide can contribute to binding or recognition of the antigen.
In one embodiment, the nucleic acid molecule encoding an APC tagging molecule specific for binding to a CD19 B cell marker comprises a nucleotide sequence encoding an scFv antibody fragment comprising a heavy chain variable region comprising an amino acid sequence as set forth in SEQ NO:4, or a fragment or variant thereof, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:8, or a fragment or variant thereof. A fragment or variant of SEQ ID NO:4 or SEQ ID NO:8 may comprise a fragment or variant of SEQ ID NO:4 or SEQ ID NO:8 in which the CDR regions of SEQ ID NO:1-3 and SEQ ID NO:5-7 respectively are retained, but non-CDR sequences are modified or varied. In one embodiment, the nucleic acid molecule encoding an APC tagging molecule specific for binding to a CD20 B cell marker comprises a nucleotide sequence encoding SEQ ID NO:33, SEQ ID NO:39 or SEQ ID NO:41. In one embodiment, the nucleic acid molecule encoding an APC tagging molecule specific for binding to a CD20 B cell marker comprises a nucleotide sequence as set forth in SEQ ID NO:40 or SEQ ID NO:38.
In one embodiment, the nucleic acid molecule encoding an APC tagging molecule specific for binding to a CD19 B cell marker comprises a nucleotide sequence encoding an scFv antibody fragment comprising a heavy chain variable region comprising an amino acid sequence as set forth in SEQ NO:12, or a fragment or variant thereof, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:16, or a fragment or variant thereof. A fragment or variant of SEQ ID NO:12 or SEQ ID NO:16 may comprise a fragment or variant of SEQ ID NO:12 or SEQ ID NO:16 in which the CDR regions of SEQ ID NO:9-11 and SEQ ID NO:13-15 respectively are retained, but non-CDR sequences are modified or varied.
In one embodiment, the nucleic acid molecule encoding an APC tagging molecule specific for binding to a CD20 B cell marker comprises a nucleotide sequence encoding an scFv antibody fragment comprising a heavy chain variable region comprising an amino acid sequence as set forth in SEQ NO:20, or a fragment or variant thereof, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:24, or a fragment or variant thereof. A fragment or variant of SEQ ID NO:20 or SEQ ID NO:24 may comprise a fragment or variant of SEQ ID NO:20 or SEQ ID NO:24 in which the CDR regions of SEQ ID NO:17-19 and SEQ ID NO:21-23 respectively are retained, but non-CDR sequences are modified or varied. In one embodiment, the nucleic acid molecule encoding an APC tagging molecule specific for binding to a CD20 B cell marker comprises a nucleotide sequence encoding SEQ ID NO:33, SEQ ID NO:35 or SEQ ID NO:37. In one embodiment, the nucleic acid molecule encoding an APC tagging molecule specific for binding to a CD20 B cell marker comprises a nucleotide sequence as set forth in SEQ ID NO:34 or SEQ ID NO:36.
In one embodiment, the nucleic acid molecule encoding an APC tagging molecule specific for binding to a CD20 B cell marker comprises a nucleotide sequence encoding an scFv antibody fragment comprising a heavy chain variable region comprising an amino acid sequence as set forth in SEQ NO:28, or a fragment or variant thereof, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO:32, or a fragment or variant thereof. A fragment or variant of SEQ ID NO:28 or SEQ ID NO:32 may comprise a fragment or variant of SEQ ID NO:28 or SEQ ID NO:32 in which the CDR regions of SEQ ID NO:25-27 and SEQ ID NO:29-31 respectively are retained, but non-CDR sequences are modified or varied.
A variant may be a sequence that is substantially identical over the full-length to a parental sequence or a fragment thereof. A variant of a nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full-length of the gene sequence or a fragment thereof to a parental gene sequence.
The recombinant nucleic acid sequence construct can include one or more linker sequences as described above. The linker sequence can spatially separate or link the one or more components described herein. In some embodiments, the linker sequence can encode an amino acid sequence that spatially separates or links a heavy chain and a light chain of an scFv antibody fragment. In some embodiments, the linker sequence can encode an amino acid sequence that spatially separates or links two or more tandem scFv antibody fragments.
The recombinant nucleic acid sequence construct can include one or more promoters. The one or more promoters may be any promoter that is capable of driving gene expression and regulating gene expression. Such a promoter is a cis-acting sequence element required for transcription via a DNA dependent RNA polymerase. Selection of the promoter used to direct gene expression depends on the particular application. The promoter may be positioned about the same distance from the transcription start in the recombinant nucleic acid sequence construct as it is from the transcription start site in its natural setting. However, variation in this distance may be accommodated without loss of promoter function.
The promoter may be operably linked to the heterologous nucleic acid sequence encoding the heavy chain polypeptide and/or light chain polypeptide. The promoter may be a promoter shown effective for expression in eukaryotic cells. The promoter operably linked to the coding sequence may be a CMV promoter, a promoter from simian virus 40 (SV40), such as SV40 early promoter and SV40 later promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter such as the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter may also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, human polyhedrin, or human metallothionein. In various embodiments, the constitutive promoter is the EFlalpha promoter, the ActB promoter, the Gapdh promoter, the IGKV1-39 promoter, the Rp141 promoter, the Rps2 promoter, the Rps18 promoter or the Tsmb4× promoter. The promoter may also be a synthetic, human-designed nucleotide sequence intended to achieve the desired effect on expression. The promoter may also be comprised of fragments of promoters from any or all of the above examples.
The promoter can be a constitutive promoter or an inducible promoter, (e.g., a promoter which initiates transcription only when a TCR expressed by the T cell is bound to an antigen presented by an APC, as described in more detail below.)
The promoter can be associated with an enhancer. The enhancer can be located upstream or downstream of the coding sequence. Exemplary enhancers include, but are not limited to, those described in more detail below.
In one embodiment, the invention relates to the development of a system comprising a T cell that expresses a target protein or peptide under the control of a TCR responsive promoter, wherein the target protein or peptide is expressed when a TCR is bound to an antigenic protein or peptide. In one embodiment, exemplary target proteins or peptides that can be expressed using the TCR responsive promoter of the invention include, but are not limited to, an antibody or fragment thereof, a detectable marker, a fluorescent protein, a transcription factor, and an enzyme. In one embodiment, the invention relates to the development of a system comprising a T cell that expresses an APC tagging molecule under the control of an inducible promoter such that the APC tagging molecule is expressed upon binding of a TCR of the T cell to an antigen presented by the APC. Therefore, in some embodiments, the invention provides compositions comprising TCR responsive promoters.
In some embodiments, the TCR responsive promoter comprises a sequence selected from SEQ ID NO:50-SEQ ID NO:74. In one embodiment, therefore, the invention provides nucleic acid molecules comprising a TCR responsive promoter of SEQ ID NO:50-SEQ ID NO:74 operably linked to a sequence encoding a B cell tagging molecule of the invention.
In some embodiments, the TCR responsive promoter of the invention is inducible by NFAT. In some embodiments, the TCR responsive promoter of the invention is an NBV promoter which is inducible by a NBV transcription factor comprising a fusion of the following elements: the cytoplasmic retention and DNA-binding domains from the N′-terminus of the nuclear factor in activated T cells (NFAT), the octamer motif (‘ATGCAAAT’)-binding domain from the transcriptional co-activator Bob1, and the C′-terminal transactivation domain (TAD) from the herpesvirus VP16 protein. In one embodiment, the NBV promoter comprises a sequence as set forth in SEQ ID NO:50, and the NBV transcription factor comprises a sequence as set forth in SEQ ID NO:76. In one embodiment, the NBV transcription factor is encoded by a sequence as set forth in SEQ ID NO:75.
In one embodiment, the invention provides a modified cell containing a nucleic acid molecule comprising a nucleotide sequence encoding a heterologous sequence for expression under the control of a TCR responsive promoter. In one embodiment, the heterologous sequence encodes an antibody or fragment thereof, a detectable marker, a fluorescent protein, a transcription factor, or an enzyme. For example, in one embodiment, the invention provides a modified T cell containing a nucleic acid molecule comprising a nucleotide sequence encoding an APC tagging molecule operably linked to a TCR responsive promoter.
In some embodiments, the T cell of the invention comprises a deletion of the native TCR and further comprises a nucleic acid molecule encoding a non-native TCR. In some embodiments, the non-native TCR is a subject or patient derived TCR.
In one embodiment, the invention provides a T cell comprising a nucleic acid molecule comprising a nucleotide sequence encoding a B cell tagging molecule under the control of an NBV promoter and further comprises a nucleic acid molecule encoding an NBV transcription factor under the control of a TCR responsive promoter. In one embodiment, the nucleic acid molecule encoding an NBV transcription factor is operably linked to a promoter having a sequence as set forth in SEQ ID NO:50-SEQ ID NO:74.
In some embodiments, the nucleic acid molecule encoding the NBV transcription factor further comprises one or more additional regulatory elements, such as an insulator element or an enhancer element, which functions to further regulate expression of the NBV transcription factor.
In some embodiments, the nucleic acid molecule encoding the NBV transcription factor further comprises one or more nucleotide sequence encoding at least one co-stimulatory molecule to amplify expression of NFAT, thus increasing expression of the B cell tagging molecule. In some embodiments, the co-stimulatory molecule is CD2, CD226, CD40L, ICOS, OX40, and 41BB, or a combination thereof. In one embodiment, one or more nucleotide sequence encoding at least one co-stimulatory molecule is operably linked to a constitutive promoter.
Any method known in the art for introducing nucleic acid sequences into cells can be used to generate the modified T cells of the invention. Exemplary methods of introducing nucleic acid molecules into cells include, but are not limited to, electroporation, cell squeezing, sonoporation, optical transfection, protoplast fusion, impalefection, hydrodynamic delivery, fusion, magnetofection, particle bombardment, nucleofection, heat shock, lipofection, viral transduction, nonviral transfection, lithium acetate/PEG chemical transformation, or any combination thereof.
In various embodiments, the invention relates to methods of screening an antigen-presenting cell (APC) library, wherein each antigen-presenting cell displays one or more antigenic sequence. In some embodiments, the APC library comprises a plurality of cells, wherein together the plurality of cells displays at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 20,000 or more than 20,000 different polypeptides on the surface of the cells.
In some embodiments, the polypeptides for display are fusion proteins with linker molecules that allow display of multiple linked antigenic sequences on an APC.
In one embodiment, nucleic acid molecules encoding one or more antigen for display can be cloned into a minigene vector. In some embodiments, the antigen encoded on the minigene vector is expressed intracellularly, processed into small fragments, and presented as an antigen-MHC complex on the outer surface of an APC cell.
In one embodiment, nucleic acid molecules encoding one or more antigen for display can be cloned into a vector is designed to express the one or more encoded antigen on the outer surface of an APC cell containing the vector. Thereafter, the APC library can be screened for TCR reactivities with the displayed antigen(s).
Thus, in various embodiments, the present invention also includes a vector in which a nucleotide sequence encoding a polypeptide for display of the present invention is inserted. The art is replete with suitable vectors that are useful in the present invention.
In one embodiment, the expression of a nucleotide construct is typically achieved by operably linking a nucleic acid sequence comprising a promoter to a nucleic acid sequence encoding at least one antigen or fragment thereof, and incorporating the construct into an expression vector. In one embodiment, the vectors to be used are suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and other regulatory sequences useful for regulation of the expression of the desired nucleic acid sequence.
The recombinant nucleotide sequences encoding one or more antigen for display of the invention can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid or a viral vector.
In one embodiment, the vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used.
