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Definitions

• This disclosure relates to compounds, compositions, and methods for treating a subject with cancer and reducing off-target toxicity of T cells expressing chimeric antigen receptors (CAR-T cells) and/or providing enhanced control of CAR-T cell activation.
• CAR-T cells T cells expressing chimeric antigen receptors
• targeting moieties are employed with active compounds in combination with CAR-T cell therapies to direct the CAR-T cells and modify the activity thereof as desired.
• Immunotherapy based on adoptive transfer of lymphocytes (e.g., T cells) into a patient is a valuable therapy in the treatment of cancer and other diseases.
• Important advancements have been made in the development of immunotherapies based on adoptive transfer of lymphocytes.
• T cells expressing chimeric antigen receptors (CAR-T cells) are examples of T cells expressing chimeric antigen receptors (CAR-T cells).
• T cells have been genetically engineered to produce artificial T cell receptors on their surface called chimeric antigen receptors, or CARs.
• CARs are proteins that allow T cells to recognize a specific, pre-selected protein, or antigen, found on targeted tumor cells.
• CAR-T cells can be cultured and expanded in the laboratory, then re-infused into the autologous subject. Through the guidance of the engineered T cell receptor, CAR-T cells recognize and destroy the cancer cells that display the specific antigen on their surfaces.
• CARs include a recognition region (e.g., a single chain fragment variable (scFv) region derived from an antibody) for recognition and binding to the antigen expressed by the tumor.
• the recognition region can be fused to the exoplasmic domain of a T cell receptor to enhance engagement of the T cell with the cancer cell.
• the CAR can be further modified to contain an activation signaling domain that, for example, can be derived from O ⁇ 3z, a Fc receptor gamma signaling domain, or one or more costimulatory domains such as CD28, 4-1BB, ICOS, 0X40, etc.
• CAR-T cells have shown positive results in vitro, use of these genetically engineered T cells to treat human cancers has introduced challenges associated with control of the CAR-T cell’s activity.
• One such challenge especially in solid tumor cancers, is identifying target antigens expressed homogeneously throughout the tumor or other malignant target and not on normal tissues.
• target antigens expressed homogeneously throughout the tumor or other malignant target and not on normal tissues.
• CAR-T cells can potentially damage normal tissues by targeting a tumor- associated antigen that is also expressed on normal tissues. This can lead to toxicities that can harm or even kill the cancer patient.
• toxicities can include, for example, neurologic toxicity, “on target/off tumor” recognition, and anaphylaxis.
• cytokine-associated toxicity also referred to as a “cytokine storm” or cytokine release syndrome (CRS)
• CRS cytokine release syndrome
• CRS is a non-antigen specific toxicity that can occur as a result of the high-levels of CAR-T cell expansion and immune activation typically required to mediate clinical benefit using modem immunotherapies such as CAR-T cell transfer.
• Interaction between conventional CAR-T cells and its target causes the activation and expansion of the CAR-T cells and lysis of both normal and tumor cells.
• cytokines such as interferon gamma (IFN-g) and tumor-necrosis factor alpha (TNFa).
• IFN-g interferon gamma
• TNFa tumor-necrosis factor alpha
• IL-6 interleukin 6
• IL-1 interleukin 1
• IL-10 interleukin 10
• iNOS inducible nitric oxide synthase
• CAR-T cell therapies have great promise as a tool in the treatment of diseases, such as cancer
• additional CAR-T cell therapies are needed that provide reduced off-target toxicity and more precise control of CAR- T cell activation.
• CAR-T cells chimeric antigen receptors
• a small molecule ligand linked to a first targeting moiety is used as an adaptor compound (i.e., a bridge) between one or more cancer cells and CAR-T cells that, in use, directs the CAR-T cells to the cancer for treatment of the cancer.
• the small molecule ligand that is part of the adaptor compound can be, for example, a folate, a 2-[3-(l,3- dicarboxypropySjureidojpentanedioic acid (DUPA) ligand, a neurokinin 1 receptor (NK-1R) ligand, a carbonic anhydrase IX (CAIX) ligand, a ligand of gamma glutamyl transpeptidase, a natural killer group 2D receptor (NKG2D) ligand, or a cholecystokinin B receptor (CCKBR or CCK2) ligand, each of which is a small molecule ligand that binds specifically a receptor that is overexpressed on certain types of cancer cells (i.e., the receptor for these ligands is overexpressed on the target cancer cells as compared to normal tissues and non-target cancer cells).
• DUPA neurokinin 1 receptor
• CAIX carbonic anhydra
• the small molecule ligand can be linked to a first targeting moiety that binds to the chimeric antigen receptors (CAR) expressed by CAR-T cells.
• the first “targeting moiety” can be selected, for example, from 2,4-dinitrophenol (DNP), 2,4,6- trinitrophenol (TNP), biotin, digoxigenin, fluorescein, fluorescein isothiocyanate (FITC), NHS- fluorescein, pentafluorophenyl ester (PFP), tetrafluorophenyl ester (TFP), a knottin, a centyrin, and a designed ankyrin repeat protein (DARPin).
• DNP 2,4-dinitrophenol
• TNP 2,4,6- trinitrophenol
• biotin biotin
• digoxigenin fluorescein, fluorescein isothiocyanate
• FITC fluorescein isothiocyanate
• NHS- fluorescein pentaflu
• the first targeting moiety binds to the recognition region of the genetically engineered CAR expressed by CAR-T cells. Accordingly, the recognition region of the CAR (e.g., a single chain fragment variable region (scFv) of an antibody, a Fab, Fv, Fc, (Fab’) 2 fragment, and the like) is directed to the first targeting moiety.
• the adaptor compound comprising the small molecule ligand linked to the first targeting moiety acts as a bridge between the cancer and the CAR-T cells, directing the CAR-T cells to the cancer for treatment of the cancer.
• this CAR-T cell therapy can provide reduced off-target toxicity, and more precise control of CAR- T cell activation because the small molecule ligand in the adaptor compound binds specifically to targeted cancer cells and not to non-targeted tissues resulting in reduced off-target toxicity, and the adaptor compound has a short half-life in circulation which allows for rapid changes in the concentration of the adaptor compound to control precisely CAR-T cell activation.
• At least one of the one or more adaptor compounds, or the pharmaceutically acceptable salts thereof, is not an antibody nor comprises a fragment of an antibody.
• the therapy described above also has the advantage that a cocktail of different tumor-specific adaptor compounds (e.g., different small molecule ligands but the same targeting moiety) can be administered.
• a cocktail of different tumor-specific adaptor compounds e.g., different small molecule ligands but the same targeting moiety
• heterogeneous solid tumors that have mutated and have lost their primary tumor antigen can still be eradicated because multiple tumor-specific adaptor compounds are used and a different tumor-specific adaptor compound(s) can act as a bridge(s) between the cancer and the CAR-T cells when a tumor mutates and loses its primary tumor antigen.
• adaptor compounds also provides ‘universality’ because a single type of CAR- T cell, with a single type of recognition region, can be used to eradicate multiple tumor types.
• the CAR-T cell has a single type of recognition region directed to the first targeting moiety in the adaptor compounds, but different adaptor compounds have different small molecule ligands (i.e., tumor targeting ligands) directed to multiple tumor types.
• the adaptor compounds make the CAR-T cells ‘universal’ CAR-T cells for killing tumors that express different antigens because the different small molecule ligands in the adaptor compounds bind to different tumor types, but only one type of CAR-T cell with one type of recognition region is used.
• CAR-T cell therapies may become dysfunctional or “exhausted” or reduced proliferation may result upon chronic exposure to tumor antigens or immunosuppressive factors (e.g., myeloid-derived suppressor cells (MDSCs), tumor- associated macrophages (TAMs), regulatory T cells (Tregs), and inhibitory cytokines) in the tumor microenvironment.
• immunosuppressive factors e.g., myeloid-derived suppressor cells (MDSCs), tumor- associated macrophages (TAMs), regulatory T cells (Tregs), and inhibitory cytokines
• CRS cytokine release syndrome
• the endocytosis of the recognition region e.g., a scFV fragment
• the recognition region e.g., a scFV fragment
• the activity modifying compound, or a pharmaceutically acceptable salt thereof is linked to a second targeting moiety, either directly or by a second linker.
• the second targeting moiety is selected from a group consisting of: DNP, TNP, biotin, digoxigenin, fluorescein, FITC, NHS-fluorescein, pentafluorophenyl ester, tetrafluorophenyl ester, knottin, centyrin, and DARPin.
• one or both of the first targeting moiety and the second targeting moiety do not comprise a peptide epitope.
• the activity modifying compound, or a pharmaceutically acceptable salt thereof can comprise a rejuvenating compound, or a pharmaceutically acceptable salt thereof.
• the rejuvenating compound, or the pharmaceutically acceptable salt thereof can be a compound, drug or active agent formulated to rejuvenate exhausted CAR-T cells (e.g., by blocking inhibitory signaling or exhausted CAR-T cells, reactivating the exhausted CAR-T cells through an antigen independent pathway, performing both of the foregoing, or via other methods).
• the rejuvenating compound, or the pharmaceutically acceptable salt thereof is selected from a group consisting of a Toll-Like Receptor (TLR) agonist (e.g., a TLR1, TLR2, TLR7, TLR8, TLR7/8, TLR9, TLR3, TLR4, etc.), a stimulator of interferon genes (STING) agonist, a Nod-like receptor stimulant (NLRs), an absent in melanoma 2 (AIM2)-like receptor (ALRs) agonist, a kinase inhibitor targeting kinases such as GSK-3beta, PI3K, etc., and a phosphatase inhibitor.
• TLR Toll-Like Receptor
• STING stimulator of interferon genes
• NLRs Nod-like receptor stimulant
• AIM2 absent in melanoma 2
• AIM2 melanoma 2
• kinase inhibitor targeting kinases such as GSK-3beta, PI3
• the rejuvenating drug When administered, the rejuvenating drug is delivered into the CAR-T cell (e.g., via the second targeting moiety and the CAR recognition region).
• the rejuvenating drug By concentrating the rejuvenating drug within the CAR-T cell, exhausted or dysfunctional CAR-T cells can be rejuvenated to functional and tumor-killing CAR-T cells, leading to renewed eradication of a solid tumor.
• the administration of the rejuvenating compounds to target CAR-T cells can reverse exhaustion or dysfunction of CAR-T cells induced by the tumor microenvironment.
• the rejuvenating compound or pharmaceutically acceptable salt thereof has the following formula:
• the activity modifying compound, or a pharmaceutically acceptable salt thereof can comprise an immunosuppressive compound, or a pharmaceutically acceptable salt thereof.
• the immunosuppressive compound or pharmaceutically acceptable salt thereof can be a compound, drug or active agent formulated to reduce the activity of the CAR-T cells.
• the immunosuppressive compound or pharmaceutically acceptable salt thereof is selected from a group consisting of tacrolimus, sirolimus, and cyclosporine.
• the immunosuppressive drug is delivered into the CAR-T cell (e.g., via the second targeting moiety and the CAR recognition region).
• a method of modifying T cell activity in a subject with cancer comprises administering to a subject a composition comprising: a vector (e.g., a lentiviral vector) comprising a promoter operatively linked to a nucleic acid sequence encoding a CAR, or CAR-T cells expressing the CAR, wherein the CAR is directed to a first targeting moiety, a second targeting moiety, or both the first and second targeting moieties; administering to the patient one or more adaptor compounds, or pharmaceutically acceptable salts thereof, wherein each adaptor compound, or pharmaceutically acceptable salt thereof, comprises a small molecule ligand linked to the first targeting moiety; and administering to the subject an activity modifying compound linked to a second targeting moiety (e.g., a rejuvenating compound or a pharmaceutically acceptable salt thereof or an immunosuppressive compound or a pharmaceutically acceptable salt thereof).
• the method can further comprise imaging the subject prior
• the small molecule ligand of each adaptor compound can be linked to the first targeting moiety thereof by a first linker.
• the activity modifying compound can be linked to the second targeting moiety by a second linker.
• the first and second linkers can have the same structure or different structures.
• the first and second linker can each independently be a releasable linker or a non- releasable linker.
• the first linker, the second linker, or both can independently comprise a C1-C20 alkyl, a polyethylene glycol (PEG), a polyproline, an oligo-(4- piperidine carboxylic acid, an oligo piperidine, a peptide, a saccharo-peptide, a hydrophilic amino acid, a sugar, an unnatural peptidoglycan, a polyvinylpyrrolidone, pluronic F-127, or a combination thereof.
• the first linker or the second linker comprises a PEG.
• the one or more adaptor compounds, or the pharmaceutically acceptable salts thereof each have the formula: where B represents the small molecule ligand, L represents the first linker, and T represents the first targeting moiety, and L comprises a structure having the formula:
• the second linker can have a structure having the formula:
• the first linker in the one or more adaptor compounds can be positioned between the small molecule ligand and the first targeting moiety.
• the second linker in the activity modifying compound can be positioned between the second targeting moiety and the activity modifying compound.
• the first linker and/or the second linker can each comprise a chemical moiety having a structure independently selected from the following formulae:
• n is an integer from 0 to 200.
