US20200197497A1

US20200197497A1 - Peptidic chimeric antigen receptor t cell switches and uses thereof

Info

Publication number: US20200197497A1
Application number: US16/532,860
Authority: US
Inventors: Travis Young; Chanhyuk Kim; Peter G. Schultz
Original assignee: Scripps Research Institute
Current assignee: Scripps Research Institute
Priority date: 2013-10-15
Filing date: 2019-08-06
Publication date: 2020-06-25
Also published as: US20150307564A1; EP3057994A4; JP2016533174A; AU2020207804A1; EP3057994B1; KR20160062760A; US10391155B2; WO2015057834A1; CN105829349A; JP6734774B2; JP2020096644A; CN105829349B; AU2014337367B2; KR102339240B1; EP3057994A1; ES2845924T3; US20230355728A1; CA2927543C; US9624276B2; US20170246270A1

Abstract

Disclosed herein are chimeric antigen receptor effector cells (CAR-ECs) and CAR-EC switches. The switchable CAR-ECs are generally T cells. The one or more chimeric antigen receptors may recognize a peptidic antigen on the CAR-EC switch. The CAR-ECs and switches may be used for the treatment of a condition in a subject in need thereof.

Description

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No. 15/448,477, filed on Mar. 2, 2017, now allowed, which is a continuation of U.S. application No. 14/688,894, filed Apr. 16, 2015, issued as U.S. Pat. No. 9,624,276, which is a continuation of International Application No. PCT/US14/60684, filed Oct. 15, 2014, which claims the benefit of U.S. provisional application Ser. No. 61/891,347, filed Oct. 15, 2013; U.S. provisional application Ser. No. 61/895,704, filed Oct. 25, 2013; U.S. provisional application Ser. No. 62/009,054, filed Jun. 6, 2014; U.S. provisional application Ser. No. 62/030,526, filed Jul. 29, 2014; and U.S. provisional application Ser. No. 62/030,514, filed Jul. 29, 2014; which are all incorporated by reference in their entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is TSRI_003_04US_ST25.txt. The text file is about 82 KB, created on Jul. 31, 2019, and is being submitted electronically via EFS-Web.

BACKGROUND OF THE INVENTION

Immunotherapies are becoming attractive alternatives to chemotherapies, including immunotherapies that use adoptive transfer of genetically modified T cells to “reteach” the immune system to recognize and eliminate malignant tumor cells. Genetically modified T cells express chimeric antigen receptors, which generally consist of a signaling endodomain, a CD3-zeta transmembrane domain and an extracellular single-chain variable fragment (scFv) derived from a monoclonal antibody which gives the receptor specificity for a tumor-associated antigen on a target malignant cell. Upon binding the tumor-associated antigen via the chimeric antigen receptor, the chimeric antigen receptor expressing T cell (CAR T-cell) mounts an immune response that is cytotoxic to the malignant cell. Such therapies can circumvent chemotherapy resistance and have been shown to be active against relapsed/refractory disease, resulting in sustained remissions for some chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL) patients. However, these therapies require further investigation and optimization, as they caused undesirable effects such as toxic lymphophenia, chronic hypogammaglobulinemia for hematological targets, fatal off-target cytolysis for solid tumor targets, persistent B cell aplasia with the use of anti-CD19 antibody expressing CAR T-cells, and, in some cases, death.
Introduction of a switch, which controls the activity of the CAR T-cell, would allow CAR T-cell activity and associated immune responses to be turned off after neoplastic cells are eliminated and would allow B cells to reproliferate. Recent preclinical studies have demonstrated that CAR T-cell systems can be controlled through an antibody-based switch, wherein the antibody binds the target cell (e.g. cancer cell), blocking the CAR T-cell from binding the target cell and “switching off” CAR-T activity. While these systems conceptually allow for switchable targeting of tumors using CAR T-cells, they may suffer from a series of limitations. Non-specific labeling of antibodies using cysteines or lysines produces heterogeneous products which includes variants that may be non-functional, have unpredictable pharmacokinetics and/or immunogenicity, and that may be difficult to optimize.

SUMMARY OF THE INVENTION

Disclosed herein are chimeric antigen receptor-effector cell switches comprising: a peptidic antigen that binds a chimeric antigen receptor on an effector cell; and a targeting moiety that binds a cell surface molecule on a target cell. The peptidic antigen may be based on or derived from a naturally occurring peptide. The peptidic antigen may be based on or derived from a non-human peptide. The peptidic antigen may be based on or derived from a eukaryotic peptide. The peptidic antigen may be based on or derived from a peptide, wherein the peptide is expressed by a yeast. The peptidic antigen may be based on or derived from a yeast transcription factor GCN4. The peptidic antigen may comprise a non-naturally occurring peptide. The peptidic antigen may comprise a synthetic peptide tag. The peptidic antigen may be based on or derived from a sequence selected from SEQ ID NOs: 2-7. The peptidic antigen may comprise a sequence that is at least about 50% homologous to a peptide sequence selected from SEQ ID NOs: 2-7. The targeting moiety may comprise a targeting peptide. The targeting moiety may comprise a targeting antibody or antibody fragment. The targeting antibody or antibody fragment may be selected from the group consisting of: an immunoglobulin, an Fc null immunoglobulin, and a Fab, and fragments thereof. The targeting moiety may be selected from the group consisting of: an anti-EGFR antibody, an anti-Her2 antibody, anti-EGFRvIII antibody, an anti-CD33 antibody, an anti-CLL-1 antibody, an anti-CEA antibody, an anti-CD19 antibody, an anti-CD22 antibody, an anti-BCMA antibody, and an anti-CS1 antibody, and fragments thereof. The targeting antibody or antibody fragment may comprise a light chain and a heavy chain pair, wherein the light chain and heavy chain are encoded by nucleic acid sequences based on or derived from nucleic acid sequence pairs selected from the group consisting of: SEQ ID NOs: 8 and 9; SEQ ID NOs: 8 and 10; SEQ ID NOs: 11 and 12; SEQ ID NOs. 13 and 14; SEQ ID NOs: 15 and 16; SEQ ID NOs: 17 and 18; and SEQ ID NOs: 19 and 20. The targeting antibody or antibody fragment may comprise a light chain and a heavy chain pair, wherein the light chain and heavy chain are encoded by amino acid sequences based on or derived from amino acid sequence pairs selected from the group consisting of: SEQ ID NOs: 21 and 22; SEQ ID NOs: 23 and 24; SEQ ID NOs. 25 and 26; SEQ ID NOs: 27 and 28; and SEQ ID NOs: 27 and 29. The chimeric antigen receptor-effector cell switch may comprise a light chain and a heavy chain pair, wherein the light chain and heavy chain are encoded by amino acid sequences based on or derived from amino acid sequence pairs selected from the group consisting of: SEQ ID NOs: 30 and 29; SEQ ID NOs: 36 and 29; SEQ ID NOs: 31 and 28; SEQ ID NOs: 27 and 32; SEQ ID NOs: 27 and 33; SEQ ID NOs: 27 and 34; and SEQ ID NOs: 27 and 35. The peptidic antigen may be fused to a terminus of the targeting antibody or antibody fragment. The peptidic antigen may be fused to a region of the targeting antibody or antibody fragment selected from the group consisting of: an N terminus of a light chain, a C terminus of a light chain, an N terminus of a heavy chain, a C terminus of a Fab heavy chain and a C terminus of a constant region heavy chain. The peptidic antigen may be grafted into the targeting moiety. The targeting moiety may comprise a targeting antibody or antibody fragment. The peptidic antigen may be grafted into a region of the targeting antibody or antibody fragment selected from a CH₁domain, a CH₂domain, a CH₃domain, a CL domain, a VH domain, a VL domain and a hinge region. The peptidic antigen may be grafted between two regions of the targeting antibody or antibody fragment selected from a CH₁domain, a CH₂domain, a CH₃domain, a CL domain, a VH domain, a VL domain, a heavy chain, a light chain and a hinge region, wherein the two regions are adjacent. The peptidic antigen may be grafted into a loop of the targeting antibody or antibody fragment. The peptidic antigen may be grafted into a loop of a constant domain of the targeting antibody or antibody fragment. The peptidic antigen may be grafted between the hinge region and a heavy chain constant domain of the targeting antibody or antibody fragment. The peptidic antigen may replace one or more amino acids of the targeting antibody or antibody fragment. The peptidic antigen may be grafted into the targeting antibody or antibody fragment without replacing an amino acid. The chimeric antigen receptor-effector cell may further comprise a linker that links the peptidic antigen and the targeting moiety. The linker may be a peptide that links the peptidic antigen and the targeting moiety, wherein the targeting moiety comprises a targeting polypeptide. The linker may comprise about 1 to about 20 amino acids. The linker may comprise a sequence based on or derived from a sequence selected from SEQ ID NOs: 38-42. The peptidic antigen may comprise a yeast transcription factor GCN4 or homolog thereof and the targeting moiety is selected from the group consisting of: an anti-Her2 antibody, an anti-BCMA antibody, an anti-CD19 antibody, an anti-CEA antibody and fragments thereof. The target cell may be a cancer cell. The cell surface molecule may be a tumor associated antigen. The cell surface molecule may be selected from the group consisting of: a cluster of differentiation protein, a receptor, an integral membrane protein and a glycoprotein. The homogeneity of the chimeric antigen receptor-effector cell switch may be at least about 90%.
Further disclosed herein are pharmaceutical compositions comprising: a chimeric antigen receptor-effector cell switch comprising: a peptidic antigen that binds a chimeric antigen receptor on an effector cell; and a targeting moiety that binds a cell surface molecule on a target; and a pharmaceutically acceptable salt, excipient and/or vehicle.
Disclosed herein are kits comprising: a chimeric antigen receptor-effector cell switch comprising: a peptidic antigen that binds a chimeric antigen receptor on an effector cell; and a targeting moiety that binds a cell surface molecule on a target cell; and a chimeric antigen receptor-effector cell comprising a chimeric antigen receptor that binds to the peptidic antigen of the chimeric antigen receptor-effector cell switch. The targeting moiety may comprise a targeting peptide. The targeting moiety comprises a targeting antibody or antibody fragment. The peptidic antigen is grafted within the targeting moiety. The kit may comprise a first chimeric antigen receptor-effector cell switch and a second chimeric antigen receptor-effector cell switch, wherein the first chimeric antigen receptor-effector cell switch comprises a first peptidic antigen and a first targeting moiety and the second chimeric antigen receptor-effector cell switch comprises a second peptidic antigen and a second targeting moiety. The first peptidic antigen and the second peptidic antigen may be the same. The first targeting moiety may bind a first cell surface molecule on a first target cell and the second targeting moiety may bind a second cell surface molecule on a second target cell, wherein the first cell surface molecule and the second cell surface molecule are different. The effector cell may be selected from a T cell, an effector B cell, a natural killer cell, a macrophage and a progenitor thereof. The effector cell may be selected from a naive T cell, a memory stem cell T cell, a central memory T cell, an effector memory T cell, a helper T cell, a CD4+ T cell, a CD8+ T cell, a CD8/CD4+ T cell, an αβ T cell, a γδ T cell, a cytotoxic T cell, a natural killer T cell, a natural killer cell, a macrophage.
Further disclosed herein are chimeric antigen receptors that bind a peptidic antigen of a chimeric antigen receptor-effector cell switch. The chimeric antigen receptor may comprise an antibody or antibody fragment that binds the peptidic antigen of a chimeric antigen receptor-effector cell switch. The antibody fragment or antibody fragment may bind a eukaryotic antigen. The antibody or antibody fragment may bind a non-naturally occurring peptide. The antibody fragment may be an scFv. The antibody or antibody fragment may be selected from an anti-yeast transcription factor GCN4 antibody, an anti-FLAG® antibody, an anti-HTP antibody and fragments thereof. The chimeric antigen receptor may be encoded by a polynucleotide based on or derived from SEQ ID NO: 1.
Disclosed herein are effector cells comprising a chimeric antigen receptor, wherein the chimeric antigen receptor that binds a peptidic antigen of a chimeric antigen receptor-effector cell switch. The effector cells may be T cells. The effector cells may comprise one or more polynucleotides based on or derived from SEQ ID NO: 1.
Further disclosed herein are vectors comprising a polynucleotide having a sequence that encodes a chimeric antigen receptor-effector cell switch, wherein the chimeric antigen receptor-effector cell switch comprises peptidic antigen and a targeting moiety, wherein the targeting moiety comprises a peptide and binds a cell surface molecule on a target cell.
Disclosed herein are vectors comprising a first polynucleotide having a first sequence that encodes a heavy chain of a targeting antibody or antibody fragment; a second polynucleotide having a second sequence that encodes a light chain of a targeting antibody or antibody fragment; and a third polynucleotide having a third sequence that encodes a peptidic antigen, wherein expression of the vector produces a chimeric antigen receptor-effector cell switch. The third sequence may be adjacent to a sequence selected from the first sequence and the second sequence. The third sequence may be located within a sequence selected from the first sequence and the second sequence.
Further disclosed herein are methods of producing a chimeric antigen receptor-effector cell switch, comprising expressing from one or more polynucleotide vectors: a first sequence that encodes a heavy chain of a targeting antibody or antibody fragment; a second sequence that encodes a light chain of a targeting antibody or antibody fragment; and a third sequence that encodes a peptidic antigen, wherein expression of the vector produces a chimeric antigen receptor-effector cell switch.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates a general overview of chimeric antigen receptor-T cell (CAR T-cell) and CAR T-cell switch therapy with switches disclosed herein. Lymphocytes are isolated from a subject and an expression vector encoding a chimeric antigen receptor is subsequently introduced to the lymphocytes to produce chimeric antigen receptor expressing cells. Resulting engineered lymphocytes are administered to the subject, along with a CAR T-cell switch.

FIG. 1B illustrates a CAR T-cell switch, comprising a peptide that is bound by the chimeric antigen receptor of the CAR T-cell and a targeting antibody that is selective for a target cell. Binding of the CAR T-cell switch to the CAR T-cell induces an immune response that would be cytotoxic to the malignant cell also bound to the CAR T-cell switch.

FIG. 2 depicts a PDB 1P4B crystal structure of an affinity matured scFv (light and medium gray represent light chain and heavy chain) bound to a peptide derived from the yeast transcription factor GCN4 (7P-14P) (dark grey represents the GCN4 peptide).

FIG. 3 shows mass spectrometry of an anti-CD19-Fab-GCN4_CL1CAR-EC switch. Calculated: 49533, found: 49537.09.

FIG. 4 shows cytotoxicity of an anti-GCN4 CAR T-cell with various anti-CD19 antibodies or antibody fragments with a GCN4 peptide grafted or fused to various regions or domains of the anti-CD19 antibodies or antibody fragments.

FIG. 5A shows a non-reducing SDS-PAGE gel of anti-CD19 antibodies or antibody fragments with a GCN4 peptide grafted or fused to various regions or domains of the antibodies or antibody fragments.

FIG. 5B shows a reducing SDS-PAGE gel of anti-CD19 antibodies or antibody fragments with a GCN4 peptide grafted or fused to various regions or domains of the antibodies or antibody fragments.

FIG. 6 depicts a yeast GCN4 peptide grafting positions in an anti-CD19 Fab (FMC63).

FIG. 7 shows in vivo efficacy of an anti-CD19 Fab-GCN4 peptide CAR T-cell switch and an anti-GCN4 CAR T-cell in a xenograft tumor mouse model. FIG. 7A shows quantification of tumors in untreated versus treated mice. FIG. 7B depicts in vivo treatment regimen and visualization of tumor cells in untreated versus treated mice.

FIG. 8 shows cytotoxicity of an anti-GCN4 CAR T-cell and CAR T-cell switch (anti-BCMA antibody -GCN4 peptide grafted into the light chain constant domain) against BCMA-positive cells (OPM2).

DETAILED DESCRIPTION OF THE INVENTION

Current chimeric antigen receptor T cell (CAR T-cell) therapies can be unreliable due to lack of means to control CAR T-cell activity. Disclosed herein are compositions and methods for selectively activating and deactivating chimeric antigen receptor T cells, which may provide for safer and more versatile immunotherapies than those currently being tested and administered. Disclosed herein are switchable chimeric antigen receptor effector cells (CAR-ECs) and chimeric antigen receptor effector cell (CAR-EC) switches, wherein the CAR-EC switches have a first region that is bound by a chimeric antigen receptor on the CAR-EC and a second region that binds a cell surface molecule on target cell, thereby stimulating an immune response from the CAR-EC that is cytotoxic to the bound target cell. In general, the CAR-EC is a T cell. In this way, the CAR-EC switch may act as an “on-switch” for CAR-EC activity. Activity may be “turned off' by reducing or ceasing administration of the switch. These CAR-EC switches may be used with CAR-ECs disclosed herein, as well as existing CAR T-cells, for the treatment of a disease or condition, such as cancer, wherein the target cell is a malignant cell. Such treatment may be referred to herein as switchable immunotherapy, for which an exemplary schematic overview is depicted in FIG. 1.
The CAR-EC switches disclosed herein comprise a first region that binds a cell surface molecule on a target cell, and a second region that is bound by a chimeric antigen receptor. In general the first region is a targeting polypeptide. The targeting polypeptide may be a targeting antibody or antibody fragment that binds an antigen on the target cell. Alternatively or additionally, the first region may comprise a non-peptide small molecule (e.g. vitamin, metabolite). The second region, referred to herein as a chimeric antigen binding peptidic antigen (CAR-BP), comprises a peptide. For simplicity, the term chimeric antigen binding peptidic antigen may simply be referred to herein as a peptidic antigen. In general, the CAR-BP is fused to a terminus of the targeting polypeptide or grafted within the targeting polypeptide. Fusing or grafting the CAR-BP to the targeting polypeptide may be carried out by cloning one or more polynucleotides encoding the first region and the second region into a polynucleotide expression vector, in a desired order or combination.
Methods of treating a disease or condition comprising administering the CAR-EC switches, disclosed herein, may provide for a titratable response, improved safety and/or cessation of CAR-EC activity by reducing or ceasing administration of the CAR-EC switch. In contrast to other approaches of controlling CAR-EC activity, which “turn off” CAR-EC activity by competing with the target cell surface molecule for binding the CAR, the CAR-EC switches disclosed herein, generally function as CAR-EC activators or “on” switches.
Further disclosed herein are CAR-EC platforms including CAR-EC switches and effector cells comprising universal chimeric antigen receptors (CAR) that can bind multiple CAR-EC switches, providing for sequential targeting of one or more types of target cells (e.g. treatment of heterogeneous tumors). The CAR may comprise an ultra-high affinity antibody or antibody fragment (e.g. scFv) to the switch. Methods of producing the CAR-EC switches disclosed herein may advantageously provide for control of CAR-EC cell activity, titration of off-target reactivity, abrogation of tumor lysis syndrome (TLS), attenuation of cytokine release syndrome (CRS), and/or optimization of CAR-EC switch binding by affinity, valency, geometry, length and/or chemistry through site-specific grafting/fusing of CAR-EC switch peptides/antibodies.
Unless otherwise specified, the terms “switch” and “CAR-EC switch”, as used herein, are used interchangeably and may refer to a peptide switch. The antibody portion of the peptide antibody switch may comprise at least a portion of an antibody or an entire antibody. For example, the antibody portion of the peptide antibody switch may comprise at least a portion of a heavy chain, a portion of a light chain, a portion of a variable region, a portion of a constant region, a portion of a complementarity determining region (CDR), or a combination thereof. The antibody portion of the peptide antibody switch and/or hapten antibody switch may comprise at least a portion of the Fc (fragment, crystallizable) region. The antibody portion of the peptide antibody switch may comprise at least a portion of the complementarity determining region (e.g., CDR1, CDR2, CDR3). The antibody portion of the peptide antibody switch may comprise at least a portion of the Fab (fragment, antigen-binding) region. The peptide switch may be a peptide-Fab switch.
Before the present methods, kits and compositions are described in greater detail, it is to be understood that this invention is not limited to particular method, kit or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
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, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.
It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the peptide” includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
Methods, kits and compositions are provided for producing CAR-EC platforms and CAR-EC switches used to bring an effector cell together with a target in a subject. These methods, kits and compositions find therapeutic use in a number of diseases. For example, heterogeneous tumors and blood cell malignancies (e.g. acute lymphoblastic leukemia and chronic lymphocytic leukemia) may be more effectively treated with a CAR-EC platform when the length, valency and orientation of the CAR-EC switch linkage as well as the CAR-EC switch cell targeting moiety is optimized. Heterogeneous tumors may be more effectively treated with multiple CAR-EC switches that target more than one tumor antigens. Advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the compositions and methods as more fully described below.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

IA. Peptide Switch

Disclosed herein are chimeric antigen receptor-effector cell switches comprising: a peptidic antigen that binds a chimeric antigen receptor on an effector cell; and a targeting moiety that binds a cell surface molecule on a target. The targeting moiety may be a targeting polypeptide, comprising a targeting peptide that binds the cell surface molecule. The targeting moiety may be a targeting antibody or antibody fragment comprising the targeting peptide, wherein the targeting peptide is an antigen binding site of the targeting antibody or antibody fragment. The targeting peptide may be at least a portion of an antibody fragment and the cell surface molecule may be an antigen. The targeting moiety may comprise one or more peptides that recognize and/or bind one or more antigens. The targeting moiety may comprise one or more peptides that recognize and/or bind only one antigen. The peptidic antigen may not comprise an antibody or antibody fragment that recognizes and/or binds an antigen.
Further disclosed herein are CAR-EC switches comprising: a peptidic antigen that binds a CAR (CAR-BP) on an effector cell, wherein the CAR-BP; and a targeting polypeptide that binds a cell surface molecule on a target cell. The peptidic antigen may be fused to a terminus of the targeting polypeptide. The peptidic antigen may be grafted into the targeting polypeptide (e.g. between chosen amino acids of the targeting polypeptide). The targeting polypeptide may be fused to a terminus of the peptidic antigen. The targeting polypeptide may be grafted into the peptidic antigen (e.g. between chosen amino acids of the peptidic antigen).
Disclosed herein are CAR-EC switches comprising: a peptidic antigen that binds a CAR (CAR-BP) on an effector cell; and a targeting antibody or antibody fragment that binds an antigen on a target. The targeting antibody or antibody fragment may be selected from an immunoglobulin, a Fab, a Fab′, a F(ab′)₂and an scFv. The targeting antibody or antibody fragment may comprise a light chain. The targeting antibody or antibody fragment may comprise a heavy chain.
The peptidic antigen may be fused to an N terminus of the light chain of the targeting antibody or antibody fragment. The peptidic antigen may be fused to a C terminus of the light chain of the targeting antibody or antibody fragment. The peptidic antigen may be fused to an N terminus of the heavy chain of the targeting antibody or antibody fragment. The peptidic antigen may be fused to a C terminus of the heavy chain of the targeting antibody or antibody fragment. The peptidic antigen may be fused to an N terminus of a VL domain of the targeting antibody or antibody fragment. The peptidic antigen may be fused to an N terminus of a VH domain of the targeting antibody or antibody fragment. The peptidic antigen may be fused to a C terminus of a CL domain of the targeting antibody or antibody fragment. The peptidic antigen may be fused to a C terminus of an Fc domain of the targeting antibody or antibody fragment. The peptidic antigen may be fused to an N terminus of a VL domain of an IgG. The peptidic antigen may be fused to an N terminus of a VH domain of an IgG. The peptidic antigen may be fused to a C terminus of a CL domain of an IgG. The peptidic antigen may be fused to a C terminus of an Fc domain of an IgG. The peptidic antigen may be fused to an N terminus of a VL domain of a Fab. The peptidic antigen may be fused to an N terminus of a VH domain of a Fab. The peptidic antigen may be fused to a C terminus of a CL domain of a Fab. The peptidic antigen may be fused to a C terminus of a CHi domain of the Fab.
The peptidic antigen may be grafted into an internal site of a targeting antibody or antibody fragment (e.g. between chosen amino acids of the targeting antibody or antibody fragment). The peptidic antigen may be grafted into a heavy chain of a targeting antibody or antibody fragment. The peptidic antigen may be grafted into a light chain of a targeting antibody or antibody fragment. The peptidic antigen may be grafted into a constant domain/region of a targeting antibody or antibody fragment. The peptidic antigen may be grafted into a variable domain/region of a targeting antibody or antibody fragment. The peptidic antigen may be grafted into an internal site of a Fab. The peptidic antigen may be grafted into an internal site of an immunoglobulin (e.g. IgG). The peptidic antigen may be grafted into a domain of the targeting antibody or fragment thereof selected from a CL domain, a CH₁domain, a CH₂domain, a CH₃domain, a VL domain, a VH domain and a hinge domain. The peptidic antigen may be grafted between two domains of the antibody or fragment thereof selected from a CL domain, a CH₁domain, a CH₂domain, a CH₃domain, a VL domain, a VH domain and a hinge domain, wherein the two domains are adjacent. The peptidic antigen may be grafted into a CL domain of the antibody or fragment thereof. The peptidic antigen may be grafted into a CH₁domain of the antibody or fragment thereof. The peptidic antigen may be grafted into a hinge domain of the antibody or fragment thereof. The peptidic antigen may be grafted into a loop of the antibody or fragment thereof. The peptidic antigen may be grafted into a CL domain loop of the antibody or fragment thereof.
The CAR-BP may be grafted into the C terminus of the antibody or antibody fragment and therefore the distance between the chimeric antigen receptor and the target may differ substantially depending on the size of CAR-EC switch (approximately 40 Å for scFv, 70 Å for Fab, and 120 Å for IgG). While a larger distance may negatively impact efficacy in vitro, the increased residence time of the full length antibody may be superior in vivo.
The CAR-BP may further comprise a linker. The linker may provide the CAR-EC switch flexibility, length or geometry optimal for facilitating an interaction or effect of the CAR-EC on the target cell. The CAR-BP may further comprise one or more linkers. The CAR-BP may comprise two linkers. The linker may comprise a peptide. The linker may be at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9 or at least about 10 amino acids in length. The one or more linkers may comprise about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 80, about 90 or about 100 amino acids. The linker may be located at the N terminus or the C terminus of the CAR-BP to graft the CAR-BP to the targeting polypeptide. A first linker may be fused to the N terminus of the CAR-BP and a second linker may be fused to the C terminus of the CAR-BP. The linker may be comprised of the sequence (GGGGS)_n, (SEQ ID NO.40), wherein n may be 1, 2, 3, 4, 5 or more. The linker may be comprised of the sequence (GGS)_n, (SEQ ID NO.40), wherein n may be 1, 2, 3, 4, 5 or more. The CAR-BP may be grafted into an internal site of the targeting polypeptide with a linker on either end of the CAR-BP. The linker may comprise a sequence selected from SEQ ID NOS: 40-44.