For example, vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. In one embodiment, the composition includes a vector derived from an adeno-associated virus (AAV). Adeno-associated viral (AAV) vectors have become powerful gene delivery tools for the treatment of various disorders. AAV vectors possess a number of features that render them ideally suited for gene therapy, including a lack of pathogenicity, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner. Expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells by choosing the appropriate combination of AAV serotype, promoter, and delivery method
In certain embodiments, the vector also includes conventional control elements which are operably linked to the encoded antigen sequence in a manner which permits its transcription, translation and/or expression in a cell transfected with the plasmid vector or infected with the virus produced by the invention. As used herein, “operably linked” sequences include both expression control sequences that are contiguous with the reporter molecule and expression control sequences that act in trans or at a distance to control the expression of the reporter molecule. Expression control sequences include appropriate transcription initiation, termination, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. All of the above-described functional elements can be used in any combination to produce a suitable display vector. In one embodiment, a display vector comprises an origin of replication capable of initiating DNA synthesis in a suitable APC.
In one embodiment, a display vector comprises a selection marker gene to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Selectable marker genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
A selection marker sequence can be used to eliminate host cells in which the display vector has not been properly transfected. A selection marker sequence can be a positive selection marker or negative selection marker. Positive selection markers permit the selection for cells in which the gene product of the marker is expressed. This generally comprises contacting cells with an appropriate agent that, but for the expression of the positive selection marker, kills or otherwise selects against the cells.
Examples of selection markers also include, but are not limited to, proteins conferring resistance to compounds such as antibiotics, proteins conferring the ability to grow on selected substrates, proteins that produce detectable signals such as luminescence, catalytic RNAs and antisense RNAs. A wide variety of such markers are known and available, including, for example, a Zeocin™ resistance marker, a blasticidin resistance marker, a neomycin resistance (neo) marker (Southern & Berg, J. Mol. Appl. Genet. 1: 327-41 (1982)), a puromycin (puro) resistance marker; a hygromycin resistance (hyg) marker (Te Riele et al., Nature 348:649-651 (1990)), thymidine kinase (tk), hypoxanthine phosphoribosyltransferase (hprt), and the bacterial guanine/xanthine phosphoribosyltransferase (gpt), which permits growth on MAX (mycophenolic acid, adenine, and xanthine) medium. See Song et al., Proc. Nat'l Acad. Sci. U.S.A. 84:6820-6824 (1987). Other selection markers include histidinol-dehydrogenase, chloramphenicol-acetyl transferase (CAT), dihydrofolate reductase (DHFR), β-galactosyltransferase and fluorescent proteins such as GFP.
Expression of a fluorescent protein can be detected using a fluorescent activated cell sorter (FACS). Expression of β-galactosyltransferase also can be sorted by FACS, coupled with staining of living cells with a suitable substrate for β-galactosidase. A selection marker also may be a cell-substrate adhesion molecule, such as integrins, which normally are not expressed by the host cell. In one embodiment, the cell selection marker is of mammalian origin, for example, thymidine kinase, aminoglycoside phosphotransferase, asparagine synthetase, adenosine deaminase or metallothionien. In one embodiment, the cell selection marker can be neomycin phosphotransferase, hygromycin phosphotransferase or puromycin phosphotransferase, which confer resistance to G418, hygromycin and puromycin, respectively.
Suitable prokaryotic and/or bacterial selection markers include proteins providing resistance to antibiotics, such as kanamycin, tetracycline, and ampicillin. In one embodiment, a selection marker includes a protein capable of conferring selectable traits to both a prokaryotic host cell and a mammalian target cell.
In one embodiment, the invention provides a plurality of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 20,000 or more than 20,000 recombinant nucleic acid molecules (i.e. a minigene library), wherein together the plurality of recombinant nucleic acid molecules encode at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 20,000 or more than 20,000 different antigenic sequences for presentation by an APC library.
In some embodiments, the antigen encoding region of each nucleic acid molecule in the minigene library is designed to be a predetermined length. For example, in one embodiment, the minigene library is designed to comprise antigen encoding regions of 900 nucleotides. In one embodiment, the minigene library is designed to comprise antigen encoding regions of 1050 nucleotides. In one embodiment, the minigene library is designed to comprise antigen encoding regions of 1200 nucleotides. In such embodiments, antigen encoding regions are designed to comprise a combination of nucleotide sequences encoding fragments of proteins, wherein each fragment has the predetermined length (e.g., 990 or 1200 nucleotides) and nucleotide sequences encoding multiple peptides linked by one or more linker sequences such that the full-length sequence comprises the predetermined length (e.g., 990 or 1200 nucleotides).
In one embodiment, each of the recombinant nucleic acid molecules in the plurality of recombinant nucleic acid molecules encodes one or more polypeptide sequence for presentation by an APC and further comprises a unique nucleotide sequence encoding a set or shared amino acid barcode sequence. The unique nucleotide sequence is then associated with the encoded antigen sequence(s). In various embodiments, the unique barcode sequence comprises a nucleotide sequence of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 nucleotides which is non-redundant with other nucleotide sequences included in the library, but which encodes the same amino acid sequences as other nucleotide barcode sequences included in the library.
In some embodiments, the nucleic acid molecule encoding the minigene antigen expression cassette further comprises a sequence encoding at least one transmembrane domain for class II antigen presentation, or fragment thereof. Exemplary transmembrane domains that can be encoded include, but are not limited to, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO: 122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, and SEQ ID NO:135.
In some embodiments, the nucleic acid molecule encoding the minigene antigen expression cassette further encodes a flag-based surface receptor, a ligand for a co-stimulatory molecule, or a combination thereof. In some embodiments, the co-stimulatory molecule is CD40, CD58, CD80, CD83, CD86, OX40L, 4-1BBL, or a combination thereof.
In some embodiments, the invention relates to methods of generating a minigene library for expression of a plurality of antigens on the surface of a plurality of APCs. In some embodiments, the method comprises obtaining or generating a library of barcoded nucleic acid molecules, wherein each nucleic acid molecule comprises a nucleotide sequence encoding one or more polypeptide for presentation on the surface of an APC; and introducing the plurality of recombinant nucleic acid molecules into APC cells for expression and/or display of the recombinant nucleic acid molecules. In one embodiment, the nucleotide sequence encoding one or more polypeptide for presentation on the surface of an APC is operably linked to a unique nucleotide barcode sequence encoding a predetermined or shared amino acid sequence.
Any method known in the art for introducing nucleic acid sequences into cells can be used to generate the APC library of the invention. Exemplary methods of introducing nucleic acid molecules into cells include, but are not limited to, electroporation, cell squeezing, sonoporation, optical transfection, protoplast fusion, impalefection, hydrodynamic delivery, fusion, magnetofection, particle bombardment, nucleofection, heat shock, lipofection, viral transduction, nonviral transfection, lithium acetate/PEG chemical transformation, or any combination thereof.
In one embodiment, the method comprises generating a library of cells for displaying polypeptides which function as antigens for TCR binding. Thus, in one embodiment, the method comprises generating a library of cells, wherein the library comprises cells comprising barcode-labeled nucleic acid sequences, wherein the barcode-labeled nucleic acid sequences encode polypeptides which function as antigens for TCR binding.
In one embodiment, the sequences of the recombinant nucleic acid molecules for inclusion in the minigene library of the invention, or sequences for expression by the APCs of the invention, are determined using the Fast ALgorithm for Codon OptimizatioN (herein referred to as FALCON), which optimizes nucleotide sequences according to multiple parameters for a high-yield protein expression. The FALCON codon optimization process starts with a list of amino acid sequences corresponding to the genes that will be optimized. The system in which the genes will be expressed is chosen, and the sequences are back-translated using a weighted random codon selection function. In the process of codon selection, a codon usage table specific for the expression system is used. In this table, each codon is stored along its particular weight or selection probability. These weights reflect the single and bi-codon usage pattern in highly expressed genes of the expression system.
In one embodiment, the codon usage table selected is one designed in which codons with a higher representation have higher weights. Additionally, the codon weights also reflect the abundance of their cognate tRNAs. Thus, in one embodiment, the more commonly used codons for which respective tRNAs abound are more likely to be selected. This supports efficient protein translation in cells, as it promotes the usage of tRNAs that are more readily available. In some embodiments, the codon selection process includes a series of adjustment layers that modifies the codon weights according to the GC content of the growing nucleotide sequence and codon autocorrelation (i.e., the bias that exists in a number of codons that are correlated with themselves and other codons). For example, during the back-translation process, if a codon was already used by FALCON within the last 25 nucleotide triplets, its likelihood to be selected again will be proportionally higher to the distance of its last occurrence (distance in codons).
In some embodiments, the minigene library is optimized for a desired GC content. In some embodiments, the GC content optimization is performed using a 4-parameter logistic function (4PL). In this function, the four parameters; A, B, C and D, and the independent variable ‘x’; are used to calculate the correction coefficient ‘y’, according to equation 1 (
In some embodiments, after 10 codons have been selected, the sequence is subjected to several motif avoidance steps. In some embodiments, restriction sites as well as other undesired patterns are eliminated.
In some embodiments, the process of back-translation and motif avoidance is repeated continuously until an optimized minigene library is obtained.
In some embodiments, to promote efficient translation initiation, FALCON optimizes the 5′ start of the first 20 codons, by generating 10 candidate partial sequences using back-translation, and then selecting the sequence with the highest minimum free energy (MFE). A higher MFE is indicative of a less stable mRNA secondary structure, which correlates with higher expression levels (Jia & Li, 2005, FEBS Letters, 579(24), 5333-5337). The MFE is calculated using the open-source ‘seqfold’ package, developed by JJTimons (pypi.org/project/seqfold/). The optimized 5′ start will then be used by FALCON as a starting point to generate 10 full candidate sequences. Subsequently, a step termed “Tournament style selection” follows.
In “Tournament style selection” the utilization of a weighted, yet random, codon selection approach is used such that if a gene is optimized multiple times, virtually every resulting sequence will differ to a degree from each other. This characteristic gives FALCON the advantage of generating multiple contesting full sequences, which can then be evaluated according to specific quality criteria. For each gene, FALCON creates 10 candidates, and the highest-ranking gene is selelected according to the following three parameters:
The CAI is a measure widely used to quantify codon preferences toward most common codons. A sequence that only uses the most common codons for a particular organism will have a CAI=1. Deviations from this usage will result in lower CAI values. FALCON first calculates the codon relative adaptiveness (CRA) of each candidate sequence (equation 2;
Therefore, in some embodiments, the minigene library comprises sequences that have been optimized for expression using the FALCON system comprising the steps of:
In some embodiments, the invention is based, in part, on the development of transmembrane domain for enhanced antigen presentation. In some embodiments, the transmembrane domains of the invention enhance the level or stability of class II MHC presentation of antigens. Exemplary enhanced presentation transmembrane domains include, but are not limited to, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO: 122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO: 125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, and SEQ ID NO:135.
In some embodiments, the invention relates to fusion molecules comprising an enhanced transmembrane domain of the invention linked to an antigen for presentation on a cell surface. In some embodiments, the invention relates to nucleic acid molecules encoding fusion molecules comprising an enhanced transmembrane domain of the invention linked to an antigen for presentation on a cell surface.
In some embodiments, the invention relates to compositions comprising fusion molecules comprising an enhanced transmembrane domain of the invention linked to an antigen for presentation on a cell surface. In some embodiments, the invention relates to compositions comprising nucleic acid molecules encoding fusion molecules comprising an enhanced transmembrane domain of the invention linked to an antigen for presentation on a cell surface.
For example, in some embodiments, the invention provides compositions comprising a viral antigen linked to a transmembrane domain for presentation on the surface of a cell. Such an embodiment can be used to induce an immune response against the displayed antigen or to increase the efficacy of a vaccine. However, the invention is not limited by to any specific antigen or use, as any antigen or library of antigens can be linked to a transmembrane domain of the invention for enhanced display.
In some embodiments, invention provides a method of co-culturing at least one T cell of the invention with an APC library expressing antigens encoded by the minigene library invention, to identify the antigenic polypeptide target of the TCR of the T cell. The co-culture can be performed using any co-culture system including, but not limited to, cell culture plate-based systems, slide based systems, emulsion droplet based systems or other systems for co-culture of cells. In some embodiments, the co-culture is performed using a microfluidic co-culture device. An exemplary microfluidic co-culture device that can be used to co-culture an APC library of the invention and a T cell of the invention is described below, however, the invention should not be seen as being limited to the use of the described microfluidic co-culture device.
