Invention for Smart Car Devices, DE car Polypeptides, Side CARs and Uses Thereof

Invented by Benjamin Wang, Gusti Zeiner, Chimera Bioengineering Inc

The Chimera Bioengineering Inc invention works as follows

RNA Control Devices or destabilizing elements can regulate the expression Chimeric Antigen Receptors in eukaryotic cell. DEs, RNA Control Devices and/or Side-CARs are used in conjunction with small molecule ligands for the regulation of Chimeric Antigen Receptors. These DE-CARs or Smart CARs are a small molecule-activated RNA trigger. Smart-DE CARs and/or Side CARs may be used to treat disease.

Background for Smart Car Devices, DE car Polypeptides, Side CARs and Uses Thereof

The CAR T cell therapy is effective in achieving complete responses to acute lymphoblastic and B-cell related malignancies. It has also been proven to be effective for refractory/relapsed ASL (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 an inducible “kill switch.” 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 the apoptosis inducing proteins will be translated fused. These kill switches are designed to sacrifice CAR’s long-term monitoring benefit 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-prone copying associated with insertion of stable T-cell transgenes. 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 some embodiments the invention is related to Smart-CARs, DE-CARs, Side-CARs and/or combinations of these constructs for use in eukaryotic cell. In some embodiments the CAR constructs include a nucleic acids encoding CARs (chimeric antigens receptors) and a destabilizing element or an RNA control device. The invention can be embodied in CARs (smart CARs), DE-CARs and/or Smart DE-CARs which are composed of two or more parts that combine to form the CARs.

In some embodiments, an RNA control device consists of a sensor, a linker, and a actuator. In some embodiments the nucleic acid that encodes the CAR includes nucleic acids that code an extracellular antigen-binding element, a membrane element, an intracellular communication element and a costimulatory component. In some embodiments the nucleic acid that encodes the CAR includes nucleic acids that code an extracellular antigen-binding element, a membrane element, and an internal signaling element. In some embodiments, the intracellular element includes a costimulatory function and sequence. In certain embodiments, Smart CAR nucleic acid of the invention is placed in an expression vector that can be used by eukaryotic cells. In some embodiments the nucleic acids are DNA or RNA. In some embodiments the RNA device inhibits CAR expression, and CAR expression increases when the sensor element binds the ligand. “In some embodiments, RNA control devices inhibit CAR expression when ligand binds to the sensor elements, but when ligand does not bind to the sensors, CAR expression increases.

In some embodiments the Smart CAR or DE-CAR and/or Smart-DECAR nucleic acid of the invention is placed in an expression vector that can be used by a eukaryotic cellular. In some embodiments the nucleic acids are DNA or RNA. In certain embodiments, the Destabilizing Element targets DE-CAR for proteolysis within the eukaryotic cells. In certain embodiments, DE can bind a ligand. In certain embodiments, the binding of ligand reduces the proteolysis in the eukaryotic cells of the DE polypeptide. Binding of ligand can increase proteolysis in some embodiments. In some embodiments the DE-CAR is not proteolyzed when ligand has not been bound. In some embodiments the RNA-control device increases CAR-, Side-CAR-, or DE-CAR-polypeptide production when it binds the ligand. “In some embodiments, RNA control devices inhibit CAR DE-CAR and/or side-CAR expression when the ligand binds to the sensor elements, but when the ligand does not bind to the sensors, CAR DE-CAR and/or side-CAR expression increases.

In some embodiments the two parts of a CAR or Smart CAR are associated in response to small molecules, polypeptides, or any other stimuli (e.g. light, heat etc.). These split constructs may be referred to in some embodiments as Side-CARs. In certain embodiments, a part of a CAR, DECAR or Smart-DECAR is membrane-bound through a polypeptide transmembrane segment, while the other is not. In some embodiments the non-membranebound part is not bound to the membrane. In some embodiments the membrane is linked to the non-membrane part through a tether. In some embodiments, the tether is through a glycophosphatidylinositol (GPI). In this embodiment, a GPI sequence is included on the C-terminal of the non-membrane-bound part. In some embodiments, the CAR or DECAR is formed by a functional CAR and side-CARs that are primed to interact through a binding interaction. In some embodiments an extracellular CAR element or DECAR binds the target antigen, causing a conformational shift in the extracellular component. This allows it to be associated with other parts of CAR or DECAR. In some embodiments the molecule that binds the two components together is an antigen. In some embodiments the molecule responsible for the association between the two components is a small molecular.

