Invented by Benjamin Wang, Gusti Zeiner, Chimera Bioengineering Inc
The Chimera Bioengineering Inc invention works as followsControl Devices include RNA destabilizing element (RDE), RNA controls devices, and destabilizing element (DE) in combination with Chimeric Antigen Receptors or other transgenes within eukaryotic cell. The control devices can be used to engineer host eukaryotic cell with CARs or transgenes. These control devices are used to optimize the expression of CARs within eukaryotic cell so that, for instance, effector functions can be optimized. The CARs, as well as the transgenes that carry them, can be engineered in eukaryotic cellular systems so that they are delivered and expressed after the stimulation of the CAR.
Background for Gold optimized CAR-T-cells
Chimeric Antigen receptors are human-engineered receptors which may direct a cell to attack a specific target that is recognized by CAR. CAR T-cell therapy, for example, has shown to induce complete responses against acute leukemia, other B-cell related malignancies, and to achieve and sustain remissions in refractory/relapsed A-L-L (Maude et al. NEJM 371:1507 2014). Some patients have experienced dangerous side effects such as cytokine-release syndrome (CRS), tumour lysis syndromes (TLS), B cell aplasia, and off-target, on-tumor toxicities.
There are two strategies that exist to control CAR technologies. First, there is a ‘kill switch? that can be induced. This approach involves one or more suicide? In this approach, one or more?suicide? PLoS1, 2013 doi:10.1371/journal.pone.0082742). The addition of AP1903 is what activates these suicide genes. It’s a lipid permeable tachrolimus analogue that homodimerizes the human protein FKBP12(Fv) to which apoptosis inducing proteins ace are translationally fused. These kill switches are designed to sacrifice CAR’s long-term monitoring benefit in order to protect against toxicity. In vivo however, these suicide switch are unlikely to achieve this goal as they operate against strong selection pressures on CAR T cells that do not react to AP1903, a problem made worse by the error-prone, retroviral-copying-incompatible copying of stable transgenes in patient T-cells. In this scenario, CAR T cell clones that are not responsive will continue to multiply and kill target cells based on antigen. The kill switch technology will not provide adequate protection against toxicity.
The second CAR regulatory strategy is transient expression of CAR, which can be achieved several different ways. In one method, T-cells from unrelated donors are harvested, the HLA gene is deleted using genome-editing technologies, and CAR-encoding genes are inserted into their genome. These CAR T cells will be destroyed by the recipient immune system after adoptive transfer. This is why the CAR exposure occurs in this system. In a second transient CAR approach, the mRNA of CAR-encoding genes is introduced into patient T-cells. (Beatty, G. L. 2014. Cancer Immunology Research 2 (2): 112-20. doi:10.1158/2326-6066.CIR-13-0170). The mRNA is short-lived and does not replicate in the cells or are stable. Therefore, the CAR will only be expressed and active for a brief period. These transient CAR-exposure approaches, like the kill switch approach, sacrifice the surveillance benefits of CARs. These transient systems can also cause acute toxicity, which is difficult to control.
In one aspect, the description discloses a eukaryotic cellular system with a CAR or T-cell receptor and a transgene controlled by an RNA Destabilizing Element.” The RDE can control multiples transgenes, or multiple RDEs can control multiples transgenes. Multiple transgenes can be arranged in a serial manner, as a concatemer or other ways. Multiple RDEs are used to regulate transgenes. These multiple RDEs can either be arranged as a concatemer or interspersed in a particular region of the transcription, or they can be located at different locations within the transcript. An RDE, or a combination RDEs, can regulate multiple transgenes. RDEs may be located in the 5?UTR, 3?UTR or intron. The RDE can, in one aspect, be engineered to increase the binding affinity of the RNA binding proteins that interact with it. The RNA-binding protein regulates transgene expression and can be altered by altering its affinity. In one aspect, the RDE’s metabolic state affects the RNA-binding protein binding. Changing the RDE’s binding affinity for the RNA-binding protein can alter the response and/or timing to transgene expression. “In an aspect, the RDE RNA binding is affected by the redox condition of the cells and changing the affinity of the RDE to the RNA-binding protein changes the response and/or timing for transgene expression.
In one aspect, a CAR, T cell receptor, B cell receptor, innate immune receptor, or another targeting receptor or targeting peptide recognizes a second antigen at th target site (e.g. tumor cell, other diseased tissues/cells) and activates the cells. The transgene could be another CAR which recognizes a secondary antigen at the site of interest. Activation of the eukaryotic cells by the first CAR or T-cell receptor, or any other targeting polypeptide, induces this second CAR and allows the cell to recognize a target site using a second molecule. In one aspect, an eukaryotic cells has a CAR that detects an antigen on a target site. This activates a RDE that encodes a peptide which directly or indirectly reduces activation of the cell. The transgene, for example, may encode a CAR that recognizes antigens on healthy tissues so that, when the first CAR reacts to antigens at nontarget cells, the eukaryotic will be deactivated by the interaction of the second CAR with the healthy cell’s antigen.