Referring now to
Device 100 can be fabricated as recesses in a substrate and sealed with a cover to form fluidly connected channels and structures. Contemplated substrate and cover materials include but are not limited to: silicon, glass, ceramic, metals, composites, inorganic materials and organic polymers, such as polydimethylsiloxane (PDMS), thermoset polyester, polystyrene, polycarbonate, poly-methyl methacrylate (PMMA), poly-ethylene glycol diacrylate (PEGDA), perfluorinated polymers (e.g., fluorinated ethylene propylene, perfluoroalkoxy, perfluoropolyether, etc.), polyurethane, and the like. In some embodiments, substrate materials may be selected based on a fabrication method of device 100. For example, in some embodiments a PDMS or thermoset polymer substrate material may be selected to fabricate a microfluidic device through casting with a mold, and in some embodiments a silicon, glass, or ceramic substrate material may be selected to fabricate a microfluidic device through lithography or etching. In some embodiments, certain components or portions of certain components can be constructed from a transparent or translucent material.
Referring now to
Downstream from stream separators 120, filter 108 can further comprise a filtering region comprising a plurality of pillars 122. Pillars 122 are spaced apart from each other such that individual particles are encouraged to separate from each other while minimizing the likelihood of particles clumping and clogging between each pillar 122. While pillars in microfluidic devices are generally understood as filtering elements, wherein decreasing spacing between pillars increases particle separation, the present invention is based in part on the surprising and unexpected discovery that increasing spacing between pillars achieves effective particle separation while minimizing the probability or occurrences of clogging. Pillars 122 can be spaced apart by any desired distance, such as between about 50 μm and 5000 μm. Spacing can be regular or irregular. Pillars 122 can have any desired cross-sectional shape, such as a circle, oval, triangle, square, rectangle, diamond, polygon, V-shape, U-shape, and the like. Pillars 122 can have any desired size, such as a width or diameter between about 1 μm and 100 μm.
The bottom image in
While the focusing loops 112 are depicted in
While the focusing loops 112 are depicted in
In various embodiments, device 100 can comprise one or more additional structures configured to augment its function. For example, device 100 can comprise one or more flow modulators configured to alter fluid flow within device 100. Contemplated flow modulators include but are not limited to valves, fluid resistors, expandable elements, contractible elements, pumps, membranes, and the like. Exemplary fluid resistors 110 are depicted in
In some embodiments, device 100 can further comprise one or more additional inlets, such as inlet 106b shown in
Device 100 can have any suitable dimensions. For example, device 100 can have an overall length between about 5000 μm to about 50000 μm. The fluid passageways of device 100 can have a channel height of between about 10 μm and 100 μm, or about 35 μm. The fluid passageways of device 100 can have a channel width of between about 5 μm and 100 μm. Channel height and channel width can be constant throughout device 100 or different in certain sections of device 100.
The microfluidic devices of the present invention can be fabricated using any suitable method known in the art. The method of making may vary depending on the materials used. For example, devices substantially comprising a metal may be milled from a larger block of metal or may be cast from molten metal. Likewise, components substantially comprising a plastic or polymer may be milled from a larger block, cast, or injection molded. In some embodiments, the devices may be made using 3D printing or other additive manufacturing techniques commonly used in the art. In some embodiments, the methods can embed additional components, such as circuitry, electrodes, magnets, diodes, and the like, such that the resulting device can be electrified to support electroporation, photoporation, magnetic fields, and the like. In various embodiments, coatings, patterns, and other finely detailed features can be applied using techniques such as etching, lithography, deposition, spin coating, dip coating, and the like. In some embodiments, coatings and patterns can include one or more capture agents immobilized on an inner surface of the device. Capture agents have an affinity to analytes of interest and can be used for a number of purposes, including but not limited to detecting analyte presence, quantifying analyte amount, and filtering analytes from a solution. In some embodiments, certain capture agents employ nonspecific binding. In some embodiments, certain capture agents employ size-specific filtration and do not rely on binding. Contemplated capture agents include at least antibodies, antigens, aptamers, affibodies, proteins, peptides, nucleic acids, carbon nanotubes, nanowires, magnetic beads, and fragments thereof. The capture molecules can be provided in a uniform coating, in an array pattern, or in any shape or form desired.
In certain embodiments, the devices are cast from molds. For example, a positive mold can be fabricated through patterning with SU-8 photoresist and soft lithography. For an exemplary device, a positive mold can comprise a channel layer having an exemplary 35 μm height. Curing a polymer with the mold would result in a substrate having a top surface with corresponding embedded channels having an exemplary 35 μm depth be included in the mold. Inlets, outlets, reservoirs, and the like can be added by punching or milling. The top surface of the substrate can be sealed using a layer of metal, glass, or polymer. In various embodiments, the device can be cleaned or sterilized using any sterilization techniques, including but not limited to autoclaving, gamma ray sterilization, electron beam sterilization, and the application of any sterilizing gas, plasma, or solution, such as ethylene oxide, chlorine dioxide, hydrogen peroxide, oxygen plasma, and the like.
In some embodiments, the devices are fabricated in the form of an array on a microfluidic chip. Referring now to
In some embodiments, the present invention relates to particle suspension reservoirs configured to supply input fluids to or receive output fluids from microfluidic devices described elsewhere herein. Referring now to
In some embodiments, the microfluidic device of the invention is used to co-culture an APC of the invention and a T cell of the invention. In such an embodiment, at least one T cell of the invention is applied to one inlet 106 of the microfluidic device 100 and at least one APC of the invention is applied to a second inlet 106 of the microfluidic device 100. The APC and the T cell of the invention then are flowed through the microfluidic device and combined at the nozzle of the microfluidic device such that each emulsion droplet comprises a number (n) of APCs, where n is 0, 1, 2, 3, 4 or 5, and a number (m) of T cells where m is 0, 1, 2, 3, 4 or 5.
In one embodiment, the APC applied to the microfluidic device comprises a B cell. In one embodiment, a B cell suspension applied to the microfluidic device comprises EDTA.
In one embodiment, the T cell suspension applied to the microfluidic device comprises Ca2+. In one embodiment, the Ca2+ is present in the T cell suspension at least at an equimolar concentration as that of the EDTA present in the B cell suspension.
The present invention also includes methods of using microfluidic devices for the co-encapsulation of individual particles (such as individual cells). As described elsewhere herein, the microfluidic devices of the present invention are capable of generating longitudinal flows of individual particles from fluid suspensions, combining two or more individual particle flows into a single stream of individual particles, and segmenting the single stream of individual particles using an isolation fluid such that each stream segment co-encapsulates an individual particle from each fluid suspension.
Contemplated particles can include but are not limited to: cells, viruses, bacteria, amoeba, protozoa, paramecium, microparticles, nanoparticles, beads, microorganisms, vesicles, nucleic acid oligonucleotides, proteins, polypeptides, carbohydrates, and fragments thereof. The particles can be provided in any desired suspension fluid, including but not limited to water, cell growth media, serum, plasma, oil, and the like. Isolation fluids can be any desired fluid, including but not limited to oils, gels, liquid metals, liquid polymers, glues, and the like. Isolation fluids and can be selected such that the isolation fluid is immiscible with the particle suspension fluids. For example, oil-based isolation fluids can be selected for aqueous suspension fluids, and vice versa (where aqueous isolation fluids can be selected for oil-based suspension fluids).
As described elsewhere herein, particle suspensions can include one or more additives that enhance particle separation into an individual particle flow. The microfluidic devices can also accept one or more reagents that are added into an individual particle flow or a combined individual particle stream for co-encapsulation. In various embodiments, the additives and reagents can include natural or synthetic drugs, including but not limited to: analgesics, anesthetics, antifungals, antibiotics, anti-inflammatories, nonsteroidal anti-inflammatory drugs (NSAIDs), anthelmintics, antidotes, antiemetics, antihistamines, anti-cancer drugs, antihypertensives, antimalarials, antimicrobials, antipsychotics, antipyretics, antiseptics, antiarthritics, antituberculotics, antitussives, antivirals, cardioactive drugs, cathartics, chemotherapeutic agents, a colored or fluorescent imaging agent, corticoids (such as steroids), antidepressants, depressants, diagnostic aids, diuretics, enzymes, expectorants, hormones, hypnotics, minerals, nutritional supplements, parasympathomimetics, potassium supplements, radiation sensitizers, a radioisotope, fluorescent nanoparticles such as nanodiamonds, sedatives, sulfonamides, stimulants, sympathomimetics, tranquilizers, urinary anti-infectives, vasoconstrictors, vasodilators, vitamins, xanthine derivatives, and the like. The therapeutic agent may also be other small organic molecules, naturally isolated entities or their analogs, organometallic agents, chelated metals or metal salts, peptide-based drugs, or peptidic or non-peptidic receptor targeting or binding agents. The therapeutic agent may also be a large molecule, such as a monoclonal antibody or other recombinant protein.
In some embodiments, the additives include a chelating agent. For example, B cells have a natural tendency to clump together in suspension, which impedes the generation of a flow of individual B cells and increases the likelihood of clogging. A chelator, such as a calcium ion chelator, can be added to a B-cell suspension to inhibit clumping of B cells. Contemplated chelating agents include but are not limited to EDTA and EGTA. In some embodiments, the additives include an ion solution to compensate for chelated ions. For example, T-cell activation signaling relies on extracellular calcium. However, co-encapsulating a B-cell suspension having a calcium ion chelator with a T-cell suspension would inhibit T-cell activation. Accordingly, calcium can be added to the T-cell suspension to compensate, thereby restoring a baseline calcium concentration of a resultant co-encapsulated B cell and T cell.
In various embodiments, the additives and reagents can include natural peptides, such as glycyl-arginyl-glycyl-aspartyl-serine (GRGDS), arginylglycylaspartic acid (RGD), and amelogenin. In some embodiments, the surface treatments can include sucrose, fructose, cellulose, or mannitol. In some embodiments, the surface treatments can include nutrients, such as bovine serum albumin. In some embodiments, the surface treatments can include vitamins, such as vitamin B2, vitamin Ad, Vitamin D, Vitamin E, and Vitamin K. In some embodiments, the surface treatments can include nucleic acids, such as mRNA and DNA. In some embodiments, the surface treatments can include natural or synthetic steroids and hormones, such as dexamethasone, hydrocortisone, estrogens, and its derivatives. In some embodiments, the surface treatments can include growth factors, such as fibroblast growth factor (FGF), transforming growth factor beta (TGF-β), and epidermal growth factor (EGF). In some embodiments, the surface treatments can include a delivery vehicle, such as nanoparticles, microparticles, liposomes, viral and non-viral transfection systems.
Additional contemplated additives and reagents include but are not limited to: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; stabilizing agents; enzymes; nucleic acids; and the like.
The present invention relates, in part, to methods of identifying binding partners of T cell receptors (TCRs). In one embodiment, the method comprises identifying an antigenic polypeptide-HLA complex that specifically binds to a TCR of interest. In one embodiment, the method comprises identifying novel TCR:antigenic peptide-HLA complex interactions.
In one embodiment, the invention relates to a screening method for TCR-antigen interactions, wherein the method comprises generating a library of polypeptides that are then screened for interactions with at least one TCR. Therefore, in one embodiment, the invention relates to a polypeptide display library and methods of use thereof for screening for TCR:antigenic peptide-HLA complex interactions.