• the small molecule ligand of the one or more adaptor compounds, or pharmaceutically acceptable salts thereof comprises a structure having the formula: wherein:
• X 1 and Y 1 are each independently selected from the group consisting of a halo, R 2 , OR 2 , SR 3 , and NR 4 R 5 ;
• Q is selected from the group consisting of C and CH;
• X 2 and X 3 are each independently selected from the group consisting of oxygen, sulfur, -C(Z)-, -C(Z)O-, -OC(Z)-, -N(R 4b )-, -C(Z)N(R 4b )-, -N(R 4b )C(Z)-, -OC(Z)N(R 4b )- , -N(R 4b )C(Z)O-, -N(R 4b )C(Z)N(R 5b )-, -S(O)-, -S(O) 2 -, -N(R 4a )S(O) 2 -, -C(R 6b )(R 7b )-, -N(C ⁇ CH)- , -N(CH 2 C ⁇ CH)-, C 1 -C 12 alkylene, and C 1 -C 12 alkyeneoxy, where Z is oxygen or sulfur;
• R 1 is selected-from the group consisting of hydrogen, halo, C 1 -C 12 alkyl, and C 1 -C 12 alkoxy;
• R 2 , R 3 , R 4 , R 4a , R 4b , R 5 , R 5b , R 6b , and R 7b are each independently selected from the group consisting of hydrogen, halo, C 1 -C 12 alkyl, C 1 -C 12 alkoxy, C 1 -C 12 alkanoyl, C 1 -C 12 alkenyl, C 1 -C 12 alkynyl, (C 1 -C 12 alkoxy)carbonyl, and (C 1 -C 12 alkylamino)carbonyl;
• R 6 and R 7 are each independently selected from the group consisting of hydrogen, halo, C 1 -C 12 alkyl, and C 1 -C 12 alkoxy; or, R 6 and R 7 are taken together to form a carbonyl group;
• R 6a and R 7a are each independently selected from the group consisting of hydrogen, halo, C 1 -C 12 alkyl, and C 1 -C 12 alkoxy; or R 6a and R 7a are taken together to form a carbonyl group; p, r, s, and t are each independently either 0 or 1; and
• * represents a covalent bond, if the one or more adaptor compound, or pharmaceutically acceptable salt thereof, comprises a chemical moiety.
• the one or more adaptor compounds, or pharmaceutically acceptable salts thereof comprises at least: a first set of adaptor compounds or pharmaceutically acceptable salts thereof, each adaptor compound or pharmaceutically acceptable salt thereof of the first set comprising a first small molecule ligand linked to the first targeting moiety; and a second set of adaptor compounds or pharmaceutically acceptable salts thereof, each adaptor compound or pharmaceutically acceptable salt thereof of the second set comprising a second small molecule ligand linked to the first targeting moiety; wherein the first small molecule ligand is specific to a receptor overexpressed on a first type of cancer cell and the second small molecule ligand is specific to a receptor overexpressed on a second type of cancer cell.
• the CAR can have a recognition region comprising a scFv region of an antibody that binds to the first targeting moiety and/or the second targeting moiety with high affinity (e.g., in the sub-nanomolar range).
• the scFv region can be a scFv region of an anti-FITC antibody.
• the CAR can further comprise a co-stimulation domain and/or an activation signaling domain.
• the co-stimulation domain is selected from a group consisting of: CD28, CD137 (4-1BB), CD134 (0X40), and CD278 (ICOS).
• the activation signaling domain is a T cell CD3z chain or an Fc receptor g.
• a method for treating a subject having received CAR-T cell therapy is also provided using the disclosed compositions, cells, vectors, and compounds.
• the method of treating cancer and/or killing cancer cells in a subject comprises: administering to the subject one or more adaptor compounds, or pharmaceutically acceptable salts thereof, each adaptor compound or pharmaceutically acceptable salt thereof comprising a small molecule ligand linked to a first targeting moiety, wherein, prior to the administering step, the subject has received at least a dose of T cells expressing a CAR that recognizes and binds to the first targeting moiety.
• the method further comprises the step of administering to the subject an activity modifying compound linked to a second targeting moiety, wherein the CAR recognizes and binds to the second targeting moiety.
• a combination for modifying T cell activity (which, for example, can result in treating a cancer).
• the combination comprises one or more adaptor compounds, or pharmaceutically acceptable salts thereof, wherein each adaptor compound, or pharmaceutically acceptable salt thereof, comprises a small molecule ligand linked to the first targeting moiety; and an activity modifying compound, the activity modifying compound comprising: a rejuvenating compound, or a pharmaceutically acceptable salt thereof, or an immunosuppressive compound, or a pharmaceutically acceptable salt thereof.
• the one or more adaptor compounds (or pharmaceutically acceptable salts thereof) and the activity modifying compound can be any of the compounds described herein and include, without limitation, a first linker and a second linker, respectively.
• Other combinations for modifying T cell activity in a subject with cancer and/or treating cancer comprise one or more adaptor compounds, or pharmaceutically acceptable salts thereof, wherein each adaptor compound, or pharmaceutically acceptable salt thereof, comprises a small molecule ligand linked to the first targeting moiety; an activity modifying compound, the activity modifying compound comprising: a rejuvenating compound, or a pharmaceutically acceptable salt thereof, or an immunosuppressive compound, or a pharmaceutically acceptable salt thereof; and a composition comprising CAR-T cells, wherein the CAR-T cells comprise a CAR directed to the first targeting moiety, the second targeting moiety or both the first and second targeting moiety.
• the CAR can have a recognition region that binds to the first targeting moiety and/or the second targeting moiety with high affinity (e.g., in a sub-nanomolar range).
• the one or more adaptor compounds (or pharmaceutically acceptable salts thereof), the activity modifying compound, and the CAR-T cell composition can be any of the compounds and compositions described herein.
• Combinations for modifying T cell activity/treating cancer comprising a vector composition are also provided.
• the combination comprises one or more adaptor compounds, or pharmaceutically acceptable salts thereof, wherein each adaptor compound, or pharmaceutically acceptable salt thereof, comprises a small molecule ligand linked to the first targeting moiety; an activity modifying compound, the activity modifying compound comprising: a rejuvenating compound, or a pharmaceutically acceptable salt thereof, or an immunosuppressive compound, or a pharmaceutically acceptable salt thereof; and a vector composition comprising a promoter operatively linked to a nucleic acid sequence encoding a CAR.
• the one or more adaptor compounds (or pharmaceutically acceptable salts thereof), the activity modifying compound, the vector composition, and the CAR can be any of the compounds, compositions, or CARs (as appropriate) described herein and include, without limitation, a first linker and a second linker, respectively.
• the vector composition can comprise a lenti viral vector.
• the vector composition can comprise lentiviral particles comprising the nucleic acid vector.
• the vector composition comprises a therapeutically effective amount of the disclosed viral particles.
• a compound for rejuvenating CAR-T cells comprises a rejuvenating compound, or a pharmaceutically acceptable salt thereof, selected from a group comprising a TLR agonist, a STING agonist, a NLR, an ALR agonist, a kinase inhibitor targeting kinase, a RLR, a RAGE, a phosphatase inhibitor, and any other pattern recognition receptor that is located in the endosome or cytoplasm of the targeted CAR-T cell.
• the compound for rejuvenating CAR-T cells, or the pharmaceutically acceptable salt thereof has a structure of the following formula:
• the compound for rejuvenating CAR-T cells has the following formula: BRIEF DESCRIPTION OF THE DRAWINGS
• Fig. 1 shows a general diagram of constructs that can be used for CAR-T cell transduction.
• Fig. 2A shows an illustration of the in vitro exhaustion model.
• Fig. 2B shows that anti-FITC CAR-T cells became exhausted after being stimulated twice with fresh MDA-MB-231 cells in vitro as shown by the decreased killing effect and Fig. 2C shows the corresponding increased expression of T cell exhaustion markers (PD-1+Tim3+LAG3+).
• Fig. 3 shows the structure of TLR7 agonist-FITC conjugates.
• Fig. 3B shows the rejuvenation effect of the TLR7 agonist-FITC conjugates on exhausted anti-FITC CAR-T cells in vitro in the exhaustion model, shown as the increased killing effect;
• Fig. 4A shows the rejuvenation effect of the FITC-TLR7 agonist conjugates in a KB xenograft model, shown as decreased tumor size.
• Fig. 4B and Fig. 4C show the rejuvenation effect of the FITC-TLR7 agonist conjugates in a KB xenograft model, shown as decreased expression of exhaustion markers (PD-1 + Tim3 + ).
• Fig. 4D shows the change in CAR T cell population.
• Fig. 4E shows the change in mice body weight.
• SEQ ID NO: 1 is an examplary nucleic acid sequence for encoding a chimeric antigen receptor (CAR);
• SEQ ID NO: 2 is an exemplary CAR amino acid sequence encoded by SEQ ID NO: 1 or 3;
• SEQ ID NO: 3 is an examplary nucleic acid sequence for encoding a CAR.
• the present disclosure relates to the preparation and use of compounds and compositions that reduce the propensity for off-target toxicity following administration of T cells expressing a chimeric antigen receptor (CAR-T cells) to a subject as compared to conventional CAR-T cell therapies.
• off-target toxicity means organ or tissue damage or a reduction in the subject’s weight that is not desirable to the physician treating the subject, or any other effect on the subject that are potential adverse indicators to the treating physician, for example, B cell aplasia, a fever, a drop in blood pressure, or pulmonary edema.
• treat is an approach for obtaining beneficial or desired results including and preferably clinical results and includes, but is not limited to, one or more of the following: improving a condition associated with a disease, curing a disease, lessening severity of a disease, delaying progression of a disease, alleviating one or more symptoms associated with a disease, increasing the quality of life of one suffering from a disease, prolonging survival and/or prophylactic or preventative treatment.
• the terms “treat,” “treating,” “treated,” or “treatment” can additionally mean reducing the size of a tumor, completely or partially removing the tumor (e.g. , a complete or partial response), causing stable disease, preventing progression of the cancer (e.g., progression free survival), or any other effect on the cancer that would be considered by a physician to be a therapeutic, prophylactic, or preventative treatment of the cancer.
• the compositions comprise genetically engineered CAR-T cells and at least one adaptor compound.
• the adaptor compound can have specificity to one or more targeted cancer cells and is adapted to bind with a chimeric antigen receptor (CAR) of the CAR- T cells via a targeting moiety such that, when administered, the adaptor compound forms abridge between the targeted cancer cell and the CAR-T cell.
• CAR chimeric antigen receptor
• the CAR of the CAR-T cells is directed to the targeting moiety of the adaptor compound such that it binds therewith with specificity and, thus, reduces off-target toxicity and other interactions.
• the CAR-T cell can bind to the adaptor compound and, when so bound, the adaptor compound can direct the CAR-T cells to the targeted cancer cells for treatment of the cancer.
• the adaptor compound can direct the CAR-T cells to the targeted cancer cells for treatment of the cancer.
• Various embodiments are formulated to enhance control of CAR-T cell activation in vivo following administration of CAR-T cells to a subject.
• embodiments of such compositions can comprise genetically engineered CAR-T cells and at least one activity modifying compound (e.g., a rejuvenating compound, an immunosuppressive compound, or a pharmaceutically acceptable salt of either of the foregoing).
• the CAR-T cell can comprise a CAR directed to a targeting moiety of the activity modifying compound such that when the CAR-T cell and the activity modifying compound are administered to a subject, the CAR-T cell links (e.g., with specificity) to the targeting moiety coupled with the activity modifying compound.
• the recognition region of the CAR can additionally be exploited to deliver the activity modifying compound into the CAR-T cell. Such embodiments can be used in lieu of, or in conjunction with, the adaptor compounds.
• CAR-T cells refer to a T cell or population thereof that has been modified through molecular biological methods to express a chimeric antigen receptor (CAR) on the T cell surface.
• the CAR is a polypeptide having a pre-defmed binding specificity to a desired target and is operably connected to (e.g., as a fusion, separate chains linked by one or more disulfide bonds, etc.) the intracellular part of a T cell activation domain.
• CAR engineered T cells of both CD8+ and CD4+ subsets can be recruited for redirected target cell recognition.
• a CAR can additionally include an activation signaling domain that, for example, can be derived from a T cell CD3-zeta (O ⁇ 3z) chain, a Fc receptor gamma signaling domain or a Fc receptor g, or one or more costimulatory domains such as CD28, CD137 (4-1BB), CD278 (ICOS), or CD134 (0X40).
• an activation signaling domain that, for example, can be derived from a T cell CD3-zeta (O ⁇ 3z) chain, a Fc receptor gamma signaling domain or a Fc receptor g, or one or more costimulatory domains such as CD28, CD137 (4-1BB), CD278 (ICOS), or CD134 (0X40).
• Certain CARs are fusions of binding functionality (e.g. , as a single-chain variable fragment (scFv) derived from a monoclonal antibody) to CD3z transmembrane and endodomain.
• binding functionality e.g. , as a single-chain variable fragment (scFv) derived from a monoclonal antibody
• Such molecules result in the transmission of a zeta signal in response to recognition by the recognition receptor binding functionality of its target.
• an antigen recognition domain from native T cell receptor (TCR) alpha and beta single chains can be used as the binding functionality.
• receptor ectodomains e.g., CD4 ectodomain
• All that is required of the binding functionality is that it can bind a given target with high affinity in a specific manner.
• binds with specificity “binds with high affinity,” or “specifically” or “selectively” binds, when referring to a ligand/receptor, a recognition region/targeting moiety, a nucleic acid/complementary nucleic acid, an antibody/antigen, or other binding pair indicates a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies.
• a specified ligand or recognition region binds to a particular receptor (e.g., one present on a cancer cell) or targeting moiety, respectively, and does not bind in a significant amount to other proteins present in the sample (e.g., those associated with normal, healthy cells).
• Specific binding or binding with high affinity can also mean, for example, that the binding compound, ligand, antibody, or binding composition derived from the antigen-binding site of an antibody, of the contemplated method binds to its target with an affinity that is often at least 25% greater, more often at least 50% greater, most often at least 100% (2-fold) greater, normally at least ten times greater, more normally at least 20-times greater, and most normally at least 100-times greater than the affinity with any other binding compound.
• certain compounds comprise at least one adaptor compound (or a pharmaceutically acceptable salt thereof) adapted to form a bridge between a cancer cell and a CAR-T cell, where the CAR-T cell comprises a CAR directed to a targeting moiety of the adaptor compound.
• the CAR-T cells can bind to the targeting moiety of the adaptor compound and, when so bound, the adaptor compound can direct the CAR-T cells to the cancer for treatment of the cancer.
• the adaptor compound is a small molecule ligand linked to a first targeting moiety, via a first linker or otherwise.
• the small molecule ligand can be any small molecule ligand that binds with specificity to a cancer cell type (i.e., wherein the receptor for such ligand is overexpressed on targeted cancer cells as compared to normal tissues or other non-targeted types of cancer).
• a “ligand” is a molecule, ion, or atom that is attached to the central atom or ion (e.g., a drug) of a compound.
• Ligand also encompasses a binding agent that is not an agonist or antagonist and has no agonist or antagonist properties.