The Peptidic Antigen

The peptidic antigen (CAR-BP) may be a peptide that is bound by a chimeric antigen receptor (CAR). The peptidic antigen may have high proteolytic stability and low immunogenicity in humans relative to peptides in general. The CAR-BP may be selected from a hormone, a cytokine, a chemokine, a growth factor, a cell adhesion molecule, a signaling peptide, a receptor, a cell surface peptide and fragments thereof. The CAR-BP may be a peptoid. The CAR-BP may be a peptide nucleic acid (PNA). The CAR-BP may be a ligand or a fragment thereof. The ligand may be a hormonal ligand. The ligand may be a peptide ligand. The CAR-BP may be a cyclic peptide. The CAR-BP may be a linear peptide. The CAR-BP may have a length of between about 2 and about 10, about 10 and about 20, about 20 and about 30, about 30 and about 40, about 40 and about 50, about 50 and about 60, about 60 and about 70, about 70 and about 80, and about 80 and about 90 amino acids. The CAR-BP may be an antigen. The CAR-BP may be an epitope. The CAR-BP may be a nonlinear epitope. The CAR-BP may further comprise a second peptide.
The peptidic antigen may not comprise an antibody or antibody fragment. The peptidic antigen may comprise less than 10 amino acids of an antibody or antibody fragment. The peptidic antigen may comprise less than 12 amino acids of an antibody or antibody fragment. The peptidic antigen may comprise less than 15 amino acids of an antibody or antibody fragment. The peptidic antigen may comprise less than 20 amino acids of an antibody or antibody fragment. The peptidic antigen may comprise less than 22 amino acids of an antibody or antibody fragment. The peptidic antigen may comprise less than 30 amino acids of an antibody or antibody fragment. The peptidic antigen may not comprise a paratope of an antibody or antibody fragment.
Disclosed herein are chimeric antigen receptor effector cell switches comprising a targeting moiety and a peptidic antigen, wherein the targeting moiety is a targeting polypeptide. The targeting polypeptide may comprise a targeting antibody or antibody fragment. The targeting antibody or antibody fragment may comprise a variable domain. The variable domain may be selected from a VH domain and a VL domain. The peptidic antigen may not be located at or near the N terminus of the VH domain. The peptidic antigen may not be located at or near the N terminus of the VL domain.
The peptidic antigen may comprise a non-naturally occurring peptide. The peptidic antigen may comprise a synthetic peptide. The peptidic antigen may comprise a non-animal peptide (e.g. a peptide not expressed in an animal). The peptidic antigen may comprise a non-mammalian peptide. The peptidic antigen may comprise a non-human peptide. The peptide may comprise a peptide derived from a plant, a yeast, a bacteria, a reptile, a bird or an insect.
The peptidic antigen may comprise a myc-tag. The peptidic antigen may comprise His-tag. The peptidic antigen may comprise an HA-tag. The peptidic antigen may comprise peridinin chlorophyll protein complex. The peptidic antigen may comprise green fluorescent protein (GFP). The peptidic antigen may comprise red fluorescent protein (RFP). The peptidic antigen may comprise phycoerythrin (PE). The peptidic antigen may comprise streptavidin. The peptidic antigen may comprise avidin. The peptidic antigen may comprise horse radish peroxidase (HRP). The peptidic antigen may comprise alkaline phosphatase. The peptidic antigen may comprise glucose oxidase. The peptidic antigen may comprise glutathione-S-transferase (GST). The peptidic antigen may comprise maltose binding protein. The peptidic antigen, by non-limiting example, may be a c-myc tag, polyhistidine tag, V5, VSVG, softag 1, softag 3, express tag, S tag, palmitoylation, nitrosylation, SUMO tag, thioredoxin, poly(NANP), poly-Arg, calmodulin binding protein, PurF fragment, ketosteroid isomerase, PaP3.30, TAF12 histone fold domain, FKBP-tag, SNAP tag, Halo-tag, peptides from RNAse I. The peptidic antigen may comprise a protease cleavage site. The protease cleavage site may be recognized by thrombin, factor Xa, TEV protease or enterokinase.
The peptidic antigen may be a small linear hydrophilic peptide. The small linear hydrophilic peptide may comprise a linker. The small linear hydrophilic peptide may be a hydrophilic target peptide (HTP). The small linear hydrophilic peptide may comprise the sequence GGGGSDYKDDDDK (SEQ ID NO: 5). The small linear hydrophilic peptide may comprise the sequence GGGGSDYKDDDDKP (SEQ ID NO: 6). The small linear hydrophilic peptide may consist essentially of the sequence GGGGSDYKDDDDK (SEQ ID NO: 5). The small linear hydrophilic peptide may consist essentially of the sequence GGGGSDYKDDDDKP (SEQ ID NO: 6). The small linear hydrophilic peptide may be at least about 50% homologous to SEQ ID NOs: 5 or 6. The small linear hydrophilic peptide may be at least about 60% homologous to SEQ ID NOs: 5 or 6. The small linear hydrophilic peptide may be at least about 70% homologous to SEQ ID NOs: 5 or 6. The small linear hydrophilic peptide may be at least about 80% homologous to SEQ ID NOs: 5 or 6. The small linear hydrophilic peptide may be at least about 85% homologous to SEQ ID NOs: 5 or 6. The small linear hydrophilic peptide may be at least about 90% homologous to SEQ ID NOs: 5 or 6. The small linear hydrophilic peptide may have reduced non-specific binding relative to other peptides known in the art. The small linear hydrophilic peptide may have reduced non-specific binding and reduced fusion protein instability relative to other peptides disclosed herein. The peptidic antigen may comprise a FLAG® tag (SEQ ID NO: 7) or a derivative or a homolog thereof.
The peptide may be based on or derived from a naturally occurring peptide. The peptide may be based on or derived from a human peptide. The peptide may be based on or derived from an peptide expressed in animal selected from a chimpanzee, a monkey, a rat, a mouse, a bird, a fish, a pig, a horse, a cow, a goat, a chicken, a rabbit and a guinea pig. The peptide may be based on or derived from a mammalian peptide. The peptide may be based on or derived from a non-mammalian peptide. The peptide may be based on or derived from a peptide expressed in a plant. The peptide may be based on or derived from a peptide expressed in a bacterium. The peptide may be based on or derived from a prokaryotic peptide. The peptide may be based on or derived from a eukaryotic peptide. The peptide may be based on or derived from a peptide expressed by a yeast.The peptidic antigen may comprise a yeast transcription factor GCN4 peptide or a derivative or a homolog thereof. The yeast transcription factor GCN4 peptide may comprise the sequence RMKQLEPKVEELLPKNYHLENEVARLKKLVGER (SEQ ID NO: 2). The yeast transcription factor GCN4 peptide may comprise the sequence NYHLENEVARLKKL (SEQ ID NO: 3). The yeast transcription factor GCN4 peptide may consist essentially of the sequence RMKQLEPKVEELLPKNYHLENEVARLKKLVGER (SEQ ID NO: 2). The yeast transcription factor GCN4 peptide may consist essentially of the sequence NYHLENEVARLKKL (SEQ ID NO: 3). The yeast transcription factor GCN4 peptide may comprise a portion of SEQ ID NO. 2. The portion of SEQ ID NO. 2 may be at least 4 amino acids long. The portion of SEQ ID NO. 2 may be about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12 or about 13 amino acids long. The yeast transcription factor GCN4 peptide may be at least about 50% homologous to SEQ ID NOs: 2 or 3. The yeast transcription factor GCN4 peptide may be at least about 60% homologous to SEQ ID NOs: 2 or 3. The yeast transcription factor GCN4 peptide may be at least about 70% homologous to SEQ ID NOs: 2 or 3. The yeast transcription factor GCN4 peptide may be at least about 80% homologous to SEQ ID NOs: 2 or 3. The yeast transcription factor GCN4 peptide may be at least about 85% homologous to SEQ ID NOs: 2 or 3. The yeast transcription factor GCN4 peptide may be at least about 90% homologous to SEQ ID NOs: 2 or 3. The CAR-EC switch may comprise a yeast GCN4 peptide and one or more linkers. The CAR-EC switch may comprise SEQ ID NO. 4.

The Targeting Moiety

The targeting moiety may bind to a cell surface molecule on a target. The cell surface molecule may comprise an antigen. The cell surface molecule may be selected from a protein, a lipid moiety, a glycoprotein, a glycolipid, a carbohydrate, a polysaccharide, a nucleic acid, an MHC-bound peptide, or a combination thereof. The cell surface molecule may comprise parts (e.g., coats, capsules, cell walls, flagella, fimbrae, and toxins) of bacteria, viruses, and other microorganisms. The cell surface molecule may be expressed by the target cell. The cell surface molecule may not be expressed by the target cell. By way of non-limiting example, the cell surface molecule may be a ligand expressed by a cell that is not the target cell and that is bound to the target cell or a cell surface molecule of the target cell. Also, by non-limiting example, the cell surface molecule may be a toxin, exogenous molecule or viral protein that is bound to a cell surface or cell surface receptor of the target cell.
The targeting polypeptide may be a targeting antibody or antibody fragment. The targeting antibody or antibody fragment may be an immunoglobulin (Ig). The immunoglobulin may selected from an IgG, an IgA, an IgD, an IgE, an IgM, a fragment thereof or a modification thereof. The immunoglobulin may be IgG. The IgG may be IgG1. The IgG may be IgG2. The IgG may have one or more Fc mutations for modulating endogenous T cell FcR binding to the CAR-EC switch. The IgG may have one or more Fc mutations for removing the Fc binding capacity to the FcR of FcR-positive cells. Removal of the Fc binding capacity may reduce the opportunity for crosslinking of the CAR-EC to FcR positive cells, wherein crosslinking of the CAR-EC to FcR positive cells would activate the CAR-EC in the absence of the target cell. As such, modulating the endogenous T cell FcR binding to the CAR-EC switch may reduce an ineffective or undesirable immune response. The one or more Fc mutations may remove a glycosylation site. The one or more Fc mutations may be selected from E233P, L234V, L235A, delG236, A327G, A330S, P331S, N297Q and any combination thereof. The one or more Fc mutations may be in IgG1. The one or more Fc mutations in the IgG1 may be L234A, L235A, or both. Alternatively, or additionally, the one or more Fc mutations in the IgG1 may be L234A, L235E, or both. Alternatively, or additionally, the one or more Fc mutations in the IgG1 may be N297A. Alternatively, or additionally, the one or more mutations may be in IgG2. The one or more Fc mutations in the IgG2 may be V234A, V237A, or both.
The targeting antibody or antibody fragment may be an Fc null immunoglobulin or a fragment thereof.
As used herein, the term “antibody fragment” refers to any form of an antibody other than the full-length form. Antibody fragments herein include antibodies that are smaller components that exist within full-length antibodies, and antibodies that have been engineered. Antibody fragments include, but are not limited to, Fv, Fc, Fab, and (Fab′)2, single chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy chains, light chains, alternative scaffold non-antibody molecules, and bispecific antibodies. Unless specifically noted otherwise, statements and claims that use the term “antibody” or “antibodies” may specifically include “antibody fragment” and “antibody fragments.”
The targeting antibody fragment may be human, fully human, humanized, human engineered, non-human, and/or chimeric antibody. The non-human antibody may be humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Chimeric antibodies may refer to antibodies created through the joining of two or more antibody genes which originally encoded for separate antibodies. A chimeric antibody may comprise at least one amino acid from a first antibody and at least one amino acid from a second antibody, wherein the first and second antibodies are different. At least a portion of the antibody or antibody fragment may be from a bovine species, a human species, or a murine species. At least a portion of the antibody or antibody fragment may be from a rat, a goat, a guinea pig or a rabbit. At least a portion of the antibody or antibody fragment may be from a human. At least a portion of the antibody or antibody fragment antibody may be from cynomolgus monkey.
The targeting antibody or antibody fragment may be based on or derived from an antibody or antibody fragment from a mammal, bird, fish, amphibian, reptile. Mammals include, but are not limited to, carnivores, rodents, elephants, marsupials, rabbits, bats, primates, seals, anteaters, cetaceans, odd-toed ungulates and even-toed ungulates. The mammal may be a human, non-human primate, mouse, sheep, cat, dog, cow, horse, goat, or pig.
The targeting antibody or an antibody fragment may target an antigen selected from, by non-limiting example, CD19, Her2, CLL-1, CD33, EGFRvIII, CD20, CD22, BCMA or a fragment thereof. The antigen may comprise a wildtype antigen. The antigen may comprise one or more mutations.
The targeting antibody or antibody fragment may be an anti-CD19 antibody or a fragment thereof. The targeting polypeptide may be an anti-CD22 antibody. The targeting polypeptide may be an anti-BCMA antibody or a fragment thereof. The targeting polypeptide may be an anti-CS1 antibody or a fragment thereof. The targeting polypeptide may be an anti-EGFRvIII antibody or a fragment thereof. The targeting polypeptide may be an anti-Her2 antibody or a fragment thereof. The targeting polypeptide may comprise an anti-CD20 antibody or antibody fragment. The targeting polypeptide may comprise rituximab. The targeting polypeptide may comprise an anti-EGFR antibody or antibody fragment. The targeting polypeptide may comprise an anti-CEA antibody or antibody fragment. The targeting polypeptide may comprise an anti-CLL-1 antibody or antibody fragment. The targeting polypeptide may comprise an anti-CD33 antibody or antibody fragment. The targeting polypeptide may not comprise an anti-EpCAM antibody or fragment thereof.
The targeting antibody or antibody fragment may be selected any commercially available antibody. The targeting antibody or antibody fragment may be selected from ado-trastuzumab emtansine, alemtuzumab, bevacizumab, brentuximab, vedotin, gemtuzumab, ozogamicin, ipilimumab, ibritumomab, tiuxetan, panitumumab, cetuximab, erbitux, rituximab, trastuzumab and fragments thereof.
The targeting antibody or antibody fragment may comprise an anti-CD19 antibody or fragment thereof. The targeting antibody or fragment thereof may comprise a light chain of the anti-CD19 antibody or fragment thereof. The light chain of the anti-CD19 antibody or fragment thereof may be encoded by a nucleotide sequence based on or derived from SEQ ID NO. 8. The nucleotide sequence may be about 99%, about 98%, about 97%, about 96%, about 95%, about 92%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5% or about 2% homologous to SEQ ID NO. 8. The targeting antibody or fragment thereof may comprise a heavy chain of the anti-CD19 antibody or fragment thereof. The heavy chain of the anti-CD19 antibody or fragment thereof may be encoded by a sequence based on or derived from SEQ ID NO. 9. The nucleotide sequence may be about 99%, about 98%, about 97%, about 96%, about 95%, about 92%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5% or about 2% homologous to SEQ ID NO. 9.
The targeting antibody or antibody fragment may comprise an anti-CD19 antibody or fragment thereof. The targeting antibody or fragment thereof may comprise a light chain of the anti-CD19 antibody or fragment thereof. The light chain of the anti-CD19 antibody or fragment may comprise an amino acid sequence based on or derived from SEQ ID NO. 27. The amino acid sequence may be about 99%, about 98%, about 97%, about 96%, about 95%, about 92%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5% or about 2% homologous to SEQ ID NO. 27. The targeting antibody or fragment thereof may comprise a heavy chain of the anti-CD19 or fragment thereof. The targeting antibody or fragment thereof may comprise a heavy chain of an anti-CD19 IgG. The heavy chain of the anti-CD19 IgG may comprise a sequence based on or derived from SEQ ID NO. 28. The amino acid sequence may be about 99%, about 98%, about 97%, about 96%, about 95%, about 92%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5% or about 2% homologous to SEQ ID NO. 28. The targeting antibody or fragment thereof may comprise a heavy chain of an anti-CD19 Fab. The heavy chain of the anti-CD19 Fab may comprise a sequence based on or derived from SEQ ID NO. 29. The amino acid sequence may be about 99%, about 98%, about 97%, about 96%, about 95%, about 92%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5% or about 2% homologous to SEQ ID NO. 29.
The targeting antibody or antibody fragment may comprise a nucleotide sequence selected from SEQ ID NOs: 8-20. The targeting polypeptide may be based on or derived from a nucleotide selected from SEQ ID NOs: 8-20. The targeting antibody or antibody fragment may comprise an amino acid sequence selected from SEQ ID NOs: 21-29. The targeting polypeptide may be based on or derived from an amino acid sequence selected from SEQ ID NOs: 21-29.
Disclosed herein are chimeric antigen receptor effector cell (CAR-EC) switches comprising a peptidic antigen and a targeting moiety that binds a cell surface molecule on a target cell. Generally, binding of the effector cell and the target cell to the CAR-EC switch construct brings the target cell into proximity with the effector cell sufficiently close for an activity of the effector cell to have an effect on the target cell. For example, when the T cell and the target cell are bound to the CAR-EC switch, the T cell may produce an immune response that has a cytotoxic effect on the target cell.
The CAR-EC switches may interact with a plurality of target cells. The target cell may be an infected cell. The target cell may be a pathogenically infected cell. The target cell may be a diseased cell. The target cell may be a genetically-modified cell. The target cell may not be a host cell. The target cell may come from an invading organism (e.g. yeast, worm, bacteria, fungus). Further disclosed herein are CAR-EC switches that interact with a molecule on a non-cell target. The non-cell target may be a virus or a portion thereof. The non-cell target may be a fragment of a cell. The non-cell target may be an extracellular matrix component or protein.
The target cell may be derived from a tissue. The tissue may be selected from brain, esophagus, breast, colon, lung, glia, ovary, uterus, testes, prostate, gastrointestinal tract, bladder, liver, thymus, bone and skin. The target cell may be derived from one or more endocrine glands. Alternatively, or additionally, the target cell may be derived from one or more endocrine glands. The endocrine gland may be a lymph gland, pituitary gland, thyroid gland, parathyroid gland, pancreas, gonad or pineal gland.
The target cell may be selected from a stem cell, a pluripotent cell, a hematopoietic stem cell or a progenitor cell. The target cell may a circulating cell. The target cell may be an immune cell.
The target cell may be a cancer stem cell. The target cell may be a cancer cell. The cancer cell may be derived from a tissue. The tissue may be selected from, by way of non-limiting example, a brain, an esophagus, a breast, a colon, a lung, a glia, an ovary, a uterus, a testicle, a prostate, a gastrointestinal tract, a bladder, a liver, a thyroid and skin. The cancer cell may be derived from bone. The cancer cell may be derived from blood. The cancer cell may be derived from a B cell, a T cell, a monocyte, a thrombocyte, a leukocyte, a neutrophil, an eosinophil, a basophil, a lymphocyte, a hematopoietic stem cell or an endothelial cell progenitor. The cancer cell be derived from a CD19-positive B lymphocyte. The cancer cell may be derived from a stem cell. The cancer cell may be derived from a pluripotent cell. The cancer cell may be derived from one or more endocrine glands. The endocrine gland may be a lymph gland, pituitary gland, thyroid gland, parathyroid gland, pancreas, gonad or pineal gland.
The cancer cell may be a CD19-positive cell. The cancer cell may be a CD19-positive B lymphocyte. The cancer cell may be a Her2-positive cell. The Her2-positive cell may be a Her2-positive breast cancer cell. The target cell may be a BCMA-positive cell. The cancer cell may be a BCMA-positive multiple myeloma cell. The cancer cell may be a CS1-positive cell. The CS1-positive cell may be a multiple myeloma cell. The cancer cell may be a EGFRvIII-positive cell. The cancer cell may be a EGFRvIII-positive glioblastoma cell. The cancer cell may be a CD20-positive cell. The cancer cell may be a CD22-positive cell.
The cell surface molecule may be an antigen. The antigen may be at least a portion of a surface antigen or a cell surface marker on a cell. The antigen may be a receptor or a co-receptor on a cell. The antigen may refer to a molecule or molecular fragment that may be bound by a major histocompatibility complex (MHC) and presented to a T-cell receptor. The term “antigen” may also refer to an immunogen. The immunogen may provoke an adaptive immune response if injected on its own into a subject. The immunogen may induce an immune response by itself. The antigen may be a superantigen, T-dependent antigen or a T-independent antigen. The antigen may be an exogenous antigen. Exogenous antigens are typically antigens that have entered the body from the outside, for example by inhalation, ingestion, or injection. Some antigens may start out as exogenous antigens, and later become endogenous (for example, intracellular viruses). The antigen may be an endogenous antigen. The endogenous antigen may be an antigen that has been generated within cells as a result of normal cell metabolism, or because of pathogenic infections (e.g., viral, bacterial, fungal, parasitic). The antigen may be an autoantigen. The autoantigen may be a normal protein or complex of proteins (and sometimes DNA or RNA) that is recognized by the immune system of patients suffering from a specific autoimmune disease. These antigens should, under normal conditions, not be the target of the immune system, but, due to genetic and/or environmental factors, the normal immunological tolerance for such an antigen is not present in these patients. The antigen may be present or over-expressed due to a condition or disease. The condition or disease may be a cancer or a leukemia. The condition may be an inflammatory disease or condition. The condition or disease may be a metabolic disease. The condition may be a genetic disorder.
The cell surface molecule may be an antigen that has been designated as a tumor antigen. Tumor antigens or neoantigens may be antigens that are presented by MHC I or MHC II molecules on the surface of tumor cells. These antigens may sometimes be presented by tumor cells and never by the normal ones. In this case, they are called tumor-specific antigens (TSAs) and, in general, result from a tumor-specific mutation. More common are antigens that are presented by tumor cells and normal cells, and they are called tumor-associated antigens (TAAs). Cytotoxic T lymphocytes that recognize these antigens may be able to destroy the tumor cells before they proliferate or metastasize. Tumor antigens may also be on the surface of the tumor in the form of, for example, a mutated receptor, in which case they may be recognized by B cells. Unless otherwise specified, the terms “tumor antigen,” “tumor specific antigen” and “tumor associated antigen,” are used interchangeably herein.
The cell surface molecule may be a receptor. The receptor may be an extracellular receptor. The receptor may be a cell surface receptor. By way of non-limiting example, the receptor may bind a hormone, a neurotransmitter, a cytokine, a growth factor or a cell recognition molecule. The receptor may be a transmembrane receptor. The receptor may be an enzyme-linked receptor. The receptor may be a G-protein couple receptor (GPCR). The receptor may be a growth factor receptor. By way of non-limiting example, the growth factor receptor may be selected from an epidermal growth factor receptor, a fibroblast growth factor receptor, a platelet derived growth factor receptor, a nerve growth factor receptor, a transforming growth factor receptor, a bone morphogenic protein growth factor receptor, a hepatocyte growth factor receptor, a vascular endothelial growth factor receptor, a stem cell factor receptor, an insulin growth factor receptor, a somatomedin receptor, an erythropoietin receptor and homologs and fragments thereof. The receptor may be a hormone receptor. The receptor may be an insulin receptor. By way of non-limiting example, the receptor may selected from an eicosanoid receptor, a prostaglandin receptor, an estrogen receptor, a follicle stimulating hormone receptor, a progesterone receptor, a growth hormone receptor, a gonadotropin-releasing hormone receptor, homologs thereof and fragments thereof. The receptor may be an adrenergic receptor. The receptor may be an integrin. The receptor may be an Eph receptor. The receptor may be a luteinizing hormone receptor. The cell surface molecule may be at least about 50% homologous to a luteinizing hormone receptor. The receptor may be an immune receptor. By way of non-limiting example, the immune receptor may be selected from a pattern recognition receptor, a toll-like receptor, a NOD like receptor, a killer activated receptor, a killer inhibitor receptor, an Fc receptor, a B cell receptor, a complement receptor, a chemokines receptor and a cytokine receptor. By way of non-limiting example, the cytokine receptor may be selected from an interleukin receptor, an interferon receptor, a transforming growth factor receptor, a tumor necrosis factor receptor, a colony stimulating factor receptor, homologs thereof and fragments thereof. The receptor may be a receptor kinase. The receptor kinase may be a tyrosine kinase receptor. The receptor kinase may be a serine kinase receptor. The receptor kinase may be a threonine kinase receptor. By way of non-limiting example, the receptor kinase may activate a signaling protein selected from a Ras, a Raf, a PI3K, a protein kinase A, a protein kinase B, a protein kinase C, an AKT, an AMPK, a phospholipase, homologs thereof and fragments thereof. The receptor kinase may activate a MAPK/ERK signaling pathway. The receptor kinase may activate Jak, Stat or Smad.
The cell surface molecule may be a non-receptor cell surface protein. The cell surface molecule may be a cluster of differentiation proteins. By way of non-limiting example, the cell surface molecule may be selected from CD34, CD31, CD117, CD45, CD11b, CD15, CD24, CD114, CD182, CD14, CD11a, CD91, CD16, CD3, CD4, CD25, CD8, CD38, CD22, CD61, CD56, CD30, CD13, CD33, fragments thereof, and homologs thereof.
The cell surface molecule may be a molecule that does not comprise a peptide. The cell surface molecule may comprise a lipid. The cell surface molecule may comprise a lipid moiety or a lipid group. The lipid moiety may comprise a sterol. The lipid moiety may comprise a fatty acid. The antigen may comprise a glycolipid. The cell surface molecule may comprise a carbohydrate.
Disclosed herein are CAR-EC switches comprising (a) a chimeric antigen receptor binding peptidic antigen comprising a peptide from a yeast transcription factor peptide; and (b) a targeting polypeptide. The yeast transcription factor peptide may be a GCN4 peptide. The targeting polypeptide may comprise a targeting antibody or antibody fragment. The targeting antibody or antibody fragment may comprise a heavy chain of an antibody. The targeting antibody or antibody fragment may comprise a light chain of an antibody. The targeting antibody or antibody fragment may comprise a Fab of an antibody. The targeting antibody or antibody fragment may comprise an anti-CD19 antibody or a fragment thereof. The targeting antibody or antibody fragment may comprise an anti-Her2 antibody or a fragment thereof. The targeting antibody or antibody fragment may be selected from an anti-CS1 antibody, an anti-BCMA antibody, an anti-EGFRvIII antibody, an anti-CD20 antibody, an anti-EGFR antibody, an anti-CEA antibody, an anti-CLL-1 antibody, an anti-CD33 antibody and fragments thereof.
Further disclosed herein are CAR-EC switches comprising (a) a CAR binding region comprising a hydrophilic target peptide (HTP) tag; and (b) a targeting polypeptide. The targeting polypeptide may comprise a targeting antibody or antibody fragment. The targeting antibody or antibody fragment may comprise a heavy chain of an antibody. The targeting antibody or antibody fragment may comprise a light chain of an antibody. The targeting antibody or antibody fragment may comprise a Fab of an antibody. The targeting antibody or antibody fragment may comprise an anti-CD19 antibody or a fragment thereof. The targeting antibody or antibody fragment may comprise an anti-Her2 antibody or a fragment thereof. The targeting antibody or antibody fragment may be selected from an anti-CS1 antibody, an anti-BCMA antibody, an anti-EGFRvIII antibody, an anti-CD20 antibody, an anti-EGFR antibody, an anti-CEA antibody, an anti-CLL-1 antibody, an anti-CD33 antibody and fragments thereof.
The chimeric antigen receptor-effector cell switch may comprise a heavy chain selected from SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35 and SEQ ID NO. 38. The chimeric antigen receptor-effector cell switch may comprise a heavy chain that is at least 50% homologous to a sequence selected from SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35 and SEQ ID NO. 38. The chimeric antigen receptor-effector cell switch may comprise a heavy chain that is at least 60% homologous to a sequence selected from SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35 and SEQ ID NO. 38. The chimeric antigen receptor-effector cell switch may comprise a heavy chain that is at least 70% homologous to a sequence selected from SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35 and SEQ ID NO. 38. The chimeric antigen receptor-effector cell switch may comprise a heavy chain that is at least 80% homologous to a sequence selected from SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35 and SEQ ID NO. 38. The chimeric antigen receptor-effector cell switch may comprise a heavy chain that is at least 90% homologous to a sequence selected from SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35 and SEQ ID NO. 38.
The chimeric antigen receptor-effector cell switch may comprise a light chain selected from SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 36 and SEQ ID NO. 37. The chimeric antigen receptor-effector cell switch may comprise a light chain that is at least 50% homologous to a sequence selected from SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 36 and SEQ ID NO. 37. The chimeric antigen receptor-effector cell switch may comprise a light chain that is at least 60% homologous to a sequence selected from SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 36 and SEQ ID NO. 37. The chimeric antigen receptor-effector cell switch may comprise a light chain that is at least 70% homologous to a sequence selected from SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 36 and SEQ ID NO. 37. The chimeric antigen receptor-effector cell switch may comprise a light chain that is at least 80% homologous to a sequence selected from SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 36 and SEQ ID NO. 37. The chimeric antigen receptor-effector cell switch may comprise a light chain that is at least 90% homologous to a sequence selected from SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 36 and SEQ ID NO. 37.
The chimeric antigen receptor-effector cell switch may comprise a heavy chain of SEQ ID NO. 29 and a light chain of SEQ ID NO. 30. The chimeric antigen receptor-effector cell switch may comprise a heavy chain of SEQ ID NO. 29 and a light chain of SEQ ID NO. 36. The chimeric antigen receptor-effector cell switch may comprise a heavy chain of SEQ ID NO. 28 and a light chain of SEQ ID NO. 31. The chimeric antigen receptor-effector cell switch may comprise a heavy chain of SEQ ID NO. 32 and a light chain of SEQ ID NO. 27. The chimeric antigen receptor-effector cell switch may comprise a heavy chain of SEQ ID NO. 33 and a light chain of SEQ ID NO. 27. The chimeric antigen receptor-effector cell switch may comprise a heavy chain of SEQ ID NO. 34 and a light chain of SEQ ID NO. 27. The chimeric antigen receptor-effector cell switch may comprise a heavy chain of SEQ ID NO. 35 and a light chain of SEQ ID NO. 27. The chimeric antigen receptor-effector cell switch may comprise a heavy chain of SEQ ID NO. 38 and a light chain of SEQ ID NO. 37.