The present invention provides, in part, a method of identifying TCR-antigen interactions that are associated with a disease or disorder. In one embodiment, the APC library of the invention comprises a plurality of APCs purified from a biological sample obtained from a subject having a disease or disorder, and transfected with a minigene library of the invention. In one embodiment, the TCR is a recombinant TCR generated based on genetic information from a TCR of a subject having a disease or disorder. In one embodiment, the method comprises contacting one or more cells of an APC library of the invention with a T cell expressing a TCR. In some embodiments, the step of contacting one or more cells of an APC library of the invention with a T cell is performed using a plate-based co-culture system, a slide-based co-culture system, an emulsion based co-culture system, a chip-based co-culture system, a microfluidic co-culture device, or any appropriate device or system for co-culturing an APC and T cell of the invention. For example, in one embodiment, the contacting of an APC and a T cell is performed using the microfluidic co-culture device described elsewhere herein. However, the present invention is not limited to the device described herein. In one embodiment, the contacting of an APC and a T cell is performed in a single-well or multi-well cell culture plate.
In certain embodiments, the TCR expressed by the T ell is derived from a subject. In one embodiment, the subject has been diagnosed as having a disease or disorder. In one embodiment, the APC library is generated from immortalized B cells derived from a subject having a disease or disorder. In one embodiment, the disease or disorder is selected from an autoimmune disease or disorder, cancer, an inflammatory disease or disorder, a metabolic disease or disorder, a neurodegenerative disease or disorder, a disease or disorder associated with an infectious agent, or any combination thereof.
In one embodiment, the TCR is identified to be reactive with an antigen expressed by an APC based on expression of a marker operably linked to a TCR responsive promoter. For example, in some embodiments, the TCR is identified to be reactive with an antigen expressed by an APC based on the induction of expression of a fluorescent marker (e.g. GFP) operably linked to a TCR responsive promoter. In some embodiments, the TCR is identified to be reactive with an antigen expressed by an APC based on the induction of expression of an APC tagging molecule operably linked to a TCR responsive promoter.
In one embodiment, the TCR is identified to be reactive with an antigen expressed by an APC when the APC is tagged with an APC tagging molecule of the invention (e.g., a CD19 or CD20 scFv molecule expressed by the T cell).
In one embodiment, an APC bound by an APC tag of the invention (e.g. a CD19 or CD20 scFv) is identified using any appropriate sorting or selection method. Exemplary sorting and selection methods include, but are not limited to, affinity-based selection methods, pull down assays using a molecule having affinity to a tag (e.g., Flag or HA tags), biotinylated labeled anti-immunoglobulin antibody, fluorescence activated cell sorting (FACS), fluorescently labeled anti-immunoglobulin antibody, magnetic bead-based selection, magnetic bead conjugated to an anti-immunoglobulin antibody, or any combination thereof. In one embodiment, the method comprises isolating and enriching at least one antibody-bound APC.
In one embodiment, the method comprises amplifying the barcoded recombinant nucleic acid molecule (minigene) of the antibody-bound APC. In one embodiment, the method comprises sequencing the barcoded recombinant nucleic acid molecule (minigene) to identify the antigen associated with the TCR. In one embodiment, the screening methods of the invention include a step of isolating and sequencing barcoded nucleic acid molecules from a plurality of antibody-bound (tagged) APCs.
In one embodiment, the methods described herein can utilize next-generation sequencing technologies that allow multiple samples to be sequenced individually as genomic molecules (i.e., singleplex sequencing) or as pooled samples comprising indexed genomic molecules (e.g., multiplex sequencing) on a single sequencing run. These methods can generate up to several hundred million reads of DNA sequences. In various embodiments, the sequences of nucleic acid sequence barcodes, and thus the associated antigen sequence(s) can be determined using, for example, the next generation sequencing technologies described herein. In various embodiments, analysis of the massive amount of sequence data obtained using next-generation sequencing can be performed using one or more processors.
In some embodiments, the nucleic acid product can be sequenced by next generation sequencing methods. In some embodiments, the next generation sequencing method comprises a method selected from the group consisting of Ion Torrent, Illumina, SOLiD, 454; Massively Parallel Signature Sequencing, solid phase reversible dye terminator sequencing; and DNA nanoball sequencing may be included. In some embodiments, the first and second sequencing primers are compatible with the selected next generation sequencing method.
In some embodiments, sequencing can be performed by next generation sequencing methods. As used herein, “next generation sequencing” refers to methods that were developed to increase the speed, capacity and accuracy of generating sequence data over those that were possible with conventional sequencing methods (e.g., Sanger sequencing) by reading thousands of millions of sequencing reactions simultaneously. Non-limiting examples of next generation sequencing methods/platforms include Massively Parallel Signature Sequencing (Lynx Therapeutics); pyrophosphate sequencing/454; 454 Life Sciences/Roche Diagnostics; Solid Phase Reversible Dye Terminator Sequencing (Solexa/illumina): SOLiD technology (Applied Biosystems); ion semiconductor sequencing (ION Torrent.); DNA nanoball sequencing (Complete Genomics); and technologies available from Pacific Biosciences, Intelligen Bio-systems, Oxford Nanopore Technologies, and Helicos Biosciences. In some embodiments, the sequencing primer may comprise a moiety that is compatible with the selected next generation sequencing method.
Next generation sequencing techniques and related sequencing primer constraints and design parameters are well known in the art (e.g., Shendure et al., 2008, Nature, 26:1135-1145; Mardis, 2007, Trends in Genetics, 24:133-141; Su et al., 2011, Expert. Rev. Mol. Diagn., 11:333-43; Zhang et al., 2011, J. Genet. Genomics, 38:95-109; Nyren P et al. 1993, Anal. Biochem., 208:17175; Bentley et al., 2006, Curr. Opin. Genet. Dev., 16:545-552; Strausberg et al., 2008, Drug Disc. Today, 13:569-577; U.S. Pat. Nos. 7,282,337; 7,279,563; 7,226,720; 7,220,549; 7,169,560; U.S. Patent Application Publication No. 20070070349; U.S. Pat. Nos. 6,818,395; 6,911,345; U.S. Patent Application Publication No. 2006/0252077; No. 2007/0070349).
Several targeted next generation sequencing methods are described in the literature (for review see e.g., Teer and Mullikin, 2010, Human Mol. Genet. 19:R145-151), all of which can be used in conjunction with the present invention. Many of these methods (described e.g. as genome capture, genome partitioning, genome enrichment etc.) use hybridization techniques and include array-based (e.g., Hodges et al., 2007, Nat. Genet., 39:1522-1527) and liquid based (e.g., Choi et al., 2009, Proc. Natl. Acad. Sci USA, 106:19096-19101) hybridization approaches. Commercial kits for DNA sample preparation are also available: for example, Illumina Inc. (San Diego, California) offers the TruSeq™ DNA Sample Preparation Kit and the Exome Enrichment Kit TruSeq™ Exome Enrichment Kit.
There are many methods known in the art for the detection, identification, and quantification of specific nucleic acid sequences (e.g., nucleic acid sequence barcodes) and new methods are continually reported. A great majority of the known specific nucleic acid detection, identification, and quantification methods utilize nucleic acid probes in specific hybridization reactions. Many methods useful for the detection and quantification of nucleic acid take advantage of the polymerase chain reaction (PCR). The PCR process is well known in the art (U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159). To briefly summarize PCR, nucleic acid primers, complementary to opposite strands of a nucleic acid amplification target sequence, are permitted to anneal to the denatured sample. A DNA polymerase (typically heat stable) extends the DNA duplex from the hybridized primer. The process is repeated to amplify the nucleic acid target. If the nucleic acid primers do not hybridize to the sample, then there is no corresponding amplified PCR product. In this case, the PCR primer acts as a hybridization probe. In one embodiment, the method of sequencing the minigene comprises one or more PCR step.
The invention also includes a kit comprising components useful within the methods of the invention and an instructional material that describes, for instance, an APC library of the invention, a modified T cell of the invention, a microfluidic co-encapsulation devices, and methods of use of the APC library, the T cell and/or methods of using the microfluidic co-encapsulation device as described elsewhere herein. The kit may comprise components and materials useful for performing a co-culture assay of the invention. The kit may also comprise a premade microfluidic co-encapsulation device suitable for separating and co-encapsulating a T cell and APC cell of the invention.
The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically illustrate the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.
This invention outlines the establishment of a functional, high-throughput TCR screening platform. A system to de-orphanize autoreactive TCRs needed to be able to de-orphanize TCRs that are patient-specific (autologous HLA haplotypes), detect both class I and class II-presented peptides, not be restricted to known autoantigenic peptides, detect post-translationally modified peptides, and be agnostic to the presence of autoantibodies.
The system of the invention provides a cellular interaction screening method in which a TCR activity reporter cell expressing a patient-derived TCR is co-cultured with a patient-derived antigen-presenting cell expressing a disease-derived antigen library. The interacting cells are then isolated and the TCR-interacting antigen is sequenced (
The screening method includes multiple components including a T cell secreted αCD19 scFv as a B cell targeting reporter (
T lymphocytes comprise approximately 20% of all leukocytes in human peripheral blood and are a major component of the adaptive immune system. A unique feature of each newly formed T cell is its T cell receptor (TCR), which is formed during T cell genesis in the bone marrow and thymus via somatic recombination of genomic elements. As such, each TCR has a unique ligand-binding site, forming a receptor repertoire with the potential to bind up to 1022 distinct ligands. In order to aid in the diagnosis, prevention, and treatment of infectious disease, autoimmune disorders, and cancer, it is crucial to catalog functional relationships between TCRs and their ligands.
A platform was developed for the rapid deconvolution of orphaned TCR sequences. The basis of the system is a cellular interaction screen between T cell lines transgenically modified to express TCRs of interest, as well as genetic response elements (“promoters”) which are inducible upon TCR:cognate ligand binding, and antigen-presenting cells (APCs) which have been transgenically modified to ectopically express a potential cognate ligand. Induction of the TCR signal upon ligand binding indicates a functional “match” between the interrogated TCR sequence and the ectopic antigen expressed by the APC. However, the APC is the cell containing the genetic information required to identify the cognate antigen; therefore, it is necessary to transmit the induced T cell signal onto the APC for isolation.
The invention is based in part on the development of a secreted protein that specifically binds to the APCs being used. In this platform, the APCs are Epstein-Barr virally immortalized B cells, which specifically express the proteins CD19 and CD20 on their surface. Single-chain variable fragments (scFv) from CD19- and CD20-binding monoclonal antibodies have been engineered to be produced by activated T cells, which subsequently bind to and label the interacting B cell during co-culture. Because the scFv expression is under the control of a TCR-signaling induced response element, non-cognate B cells will not elicit secreting of the signal from interacting T cells, and will therefore not be labeled. B cells in the co-culture can then be isolated from the T cells and sorted based on whether or not the labeling antibody fragments have bound to their surface, providing an enriched population of B cells which express cognate antigens.
The experimental results are now described.