• the terms “overexpressed,” “overexpression,” and their formatives (when used in connection with receptor expression and the like) have the meaning ascribed thereto by one of ordinary skill in the relevant arts, which includes (without limitation) the increased presence of a particular receptor on a target cell (e.g., a tumor cell) as compared to normal tissues or other non-targeted types of cells.
• the small molecule ligand of the adaptor compound is a folate, a carbonic anhydrase IX (CAIX) ligand, a 2-[3-(],3-dicarboxypropyl)ureido]pentanedioic acid (DUPA) ligand, a neurokinin 1 receptor (NK-1R) ligand, a ligand of gamma glutamyl transpeptidase, a natural killer group 2D receptor (NKG2D) ligand, or a cholecystokinin B receptor (CCKBR or CCK2) ligand.
• CAIX carbonic anhydrase IX
• DUPA 2-[3-(],3-dicarboxypropyl)ureido]pentanedioic acid
• DUPA 2-[3-(],3-dicarboxypropyl)ureido]pentanedioic acid
• DUPA 2-[3-(],3
• Each of the aforementioned is a small molecule ligand that binds specifically to a receptor that is overexpressed on a certain cancer cell type (i.e., the receptor for each of these ligands is overexpressed on cancers compared to expression of such receptor on normal tissues or, potentially, in diseased tissue not experiencing the targeted cancer type).
• receptors for the CAIX ligand are found, for example, on renal, ovarian, vulvar, and breast cancer; receptors for the NK-1R ligand are found, for example, on cancers of the colon and pancreas;
• DUPA ligand is a ligand bound by PSMA-positive human prostate cancer cells and other cancer cell types; receptors for the NKG2D ligand are found, for example, on cancers of the lung, colon, kidney, prostate, and on T and B cell lymphomas; receptors for the CCKBR ligand are found on cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stroma, and colon, among others; and the transpeptidase is overexpressed, for example, in ovarian cancer, colon cancer, liver cancer, astrocytic gliomas, melanomas, and leukemias.
• the adaptor compound, or the pharmaceutically acceptable salt thereof is not an antibody, and does not comprise a fragment of an antibody.
• the first targeting moiety does not comprise a peptide epitope.
• the small molecule ligand can have a mass of less than about 10,000 Daltons, less than about 9,000 Daltons, less than about 8,000 Daltons, less than about 7,000
• the small molecule ligand can have a mass of about 1 to about 10,000 Daltons, about 1 to about 9,000 Daltons, about 1 to about 8,000 Daltons, about 1 to about 7,000 Daltons, about 1 to about
• these masses can apply to the targeting moieties as well.
• a DUPA derivative can be the ligand of the small molecule ligand linked to the first targeting moiety in the adaptor compound, or the pharmaceutically salt thereof.
• DUPA derivatives are described in International Publication No. WO 2015/057852, which is incorporated herein by reference in its entirety for its teachings regarding same.
• the small molecule ligand is a folate.
• “Folate” can be folic acid, a folic acid analog, or another folate receptor-binding molecule, including for example, analogs and derivatives of folic acid such as, without limitation, folinic acid (e.g., leucovorin), pteroylpoly glutamic acid, pteroyl-D-glutamic acid, and folate receptor-binding pterdines such as tetrahydropterins, dihydrofolates, tetrahydrofolates, and their deaza and dideaza analogs.
• folinic acid e.g., leucovorin
• pteroylpoly glutamic acid pteroyl-D-glutamic acid
• folate receptor-binding pterdines such as tetrahydropterins, dihydrofolates, tetrahydrofolates, and their deaza and dideaza analogs.
• an “analog” or “derivative” with reference to a peptide, polypeptide or protein refers to another peptide, polypeptide or protein that possesses a similar or identical function as the original peptide, polypeptide or protein, but does not necessarily comprise a similar or identical amino acid sequence or structure of the original peptide, polypeptide or protein.
• An analog preferably satisfies at least one of the following: (a) a proteinaceous agent having an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the original amino acid sequence; (b) a proteinaceous agent encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding the original amino acid sequence; or (c) a proteinaceous agent encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the nucleotide sequence
• the terms “deaza” and “dideaza” analogs refer to the art-recognized analogs having a carbon atom substituted for one or two nitrogen atoms in the naturally occurring folic acid structure, or analog or derivative thereof.
• the deaza analogs can include the 1 -deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza analogs of folate, folinic acid, pteropolyglutamic acid, and folate receptor-binding pteridines such as tetrahydropterins, dihydrofolates, and tetrahydrofolates.
• the dideaza analogs include, for example, 1,5-dideaza, 5,10-dideaza, 8,10- dideaza, and 5,8-dideaza analogs.
• the foregoing folic acid analogs are conventionally termed “folates,” reflecting their capacity to bind to folate receptors.
• Other folate receptor-binding analogs include aminopterin, amethopterin (methotrexate), N10-methylfolate, 2-deamino- hydroxyfolate, deaza analogs such as 1-deazamethopterin or 3-deazamethopterin, and 3', 5'- dichloro-4-amino-4-deoxy-N 10 -methylpteroylglutamic acid (dichloromethotrexate).
• folate the folate
• folates reflecting their ability to bind to folate-receptors.
• Such molecules when conjugated with exogenous molecules, are effective to enhance transmembrane transport, such as via folate- mediated endocytosis.
• the foregoing can be used in the folate receptor-binding ligands described herein.
• the small molecule ligand of the adaptor compound hereof can have the formula: wherein:
• X 1 and Y 1 are each independently selected from the group consisting of a halo, R 2 , OR 2 , SR 3 , and NR 4 R 5 ;
• Q is selected from the group consisting of C and CH;
• X 2 and X 3 are each independently selected from the group consisting of oxygen, sulfur, -C(Z)-, -C(Z)O-, -OC(Z)-, -N(R 4b )-, -C(Z)N(R 4b )-, -N(R 4b )C(Z)-, -OC(Z)N(R 4b )- , -N(R 4b )C(Z)O-, -N(R 4b )C(Z)N(R 5b )-, -S(O)-, -S(O) 2 -, -N(R 4a )S(O) 2 -, -C(R 6b )(R 7b )-, -N(C ⁇ CH)- , -N(CH 2 C ⁇ CH)-, C 1 -C 12 alkylene, and C 1 -C 12 alkyeneoxy, where Z is oxygen or sulfur;
• R 1 is selected-from the group consisting of hydrogen, halo, C 1 -C 12 alkyl, and C 1 -C 12 alkoxy;
• R 2 , R 3 , R 4 , R 4a , R 4b , R 5 , R 5b , R 6b , and R 7b are each independently selected from the group consisting of hydrogen, halo, C1-C12 alkyl, C1-C12 alkoxy, C1-C12 alkanoyl, C1-C12 alkenyl, C 1 - C 12 alkynyl, (C 1 -C 12 alkoxy)carbonyl, and (C 1 -C 12 alkylamino)carbonyl;
• R 6 and R 7 are each independently selected from the group consisting of hydrogen, halo, C 1 -C 12 alkyl, and C 1 -C 12 alkoxy; or, R 6 and R 7 are taken together to form a carbonyl group;
• R 6a and R 7a are each independently selected from the group consisting of hydrogen, halo, C1-C12 alkyl, and C1-C12 alkoxy; or R 6a and R 7a are taken together to form a carbonyl group; p, r, s, and t are each independently either 0 or 1; and
• * represents an optional covalent bond to the rest of the conjugate, if any additional chemical moieties are part of or coupled with the folate.
• the cancer may overexpress (as compared to a normal tissue or cell or another type of cancer) a receptor for the small molecule ligand.
• the adaptor compound comprises fluorescein isothiocyanate (FITC) linked to the small molecule ligand.
• FITC fluorescein isothiocyanate
• cytotoxic T cells or another type of T cell, can be transformed to express a CAR that comprises anti-FITC scFv.
• the CAR can target FITC decorating the cancer as a result of binding of the small molecule ligand in the adaptor compound to the cancer.
• toxicity to normal, non-target cells can be avoided.
• the anti-FITC CAR-expressing T cells bind FITC, the CAR-T cells are activated and the cancer is treated.
• the adaptor compound is linked to a first targeting moiety (directly or, for example, via a first linker).
• the first targeting moiety of the adaptor compound is configured to bind to a recognition region of a genetically engineered CAR expressed by a population of CAR-T cells.
• the first targeting moiety can comprise 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), biotin, digoxigenin, fluorescein, FITC, NHS-fluorescein, pentafluorophenyl ester, tetrafluorophenyl ester, a knottin, a centyrin, a DARPin, an affibody, an affilin, an anticalin, an atrimer, an a bicicyclic peptide, an FN3 scaffold, a cys-knot, a fynomer, a Kunitz domain, or an Obody.
• DNP 2,4-dinitrophenol
• TNP 2,4,6-trinitrophenol
• biotin digoxigenin
• fluorescein fluorescein
• FITC fluorescein
• NHS-fluorescein pentafluorophenyl ester
• tetrafluorophenyl ester a knot
• the small molecule ligand is linked to the first targeting moiety via a first linker and comprises the following structure: wherein B represents the small molecule ligand, L represents the first linker, and T represents the first targeting moiety.
• B represents the small molecule ligand
• L represents the first linker
• T represents the first targeting moiety.
• the small molecule ligand linked to the first targeting moiety acts as a bridge between the target cancer cell and the CAR-T cell and directs the CAR-T cells to the cancer for treatment thereof.
• the bridge i.e., the adaptor compound, or a pharmaceutically acceptable salt thereof
• the adaptor compound, or a pharmaceutically acceptable salt thereof can be any of the adaptor compounds described herein or shown in the Examples.
• the adaptor compound (or pharmaceutically acceptable salt thereof) is a small organic molecule so clearance from the bloodstream following administration can be rapidly achieved (i.e., in about 20 minutes or less).
• the CAR-T cell population with which the at least one adaptor compound is to be administered comprises a CAR having a recognition region (e.g., a scFv of an antibody, a Fab fragment, a variable region (Fv), a Fc region, a (Fab’)2 fragment, or the like) directed to the first targeting moiety.
• a recognition region e.g., a scFv of an antibody, a Fab fragment, a variable region (Fv), a Fc region, a (Fab’)2 fragment, or the like
• the recognition region of the CAR is a scFv region of an anti-FITC antibody.
• the recognition region of the CAR binds to the first targeting moiety with high affinity such as, for example, in the sub-nanomolar range. Accordingly, when administered, the CAR-T cells will bind to the targeting moiety of the adaptor compound (or pharmaceutically acceptable salt thereof) and the CAR-T cell response can be targeted to only those cancer cells expressing a receptor for the small molecule ligand portion of the ‘bridge,’ thereby reducing off-target toxicity to normal tissues. Indeed, the small molecule ligand can direct any CAR-T cell linked thereto to a target cell with specificity.
• the at least one adaptor compound provides ‘universality’ because a single type of CAR-T cell, with a single type of recognition region, can be used with multiple adaptor compounds to eradicate multiple tumor types.
• the targeting moiety of each adaptor compound as recognized by the CAR-T cell can remain constant so that one type of CAR-T cell construct can be used, while the small molecule ligand that binds to the cancer can be altered between adaptor compounds to allow for targeting of a wide variety of cancers.
• a first set and a second set of adaptor compounds may be administered to a subject, wherein the first set comprises a first small molecule ligand linked to the first targeting moiety and the second set comprises a second small molecule ligand linked to the first targeting moiety.
• the first small molecule ligand is specific to a receptor overexpressed on a first type of cancer cell (as compared to a baseline expression of such receptor on a healthy tissue or a different type of cancer cell, collectively, a “non-targeted cell”) and the second small molecule ligand is specific to a receptor overexpressed on a second type of cancer cell (as compared to a baseline expression of such receptor on a non-targeted cell).
• both sets of adaptor compound are linked to the same targeting moiety (i.e., the first targeting moiety) such that the CAR is directed to bind therewith
• the different small molecule ligands are targeted to, and can be used to treat, different cancer cell types.
• the adaptor compounds make the CAR-T cells ‘universal’ CAR-T cells for killing tumors that express different antigens because the different small molecule ligands in the adaptor compounds bind to different tumor types, but only one type of CAR-T cell with one type of recognition region need be used.
• the small molecule ligand linked to the first targeting moiety by a first linker can have any of the following structures:
• CAR-T cell therapies A common problem faced by conventional CAR-T cell therapies is that the CAR-T cells may become dysfunctional or “exhausted” such that reduced proliferation results upon chronic exposure to tumor antigens or immunosuppressive factors (e.g., myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), regulatory T cells (Tregs), and inhibitory cytokines) in the tumor microenvironment.
• tumor antigens or immunosuppressive factors e.g., myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), regulatory T cells (Tregs), and inhibitory cytokines
• T cell exhaustion is defined by poor effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of tumors and reduced T cell exhaustion can result in improved clinical responses.
• CAR-T cells can also become overactive resulting in unwanted side effects of conventional CAR-T cell therapies, such as cytokine release syndrome (CRS) and other toxi cities, which can be fatal to the patient.
• CRS cytokine release syndrome
• CAR-T cell expansion and immune activation which ultimately causes lysis of both normal and tumor cells and the release of several cytokines such as interferon gamma (IFN-g) and tumor-necrosis factor alpha (TNFa).
• IFN-g interferon gamma
• TNFa tumor-necrosis factor alpha
• IL-6 interleukin 6
• IL-1 interleukin 1
• IL-10 interleukin 10
• iNOS inducible nitric oxide synthase
• certain compounds and compositions hereof comprise an activity modifying compound or a pharmaceutically acceptable salt thereof for targeting and modifying activity of CAR-T cells when administered in conjunction therewith.
• the activity modifying compound comprises a rejuvenating compound for rejuvenating CAR-T cells, an immunosuppressive compound for reducing activity of CAR-T cells, or a pharmaceutically acceptable salt of either of the foregoing.
• the recognition region of a CAR e.g., a scFv fragment
• the recognition region of a CAR can be modified to exploit endocytosis and facilitate the internalization of the activity modifying compounds into the CAR-T cell.