Multivalent CAR-EC Switches

Exemplified herein are CAR-EC switches comprising a chimeric antigen receptor binding peptidic antigen (CAR-BP) and a targeting polypeptide. However, one skilled in the art would understand that these switches could further comprise additional targeting polypeptides and/or additional CAR-BPs. One or more CAR-BPs may be grafted into one or more grafting sites of the targeting polypeptide. One or more CAR-BPs may be fused to one or more termini of the targeting polypeptide. This may be advantageous, as several grafting/fusing sites may be predicted to provide optimal binding of the CAR-BP to the CAR. For example, a first CAR-BP may be grafted into a first domain of the targeting polypeptide and a second CAR-BP may be grafted into a second domain of the targeting polypeptide. The first domain and the second domain may be the same. The first domain and the second domain may be different. By way of non-limiting example, the first CAR-BP may be grafted into a light chain of a targeting antibody or antibody fragment and a second CAR-BP may be grafted into heavy chain of the targeting antibody or antibody fragment. The first CAR-BP may be fused to a first terminus of the targeting polypeptide and a second CAR-BP may be fused to a second terminus of the targeting polypeptide. By way of non-limiting example, the first CAR-BP may be fused to a C terminus of a light chain of a targeting antibody or antibody fragment and a second CAR-BP may be fused to an N terminus of a heavy chain of the targeting antibody or antibody fragment. The first CAR-BP may be fused to a terminus of the targeting polypeptide and a second CAR-BP may be grafted within a domain of the targeting polypeptide. The first CAR-BP and the second CAR-BP may be the same or similar, such that the CAR-EC switch may be used with a CAR-EC cell that expresses one CAR. The first CAR-BP and the second CAR-BP may be different, such that the CAR-EC switch may be used with a CAR-EC cell that expresses one or more CARs or multiple CAR-EC cells that express different CARs.
The peptide switches disclosed herein may comprise one or more CAR-BPs. The peptide switches disclosed herein may comprise two or more CAR-BPs. The peptide switches disclosed herein may comprise three or more CAR-BPs. The peptide switches disclosed herein may comprise 1, 2, 3, 4, 5, 6, 7 or more CAR-BPs. The one or more CAR-BPs may be fused or grafted to the targeting polypeptide via one or more linkers. Thus, the peptide switches disclosed herein may comprise one or more linkers (e.g., L1, L2). The peptide switches disclosed herein may comprise two or more linkers. The peptide switches disclosed herein may comprise three or more linkers. The peptide switches disclosed herein may comprise 1, 2, 3, 4, 5, 6, 7 or more linkers.

IB. Peptide-Small Molecule Switch

Further disclosed herein are CAR-EC switches comprising a CAR binding region and a targeting moiety, wherein the CAR binding region is a CAR-binding peptidic antigen and the targeting moiety is a non-peptidic small molecule. The non-peptidic small molecule may be a cell-targeting molecule, a chemical ligand, a nucleic acid, a vitamin, a substrate or a substrate analog. The non-peptidic small molecule may not comprise two amino acids, wherein the two amino acids are connected by an amide bond. The CAR-EC switch may further comprise a linker. The CAR-binding peptidic antigen (CAR-BP) and the small molecule may be site-specifically linked. The CAR-binding peptidic antigen may comprise an unnatural amino acid. The CAR-binding peptidic antigen and the small molecule may be site-specifically linked by the unnatural amino acid. The small molecule may bind a cell surface molecule on a target cell. The cell surface molecule may be selected from an antigen, a protein, a peptide, a lipid, a sterol, a glycolipid and a cell surface marker. The CAR-binding peptidic antigen may be selected from FLAG® tag, yeast transcription factor GCN4 and a hydrophilic target peptide (HTP). The small molecule may be 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid. The small molecule may be folate. The CAR-EC switch may further comprise a linker.
Disclosed herein are methods of producing CAR-EC switches comprising conjugating the CAR binding region to the targeting moiety, wherein the CAR-EC switches comprise a CAR binding region and a targeting moiety, wherein the CAR binding region is a CAR-binding peptidic antigen and the targeting moiety is a small molecule. The method may further comprise conjugating the small molecule to the linker to create a small molecule-linker intermediate. The small molecule or the small molecule-linker intermediate may comprise one or more reactive functional groups that may react with a complementary reactive functional group on the CAR-BP, previous to incorporation into the CAR-EC switch. The linker or the small molecule-linker intermediate may be bifunctional. The linker or the small molecule-linker intermediate may be heterobifunctional.
The small molecule-linker intermediate or the CAR-EC switch may be the product of a bioorthogonal reaction, non-limiting examples of which are reviewed in Kim et al., Curr Opin Chem Bio 17:412-419 (2013). The small molecule-linker intermediate, linker or the CAR-EC switch may comprise an oxime, a tetrazole, a Diels Alder adduct, a hetero Diels Alder adduct, an aromatic substitution reaction product, a nucleophilic substitution reaction product, an ester, an amide, a carbamate, an ether, a thioether, or a Michael reaction product. The small molecule-linker intermediate, linker or the CAR-EC switch be a cycloaddition product, a metathesis reaction product, a metal-mediated cross-coupling reaction product, a radical polymerization product, an oxidative coupling product, an acyl-transfer reaction product, or a photo click reaction product. The cycloaddition may be a Huisgen-cycloaddition. The cycloaddition may be a copper-free [3+2] Huisgen-cycloaddition. The cycloaddition may be a Diels-Alder reaction. The cycloaddition may be a hetero Diels-Alder reaction. The small molecule-linker intermediate may be the product of an enzyme-mediated reaction. The small molecule-linker intermediate may be a product of a transglutaminase-mediated reaction, non-limiting examples of which are described in Lin et al., J. Am. Chem. Soc. 128:4542-4543 (2006) and WO 2013/093809. The small molecule-linker intermediate, linker or the CAR-EC switch may comprise a disulfide bridge that connects two cysteine residues, such as ThioBridge™ technology by PolyTherics. The small molecule-linker intermediate, linker or the CAR-EC switch may comprise a maleimide bridge that connects two amino acid residues. The small molecule-linker intermediate, linker or the CAR-EC switch may comprise a maleimide bridge that connects two cysteine residues.
The small molecule-linker intermediate or linker may comprise an alkoxy-amine (or aminooxy) group, azide group and/or cyclooctyne group at one or more termini. The small molecule-linker intermediate or linker may comprise an alkoxy-amine at one terminus and an azide group at the other terminus. The small molecule-linker intermediate or linker may comprise an alkoxy-amine at one terminus and a cyclooctyne group at the other terminus. The alkoxy-amine may form a stable oxime with a ketone group on an amino acid. The alkoxy-amine may form a stable oxime with a ketone group on an unnatural amino acid. The ketone group may be on ap-acetyl phenylalanine (pAcF).

II. Chimeric Antigen Receptor (CAR)

Disclosed herein are CAR-EC switches that regulate the activities of a cell expressing a chimeric antigen receptor (CAR). The chimeric antigen receptor may comprise an extracellular domain, transmembrane domain and intracellular domain. The extracellular domain may bind to the peptidic antigen (e.g. CAR-BP) of the CAR-EC switch. The extracellular domain may comprise an antibody or antibody fragment that binds to the CAR-BP of the CAR-EC switch (a CAR-antibody). The CAR-antibody may comprise at least a portion of an antibody. In some instances, the CAR-antibody is not a full-length antibody. The CAR-antibody may comprise at least a portion of an immunoglobulin or fragment thereof. The immunoglobulin or fragment thereof may be selected from the group consisting of an scFv, a di-scFv, a bi-scFv, a Fab, an Fc, an F(ab′)2, a pFc′, a nanobody, an affibody, a DARPin, a diabody, a camelid, an engineered T cell receptor and a monobody. The immunoglobulin may be selected from the group consisting of an IgAl, an IgA2, an IgD, an IgM, an IgE, an IgG1, an IgG2, an IgG3, and an IgG4. The CAR-antibody may comprise at least a portion of a single chain variable fragment (scFv). The CAR-antibody may be human, fully human, humanized, human engineered, non-human, and/or chimeric antibody.
The CAR-antibody may have a binding affinity for the CAR-BP of less than about 0.01 pM, about 0.02 pM, about 0.03 pM, about 0.04 pM, 0.05 pM, about 0.06 pM, about 0.07 pM, about 0.08 pM, about 0.09 pM, about 0.1 pM, about 0.2 pM, 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM or about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, about 10 pM, about 0.01 nM, about 0.02 nM, about 0.03 nM, about 0.04 nM, about 0.05 nM, about 0.06 nM, about 0.07 nM, about 0.08 nM, about 0.09 nM, about 0.1 nM, about 0.2 nM, about 0.3 nM, about 0.4 nM, about 0.5 nM, about 0.6 nM, about 0.7 nM, about 0.8 nM, about 0.9 nM, about 1 nM, about 2 nM, about 2.5 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 12nM, about 14 nM, about 16 nM, about 18 nM, about 20 nM, about 22 nM, about 24 nM, about 26 nM, about 28 nM or about 30 nM.
The CAR-antibody may recognize a synthetic (non-naturally-occurring) peptide. The CAR-antibody may comprise an antibody or antibody fragment that recognizes a FLAG® tag or a fragment thereof. The CAR-antibody may comprise an antibody or antibody fragment that recognizes a yeast transcription factor GCN4 or a fragment thereof. The CAR-antibody may comprise an anti-HTP antibody or a fragment thereof.
The transmembrane domain and/or the intracellular domain of the CAR may comprise at least a portion of a cytoplasmic signaling domain. The intracellular domain may comprise at least a portion of a signaling molecule selected from the group comprising CD3zeta, CD28, and 4-1BB. The intracellular domain may comprise an Fc receptor or a portion thereof. The Fc receptor or portion thereof may be CD16 or a portion thereof. The signaling molecule may comprise CD3zeta. The signaling molecule may comprise CD28. The signaling molecule may comprise 4-1BB. The intracellular domain may comprise at least a portion of CD3zeta. The intracellular domain may comprise at least a portion of CD28, The intracellular domain may comprise at least a portion of 4-1BB, The intracellular domain may comprise at least a portion of OX-40, The intracellular domain may comprise at least a portion of CD30, The intracellular domain may comprise at least a portion of CD40, The intracellular domain may comprise at least a portion of CD2. The intracellular domain may comprise at least a portion of CD27. The intracellular domain may comprise at least a portion of PD-1. The intracellular domain may comprise at least a portion of ICOS. The intracellular domain may comprise at least a portion of lymphocyte function-associated antigen-1 (LFA-1). The intracellular domain may comprise at least a portion of CD7. The intracellular domain may comprise at least a portion of LIGHT. The intracellular domain may comprise at least a portion of NKG2C. The intracellular domain may comprise at least a portion of B7-H3. The intracellular domain may comprise at least a portion of a cytoplasmic signaling domain from one or more signaling molecules. The intracellular domain may comprise at least a portion of two or more cytoplasmic signaling domains. The two or more cytoplasmic signaling domains may be from two or more different signaling molecules. The intracellular domain may comprise at least a portion of three or more cytoplasmic signaling domains. The intracellular domain may comprise at least a portion of four or more cytoplasmic signaling domains. The intracellular domain may comprise at least a portion of a ligand that binds to one or more signaling molecules. The intracellular domain may comprise at least a portion of a ligand that binds to CD83.

III. Chimeric Antigen Receptor Effector Cells (CAR-EC)

The methods, platforms and kits disclosed herein may comprise one or more chimeric antigen receptor effector cells (CAR-EC) or uses thereof. The chimeric antigen receptor effector cells disclosed herein express a chimeric antigen receptor. The chimeric antigen receptor (CAR) may be any CAR disclosed herein. Wherein the methods, platforms or kits comprise two or more effector cells, the two or more effector cells may be of the same cell type. The two or more effector cells may be of a different cell type. The two or more effector cells may be of the same cell lineage. The two or more effector cells may be of different cell lineages. The two or more effector cells may comprise two or more identical CARs. The two or more effector cells may comprise two or more different CARs. The two or more effector cells may comprise two or more similar CARs.
The effector cell may be a T cell. The effector cell may be a cell of a T cell lineage. The effector cell may be a mature T cell. The effector cell may be a precursor T cell. The effector cell may be a cytotoxic T cell. The effector cell may be a naive T cell. The effector cell may be a memory stem cell T cell (T_MSC). The effector cell may be a central memory T cell (T_CM). The effector cell may be an effector T cell (TE). The effector cell may be a CD4+ T cell. The T cell may be a CD8+ T cell. The effector cell may be a CD4+ and CD8+ cell. The effector cell may be an alpha-beta T cell. The effector cell may be a gamma-beta T cell. The effector cell may be a natural killer T cell. The effector cell may be a helper T cell.
While preferred embodiments of the present disclosure describe methods, kits and platforms comprising T cells, one skilled in the art may also understand that other cell types may be used in place of a T cell. The effector cell may be an effector cell that has an effect on a target or target cell when brought into proximity of the target or target cell. The effector cell may be a cell that has a cytotoxic effect on a target or target cell when brought into proximity of the target or target cell. The effector cell may be an immune cell. The effector cell may be selected from a B cell, a monocyte, a thrombocyte, a leukocyte, a neutrophil, an eosinophil, a basophil, or a lymphocyte. The effector cell may be a lymphocyte. The effector cell may be a macrophage. The effector cell may be a phagocytic cell. The effector cell may be an effector B cell. The effector cell may be a natural killer cell. The effector cell may isolated or derived from a subject suffering from a disease or condition. The effector cell may be a cell derived from a subject to be treated with a CAR-EC switch or CAR-EC platform disclosed herein.
The T cell may express a chimeric antigen receptor encoded by one or more polynucleotides based on or derived from SEQ ID NO: 1. The polynucleotide may be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to one or more polynucleotides based on or derived from SEQ ID NO: 1. The polynucleotide may be at least about 70% identical to one or more polynucleotides based on or derived from SEQ ID NO: 1. The polypeptide encoded by one or more polynucleotides may be based on or derived from SEQ ID NO: 1. The polypeptide may be encoded by a polynucleotide that is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to one or more polynucleotides based on or derived from SEQ ID NO: 1. The polynucleotide may be constitutively expressed. The polynucleotide may be conditionally expressed.
Disclosed herein are methods of producing a chimeric antigen receptor effector cell (CAR-EC), the methods comprising introducing one or more polynucleotides encoding a chimeric antigen receptor or a chimeric antigen receptor complex into an effector cell. The effector cell may be a T cell. Introducing one or more polynucleotides encoding a chimeric antigen receptor or a chimeric antigen receptor complex into an effector cell may comprise transfecting the effector cell with the one or more polynucleotides. Introducing one or more polynucleotides encoding a chimeric antigen receptor or a chimeric antigen receptor complex into an effector cell may comprise virally infecting the effector cell with one or more viruses comprising the one or more polynucleotides encoding a chimeric antigen receptor disclosed herein. The virus may be a lentivirus. The virus may be an adenovirus. The virus may be a retrovirus. The virus may be an adeno-associated virus. The virus may be a self-complementary adeno-associated virus (scAAV). The virus may be a modified human immunodeficiency (HIV) virus. The virus may be a modified herpes simplex virus (HSV) virus. Other methods of producing the CAR-EC may comprise a method of transferring one or more polynucleotides encoding a chimeric antigen receptor into a cell, wherein the methods comprise adding a transposon, a zinc finger nuclease, a TALEN or a CRISPR to the cell. The transposon may be a sleeping beauty transposon. The one or more polynucleotides may be based on or derived from SEQ ID NO: 1.

IV. CAR-EC Platform

Disclosed herein are chimeric antigen receptor effector cell (CAR-EC) platforms comprising a an effector cell, wherein the effector cell comprises a polynucleotide encoding a chimeric antigen receptor (CAR); and a chimeric antigen receptor effector cell (CAR-EC) switch, wherein the CAR-EC switch comprises a CAR binding peptidic antigen and a targeting polypeptide and wherein the CAR-EC switch binds a cell surface molecule on a target cell. The CAR-EC switch may be selected from any CAR-EC switches disclosed herein.
The CAR-EC platforms may comprise two or more CAR-EC switches. The CAR-EC platforms may comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more CAR-EC switches. The CAR-EC platforms may comprise may comprise more than 20, more than 25, more than 30, more than 35, more than 40, more than 45 or more than 50 CAR-EC switches. The two or more switches may be selected from one or more CAR-EC switches disclosed herein or a combination thereof.
The CAR-EC platforms disclosed herein may further comprise a first CAR-EC switch and a second CAR-EC switch, wherein the first CAR-EC switch comprises a first CAR-BP and a first targeting polypeptide and the second CAR-EC switch comprises a second CAR-BP and a second targeting polypeptide. The first CAR-BP and the second CAR-BP may be the same. The first CAR-BP and the second CAR-BP may be different. The first CAR-BP and the second CAR-BP may be about 99%, about 98%, about 97%, about 96%, about 95%, about 92%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5% or about 2% homologous. The first targeting polypeptide and the second targeting polypeptide may be the same. The first targeting polypeptide and the second targeting polypeptide may be different. The first targeting polypeptide and the second targeting polypeptide may be about 99%, about 98%, about 97%, about 96%, about 95%, about 92%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5% or about 2% homologous.

V. Kits, Vectors and Polynucleotides

Disclosed herein are kits comprising one or more CAR-EC switches disclosed herein. The kit may further comprise two or more CAR-EC switches. The kit may comprise three CAR-EC switches. The kit may comprise about 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 24, 30, 35, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 120, 150, 200, 300, 384, 400, 500, 600, 700, 800, 900 or 1000 CAR-EC switches. The kit may be employed for biological research. The kit may be used for diagnosing a disease or a condition. The kit may be used for treating a disease or condition. The CAR-EC switches of the kit may be used with CAR-EC cells disclosed herein or existing CAR T-cells clinically used or tested. The kit may further comprise one or more effector cells. The kit may further comprise one or more CAR-EC cells. The CAR-EC cell may be a T cell. The T cell may express one or more CARs. The kit may further comprise a polynucleotide encoding one or more CARs. The kit may further comprise a vector comprising a polynucleotide encoding one or more CARs. The CAR may be selected from any of the CARs disclosed herein. The kit may comprise one or more polynucleotide encoding a CAR-EC switch disclosed herein or a portion thereof (e.g. antibody, antibody fragment, peptide).
Further disclosed herein are and vectors and polynucleotides encoding CAR-EC switches or portions thereof, wherein the CAR-EC switch comprises a chimeric antigen receptor binding peptidic antigen and a targeting polypeptide, wherein the targeting peptide binds a cell surface molecule on a target cell. The polynucleotides may be DNA. The polynucleotides may be RNA. Unless otherwise specified, the terms “polynucleotide” and “vector,” as used herein, are used interchangeably. The targeting polypeptide may be an antibody or antibody fragment. The vector may comprise a sequence encoding a heavy chain of the antibody or antibody fragment. The vectors may comprise a sequence encoding a light chain of the antibody or antibody fragment. The vectors may comprise the sequence encoding the light chain of the antibody or antibody fragment and the sequence encoding the heavy chain of the antibody or antibody fragment. The light chain and the heavy chain may be expressed from the same vector. The light chain and the heavy chain may be expressed from two separate vectors.
Disclosed herein are vectors and polynucleotides encoding chimeric antigen receptors, wherein the chimeric antigen receptors comprise an extracellular domain that binds to a peptide of a chimeric antigen receptor effector cell switch. The extracellular domain may comprise an antibody or antibody fragment. The antibody or antibody fragment may bind a peptidic antigen of a chimeric antigen receptor effector cell switch . The peptidic antigen may be a yeast peptide. The yeast peptide may be GCN4. f a or portions thereof may be encoded by one or more polynucleotides based on or derived from SEQ ID NO: 1. CARs or portions thereof may be encoded by a polynucleotide at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to one or more polynucleotides based on or derived from SEQ ID NO: 1. CARs or portions thereof encoded by a polynucleotide may be at least about 70% identical to one or more polynucleotides based on or derived from SEQ ID NO: 1. Disclosed herein are vectors comprising one or more polynucleotides based on or derived from SEQ ID NO: 1.
Vectors comprising sequences encoding chimeric antigen receptors and/or chimeric antigen receptor effector cell switches and portions thereof, disclosed herein, may be selected from any commercially available expression vector. The expression vector may be a prokaryotic expression vector. The expression vector may be a eukaryotic expression vector. The expression vector may be a mammalian expression vector. The expression vector may be a viral expression vector. The expression vector may have a constitutive promoter for constitutive expression of the CAR and/or CAR-EC switch encoding sequences. The expression vector may have an inducible promoter for conditional expression of the CAR and/or CAR-EC switch encoding sequences.