Current generation T cell reporter systems consists of a cis-regulatory element comprising multiple copies of a synthetic promoter sequence (‘NBV’). This promoter was observed to sensitively respond to T cell receptor engagement and drives the expression of downstream reporter elements consisting of a CD19-specific secreted single-chain variable fragment and cytoplasmic sfGFP. The scFv is derived from a monoclonal αCD19-antibody (clone B43) binding to the B cell-specific transmembrane protein CD19, which is commonly used in monoclonal antibody-mediated immunotherapy (Uckun et al., 1988, Blood, 71(1):13-29). It is tagged with six HA epitope tags at its C-terminus, which facilitates its detection by flow cytometry. To optimize the current reporter and B cell labeling strategy, the potential of additional scFvs was tested. In particular, an αCD20-scFv derived from a human monoclonal antibody was evaluated that recognizes an epitope encompassing the membrane-proximal small-loop on the CD20 molecule, known as ofatumumab (clone 2F2) (Teeling et al., 2004, Blood, 104(6):1793-800). In order to test its functionality, scFv constructs were assembled in both the VH-VL and VL-VH configuration. The heavy and light chain domains were separated by a flexible polypeptide linker (G4S)3. To confirm that ofatumumab, as an scFv tagged with four copies of the V5 label, a small epitope-tag found on P and V proteins of the paramyxovirus, can efficiently label BOLETH cells and be detected by flow cytometry, the coding region was inserted into a CMV expression plasmid. HEK293 cells were transfected with the construct using TurboFect. As a negative control, HEK293 cells were transfected with a sfGFP-containing construct. Medium was exchanged 24 hours post-transfection. The scFv-containing supernatant (SN) was collected 72 hours post-transfection and was used to label BOLETH cells. Using fluorescent dye-conjugated antibodies recognizing the tagged scFvs, the amount of B cell bound scFv was measured by flow cytometry (
In comparison to the previously used αCD19-scFv (clone B43), the ofatumumab derived αCD20-scFv (clone 2F2) labels BOLETH cells equally well and detection by flow cytometry provides similar signal strength measured by median fluorescence intensity (MFI) (
To further optimize the potential use of the αCD20-scFv, various peptide tags and configurations were tested. First, the quadruple V5-tag was exchanged with the 6×HA from the αCD19-scFv using InFusion cloning. Determination of signal strength by flow cytometry showed that the MFI obtained with V5-tagged αCD20-scFv is moderately higher than detection of the six-times HA-tagged scFv (
Since these changes did not meet the anticipated signal optimization, the configuration of αCD20-scFv (clone 2F2) was changed in the expression plasmid from heavy-chain linker light-chain (VHVL) to light-chain followed by linker and heavy chain (VLVH). This configuration change to VLVH leads to the highest signal strength detected across all tested scFvs (
The current reporter only allows for one-dimensional sorting of αCD19-HA labeled B cells. To improve the reporter to achieve a dual labeling of B cells that present a cognate antigen and to allow sorting of the labeled B cells in two dimensions, tandem scFv reporters were designed and cloned and their functionality was tested with the labeling protocol described above. Both scFvs were connected by T2A peptide sequences that induce ribosomal skipping during translation to produce two independent proteins. Adding both αCD19-HA (B43) and αCD20-V5 (2F2) into such expression constructs drastically reduces the signal strength coming from the αCD20 scFv (
To investigate the reason behind the significantly reduced signal from αCD20-V5 scFv in tandem constructs compared to single scFv constructs, such as expression of the scFvs or steric hindrance during binding to the receptors, an experiment was performed in which the SN from HEK cells transfected with either single constructs, a tandem construct or single constructs in a co-transfection was used to label BOLETH cells. An additional condition in which the SN of single transfected single-scFv constructs was mixed was performed to provide information about the level in which the signal from the αCD20 is inhibited. Again, it was observed that the αCD19-scFv signal is not affected by any of the conditions and constructs used (
To design a reporter that allows for a double labeling and therefore for a 2-dimensional sorting of scFv-bound B cells, a bispecific diabody consisting of both, the αCD19- and αCD20-scFv, connected by a flexible-rigid-flexible linker sequence (GGGGSAEAAAKEAAAKAGGGGS; SEQ ID NO:49) was tested for its ability to label B cells. The bispecific diabody was N-terminally HA- and C-terminally V5-tagged (
In summary, the experiments demonstrate that using the ofatumumab-derived αCD20-scFv in VLVH configuration could significantly improve the signal detected from B cell-bound tagged-scFvs.
Finally, to further increase the signal strength of the previously tested reporters, the leader sequence of ofatumumab-αCD20 scFv was exchanged with the leader sequence of αCD19 (B43) (
Testing of the Optimized scFv-Reporter Constructs Secreted by T Cells
Since previous experiments only represent an optimal setting of B cell labeling in the presence of saturating scFv concentrations, certain tested scFvs were cloned into lenti-viral reporter constructs (pLVX) consisting of a double NBV promoter element and a downstream T2A sequence connecting sfGFP. αCD19- (clone B43) and αCD20-scFv (clone 2F2, VLVH) both, HA and V5-tagged, as well as a tandem construct containing αCD19-HA and αCD20-V5 and the bispecific diabody were cloned into pLVX using InFusion cloning (
To conclusively answer whether the αCD20-scFv (clone 2F2) or αCD19-scFv (clone B43) or the tags (HA vs. V5) recognized by the fluorophore labeled antibodies are responsible for the variation in signal intensity, six-times HA was added to the αCD20-scFv (VLVH), while four-times V5 was cloned onto αCD19-scFv (
Optimization of scFv Design
ScFv inserts αCD20-Rituximab (C2B8) and Ofatumumab (2F2) were cloned into pLVX-PGK-BSD containing αCD19-HA-scFv (B43) and were tested with two different tags (V5 and FLAG) (
Transient expression plasmids were tested for optimizing B cell scFv labeling. ScFv inserts αCD20-Ofatumumab (2F2) or αCD19 (B43) were cloned into pTwist CMV BetaGlobin WPRE Neo (
A comparison between HA-tag on αCD19-scFv (B43) and αCD20-scFv (2F2) was performed. HA signal was evaluated on B cells stained with the supernatant of transfected HEK cells secreting the respective scFvs (
A comparison between HA- and V5-tag on αCD20-scFv(2F2) was performed. HA and V5 signals were evaluated on B cells stained with the supernatant of transfected HEK cells secreting the respective scFvs (
A comparison between the intensity resulting from single scFv constructs and double scFv constructs was performed. HA and V5 signals were evaluated on B cells stained with the supernatant of transfected HEK cells secreting the respective scFvs (
A comparison of locating the scFvs upstream or downstream of the T2A sequence was performed. HA and V5 signals were evaluated on B cells stained with the supernatant of transfected HEK cells secreting the respective scFvs (
A comparison of αCD20-V5 scFv light and heavy chain arrangement was performed. The V5 signal was evaluated on B cells stained with the supernatant transfected HEK cells secreting the respective scFvs (
The materials and methods are now described.
Sequences encoding anti-CD19 [clone FMC63 (Nicholson, et al., 1997, Mol Immunol, 34(16-17):1157-65) and clone B43 (Uckun et al., 1988, Blood, 71(1):13-29)] and anti-CD20 [clone 2F2 (Ofatumumab, Teeling et al., 2004, Blood, 104(6):1793-800) and clone C2B8 (Rituximab, Reff et al., 1994, Blood, 83(2):435-45)] variable heavy and variable light chain domains were identified and isolated. All amino acid sequences were back-translated to nucleotide sequences and synthesized by Twist Biosciences.
In order to test functionality, scFv constructs were assembled in both VH-VL and VL-VH configurations. The heavy and light domains were separated by a flexible polypeptide linker, e.g. (G4S)3 or GSTSGSGKPGSGEGSTKG (SEQ ID NO:42) (for the latter, cf Whitlow et al., 1993, Protein Eng, 6(8):989-95). An immunoglobulin domain signal peptide for the secretory pathway was included at the N-terminus of each construct (MEFGLSWLFLVAILKGVQC; SEQ ID NO:43). For identification of labeled cells with dye-conjugated antibodies, amino acid tags were included at either the N-terminus or the C-terminus of each construct. Examples of these tags are the 4×V5
For a tandem scFv, fragments for CD19 and CD20 were separated by the flexible-rigid-flexible linker sequence GGGGSAEAAAKEAAAKAGGGGS (SEQ ID NO:49).
Co-culture experiments were performed using round-bottom 96-well plates. Jurkat TT2 T cells expressing the scFv reporter constructs and B cells are seeded at a 3-to-1 ratio into each well. Either 1 μM of cognate or negative control peptide was added to each well and incubated for 16 hours. Subsequently, cells are spun down and stained for flow cytometry.
For optimizing scFv reporter constructs, constructs containing combinations of B43 and 2F2 tagged with either HA or V5 were cloned in pTwist CMV WPRE Neo BetaGlobin. For testing, HEK cells were transiently transfected with the plasmids using TurboFect (Fermentas). At 24 hours post-transfection, medium was replaced with normal HEK culture medium. At 72 hours post-transfection, the supernatant was recovered, centrifuged, and used to stain BOLETH cells. Subsequently, cells were centrifuged and stained with fluorescent dye-conjugated antibodies for flow cytometric analysis.
A T cell reporter system has been developed that is activated upon TCR engagement by its cognate HLA:peptide complex. This system is based on a potent transgenic cassette introduced into reporter T cell lines using the Sleeping Beauty transposon system, a nonviral DNA tool that can stably integrate DNA sequences into the eukaryotic cells.
The T cell reporter system developed and shown here has several important components: the cis-regulatory nucleotide sequences for recruiting transcription factors in response to T cell activation herein called “promoters”, a synthetic transcriptional co-activator to amplify endogenous signal transduction, tagged signaling accessory molecules CD19 single-chain variable fragment tagged with HA, and sfGFP.
Upon TCR engagement by its cognate HLA:peptide complex, endogenous NFAT and other transcription factors are activated, as well as the synthetic transcriptional co-activator (NBV eTF), and recruited to the promoters that contains their binding sites. The T cell reporter system's promoters drive the expression of CD-19 scFV with the 3 HA tags, and sfGFP. Expressed sfGFP marks the T cell that is being activated; whereas the secreted CD19-scFV with 3 HA tags can bind to the activating B cell, the antigen-presenting cell, which have the cognate peptide on its HLAs.
The ectopic transcription factor NBV (‘NFAT-Bob1-VP16’) is a fusion protein consisting of the following elements: the cytoplasmic retention and DNA-binding domains from the N′-terminus of the nuclear factor in activated T cells (NFAT), the octamer motif (‘ATGCAAAT’)-binding domain from the transcriptional co-activator Bob1, and the C′-terminal transactivation domain (TAD) from the herpesvirus VP16 protein. The functional principle is that this transcription factor is recruited to the nucleus in response to calcium-induced signaling from the TCR by virtue of its NFAT-derived N′-terminus, upon which it can bind to NFAT and octamer motifs in synthetic promoters. It can then recruit the transcription factors Oct1/2 through its central Bob1-derived domain, as well as drive transcription on its own via the C′-terminal VP16 TAD.
When introducing transgenic constructs via transposable elements, construct size is not as absolute a constraint as when working with other systems (e.g., lentiviral particles). Therefore, several additional design features into the construct in order to optimize its TCR signaling-dependent response. These features include:
T cell activation is defined primarily by the interaction between the TCR itself and its the peptide-binding HLA molecule on the surface of the APC. However, this interaction and its downstream signaling cascades are mediated by additional surface molecules found on both the T cell and the APC. The strongest and best-studied interaction is that of the CD28 surface molecule on the T cells with members of the B7 family expressed on the surface of the APCs.
As a means to further potentiate minigene-based T cell activation in T cell lines used, surface receptors known to be involved in T cell activation were ectopically expressed and screened for functional synergy with the TCR-mediated signal. Constitutive expression of CD2, CD226, CD40L, ICOS, OX40, and 41BB was thus induced in TCR-expressing Jurkat and SKW-3 cells and then they were co-cultured with cognate minigene-transduced B cells. These were instrumental in potentiating T cell activation.
The materials and methods are now described:
BOLETH cells were used as B cells for antigen-presenting cells and they are maintained in RPMI 1640 medium supplemented with 10% FCS, Pen/Strep, Kanamycin, 1 mM sodium pyruvate, MEM non-essential amino acids, and 50 uM β-mercaptoethanol. T cell lines used were Jurkat and SKW-3 cells, maintained in RPMI 1640 medium supplemented with 10% FCS and Pen/Strep.
T cell receptors in lentiviral constructs are used to transfect HEK cells using the TransIT protocol. After 2 days, virus is collected and T cells are transduced. T cell medium is changed 3 days later and medium with 1 ug/ul puromycin is used for selection.
Transfection of T Cells with Reporters
Sleeping beauty transposon reporter constructs were transfected together with transposases into T cells, 5 to 1 ratio, using the Neon transfection system following the manufacturer's protocol. The voltages used for Jurkat cells were 1350V/10 W/3 pulse, and for SKW-3 cells 1500V, 30 W and 1 pulse were used as settings. 48-72 hours after transfection, the cells were selected using 10 μg/μl Blasticidin for Jurkat and 6 μg/μl Blasticidin for SKW-3 cells.