• the immunosuppressive compound, or the pharmaceutically acceptable salt thereof can be internalized into the CAR-T cells in this manner such that the immunosuppressive compound is concentrated in the CAR-T cell, and, as a result, CAR-T cell activation is reduced, thus leading to control of adverse side effects of excessive CAR- T cell activation such as CRS.
• an immunosuppressive compound, or a pharmaceutically acceptable salt thereof can be used with the CAR-T cell compositions to target CAR-T cells and can reduce CAR-T cell activation.
• a rejuvenating compound, or a pharmaceutically acceptable salt thereof can be internalized into the CAR-T cells via the recognition region of the CAR.
• a rejuvenating compound or, where applicable, an active component thereof
• exhausted or dysfunctional CAR-T cells can be rejuvenated to functional and tumor-killing CAR-T cells, leading to renewed eradication of tumors.
• a rejuvenating compound, or a pharmaceutically acceptable salt thereof can be used with the CAR-T cell compositions to target CAR-T cells and can reverse exhaustion or dysfunction of the CAR-T cells induced by the tumor microenvironment.
• the activity modifying compound (or a pharmaceutically acceptable salt thereof) can be linked to a second targeting moiety and at least a portion of the recognition region of a CAR of the CAR-T cells is directed to the second targeted moiety.
• Various embodiments of available targeting moieties are described below and any of these embodiments may be employed with the second targeting moiety of the activity modifying compound. Accordingly, the CAR-T cells can bind with high affinity to the second targeting moiety of the activity modifying compound.
• the activity modifying compound is linked to the second targeting moiety via a second linker.
• the first linker in the adaptor compound and the second linker in the activity modifying compound can have the same structures or different structures.
• the second linker in the activity modifying compound, or the pharmaceutically acceptable salt thereof can be a releasable linker or a non-releasable linker
• the first linker in the adaptor compound, or the pharmaceutically acceptable salt thereof can be a non-releasable linker.
• available linkers are described below and any of these embodiments may be employed with the second linker of the activity modifying compound.
• “Rejuvenating CAR-T cells” means activating CAR-T cells, increasing proliferation of CAR-T cells, blocking the inhibitory signaling of exhausted or dysfunctional CAR-T cells, re activating CAR-T cells through an antigen-independent pathway, or otherwise increasing the function of CAR-T cells.
• the activity modifying compound comprises a rejuvenating compound (or a pharmaceutically acceptable salt thereof)
• embodiments of the present compounds can be particularly useful in preventing or reversing T cell exhaustion or dysfunction, reduced proliferation, and like conditions induced by the tumor microenvironment.
• the rejuvenating compound or pharmaceutically acceptable salt thereof can comprise any drug or other compound that can rejuvenate T cells such as, for example, a toll-like receptor (TLR) agonist (e.g., agonists of TLR1, TLR2, TLR3, TLR4, TLR7, TLR8, TLR7/8, TLR9, etc ), a stimulator of interferon genes (STING) agonist, a Nod-like receptor (NLR) stimulant, an absent in melanoma 2 (AIM2)-like receptor (ALR) agonist, a kinase inhibitor targeting kinases such as GSK-3beta, PI3K, etc., and/or a phosphatase inhibitor.
• TLR toll-like receptor
• STING stimulator of interferon genes
• NLR Nod-like receptor
• AIM2 absent in melanoma 2
• AIM2 melanoma 2
• AIM2 kinase inhibitor targeting kinases
• the rejuvenating compound or pharmaceutically acceptable salt thereof comprises a retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR), a receptor for advanced gly cation end products (RAGE), or any other pattern recognition receptor that is located in the endosome or cytoplasm of a cell.
• RLR retinoic acid-inducible gene-I
• RAGE receptor for advanced gly cation end products
• pattern recognition receptors means any immune receptors that are expressed on the membranes of leukocytes and are capable of binding specific ligands that activate the receptor and ultimately lead to an innate immune response.
• TLRs are a class of proteins that play a key role in the innate immune system and are an example of pattern recognition receptors.
• TLRs are typically single, membrane-spanning receptors that recognize structurally conserved molecules derived from microbes and are typically expressed on the membranes of leukocytes including dendritic cells, macrophages, natural killer (NK) cells, cells of adaptive immunity (i.e. T and B lymphocytes) and non-immune cells (epithelial and endothelial cells and fibroblasts).
• the rejuvenating compound, or the pharmaceutically acceptable salt thereof has a structure of one of the following formulae: and [0096] In some embodiments, the rejuvenating compound, or pharmaceutically acceptable salt thereof, has a structure of one of the following formulae:
• n 0 to 200
• the activity modifying compound comprises an immunosuppressive compound, or a pharmaceutically acceptable salt thereof, which can target and reduce the activity of CAR-T cells.
• the “reducing activity” of CAR-T cells means suppressing any activity of CAR-T cells, killing CAR-T cells, reducing the number of CAR-T cells, or preventing or reducing proliferation of CAR-T cells.
• Such compositions can be useful in treating or preventing CRS or other toxi cities.
• the immunosuppressive compound or pharmaceutically acceptable salt thereof can comprise any drug or other compound capable of reducing the activity of T cells such as, for example, tacrolimus, sirolimus, cyclosporine and/or other immunosuppressive compounds.
• a “pharmaceutically acceptable salt” of a small molecule ligand linked to the first targeting moiety (i.e., the adaptor compound) or of an activity modifying compound (i.e., the rejuvenating compound or the immunosuppressive compound) linked to a second targeting moiety are contemplated.
• the term “pharmaceutically acceptable salt” refers to those salts whose counter ions may be used in pharmaceuticals.
• such salts include, but are not limited to 1) acid addition salts, which can be obtained by reaction of the free base of the parent compound with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, and perchloric acid and the like, or with organic acids such as acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methane sulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid or malonic acid and the like; or 2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, trimethamine,
• suitable acid addition salts are formed from acids which form non-toxic salts.
• Illustrative examples include the acetate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate, stearate, succinate,
• suitable base salts are formed from bases which form non-toxic salts.
• bases include the arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.
• Hemisalts of acids and bases also can be formed, for example, hemisulphate and hemicalcium salts.
• first or second targeting moiety is limited only in that each should be recognized and bound by the CAR, preferably with specificity, have a relatively low molecular weight, and bind to the CAR with high affinity, such as, and without limitation, in a sub-nanomolar range.
• the first targeting moiety in the adaptor compound and the second targeting moiety in the activity modifying compound have the same structure. In other embodiments, the first targeting moiety and the second targeting moiety have different structures.
• Examples of the first targeting moiety of the one or more adaptor compounds (or the pharmaceutically acceptable salts thereof) and the second targeting moiety in the activity modifying compound that bind to the CAR expressed by CAR-T cells can include, for example, DNP, TNP, biotin, digoxigenin, fluorescein, FITC, NHS -fluorescein, pentafluorophenyl ester, tetrafluorophenyl ester, a knottin, a centyrin, a DARPin, an affibody, an affilin, an anticalin, an atrimer, an avimer, a bici cyclic peptide, an FN3 scaffold, a cys-knot, a fynom
• the first and second targeting moieties hereof can have the same or different structures.
• the activity modifying compound comprises a rejuvenating compound
• the first targeting moiety in the one or more adaptor compound (or pharmaceutically acceptable salt(s) thereof) and the second targeting moiety in the activity modifying compound have the same structure.
• the activity modifying compound comprises an immunosuppressive compound
• the first targeting moiety in the one or more adaptor compound (or the pharmaceutically acceptable salt(s) thereof) and the second targeting moiety in the activity modifying compound have the same structure.
• the first and/or the second targeting moieties can have the following illustrative structure: where:
• X is oxygen, nitrogen, or sulfur, and where X is attached to linker L;
• Y is OR a , NR3 ⁇ 4, or NRV; and Y' is O, NR a , or NRV: each R is independently selected in each instance from H, a fluoro, sulfonic acid, sulfonate, and salts thereof, and the like; and R a is hydrogen or alkyl.
• both the adaptor compound and the activity modifying compound can have a first linker and a second linker, respectively.
• the first and second linkers can have the same or different structures.
• linker includes a chain of atoms that is bio-functionally adapted to form a chemical bond with a small molecule ligand of an adaptor compound or an activity modifying compound (each an “active compound”) and/or a targeting moiety (e.g., the first or second targeting moieties) and connects two or more parts of a molecule to form a compound.
• the chain of atoms may be selected from carbon (C), nitrogen (N), oxygen (O), sulfur (S), silicon (Si), and phosphorus (P), or C, N, O, S, and P, C, N, O, and S.
• the chain of atoms may covalently connect different functional capabilities, such as the small molecule ligand and the targeting moiety.
• the linker e.g., the first or second linker
• the linker may comprise a wide variety of links, such as in the range from about 2 to about 2,000 atoms in the contiguous backbone and can comprise a releasable or non-releasable linker.
• linker in the context of a linker means a linker that includes at least one bond that can be broken (e.g., chemically or enzymatically hydrolyzed) under physiological conditions, such as, for example, by reducing agent-labile, pH-labile, acid-labile, base-labile, oxidatively labile, metabolically labile, biochemically labile, enzyme-labile or p-aminobenzylic based multivalent releasable bond.
• the physiological conditions resulting in bond breaking do not necessarily include a biological or metabolic process and instead may include a standard chemical reaction, such as a hydrolysis reaction for example, at physiological pH or as a result of compartmentalization into a cellular organelle, such as an endosome, having a lower pH than cytosolic pH.
• a cleavable bond can connect two adjacent atoms within the releasable linker and/or connect other linker portions or the targeting moiety and/or active component, as described herein, for example, at either or both ends of the releasable linker.
• the releasable linker is broken into two or more fragments.
• the releasable linker is separated from the targeting moiety.
• non-releasable in the context of a linker means a linker that includes at least one bond that is not easily or quickly broken under physiological conditions.
• a non-releasable linker comprises a backbone that is stable under physiological conditions (e.g., the backbone is not susceptible to hydrolysis (e.g., aqueous hydrolysis or enzymatic hydrolysis)).
• an activity modifying compound or adaptor compound provided herein comprising a non-releasable linker does not release from the targeting moiety.
• the non-releasable linker lacks a disulfide bond (e.g., S-S) or an ester in the backbone.
• the compounds comprise a targeting moiety and an active component connected by a backbone that is substantially stable for the entire duration of the compound’s circulation.
• the non-releasable linker can comprise: an amide, ester, ether, amine, and/or thioether (e.g., thio-maleimide). While specific examples are provided herein, it will be understood that any molecule(s) can be used in the non-releasable linker provided that at least one bond that is not easily or quickly broken under physiological conditions is formed.
• Both releasable and non-releasable linkers can be engineered to optimize biodistribution, bioavailability, and PK/PD (e.g., of the respective compound) and/or to increase uptake (e.g., of the respective compound) into the targeted cell pursuant to methodologies commonly known in the art or hereinafter developed such as through PEGlaytion and the like.
• PK/PD e.g., of the respective compound
• uptake e.g., of the respective compound
• the first linker in each adaptor compound (or the pharmaceutically acceptable salt thereof) or the second linker in the activity modifying compound (or the pharmaceutically acceptable salt thereof) can comprise a C1-C20 alkyl, a polyethylene glycol (PEG), a polyproline, an oligo-(4- piperidine) carboxylic acid, an oligo piperidine, a peptide, a saccharo-peptide, a hydrophilic amino acid, a sugar, an unnatural peptidoglycan, a polyvinylpyrrolidone, a pluronic F-f27, or a combination thereof.
• the linker does not comprise a peptide epitope.
• the linker can further comprise a chemical moiety between the small molecule ligand and the first targeting moiety.
• the first linker (L) of an adaptor compound (or pharmaceutically acceptable salt thereof) and/or the second linker of the activity modifying compound (or pharmaceutically acceptable salt thereof) comprises a structure having the formula: wherein n is an integer from 0 to 200.
• n can be an integer from 0 to 150, 0 to 110, 0 to 100, 0 to 90, 0 to 80, 0 to 70, 0 to 60, 0 to 50, 0 to 40, 0 to 30, 0 to 20, 0 to 15, 0 to 14, 0 to 13, 0 to 12, 0 to 11, 0 to 10, 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, 0 to
• the first linker in the adaptor compound, or the pharmaceutically acceptable salt thereof, and/or the second linker in activity modifying compound, or the pharmaceutically acceptable salt thereof can be a direct linkage (e.g., a reaction between the isothiocyanate group of FITC and a free amine group of a small molecule ligand for the adaptor compound) or the linkage can be through an intermediary linker.
• an intermediary linker can be any biocompatible linker known in the art, such as a divalent linker.
• the divalent linker can comprise about 1 to about 30 carbon atoms.
• the divalent linker can comprise about 2 to about 20 carbon atoms.
• lower molecular weight divalent linkers i.e., those having an approximate molecular weight of about 30 Daltons to about 300 Daltons
• linker lengths that are suitable include, but are not limited to, linkers having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
• the first or the second linker can be a divalent linker that may include one or more linkers.
• Illustrative linkers are shown in the following table, where * indicates the point of attachment to the small molecule ligand of the adaptor compound, to the first targeting moiety of the adaptor compound, to the second targeting moiety of the activity modifying compound, to the rejuvenating compound, to the immunosuppressive compound, or to other divalent first or second linker portions.
• first linker in the adaptor compound, or the pharmaceutically acceptable salt thereof, or the second linker in the activity modifying compound (a rejuvenating compound or an immunosuppressive compound), or the pharmaceutically acceptable salt thereof can each comprise a linker moiety that has a structure selected from the following formulae:
• n is an integer from 0 to 200.
• the compounds can be prepared by conventional methods of organic synthesis practiced by those skilled in the art. Descriptions of compounds are limited by principles of chemical bonding known to those skilled in the art. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding and to give compounds which are not inherently unstable and/or would be known to one of ordinary skill in the art as likely to be unstable under ambient conditions, such as aqueous, neutral, and several known physiological conditions. For example, a heterocycloalkyl or heteroaryl is attached to the remainder of the molecule via a ring heteroatom in compliance with principles of chemical bonding known to those skilled in the art, thereby avoiding inherently unstable compounds.