VI. Therapeutic Use

Disclosed herein are methods, platforms and kits for treating a disease or condition in a subject in need thereof, the method comprising administering a chimeric antigen receptor effector cell (CAR-EC) switch to the subject, wherein the CAR-EC switch comprises: a CAR-binding peptidic antigen; and a targeting moiety. Disclosed herein are methods of treating a disease or condition in a subject in need thereof, the method comprising administering any one of the CAR-EC switches disclosed herein.
The methods may comprise administering a CAR-EC cell and one or more CAR-EC switches. The methods may comprise administering about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 24, 30, 35, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 120, 150, 200, 300, 384, 400, 500, 600, 700, 800, 900, 1000 or more CAR-EC switches. The methods may comprise administering two or more CAR-EC switches. The two or more CAR-EC switches may comprise the same CAR-binding peptidic antigen. The two more CAR-EC switches may comprise the same cell targeting polypeptide. The two or more CAR-EC switches may comprise one or more different CAR-binding peptidic antigens. The two more CAR-EC switches may comprise one or more different cell targeting polypeptides. The methods may comprising a plurality of CAR-EC cells and one or more CAR-EC switches.
Disclosed herein are methods of treating a disease or condition in a subject in need thereof, the method comprising administering a chimeric antigen receptor effector cell (CAR-EC) switch to the subject, wherein the CAR-EC switch comprises: a chimeric antigen receptor binding peptidic antigen (CAR-BP); and a targeting moiety that binds an antigen on a target. The CAR-BP, by non-limiting example, may be selected from a FLAG® tag, a yeast transcription factor GCN4 and a hydrophilic target peptide (HTP). The targeting moiety, by non-limiting example may be selected from an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-EGFR antibody, an anti-EGFRvIII antibody, an anti-Her2 antibody, an anti-CS1 antibody, an anti-BCMA antibody, an anti-CEA antibody, an anti-CLL-1 antibody and an anti-CD33 antibody.
The methods may comprise administering one or more chimeric antigen receptor effector cells. The methods may comprise administering one or more T cells. The one or more effector cells may be selected from T cell is selected from a naive T cell, a memory stem cell T cell, a central memory T cell, an effector memory T cell, a helper T cell, a CD4+ T cell, a CD8+ T cell, a CD8/CD4+ T cell, an αβ T cell, a γδ T cell, a cytotoxic T cell, a natural killer T cell, a natural killer cell, a macrophage.
The CAR-EC switch may have a therapeutic effect that is at least partially dependent on bringing an effector cell in proximity of a target cell. The therapeutic effect on the intended indication of the CAR-EC switch may be at least partially due to the CAR-EC switch recruiting an effector cell to the target cell. The therapeutic effect on the intended indication of the CAR-EC switch may be predominantly due to the CAR-EC switch recruiting an effector cell to the target cell. The therapeutic effect of the CAR-EC switch may be at least partially dependent on stimulating an immune response in the CAR-EC cell.
Administering the CAR-EC switch may not have any therapeutic effect without further administering an effector cell. The CAR-EC switch may not have a significant, desirable and/or intended therapeutic effect without further administering an effector cell. The CAR-EC switch may not have any therapeutic effect towards an intended indication of the CAR-EC platform without further administering an effector cell. A portion or component of the CAR-EC switch (e.g. CAR-BP or targeting moiety) may not have a therapeutic effect towards the intended indication of the CAR-EC switch without being conjugated to a second portion or component of the CAR-EC switch (e.g. CAR-BP or targeting moiety). The dose of a portion or component of the CAR-EC switch (e.g. CAR-BP or targeting moiety) when administered as part of the CAR-EC platform to provide a therapeutic effect may not have a therapeutic effect when the portion or component of the CAR-EC switch is administered alone at that dose. The portion or component of the CAR-EC switch may not be intended to have any therapeutic effect besides recruiting the T cell to the target cell. Administering the portion or component of the CAR-EC switch alone may have a therapeutic effect on the target cell, wherein the therapeutic effect is negligible relative to the therapeutic effect of administering the CAR-EC switch and the CAR-EC cell. Administering the portion or component of the CAR-EC switch may have a therapeutic effect on the target cell, wherein the therapeutic effect is less than the therapeutic effect of administering the CAR-EC switch and the CAR-EC cell.
Disclosed herein are uses of CAR-EC switches disclosed herein to treat a disease or condition in a subject in need thereof. Further disclosed herein are uses of CAR-EC switches disclosed herein in the manufacture of a medicament for the treatment of a disease.
Disclosed herein is use of a switch comprising a peptidic antigen that binds a CAR (CAR-BP) on an effector cell; and a targeting polypeptide that binds an antigen on a target to treat a disease or condition in a subject in need thereof. Further disclosed herein is use of a switch comprising a peptidic antigen (CAR-BP) that binds a CAR on an effector cell, wherein the CAR-BP; and a targeting polypeptide that binds an antigen on a target in the manufacture of a medicament for the treatment of a disease.
Disclosed herein is use of a CAR-EC switch comprising a CAR-BP, wherein the CAR-BP comprises a hydrophilic target peptide (HTP) or derivative thereof and a targeting polypeptide, wherein the targeting polypeptide comprises an anti-CD19 antibody or fragment thereof and an effector cell comprising a CAR, wherein the CAR comprises an anti-HTP antibody, wherein the anti-CD19 antibody or fragment thereof binds CD19 on a B cell to treat a multiple myeloma.
Disclosed herein is use of a CAR-EC switch comprising a CAR-BP, wherein the CAR-BP comprises a yeast transcription factor GCN4 or derivative thereof and a targeting polypeptide, wherein the targeting polypeptide comprises an anti-CD19 antibody or fragment thereof; and an effector cell comprising a CAR, wherein the CAR comprises an anti-GCN4 antibody, wherein the anti-CD19 antibody or fragment thereof binds CD19 on a lymphoblast, lymphocyte or B cell, to treat an acute lymphoblastic leukemia, a chronic lymphocytic leukemia or a B-cell lymphoma.
The disease or condition may be a cell proliferative disorder. The cell proliferative disorder may be selected from a solid tumor, a lymphoma, a leukemia and a liposarcoma. The cell proliferative disorder may be acute, chronic, recurrent, refractory, accelerated, in remission, stage I, stage II, stage III, stage IV, juvenile or adult. The cell proliferative disorder may be selected from myelogenous leukemia, lymphoblastic leukemia, myeloid leukemia, an acute myeloid leukemia, myelomonocytic leukemia, neutrophilic leukemia, myelodysplastic syndrome, B-cell lymphoma, burkitt lymphoma, large cell lymphoma, mixed cell lymphoma, follicular lymphoma, mantle cell lymphoma, hodgkin lymphoma, recurrent small lymphocytic lymphoma, hairy cell leukemia, multiple myeloma, basophilic leukemia, eosinophilic leukemia, megakaryoblastic leukemia, monoblastic leukemia, monocytic leukemia, erythroleukemia, erythroid leukemia and hepatocellular carcinoma. The cell proliferative disorder may comprise a hematological malignancy. The hematological malignancy may comprise a B cell malignancy. The cell proliferative disorder may comprise a chronic lymphocytic leukemia. The cell proliferative disorder may comprise an acute lymphoblastic leukemia. The cell proliferative disorder may comprise a CD19-positive Burkitt's lymphoma.
The disease or condition may be a cancer, a pathogenic infection, autoimmune disease, inflammatory disease, or genetic disorder.
In some instances, the one or more diseases comprises a cancer. The cancer may comprise a recurrent and/or refractory cancer. Examples of cancers include, but are not limited to, sarcomas, carcinomas, lymphomas or leukemias.
The cancer may comprise a neuroendocrine cancer. The cancer may comprise a pancreatic cancer. The cancer may comprise an exocrine pancreatic cancer. The cancer may comprise a thyroid cancer. The thyroid cancer may comprise a medullary thyroid cancer. The cancer may comprise a prostate cancer.
The cancer may comprise an epithelial cancer. The cancer may comprise a breast cancer. The cancer may comprise an endometrial cancer. The cancer may comprise an ovarian cancer. The ovarian cancer may comprise a stromal ovarian cancer. The cancer may comprise a cervical cancer.
The cancer may comprise a skin cancer. The skin cancer may comprise a neo-angiogenic skin cancer. The skin cancer may comprise a melanoma.
The cancer may comprise a kidney cancer.
The cancer may comprise a lung cancer. The lung cancer may comprise a small cell lung cancer. The lung cancer may comprise a non-small cell lung cancer.
The cancer may comprise a colorectal cancer. The cancer may comprise a gastric cancer. The cancer may comprise a colon cancer.
The cancer may comprise a brain cancer. The brain cancer may comprise a brain tumor. The cancer may comprise a glioblastoma. The cancer may comprise an astrocytoma.
The cancer may comprise a blood cancer. The blood cancer may comprise a leukemia. The leukemia may comprise a myeloid leukemia. The cancer may comprise a lymphoma. The lymphoma may comprise a non-Hodgkin's lymphoma.
The cancer may comprise a sarcoma. The sarcoma may comprise an Ewing's sarcoma.
Sarcomas are cancers of the bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Sarcomas include, but are not limited to, bone cancer, fibrosarcoma, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, bilateral vestibular schwannoma, osteosarcoma, soft tissue sarcomas (e.g. alveolar soft part sarcoma, angiosarcoma, cystosarcoma phylloides, dermatofibrosarcoma, desmoid tumor, epithelioid sarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, and synovial sarcoma).
Carcinomas are cancers that begin in the epithelial cells, which are cells that cover the surface of the body, produce hormones, and make up glands. By way of non-limiting example, carcinomas include breast cancer, pancreatic cancer, lung cancer, colon cancer, colorectal cancer, rectal cancer, kidney cancer, bladder cancer, stomach cancer, prostate cancer, liver cancer, ovarian cancer, brain cancer, vaginal cancer, vulvar cancer, uterine cancer, oral cancer, penile cancer, testicular cancer, esophageal cancer, skin cancer, cancer of the fallopian tubes, head and neck cancer, gastrointestinal stromal cancer, adenocarcinoma, cutaneous or intraocular melanoma, cancer of the anal region, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, cancer of the urethra, cancer of the renal pelvis, cancer of the ureter, cancer of the endometrium, cancer of the cervix, cancer of the pituitary gland, neoplasms of the central nervous system (CNS), primary CNS lymphoma, brain stem glioma, and spinal axis tumors. In some instances, the cancer is a skin cancer, such as a basal cell carcinoma, squamous, melanoma, nonmelanoma, or actinic (solar) keratosis.
In some instances, the cancer is a lung cancer. Lung cancer may start in the airways that branch off the trachea to supply the lungs (bronchi) or the small air sacs of the lung (the alveoli). Lung cancers include non-small cell lung carcinoma (NSCLC), small cell lung carcinoma, and mesotheliomia. Examples of NSCLC include squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. The mesothelioma may be a cancerous tumor of the lining of the lung and chest cavity (pleura) or lining of the abdomen (peritoneum). The mesothelioma may be due to asbestos exposure. The cancer may be a brain cancer, such as a glioblastoma.
Alternatively, the cancer may be a central nervous system (CNS) tumor. CNS tumors may be classified as gliomas or nongliomas. The glioma may be malignant glioma, high grade glioma, diffuse intrinsic pontine glioma. Examples of gliomas include astrocytomas, oligodendrogliomas (or mixtures of oligodendroglioma and astocytoma elements), and ependymomas. Astrocytomas include, but are not limited to, low-grade astrocytomas, anaplastic astrocytomas, glioblastoma multiforme, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and subependymal giant cell astrocytoma. Oligodendrogliomas include low-grade oligodendrogliomas (or oligoastrocytomas) and anaplastic oligodendriogliomas. Nongliomas include meningiomas, pituitary adenomas, primary CNS lymphomas, and medulloblastomas. In some instances, the cancer is a meningioma.
The leukemia may be an acute lymphocytic leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia, or chronic myelocytic leukemia. Additional types of leukemias include hairy cell leukemia, chronic myelomonocytic leukemia, and juvenile myelomonocytic leukemia.
Lymphomas are cancers of the lymphocytes and may develop from either B or T lymphocytes. The two major types of lymphoma are Hodgkin's lymphoma, previously known as Hodgkin's disease, and non-Hodgkin's lymphoma. Hodgkin's lymphoma is marked by the presence of the Reed-Sternberg cell. Non-Hodgkin's lymphomas are all lymphomas which are not Hodgkin's lymphoma. Non-Hodgkin lymphomas may be indolent lymphomas and aggressive lymphomas. Non-Hodgkin's lymphomas include, but are not limited to, diffuse large B cell lymphoma, follicular lymphoma, mucosa-associated lymphatic tissue lymphoma (MALT), small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt's lymphoma, mediastinal large B cell lymphoma, Waldenstrom macroglobulinemia, nodal marginal zone B cell lymphoma (NMZL), splenic marginal zone lymphoma (SMZL), extranodal marginal zone B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, and lymphomatoid granulomatosis.
The cancer may comprise a solid tumor. The cancer may comprise a sarcoma. The cancer may be selected from a group consisting of a bladder cancer, a breast cancer, a colon cancer, a rectal cancer, an endometrial cancer, a kidney cancer, a lung cancer, melanoma, a myeloma, a thyroid cancer, a pancreatic cancer, a glioma, a malignant glioma of the brain, a glioblastoma, an ovarian cancer, and a prostate cancer. The cancer may have non-uniform antigen expression. The cancer may have modulated antigen expression. The antigen may be a surface antigen. The cancer may not comprise a myeloma. The cancer may not comprise a melanoma. The cancer may not comprise a colon cancer. The cancer may be acute lymphoblastic leukemia (ALL). The cancer may be relapsed ALL. The cancer may be refractory ALL. The cancer may be relapsed, refractory ALL. The cancer may be chronic lymphocytic leukemia (CLL). The cancer may be relapsed CLL. The cancer may be refractory CLL. The cancer may be relapsed, refractory CLL.
The cancer may comprise a breast cancer. The breast cancer may be triple positive breast cancer (estrogen receptor, progesterone receptor and Her2 positive). The breast cancer may be triple negative breast cancer (estrogen receptor, progesterone receptor and Her2 negative). The breast cancer may be estrogen receptor positive. The breast cancer may be estrogen receptor negative. The breast cancer may be progesterone receptor positive. The breast cancer may be progesterone receptor negative. The breast cancer may comprise a Her2 negative breast cancer. The breast cancer may comprise a low-expressing Her2 breast cancer. The breast cancer may comprise a Her2 positive breast cancer. Cell lines expressing Her2 have been well-characterized for antigen density, reflecting clinical immunohistochemistry characterization which classifies malignancies as 0 (<20,000 Her2 antigens per cell), 1+ (100,000 Her2 antigens per cell), 2+ (500,000 Her2 antigens per cell), and 3+ (>2,000,000 Her2 antigens per cell). The present invention provides for methods of treating breast cancers of these classifications. The breast cancer may comprise a breast cancer classified as Her2 0. The breast cancer may comprise a breast cancer classified as Her2 1+. The breast cancer may comprise a breast cancer classified as Her2 2+. The breast cancer may comprise a breast cancer classified as a Her2 3+.
The disease or condition may be a pathogenic infection. Pathogenic infections may be caused by one or more pathogens. In some instances, the pathogen is a bacterium, fungi, virus, or protozoan.
Exemplary pathogens include but are not limited to: Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio, or Yersinia. In some cases, the disease or condition caused by the pathogen is tuberculosis and the heterogeneous sample comprises foreign molecules derived from the bacterium Mycobacterium tuberculosis and molecules derived from the subject. In some instances, the disease or condition is caused by a bacterium is tuberculosis, pneumonia, which may be caused by bacteria such as Streptococcus and Pseudomonas, a foodborne illness, which may be caused by bacteria such as Shigella, Campylobacter and Salmonella, and an infection such as tetanus, typhoid fever, diphtheria, syphilis and leprosy. The disease or condition may be bacterial vaginosis, a disease of the vagina caused by an imbalance of naturally occurring bacterial flora. Alternatively, the disease or condition is a bacterial meningitis, a bacterial inflammation of the meninges (e.g., the protective membranes covering the brain and spinal cord). Other diseases or conditions caused by bacteria include, but are not limited to, bacterial pneumonia, a urinary tract infection, bacterial gastroenteritis, and bacterial skin infection. Examples of bacterial skin infections include, but are not limited to, impetigo which may be caused by Staphylococcus aureus or Streptococcus pyogenes; erysipelas which may be caused by aStreptococcus bacterial infection of the deep epidermis with lymphatic spread; and cellulitis which may be caused by normal skin flora or by exogenous bacteria.
The pathogen may be a fungus, such as, Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis, and Stachybotrys. Examples of diseases or conditions caused by a fungus include, but are not limited to, jock itch, yeast infection, ringworm, and athlete's foot.
The pathogen may be a virus. Examples of viruses include, but are not limited to, adenovirus, coxsackievirus, Epstein-Barr virus, Hepatitis virus (e.g., Hepatitis A, B, and C), herpes simplex virus (type 1 and 2), cytomegalovirus, herpes virus, HIV, influenza virus, measles virus, mumps virus, papillomavirus, parainfluenza virus, poliovirus, respiratory syncytial virus, rubella virus, and varicella-zoster virus. Examples of diseases or conditions caused by viruses include, but are not limited to, cold, flu, hepatitis, AIDS, chicken pox, rubella, mumps, measles, warts, and poliomyelitis.
The pathogen may be a protozoan, such as Acanthamoeba (e.g., A. astronyxis, A. castellanii, A. culbertsoni, A. hatchetti, A. polyphaga, A. rhysodes, A. healyi, A. divionensis), Brachiola (e.g., B. connori, B. vesicularum), Cryptosporidium (e.g., C. parvum), Cyclospora (e.g., C. cayetanensis), Encephalitozoon (e.g., E. cuniculi, E. hellem, E. intestinalis), Entamoeba (e.g., E. histolytica), Enterocytozoon (e.g., E. bieneusi), Giardia (e.g., G. lamblia), Isospora (e.g, I. belli), Microsporidium (e.g., M. africanum, M. ceylonensis), Naegleria (e.g., N. fowleri), Nosema (e.g., N. algerae, N. ocularum), Pleistophora, Trachipleistophora (e.g., T. anthropophthera, T. hominis), and Vittaforma (e.g., V. corneae).
The disease or condition may be an autoimmune disease or autoimmune related disease. An autoimmune disorder may be a malfunction of the body's immune system that causes the body to attack its own tissues. Examples of autoimmune diseases and autoimmune related diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome (APS), autoimmune aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, Behcet's disease, celiac sprue, Crohn's disease, dermatomyositis, eosinophilic fasciitis, erythema nodosum, giant cell arteritis (temporal arteritis), Goodpasture's syndrome, Graves' disease, Hashimoto's disease, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, juvenile arthritis, diabetes, juvenile diabetes, Kawasaki syndrome, Lambert-Eaton syndrome, lupus (SLE), mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, pemphigus, polyarteritis nodosa, type I, II, & III autoimmune polyglandular syndromes, polymyalgia rheumatica, polymyositis, psoriasis, psoriatic arthritis, Reiter's syndrome, relapsing polychondritis, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, sperm & testicular autoimmunity, stiff person syndrome, Takayasu's arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis.
The disease or condition may be an inflammatory disease. Examples of inflammatory diseases include, but are not limited to, alveolitis, amyloidosis, angiitis, ankylosing spondylitis, avascular necrosis, Basedow's disease, Bell's palsy, bursitis, carpal tunnel syndrome, celiac disease, cholangitis, chondromalacia patella, chronic active hepatitis, chronic fatigue syndrome, Cogan's syndrome, congenital hip dysplasia, costochondritis, Crohn's Disease, cystic fibrosis, De Quervain's tendinitis, diabetes associated arthritis, diffuse idiopathic skeletal hyperostosis, discoid lupus, Ehlers-Danlos syndrome, familial mediterranean fever, fascitis, fibrositis/fibromyalgia, frozen shoulder, ganglion cysts, giant cell arteritis, gout, Graves' Disease, HIV-associated rheumatic disease syndromes, hyperparathyroid associated arthritis, infectious arthritis, inflammatory bowel syndrome/irritable bowel syndrome, juvenile rheumatoid arthritis, lyme disease, Marfan's Syndrome, Mikulicz's Disease, mixed connective tissue disease, multiple sclerosis, myofascial pain syndrome, osteoarthritis, osteomalacia, osteoporosis and corticosteroid-induced osteoporosis, Paget's Disease, palindromic rheumatism, Parkinson's Disease, Plummer's Disease, polymyalgia rheumatica, polymyositis, pseudogout, psoriatic arthritis, Raynaud's Phenomenon/Syndrome, Reiter's Syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, sciatica (lumbar radiculopathy), scleroderma, scurvy, sickle cell arthritis, Sjogren's Syndrome, spinal stenosis, spondyloisthesis, Still's Disease, systemic lupus erythematosis, Takayasu's (Pulseless) Disease, Tendinitis, tennis elbow/golf elbow, thyroid associated arthritis, trigger finger, ulcerative colitis, Wegener's Granulomatosis, and Whipple's Disease.
Methods of treatment disclosed herein may comprise off-target activity as measured by cytokine levels. The method may reduce the off-target activity, as measured by cytokine levels, when compared to other CAR-EC therapies. The method may reduce the off-target activity as measured by interferon gamma levels. Other off-target activities that may be reduced include toxic lymphophenia, fatal cytolysis of solid tumor targets and chronic hypogammaglobulinemia for hematological targets. Methods of treatment and compositions disclosed herein may be used to treat a cancer comprising CD19-mediated B cell aplasia. The methods and compositions may minimize the CD19-mediated B cell aplasia. The method may avoid long-term B-cell aplasia.
The CAR-EC platforms, methods and compositions disclosed herein may be used to treat a heterogeneous tumor or a heterogeneous blood cell malignancy in a subject in need thereof. The “pan-B cell” marker CD20 is the most prevalently targeted antigen for B cell neoplasms and the FDA-approved antibody rituximab is a vital component in the treatment of many leukemias and lymphomas. However, resistance mechanisms related to modulation of CD20 antigen expression occurs in a significant number of patients. It is clear that targeting with either CD19 or CD20 antigen alone is insufficient for a curative therapy. The methods disclosed herein provide for construction and administration of two or more switches with different specificities (e.g. an anti-CD19 antibody CAR-EC switch and an anti-CD20 antibody CAR-EC switch). The methods disclosed herein provide for construction and administration of two or more switches with different specificities (e.g. an anti-CD19 antibody CAR-EC switch and an anti-CD22 antibody CAR-EC switch). This methodology may offer a significant advantage against the propensity for relapse in the clinic while avoiding persistent loss of B cells. A heterogeneous tumor or heterogeneous blood cell malignancy may also be treated with an anti-CD19 antibody CAR-EC switch and an anti-CD22 antibody CAR-EC switch. One or more CAR-EC switches may be administered sequentially or simultaneously.
The CAR-EC switch may be administered with one or more additional therapeutic agents. The one or more additional therapeutic agents may be selected from a group consisting of an immunotherapy, a chemotherapy and a steroid. The one or more additional therapeutic agents may be a chemotherapy drug. The chemotherapy drug may be an alkylating agent, an antimetabolite, an anthracycline, a topoisomerase inhibitor, a mitotic inhibitor, a corticosteroid or a differentiating agent. The chemotherapy drug may be selected from actinomycin-D, bleomycin, altretamine, bortezomib, busulfan, carboplatin, capecitabine, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, estramustine, floxuridine, fludarabine, fluorouracil, gemcitbine (Gemzar), hydroxyurea, idarubicin, ifosfamide, irinotecan (Camptosar), ixabepilone, L-asparaginase, lomustine, mechlorethamine, melphalan, 6-mercaptopurine, methotrexate, mitomycin-C, paclitaxel (Taxol), pemetrexed, pentostatin, streptozocin, temozolomide, teniposide, thioguanine, thiotepa, topotecan (Hycamtin), vincristine, vinblastine, vinorelbine, retinoids, tretinoin (ATRA or Atralie), bexarotene (Targretin®) and arsenic trioxide (Arsenox®). The chemotherapy may be administered as a pill to swallow, as an injection into the muscle or fat tissue, intravenously, topically or directly into a body cavity.
The one or more additional therapeutic agents may comprise an angiogenesis inhibitor. The angiogenesis inhibitor may be selected from bevacizumab, itraconazole, carboxyamidotriazole, TNP-470, CM101, IFN alpha, IL-12, platelet factor 4, suramin, SU5416, thrombospondin, a VEGFR antagonist, an angiostatic steroid with heparin, CAR-ECilage-derived angiogenesis inhibitory factor, matrix metalloprotease inhibitors, angiostatin, endostatin, sorafenib, sunitinib, pazopanib, everolimus, 2-methoxyestradiol, tecogalan, tetrathiomolybdate, thalidomide, prolactin, αvβ₃inhibitor, linomide, tasquinimod, soluble VEGFR-1, soluble NRP-1, angiopoietin 2, vasostatin, calreticulin, TIMP, CDAI, Meth-1, Meth-2, interferon-alpha, interferon-beta, interferon-gamma, CXCL10, IL-4, IL-12, IL-18, prothrombin, antithrombin III fragment, prolactin, VEGI, SPARC, osteopontin, maspin, canstatin, proliferin-related protein and restin.
The one or more additional therapeutic agents may comprise a hormone therapy. The hormone therapy may be selected from an anti-estrogen (e.g. fulvestrant (Faslodex®), tamoxifen, toremifene (Fareston®)); an aromatase inhibitor (e.g. anastrozole (Arimidex®), exemestane (Aromasie), letrozole (Femara®)); a progestin (e.g. megestrol acetate (Megace®)); an estrogen; an anti-androgen (e.g. bicalutamide (Casodex®), flutamide (Eulexin®), nilutamide (Nilandron®)); a gonadotropin-releasing hormone (GnRH) or luteinizing hormone-releasing hormone (LHRH) agonist or analog (e.g. leuprolide (Lupron®), goserelin (Zoladex®)).
The one or more additional therapeutic agents may comprise a steroid. The steroid may be a corticosteroid. The steroid may be cortisol or a derivative thereof. The steroid may be selected from prednisone, methylprednisolone (Solumedrol®) or dexamethasone.
The CAR-EC switch may be administered with one or more additional therapies. The one or more additional therapies may comprise laser therapy. The one or more additional therapies may comprise radiation therapy. The one or more additional therapies may comprise surgery.
Disclosed herein are platforms, kits and methods for treating a disease or condition in a subject. The subject may be a healthy subject. The subject may be suffering from a disease or condition. The subject may be suffering from more than one disease or condition. The subject may be suffering from chronic lymphocytic leukemia. The subject may be suffering from acute lymphoblastic leukemia. The subject may be an animal. The subject may be a mammal. The mammal may be a human, a chimpanzee, a gorilla, a monkey, a bovine, a horse, a donkey, a mule, a dog, a cat, a pig, a rabbit, a goat, a sheep, a rat, a hamster, a guinea pig or a mouse. The subject may be a bird or a chicken. The subject may be a human. The subject may be a child. The child may be suffering from acute lymphoblastic leukemia. The subject may be less than 6 months old. The subject may be about 1 year old, about 2 years old, about 3 years old, about 4 years old, about 5 years old, about 6 years old, about 7 years old, about 8 years old, about 9 years old, about 10 years old, about 11 years old, about 12 years old, about 13 years old, about 14 years old, about 15 years old, about 18 years old, about 20 years old, about 25 years old, about 30 years old, about 35 years old, about 40 years old, about 45 years old, about 50 years old, about 55 years old, about 60 years old, about 65 years old, about 70 years old, about 75 years old, about 80 years old, about 85 years old, about 90 years old, about 95 years old, about 100 years old or about 105 years old.