In some aspects, an eukaryotic cellular system is an immune cell. For example, it can be a T cell, natural killer cell (NK), B cell, macrophage or dendritic cells. In these aspects activation of a cell by the RDE or altering the metabolic state of an immune cell can induce the expression of the transgene. The RDE can contain microRNA-binding sites, and it can be engineered so that one or more microRNA-binding sites are removed. Hu Protein R can bind to the RDE. It is believed that HuR can bind some RDEs and stabilize the mRNA leading to increased translation. The glycolytic state of an eukaryotic cellular can be determined by the enzymes glyceraldehyde-3-phosphate dehydrogenase, other dehydrogenases or oxidoreductases. The RDE can be affected by GAPDH, or other enzymes that bind the RDE. This will reduce the half-life of RNA in the RDE. CAR activation in eukaryotic cells (e.g. T-lymphocytes) can cause glycolysis, which increases the half-life of RNA. This leads to increased expression of transgene encoded by the RNA controlled by RDE. As GAPDH leaves the RDE HuR or RDE-binding proteins can subsequently bind the same RDE or an RDE that was previously inaccessible (sterically blocked by presence of GAPDH), stabilizing mRNA further, increasing the half-life and producing increased expression of transgene encoded and controlled by RDE. CAR activation may induce the expression of transgene. Other activation of immune cells can also cause GAPDH engage in glycolysis, and thus induce expression of transgenes under the control RDE.
Expression of the transcript(s) with the RDE (s) can respond the metabolic state in the cell. The RDE, for example, can be bound to metabolic or glycolytic proteins which couples the expression of the transgene with the activation state through these metabolic and glycolytic proteins. Some metabolic or glycolytic proteins bind to RDEs within the transcript, and then degrade the transcript. The enzymes will no longer bind the RDEs when they become active. This allows the transcripts to be stable for longer periods of time and can also be translated. The cells that express transgenes controlled by such RDEs may also be engineered so they express a CAR which can change the metabolic state of the cells at specific times, resulting in the expression of the transgene. Other stimuli may be used to change the metabolic state of a eukaryotic cellular resulting in the expression of the transgene. “For example, the metabolic status of the cell can either be altered (or inhibited) by stimuli such as small molecules (e.g. PMA/ionomycin), TCR and costimulatory-domain engagement with ligands, oxygen levels, temperature, light/radiation, or cellular stress.
GAPDH binding to the RDE can be increased by introducing into the cell a small molecule that inhibits glycolysis such as, for example, rapamycin, 2-deoxyglucose, 3-bromophyruvic acid, iodoacetate, fluoride, oxamate, ploglitazone, dichloroacetic acid, or other metabolism inhibitors such as, for example, dehydroepiandrosterone. You can also use other small molecules to reduce GAPDH’s binding to the RDE. These small molecules can block GAPDH’s RDE binding site, such as CGP 3466B Maleate, Heptelidic Acid (both sold by Santa Cruz Biotechnology, Inc.), Pentalenolactone, and 3-bromopyruvic acids. Other small molecules may be used to inhibit RDE binding by other enzymes and polypeptides. Other small molecules may be used to alter the redox status of GAPDH. This will lead to a change in the affinity of GAPDH to the RDE.
In one aspect, activation (of the immune cell) induces the expression of the transgene which can encode a load to be delivered at target site. The transgene encodes a payload that is delivered at the site of immune cell activation or CAR activation. Payloads can include cytokines, antibodies, reporters (e.g. for imaging), receptors (such as CARs), or polypeptides that have an effect on the target site. The payload may remain inside the cell or be applied to the surface of the cell in order to change the behavior. Payloads can include intracellular proteins such as kinase or phosphatase enzymes, metabolic enzymes, epigenetic modifiers, gene editing enzymes, etc. Payloads can include gene regulatory RNAs such as antisense, microRNAs and ribozymes or guide RNAs that are used with CRISPR. Payloads can be membrane-bound proteins such as GPCRs, transporters, etc. The payload could be an imaging agent which allows the target site to imaged. (The target site contains a certain amount of antigen that is bound by CAR). Payloads can include a checkpoint inhibitor, where the CAR or other binding proteins (e.g. T-cell receptors, antibodies, or innate immune receptors) recognize a cancer associated antigen to deliver the checkpoint suppressor at the tumor. Payloads can include a cytotoxic compound such as a complement, granzyme or apoptosis-inducing small molecules. In certain aspects, the expression of CAR can be controlled by an inducible promotor, an RNA-control device, a DE or Side-CAR and/or RDE. The amount of antigen on the surface can be used to activate the eukaryotic cells at the tumor and not the normal tissue. This is because the tumor has higher levels of target antigen. This regulatory control over CAR expression also provides an additional level of control for the eukaryotic cells and their delivery of payload. The payload may remain within the cell, or even on its surface (rather then being secreted into the target) to alter the behavior of the cellular.