In order to induce minigene expression and subsequent presentation on HLA molecules, lentiviral particles driving expression of the respective minigene from a constitutive promoter were generated. Specifically, cognate or mismatch minigene were restriction-based cloned into the pLVX-EF1a-IRES-Puro vector (TAKARA). This vector was further modified to include a T2A polycistronic cassette in frame with the minigene, directly followed by a TagBFP2 fluorescent reporter. Moreover, a CMA sequence was introduced immediately upstream of the minigene's coding sequence to facilitate chaperon-mediated autophagy and subsequent peptide presentation on ClassII HLA molecules.
BOLETH cells (Sigma, 88052031-1VL) were cultured in 6-well cell culture plates in RMPI medium (Gibco, #61870143) supplemented with 10% FBS (HyClone), 1% penicillin-streptomycin, 1% kanamycin, 50 μM β-mercaptoethanol, 1 mM sodium pyruvate, and 1 mM NEAA (Gibco, #11140050) at 37° C. humidified incubator with 5% CO2. Cells were passaged every two to three days at a ratio of 1:2.
In order to induce minigene expression and presentation, lentiviral particles were generated via co-transfection into HEK 293FT cells in 6-well plates of 1.33 μg of the transfer vector alongside 1 ug of psPAX2 and 0.67 ug of pMD2.G accessory vectors using the TransIT VirusGEN transfection reagent (Mirus). Following 48 hours of incubation, supernatants were collected, filtered through a 0.45 μm PES filter. BOLETH cells were subsequently transduced by resuspending 1.5×106/6 well plate in a 1:1 viral supernatant dilution in the presence of 8 μg/ml of Polybrene (Millipore) and 2 mM BX795 (Sigma). After 24 hours of incubation cells were washed from virus-containing medium and resuspended with full B cell medium. Functional experiments were conducted 4-5 days following transduction.
The endogenous coding region of the human surface molecules CD2, CD226, CD40L, ICOS, OX40 and 41BB was ordered from TWIST bioscience directly cloned into the pTwist Lenti SFFV lentiviral vector. In these vectors, expression of each candidate is controlled by the SFFV-derived promoter sequence. Lentiviral supernatants were generated via co-transfection into HEK 293FT cells in 6-well plates of 1.33 μg of the transfer vector alongside 1 ug of psPAX2 and 0.67 μg of pMD2.G vector using the TransIT VirusGEN transfection reagent (Mirus). Following 48 h of incubation, supernatants were collected, filtered through a 0.45 μm PES filter. TCR-expressing Jurkat cells were then transduced in a 1:4 supernatant dilution ratio and TCR-expressing SKW3 cells were comparably transduced in a 1:2 dilution ratio. After 24 hours of incubation, cells were washed from virus-containing media and resuspended in full RPMI medium. Expression of above-mentioned surface receptors was then validated using flow-cytometry grade fluorescent dye-conjugated antibodies. Functional experiments were conducted 1 week post-transduction.
Co-culture experiments were performed using round-bottom 96-well plates. P10-NBV Jurkat and SKW-3 reporter cells and B cells were seeded at a 1:1 ratio into each well. For peptide co-culture experiments, either cognate peptide or mismatched peptide is added to each well and incubated for 16 hours. For minigene-based co-culture experiments, minigene-transduced B cells harboring either the cognate of mismatch minigenes were used. Next day the cells are spun down and stained for Flow cytometry.
The experimental results are now described.
Sleeping beauty-based transposon constructs were designed with TCR inducible reporter genes cassettes upstream of a constitutively expressed blasticidin resistance gene (
The sensitivity of the Jurkat-P10NBV reporter line with various TCRs targeting DR4-presented peptides from the tetanus toxin protein is shown in
The GFP and HA median intensities of B cells co cultured with 1 μM cognate or mismatched peptide and SKW-3 cells expressing sleeping beauty transposon system reporters is shown in
A 4×NFAT and NBV promoter comparison in sleeping beauty transposon system via co-culturing with B cells using different concentrations of cognate peptide is shown in
The GFP and HA median intensities of B cells co cultured with 1 μM cognate or mismatched peptide and Jurkat cells expressing sleeping beauty transposon system reporters is shown in
The ectopic expression of co-stimulatory molecules from lentiviral constructs can further increase reporter gene expression in Jurkat-NBV58-TT11 cells (
The TCR responsive promoter-driven synthetic transcription factor NBVtf can further increase reporter gene expression (
Surface expression of co-stimulatory proteins potentiates minigene-based T cell activation. TT2-expressing P10-NBV reporter Jurkat and SKW3 co-culture experiment summary depicting CD69 and GFP upregulation in T cells and anti CD19 scFv marking on cognate minigene-transduced B cells (
Element 1: synthetic transcription factor NBVtf, which is TCR signaling responsive expressed under the promoter P32.
Element 2: co-stimulatory molecules, which are constitutively expressed under commercial/published promoter PGK.
With the sleeping beauty construct shown in
The identification of foreign, self-, and neo-antigens presented by cells is an essential step in the prevention, diagnosis, and therapy of not only infectious disease, but also cancer and autoimmunity. Here, new methodologies are presented for generating unbiased in-frame synthetic antigen-presenting libraries for application in high-throughput TCR: peptide screening approaches. The antigens will be processed by the intracellular machinery, while avoiding the potential restrictions on spliced peptides and haplotype-specific presentation events. Once established, these libraries can be exploited in high-throughput screens to functionally identify neo-antigens together with their corresponding T cell receptor. The antigen-presenting library design presented here has four major advantages compared to published methods: HLA-haplotype independent, genome-wide, employs natural antigen processing, and simultaneously includes MHC class I and class II antigens in the screening.
B cells purified from human peripheral blood are incubated with infectious supernatant from the Epstein-Barr virus-producing cell line B-95. After several weeks of continuous culture, outgrown cells are passaged and expanded. It has been demonstrated that these cells continue to express both class II and class I HLA molecules and are functionally professional antigen-presenting cells.
The main advantage of using a synthetic library is to minimize sequence-derived bias during library cloning and transduction. To solve these problems, selected genes will process to generate gene fragments with the same length and normalized GC content. Each gene fragment that resulted from this process is called ‘minigene’.
The first step in the minigene generation process is to break down the amino-acid sequence up to the maximum fragment length. Fragments derived from the same protein will have an overlap of a 30 amino-acid length. Fragments shorter than the maximum length will be linked together using ‘GSGSGG’ sequence. This iterative process of break down and build-up will ensure that generated fragment will have the same amino-acid length. In the remaining fragments, a protein domain will be linked to reach the maximum fragment length.
After the back-translation steps the minigene sequences are generated. A scheme of the process is illustrated in
A peptide barcode is added at the 3′ end to enable library NGS analysis. In
To test the ability of minigene containing antigen to activated T cells, a co-culture experiment was performed comparing minigene sequence to natural open reading frame (ORF). The DMF5 TCR and MLANA gene were selected for the experiment. As shown in
Because minigenes in a screening library are large (1200 bp) and diverse, full-amplicon sequencing for the identification of positive interactions can be difficult. In particular, sequencing and amplification biases negatively influence the experimenter's ability to isolate the correct sequence, even when the cellular interaction was present in the screen. In order to reduce this bias, each minigene is provided in a library with a unique molecular barcode using DNA. Nucleotide barcodes have been in use for some time to track DNA molecules; however, approaches that rely heavily on the sequence of the translated peptide, and using random nucleotide barcodes could introduce further unwanted effects. Therefore, a system was developed for barcoding individual members of a screening library that maintains an identical translated sequence across the entire library. Furthermore, the barcode generator ensures that a minimal Levenshtein distance between any two members of the library is maintained (as shown for example,
Expression of minigenes in EBV-transformed B cell lines is mediated by lentiviral-based overexpression. The number of copies of minigenes that subsequently integrate into the genome is defined by the viral multiplicity-of-infection (MOI), and occurs stochastically based on Poisson distributions. Thus, transduction of complex libraries is expected to result in either several integration events per cell when using maximal MOI, or result in few integration events alongside a large fraction of non-transduced cells upon diluting of viral supps, resulting in high inefficacy of any subsequent screen.
In order to minimize the amount of irrelevant integration events in cells containing the cognate minigene while at the same time conducting screens with a homogenously-transduced B cell populations, a method was developed in which a Flag-tagged surface receptor is expressed in tandem with the minigene. B cells are thus transduced to achieve 30% Flag+ cells, ensuring an average of a single integration event, and subsequently are enriched for Flag expression via magnetic-based cell enrichment.
Candidate surface receptors were chosen based on potential ability to further increase minigene-based T cell activation by TCR-cross activation. Thus, candidate surface receptors were screened both of ability to subsequently mediate successful enrichment of Flag+ minigene-expressing cells as well as their respective potency in mediating T cell activation. Combined, these were instrumental at highlighting OX40L-Flag and 41BBL-Flag as two surface receptors who are effectively expressed on transduced B cells, whose Flag-expression enables cell enrichment, and whose expression further potentiates minigene-based T cell activation.
In order to induce minigene expression and subsequent presentation on HLA molecules, lentiviral particles driving expression of the respective minigene from a constitutive promoter were generated. Specifically, cognate or mismatch minigene were restriction-based cloned into the pLVX-EF1a-IRES-Puro vector (TAKARA). This vector was further modified to include a T2A polycistronic cassette in frame with the minigene, directly followed by an mTagBFP2 fluorescent reporter. Moreover, a CMA sequence was introduced immediately upstream of the minigene's coding sequence to facilitate chaperon-mediated autophagy and subsequent peptide presentation on Class II HLA molecules. This CMA-T2A-BFP backbone vector is thus named C2B (SEQ ID NO:77).
For the purpose of testing the different Flag-tagged surface receptors, InFusion-based cloning (TAKARA) was used to replace the mTagBFP2 reporter with the human codon optimized coding sequence of each candidate surface receptor, N-terminally or C-terminally tagged with a flexible 6× Flag to face the extracellular space. Specifically, the coding region of CD40, CD58, CD80, CD83, CD86, OX40L and 4-1BBL were cloned and tested.
BOLETH cells (Sigma, 88052031-1VL) were cultured in 6-well cell culture plates in RMPI medium (Gibco, #61870143) supplemented with 10% FBS (HyClone), 1% penicillin-streptomycin, 1% kanamycin, 50 μM β-mercaptoethanol, 1 mM sodium pyruvate, and 1 mM NEAA (Gibco, #11140050) at 37° C. humidified incubator with 5% CO2. Cells were passaged every two to three days at a ratio of 1:2.
In order to induce minigene expression and presentation, lentiviral particles were generated via co-transfection into HEK 293FT cells in 6-well plates of 1.33 ug of the transfer vector alongside 1 ug of psPAX2 and 0.67 ug of pMD2.G accessory vectors using the TransIT VirusGEN transfection reagent (Mirus). Following 48 hours of incubation, supernatants were collected, filtered through a 0.45 μm PES filter. BOLETH cells were subsequently transduced by resuspending 1.5×106/6 well plate in a 1:1 viral supernatant dilution in the presence of 8 μg/ml of Polybrene (Millipore) and 2 mM BX795 (Sigma). After 24 hours of incubation cells were washed from virus-containing medium and resuspended with full B cell medium. Functional experiments were conducted 4-5 days following transduction.
Minigene-transduced B cells were removed from culture plates and washed with cold Enrichment buffer (PBS+2% FBS+1 mM EDTA) to remove any residual media. Cell pellets were resuspended in same buffer supplemented with 0.05 μg of PE-conjugated aDYKDDDDK antibody (Biolegend) and 2 μl of human FcR blocker (Miltenyi) per 106 total B cells. Following 20 minutes incubation, cells were washed with same buffer. Enrichment for PE-bound B cells was conducted using EasySep™ PE Positive Selection Kit II (STEMCELL) according to manufactures' instructions. Respective enrichment compared to the input cell fraction was subsequently determined using a FACS Aria II (BD) by probing for the PE signal.