VII. Method of Clearing Effector Cells

Further disclosed herein are methods of clearing CAR-EC cells in a subject, comprising administering a CAR-EC off switch. The CAR-EC off switch may comprise an antibody or antibody fragment that targets a cell surface marker on the effector cell. The CAR-EC off switch may comprise a peptide that is bound by the CAR of the CAR-EC. The CAR-EC off switch may comprise a CAR-BP that is bound by the CAR of the CAR-EC.
The antibody, antibody fragment or peptide of the CAR-EC off switch may be conjugated to a drug or a toxin. The drug or toxin may be selected from maytasine (e.g. DM1, DM4), monomethylauristatin E, monomethylauristatin F, Ki-4.dgA, dolastatin 10, calicheamicin, SN-38, duocarmycin, irinotecan, ricin, saporin, gelonin, poke weed antiviral protein, pseudomonas aeruginosa exotoxin A or diphtheria toxin. The toxin may comprise a poison, a bacterial toxin (e.g. bacterial toxins causing tetanus, diphtheria), a plant toxin or animal toxin. The toxin may be a snake venom. The toxin may comprise vinblastine. The toxin may comprise auristatin. The toxin may be contained in a liposome membrane-coated vesicle. Wherein the toxin is contained in a liposome membrane-coated vesicle, the antibody is attached to the vesicle.
The cell surface marker may be a viral protein or fragment thereof. Alternatively or additionally, the effector cell expresses a viral protein or fragment thereof that is not a cell surface marker. The effector cell expressing a viral protein or fragment thereof may be targeted with a drug. Wherein the effector cell comprises a viral protein or fragment thereof, the drug may be selected from a group comprising abacavir, acyclovir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla, balavir, boceprevirertet, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, an entry inhibitor, famciclovir, a fixed dose combination antiretroviral drug, fomivirsen, fosamprenavir, foscarnet, fosfonet, a fusion inhibitor, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitor, interferon type III, interferon type II, interferon type I, interferon, lamivudine, lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfinavir, nevirapine, nexavir, nucleoside analogue, oseltamivir, peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibiro, raltegravir, a reverse transcriptase inhibitor, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, sofosbuvir, stavudine, a synergistic enhancer retroviral durg, tea tree oil, telaprevir, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, vicriviroc, vidarabine, viramidine, zacitabine, zanamivir or zidovudine. The drug may be ganciclovir. The drug may be acyclovir.

VIII. Pharmaceutical Compositions

Disclosed herein is a pharmaceutical composition comprising one or more of the CAR-EC switches disclosed herein. The compositions may further comprise one or more pharmaceutically acceptable salts, excipients or vehicles. Pharmaceutically acceptable salts, excipients, or vehicles for use in the present pharmaceutical compositions include carriers, excipients, diluents, antioxidants, preservatives, coloring, flavoring and diluting agents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobials, and surfactants.
Neutral buffered saline or saline mixed with serum albumin are exemplary appropriate carriers. The pharmaceutical compositions may include antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, pluronics, or polyethylene glycol (PEG). Also by way of example, suitable tonicity enhancing agents include alkali metal halides (preferably sodium or potassium chloride), mannitol, sorbitol, and the like. Suitable preservatives include benzalkonium chloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid and the like. Hydrogen peroxide also may be used as preservative. Suitable cosolvents include glycerin, propylene glycol, and PEG. Suitable complexing agents include caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxy-propyl-beta-cyclodextrin. Suitable surfactants or wetting agents include sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapal, and the like. The buffers may be conventional buffers such as acetate, borate, citrate, phosphate, bicarbonate, or Tris-HCl. Acetate buffer may be about pH 4-5.5, and Tris buffer may be about pH 7-8.5. Additional pharmaceutical agents are set forth in Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company, 1990.
The composition may be in liquid form or in a lyophilized or freeze-dried form and may include one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives and/or bulking agents (see, for example, U.S. Pat. Nos. 6,685,940, 6,566,329, and 6,372,716). In one embodiment, a lyoprotectant is included, which is a non-reducing sugar such as sucrose, lactose or trehalose. The amount of lyoprotectant generally included is such that, upon reconstitution, the resulting formulation will be isotonic, although hypertonic or slightly hypotonic formulations also may be suitable. In addition, the amount of lyoprotectant should be sufficient to prevent an unacceptable amount of degradation and/or aggregation of the protein upon lyophilization. Exemplary lyoprotectant concentrations for sugars (e.g., sucrose, lactose, trehalose) in the pre-lyophilized formulation are from about 10 mM to about 400 mM. In another embodiment, a surfactant is included, such as for example, nonionic surfactants and ionic surfactants such as polysorbates (e.g., polysorbate 20, polysorbate 80); poloxamers (e.g., poloxamer 188); poly(ethylene glycol) phenyl ethers (e.g., Triton); sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-or stearyl-sarcosine; linoleyl, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl ofeyl-taurate; and the MONAQUAT™. series (Mona Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68 etc). Exemplary amounts of surfactant that may be present in the pre-lyophilized formulation are from about 0.001-0.5%. High molecular weight structural additives (e.g., fillers, binders) may include for example, acacia, albumin, alginic acid, calcium phosphate (dibasic), cellulose, carboxymethylcellulose, carboxymethylcellulose sodium, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, dextran, dextrin, dextrates, sucrose, tylose, pregelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose, disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfite, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, compressible sugar, magnesium aluminum silicate, maltodextrin, polyethylene oxide, polymethacrylates, povidone, sodium alginate, tragacanth microcrystalline cellulose, starch, and zein. Exemplary concentrations of high molecular weight structural additives are from 0.1% to 10% by weight. In other embodiments, a bulking agent (e.g., mannitol, glycine) may be included.
Compositions may be suitable for parenteral administration. Exemplary compositions are suitable for injection or infusion into an animal by any route available to the skilled worker, such as intraarticular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, or intralesional routes. A parenteral formulation typically will be a sterile, pyrogen-free, isotonic aqueous solution, optionally containing pharmaceutically acceptable preservatives.
Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringers' dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, anti-microbials, anti-oxidants, chelating agents, inert gases and the like. See generally, Remington's Pharmaceutical Science, 16th Ed., Mack Eds., 1980.
Pharmaceutical compositions described herein may be formulated for controlled or sustained delivery in a manner that provides local concentration of the product (e.g., bolus, depot effect) and/or increased stability or half-life in a particular local environment. The compositions may comprise the formulation of CAR-EC switches, polypeptides, nucleic acids, or vectors disclosed herein with particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., as well as agents such as a biodegradable matrix, injectable microspheres, microcapsular particles, microcapsules, bioerodible particles beads, liposomes, and implantable delivery devices that provide for the controlled or sustained release of the active agent which then may be delivered as a depot injection. Techniques for formulating such sustained-or controlled-delivery means are known and a variety of polymers have been developed and used for the controlled release and delivery of drugs. Such polymers are typically biodegradable and biocompatible. Polymer hydrogels, including those formed by complexation of enantiomeric polymer or polypeptide segments, and hydrogels with temperature or pH sensitive properties, may be desirable for providing drug depot effect because of the mild and aqueous conditions involved in trapping bioactive protein agents (e.g., antibodies comprising an ultralong CDR3). See, for example, the description of controlled release porous polymeric microparticles for the delivery of pharmaceutical compositions in WO 93/15722. Suitable materials for this purpose include polylactides (see, e.g., U.S. Pat. No. 3,773,919), polymers of poly-(a-hydroxycarboxylic acids), such as poly-D-(−)-3-hydroxybutyric acid (EP 133,988A), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers, 22: 547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981), and Langer, Chem. Tech., 12: 98-105 (1982)), ethylene vinyl acetate, or poly-D(−)-3-hydroxybutyric acid. Other biodegradable polymers include poly(lactones), poly(acetals), poly(orthoesters), and poly(orthocarbonates). Sustained-release compositions also may include liposomes, which may be prepared by any of several methods known in the art (see, e.g., Eppstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-92 (1985)). The carrier itself, or its degradation products, should be nontoxic in the target tissue and should not further aggravate the condition. This may be determined by routine screening in animal models of the target disorder or, if such models are unavailable, in normal animals. Microencapsulation of recombinant proteins for sustained release has been performed successfully with human growth hormone (rhGH), interferon-(rhIFN-), interleukin-2, and MN rgp120. Johnson et al., Nat. Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993); Hora et al., Bio/Technology. 8:755-758 (1990); Cleland, “Design and Production of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems,” in Vaccine Design: The Subunit and Adjuvant Approach, Powell and Newman, eds, (Plenum Press: New York, 1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat. No. 5,654,010. The sustained-release formulations of these proteins were developed using poly-lactic-coglycolic acid (PLGA) polymer due to its biocompatibility and wide range of biodegradable properties. The degradation products of PLGA, lactic and glycolic acids may be cleared quickly within the human body. Moreover, the degradability of this polymer may be depending on its molecular weight and composition. Lewis, “Controlled release of bioactive agents from lactide/glycolide polymer,” in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1-41. Additional examples of sustained release compositions include, for example, EP 58,481A, U.S. Pat. No. 3,887,699, EP 158,277A, Canadian Patent No. 1176565, U. Sidman et al., Biopolymers 22, 547 [1983], R. Langer et al., Chem. Tech. 12, 98 [1982], Sinha et al., J. Control. Release 90, 261 [2003], Zhu et al., Nat. Biotechnol. 18, 24 [2000], and Dai et al., Colloids Surf B Biointerfaces 41, 117 [2005].
Bioadhesive polymers are also contemplated for use in or with compositions of the present disclosure. Bioadhesives are synthetic and naturally occurring materials able to adhere to biological substrates for extended time periods. For example, Carbopol and polycarbophil are both synthetic cross-linked derivatives of poly(acrylic acid). Bioadhesive delivery systems based on naturally occurring substances include for example hyaluronic acid, also known as hyaluronan. Hyaluronic acid is a naturally occurring mucopolysaccharide consisting of residues of D-glucuronic and N-acetyl-D-glucosamine. Hyaluronic acid is found in the extracellular tissue matrix of vertebrates, including in connective tissues, as well as in synovial fluid and in the vitreous and aqueous humor of the eye. Esterified derivatives of hyaluronic acid have been used to produce microspheres for use in delivery that are biocompatible and biodegradable (see, for example, Cortivo et al., Biomaterials (1991) 12:727-730; EP 517,565; WO 96/29998; Illum et al., J. Controlled Rel. (1994) 29:133-141).
Both biodegradable and non-biodegradable polymeric matrices may be used to deliver compositions of the present disclosure, and such polymeric matrices may comprise natural or synthetic polymers. Biodegradable matrices are preferred. The period of time over which release occurs is based on selection of the polymer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable. Exemplary synthetic polymers which may be used to form the biodegradable delivery system include: polymers of lactic acid and glycolic acid, polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyanhydrides, polyurethanes and co-polymers thereof, poly(butic acid), poly(valeric acid), alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinyl chloride, polystyrene and polyvinylpyrrolidone. Exemplary natural polymers include alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion. The polymer optionally is in the form of a hydrogel (see, for example, WO 04/009664, WO 05/087201, Sawhney, et al., Macromolecules, 1993, 26, 581-587) that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multi-valent ions or other polymers.
Delivery systems also include non-polymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di-and tri-glycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the product is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,675,189 and 5,736,152 and (b) diffusional systems in which a product permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686. Liposomes containing the product may be prepared by methods known methods, such as for example (DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; JP 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324).
Alternatively or additionally, the compositions may be administered locally via implantation into the affected area of a membrane, sponge, or other appropriate material on to which a CAR-EC switch disclosed herein has been absorbed or encapsulated. Where an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of a CAR-EC switch, nucleic acid, or vector disclosed herein may be directly through the device via bolus, or via continuous administration, or via catheter using continuous infusion.
A pharmaceutical composition comprising a CAR-EC switch disclosed herein may be formulated for inhalation, such as for example, as a dry powder. Inhalation solutions also may be formulated in a liquefied propellant for aerosol delivery. In yet another formulation, solutions may be nebulized. Additional pharmaceutical composition for pulmonary administration include, those described, for example, in WO 94/20069, which discloses pulmonary delivery of chemically modified proteins. For pulmonary delivery, the particle size should be suitable for delivery to the distal lung. For example, the particle size may be from 1 μm to 5 μm; however, larger particles may be used, for example, if each particle is fairly porous.
Certain formulations containing CAR-EC switches disclosed herein may be administered orally. Formulations administered in this fashion may be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. For example, a capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to facilitate absorption of a selective binding agent. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders also may be employed.
Another preparation may involve an effective quantity of a CAR-EC switch disclosed herein in a mixture with non-toxic excipients which are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions may be prepared in unit dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
Suitable and/or preferred pharmaceutical formulations may be determined in view of the present disclosure and general knowledge of formulation technology, depending upon the intended route of administration, delivery format, and desired dosage. Regardless of the manner of administration, an effective dose may be calculated according to patient body weight, body surface area, or organ size. Further refinement of the calculations for determining the appropriate dosage for treatment involving each of the formulations described herein are routinely made in the art and is within the ambit of tasks routinely performed in the art. Appropriate dosages may be ascertained through use of appropriate dose-response data.

IX. CAR-EC Switch Production Methods

Disclosed herein are methods of producing CAR-EC switches comprising expressing one or more polypeptides from one or more vectors comprising one or more polynucleotide having one or more sequences that encode a chimeric antigen receptor-effector cell switch or a portion thereof, wherein the chimeric antigen receptor-effector cell switch comprises a peptidic antigen (CAR-BP) and a targeting polypeptide. The targeting moiety may comprise a targeting polypeptide. In general, the methods comprise fusing or grafting a polynucleotide encoding the CAR-BP to a polynucleotide encoding the targeting polypeptide. Fusing or grafting may be carried out by any standard cloning method known to one skilled in the art. Fusing or grafting the polynucleotides encoding the CAR-BP and targeting polypeptide may comprise enzymatic digestion of the polynucleotides, ligation of the polynucleotides and/or amplification of the polynucleotides.
The peptidic antigen may be fused to an N terminus of the targeting polypeptide. The peptidic antigen may be fused to a C terminus of the targeting polypeptide. The peptidic antigen may be grafted within the targeting polypeptide. The targeting polypeptide may comprise a targeting antibody or antibody fragment. The peptidic antigen may be fused to an N terminus of the targeting antibody or antibody fragment. The peptidic antigen may be fused to a C terminus of the targeting antibody or antibody fragment.
As used herein, the term “fused” may refer to adjoining a terminus of the CAR-BP with a terminus of the targeting polypeptide. The CAR-BP may be fused to the terminus of the targeting polypeptide without replacing or removing any amino acids of the targeting polypeptide. Fusing the CAR-BP to the terminus of the targeting polypeptide may comprise removing or replacing amino acids at the terminus of the targeting polypeptide. Removing or replacing amino acids at the terminus of the targeting polypeptide may comprise removing or replacing about 1 to about 20 amino acids at the terminus of the targeting polypeptide. The CAR-BP may be fused to the terminus of the targeting polypeptide via a linker. The linker may be fused to the CAR-BP to produce a CAR-BP-linker intermediate. The linker may be fused to a CAR-BP N terminus to produce the CAR-BP-linker intermediate. The linker may be fused to a CAR-BP C terminus to produce the CAR-BP-linker intermediate. The CAR-BP-linker intermediate may be fused to the targeting polypeptide. The CAR-BP-linker intermediate may be fused to the N terminus of the targeting polypeptide. The CAR-BP-linker intermediate may be fused to the C terminus of the targeting polypeptide. A first CAR-BP linker intermediate may be fused to the N terminus of the targeting polypeptide and a second CAR-BP linker intermediate may be fused to the C terminus of the targeting polypeptide. The CAR-BP of the first CAR-BP linker intermediate may be the same or similar to the CAR-BP of the second CAR-BP linker intermediate. The CAR-BP of the first CAR-BP linker intermediate may be different from the CAR-BP of the second CAR-BP linker intermediate.
As used herein, the term “grafted” may refer to inserting a CAR-BP within a targeting polypeptide (e.g. between two amino acids of the targeting polypeptide). The CAR-BP may be grafted within the targeting polypeptide without replacing or removing any amino acids of the targeting polypeptide. Grafting the CAR-BP within the targeting polypeptide may comprise removing or replacing amino acids within the targeting polypeptide. Removing or replacing amino acids within the targeting polypeptide may comprise removing or replacing about 1 to about 20 amino acids within the targeting polypeptide. The CAR-BP may be grafted within the targeting polypeptide via one linker. The CAR-BP may be grafted within the targeting polypeptide via two linkers. The linker may be fused to the CAR-BP N terminus to produce a CAR-BP-linker intermediate. The linker may be fused to the CAR-BP C terminus to produce a CAR-BP-linker intermediate. A first linker may be fused to the CAR-BP N terminus and a second linker may be fused to the CAR-BP C terminus to produce a CAR-BP-linker intermediate. The CAR-BP linker intermediate may be grafted with in the targeting polypeptide. A first CAR-BP linker intermediate may be grafted within the targeting polypeptide and a second CAR-BP linker intermediate may be grafted within the targeting polypeptide. The first CAR-BP linker intermediate may be grafted within a first domain of the targeting polypeptide and a second CAR-BP linker intermediate may be grafted within a second domain of the targeting polypeptide. The first domain of the targeting polypeptide may be the same as the second domain of the targeting polypeptide. The first domain of the targeting polypeptide may be different from the second domain of the targeting polypeptide. The CAR-BP of the first CAR-BP linker intermediate may be the same or similar to the CAR-BP of the second CAR-BP linker intermediate. The CAR-BP of the first CAR-BP linker intermediate may be different from the CAR-BP of the second CAR-BP linker intermediate. Unless otherwise specified, the terms “graft” and “insert”, as used herein, are used interchangeably.
The targeting moiety may comprise an antibody or antibody fragment. The antibody or antibody fragment may comprise a heavy chain and a light chain or fragments thereof. The methods may comprise expressing a heavy chain wherein the peptidic antigen is fused to a terminus of the heavy chain. The methods may comprise expressing a heavy chain wherein the peptidic antigen is grafted within the heavy chain. The methods may comprise expressing a light chain wherein the peptidic antigen is fused to a terminus of the light chain. The methods may comprise expressing a light chain wherein the peptidic antigen is grafted within the light chain.
The methods may further comprise cloning one or more polynucleotides encoding the targeting polypeptide and/or the peptidic antigen into an expression vector. The methods may further comprise ligation of the one or more polynucleotides encoding the targeting polypeptide and/or peptidic antigen into an expression vector. The expression vector may be a prokaryotic expression vector. The expression vector may be a eukaryotic expression vector. The expression vector may be a mammalian expression vector. The expression vector may be a viral expression vector. The methods may further comprise validating the cloning of the one or more polynucleotides encoding the targeting polypeptide and/or peptidic antigen into the expression vector comprising sequencing the expression vector, running gel electrophoresis of the vector and/or viewing the targeting polypeptide and/or peptidic antigen on an SDS page gel.
The methods may further comprise amplifying a polynucleotide encoding the targeting polypeptide and/or peptidic antigen and cloning the targeting polypeptide and/or peptidic antigen into the expression vector. Amplifying the polynucleotide encoding the targeting polypeptide and/or the peptidic antigen may comprise synthesizing oligonucleotides at least partially complementary to the gene. The oligonucleotides may be sufficiently complementary to the gene to anneal to the polynucleotide. The oligonucleotides may comprise linker sequences. The linker sequences may be selected from SEQ ID NOs: 40-44.
The methods may further comprise transfecting or infecting a cell with the expression vector. The methods may further comprise expressing the targeting polypeptide and/or peptidic antigen in the cell. The methods may further comprise expressing the targeting polypeptide and/or peptidic antigen in a cell free system. The methods may further comprise producing a virus comprising the expression vector. The methods may further comprise propagating the virus. The methods may further comprise infecting a cell with the virus comprising the expression vector. The methods may further comprise propagating the cell.
Disclosed herein are methods of grafting the antibody or antibody fragment, the peptidic antigen or the targeting peptide to produce a CAR-EC switch. The method may comprise grafting the CAR-BP to the antibody or antibody fragment. The method may comprise grafting the CAR-BP to an N terminus, C terminus or internal site of the antibody or antibody fragment. The CAR-BP may be grafted to a CL domain of the antibody or antibody fragment. The CAR-BP may be grafted to a loop of the CL domain of the antibody or antibody fragment. The method may comprise grafting the antibody or antibody fragment to the CAR-BP. The method may comprise grafting the antibody or antibody fragment to an N terminus, C terminus or internal site of the CAR-BP. The method may comprise grafting the CAR-BP to the targeting peptide. The method may comprise grafting the CAR-BP to an N terminus, C terminus or internal site of the targeting peptide. The method may comprise grafting the targeting peptide to the CAR-BP. The method may comprise grafting the targeting peptide to an N terminus, C terminus or internal site of the CAR-BP.
The CAR-BP, targeting peptide, antibody or antibody fragment may comprise one or more linkers, wherein the linker is located at the N terminus and/or C terminus of the CAR-BP, targeting peptide, antibody or antibody fragment. The method may comprise grafting the antibody or antibody fragment, the CAR-BP or the targeting peptide through the linker. The linker may comprise (GSSSS)_n.
Grafting may comprise producing a CAR-EC switch encoding nucleic acid. Producing the CAR-EC switch encoding nucleic acid may comprise one or more polymerase chain reactions. Producing the CAR-EC switch encoding nucleic acid may comprise one or more nucleic acid enzymatic digestions. The enzymatic digestion may be site specific. Producing the CAR-EC switch encoding nucleic acid may comprise one or more ligations. The methods of producing the CAR-EC switch may comprise incorporating the CAR-EC switch encoding nucleic acid into a CAR-EC switch vector. The vector may be an expression vector. The expression vector may comprise a constitutive promoter, an inducible promoter and/or a conditional promoter. The CAR-EC switch encoding nucleic acid or CAR-EC switch vector may be expressed in a cell and the resulting CAR-EC switch isolated and purified. The cell may be a prokaryotic cell. The cell may be an E. coli. The cell may be a eukaryotic cell. The cell may be a mammalian cell. The CAR-EC switch encoding nucleic acid or CAR-EC switch vector may be expressed in a cell-free system. Alternatively or additionally the CAR-EC switch may be synthesized from free amino acids.