• the adaptor compound(s), the activity modifying compound, and/or the pharmaceutically acceptable salt of either of the foregoing can contain one or more chiral centers, or can exist as multiple stereoisomers. Accordingly, various embodiments include pure stereoisomers as well as mixtures of stereoisomers, such as enantiomers, diastereomers, and enantiomerically or diastereomerically enriched mixtures.
• the adaptor compound(s), the activity modifying compound, and/or the pharmaceutically acceptable salt of either of the foregoing can exist as geometric isomers. Accordingly, various embodiments can include pure geometric isomers or mixtures of geometric isomers of the compounds. [0121]
• the adaptor compound(s), the activity modifying compound, and/or the pharmaceutically acceptable salt of either of the foregoing can also exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present disclosure.
• compositions and methods can include engineered CAR-T cell compositions.
• T lymphocytes e.g., cytotoxic T lymphocytes
• the first targeting moiety e.g., FITC, DNP, or TNP
• the bridge i.e., the adaptor compound or the pharmaceutically acceptable salt thereof
• the CARs comprise three domains including 1) a recognition region (e.g., a scFv region of an antibody, a Fab fragment, or the like) that recognizes and binds to the first or second targeting moiety with specificity, 2) a co-stimulation domain that enhances the proliferation and survival of the T lymphocytes, and 3) an activation signaling domain that generates a T lymphocyte activation signal.
• a recognition region e.g., a scFv region of an antibody, a Fab fragment, or the like
• co-stimulation domain that enhances the proliferation and survival of the T lymphocytes
• an activation signaling domain that generates a T lymphocyte activation signal.
• the scFv region can be prepared from (i) an antibody known in the art that binds a first or a second targeting moiety, (ii) an antibody newly prepared using a selected first or second targeting moiety, such as a hapten, and (iii) sequence variants derived from the scFv regions of such antibodies, e.g., scFv regions having at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity with the amino acid sequence of the scFv region from which they are derived.
• Percent (%) sequence identity with respect to a reference to a polypeptide sequence is defined as the percentage of amino acid or nucleic acid residues, respectively, in a candidate sequence that are identical with the residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill of the art, for instance, using publicly available computer software.
• determination of percent identity or similarity between sequences can be done, for example, by using the GAP program (Genetics Computer Group, software; now available via Accelrys on http://www.accelrys.com), and alignments can be done using, for example, the ClustalW algorithm (VNTI software, InforMax Inc.) ⁇
• a sequence database can be searched using the nucleic acid or amino acid sequence of interest. Algorithms for database searching are typically based on the BLAST software (Altschul et al., 1990), but those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
• the percent identity can be determined along the full-length of the nucleic acid or amino acid sequence.
• the co-stimulation domain of a CAR can serve to enhance the proliferation and survival of the cytotoxic T lymphocytes upon binding of the CAR to a first or a second targeting moiety.
• Suitable co-stimulation domains include, but are not limited to, CD28, CD137 (4-1BB), a member of the tumor necrosis factor (TNF) receptor family, CD134 (0X40), a member of the TNFR-superfamily of receptors, CD27, CD30, CD 150, a DN AX-activating protein of lOKDa (DAP 10), NKG2D, and CD278 (ICOS), a CD28-superfamily co-stimulatory molecule expressed on activated T cells, or combinations thereof.
• sequence variants of these co-stimulation domains can be used without adversely impacting the invention, where the variants have the same or similar activity as the domain upon which they are modeled.
• such variants can have at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the amino acid sequence of the domain from which they are derived.
• the activation signaling domain serves to activate T lymphocytes (e.g., cytotoxic T lymphocytes) upon binding of the CAR to a first or second targeting moiety.
• Suitable activation signaling domains include, without limitation, the T cell CD3z chain, CD3 delta receptor protein, mbl receptor protein, B29 receptor protein, and Fc receptor g. The skilled artisan will understand that sequence variants of these activation signaling domains can be used where the variants have the same or similar activity as the domain upon which they are modeled.
• the variants have at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity with the amino acid sequence of the domain from which they are derived.
• Constructs encoding the CARs can be prepared using genetic engineering techniques. Such techniques are described in detail in Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 3rd Edition, Cold Spring Harbor Laboratory Press, (2001), and Green and Sambrook, “Molecular Cloning: A Laboratory Manual,” 4th Edition, Cold Spring Harbor Laboratory Press, (2012), which are both incorporated herein by reference in their entireties (collectively, the “Protocols”).
• a plasmid or viral expression vector e.g., a lentiviral vector, a retrovirus vector, sleeping beauty, and piggyback (transposon/transposase systems that include a non-viral mediated CAR gene delivery system)
• a plasmid or viral expression vector e.g., a lentiviral vector, a retrovirus vector, sleeping beauty, and piggyback (transposon/transposase systems that include a non-viral mediated CAR gene delivery system)
• a fusion protein comprising a recognition region, one or more co-stimulation domains, and an activation signaling domain, in frame and linked in a 5' to 3' direction.
• vector means any nucleic acid that functions to carry, harbor, or express a nucleic acid of interest.
• Nucleic acid vectors can have specialized functions such as expression, packaging, pseudotyping, or transduction. Vectors can also have manipulatory functions if adapted for use as a cloning or shuttle vector.
• the structure of the vector can include any desired form that is feasible to make and desirable for a particular use. Such for can include, for example, circular forms such as plasmids and phagemids, as well as linear or branched forms.
• a nucleic acid vector can be composed of, or example, DNA or RNA, as well as contain partially or fully, nucleotide derivatives, analogs or mimetics. Such vectors can be obtained from natural sources, produced recombinantly or chemically synthesized.
• the placement of the recognition region in the fusion protein will generally be such that display of the region on the exterior of the cell is achieved.
• the CARs can also include additional elements, such as a signal peptide (e.g., CD8a signal peptide) to ensure proper export of the fusion protein to the cell surface, a transmembrane domain to ensure the fusion protein is maintained as an integral membrane protein (e.g., CD8a transmembrane domain, CD28 transmembrane domain, or O ⁇ 3z transmembrane domain), and a hinge domain (e.g., CD8a hinge) that imparts flexibility to the recognition region and allows strong binding to the targeting moiety.
• a signal peptide e.g., CD8a signal peptide
• a transmembrane domain to ensure the fusion protein is maintained as an integral membrane protein (e.g., CD8a transmembrane domain, CD28 transmembrane domain, or O ⁇ 3z transmembrane domain)
• FIG. 1 A diagram of an exemplary CAR is shown in Figure 1 where the fusion protein sequence is incorporated into a lentivirus expression vector and where “SP” is a signal peptide, the CAR is an anti-FITC CAR, a CD8a hinge and a CD8a transmembrane domain are present, the co stimulation domain is 4-1BB, and the activation signaling domain is CD3z.
• Exemplary nucleic acid sequences of a CAR insert are provided as SEQ ID NOS: 1 and 3, and the encoded amino acid sequence is provided as SEQ ID NO: 2.
• SEQ ID NO: 2 can comprise or consist of humanized, or human amino acid sequences.
• the CAR has a recognition region comprising a scFv region of an anti-FITC antibody, a co-stimulation domain and the co-stimulation domain is CD 137 (4- 1BB), and an activation signaling domain and the activation signaling domain is a T cell CD3z chain. It is well-known to the skilled artisan that an anti-FITC scFv and an anti-fluorescein scFv are equivalent terms.
• T lymphocytes e.g., cytotoxic T lymphocytes
• T lymphocytes can be genetically engineered to express CAR constructs by transfecting a population of the T lymphocytes with an expression vector encoding the CAR construct.
• Suitable methods for preparing a transduced population of T lymphocytes expressing a selected CAR construct are well-known to the skilled artisan and are described in at least the Protocols.
• CAR-T cells comprising a nucleic acid of SEQ ID NO: 1 or 3 are used.
• CAR-T cells comprising a polypeptide of SEQ ID NO: 2 are used.
• a lentiviral vector is used comprising SEQ ID NO: 1 or 3.
• SEQ ID NO: 2 can comprise or consist of humanized or human amino acid sequences.
• variant nucleic acid sequences or amino acid sequences having at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to SEQ ID NOS: 1 to 3 can be used.
• the nucleic acid sequence can be a variant nucleic acid sequence having at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to SEQ ID NOS: 1 or 2 as long as the variant sequence encodes a polypeptide of SEQ ID NO: 2.
• the nucleic acid sequence or the amino acid sequence can be a variant nucleic acid or amino acid sequence having at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to SEQ ID NO: 1 or 3 along a stretch of 200 nucleic acids or, for SEQ ID NO: 2, along a stretch of 200 amino acids.
• the T lymphocytes e.g., cytotoxic T lymphocytes used to prepare CAR-T cells
• allogenic cells can be used.
• T lymphocytes can be obtained from a subject by means well-known in the art.
• T cells e.g., cytotoxic T cells
• T cells can be obtained by collecting peripheral blood from the subject, subjecting the blood to Ficoll density gradient centrifugation, and then using a negative T cell isolation kit (such as EasySepTM T Cell Isolation Kit) to isolate a population of T cells from the peripheral blood.
• a negative T cell isolation kit such as EasySepTM T Cell Isolation Kit
• the population of T lymphocytes need not be pure and may contain other cells, such as other types of T cells (in the case of cytotoxic T cells, for example), monocytes, macrophages, natural killer cells, and B cells.
• the population being collected can comprise at least about 90% of the selected cell type, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the selected cell type.
• the cells are cultured under conditions that promote the activation of the cells.
• the culture conditions are such that the cells can be administered to a subject without concern for reactivity against components of the culture medium.
• the culture conditions may not include bovine serum products, such as bovine serum albumin.
• the activation can be achieved by introducing known activators into the culture medium, such as anti-CD3 antibodies in the case of cytotoxic T cells. Other suitable activators are generally known and include, for example, anti- CD28 antibodies.
• the population of lymphocytes can be cultured under conditions promoting activation for about 1 to about 4 days, for example.
• the appropriate level of activation can be determined by cell size, proliferation rate, or activation markers determined by flow cytometry.
• the cells are transfected with an expression vector encoding a CAR. Suitable vectors and transfection methods for use in various embodiments are described above. After transfection, the cells can be immediately administered to the patient or the cells can be cultured for a time period to allow time for the cells to recover from the transfection, for example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more days, or between about 5 and about 12 days, between about 6 and about 13 days, between about 7 and about 14 days, or between about 8 and about 15 days.
• suitable culture conditions can be similar to the conditions under which the cells were cultured for activation either with or without the agent that was used to promote activation.
• the methods of treatment described herein can further comprise 1) obtaining a population of autologous or heterologous T lymphocytes (e.g., cytotoxic T lymphocytes used to prepare CAR-T cells), 2) culturing the T lymphocytes under conditions that promote the activation of the cells, and 3) transfecting the lymphocytes with an expression vector encoding a CAR to form CAR-T cells.
• a composition comprising the CAR-T cells can be prepared and administered to the subject.
• culture media that lacks any animal products, such as bovine serum, can be used to culture the CAR-T cells.
• tissue culture conditions typically used by the skilled artisan to avoid contamination with bacteria, fungi and mycoplasma can be used.
• the cells e.g., CAR-T cells
• the cells prior to being administered to a patient, are pelleted, washed, and are resuspended in a pharmaceutically acceptable carrier or diluent.
• compositions comprising CAR-expressing T lymphocytes include compositions comprising the cells in sterile 290 mOsm saline, in infusible cryomedia (containing Plasma-Lyte A, dextrose, sodium chloride injection, human serum albumin and DMSO), in 0.9% NaCl with 2% human serum albumin, or in any other sterile 290 mOsm infusible materials.
• the CAR-T cells can be administered in the culture media as the composition, or concentrated and resuspended in the culture medium before administration.
• the CAR-T cell composition can be administered to the subject via any suitable means, such as parenteral administration, e.g., intradermally, subcutaneously, intramuscularly, intraperitoneally, intravenously, or intrathecally.
• parenteral administration e.g., intradermally, subcutaneously, intramuscularly, intraperitoneally, intravenously, or intrathecally.
• the total number of CAR-T cells and the concentration of the cells in the composition administered to the patient will vary depending on a number of factors including the type of T lymphocytes (e.g., cytotoxic T lymphocytes) being used, the binding specificity of the CAR, the identity of the first or second or third targeting moiety and the small molecule ligand, the rejuvenating compound, or the immunosuppressive compound, the identity of the cancer, the location of the cancer in the patient, the means used to administer the compositions to the patient, and the health, age and weight of the patient being treated.
• suitable compositions comprising transduced CAR-T cells include those having a volume of about 0.1 ml to about 200 ml and about 0.1 ml to about 125 ml.
• compositions and methods can include vector compositions.
• the composition comprises a vector comprising a promoter operatively linked to a nucleic acid sequence encoding a CAR construct described herein (for example, and without limitation, SEQ ID NO: 1 or 3).
• the vector composition comprises lentiviral particles that carry a nucleic acid sequence encoding a CAR described herein.
• the vector composition comprises a therapeutically effective amount of such lentiviral particles.
• a lentivirus is a non-limiting example of a vector system that can be used.
• Lentiviruses are complex retroviruses that, in addition to the common retroviral genes Gag, Pol and Env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection.
• Some examples of lentivirus include the Human Immunodeficiency Viruses (HIV-1 and HIV -2) and the Simian Immunodeficiency Virus (SIV).
• Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes Env, Vif, Vpr, Vpu and Nef are deleted, making the vector biologically safe.
• Lentiviral vectors offer many advantages for gene therapy. Unless engineered to be non integrating, lentiviral vectors integrate stably into chromosomes of target cells, permitting long term expression of delivered transgenes. Further, they do not transfer viral genes thus avoiding the problem of generating transduced cells that can be destroyed by cytotoxic T cells. Further, they have a relatively large cloning capacity, sufficient for most envisioned clinical applications. Among retroviruses, lentiviruses have the unique ability to integrate their genome into the chromatin of nondividing cells. This is especially important in the context of gene-therapy for tissues, for example, in the hematopoietic system, the brain, liver, lungs, and muscle.