In certain aspects, expression of CAR or DE-CAR is at least partially controlled by an RDE which interacts with GAPDH, a glycolytic with RDE-binding activity. The glycolytic enzyme may bind to RDEs and reduce the production of CAR, De-CAR, or Side-CAR polypeptides, as well as other transgene products. The reduction in polypeptide can be due to an inhibition of translation or an increase in mRNA degradation rate (RDE binding may shorten the half life of mRNA). RDE-binding proteins can reduce polypeptide production by enhancing RNA degradation and reducing translation. RDE is an AU-rich element of the 3? The RDE can be an AU-rich element from the 3? It can also be a modified UTR (3? The modified 3?UTR can be engineered to remove microRNA sites, e.g. the modified 3?UTR of IL-2 and IFN -?). In one aspect, the expression is the transgene CAR, Side-CAR, or DE-CAR under the control of a RDE bound to a glycolytic (e.g. GAPDH) enzyme. This can be increased by increasing activity of enzymes in glycolysis. Increased glucose in cell medium can increase the activity of glycolysis enzymes. Also, the activity of triose isomerase in cells can be increased. RDE can bind Hu Protein R. It is believed that HuR binds some AU rich RDEs and U rich RDEs. This can stabilize mRNA and lead to increased translation. HuR can be expressed in cells under conditions that are favorable for transgenes that contain AU-rich and/or Urich elements. Conversely, conditions that inhibit HuR can reduce the expression of these transgenes. HuR interaction with 3? The UTR (or native genes of the transgene) can be altered as well by expressing a transcript that contains HuR binding sites. These transcripts are expressed to reduce the amount HuR that is available to bind the transgene or native HuR-regulated transcripts. They also reduce the half lives of these transcriptions, resulting in decreased gene expression.
In one aspect, bicistronic vectors (or multicistronic vectors) are used to introduce transgene-RDE constructions. These bicistronic vectors can be derived by lentivirus. The control region located between two nucleic acid encoding transgenes can be used to express the two transgenes in opposite directions. If more than two (multicistronic) transgenes have been placed in the construct, the third and additional transgene can be placed in a series. One or both transgenes may then be expressed in opposite direction. These additional transgenes can be expressed either from the same control area or separate control areas. The CAR may be encoded by one transgene, and the payload delivered to the cell when the CAR activates may be encoded by the other(s). The RDE can control the nucleic acids that encode the payload of the multicistronic construct or bicistronic construction based on the glycolytic state or energy level of the cell. The transgene that encodes the CAR may be operably paired with a promoter that is either inducible or has a low transcription level. The transgene that encodes the payload, on the other hand, can be linked to an inducible, high transcription level, or a lower transcription level control region. The CAR may be operably connected to a region with a high level of transcription and/or inducible, while the payload transgene can be linked to an area that has lower transcription levels (and/or inducible). The CAR can be linked to a region with a lower transcription level (and/or inducible), and the transgene that encodes the payload to a region that is higher in transcription.
Nucleic acids are a way to increase the response of the immune cells when they are stimulated. The immune cell, for example, can produce greater amounts of immune polypeptides with faster production kinetics. Immune polypeptides include, for instance, cytokines perforins granzymes apoptosis-inducing polypeptides etc. Nucleic acids that boost immune responses can include control regions operably coupled to nucleics encoding RDEs of selected RDE-binding proteins. This allows the RDEs of the RNA to bind RDE binding protein that represses expression of a specific polypeptide such as cytokines perforins granzymes and other immune polypeptides. The expression of RNAs with RDEs can prepare the eukaryotic cells for the expression of polypeptides controlled by RDEs. “For example, RNAs and RDEs can be expressed in immune cells so that the cell is ready to express immune polypeptides when the immune cell is stimulated.
Certain RDEs are associated with specific disease states within a subject. By comparing RDEs found in normal tissue (cells) with RDEs found in diseased or abnormal tissues (cells), some disease-associated RDEs can easily be identified. RDEs from normal (healthy), and their RNA-binding proteins can be compared with those found in diseased or abnormal cells by trapping RDEs along with the RNA-binding proteins, using the methods described in Castello et al. Molc. Cell 63:696-710, is incorporated in its entirety by reference for all purposes. RDEs with aberrant interactions can be linked to disease states and sequencing RDEs within the genes or transcripts of an individual could show their susceptibility to diseases and/or disease state.