Purification of CAR-EC Switches and Portions Thereof

Disclosed herein are methods of purifying CAR-EC switches disclosed herein, comprising separating the CAR-EC switches disclosed herein from components of a CAR-EC switch production system (e.g. cellular debris, free amino acids). Purifying the CAR-EC switch may comprise use of one or more concentrator columns, electrophoresis, filtration, centrifugation, chromatography or a combination thereof. Chromatography may comprise size-exclusion chromatography. Additional chromatography methods include, but are not limited to, hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatography, metal binding, immunoaffinity chromatography, and high performance liquid chromatography or high pressure liquid chromatography. Electrophoresis may comprise denaturing electrophoresis or non-denaturing electrophoresis.
The CAR-EC switches may comprise one or more peptide tags. The methods of purifying CAR-EC switches may comprise binding one or more peptide tags of the CAR-EC switches to a capturing agent. The capturing agent may be selected from an antibody, a column, a bead and a combination thereof. The one or more tags may be cleaved by one or more proteases. Examples of tags include, but are not limited to, polyhistidine, FLAG® tag, HA, c-myc, V5, chitin binding protein (CBP), maltose binding protein (MBP), and glutathione-S-transferase (GST). The peptide tag may be the CAR-BP. The peptide tag may be HTP. The peptide tag may be yeast transcription factor GCN4.
The methods may further comprise lyophilization or ultracentrifugation of the CAR-BPs, targeting polypeptides and/or the CAR-EC switches.
The purity of the CAR-BPs, targeting polypeptides and/or the CAR-EC switches may be equal to or greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. The purity of the CAR-BPs, targeting polypeptides and/or the CAR-EC switches may be equal to or greater than 85%. The purity of the CAR-BPs, targeting polypeptides and/or the CAR-EC switches may be equal to or greater than 90%. The purity of the CAR-BPs, targeting polypeptides and/or the CAR-EC switches may be equal to or greater than 95%. The purity of the CAR-BPs, targeting polypeptides and/or the CAR-EC switches may be equal to or greater than 97%.
The methods of producing CAR-EC switches disclosed herein may comprise producing CAR-EC switches that are structurally homogeneous. The method of producing the CAR-EC switch from a polynucleotide may result in one or more CAR-EC switches that have the same or similar form, features, binding affinities (e.g. for the CAR or the target), geometry and/or size. The homogeneity of the CAR-EC switches may be equal to or greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. The homogeneity of the CAR-EC switches may be equal to or greater than 85%. The homogeneity CAR-EC switches may be equal to or greater than 90%. The homogeneity of the CAR-EC switches may be equal to or greater than 95%. The homogeneity of the CAR-EC switches may be equal to or greater than 97%. The homogeneity may be a structural homogeneity. The homogeneity may be a structural homogeneity prior to administering the cell to a subject. The homogeneity may be a structural homogeneity prior to modifications to the CAR-EC switch by cellular activities (methylation, acetylation, glycosylation, etc.). These high percentages of homogeneity may provide a more predictable effect of the CAR-EC switch. These high percentages of homogeneity may provide for less off-target effects of the CAR-EC switch, when combined with a CAR-EC to treat a condition in a subject.

EXAMPLES

The following illustrative examples are representative of embodiments of the software applications, systems, and methods described herein and are not meant to be limiting in any way.

Example 1

Production and Evaluation of a Switchable CAR-T Platform

The solubility, stability, affinity, and potential for cross reactive epitopes in the human proteome of developed antibodies were considered in choosing a CAR-EC switch peptidic antigen. Based on these criteria, a linear amino acid epitope from the yeast transcription factor GCN4 (7P14P) was chosen. Single chain antibodies with affinities varying from 2.6 nM to 5.2 pM enable optimization of the CAR-EC through binding kinetics. Additionally, these antibodies are among the highest affinity anti-peptide single chain antibodies for linear epitopes. The dissociation constant (Kd) for the chosen GCN4 epitope (7P14P) having a sequence of NYHLENEVARLKKL (SEQ ID NO. 3) and GCN4 binding scFv (52SR4) is 5.2 pM.
A small hydrophilic target peptide (HTP), based on the commonly used FLAG® tag, was developed. FLAG® has low antigenicity, is highly soluble, and has been fused to numerous proteins with little impact on protein folding or stability. In modifying FLAG to HTP, a proline residue was incorporated after the terminal lysine in an effort to increase proteolytic stability. Antibodies to this epitope are developed by traditional mouse immunization and subsequent humanization or by phage panning of a human library. Binding kinetics of evolved scFv's are fully characterized and peptide-CAR-ECs are created and tested for off-target specificity as described.

Evaluation of a Switchable CAR-T Platform in a Xenograft Model

To evaluate efficacy, mouse xenograft models are used to compare these switchable platforms to previously developed by CAR-T switch platforms. Towards this end, RS4;11, NALM-6, Raji or other CD19 positive cell lines are used to establish tumor models in non-obese diabetic-severe combined immunodeficiency (NOD-SCID-_γ ^−/−, NSG) mice. CAR-Ts are delivered by intravenous administration. Dose-range finding is carried out for the peptide anti-CD19 switch, and is compared to a wild type CD19 Fab control. Efficacy is judged based on tumor burden and overall survival. Mice are monitored with weekly blood draws to monitor proliferation of CAR-ECs in peripheral blood. Detailed immunophenotypic characterization of CAR-ECs focus on effector, memory, senescent (terminally differentiated), or anergized phenotypes are defined according to standard phenotypic parameters using multi-channel flow cytometry.
Efficacy of the Fab, and IgG based switches are delivered at appropriate dosages per observed PK data and compared. IgG is most efficacious in this model for its long residence time in vivo. Further exploration on this idea is carried out in the syngeneic model.
Primary patient-derived ALL or CLL samples are obtained and for generating xenograft models in NSG mice. Primary samples are characterized for CD19 expression by flow cytometry. Leukemia is established in mice for 2-3 weeks prior to administration of therapy. Efficacy versus CAR-T-19 is judged by monitoring CD19⁺ ALL blast counts in peripheral blood. In the event that leukemia is not controlled or eliminated, proliferated blasts are immunophenotyped (specifically looking for loss of CD19 antigen expression, vida infra for further study). Persistence of CAR-ECs is also monitored (although the latter is not expected to differ substantially from RS4;11-based xenografts).

Evaluation of a Switchable CAR-T Platform in a Syngeneic Model

Although the xenograft models in immunodeficient mice allow measurement of the efficacy of the switchable platform, this model is not optimal to assess a method for alleviating the long-term lymphopenia associated with CAR-T-19 therapy. Switchable CAR-ECs are tested for the ability to reverse B cell aplasia in an immunocompetent B cell lymphoma mouse model. To create a murine surrogate CAR-T, the engineered peptide-based chimeric receptor is cloned to a Moloney murine leukemia-based retroviral vector for transduction into murine splenocytes. The murine-derived signaling domains CD28 and CD3z are used. The anti-human CD19 antibody does not cross-react with mouse CD19; therefore, the rat anti-mouse CD19 hybridoma 1D3 is obtained (from ATCC) and variable regions sequenced. This sequence is cloned into an expression vector for peptide fusion to create the switch and is cloned into a chimeric antigen receptor to create a CAR-T-19 mouse surrogate.
After optimization of transduction and assessment of efficacy in vitro, the Myc5-CD19 cell line is used to establish B cell lymphoma in wild type C57BL/6 mice. CAR-ECs and switches are administered with dosing schedules based on xenograft studies and in vitro assays with surrogate system. Of particular interest in this model is to compare Fab, and IgG based switches on the rate of Myc5-CD19 disappearance and B cell ablation. As with xenografts studies, CAR-T proliferation is monitored and immunophenotypic characterization is carried out ex vivo. After eradication of lymphoma cells, switch administration is halted and the reproliferation of B cells in peripheral blood is monitored. Both the surrogate CAR-T-19 and the surrogate switchable CAR-T are expected to enable long-term remission, but only the switchable platform enables repopulation of B cells. CAR-T infiltration to major organs is monitored via histology on predefined cohorts and cellular analysis is carried out post-therapy. Long-term persistence of CAR-ECs in the absence of stimulation is followed.

Evaluation of a Switchable CAR-T Platform in a Heterogeneous Cancer Model

A first switch containing an anti-CD19 targeting antibody and a second switch containing the anti-CD20 targeting antibody rituximab are used sequentially or simultaneously to target different antigens in the same patient using a single adoptively transferred CAR-T in an effort to combat ALL relapse attributed to a CD19 escape variant during CAR-T-19 therapy.
An anti-CD20 switch is created in analogous fashion to the anti-CD19 switch using the optimal characteristics determined in Example 3. A CAR-T-20 based on rituximab is constructed for comparison. Efficacy is tested in vitro against CD20-positive IM-9 and Daudi cells lines. To create a heterogeneous B-cell lymphoblast, the chronic myelogenous leukemia-derived K562 cell line (which is negative for CD20 and CD19) is stably transduced with the CD19 antigen using a lentiviral vector. Single cell clones are obtained via flow-sorting to obtain a population with homogenous CD19 expression. This cell line is then be transduced with CD20 and sorted by high (CD20^hi) or low (CD20^low) level of antigen expression. The activation and cytotoxicity of the switchable CAR-T on mixtures of CD19⁺CD20⁻ and CD19⁺CD20^hior CD19⁺CD²⁰ ^loware assessed in vitro using the CD19 and CD20 switches (simultaneous or sequential administration). The method provides an opportunity to study the lowest percentage of CD20^hior CD20^lowcells in a population that are necessary to stimulate the CAR-T with the rituximab switch. This may be more physiologically relevant than a homogeneous population. This system is then tested in a xenograft mouse model. A mixture of CD19⁺CD20⁻ and CD19⁺CD20⁺ are used to establish the xenograft. Alternatively, primary patient derived ALL samples are used for this experiment if found to be heterogeneous for CD19 or CD20 expression in our initial xenograft study. Switchable CAR-ECs with the anti-CD20 switch are administered to eliminate the CD19⁺CD20⁺ population and allow outgrowth of CD19⁺CD20⁻ cells. To demonstrate the feasibility of retargeting the same CAR-T, the anti-CD19 switch is subsequently dosed and growth of remaining xenograft monitored. Tumors are evaluated for antigen expression in cohorts of sacrificed mice or in primary blasts. Simultaneous targeting is also assessed. Treatment is compared with CAR-T-19, CAR-T-20, or both simultaneously.

Example 2

CAR Construction

The CARs were constructed as follows:
LV-EF1a-GCN4-BBZ was designed to target the 7P14P epitope of the yeast transcription factor GCN4 (sequence RMKQLEPKVEELLPKNYHLENEVARLKKLVGER (SEQ ID NO. 2) where the underlined amino acids have been shown to bind to the c11L32Ser scFv in the 1P4B crystal structure from PDB. The scFv was constructed from the 52SR4 (high affinity mutant with similar sequence to c11L32Ser) antibody scFv from reference: Zahnd, C., Spinelli, S., Luginbuhl, B., Amstutz, P., Cambillau, C., and Pluckthun, A. (2004) Directed in vitro evolution and crystallographic analysis of a peptide-binding single chain antibody fragment (scFv) with low picomolar affinity, The Journal of Biological Chemistry 279, 18870-18877.

Example 3

Cloning, Expression and Purification of Anti-CD19-Fab-GCN4

Cloning: Mammalian expression vector of CD19 Fab heavy chain was generated by ligation of amplified CD19 Fab heavy chain (VH and CH1) to pFuse-hIgG1-Fc backbone vector (InvivoGen, CA) without Fc fragment. A gene encoding antibody CD19 light chain was amplified and cloned into the pFuse vector without hIgG1 Fc fragment. A gene encoding GCN4 (NYHLENEVARLKKL=SEQ ID NO: 3) with was synthesized as oligonucleotides. Subsequently, anti-CD1-Fab-GCN4_HClfusion proteins were created by grafting GCN4 into the mature heavy chain of the CD19 Fab following S135 of the CD19 Fab heavy chain. The resulting mammalian expression vectors were confirmed by DNA sequencing.
Expression and Purification: anti-CD19-Fab-GCN4_HClwas expressed through transient transfection of FreeStyle HEK 293 cells with expression vectors of CD19-Fab light chain and GCN4-CD19-HCl, according to the manufacturer's protocol. Briefly, 28 mL FreeStyle HEK 293 cells containing 3×107 cells were seeded in a 125 mL shaking flask. 15 μg light chain plasmid and 15 μg heavy chain plasmid diluted in 1 mL Opti-MEM medium were added in 1 mL Opti-MEM containing 60 μL 293fectin (Invitrogen, Inc). After the plasmids were incubated with 293fectin for 30 min, the lipoplex mixture was added to the cell suspension. Cells were then shaken at 125 rpm in a 5% CO2 environment at 37° C. Culture medium containing secreted proteins was harvested at 48 and 96 hours after transfection. The anti-CD1-Fab-GCN4_HClwas purified by Protein G chromatography (Thermo Fisher Scientific, IL). Purified proteins were analyzed by SDS-PAGE gels. FIGS. 5A and 5B show SDS gel images of anti-CD1-Fab-GCN4_HCl(Lane 7) in non-reducing and reducing (with 50 mM DTT) conditions, respectively.

Example 4

Cloning, Expression and Purification of Anti-CD19-IgG-GCN4_HCl

Cloning: Mammalian expression vector of CD19 IgG heavy chain was generated by in-frame ligation of amplified CD19 Fab heavy chain (VH and CH1) to pFuse-hIgG1-Fc backbone vector (InvivoGen, CA). A gene encoding antibody CD19 light chain was amplified and cloned into the pFuse vector without hIgG1 Fc fragment. A gene encoding GCN4 (NYHLENEVARLKKL=SEQ ID NO: 3) was synthesized as oligonucleotides. Subsequently, anti-CD19-IgG-GCN4_HClfusion proteins were created by inserting GCN4 following S135 of the mature heavy chain of the CD19 IgG. The resulting mammalian expression vectors were confirmed by DNA sequencing.
Expression and Purification: anti-CD19-IgG-GCN4_HClwas expressed through transient transfection of FreeStyle HEK 293 cells with expression vectors of CD19-IgG light chain and GCN4-CD19 heavy chain, according to the manufacturer's protocol. Briefly, 28 mL FreeStyle HEK 293 cells containing 3×107 cells were seeded in a 125 mL shaking flask. 15 μg light chain plasmid and 15 μg heavy chain plasmid diluted in 1 mL Opti-MEM medium were added in 1 mL Opti-MEM containing 60 μL 293fectin (Invitrogen, Inc). After the plasmids were incubated with 293fectin for 30 min, the lipoplex mixture was added to the cell suspension. Cells were then shaken at 125 rpm in a 5% CO2 environment at 37° C. Culture medium containing secreted proteins was harvested at 48 and 96 hours after transfection. GCN4-CD19 heavy chain was purified by Protein G chromatography (Thermo Fisher Scientific, IL). Purified proteins were analyzed by SDS-PAGE gels. FIGS. 5A & 5B show SDS gel images of anti-CD19-IgG-GCN4_HCl(Lane 3) in non-reducing and reducing (with 50 mM DTT) conditions, respectively.

Example 5

Cloning, Expression and Purification of Anti-CD19-Fab-GCN4_C-term

Cloning: Mammalian expression vector of CD19 Fab heavy chain was generated by ligation of amplified CD19 Fab heavy chain (VH and CH1) to pFuse-hIgG1-Fc backbone vector (InvivoGen, CA) without Fc fragment. A gene encoding antibody CD19 light chain was amplified and cloned into the pFuse vector without hIgG1 Fc fragment. A gene encoding GCN4 (NYHLENEVARLKKL=SEQ ID NO: 3) with GGGGS (SEQ ID NO: 40, wherein n=1) linker at N-terminal end of GCN4 with was synthesized as oligonucleotides. Subsequently, anti-CD19-Fab-GCN4_C-termfusion proteins were created by fusing the linker-GCN4 to the C terminus of the Fab heavy chain at C223. The resulting mammalian expression vectors were confirmed by DNA sequencing.
Expression and Purification: anti-CD19-Fab-GCN4_C-termwas expressed through transient transfection of FreeStyle HEK 293 cells with expression vectors of CD19-Fab light chain and anti-CD19-Fab-GCN4_C-term, according to the manufacturer's protocol. Briefly, 28 mL FreeStyle HEK 293 cells containing 3×107 cells were seeded in a 125 mL shaking flask. 15 μg light chain plasmid and 15 μg heavy chain plasmid diluted in 1 mL Opti-MEM medium were added in 1 mL Opti-MEM containing 60 μL 293fectin (Invitrogen, Inc). After the plasmids were incubated with 293fectin for 30 min, the lipoplex mixture was added to the cell suspension. Cells were then shaken at 125 rpm in a 5% CO2 environment at 37° C. Culture medium containing secreted proteins was harvested at 48 and 96 hours after transfection. anti-CD19-Fab-GCN4_C-termwas purified by Protein G chromatography (Thermo Fisher Scientific, IL). Purified proteins were analyzed by SDS-PAGE gels. FIGS. 5A and 5B show SDS gel images of anti-CD19-Fab-GCN4_C-term(Lane 9) in non-reducing and reducing (with 50 mM DTT) conditions, respectively.

Example 6

Cloning, Expression and Purification of Anti-CD19-IgG-GCN4_hinge

Cloning: Mammalian expression vector of CD19 IgG heavy chain was generated by in-frame ligation of amplified CD19 Fab heavy chain (VH and CH1) to pFuse-hIgG1-Fc backbone vector (InvivoGen, CA). A gene encoding antibody CD19 light chain was amplified and cloned into the pFuse vector without hIgG1 Fc fragment. A gene encoding GCN4 (NYHLENEVARLKKL=SEQ ID NO: 3) with GGGGS (SEQ ID NO: 40, wherein n=1) linker at N-terminal end of GCN4 and GGS (SEQ ID NO. 42, wherein n=1) at C-terminal of GCN4 (“linker-GCN4-linker”) was synthesized as oligonucleotides. Subsequently, anti-CD19-IgG-GCN4_hingefusion proteins were created by grafting the linker-GCN4-linker between the C terminus of the Fab heavy chain at C223 and the hinge region. Thus, the linker-GCN4-linker extends the hinge region of the IgG, mimicking an IgG3 structure with an elongated hinge region. The resulting mammalian expression vectors were confirmed by DNA sequencing.
Expression and Purification: anti-CD19-IgG-GCN4_hingewas expressed through transient transfection of FreeStyle HEK 293 cells with expression vectors of CD19-IgG light chain and GCN4-CD19 hinge heavy chain, according to the manufacturer's protocol. Briefly, 28 mL FreeStyle HEK 293 cells containing 3×107 cells were seeded in a 125 mL shaking flask. 15 μg light chain plasmid and 15 μg heavy chain plasmid diluted in 1 mL Opti-MEM medium were added in 1 mL Opti-MEM containing 60 μL 293fectin (Invitrogen, Inc). After the plasmids were incubated with 293fectin for 30 min, the lipoplex mixture was added to the cell suspension. Cells were then shaken at 125 rpm in a 5% CO2 environment at 37° C. Culture medium containing secreted proteins was harvested at 48 and 96 hours after transfection. GCN4-CD19 hinge IgG was purified by Protein G chromatography (Thermo Fisher Scientific, IL). Purified proteins were analyzed by SDS-PAGE gels. FIGS. 5A & 5B show SDS gel images of anti-CD19-IgG-GCN4_hinge(Lane 5) in non-reducing and reducing (with 50 mM DTT) conditions, respectively.

Example 7

Cloning, Expression and Purification of Anti-CD19-IgG-GCN4_CL1

Cloning: Mammalian expression vector of CD19 IgG heavy chain was generated by in-frame ligation of amplified CD19 Fab heavy chain (VH and CH1) to pFuse-hIgG1-Fc backbone vector (InvivoGen, CA). A gene encoding antibody CD19 light chain was amplified and cloned into the pFuse vector without hIgG1 Fc fragment. A gene encoding GCN4 (NYHLENEVARLKKL=SEQ ID NO: 3) with GGGGS (SEQ ID NO: 40, wherein n=1) linker at both ends was synthesized as oligonucleotides. Subsequently, anti-CD19-IgG-GCN4_CL1fusion proteins were created by replacing the K169 in CL region of CD19 light chain with GCN4 with linker sequences at both ends. The resulting mammalian expression vectors were confirmed by DNA sequencing.
Expression and Purification: anti-CD19-IgG-GCN4_CL1was expressed through transient transfection of FreeStyle HEK 293 cells with expression vectors of CD19-IgG heavy chain and GCN4-CD19-CL1 light chain, according to the manufacturer's protocol. Briefly, 28 mL FreeStyle HEK 293 cells containing 3x10⁷cells were seeded in a 125 mL shaking flask. 15 μg light chain plasmid and 15 μg heavy chain plasmid diluted in 1 mL Opti-MEM medium were added in 1 mL Opti-MEM containing 60 μL 293fectin (Invitrogen, Inc). After the plasmids were incubated with 293fectin for 30 min, the lipoplex mixture was added to the cell suspension. Cells were then shaken at 125 rpm in a 5% CO2 environment at 37° C. Culture medium containing secreted proteins was harvested at 48 and 96 hours after transfection. anti-CD19-IgG-GCN4_CL1was purified by Protein G chromatography (Thermo Fisher Scientific, IL). Purified proteins were analyzed by SDS-PAGE gels. FIGS. 5A & 5B show SDS gel images of anti-CD19-IgG-GCN4_CL1(Lane 4) in non-reducing and reducing (with 50 mM DTT) conditions, respectively.

Example 8

Cloning, Expression and Purification of Anti-CD19-Fab-GCN4_CL1

Cloning: Mammalian expression vector of CD19 Fab heavy chain was generated by ligation of amplified CD19 Fab heavy chain (VH and CH1) to pFuse-hIgG1-Fc backbone vector (InvivoGen, CA) without Fc fragment. A gene encoding antibody CD19 light chain was amplified and cloned into the pFuse vector without hIgG1 Fc fragment. A gene encoding GCN4 (NYHLENEVARLKKL=SEQ ID NO: 3) with GGGGS (SEQ ID NO: 40, wherein n=1) linker at both ends was synthesized as oligonucleotides. Subsequently, anti-CD19-Fab-GCN4_CL1fusion proteins were created by replacing the K169 in CL region of CD19 light chain with GCN4 with linker sequences at both ends. The resulting mammalian expression vectors were confirmed by DNA sequencing.
Expression and Purification: anti-CD19-Fab-GCN4_CL1was expressed through transient transfection of FreeStyle HEK 293 cells with expression vectors of CD19-Fab heavy chain and GCN4-CD19-CL light chain, according to the manufacturer's protocol. Briefly, 28 mL FreeStyle HEK 293 cells containing 3×10⁷cells were seeded in a 125 mL shaking flask. 15 μg light chain plasmid and 15 μg heavy chain plasmid diluted in 1 mL Opti-MEM medium were added in 1 mL Opti-MEM containing 60 μL 293fectin (Invitrogen, Inc). After the plasmids were incubated with 293fectin for 30 min, the lipoplex mixture was added to the cell suspension. Cells were then shaken at 125 rpm in a 5% CO2 environment at 37° C. Culture medium containing secreted proteins was harvested at 48 and 96 hours after transfection. The anti-CD19-Fab-GCN4_CL1was purified by Protein G chromatography (Thermo Fisher Scientific, IL). Purified proteins were analyzed by SDS-PAGE gels. FIGS. 5A & 5B show SDS gel images of anti-CD19-Fab-GCN4_CL1(Lane 8) in non-reducing and reducing (with 50 mM DTT) conditions, respectively.

Example 9

Cloning, Expression and Purification of Anti-CD19-Fab-GCN4_LC1-N-term

Cloning: Mammalian expression vector of CD19 Fab heavy chain was generated by ligation of amplified CD19 Fab heavy chain (VH and CH1) to pFuse-hIgG1-Fc backbone vector (InvivoGen, CA) without Fc fragment. A gene encoding antibody CD19 light chain was amplified and cloned into the pFuse vector without hIgG1 Fc fragment. A gene encoding GCN4 (NYHLENEVARLKKL=SEQ ID NO: 3) with GGGGS (SEQ ID NO: 40, wherein n=1) linker at C-terminal end of GCN4 with was synthesized as oligonucleotides. Subsequently, anti-CD19-Fab-GCN4_LC1-N-termfusion proteins were created by fusing the linker-GCN4 to the N terminus of the Fab light chain. The resulting mammalian expression vectors were confirmed by DNA sequencing.
Expression and Purification: anti-CD19-Fab-GCN4_LC1-N-termwas expressed through transient transfection of FreeStyle HEK 293 cells with expression vectors of CD19-Fab light chain and GCN4-CD19-C-term, according to the manufacturer's protocol. Briefly, 28 mL FreeStyle HEK 293 cells containing 3×107 cells were seeded in a 125 mL shaking flask. 15 μg light chain plasmid and 15 μg heavy chain plasmid diluted in 1 mL Opti-MEM medium were added in 1 mL Opti-MEM containing 60 μL 293fectin (Invitrogen, Inc). After the plasmids were incubated with 293fectin for 30 min, the lipoplex mixture was added to the cell suspension. Cells were then shaken at 125 rpm in a 5% CO2 environment at 37° C. Culture medium containing secreted proteins was harvested at 48 and 96 hours after transfection. anti-CD19-Fab-GCN4_LC1-N-termwas purified by Protein G chromatography (Thermo Fisher Scientific, IL). Purified proteins were analyzed by SDS-PAGE gels. FIGS. 5A and 5B show SDS gel images of anti-CD19-Fab-GCN4_LC1-N-term(Lane 10) in non-reducing and reducing (with 50 mM DTT) conditions, respectively.