• vectors derived from HIV-1 allow efficient in vivo and ex vivo delivery, integration and stable expression of transgenes into cells such as neurons, hepatocytes, and myocytes (Blomer et al., 1997; Kafri et al., 1997; Naldini et al., 1996; Naldini et al., 1998).
• Lentiviral vectors are known in the art. For example, see Naldini et al. (1996) Science 272: 263-267; Zufferey et al. (1998) J. Virol. 72: 9873-9880; Dull et al. (1998) J. Virol. 72: 8463-8471; U.S. Pat. No. 6,013,516; U.S. Pat. No. 5,994,136. Generally, these vectors are configured to carry the essential sequences for selection of cells containing the vector, for incorporating foreign nucleic acid into a lentiviral particle, and for transfer of the nucleic acid into a target cell.
• a commonly used lentiviral vector system is the so-called third-generation system.
• Third- generation lentiviral vector systems can include four plasmids.
• the “transfer plasmid” encodes the polynucleotide sequence that is delivered by the lentiviral vector system to the target cell.
• the transfer plasmid generally has one or more transgene sequences of interest flanked by long terminal repeat (LTR) sequences that facilitate integration of the transfer plasmid sequences into the host genome.
• LTR long terminal repeat
• transfer plasmids are generally designed to make the resulting vector replication incompetent.
• the transfer plasmid lacks gene elements necessary for generation of infective particles in the host cell.
• the transfer plasmid can be designed with a deletion of the 3' LTF, rendering the virus “self-inactivating” (SIN). See Dull et al. (1998) J. Virol. 72:8463-8471; Miyoshi et al. (1998) J. Virol. 72:8150-8157.
• Third-generation systems also generally include two “packaging plasmids” and an “envelope plasmid.”
• the “envelope plasmid” generally encodes an Env gene operatively linked to a promoter.
• the Env gene is VSV-G and the promoter is the CMV promoter.
• the third-generation system uses two packaging plasmids, one encoding Gag and Pol and the other encoding Rev as a further safety feature - an improvement over the single packaging plasmid of so-called second-generation systems. Although safer, the third-generation system can be more cumbersome to use and result in lower viral titers due to the addition of an additional plasmid.
• Examplary packing plasmids including, without limitation, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.
• lentiviral vector systems rely on the use of a “packaging cell line.”
• the packaging cell line is a cell line whose cells are capable of producing infectious lentiviral particles when the transfer plasmid, packaging plasmid(s), and envelope plasmid are introduced into the cells.
• Various methods of introducing the plasmids into the cells can be used, including transfection or electroporation.
• a packaging cell line is adapted for high- efficiency packaging of a lentiviral vector system into lentiviral particles.
• lentiviral vector means a nucleic acid that encodes a lentiviral cis nucleic acid sequence required for genome packaging.
• a lentiviral vector also can encode other cis nucleic acid sequences beneficial for gene delivery, including for example, cis sequences required for reverse transcription, proviral integration or genome transcription.
• a lentiviral vector performs transduction functions of a lentiviral vector. As such, the exact makeup of a vector genome will depend on the genetic material desired to be introduced into a target cell. Therefore, a vector genome can encode, for example, additional polypeptides or functions other than that required for packaging, reverse transcription, integration, or transcription.
• Such functions generally include coding for cis elements required for expression of a nucleic acid of interest.
• the lentiviral cis sequences or elements can be derived from a lentivirus genome or other virus or vector genome so long as the lentiviral vector genome can be packaged by a packaging cell line into a lentiviral particle and introduced into a target cell.
• the target cell for the lentiviral vector is an immune cell.
• the target immune cell is a T cell or NK cell.
• the target immune cell exists in a tumor microenvironment.
• the lentiviral particles produced generally include an RNA genome (e.g., derived from a transfer plasmid), a lipid-bilayer envelope in which the Env protein is embedded, and other accessory proteins including integrase, protease, and matrix protein.
• RNA genome e.g., derived from a transfer plasmid
• lipid-bilayer envelope in which the Env protein is embedded
• other accessory proteins including integrase, protease, and matrix protein.
• the term “lentiviral particle” means a viral particle that includes an envelope, has one or more characteristics of a lentivirus, and is capable of invading a target host cell (e.g., a T cell or NK cell).
• Such characteristics can include, for example, infecting non-dividing host cells, transducing non-dividing host cells, infecting or transducing host immune cells, containing a lentiviral virion including one or more of the gag structural polypeptides p7, p24, and pi 7, containing a lentiviral envelope including one or more of the env encoded glycoproteins p41, pi 20, and pi 60, containing a genome including one or more lentivirus cis-acting sequences functioning in replication, proviral integration or transcription, containing a genome encoding a lentiviral protease, reverse transcriptase or integrase, or containing a genome encoding regulatory activities such as Tat or Rev.
• Lentiviral vectors can be used to encode T cell activation receptors.
• a “T cell activation receptor” means one or more transmembrane proteins that are configured to be expressed on the cell surface of transduced cells such that the T cell activation receptor provides a mitogenic signal to the transduced cell.
• a T cell activation receptor is used because the target cells, in most cases, are T cells. The present methods can be adapted for use with other cell types by use of an activation receptor that retains activity in another cell type.
• T cell activation receptors useful here can include a signaling domain that is a cytokine receptor signaling domain, a co-stimulatory receptor signaling domain, a T cell receptor subunit signaling domain, a growth factor receptor signaling domain, or the like (e.g., as previously described in connection with the CAR compositions).
• a signaling domain that is a cytokine receptor signaling domain, a co-stimulatory receptor signaling domain, a T cell receptor subunit signaling domain, a growth factor receptor signaling domain, or the like (e.g., as previously described in connection with the CAR compositions).
• the lentiviral vector can comprise (instead of or in addition to other genes) a polynucleotide encoding a CAR described herein (e.g., those directed to the first and/or second targeting moieties and, for example, inducibly dimerize the small molecule ligand or compound linked therewith).
• lentiviral vectors can further comprise promoters and/or enhancers specific to T cells.
• promoters can be used to control expression of the T cell activation receptor.
• lentiviral vectors can include fusion glycoproteins (e.g., for pseudotyping purposes). See, e.g., Joglekar et al. (2017) Human Gene Therapy Methods 28:291-301.
• pseudotyping a fusion glycoprotein or functional variant thereof facilitates targeting transduction of specific cell types including, without limitation, T cells.
• vectors hereof can include the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (wPRE) or a nucleic acid sequence substantially identical to wPRE.
• wPRE Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element
• Variants of the wPRE element with reduced size are known in the art.
• wPRE-0 refers to a variant of wPRE with the intermediate size.
• the wPRE sequence increases expression of genes delivered by such viral vectors.
• lentiviral vectors can comprise a polynucleotide sequence encoding the 2A peptide.
• the term “2A peptide” refers to a self-cleaving peptide configured to generate two or more proteins from a single open reading frame. 2A peptides are 18-22 residue-long viral oligopeptides that mediate “cleavage” of polypeptides during translation in eukaryotic cells. “2A peptide” can refer to peptides with various amino acid sequences. Detailed methodology for design and use of 2A peptides is provided by Szymczak-Workman et al. (2012) Cold Spring Harb. Protoc. 2012: 199-204.
• vector compositions are administered directly to the subject. In some embodiments, vector compositions are administered in conjunction with T cells. In some embodiments, vector compositions and T cells are separately administered. In some embodiments, T cells are activated and transduced in vivo by administered vector compositions.
• compositions for treating a cancer are also provided.
• the term “combination” generally refers to any product comprising more than one ingredient, including one or more of the compounds described herein (e.g., an adaptor compound, an activity modifying compound (e.g., a rejuvenating compound or an immunosuppressive compound), or a pharmaceutically acceptable salt of the foregoing).
• the compositions described herein can be prepared from isolated compounds or from salts, solutions, hydrates, solvates, and other forms of the compounds. It is appreciated that certain functional groups, such as the hydroxy, amino, and like groups, can form complexes with water and/or various solvents, in the various physical forms of the compounds.
• compositions comprising the compounds
• the compounds can be prepared from various amorphous, non-amorphous, partially crystalline, crystalline, and/or other morphological forms of the compounds
• the compositions can be prepared from various hydrates and/or solvates of the compounds.
• pharmaceutical compositions that recite the compounds described herein include each of, or any combination of, or individual forms of, the various morphological forms and/or solvate or hydrate forms of the compounds.
• a combination for modifying T cell activity in a subject having cancer and/or treating cancer comprises one or more adaptor compounds, or pharmaceutically acceptable salts thereof, and an activity modifying compound (e.g., a rejuvenating compound, an immunosuppressive compound, or a pharmaceutically acceptable salt of either of the foregoing) is also provided.
• an activity modifying compound e.g., a rejuvenating compound, an immunosuppressive compound, or a pharmaceutically acceptable salt of either of the foregoing
• each adaptor compound comprises a small molecule ligand linked to a first targeting moiety by a first linker
• the activity modifying compound comprises a rejuvenating compound or an immunosuppressive compound linked to a second targeting moiety via a second linker.
• the combination can further comprise a composition comprising CAR-T cells expressing a CAR or a vector comprising a promoter operatively linked to a nucleic acid sequence encoding the CAR (for example, SEQ ID NO: 1 or 3), with the CAR directed to the first targeting moiety, the second targeting moiety, or both the first and second targeting moieties.
• a composition comprising CAR-T cells expressing a CAR or a vector comprising a promoter operatively linked to a nucleic acid sequence encoding the CAR (for example, SEQ ID NO: 1 or 3), with the CAR directed to the first targeting moiety, the second targeting moiety, or both the first and second targeting moieties.
• a combination for treating a cancer (for example, by modifying T cell activity in a subject) is provided.
• the combination comprises one or more of the compounds and compositions of the present disclosure.
• Compounds and compositions can be administered in unit dosage forms and/or compositions containing one or more pharmaceutically acceptable carriers, adjuvants, diluents, excipients, and/or vehicles, and combinations thereof.
• the term “administering,” and its formatives, generally refer to any and all means of introducing the compounds and compositions described herein (e.g., the CAR-T cell compositions, the adaptor compound(s) or the pharmaceutically acceptable salt(s) thereof, and/or the activity modifying compounds) to a cell, tissue, organ, or biological fluid of a subject.
• salts may be appropriate.
• acceptable salts include, without limitation, alkali metal (for example, sodium, potassium or lithium) or alkaline earth metals (for example, calcium) salts; however, any salt that is generally non-toxic and effective when administered to the subject being treated is acceptable.
• a “subject” is a mammal, preferably a human, but it can also be a non human animal (including, without limitation, a laboratory, an agricultural, a domestic, or a wild animal).
• a laboratory including, without limitation, a laboratory, an agricultural, a domestic, or a wild animal.
• the methods, compounds, and compositions described herein are applicable to both human and veterinary disease and applications.
• the subject can be a laboratory animal such as a rodent (e.g., mouse, rat, hamster, etc.), a rabbit, a monkey, a chimpanzee, a domestic animal such as a dog, a cat, or a rabbit, an agricultural animal such as a cow, a horse, a pig, a sheep, or a goat, or a wild animal in captivity such as a bear, a panda, a lion, a tiger, a leopard, an elephant, a zebra, a giraffe, a gorilla, a dolphin, or a whale.
• a rodent e.g., mouse, rat, hamster, etc.
• a rabbit e.g., a monkey, a chimpanzee
• a domestic animal such as a dog, a cat, or a rabbit
• an agricultural animal such as a cow, a horse, a pig, a sheep, or a goat
• subjects are “patients,” i.e., living humans or animals that are receiving medical care for a disease or condition, which includes persons or animals with no defined illness who are being evaluated for signs of pathology.
• subjects that can be addressed using the methods hereof include subjects identified or selected as having or being at risk for having cancer. Such identification and/or selection can be made by clinical or diagnostic evaluation.
• the compounds and compositions can be formulated as pharmaceutical compositions and/or administered to a subject, such as a human patient, in a variety of forms adapted to the chosen route of administration.
• a subject such as a human patient
• the adaptor compound, or the pharmaceutically acceptable salt thereof, or the activity modifying compound, or the pharmaceutically acceptable salt thereof, or the CAR-T cell composition, or the vector composition can be administered to a subject using any suitable method known in the art.
• the adaptor compound, or the pharmaceutically acceptable salt thereof, or the rejuvenating compound, or the pharmaceutically acceptable salt thereof, or the immunosuppressive compound, or the pharmaceutically acceptable salt thereof described herein may be administered in unit dosage forms and/or formulations containing conventional nontoxic pharmaceutically-acceptable carriers, adjuvants, and vehicles.
• the adaptor compound(s), or the pharmaceutically acceptable salt(s) thereof, or the rejuvenating compound, or the pharmaceutically acceptable salt thereof, or the immunosuppressive compound, or the pharmaceutically acceptable salt thereof, or the CAR-T cell composition, or the vector composition as described herein can be administered directly into the blood stream, into muscle, or into an internal organ.
• suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intracerebroventricular, intraurethral, intrastemal, intracranial, intratumoral, intramuscular and subcutaneous delivery.
• means for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques. It will be appreciated that the compounds and compositions hereof can be formulated for the desired administration modality as well.
• parenteral formulations are typically aqueous solutions and can contain carriers or excipients such as salts, carbohydrates and buffering agents (preferably at a pH of from 3 to 9), but can also be formulated, where suitable, as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water or sterile saline.
• a suitable vehicle such as sterile, pyrogen-free water or sterile saline.
• any of the liquid formulations described herein can be adapted for parenteral administration.
• the preparation under sterile conditions, by lyophilization to produce a sterile lyophilized powder for a parenteral formulation can readily be accomplished using standard pharmaceutical techniques well-known to those skilled in the art.
• the solubility of the adaptor compound, or the pharmaceutically acceptable salt thereof, or the rejuvenating compound, or the pharmaceutically acceptable salt thereof, or the immunosuppressive compound, or the pharmaceutically acceptable salt thereof, or the activity modifying compound, or the pharmaceutically acceptable salt thereof, used in the preparation of a parenteral formulation can be increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing agents.
• the pharmaceutical dosage forms of the adaptor compound(s) and/or the activity modifying compound that are suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredients that are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
• the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage.