RDE control can be achieved by an RDE which is responsive to metabolic state in the eukaryotic cells. By changing the metabolic state in the eukaryotic cells, for example, an RDE bound to a glycolytic or other metabolic enzyme can inhibit the expression of a CAR, trangene payload and/or transgene. By binding the RDE to GAPDH, turning off glycolysis (e.g. using an inhibitor for glycolysis), the expression of CAR, payload or transgene could be inhibited. This inhibition of expression is used to reduce the adverse events that are caused by the expression of CARs, transgenes, or transgenes.
In one aspect, the Side-CAR polypeptides and/or CAR polypeptides can be directed at antigens on acute myeloid lymphoma (AML) cell surfaces, such as CD 33, 34, 38, 45, 45RA, 47, 64, 66, 123, 133, 157, LeY, PR1, CXCR4, CLL-1 (CD 168), WT1, TIM-3 (CD 168), RHAMM (CD 168), or CXCR4 (CD 168), CLL-1 (CD 168), CXCR4, CXCR4, CXCR4, CXCR4, CXCR4, CLL-1 (CD 167), CXCR4, CLL-1 (CD 158), CLL-1 (CD 168), CLL-1 (CD 168 (CD 166 (CD tim-3 (CD 68 (CD 169), TIM-3 (CD 168 (CD 168 (CD 168 (CD 165 (CD 164), PR1, TIM-3 (PR1), tim3 (CD162), PR1, TIM3 (CD As an extracellular element, the monoclonal antigen 293C3SDIE may be used for CAR, Side-CAR or DE-CAR polypeptides. (Rothfelder et al., 2015, at ash.confex.com/ash/2015/webprogram/Paper81121.html, which is incorporated by reference in its entirety for all purposes) Other antigens for AML are known in the art and may be the target of the CAR, DE-CAR, Side-CAR, and/or other receptor. In one aspect, CAR, DECAR, SideCAR polypeptides and/or another receptor can be directed at antigens on diffuse large B-cell lymphoma cells (DLBCL), including, for instance, CD19. CD20. CD22. CD79a. CD5, CD10 and CD43. “Other antigens of DLBCL may be known to the art, and could be targeted by the CAR. DE-CAR. Side-CAR and/or any other receptor.
The desired amount of CARs expression can be determined by the following factors: the concentration of the target cells, the density of the antigen target on the cells, and the affinity of the extracellular elements (antigen-binding element) to the antigen target. The parameters can be combined with others to determine the desired level of expression of CARs. It is also possible to consider inhibitory receptors expressed on eukaryotic cells, as well as inhibitory receptor ligands expressed on target and other cells when determining the desired amount of CAR. You can use the following equations, at least partially, to determine a desired CAR polypeptide amount:
The desired amount of expression of CAR can produce the desired number of CARs at the surface of an eukaryotic cellular. The desired amount CAR expression could produce up to 100,000 CARs on the surface. The eukaryotic cells can be T-lymphocytes and the CARs, DE-CARs, or Side-CARs on the surface can range from 2-100,000. The CAR, Side-CAR and/or DE-CAR may bind to the target ligand in the range of micromolars (?M), and the desired CARs, Side-CARs and/or DE-CARs can be between 100-500,000. The CAR DE-CAR and/or side-CAR can bind target ligand in the nanomolar range (nM). This can result in a desired number of CARs DE-CARs and/or side-CARs to be on the surface T-lymphocytes or natural killer cells of 2-100,000.
A nucleic-acid construct encoding transcripts with selected RDEs may be expressed in an immunity cell, such as a T lymphocyte. The recombinant transcription with selected RDEs can bind and deplete levels of RDE-binding proteins in the T lymphocyte, so that transcripts of polypeptides regulated via the depleted RDE-binding proteins are expressed at various threshold points for activation of other cellular signals. The RDE constructs may increase the expression kinetics and/or Cmax of polypeptides that are controlled by RDEs.
It is important to note that, before the different embodiments are explained, the disclosure does not limit itself to these particular embodiments. As such, the contents of the disclosure can vary. The terminology used in this document is only to describe particular embodiments and not to limit the scope of the teachings.
Unless otherwise defined, all technical and science terms used in this document have the same meaning that is commonly understood by a person of ordinary skill within the field to which the disclosure belongs. While any materials and methods similar to or equivalent to those described in this disclosure can be used for the practice or test of the present teachings as well, a few exemplary materials and methods are described now.
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