Example 10

Cytotoxicity of anti-CD19 Fab-GCN4_CL1, anti-CD19 IgG_FcNull-GCN4 and Anti-CD19 Fab-GCN4_C-termCAR-EC switches

Peptide CAR-EC switches were created by fusing 14 amino acids of the GCN4 yeast transcription factor peptide sequence 7P14P (defined in Zahnd et al. (2004) Directed in vitro evolution and crystallographic analysis of a peptide-binding single chain antibody fragment (scFv) with low picomolar affinity, The Journal of Biological Chemistry 279, 18870-18877). The 14 amino acids were chosen based on those defined in crystal structure 1P4B of GCN4 peptide 7P14P with scFv c11L32Ser. The switches were constructed by either fusing the GCN4 peptide sequence to the C-terminus of the heavy chain of the Fab antibody or by fusing the GCN4 peptide sequence in the CL loop of the light chain of the Fab or IgG antibody. All expressions were carried out in CHO or HEK cells.
To create a grafted GCN4 peptide based anti-CD19 CAR-T switch (SEQ ID NO: 30), the peptide NYHLENEVARLKKL (SEQ ID NO: 3), suggested to be the minimal binding epitope according to the crystal structure (PDB: 1P4B) (see FIG. 2) from the yeast transcription factor GCN4 peptide (7P14P) RMKQLEPKVEELLPKNYHLENEVARLKKLVGER (SEQ ID NO: 2), was grafted to the mouse anti-human CD19 Fab clone FMC63. The graft was carried out by replacing K63 (as counted from the N terminus of the constant region, which would be K169 when counting from the N terminus of the mature protein) of the light chain with the sequence GGGGSNYHLENEVARLKKLGGGGS (SEQ ID NO. 4), the GCN4 epitope flanked by GGGGS linkers (SEQ ID NO: 40, wherein n=1). The mass spec of anti-CD19 Fab_CL1-GCN4 is provided below (FIG. 3). Alternatively, the peptide is grafted to the heavy chain (SEQ ID NO: 29).
The cytotoxic activity of the anti-CD19 Fab_CL1-GCN4 switch was assessed with the human PBMCs transduced with LV-EF1a-GCN4(52SR4) to create CAR-T-GCN4 at E:T ratios of 10:1 and 24 hour incubation. Activity was assessed against NALM-6 (CD19⁺), RS4;11 (CD19⁺), or RPMI-8226 (CD19⁻) (Table 1). The activity of the IgG (FcNull) switch was assessed against RS4;11 (CD19⁺), or K562 (CD19⁻) (Table 2). The activity of the C-terminal switch was assessed against RS4;11 (CD19⁺), or K562 (CD19⁻) (Table 3).

TABLE 1

Cytotoxicity of the anti-CD19 Fab_CL1-GCN4 switch

% Cytotoxicity

Concentration	NALM-6	RS4; 11	RPMI-8226
(pM)	(CD19 positive)	(CD19 positive)	(CD19 negative)

10	86.74405	104.0488	15.20283
1	82.23607	90.00308	8.149928
0.1	77.28449	84.11992	3.819127
0.01	47.15363	45.37116	2.656606
0.001	−5.794394	−2.258805	−4.993927
0.0001	−7.191706	−8.02902	−7.662813
0.00001	−4.779683	−2.792706	−1.318068

TABLE 2

Cytotoxicity of anti-CD19 IgG_FcNull-GCN4 switch

% Cytotoxicity

Concentration	RS4; 11	K562
(pM)	(CD19 positive)	(CD19 negative)

1	53.56274	6.218475
0.1	48.75237	1.844815
0.01	38.21278	−2.777584
0.001	12.10702	−2.964143
0.0001	0.1621473	−6.301391
0.00001	−0.9188344	−4.891867

TABLE 3

Cytotoxicity of anti-CD19 Fab_{C term}-GCN4 switch

% Cytotoxicity

Concentration	RS4; 11	K562
(nM)	(CD19 positive)	(CD19 negative)

10	92.10811	1.44819
1	76.75676	−3.445695
0.1	66.59459	−2.197255
0.01	60.97298	−1.348315
0.001	8.216215	−2.147315
0.0001	−2.162161	−3.046195
0.00001	1.945946	−0.299624

Example 11

Cytotoxicity of Various Anti-CD19-GCN4 CAR-EC Switches with GCN4 Grafted/Fused to Different Regions of an Anti-CD19 Antibody or Antibody Fragment

The cytotoxic activities of various anti-CD19-GCN4 CAR-EC switches grafted/fused to different regions of anti-CD19 FMC63 antibodies or antibody fragments were assessed with the human PBMCs transduced with LV-EF1a-GCN4(52SR4) to create CAR-T-GCN4 at E:T ratios of 10:1 and 24 hour incubation. Switches tested were anti-CD19 Fab_CL1-GCN4 (“CL1 Fab), anti-CD19-GCN4 Fab_C-term(“C-term Fab), anti-CD19 IgG_HC1-GCN4 (“HC1 IgG”), anti-CD19 IgG_CL1-GCN4 (“CL1 IgG”), anti-CD19 IgG_Hinge-GCN4 (“Hinge IgG”), anti-CD19 IgG_WT-GCN4 (“Wt IgG”), anti-CD19 Fab_HC1-GCN4 (“HC1 Fab”), and anti-CD19 Fab_{N-term LC1}-GCN4 (“N-term LC1 Fab”). Activities were assessed against RS4;11 (CD19⁺) (FIG. 4, Table 4). FIG. 6 depicts the grafting positions of switches described in this example. The CL1 and HC1 grafting positions were applied to both Fab and IgG formats. The N-terminus grafting is shown as grafted to the light chain, however N-terminal grafting is not restricted to the light chain or Fab and may also be grafted to the heavy chain as well as the IgG format. The C-term position on the Fab is isosteric with the hinge IgG. In this context all Fab constructs are monovalent and all IgG constructs are bivalent, but these are not a necessary requirements for CAR-EC switches in general.

TABLE 4

Cytotoxicity of anti-CD19-GCN4 switches

Switch
Conc		C-term			Hinge			N-term
(nM)	CL1 Fab	Fab	HC1 IgG	CL1 IgG	IgG	WT IgG	HC1 Fab	LC1 Fab

10	70.10483	63.81551	47.46331	54.02444	67.4252	1.785714	41.07143	59.97437
1	58.28092	59.53878	39.91614	59.58702	52.76022	2.040816	43.87755	62.53738
0.1	60.54507	55.26205	39.16142	58.3228	40.62368	3.061224	44.38776	62.28107
0.01	46.96017	33.37526	28.09225	56.80573	35.0611	2.55102	20.66327	49.46604
0.001	4.444445	−2.09644	1.174004	24.18879	2.697009	2.55102	−0.2551	21.52926
0.0001	2.180294	−4.61216	−2.09644	1.685631	−5.14117	2.040817	−0.5102	3.075609
0.00001	1.425577	−3.60587	−1.09015	0.927097	−6.65823	1.785714	−0.7653	5.07E−07
1E−07	0.922432	−1.34172	0.419288	0.674253	−1.60135	1.27551	−1.27551	1.281505

Example 12

In Vivo Efficacy of Anti-CD19-Fab-GCN4CL1 CAR-EC Switch and Anti-GCN4 CAR T-cells (swiCAR T-cells) in a Xenograft Tumor Mouse Model

To assess swiCAR-T cell in vivo activity, a pilot study with an orthotopic (liquid) xenograft tumor model based on luciferized NALM-6 cells was conducted. In this model swiCAR T-cells demonstrated regression after just 5 days of daily treatment with 0.5 mg/kg of anti-CD19(GCN4) CL1 Fab. Treatment with the wild type anti-CD19 Fab with swiCAR T-cells were not capable of mediating tumor regression (not significant by one-way ANOVA). These results demonstrate the ability to redirect swiCAR T-cells in vivo. Experiment details: 10⁶luciferized NALM-6 cells were injected I.V. into nonobese diabetic-severe combined immunodeficiency (NOD-SCID-γ−/−, NSG) mice. Six days later, 30×10⁶swiCAR T-cells or CART-19 cells (50% transduced) were infused I.V. Dosing of αCD19-Fab-GCN4-CL1 (I.V.) began on the same day, q.d. 0.5 mg/kg. After 5 days of dosing (day 11) mice were injected with luciferin and imaged on an in vivo imaging system (IVIS), n=3 or 4, average radiance (p/s/cm²/sr) plotted measured per mouse, and plotted mean±SEM, **p≤0.05, one-way ANOVA. The difference between no treatment and swiCAR-T+WT Fab is not statistically significant. Results are shown in FIG. 7A.

Example 13

Cloning, Expression and Purification of Anti-BCMA-IgG-GCN4_CL1

Cloning: Mammalian expression vector of CD19 IgG heavy chain was generated by in-frame ligation of amplified anti-BCMA IgG heavy chain (VH and CH1) to pFuse-hIgG1-Fc backbone vector (InvivoGen, CA). A gene encoding antibody BCMA light chain was amplified and cloned into the pFuse vector without hIgG1 Fc fragment. A gene encoding GCN4 (NYHLENEVARLKKL=SEQ ID NO: 3) with GGGGS (SEQ ID NO: 40, wherein n=1) linker at both ends was synthesized as oligonucleotides. Subsequently, anti-BCMA-IgG-GCN4_CL1fusion proteins were created by grafting GCN4 with linker sequences at both ends into the CL region of the anti-BCMA light chain. The resulting mammalian expression vectors were confirmed by DNA sequencing.
Expression and Purification: anti-BCMA-IgG-GCN4_CL1was expressed through transient transfection of FreeStyle HEK 293 cells with expression vectors of BCMA-IgG heavy chain and GCN4-BCMA-CL1 light chain, according to the manufacturer's protocol. Briefly, 28 mL FreeStyle HEK 293 cells containing 3×10⁷cells were seeded in a 125 mL shaking flask. 15 μg light chain plasmid and 15 μg heavy chain plasmid diluted in 1 mL Opti-MEM medium were added in 1 mL Opti-MEM containing 60 μL 293fectin (Invitrogen, Inc). After the plasmids were incubated with 293fectin for 30 min, the lipoplex mixture was added to the cell suspension. Cells were then shaken at 125 rpm in a 5% CO2 environment at 37° C. Culture medium containing secreted proteins was harvested at 48 and 96 hours after transfection. anti-BCMA-IgG-GCN4_CL1was purified by Protein G chromatography (Thermo Fisher Scientific, IL).

Example 14

Cytotoxicity of Anti-BCMA-IgG-GCN4_CL1CAR-EC Switch with GCN4 Grafted to the Light Chain of an Anti-BCMA Antibody or Antibody Fragment

The cytotoxic activity of the anti-BCMA-IgG-GCN4_CL1CAR-EC switch was assessed with the human PBMCs transduced with LV-EF1a-GCN4(52SR4) to create CAR-T-GCN4 at E:T ratios of 10:1 and 24 hour incubation. Transduction efficiency of PBMCs was approximately 50%. Activities were assessed against OPM2 (BCMA⁺), by quantifying lactate dehydrogenase due to cytolysis of target cells (FIG. 8, Table 5).

TABLE 5

Cytotoxicity of anti-BCMA-IgG-GCN4_CL1CAR-EC switch and
anti-GCN4 CAR-Tcell

	anti-BCMA-IgG-GCN4_CL1switch
	concentration [pM]	% cytotoxicity

	10000.000	35.52758
	1000.000	35.06853
	100.000	41.44725
	10.000	31.59707
	1.000	5.575391
	0.100	1.13803
	0.010	0.812881

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

TABLE 6

Chimeric Antigen Receptor-Nucleotide Sequence

NAME	SEQ ID NO	SEQUENCE

LV-EF1a-	1	CAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTA
GCN4(52SR4)		TTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTC
-BBZ		ATGAGACAATAACCCTGATAAATGCTTCAATAATATTGA
		AAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCC
		CTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGC
		TCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGA
		TCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCT
		CAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGA
		ACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGT
		GGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAA
		CTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTG
		AGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCA
		TGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGA
		GTGATAACACTGCGGCCAACTTACTTCTGACAACGATCG
		GAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGG
		GGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGC
		TGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA
		TGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAA
		CTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAAT
		AGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCT
		GCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAA
		TCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCA
		GCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTT
		ATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGA
		AATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAG
		CATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTT
		AGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTA
		GGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCT
		TAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAG
		AAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCG
		CGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACC
		AGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTT
		TTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCA
		AATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACT
		TCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCT
		AATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTC
		GTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGA
		TAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCAC
		ACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAG
		ATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCC
		CGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCA
		GGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGG
		GGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCC
		ACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGG
		GGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTT
		TTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT
		TCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATT
		ACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGA
		ACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGA
		AGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTG
		GCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGA
		CTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAG
		TTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATG
		CTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAAC
		AATTTCACACAGGAAACAGCTATGACCATGATTACGCCA
		AGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGG
		AGCTGCAAGCTTAATGTAGTCTTATGCAATACTCTTGTAG
		TCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTA
		CAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGA
		AGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACA
		GACGGGTCTGACATGGATTGGACGAACCACTGAATTGCC
		GCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATAC
		AATAAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTG
		GGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCC
		TCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCC
		CGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGAC
		CCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCG
		AACAGGGACCTGAAAGCGAAAGGGAAACCAGAGCTCTC
		TCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAG
		AGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTT
		GACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGA
		GCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGG
		AAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATAT
		AAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGA
		ACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGA
		AGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCT
		TCAGACAGGATCAGAAGAACTTAGATCATTATATAATAC
		AGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGAT
		AAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAG
		AGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCC
		GCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAA
		TTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAAT
		TGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAA
		GAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGA
		GCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTA
		TGGGCGCAGCCTCAATGACGCTGACGGTACAGGCCAGAC
		AATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCT
		GAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCAC
		AGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGC
		TGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGAT
		TTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTG
		CCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAG
		ATTGGAATCACACGACCTGGATGGAGTGGGACAGAGAA
		ATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAG
		AATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTA
		TTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTT
		AACATAACAAATTGGCTGTGGTATATAAAATTATTCATA
		ATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTT
		GCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATT
		CACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGG
		GACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGA
		GAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGG
		ATCTCGACGGTTAACTTTTAAAAGAAAAGGGGGGATTGG
		GGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAG
		CAACAGACATACAAACTAAAGAATTACAAAAACAAATTA
		CAAAAATTCAAAATTTTATCGAGCTTTGCAAAGATGGAT
		AAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGAC
		CTTCTAGGTCTTGAAAGGAGTGCCTCGTGAGGCTCCGGT
		GCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCC
		GAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGC
		CTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATG
		TCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGA
		ACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTT
		CGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTG
		TGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCC
		CTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGT
		GATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGA
		GAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGT
		GCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGC
		GTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTT
		TCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGC
		TGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCG
		GGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCG
		CGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTT
		CGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATC
		GGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTG
		CCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCG
		GCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAA
		AGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAA
		TGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTC
		ACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGT
		CGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAG
		GCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCT
		TTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCC
		CACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGG
		CACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTT
		TGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAA
		AGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCGGT
		ACCGCGGCCGCCCGGGGATCCATGGCCTTACCAGTGACC
		GCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCA
		GGCCGGACGCCGTTGTGACCCAGGAATCCGCTCTGACCT
		CTTCTCCAGGCGAAACCGTGACTCTGACTTGCCGTAGTAG
		CACCGGGGCTGTGACCACATCTAACTATGCCAGTTGGGT
		CCAGGAAAAACCGGATCACCTGTTTACTGGCCTGATTGG
		CGGCACCAACAATCGCGCACCGGGTGTGCCCGCTCGTTT
		CAGCGGTTCCCTGATTGGGGACAAGGCAGCACTGACTAT
		CACCGGCGCCCAGACCGAAGATGAGGCGATCTATTTTTG
		CGTCCTGTGGTACAGCGACCATTGGGTGTTCGGGGGAGG
		CACCAAACTGACAGTGCTGGGCGGAGGAGGAGGTTCAG
		GAGGAGGAGGTAGCGGGGGAGGCGGTTCCGGGGGAGGC
		GGTTCTGATGTGCAGCTGCAAGAATCCGGGCCAGGACTG
		GTTGCGCCTTCTCAGAGTCTGTCAATTACATGTACTGTTA
		GTGGCTTTCTGCTGACCGACTATGGTGTGAACTGGGTTCG
		TCAGAGCCCAGGCAAGGGTCTGGAGTGGCTGGGAGTGAT
		TTGGGGGGATGGAATCACAGACTACAATAGCGCACTGAA
		ATCTCGGCTGAGTGTTACCAAAGATAACAGCAAGTCCCA
		GGTCTTCCTGAAGATGAACAGCCTGCAAAGCGGCGACTC
		CGCTCGCTATTACTGCGTTACCGGACTGTTTGATTATTGG
		GGGCAGGGGACAACTCTGACTGTTTCCTCCACCACGACG
		CCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCG
		TCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCA
		GCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTT
		CGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACT
		TGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACT
		GCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAAC
		AACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAG
		ATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAG
		GATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGAC
		GCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAAC
		GAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTG
		GACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAA
		GCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATG
		AACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAG
		ATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCA
		CGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGA
		CACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC
		TAAGTCGACAATCAACCTCTGGATTACAAAATTTGTGAA
		AGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGC
		TATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTAT
		TGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAAT
		CCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGT
		CAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGC
		AACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTC
		CTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGG
		CGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAG
		GGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTC
		GGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTT
		GCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCC
		CTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCT
		GCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGC
		CCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGC
		CTGGAATTCGAGCTCGGTACCTTTAAGACCAATGACTTAC
		AAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAG
		GGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAA
		GATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACC
		AGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACC
		CACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCA
		AGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAG
		AGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTA
		GCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATA
		ACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTT
		GTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAG
		CATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCAT
		TCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATC
		ATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCAGT
		TCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTAT
		TTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATT
		CCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTT
		TTGCGTCGAGACGTACCCAATTCGCCCTATAGTGAGTCGT
		ATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGA
		CTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGC
		AGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGA
		GGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCT
		GAATGGCGAATGGCGCGACGCGCCCTGTAGCGGCGCATT
		AAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGC
		TACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTC
		TTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCA
		AGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGT
		GCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGT
		GATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTT
		TTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTG
		GACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTC
		GGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCG
		GCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTT
		AACGCGAATTTTAACAAAATATTAACGTTTACAATTTCC

TABLE 7

CAR Binding Region -Nucleotide & Amino Acid Sequences

NAME	SEQ ID NO	SEQUENCE

yeast transcription factor GCN4	2	RMKQLEPKVEELLPKNYHLENEVARLKK
(7P14P)		LVGER

yeast transcription factor GCN4	3	NYHLENEVARLKKL
minimal binding peptide

yeast transcription factor GCN4	4	GGGGSNYHLENEVARLKKLGGGGS
minimal binding peptide with
linkers

Hydrophilic target peptide	5	GGGGSDYKDDDDK
(HTP)

Hydrophilic target peptide	6	GGGGSDYKDDDDKP
(HTP) P

FLAG ®	7	DYKDDDDK

TABLE 8

CAR-T switch targeting polypeptides-Nucleotide Sequence

	SEQ
NAME	ID NO	SEQUENCE

Light chain of	8	GACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTC
anti-CD19		TGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACA
antibody		TTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAA
		CTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGG
		AGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTA
		TTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACT
		TACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAG
		GGGGGACCAAGCTTGAGATCAAACGAACTGTGGCTGCACCAT
		CTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGG
		AACTGCCTCTGTCGTGTGCCTGCTGAATAACTTCTATCCCAGA
		GAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCG
		GGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGAC
		AGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCA
		GACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCAT
		CAGGGCCTGTCCTCGCCCGTCACAAAGAGCTTCAACAGGGGA
		GAGTGT

Heavy chain	9	GAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCC
of anti-CD19		TCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCAT
antibody Fab		TACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAA
		GGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCAC
		ATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAG
		GACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTG
		CAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATT
		ACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAA
		CCTCAGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGT
		CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACA
		GCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCG
		GTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTG
		CACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCC
		TCAGCAGCGTGGTGACTGTGCCCTCTAGCAGCTTGGGCACCCA
		GACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAA
		GGTGGACAAGAAAGTTGAGCCCAAATCTTGT

Heavy chain	10	GAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCC
of anti-CD19		TCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCAT
IgG		TACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAA
		GGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCAC
		ATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAG
		GACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTG
		CAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATT
		ACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAA
		CCTCAGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGT
		CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACA
		GCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCG
		GTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTG
		CACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCC
		TCAGCAGCGTGGTGACTGTGCCCTCTAGCAGCTTGGGCACCCA
		GACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAA
		GGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCA
		CACATGCCCACCGTGCCCAGCACCTCCAGTCGCCGGACCGTCA
		GTCTTCCTCTTCCCTCCAAAACCCAAGGACACCCTCATGATCT
		CCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCC
		ACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG
		TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGT
		ACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCA
		CCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTC
		CAACAAAGGCCTCCCAAGCTCCATCGAGAAAACCATCTCCAA
		AGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCC
		TCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGAC
		CTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAG
		TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCAC
		GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGC
		AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTC
		TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA
		CGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

Light chain of	11	ATGAAAAAGAATATCGCATTTCTTCTTGCTAGCATGTTCGTTT
Trastuzumab		TTTCTATTGCTACAAACGCATACGCTGACATCCAGATGACCCA
(anti-Her2)		GTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACC
		ATCACTTGCCGGGCAAGTCAGGATGTGAATACCGCGGTCGCA
		TGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATC
		TATTCTGCATCCTTCTTGTATAGTGGGGTCCCATCAAGGTTCA
		GTGGCAGTAGATCTGGGACAGATTTCACTCTCACCATCAGCAG
		TCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGCAT
		TACACTACCCCTCCGACGTTCGGCCAAGGTACCAAGCTTGAGA
		TCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCC
		ATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTCGTGTGC
		CTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGG
		AAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGT
		GTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGC
		AGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAA
		GTCTACGCCTGCGAAGTCACCCATCAGGGCCTGTCCTCGCCCG
		TCACAAAGAGCTTCAACAGGGGAGAGTGT

Heavy chain	12	ATGAAAAAGAATATCGCATTTCTTCTTGCATCTATGTTCGTTTT
of		TTCTATTGCTACAAACGCGTACGCTGAGGTGCAGCTGGTGGAG
Trastuzumab		TCTGGAGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCT
(anti-Her2)		CCTGTGCAGCCTCTGGGTTCAATATTAAGGACACTTACATCCA
		CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGC
		ACGTATTTATCCTACCAATGGTTACACACGCTACGCAGACTCC
		GTGAAGGGCCGATTCACCATCTCCGCAGACACTTCCAAGAAC
		ACGGCGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACG
		GCCGTGTATTACTGTTCGAGATGGGGCGGTGACGGCTTCTATG
		CCATGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTC
		AGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCC
		TCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTG
		GTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAAC
		TCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCC
		TACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACTGT
		GCCCTCTAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTG
		AATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAG
		CCCAAATCTTGTGACAAAACTCACACA

Light Chain of	13	ATGAAAAAGAATATCGCATTTCTTCTTGCTAGCATGTTCGTTT
Rituximab		TTTCTATTGCTACAAACGCATACGCTCAGATTGTGCTGAGCCA
(anti-CD20)		GAGCCCGGCGATTCTGAGCGCGAGCCCGGGCGAAAAAGTGAC
		CATGACCTGCCGCGCGAGCAGCAGCGTGAGCTATATTCATTG
		GTTTCAGCAGAAACCGGGCAGCAGCCCGAAACCGTGGATTTA
		TGCGACCAGCAACCTGGCGAGCGGCGTGCCGGTGCGCTTTAG
		CGGCAGCGGCAGCGGCACCAGCTATAGCCTGACCATTAGCCG
		CGTGGAAGCGGAAGATGCGGCGACCTATTATTGCCAGCAGTG
		GACCAGCAACCCGCCGACCTTTGGCGGCGGCACCAAGCTTGA
		GATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCG
		CCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTCGTGT
		GCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGT
		GGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGA
		GTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCA
		GCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACA
		AAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGTCCTCGCC
		CGTCACAAAGAGCTTCAACAGGGGAGAGTGT

Heavy Chain	14	ATGAAAAAGAATATCGCATTTCTTCTTGCATCTATGTTCGTTTT
of Rituximab		TTCTATTGCTACAAACGCGTACGCTCAGGTGCAGCTGCAGCAG
(anti-CD20)		CCGGGCGCGGAACTGGTGAAACCGGGCGCGAGCGTGAAAATG
		AGCTGCAAAGCGAGCGGCTATACCTTTACCAGCTATAACATG
		CATTGGGTGAAACAGACCCCGGGCCGCGGCCTGGAATGGATT
		GGCGCGATTTATCCGGGCAACGGCGATACCAGCTATAACCAG
		AAATTTAAAGGCAAAGCGACCCTGACCGCGGATAAAAGCAGC
		AGCACCGCGTATATGCAGCTGAGCAGCCTGACCAGCGAAGAT
		AGCGCGGTGTATTATTGCGCGCGCAGCACCTATTATGGCGGCG
		ATTGGTATTTTAACGTGTGGGGCGCGGGCACCACCGTGACCGT
		GAGCGCGGCGAGCACCAAGGGCCCATCGGTCTTCCCCCTGGC
		ACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGG
		CTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG
		TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCG
		GCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGG
		TGACTGTGCCCTCTAGCAGCTTGGGCACCCAGACCTACATCTG
		CAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAA
		AGTTGAGCCCAAATCTTGTGACAAAACTCACACA