• the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example and without limitation, water, ethanol, a polyol (e.g., glycerol, propylene glycol, liquid PEG(s), and the like), vegetable oils, nontoxic glyceryl esters, and/or suitable mixtures thereof.
• a polyol e.g., glycerol, propylene glycol, liquid PEG(s), and the like
• vegetable oils e.g., glycerol, propylene glycol, liquid PEG(s), and the like
• suitable mixtures thereof e.g., glycerol, propylene glycol, liquid PEG(s), and the like
• the proper fluidity can be maintained by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
• the action of microorganisms can be prevented by the addition of various antibacterial and antifungal agents, such as parab
• the injectable compositions can be desirable to include one or more isotonic agents, such as sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the incorporation of agents formulated to delay absorption, for example, aluminum monostearate and gelatin.
• Sterile injectable solutions can be prepared by incorporating the active component in the required amount of the appropriate solvent with one or more of the other ingredients set forth above, as required, followed by filter sterilization.
• the preferred methods of preparations are vacuum drying and the freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
• Useful dosages of the compounds can be determined by comparing their in vitro activity and the in vivo activity in animal models. Methods of the extrapolation of effective dosages in mice and other animals to human subjects are known in the art. Indeed, the dosage of the compound can vary significantly depending on the condition of the subject, the cancer type being treated, how advanced the pathology is, the route of administration of the compound and tissue distribution, and the possibility of co-usage of other therapeutic treatments (such as radiation therapy or additional drugs in combination therapies).
• compositions and/or compound(s) required for use in treatment e.g., the therapeutically or prophylactically effective amount or dose
• amount of the compositions and/or compound(s) required for use in treatment will vary not only with the particular application, but also with the salt selected (if applicable) and the characteristics of the subject (such as, for example, age, condition, sex, the subject’s body surface area and/or mass, tolerance to drugs) and will ultimately be at the discretion of the attendant physician, clinician, or otherwise.
• “Therapeutically effective amount” or “prophylactically effective amount” is defined as an amount of a reagent or pharmaceutical composition that is sufficient to induce a desired immune response specific for encoded heterologous antigens or show benefit in a subject (i.e., to cause a decrease, prevention, or treatment of the symptoms of the condition being treated).
• the transduced CAR-T cells administered to the subject can comprise from about 1 X 10 5 to about 1 X 10 15 or 1 X 10 6 to about 1 X 10 15 transduced CAR-T cells.
• about 1 X 10 5 to about 1 X 10 10 about 1 X 10 6 to about 1 X 10 10 , about 1 X 10 6 to about 1 X 10 9 , about 1 X 10 6 to about 1 X 10 8 , about 1 X 10 6 to about 2 X 10 7 , about 1 X 10 6 to about 3 X 10 7 , about 1 X 10 6 to about 1.5 X 10 7 , about 1 X 10 6 to about 1 X 10 7 , about 1 X 10 6 to about 9 X 10 6 , about 1 X 10 6 to about 8 X 10 6 , about 1 X 10 6 to about 7 X 10 6 , about 1 X 10 6 to about 7 X 10 6 , about 1 X 10 6 to about 6 X 10 6 , about 1 X 10 6 to about
• a single dose or multiple doses of the CAR-T cells can be administered to the subject.
• the CAR-T cell dose can be in numbers of CAR-T cells per kg of subject body weight.
• the CAR-T cells can be administered before or after the adaptor compound(s), or the pharmaceutically acceptable salt thereof.
• the dose of the CAR-T cells administered to the subject in the CAR- T cell composition is selected from the group consisting of about 1 million, about 2 million, about 3 million, about 4 million, about 5 million, about 6 million, about 7 million, about 8 million, about 9 million, about 10 million, about 11 million, about 12 million, about 12.5 million, about 13 million, about 14 million, and about 15 million of the CAR-T cells.
• the CAR-T cell dose can be in numbers of CAR-T cells per kg of subject body weight.
• the amount of the one or more adaptor compounds, or the pharmaceutically acceptable salts thereof, or the rejuvenating compound, or the pharmaceutically acceptable salt thereof, or the immunosuppressive compound, or the pharmaceutically acceptable salt thereof, or the activity modifying compound, or the pharmaceutically acceptable salt thereof, to be administered to the subject can vary significantly depending on the cancer being treated, the route of administration of the one or more adaptor compounds, or the pharmaceutically acceptable salts thereof, or the rejuvenating compound, or the pharmaceutically acceptable salt thereof, and the tissue distribution.
• the amount to be administered to a subject can be based on body surface area, mass, and physician assessment.
• amounts to be administered can range, for example, from about 0.05 mg to about 30 mg, 0.05 mg to about 25.0 mg, about 0.05 mg to about 20.0 mg, about 0.05 mg to about 15.0 mg, about 0.05 mg to about 10.0 mg, about 0.05 mg to about 9.0 mg, about 0.05 mg to about 8.0 mg, about 0.05 mg to about 7.0 mg, about 0.05 mg to about 6.0 mg, about 0.05 mg to about 5.0 mg, about 0.05 mg to about 4.0 mg, about 0.05 mg to about 3.0 mg, about 0.05 mg to about 2.0 mg, about 0.05 mg to about 1.0 mg, about 0.05 mg to about 0.5 mg, about 0.05 mg to about 0.4 mg, about 0.05 mg to about 0.3 mg, about 0.05 mg to about 0.2 mg, about 0.05 mg to about 0.1 mg, about .01 mg to about 2 mg, about 0.3 mg to about 10 mg, about 0.1 mg to about 20 mg, or about 0.8 to about 3 mg.
• 0.05 mg to about 30 mg 0.05 mg to about 25.0 mg, about
• the dose of the one or more adaptor compounds, or the pharmaceutically acceptable salts thereof, or the rejuvenating compound, or the pharmaceutically acceptable salt thereof, or the immunosuppressive compound, or the pharmaceutically acceptable salt thereof, or the activity modifying compound, or the pharmaceutically acceptable salt thereof can range, for example, from about 50 nmoles/kg to about 3,000 nmoles/kg of subject body weight, about 50 nmoles/kg to about 2,800 nmoles/kg about 50 nmoles/kg to about 2,600 nmoles/kg about 50 nmoles/kg to about 2,400 nmoles/kg about 50 nmoles/kg to about 2,200 nmoles/kg about 50 nmoles/kg to about 2,100 nmoles/kg about 50 nmoles/kg to about 2,000 nmoles/kg, about 50 nmoles/kg to about 1,000 nmoles/kg, about 50 nmoles/kg to about 50 nmoles
• the dose may be about 1 nmoles/kg, about 5 nmoles/kg, about 10 nmoles/kg, about 20 nmoles kg, about 25 nmoles/kg, about 30 nmoles/kg, about 40 nmoles/kg, about 50 nmoles/kg, about 60 nmoles/kg, about 70 nmoles/kg, about 80 nmoles/kg, about 90 nmoles/kg, about 100 nmoles/kg, about 150 nmoles/kg, about 200 nmoles/kg, about 250 nmoles/kg, about 300 nmoles/kg, about 350 nmoles/kg, about 400 nmoles/kg, about 450 nmoles/kg, about 500 nmoles/kg, about 600 nmoles/kg, about 700 nmoles/kg, about 800 nmoles/kg, about 900 nmol
• the dose may be about 0.1 nmoles/kg, about 0.2 nmoles/kg, about 0.3 nmoles/kg, about 0.4 nmoles kg, or about 0.5 nmoles/kg, about 0.1 nmoles/kg to about 1000 nmoles/kg, about 0.1 nmoles/kg to about 900 nmoles/kg, about 0.1 nmoles/kg to about 850 nmoles/kg, about 0.1 nmoles/kg to about 800 nmoles/kg, about 0.1 nmoles/kg to about 700 nmoles/kg, about 0.1 nmoles/kg to about 600 nmoles/kg, about 0.1 nmoles/kg to about 500 nmoles/kg, about 0.1 nmoles/kg to about 400 nmoles/kg, about 0.1 nmoles/kg to about 300 nmoles/kg
• the dose may be about 0.3 nmoles/kg to about 1000 nmoles/kg, about 0.3 nmoles/kg to about 900 nmoles/kg, about 0.3 nmoles/kg to about 850 nmoles/kg, about 0.3 nmoles/kg to about 800 nmoles/kg, about 0.3 nmoles/kg to about 700 nmoles/kg, about 0.3 nmoles/kg to about 600 nmoles/kg, about 0.3 nmoles/kg to about 500 nmoles/kg, about 0.3 nmoles/kg to about 400 nmoles/kg, about 0.3 nmoles/kg to about 300 nmoles/kg, about 0.3 nmoles/kg to about 200 nmoles/kg, about 0.3 nmoles/kg to about 100 nmoles/kg, about 0.3 nmoles/kg to about 50
• the dose of the one or more adaptor compounds, or the pharmaceutically acceptable salts thereof, or the rejuvenating compound, or the pharmaceutically acceptable salt thereof, or the immunosuppressive compound, or the pharmaceutically acceptable salt thereof, or the activity modifying compound, or the pharmaceutically acceptable salt thereof may range from, for example, about 10 nmoles/kg to about 10,000 nmoles/kg, from about 10 nmoles/kg to about 5,000 nmoles/kg, from about 10 nmoles/kg to about 3,000 nmoles/kg, about 10 nmoles/kg to about 2,500 nmoles/kg, about 10 nmoles/kg to about 2,000 nmoles/kg, about 10 nmoles/kg to about 1,000 nmoles/kg, about 10 nmoles/kg to about 900 nmoles/kg, about 10 nmoles/kg to about 800 nmoles/kg, about 10 nmoles/kg, about 10 nmole
• the dose of the one or more adaptor compounds, or the pharmaceutically acceptable salts thereof, or the rejuvenating compound, or the pharmaceutically acceptable salt thereof, or the immunosuppressive compound, or the pharmaceutically acceptable salt thereof, or the activity modifying compound, or the pharmaceutically acceptable salt thereof may range from, for example, about 1 nmoles/kg to about 10,000 nmoles/kg, from about 1 nmoles/kg to about 5000 nmoles/kg, from about 1 nmoles/kg to about 3000 nmoles/kg, about 1 nmoles/kg to about 2500 nmoles/kg, about 1 nmoles/kg to about 2000 nmoles/kg, about 1 nmoles/kg to about 1000 nmoles/kg, about 1 nmoles/kg to about 900 nmoles/kg, about 1 nmoles/kg to about 800 nmoles/kg, about 1 nmoles
• from about 20 pg/kg body weight to about 3 mg/kg body weight of the adaptor compound, or the pharmaceutically acceptable salt thereof, or the rejuvenating compound, or the pharmaceutically acceptable salt thereof, or the immunosuppressive compound, or the pharmaceutically acceptable salt thereof, or the activity modifying compound, or the pharmaceutically acceptable salt thereof can be administered to the subject.
• amounts can be from about 0.2 mg/kg body weight to about 0.4 mg/kg body weight or can be about 50 pg/kg body weight.
• “kg” is kilograms of body weight of the subject.
• a single dose or multiple doses of the one or more adaptor compound(s), or the pharmaceutically acceptable salt thereof, or the rejuvenating compound, or the pharmaceutically acceptable salt thereof, or the immunosuppressive compound, or the pharmaceutically acceptable salt thereof, or the activity modifying compound, or the pharmaceutically acceptable salt thereof, can be administered to the subject.
• the timing between the administration of CAR-T cells and the small molecule linked to the first targeting moiety can vary widely depending on factors that include the type of CAR-T cells being used, the binding specificity of the CAR, the identity of the first targeting moiety and the small molecule ligand, the identity of the cancer, the location in the subject of the cancer, the means used to administer to the subject the CAR-T cells and the adaptor compound, or the pharmaceutically acceptable salt thereof, and the health, age, and weight of the subject.
• the small molecule ligand linked to the first targeting moiety i.e., the adaptor compound, or the pharmaceutically acceptable salt thereof
• the CAR-T cells or composition thereof
• the one or more adaptor compounds, or pharmaceutically acceptable salts thereof can be administered to the subject at the same time as the CAR-T cell composition, but in different formulations, or in the same formulation.
• any applicable dosing schedule known in the art can be used for administration of the adaptor compound(s), or the pharmaceutically acceptable salt thereof, the activity modifying compound, or the pharmaceutically acceptable salt thereof (e.g., the rejuvenating compound, or the pharmaceutically acceptable salt thereof, or the immunosuppressive compound, or the pharmaceutically acceptable salt thereof), or for the CAR-T cell composition.
• a.k.a qd twice per day dosing
• a.k.a. bid three times per day dosing
• a.k.a. tid twice per week dosing
• twice per week dosing a.k.a. BIW
• three times per week dosing a.k.a.
• the dosing schedule selected can take into consideration the concentration of the compounds/compositions being administered (including, for example, the number of CAR-T cells administered) to regulate the cytotoxicity of the CAR-T cell composition and to control any potential adverse effects (e.g., CRS).
• CRS any potential adverse effects
• a method of treating a cancer is provided using any of the compounds and compositions set forth herein.
• the cancer can additionally be imaged prior to administration to the subject of the adaptor compound(s), or the pharmaceutically acceptable salts thereof, or the CAR-T cell composition.
• imaging can occur by positron emission tomography (PET) imaging, magnetic resonance imaging (MRI), or single-photon-emission computed tomography ( S P EC T )/ comp uted tomography (CT) imaging.
• PET positron emission tomography
• MRI magnetic resonance imaging
• S P EC T single-photon-emission computed tomography
• CT comp uted tomography
• the imaging method can be any suitable imaging method known in the art.
• the imaging method can involve the use of the small molecule ligand described herein ( e.g . , of the adaptor compound or salt thereof), but linked to an imaging agent suitable for the types of imaging described herein.
• a method of modifying the activity of CAR-T cells comprises administering to a subject a composition comprising CAR-T cells comprising a CAR directed to a first targeting moiety, a second targeting moiety, or both the first and second targeting moieties; administering to the subject one or more adaptor compounds, or pharmaceutically acceptable salts thereof, each adaptor compound comprising a small molecule ligand linked to the first targeting moiety; and administering to the subject an activity modifying compound linked to the second targeting moiety.