In some embodiments the invention relates a eukaryotic cellular nucleic acid that encodes Smart CARs, DE-CARs, Smart-DECARs and/or SideCARs. In some embodiments the eukaryotic cells contain an expression vector containing nucleic acid encoding Smart-CARs, DE-CARs, Smart-DE CARs and/or SideCARs. In some embodiments the invention’s eukaryotic cells are mammalian cells. In certain embodiments, an eukaryotic cellular is either a human or murine cell. In some embodiments the eukaryotic cells is a cell of the hematopoietic cell lineage. In some embodiments the eukaryotic cells are a T lymphocyte, a natural killing cell, a B lymphocyte or a macrophage. In certain embodiments, an eukaryotic cellular component of the invention contains a desired quantity of CAR, Side-CAR and/or DE-CAR polypeptides. In certain embodiments, eukaryotic cells have a desired quantity of CAR, Side-CAR and/or DE-CAR polypeptides on their surface. In certain embodiments, an eukaryotic containing the CAR-, DE-CAR-, and/or side-CAR-polypeptides of the invention exhibits a desired level of activity. In some embodiments the desired amount activity results in a proliferation activity. In some embodiments the desired amount is an amount that binds to a target or an amount that has an effector (e.g. killing target cells).

In some embodiments, RNA control devices are expressed in trans to the mRNA that encodes the CAR or DE-CAR. In some embodiments the RNA-control device is expressed trans to the mRNA that encodes the CAR DE-CAR and/or SideCAR. The invention can be applied to an eukaryotic cellular system with Smart CARs, DE-CARs, Smart-DE-CARs and/or SideCARs. These cells incorporate several independent orthogonal RNA controls that are responsive to different small molecules ligands. In some embodiments of the invention, a eukaryotic cellular system is provided that incorporates multiple Smart CARs, DE-CARs (or DE-CARs), Smart DE-CARs (or Smart-DE-CARs), or Side-CARs. These cells can have similar or different target specificities. In some embodiments the multiple Smart CARs, DE-CARs, Smart-DE-CARs and/or SideCARs within the eukaryotic cells respond to distinct sets of small molecule receptors. In some embodiments the multiple Smart CARs, DE-CARs, Smart-DE-CARs, and/or SideCARs in the eukaryotic cell respond to a distinct set of small molecule ligands.

In some embodiments the invention relates eukaryotic cell(s) with custom CAR expression, custom DECAR expression, and/or custom side-CAR polypeptides. The DE, RNA Control Device, and/or the Side-CAR are used in this embodiment to customize the amount CAR, DE -CAR, and/or the Side-CAR that is displayed on the eukaryotic cells surface. In some embodiments the customized DE-CAR or Side-CAR displays give a desired activity against a targeted and/or desired proliferative activities when the eukaryotic cell with the customized DE-CAR or Side-CAR is placed in a subject. In some embodiments the customized CAR DE-CAR or Side-CAR display gives a desired level of cell death. In some embodiments the customized CAR DE-CAR or Side-CAR express gives a desired proliferation rate for the eukaryotic cells with the CAR DE-CAR or Side-CAR Polypeptide. In some embodiments the customized CAR DE-CAR or Side-CAR express gives a desired memory cell formation rate when the eukaryotic cells is an immune cell. In some embodiments the customized CAR or Side-CAR expression can be used to give a desired level of effector function within an appropriate eukaryotic cellular, such as an immune cell.

In some embodiments, this invention relates methods for making eukaryotic cell with customized levels CAR, DECAR and/or side-CAR expression by using Smart CAR(s), DECAR(s), smart-DE-CARs and/or side-CARs. The invention can be used to make eukaryotic cell(s) with the desired amount of polypeptides (CAR, Side-CAR, and/or DE-CAR). In certain embodiments, the invention is related to methods of making eukaryotic cell with Smart-CAR (s), DE CAR (s), Smart DE-CAR (s), and/or side-CARs which have a desired activity level against a targeted. In certain embodiments, the invention is related to making eukaryotic cell with a targeted activity level. In some embodiments the invention relates methods of making eukaryotic cell(s) with Smart CARs, DE-CARs, Smart-DECARs and/or SideCARs which have a desired proliferation level when placed in a patient. “Other eukaryotic cells activities that can be customized include, for example: rate of memory cell production, release rate cytokines and phagocytosis.