Light chain of	15	ATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGC
Clone C225		TCCCAGGTGCACGATGTGACATCCTGCTGACCCAGTCCCCCGT
(anti-EGFR)		GATCCTGTCCGTGTCCCCTGGCGAGCGGGTGTCCTTCTCCTGC
		CGGGCCTCCCAGTCCATCGGCACCAACATCCACTGGTATCAGC
		AGCGGACCAACGGCTCCCCTCGGCTGCTGATCAAGTACGCCTC
		CGAGTCTATCTCCGGCATCCCTTCCCGGTTCTCCGGCTCCGGC
		TCTGGCACCGACTTCACCCTGTCCATCAACTCCGTGGAGTCCG
		AGGATATCGCCGACTACTACTGCCAGCAGAACAACAACTGGC
		CTACCACCTTCGGCGCTGGAACCAAGCTGGAGCTGAAGCGTA
		CGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
		GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAAT
		AACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGAT
		AACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAG
		CAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTG
		ACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCC
		TGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAG
		AGCTTCAACAGGGGAGAGTGTTGATGA

Heavy chain	16	ATGGGTTGGAGCCTCATCTTGCTCTTCCTTGTCGCTGTTGCTAC
of Clone C225		GCGTGTCCACTCCCAGGTGCAGCTGAAGCAGTCCGGCCCTGG
(anti-EGFR)		CCTGGTGCAGCCTTCCCAGTCCCTGTCCATCACCTGCACCGTG
		TCCGGCTTCTCCCTGACCAACTACGGCGTGCACTGGGTGCGCC
		AGTCCCCCGGCAAGGGCCTGGAGTGGCTGGGCGTGATCTGGT
		CCGGCGGCAACACCGACTACAACACCCCTTTCACCTCCCGGCT
		GTCCATCAACAAGGACAACTCCAAGTCCCAGGTGTTCTTCAAG
		ATGAACTCCCTGCAGTCCAACGACACCGCCATCTACTACTGCG
		CCAGAGCCCTGACCTACTATGACTACGAGTTCGCCTACTGGGG
		CCAGGGCACCCTGGTGACCGTGTCCGCCGCTAGCACCAAGGG
		CCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCT
		GGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTC
		CCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACC
		AGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGAC
		TCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTT
		GGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAG
		CAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGA
		CAAAACTCACACATGCCCACCGTGCCCA

Light chain of	17	GAGAACGTGCTCACCCAATCCCCCGCCATTATGTCCGCCTCCC
anti-CLL-1		CAGGCGAAAAGGTGACAATGACCTGCAGGGCCAGCTCCAACG
antibody		TGATCAGCTCTTACGTGCACTGGTACCAGCAACGGTCCGGCGC
		CTCCCCTAAGCTGTGGATCTATAGCACAAGCAACCTGGCTTCC
		GGCGTGCCTGCACGGTTCAGCGGAAGCGGAAGCGGAACAAGT
		TACTCCCTCACCATTTCTAGCGTTGAAGCCGAGGATGCCGCTA
		CATACTATTGTCAACAGTACAGCGGATACCCCCTGACCTTCGG
		AGCCGGCACAAAACTGGAGCTCAAGAGAGCAGCTGCAGCTCC
		CAGCGTGTTCATTTTTCCTCCCTCCGACGAACAACTGAAAAGC
		GGAACAGCCTCTGTCGTTTGCCTGTTGAACAATTTCTACCCTA
		GGGAGGCCAAGGTCCAGTGGAAAGTGGATAACGCTCTGCAAA
		GCGGAAATTCTCAGGAAAGCGTTACCGAACAGGATTCTAAGG
		ACTCTACATACTCTCTGTCTAGCACACTCACGCTGAGCAAAGC
		AGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCA
		TCAGGGCCTGTCCTCGCCCGTCACAAAGAGCTTCAACAGGGG
		AGAGTGT

Heavy chain	18	GACATCCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCC
of anti-CLL-1		AGCCAGAGCCTGAGCCTGACCTGCAGCGTGACCGGCTACAGC
antibody		ATCACCAGCGCCTATTACTGGAACTGGATCCGGCAGTTCCCCG
		GCAACAAGCTGGAGTGGATGGGCTACATCAGCTACGACGGCC
		GGAACAACTACAACCCAAGCCTGAAGAACCGGATCAGCATCA
		CCCGGGACACCAGCAAGAACCAGTTTTTCCTGAAGCTGAACA
		GCGTGACCACAGAGGACACCGCCACCTATTACTGCGCCAAGG
		AGGGAGACTACGACGTGGGCAACTACTACGCCATGGACTACT
		GGGGCCAGGGCACCAGCGTGACCGTGTCTAGCGCCCGGACCA
		AGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCTCTAAGAGCAC
		CAGCGGCGGAACCGCCGCTCTGGGCTGCCTGGTGAAGGACTA
		CTTCCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCCCT
		GACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCTCT
		GGCCTGTACAGCCTGAGCAGCGTGGTTACCGTGCCCAGTTCTT
		CCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGC
		CCAGCAACACCAAGGTGGACAAGAAAGTGGAGCCCAAGAGC
		TGC

Light chain of	19	GATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGAGC
anti-CD33		GTGGGCGATCGCGTGACCATTACCTGCCGCGCGAGCGAAAGC
antibody		GTGGATAACTATGGCATTAGCTTTATGAACTGGTTTCAGCAGA
		AACCGGGCAAAGCGCCGAAACTGCTGATTTATGCGGCGAGCA
		ACCAGGGCAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGCA
		GCGGCACCGATTTTACCCTGAACATTAGCAGCCTGCAGCCGG
		ATGATTTTGCGACCTATTATTGCCAGCAGAGCAAAGAAGTGCC
		GTGGACCTTTGGCCAGGGCACCAAAGTGGAAATTAAACGAAC
		TGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAG
		CAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATA
		ACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATA
		ACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGC
		AGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGA
		CGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCT
		GCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGA
		GCTTCAACAGGGGAGAGTGT

Heavy chain	20	CAGGTGCAGCTGGTGCAGAGCGGCGCGGAAGTGAAAAAACCG
of anti-CD33		GGCAGCAGCGTGAAAGTGAGCTGCAAAGCGAGCGGCTATACC
antibody		TTTACCGATTATAACATGCATTGGGTGCGCCAGGCGCCGGGCC
		AGGGCCTGGAATGGATTGGCTATATTTATCCGTATAACGGCGG
		CACCGGCTATAACCAGAAATTTAAAAGCAAAGCGACCATTAC
		CGCGGATGAAAGCACCAACACCGCGTATATGGAACTGAGCAG
		CCTGCGCAGCGAAGATACCGCGGTGTATTATTGCGCGCGCGG
		CCGCCCGGCGATGGATTATTGGGGCCAGGGCACCCTGGTGAC
		CGTGAGCAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTG
		GCACCCTCCTCCTAGAGCACCTCTGGGGGCACAGCGGCCCTG
		GGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT
		CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC
		CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGT
		GGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATC
		TGCAACGTGAATCACAAGCCCAGCAACACCAAGGTCGACAAG
		AAAGTTGAGCCCAAATCTTGTGGTGGCGGTCACCATCACCATC
		ATCACCACCAC

TABLE 9

CAR-T switch targeting polypeptides -Amino Acid Sequence

NAME	SEQ ID NO	SEQUENCE

Light chain of	21	DIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPG
wildtype anti-		KVPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDV
CS1 antibody		ATYYCQQYSSYPYTFGQGTKLEIK

Heavy chain	22	EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQ
of wildtype		APGKGLEWIGEINPDSSTINYAPSLKDKFIISRDNAKNSLYLQ
anti-CS1		MNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVTVSSAS
antibody Fab		TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
		GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
		NHKPSNTKVDKKVEPKSC

Light chain of	23	DIQMTQSPSSMSVSVGDRVTITCHSSQDINSNIGWLQQKPGK
anti-		SFKGLIYHGTNLDDGVPSRFSGSGSGTDYTLTISSLQPEDFAT
EGFRvIII		YYCVQYAQFPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKS
antibody		GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
(Hu806) Fab		SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
		FNRGEC

Heavy chain	24	QLQESGPGLVKPSQTLSLTCTVSGYSISSDFAWNWIRQPPGK
of anti-		GLEWMGYISYSGNTRYQPSLKSRITISRDTSKNQFFLKLNSV
EGFRvIII		TAADTATYYCVTAGRGFPYWGQGTLVTVSSASTKGPSVFP
antibody		LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH
(Hu806) Fab		TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
		VDKKVEPKSC

Light chain of	25	DIQMTQSPSSLSASVGDRVTITCRANQGISNNLNWYQQKPG
anti-BCMA		KAPKPLIYYTSNLQSGVPSRFSGSGSGTDYTLTISSLQPEDFA
antibody		TYYCQQFTSLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS
(BCMA98)		GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
Fab		SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS
		FNRGEC

Heavy chain	26	EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFDMAWVRQA
of anti-BCMA		PGKGLVWVSSITTGGGDTYYADSVKGRFTISRDNAKSTLYL
antibody		QMDSLRSEDTAVYYCVRHGYYDGYHLFDYWGQGTLVTVS
(BCMA98)		SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
Fab		NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
		NVNHKPSNTKVDKKVEPKSC

Light Chain of	27	DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPD
anti-CD 19		GTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA
antibody		TYFCQQGNTLPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLK
		SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
		DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK
		SFNRGEC

Heavy Chain	28	EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR
of anti-CD19		KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMN
antibody IgG		SLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAS
		TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
		GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
		NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPPVAGPSVFLFPP
		KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH
		NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
		NKGLPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT
		CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
		SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Heavy Chain	29	EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR
of anti-CD19		KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMN
antibody Fab		SLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAS
		TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
		GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
		NHKPSNTKVDKKVEPKSC

TABLE 10

CAR-T switches-Amino Acid Sequence

NAME	SEQ ID NO	SEQUENCE

anti-CD19	30	DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPD
Fab CL1-		GTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA
GCN4 switch		TYFCQQGNTLPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLK
Light Chain		SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
		DSGGGGS NYHLENEVARLKKL GGGGSDSTYSLSSTLTLS
		KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

anti-CD19	31	DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPD
IgG CL1-		GTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA
GCN4 switch		TYFCQQGNTLPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLK
Light Chain		SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
		DSGGGGS NYHLENEVARLKKL GGGGSDSTYSLSSTLTLS
		KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

anti-CD19	32	EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR
Fab HC1-		KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMN
GCN4 switch		SLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAS
Heavy Chain		TKGPSVFPLAPSS NYHLENEVARLKKL SGGTAALGCLVKD
		YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS
		SSLGTQTYICNVNHKPSNTKVDKKVEPKSC

anti-CD19	33	EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR
IgG HC1-		KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMN
GCN4 switch		SLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAS
Heavy Chain		TKGPSVFPLAPSS NYHLENEVARLKKL SGGTAALGCLVKD
		YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS
		SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA
		PPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK
		FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW
		LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSR
		DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
		VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY
		TQKSLSLSPGK

anti-CD19	34	EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR
Fab Cterm-		KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMN
GCN4 switch		SLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAS
Heavy Chain		TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
		GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
		NHKPSNTKVDKKVEPKSCGGGGS NYHLENEVARLKKL

anti-CD19	35	EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR
IgG hinge-		KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMN
GCN4 switch		SLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAS
Heavy Chain		TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS
		GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
		NHKPSNTKVDKKVEPKSCGGGGS NYHLENEVARLKKLG
		GSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTC
		VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
		RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG
		QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES
		NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
		SCSVMHEALHNHYTQKSLSLSPGK

anti-CD19	36	NYHLENEVARLKKL GGGGSDIQMTQTTSSLSASLGDRVTI
Fab CL1 N-		SCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRF
term Light		SGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKL
Chain		EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV
		QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE
		KHKVYACEVTHQGLSSPVTKSFNRGEC

anti-BCMA	37	DIQMTQSPSSLSASVGDRVTITCRANQGISNNLNWYQQKPG
GCN4 CL1		KAPKPLIYYTSNLQSGVPSRFSGSGSGTDYTLTISSLQPEDFA
light chain		TYYCQQFTSLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS
		GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
		SGGGGS NYHLENEVARLKKL GGGGSDSTYSLSSTLTLSK
		ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

anti-BCMA	38	EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFDMAWVRQA
heavy chain		PGKGLVWVSSITTGGGDTYYADSVKGRFTISRDNAKSTLYL
WT IgG		QMDSLRSEDTAVYYCVRHGYYDGYHLFDYWGQGTLVTVS
		SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW
		NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
		NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPPVAGPSVFL
		FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
		VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
		VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS
		LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
		LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
		GK

Bold indicates grafted region (peptide and/or linker(s). Underline indicates peptide.

TABLE 11

pBAD vector with CAR-T targeting moiety -Nucleotide Sequence

NAME	SEQ ID NO	SEQUENCE

pBAD-	39	AAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTC
CD 19wt		ACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGT
		AACCCTGATTATTTGCACGGAGTCACACTTTGCTATGCCA
		TAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACG
		CTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTT
		TTTGGGCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGA
		ATACATCAACTAGTACGCAAGTTCACGTAAAAAGGGTAT
		CTAGAGGTTGAGGTGATTTTATGAAAAAGAATATCGCAT
		TTCTTCTTGCTAGCATGTTCGTTTTTTCTATTGCTACAAAC
		GCATACGCTGACATCCAGATGACACAGACTACATCCTCC
		CTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCA
		GGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATC
		AGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACC
		ATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCA
		GTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAG
		CAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCA
		ACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGAC
		CAAGCTTGAGATCAAACGAACTGTGGCTGCACCATCTGT
		CTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGA
		ACTGCCTCTGTCGTGTGCCTGCTGAATAACTTCTATCCCA
		GAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCC
		AATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACA
		GCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGC
		TGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCT
		GCGAAGTCACCCATCAGGGCCTGTCCTCGCCCGTCACAA
		AGAGCTTCAACAGGGGAGAGTGTTAAGCTGGGGATCCTC
		TAGAGGTTGAGGTGATTTTATGAAAAAGAATATCGCATT
		TCTTCTTGCATCTATGTTCGTTTTTTCTATTGCTACAAACG
		CGTACGCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCC
		TGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGT
		CTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATT
		CGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTA
		ATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTC
		AAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGC
		CAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGAC
		ACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTG
		GTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAG
		TCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTT
		CCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCAC
		AGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGA
		ACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAG
		CGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGA
		CTCTACTCCCTCAGCAGCGTGGTGACTGTGCCCTCTAGCA
		GCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACA
		AGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCC
		AAATCTTGTGACAAAACTCACACATAATAAGTCGACCGA
		TGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTG
		GGCGCGGGGCATGACTATCGTCGCCGCACTTATGACTGT
		CTTCTTTATCATGCAACTCGTAGGACAGGTGCCAAACGGT
		CTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCA
		GCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGA
		TAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCC
		CACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTA
		GCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAG
		GGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCG
		AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGA
		ACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATT
		TGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCA
		GGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAG
		AAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAA
		CTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATC
		CGCTCATGAGACAATAACCCTGATAAATGCTTCAATAAT
		ATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTG
		TCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTT
		TTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCT
		GAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTG
		GATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCC
		GAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGC
		TATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAG
		AGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTT
		GGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGA
		TGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAAC
		CATGAGTGATAACACTGCGGCCAACTTACTTCTGACAAC
		GATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAA
		CATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACC
		GGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACA
		CCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAAC
		TATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACA
		ATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACC
		ACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCT
		GATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATC
		ATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATC
		GTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGAT
		GAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTG
		ATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATA
		TACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAG
		GATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAA
		ATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACC
		CCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTT
		TCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACC
		GCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACC
		AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCA
		GATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGC
		CACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCG
		CTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGA
		TAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTT
		ACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTC
		GTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGA
		ACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCAC
		GCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAA
		GCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTT
		CCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGG
		TTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCT
		CGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAAC
		GCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGC
		TCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATA
		ACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCG
		CAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGG
		AAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGC
		ATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAG
		TACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACA
		CTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCG
		ACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTG
		TCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTC
		TCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCAC
		CGAAACGCGCGAGGCAGCAGATCAATTCGCGCGCGAAG
		GCGAAGCGGCATGCATAATGTGCCTGTCAAATGGACGAA
		GCAGGGATTCTGCAAACCCTATGCTACTCCGTCAAGCCG
		TCAATTGTCTGATTCGTTACCAATTATGACAACTTGACGG
		CTACATCATTCACTTTTTCTTCACAACCGGCACGGAACTC
		GCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAG
		AAATAGAGTTGATCGTCAAAACCAACATTGCGACCGACG
		GTGGCGATAGGCATCCGGGTGGTGCTCAAAAGCAGCTTC
		GCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACG
		CTAATCCCTAACTGCTGGCGGAAAAGATGTGACAGACGC
		GACGGCGACAAGCAAACATGCTGTGCGACGCTGGCGATA
		TCAAAATTGCTGTCTGCCAGGTGATCGCTGATGTACTGAC
		AAGCCTCGCGTACCCGATTATCCATCGGTGGATGGAGCG
		ACTCGTTAATCGCTTCCATGCGCCGCAGTAACAATTGCTC
		AAGCAGATTTATCGCCAGCAGCTCCGAATAGCGCCCTTC
		CCCTTGCCCGGCGTTAATGATTTGCCCAAACAGGTCGCTG
		AAATGCGGCTGGTGCGCTTCATCCGGGCGAAAGAACCCC
		GTATTGGCAAATATTGACGGCCAGTTAAGCCATTCATGC
		CAGTAGGCGCGCGGACGAAAGTAAACCCACTGGTGATAC
		CATTCGCGAGCCTCCGGATGACGACCGTAGTGATGAATC
		TCTCCTGGCGGGAACAGCAAAATATCACCCGGTCGGCAA
		ACAAATTCTCGTCCCTGATTTTTCACCACCCCCTGACCGC
		GAATGGTGAGATTGAGAATATAACCTTTCATTCCCAGCG
		GTCGGTCGATAAAAAAATCGAGATAACCGTTGGCCTCAA
		TCGGCGTTAAACCCGCCACCAGATGGGCATTAAACGAGT
		ATCCCGGCAGCAGGGGATCATTTTGCGCTTCAGCCATACT
		TTTCATACTCCCGCCATTCAGAG

TABLE 12

Linker-Amino Acid Sequence

NAME	SEQ ID NO	SEQUENCE

	40	(GGGGS)_n, n is at least 1
	41	(GGGS)_n, n is at least 1
	42	(GGS)_n, n is at least 1
	43	(G_mS)_n, n is at least 1, m is at least 1
	44	(X_mS)_n, n is at least 1, m is at least 1 and X is
		an amino acid

Bold indicates grafted region (peptide and/or linker(s).
Underline indicates peptide.

Claims

1. A chimeric antigen receptor-effector cell switch comprising:

a. a peptidic antigen that binds a chimeric antigen receptor on an effector cell; and

b. a targeting moiety that binds a cell surface molecule on a target cell.

2.-62. (canceled)

63. A method of treating a cancer in a subject comprising:

a. administering to the subject a first chimeric antigen receptor-effector cell (CAR-EC) switch to a subject, wherein the first CAR-EC switch comprises:

a. a first peptidic antigen that binds a chimeric antigen receptor on an effector cell; and

b. a first targeting moiety that binds a cell surface molecule on a target cell; and

b. administering a first chimeric antigen receptor-effector cell comprising a chimeric antigen receptor that binds to the peptidic antigen of the chimeric antigen receptor-effector cell switch.

64. The method of claim 63, wherein the binding of the first peptidic antigen on the first CAR-EC switch to the first chimeric antigen receptor on an effector cell and the binding of the first targeting moiety on the first CAR-EC switch to the cell surface molecule on the target cell induces a CAR-EC-mediated immune response that is cytotoxic to the target cell.

65. The method of claim 63, wherein the first CAR-EC switch comprises two peptidic antigens.

66. The method of claim 63, wherein the first targeting moiety comprises a targeting peptide, an antibody, or an antigen binding fragment of an antibody.

67. The method of claim 66, wherein the antibody or the antigen binding fragment of the antibody is selected from the group consisting of: an immunoglobulin, an Fc null immunoglobulin, a Fab, and antigen binding fragments thereof.

68. The method of claim 66, wherein the first targeting moiety is selected from the group consisting of: an anti-EGFR antibody, an anti-Her2 antibody, an anti-EGFRvIII antibody, an anti-CD33 antibody, an anti-CLL-1 antibody, an anti-CEA antibody, an anti-CD19 antibody, an anti-CD22 antibody, an anti-BCMA antibody, and an anti-CS1 antibody, and antigen binding fragments thereof.

69. The method of claim 67, wherein the first targeting moiety is an anti-CD19 antibody or an antigen binding fragments thereof.

70. The method of claim 66, wherein the first targeting antibody or the antigen binding fragment of the antibody comprises a light chain and a heavy chain pair, wherein the light chain and heavy chain are encoded by nucleic acid sequences comprising nucleic acid sequence pairs selected from the group consisting of: SEQ ID NOs: 8 and 9; SEQ ID NOs: 8 and 10; SEQ ID NOs: 11 and 12; SEQ ID NOs. 13 and 14; SEQ ID NOs: 15 and 16; SEQ ID NOs: 17 and 18; and SEQ ID NOs: 19 and 20.

71. The method of claim 66, wherein the first targeting antibody or the antigen binding fragment of the antibody comprises a light chain and a heavy chain pair, wherein the light chain and heavy chain comprise amino acid sequence pairs selected from the group consisting of: SEQ ID NOs: 21 and 22; SEQ ID NOs: 23 and 24; SEQ ID NOs. 25 and 26; SEQ ID NOs: 27 and 28; SEQ ID NOs: 27 and 29; SEQ ID NOs: 30 and 29; SEQ ID NOs: 36 and 29; SEQ ID NOs: 31 and 28; SEQ ID NOs: 27 and 32; SEQ ID NOs: 27 and 33; SEQ ID NOs: 27 and 34; and SEQ ID NOs: 27 and 35.

72. The method of claim 66, wherein the first peptidic antigen is fused to a region of the targeting antibody or antigen binding fragment of the antibody selected from the group consisting of: (i) an N terminus of a light chain; (ii) an N terminus of a heavy chain; (iii) both an N terminus of a light chain and an N terminus of a heavy chain; (iv) an N terminus of a VL domain of an IgG; (v) an N terminus of a VH domain of an IgG; (vi) an N terminus of a VL domain of a Fab; (vii) an N terminus of a VH domain of a Fab; (viii) an N terminus of a VL domain and a VH domain of an IgG; (ix) and an N terminus of a VL domain and a VH domain of a Fab.

73. The method of claim 69, wherein the first peptidic antigen is fused to a region of the targeting antibody or antigen binding fragment of the antibody selected from the group consisting of: (i) an N terminus of a light chain; (ii) an N terminus of a heavy chain; (iii) both an N terminus of a light chain and an N terminus of a heavy chain; (iv) an N terminus of a VL domain of an IgG; (v) an N terminus of a VH domain of an IgG; (vi) an N terminus of a VL domain of a Fab; (vii) an N terminus of a VH domain of a Fab; (viii) an N terminus of a VL domain and a VH domain of an IgG; (ix) and an N terminus of a VL domain and a VH domain of a Fab.

74. The method of claim 63, wherein the first CAR-EC switch comprises a linker that links the peptidic antigen and the targeting moiety. (New) The method of claim 74 wherein (i) the linker comprises about 1 to about 20 amino acids; (ii) the linker comprises a sequence selected from SEQ ID NOs: 38-42; or (iii) the linker comprises about 1 to about 20 amino acids and the linker comprises a sequence selected from SEQ ID NOs: 38-42.

76. The method of claim 63, wherein the cancer is selected from the group consisting of leukemia, lymphoma, breast cancer, pancreatic cancer, lung cancer, glioma, and glioblastoma.

77. The method of claim 63, wherein the cell surface molecule is selected from the group consisting of: a tumor associated antigen; a cluster of differentiation protein; a receptor; an integral membrane protein and a glycoprotein. (New) The method of claim 63, wherein the first peptidic antigen comprises a length of between about 2 and about 10, about 10 and about 20, about 20 and about 30, about 30 and about 40, about 40 and about 50, about 50 and about 60, about 60 and about 70, about 70 and about 80, about 80 and about 90 amino acids, or about 2 and about 90 amino acids.

79. The method of claim 63, further comprising administering one or more second CAR-EC switch to the subject, each second CAR-EC switch comprising:

b. a second targeting moiety that binds a cell surface molecule on a target cell;

wherein the second targeting moiety of each second CAR-EC switch differs from the first targeting moiety comprised on the first CAR-EC switch; and

wherein the peptidic antigen comprised on the second CAR-EC switch is optionally

(i) the same as the first peptidic antigen comprised on the first CAR-EC switch or

(ii) a second peptidic antigen that differs from the first peptidic antigen comprised on the first CAR-EC switch;

provided that if the second CAR-EC switch comprises a second peptidic antigen, the method further comprises administering a second chimeric antigen receptor-effector cell comprising a chimeric antigen receptor that binds to the second peptidic antigen of the second CAR-EC switch.

80. The method of claim 79, wherein

(i) the first targeting moiety comprises an anti-CD20 antibody or an antigen binding portion thereof and the second targeting moiety comprises an anti-CD19 antibody or an antigen binding portion thereof; or

(ii) the first targeting moiety comprises an anti-CD19 antibody or an antigen binding portion thereof and the second targeting moiety comprises an anti-CD20 antibody or an antigen binding portion thereof.

81. The method of claim 79, wherein the second CAR-EC switch is administered to the subject after the first CAR-EC switch.