Co-culture experiments were done using round bottom 96 well plates. P10-NBV Jurkat and SKW3 reporter cells and B cells are seeded 1 to 1 ratio into each well. Next day the cells are spun down and stained for Flow cytometry.
The genetic code that dictates the translation of nucleotide triplets (i.e., codons) into amino acids is redundant in that most amino acids are coded for by several codons. Despite this apparent redundancy, non-random codon usage has been widely observed among different cell lines and organisms. The common and rare codons in a particular organism will vary from those of another (Novoa & Ribas de Pouplana, 2012, Trends in Genetics, Vol. 28, pp. 574-581). Moreover, not every codon composition will sustain the same expression level for a given protein (Plotkin & Kudla, 2011, Nature Reviews Genetics, Vol. 12, pp. 32-42). For this reason, codon optimization is used as a means to increase protein expression.
The Fast ALgorithm for Codon OptimizatioN (herein referred to as FALCON) is a tool written in Python that optimizes nucleotide sequences according to multiple parameters for a high-yield protein expression. A list of the features included in FALCON can be viewed in
The process starts with a list of amino acid sequences corresponding to the genes that will be optimized. The system in which the genes will be expressed is chosen (i.e., HEK293 cells), and the sequences are back-translated using a weighted random codon selection function. Multiple codon usage tables were developed for the expression systems that FALCON supports (see example in
FALCON optimization allows for a desired GC content to be set by the user at the beginning of the optimization process. The GC content optimization is performed using a 4-parameter logistic function (4PL). In this function, the four parameters; A, B, C and D, and the independent variable ‘x’; are used to calculate the correction coefficient ‘y’, according to equation 1 (
After 10 codons have been selected, the sequence is subjected to several motif avoidance steps. Restriction sites as well as other undesired patterns are eliminated.
The process of back-translation and motif avoidance is repeated continuously until the sequence is finished.
One of the main rate-limiting steps in protein synthesis is at the translation initiation (Shah et al., 2013, Cell, 153(7), 1589). If the mRNA folds and forms a thermodynamically-stable structure, especially at the 5′ start, the coupling of the ribosome and the translation machinery can be hindered (Kudla et al., 2009, Science, 324(5924), 255-258). Thus, the potential for any stable structure forming at this position should be reduced. In order to promote an efficient translation initiation, FALCON optimizes the 5′ start of the first 20 codons, by generating 10 candidate partial sequences using the back-translation approach described above, and then selecting the sequence with the highest minimum free energy (MFE). A higher MFE is indicative of a less stable mRNA secondary structure, which correlates with higher expression levels (Jia & Li, 2005, FEBS Letters, 579(24), 5333-5337). The MFE is calculated using the open-source ‘seqfold’ package, developed by JJTimons (pypi.org/project/seqfold/). The optimized 5′ start will then be used by FALCON as a starting point, to generate 10 full candidate sequences. Subsequently, a step termed ‘Tournament style selection’ follows.
The utilization of a weighted, yet random, codon selection approach in FALCON means that, if a gene is optimized multiple times, virtually every resulting sequence will differ to a degree from each other. This characteristic gives FALCON the advantage of generating multiple contesting full sequences, which can then be evaluated according to specific quality criteria. For each gene, FALCON creates 10 candidates, and selects the highest-ranking according to the following three parameters:
The CAI is a measure widely used to quantify codon preferences toward most common codons. A sequence that only uses the most common codons for a particular organism will have a CAI=1. Deviations from this usage will result in lower CAI values. FALCON first calculates the codon relative adaptiveness (CRA) of each candidate sequence (equation 2;
The final score is calculated according to equation 5 (
The evaluation system has been tested and calibrated with a large set of tests such that, effectually in every case, the championed sequence has scored the highest in at least one or two of the assessed criteria, and it is among the top in the third. It is thus assured that the selected sequences will reliably be the most optimal from their batch.
A comparison of the FALCON algorithm is available in
A last and important feature of FALCON algorithm is that it makes use of multiprocessing, allowing it to optimize several sequences concurrently. The adjustment-layers approach used by FALCON for sequence optimization is already in itself extremely fast. Other available algorithms such as the genetic algorithm used by COOL (Chin et al., 2014, Bioinformatics, 30(15), 2210-2212), or the simulated annealing and genetic algorithm used by EuGene (Gaspar et al., 2012, Bioinformatics, 28(20), 2683-2684), by contrast, take a considerably higher amount of time to find an optimal solution to the sequence optimization problem. For instance, Chin and colleagues report that COOL takes around 20 min to optimize a 500 amino acid sequence. Slightly faster, EuGene took over 24 minutes for a double-sized 1000 amino acid sequence. In the same machine in which the EuGene software was run, FALCON took over 4 seconds to optimize the same 1000 amino acid sequence. FALCON optimization was 360 times faster than EuGene, and included all of the parameters listed in
The experimental results are now described
Minigene sequence libraries of different lengths were designed (
A FALCON algorithm, which employs an adjusted codon usage table for EBV immortalized B cells, was developed and optimized (
A barcode generator that ensures that a minimal Levenshtein distance between any two members of the library is maintained was developed (
CMA-Minigene-BFP (C2B) based constructs and modified, surface-receptor Flag-conjugated constructs (C2F) were designed (
Flag-based surface receptor constructs can be used to successfully enrich for transduced B cells (
OX40L and 4-1BBL overexpression potentiates minigene-based T cell activation (
Codon optimized candidate surface receptor accessory molecules with Flag flexible linker
Expression of a transgene from different promoters was tested.
Described herein is a new microfluidic co-encapsulation device that enables prolonged, clog-free encapsulation with eventual distributed cell flow and improved cell encapsulation ratios. The current study investigates the benefits of an increase of overall height from 25 μm to 35 μm, an inverted oil inlet, enlarged cell filter regions, stream separator elements that ensure even distribution of cell flow along the entire cell filter area, a novel cell filter shape, an overall shape that supports laminar flow, and the inclusion of asymmetrical focusing loops that lead to a more even flow of longitudinally aligned cells (
One of the physical limitations of the microfluidic system used for this screening method is the natural behavior of Epstein-Barr virally immortalized B cells to form clumps, which are not amenable to single-cell encapsulation and are impeded by the narrow structure of the device channels. To co-encapsulate B and T cells in droplets, a microfluidic PDMS device is used (
To enable a constant flow of single cells into the microfluidic device, it is necessary to keep the cells in suspension. Therefore, magnetic stirring is needed to avoid cellular aggregation and uneven distribution. In an OB-1 microfluidic flow controller device (ELVEFLOW), pressure is applied to a fluid suspension of cells through a tube which has an adaptor for 15 ml centrifuge tubes (specifically FALCON 352095). Due to the V-bottom shape of the centrifuge tube, it is not possible to keep the cells in suspension via magnetic stirring. To overcome this problem, a 5 ml round bottom polystyrene test tube (FALCON 352052) was placed inside the 15 ml centrifuge tube. (
The materials and methods are now described.
Computer-aided photomask designs were printed on high-resolution photolithography film photomasks.
Depending on the desired channel depth, a specific SU-8 photoresist was used. All soft lithography work was conducted in clean room facilities. For a channel depth of 25 μm or 35 μm, SU-8 2025 photoresist was used. A specific guideline can be found online (https://kayakuam.com/products/su-8-2000/). Coating of the silicon wafer with photoresist was conducted by centering the wafer on a spin coater (Spin150 wafer spinner) and dispensing approximately 3 ml of photoresist onto the wafer. To equally disperse the photoresist along the wafer, the spin coater's rotational speed was set to 500 rpm (at an acceleration rate of 500 rpm/s) for 15 s, followed by an increase of the rotational speed to 3000 rpm for achieving a 25 μm coating, or 2000 rpm for achieving a 35 μm coating (at an acceleration rate of 500 rpm/s) for 30 s. Afterwards, the wafer was removed from the spin coater and baked at 65° C. for 1 min and then at 95° C. for 3 min (pre-bake) on a hotplate. Once the wafer cooled to RT, it was illuminated to constant ultraviolet (UV) light using a SUSS MICROTEC Mask Aligner MA6 with the following settings: mask aligner pressure: check if 0.8 bar or higher, wedge error compensation (WEC) pressure: adjustment to 0.15 bar (as indicated on the chuck), alignment gap: 30 μm, exposure type: hard, work type: cont (contact), upset offset: 10 s, HC wait: 10 s, exposure time: 20 s.
The film photomask was taped to a soda-lime glass plate. This was then brought in contact with the wafer and the UV source of the mask aligner was turned on. Following UV exposure, the wafer was subsequently baked at 65° C. for 1 min, then at 95° C. for 3 min (post-bake) on a hotplate. For developing the design and removing residual (non-exposed) photoresist, the wafer was submerged in PGMEA (propylene glycol monomethyl ether acetate) in a crystallizing dish and gently agitated for 4 min. The wafer was then removed from the crystallizing dish, rinsed with isopropanol and dried using a nitrogen gun. After developing, the wafer was baked at 150° C. for 10-30 min on a hotplate to fully resolve the features and remove tension-based cracks in the design.
The master wafer was rinsed with isopropanol and dried with nitrogen, then subsequently placed in the center of a plastic petri dish. PDMS base and curing agent were mixed in a 9:1 ratio (w/w) (Dow SYLGARD™ 184 Silicone Elastomer Kit), mixed well, and depressurized in a vacuum chamber to remove trapped air bubbles. Per PDMS mold, approximately 30 g of mixture were applied. After removal of a majority of air bubbles, the PDMS was poured onto the wafer in a plastic petri dish. The PDMS was cured overnight at 65° C. The next day, the PDMS (including the wafer) was cut out of the petri dish using a scalpel and carefully peeled off the wafer. The wafer was cleaned with isopropanol and stored at room temperature for future use. Using a 0.75 μm diameter biopsy punch, holes were created at each fluid inlet or outlet of the PDMS mold. 75 mm×50 mm glass slides were pre-cleaned in parallel by submerging in isopropanol and sonicating for 10 min. The PDMS mold was then positioned on the glass slide and hybridized into it using oxygen plasma with the following settings: plasma duration: 12 s, plasma power: 90%, process pressure: 0.3 mbar, gas supply duration: 60 s, gas type: O2, flushing duration: 10 s, venting duration: 30 s. Following the oxygen plasma treatment of the PDMS (channel side facing up) and the glass slide, the two parts become irreversibly attached.
For AQUAPEL treatment, the microfluidic chip containing the droplet encapsulation design was connected to a pressure control unit (ELVEFLOW OB1mk3 Flow Controller) which in turn is attached to a pressure pump. This allows flushing the channels with the desired fluid at a pre-defined pressure. The chip was placed on a microscope to enable visualization of the different processes. In order to treat the glass surface and facilitate laminar flow of aqueous fractions, the different channels were flashed with AQUAPEL prior to conducting encapsulation experiments. Specifically, AQUAPEL was filtered through a 0.22 μm filter and then flushed into all inlets and channels of the device using a syringe. Subsequently, the chip is similarly flushed with oil (3M NOVEC 7500) to remove the AQUAPEL agent. This was followed by drying of the design by flushing it with air. The chip is then left to dry overnight at room temperature prior to conducting encapsulation experiments.
For droplet encapsulation, Aquapel-treated microfluidic chips were used. The aqueous cell fractions containing the B and T cell fractions were each connected to the aqueous phase cell inlets using flexible PTFE tubing with an average diameter of 0.32 μm (ADTECH). Additionally, PICO-SURF detergent (SPHERE FLUIDICS) reconstituted to a 1% final concentration in 3M Novec 7500 oil was connected to the oil inlet using PTFE tubing. These were then connected to individual inlets of the pressure control unit. The chip was placed on a microscope to record droplet encapsulation. During encapsulation, cells were constantly mixed using a 5 mm×2 mm magnetic stir bar. Pressure was turned on for all inlets at the same time (detergent: 1000 mbar, aqueous inlets: 600 mbar). Depending on the filter design and height on the design, the pressure was adjusted to generate droplets of equal shape and diameter. Droplets were then collected in a microcentrifuge tube pre-filled with 400 μl oil and sampled after various encapsulation durations.