In some embodiments, Smart (CAR), DE (CAR), Smart-DE (CAR), and/or side-CARs are introduced to a mammalian cellular as an RNA which encodes a Smart CAR or DE CAR. In some embodiments, the RNA that encodes Smart CARs, DE-CARs, Smart-DECARs, and/or side-CARs, is a viral vector, which can be derived from retroviruses, lentiviruses, alphaviruses, other RNA viruses, adenoviruses, or DNA viruses, or a combination thereof. These vectors can harbor a library containing Smart CARs, DECARs, Smart DECARs and/or side-CARs encoded with a reporter gene. Transduced cells displaying desired Smart CARs, DECARs, Smart DECARs and/or side-CARs behavior are isolated and refined based on their activity. In some embodiments the RNA that encodes the Smart CARs, DE-CARs, Smart-DECARs, and/or side-CARs is the reverse complement strand, also known as the antisense. The RNA in some embodiments is derived either from a retrovirus, lentivirus, alphavirus, or another RNA vector vector. In these embodiments, the RNA encodes the Smart CA, DE CAR and/or Smart DE CAR. This RNA also has the antisense (or reverse complement) strand relative to the sense (or +strand) strand of the vector.

In some embodiments, a polynucleotide that encodes the Smart CAR or DE-CAR and/or Smart-DE CAR is/are integrated in a chromosome. In some embodiments the polynucleotide that encodes the Smart CAR DE-CAR Smart-DE CAR and/or Side CAR is present extrachromosomally in the eukaryotic cells. In some embodiments the polynucleotide that encodes the Smart CAR (or DE-CAR), Smart-DE CAR (or Side-CAR), or Side-CAR (or both) is integrated by a genome-editing enzyme (CRISPR TALEN Zinc-Finger nuclease), along with appropriate nucleic acid (including nucleic acid encoding Smart CAR (or DE-CAR), Smart-DE CAR (or Side CAR), etc. In one embodiment, genome editing enzymes, nucleic acids, and encoding nucleic acid are integrated at a genomic safety harbor site such as the CCR5, human ROSA26 or PSIP1 loci. In some embodiments the eukaryotic cells are human T-lymphocytes and the nucleic acids encoding Smart CAR or DE-CAR and/or Side CAR is integrated on the CCR5 loci.

In some embodiments of the invention, methods are provided for selecting RNA aptamers that have high selectivity and specificity for selected small molecule ligands. In certain embodiments, the invention offers methods to increase higher sensitivity, greater selectivity or both for a target ligand in RNA control devices. This is achieved by using bacteriophage culture. In some embodiments, the invention provides methods for increasing higher sensitivity, selectivity, or both to a target ligand of RNA control devices, achieved through the use of replication-competent murine retrovirus or lentivirus in murine cell culture. In certain embodiments, the invention provides a method for combining RNA aptamers and ribozymes to yield an RNA control device. The invention, in some embodiments provides methods to incorporate RNA control devices into mammalian, bacterial, or plant cells.

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.

It is important to note that the singular forms “a”, “an” and “the” are used in this document and the claims appended. Plural referents are included unless the context makes it clear otherwise. The claims can also be written to exclude optional elements. This statement will serve as an antecedent to the use of exclusive terms such as “only” or “only”. ?only? The use of words like?only? limitation. If the context is clear, numerical limitations are meant to be approximate. When a concentration (for example, 10 g) is stated, the intention is that it be understood as at least approximately 10 g.

As will be evident to those skilled in the art after reading this disclosure, the various embodiments can be easily separated or combined without departing from scope or spirit. The recited method may be performed in the order in which it is described or in any order that makes sense.

Definitions

The following terms are intended to have the following meanings. The following terms have been given the following definitions.

As used in this document, an “actuator element” is defined as a domain that encodes the system control function of the RNA control device. It is defined as a domain which encodes the system-control function of the RNA device. In some embodiments the actuator domain encodes gene-regulatory functions.

Click here to view the patent on Google Patents.