BOLETH cells (SIGMA, 88052031-1VL) were cultured in 6-well cell culture plates in B cell medium (RPMI 1640 (GIBCO, #61870143) supplemented with 10% FBS (HyClone), 1% penicillin-streptomycin, 1% kanamycin, 50 μM β-mercaptoethanol, 1 mM sodium pyruvate, and 1 mM NEAA (GIBCO, #11140050)) at 37° C. humidified incubator with 5% CO2. Cells were passaged every two to three days at a ratio of 1:2. Reductions in B-cell clumping were tested with the following treatments: Gibco Anti-Clumping agent (GIBCO 0010057AE); EDTA (INVITROGEN #15575020); Opti-prep density gradient medium (STEMCELL TECHNOLGIES #07820); Cell clumping was evaluated in plates by time-resolved imaging using an INCUCYTE automated microscopy system (SARTORIUS). Jurkat cells were cultured in RPMI 1640+10% heat-inactivated FBS (HYCLONE) and 1% penicillin-streptomycin at 37° C. in a humidified incubator with 5% CO2. Cells were split every two to three days at a ratio of 1:4.
For functional assays involving presentation of soluble peptides from the tetanus toxin (tetanospasmin, tetX), BOLETH cells were incubated overnight in the presence of 1 μM peptide. BOLETH and transgenic T-cell receptor-expressing Jurkat cells were collected and centrifuged at 300 g for 5 min. BOLETH cells were then resuspended in B-cell medium to a concentration of 1×106 cells/ml and the respective peptide was added at a final concentration of 1 μM. Jurkat cells were comparably resuspended in B-cell medium to a final concentration of 3×106 cells/ml. For the co-culture experiment, 50 μl of each cell suspension were mixed in a round-bottom 96-well plate to obtain a 3:1 ratio of T to B cells. Cells were incubated for 7 h and subsequently analyzed.
One day prior to encapsulation, BOLETH cells were cultured in the presence of cognate or negative control peptides at a final concentration of 1 μM. Cells were collected the next day, centrifuged at 300 g for 5 min, then resuspended in B-cell medium at a final concentration of 1.4×107 cells/ml. These were further supplemented with same peptide and with or without EDTA. TCR-expressing Jurkat cells were centrifuged at 300 g and resuspended in B-cell medium at a concentration of 4×107 cells/ml. These were then further supplemented with respective concentrations of calcium. BOLETH, Jurkat, and detergent-containing fractions were connected to the microfluidic chip as described. Pressures were applied using an OB1 MK3+ and the droplet emulsion was collected into a 1.5 ml reaction tube containing oil.
Droplets were incubated at 37° C. in a 5% CO2 humidified incubator. Following overnight incubation, droplet emulsions were disrupted by adding two volumes of Pico-Break 1 (SPHERE FLUIDICS). Tubes were quickly inverted several times and centrifuged for 15 s at 300 rpm to facilitate separation of the cell-containing fraction. This was then transferred into new tubes containing 1 ml FACS buffer (PBS+10% FBS+5 mM EDTA). Tubes were centrifuged for 5 min at 300 g and cell pellets were subsequently resuspended in 100 μl of FACS buffer and were analyzed via flow cytometry.
To quantitate the ratios of encapsulated B and T cells in each design or upon addition of EDTA, cells were stained using membrane-penetrating fluorescent dyes. Specifically, BOLETH cells were stained with a red dye (AAT BIOQUEST CYTOTRADE RED) while Jurkat cells were stained with CFSE according to manufacturer protocols. Cells were subsequently washed in B-cell medium to remove any residual unbound dye. Cell pellets were then resuspended in B-cell medium at concentrations as described above and processed for droplet generation. Following encapsulation, 3 μl of droplet emulsion was then overlayed with 5 μl of 3M NOVEC 7500 oil on standard glass microscopy slides. Representative images were taken in the brightfield, Texas Red, and FITC microscope channels. Total droplets, B cell-containing droplets, T cell-containing droplets, and droplets containing both at least one T and one B cell were then counted from the images.
At the conclusion of a co-culture experiment, cells were first washed in cold FACS buffer and centrifuged at 300 g for 5 min. Supernatants were removed and pellets were then resuspended in FACS buffer supplemented with fluorescent dye-conjugated antibodies and incubated for 20 minutes at 4° C. in the dark. Antibodies were used at the following dilutions:
Following incubation, cells were washed again with cold FACS buffer and resuspended in 100 ul cold buffer prior to analysis. Flow cytometry analysis was conducted with a FACS ARIA II (BD BIOSCIENCES). Data analysis was subsequently performed using FLOWJO (BD BIOSCIENCES).
The identification of foreign, self-, and neo-antigens presented by cells is an essential step in the prevention, diagnosis, and therapy of a spectrum of disorders, including infectious disease, cancer, and autoimmunity. Here, new methodologies are presented for generating unbiased in-frame antigen presenting libraries for application in high-throughput TCR: peptide screening approaches. This simultaneously addresses class I and class II HLA (generally, MHC) complexes by non-professional and professional antigen-presenting cells. Furthermore, the antigens will be processed by the intracellular machinery, while avoiding potential issues with spliced peptides and haplotype-specific presentation events. Once established, these libraries can be exploited in high-throughput screens to functionally identify neo-antigens together with their cognate T-cell receptor. The antigen-presenting library design presented here has three major advantages compared to published methodologies: HLA-haplotype independence, MHC class II antigen inclusion, and genome-wide screening capability (Kula et al., 2019, Cell, 178(4):1016-1028.e13).
The platform aims to present both class II and class I antigens encoded by the same transgenic construct. The construct contains a constitutively active EF1α promoter which drives expression of the open reading frame (ORF) containing the minigene linked via a 2A viral peptide linker to mTagBFP2. The ORF is transcribed as a single mRNA but two separate proteins will be produced at a stoichiometric ratio; therefore mTagBFP2 is used as a fluorescence marker for transduction control as well as verifying the reading frame of the upstream ORF. The minigene is flanked by SpeI and BamHI restriction sites to facilitate synthetic library cloning.
A MLANA A27L transgene and DMF5 TCR (Abdel-Wahab et al., 2003, Cell Immunol, 224(2):86-97) were used as positive controls for class I presentation, while for class II presentation a fragment of the tetanospasmin protein, (TetX_400_1) was used together with a cognate TCR (TT2). Both minigenes were cloned in a lentiviral construct for B-cell transduction, cells were used in a co-culture experiment with their cognate TCRs. In both samples, clear TCR activation was detected, even for the class II positive control which was not expected, because class II presentation is generally associated with extracellular antigens (
Constant mTagBFP2 expression levels in the B cells excluded the possibility of transduction biases and transgene silencing. Presentation biases were tested with a co-culture experiment in which freshly peptide-pulsed BOLETH cells were compared to re-pulsed transgenic BOLETH cells cultured over time (
Lysosomal Targeting does not Improve Class II Antigen Processing
Class II antigens are processed and loaded on HLA molecules in the lysosome. To improve class II processing, three strategies were used to target the lysosome. The chaperone-mediated autophagy (CMA) pathway was targeted using the KFERQ sequence (Koga et al., 2011, Nat Commun 2, 386), the binding motif for the HSC70 chaperone protein that will translocate the minigenes through LAMP2. DC-LAMP is another lysosomal membrane proteins, N-term and C-term domains were used to anchor the minigenes in the membrane and localize it in the lysosomal lumen (Dominguez-Bautista et al., 2015, Eur J Cell Biol. 2015 March-April; 94(3-4):148-61). LIR domains are found in cargo proteins and interact with LC3 at the level of the phagophore formation; LIR domains were used to localize the minigenes to the lysosome at this step (Johansen et al., 2020, J Mol Biol, 432(1):80-103). (
In a recent publication, a class II antigen screening experiment was performed cloning the ORF into a CD74 fusion protein instead of the CLIP peptide. When testing this construct, two key observations were made: the minigene activation was as strong as that with pulsed peptide and T-cell activation did not decrease over time (
CD74 binds the HLA class II molecule in the ER, then the complex is translocated to the lysosome, where Cathepsin S cleaves CD74, leaving the CLIP peptide bound on class II HLA. HLA-DM releases the CLIP peptide and allows another peptide to bind class II HLA. To target the antigen presentation pathway, minigenes were cloned into a fusion with HLA-DM; this construct similarly failed to improve T-cell activation in a co-culture experiment (
Bcap31 is an ER chaperone membrane protein, which is also involved in the ER-associated degradation (ERAD) pathway. The protein contains three transmembrane domains. Protein fragments were cloned and added to the N-terminus of the minigene in transgenic expression construct (
Constructs were tested with a 100 AA tetanospasmin fragment containing the target of the TT2 TCR. When minigene-expressing cells and T-cells were co-cultured, the BT1 construct induced a strong T-cell activation, similar to the positive control. Moreover, the construct overcomes antigen processing attenuation and demonstrates stable T-cell activation over time (
However, the BT1 construct did not improve the presentation of the TetX_400_1 minigene. In this case, T-cell activation was lower in the BT1 construct relative to 2B. The experiment was expanded to include the TT7 and TT11 TCRs and their 400 AA cognate minigenes. TetX_400_916 (which contains the TT7 target at its C-terminus) was included in the experiment. In this co-culture series, the BT1 construct outperformed the 2B constructs and improved antigen presentation (
The BT1 construct significantly increases T-cell activation, but still has several limitations. These observations focused our attention on ER membrane proteins as a potential source of TM-spanning sequences able to target minigenes to the class II processing and presentation pathway. Here a functional screening strategy is presented to identify additional TM sequences that can further improve the BT1 construct results.
Transmembrane domains from ER protein sequences were downloaded from UniProt. The list contains 1410 sequences with lengths between 8 and 45 AA, with a modal length (n=977) of 21 AA. 800 sequences with a length of 21 AA were randomly selected for the screening. These were extended by positions −2, −1, +1, +2 from the TM to provide cytoplasmic/luminal anchors, giving a final sequence length of AA. The amino acid composition of these TM domains was analyzed; as expected, they show a clear enrichment of hydrophobic residues. Anchor positions, in contrast, were strongly enriched in hydrophilic and charged amino acids. With this data, an R script was written to generate synthetic TM sequences which maintain this composition. From the synthetic TM domain batch, 400 sequences were randomly selected for screening. The final library is composed of 400 synthetic and 800 natural TM domains. The sequences were back-translated and ordered as an oligonucleotide pool with the restriction sites required for cloning.
To subsample the library and assess individual sequences, a microscope-based screen was performed with 1152 individually picked transformants following bulk cloning of the entire library. The transduced BOLETH cells were co-cultured with T-cells expressing the cognate TT7 TCR, and T-cell activation was measured via image-based sfGFP intensity.
In all the three independent replicates, approximately 20 transmembrane sequences induced T-cell activation in two technical replicates, meaning 10% of the library sequences were able to induce synthetic class II presentation. These sequences outperformed BT1 construct, which induced a weaker T-cell activation (data not shown). Of these, fewer TM sequences induced a positive signal at both time points, suggesting not all presentation-promoting sequences could overcome antigen processing attenuation. In an initial round of screening, 576 transformants were tested and 5 of them were able to strongly activate T-cells at both time points (
The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.
This application claims priority to U.S. Provisional Application No. 63/277,347; filed Nov. 9, 2021 and to U.S. Provisional Application No. 63/277,311; filed Nov. 9, 2021 each of which is hereby incorporated by reference herein in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/079461 | 11/8/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63277347 | Nov 2021 | US | |
| 63277311 | Nov 2021 | US |