• a method of modifying the activity of CAR-T cells and/or treating a cancer comprises administering to a subject a composition comprising a vector comprising a promoter operatively linked to a nucleic acid sequence encoding a CAR directed to a first targeting moiety, a second targeting moiety or both the first and second targeting moieties, wherein prior to, during, or after the administering step the subject received or receives a dose of one or more adaptor compounds, or pharmaceutically acceptable salts thereof, each adaptor compound comprising a small molecule ligand linked to the first targeting moiety. Additionally, in some embodiments, prior to, during, or after vector composition the administering step, the subject received or receives a dose of an activity modifying compound linked to the second targeting moiety.
• Such vector composition can comprise the vector (e.g., lentiviral particles comprising the vector) comprising the nucleic acid vector that encodes at least a CAR described herein.
• the vector composition comprises a therapeutically effective amount of the lentiviral particles according to any of the foregoing embodiments.
• the vector composition can be administered by any route, including oral, nasal, intravenous, intraarterial, intramuscularly, or intraperitoneal routes. In some cases, the vector composition is administered by intravenous injection or by intratumoral injection.
• Each of the one or more adaptor compounds, or pharmaceutically acceptable salts thereof, can be linked to the first targeting moiety via a first linker, and the activity modifying compound can be linked to the second targeting moiety via a second linker.
• the first and second linkers can comprise any of the linkers described herein and can have the same or different structures.
• the first and second targeting moieties can comprise any of the targeting moieties described herein and can have the same or different structures.
• the first and second targeting moieties have the same structure, while the first and second linkers have the same or different structures.
• the activity modifying compound can comprise any of the activity modifying compounds described herein including, for example, a rejuvenating compound or an immunosuppressive compound.
• Cancer has its plain and ordinary meaning when read in light of the specification and can include, but is not limited to, a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Numerous types of cancers can be treated using the compositions, compounds, and methods described herein including, without limitation, a carcinoma, a sarcoma, an osteosarcoma, a lymphoma, a melanoma, a mesothelioma, a nasopharyngeal carcinoma, a leukemia, an adenocarcinoma, and a myeloma.
• cancers that can be treated in accordance with the methods and/or using the compounds and compositions hereof include, but are not limited to, lung cancer (including, without limitation, non-small cell lung cancer), bone cancer (including, without limitation, osteosarcoma), pancreatic cancer, skin cancer (including, without limitation, cutaneous melanoma), cancer of the head, cancer of the neck, intraocular melanoma, uterine cancer, ovarian cancer, endometrial cancer, rectal cancer, stomach cancer, colon cancer, breast cancer, triple negative breast cancer, carcinoma of the fallopian tubes, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin’s Disease, cancer of the esophagus, 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, sarcoma of soft tissue, cancer of the urethra, prostate cancer, leukemia (including, without limitation, non-small cell lung cancer
• the cancer is a folate receptor expressing cancer, for example and without limitation, a folate receptor a-expressing cancer.
• the cancer is a folate receptor b-expressing cancer.
• the cancer is an endometrial cancer, a non-small cell lung cancer, an ovarian cancer, or a triple negative breast cancer.
• the cancer being treated can be a tumor.
• the cancer can be malignant.
• the cancer is acute myelocytic leukemia such as, for example, an acute myelocytic leukemia where the cancer expresses folate receptor-b.
• Certain embodiments of a method for modifying the activity of CAR-T cells and/or treating a cancer comprise: administering to a subject a composition comprising CAR-T cells, wherein the CAR-T cells comprise a CAR directed to a first targeting moiety, a second targeting moiety, or both the first and second targeting moieties; and administering to the subject one or more adaptor compounds, or pharmaceutically acceptable salts thereof, wherein each adaptor compound or pharmaceutically acceptable salt thereof comprises a small molecule ligand linked to the first targeting moiety via a first linker, wherein the first linker comprises a structure having the formula:
• the method can additionally comprise administering to the subject a rejuvenating compound, or a pharmaceutically acceptable salt thereof, linked to the second targeting moiety (e.g., via a second linker or directly).
• the rejuvenating compound, or the pharmaceutically acceptable salt thereof can, for example, be selected from a group comprising a TLR agonist (e.g., agonists of TLR1, TLR2, TLR3, TLR4, TLR7, TLR8, TLR7/8, TLR9, etc.), a STING agonist, aNLR, an ALR agonist, a kinase inhibitor targeting kinase, a RLR, a RAGE, a phosphatase inhibitor, and any other pattern recognition receptor that is located in the endosome or cytoplasm of the targeted cell.
• the second linker can comprise a structure having the formula: wherein n is an integer from 0 to 200.
• the rejuvenating compound, or the pharmaceutically acceptable salt thereof is a TLR agonist (e.g. , TLR 7, TLR7/8, or TLR8) having a structure of one of the following formulae:
• the rejuvenating compound or pharmaceutically acceptable salt thereof has the formula:
• the rejuvenating compound has a structure of one of the following formulae:
• Methods of treating a subject having received CAR-T cell therapy comprise administering to the subject one or more adaptor compounds, or pharmaceutically acceptable salts thereof, each adaptor compound or pharmaceutically acceptable salt thereof comprising a small molecule ligand linked to a first targeting moiety; wherein, prior to the administering step, the subject has received a dose of CAR- T cells expressing a CAR that recognizes and binds to the first targeting moiety.
• the method can further comprise administering to the subject an activity modifying compound linked to a second targeting moiety.
• the CAR-T cells can express one or more of the embodiments of CARs described herein (e.g., a CAR that recognizes and binds to the first targeting moiety, the second targeting moiety, or both the first and second targeting moieties).
• the one or more adaptor compounds, or pharmaceutically acceptable salts thereof, and the activity modifying compound of such methods can comprise any of the embodiments described herein.
• cytokine release resulting in off-target toxicity in the subject may not occur even though CAR-T cell toxicity to the cancer occurs.
• off-target tissue toxicity may not occur in the subject even though CAR-T cell toxicity to the cancer occurs.
• the cancer may comprise a tumor, and tumor size may be reduced in the subject, even though off-target toxicity does not occur.
• CRS can be reduced or prevented and the method can result in a decrease in tumor volume in the subject.
• body weight loss due to CRS can be reduced or prevented.
• the cancer can comprise a tumor and a complete response for the tumor can be obtained.
• all embodiments of the adaptor compound, or a pharmaceutically acceptable salt thereof, the activity modifying compound, the CAR-T cell compositions, and the vector compositions are applicable, including, but not limited to, the targeting moiety embodiments and the linker embodiments.
• connection or link between two components Words such as attached, linked, coupled, connected, and similar terms with their inflectional morphemes are used interchangeably, unless the difference is noted or made otherwise clear from the context. These words and expressions do not necessarily signify direct connections but include connections through mediate components. It should be noted that a connection between two components does not necessarily mean a direct, unimpeded connection, as a variety of other components may reside between the two components of note. Consequently, a connection does not necessarily mean a direct, unimpeded connection unless otherwise noted.
• the term “about,” when referring to a number or a numerical value or range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error) and thus the numerical value or range can vary between 1% and 15% of the stated number or numerical range (e.g., +/- 5 % to 15% of the recited value) provided that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result).
• a method of therapy comprises administering more than one treatment, compound, or composition to a subject
• the order, timing, number, concentration, and volume of the administration is limited only by the medical requirements and limitations of the treatment (i.e. two treatments can be administered to the subject, e.g., simultaneously, consecutively, sequentially, alternatively, or according to any other regimen).
• the disclosure may have presented a method and/or process as a particular sequence of steps. To the extent that the method or process does not rely on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. As one of ordinary skill in the art would appreciate, other sequences of steps may be possible. Therefore, the particular order of the steps disclosed herein should not be construed as limitations on the claims. In addition, the claims directed to a method and/or process should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the sequences may be varied and still remain within the spirit and scope of the present disclosure.
• the FITC-folate main peak typically eluted at 27-50 min.
• the quality of the FITC-folate fraction was monitored by analytical reverse-phase HPLC with a UV detector. Fractions with greater than 98.0% purity (LCMS) were lyophilized to obtain the final FITC-folate product.
• LCMS Chromatin
• Fmoc deprotection was carried out using 20% piperidine in DMF (3 x 10 mL). Kaiser tests were performed to assess reaction progress.
• the pure fractions were pooled and freeze-dried, providing the FITC-PEGl 2-Folate.
• Ethylenediamine, polymer-bound (200-400 mesh)-resin (50 mg) was loaded into a peptide synthesis vessel and swollen with DCM (3 mL) followed by DMF (3 mL).
• DCM 3 mL
• DMF 3 mL
• Fmoc-PEG2o-COOH solution 131 mg, 1.0 equiv
• i-PnNEt 6.0 equiv
• PyBOP 4.0 equiv
• Argon was bubbled for 6 h, the coupling solution was drained, and the resin was washed with DMF (3 x 10 mL) and i-PrOH (3 x 10 mL). Kaiser tests were performed to assess reaction progress.
• Fmoc deprotection was carried out using 20% piperidine in DMF (3 x 10 mL), before each amino acid coupling. The above sequence was repeated to complete the reaction with Fmoc-Glu-OtBu (72 mg, 2.0 equiv) and Tfa.Pteroic-acid (41 mg, 1.2 equiv) coupling steps.
• the resin was washed with 2% hydrazine in DMF 3 x 10 mL (5 min) to cleave the trifluoro- acetyl protecting group on pteroic acid and washed with i-PrOH (3 x 10 mL) followed by DMF (3 x lOmL). The resin was dried under argon for 30 min.
• the folate-peptide was cleaved from the resin using the Cleavage Solution. 10 mL of the cleavage mixture was introduced, and argon was bubbled for 1.5 h. The cleavage mixture was drained into a clean flask. The resin was washed 3 times with more cleavage mixture. The combined mixture was concentrated under reduced pressure to a smaller volume ( ⁇ 5 mL) and precipitated in ethyl ether.
• Ethylenediamine, polymer-bound (200-400 mesh)-resin (50 mg) was loaded in a peptide synthesis vessel and swollen with DCM (3 mL) followed by DMF (3 mL).
• DCM 3 mL
• DMF 3 mL
• Fmoc-PEG36-COOH solution 161 mg, 1.0 equiv
• i-PnNEt 6.0 equiv
• PyBOP 4.0 equiv
• Argon was bubbled for 6 h, the coupling solution was drained, and the resin was washed with DMF (3 x 10 mL) and i-PrOH (3 x 10 mL). Kaiser tests were performed to assess reaction progress.
• Fmoc deprotection was carried out using 20% piperidine in DMF (3 x 10 mL), before each amino acid coupling. The above sequence was repeated to complete reaction with 2X Fmoc-PEG36-COOH (161 mg, 1.0 equiv), Fmoc-Glu-OtBu (72 mg, 2.0 equiv ) and Tfa.Pteroic-acid ( 41.0 mg, 1.2 equiv) coupling steps.
• DUPA-FITC was synthesized by solid phase methodology as follows. Universal Nova TagTM resin (50 mg, 0.53 mM) was swollen with DCM (3 mL) followed by DMF 3 mL). A solution of 20% piperidine in DMF (3 x 3 mL) was added to the resin, and argon was bubbled for 5 min. The resin was washed with DMF (3 x 3 mL) and isopropyl alcohol (i-PrOH. 3 x 3 mL).
• the final compound was cleaved from the resin using the Cleavage Solution and concentrated under vacuum.
• the concentrated product was precipitated in diethyl ether and dried under vacuum.
• ACN was removed under vacuum, and purified fractions were freeze-dried to yield FITC-DUPA as a brownish-orange solid.
• Fmoc deprotection was carried out using 20% piperidine in DMF (3 x 10 mL). This procedure was repeated to complete the all coupling steps (2 x 1.5 equiv of Fmoc- Phe-OH and 1.5 equiv of 8-aminooctanoic acid and 1.2 equiv of DUPA were used on each of their respective coupling steps).
• the resin was washed with DMF (3 x 10 mL) and i-PrOH (3 x 10 mL) and dried under reduced pressure.
• the peptide was cleaved from the resin in the peptide synthesis vessel using the Cleavage Solution. 15 mL of the Cleavage Solution was added to the peptide synthesis vessel, and the reaction was bubbled under Ar for 15 min.
• the resin was treated with two additional 10 mL quantities of the Cleavage Solution for 5 min each.
• the cleavage mixture was concentrated to about 5 mL and precipitated with ethyl ether.
• the precipitate was collected by centrifugation, washed with ethyl ether (3X), and dried under high vacuum, resulting in the recovery of crude material.
• NK-1 (0.02 g, 0.0433 mmol, 1.0 eq.), O-(2-Aminoethyl)-0'-[2- (Boc-amino)ethyl]decaethylene glycol (BocNH-PEGn-ML) (Sigma, 0.0336 g, 0.0521 mmol, 1.2 eq.), Benzotriazol-l-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (0.027 g, 0.0521 mmol, 1.2 eq.) in dry CH2CI2 was added /V,/V-Diisopropylethylamine (DIPEA) (0.076 mL, 0.4338 mmol, 10 eq.) under argon at room temperature.
• DIPEA /V,/V-Diisopropylethylamine
• the pure fractions were collected, all organic solvents were evaporated and the sample was lyophilized for 48 h to provide the NK1- PEGn-NHBoc. Yield: 40.13 mg (97%).
• NKI-PEG11-NH2 (0.008 g, 0.0081 mmol, 1.0 eq.), Fluorescein isothiocyanate (FITC) (Sigma, 0.0037 g, 0.0097 mmol, 1.2 eq.) in dry dimethylsulfoxide (DMSO, 0.3 mL) was added to diisopropylethyl amine (0.0028 mL, 0.0162 mmol, 2.0 eq.) at room temperature under argon.
• DMSO dry dimethylsulfoxide
• the pure fractions were collected, all organic solvents were evaporated and the sample was lyophilized for 48 h to provide the FITC- PEG11-NK1 in a yield of 8.54 mg (77%).
• NK-1 compound was synthesized by a two-step procedure starting from the base ligand, which was prepared by using a procedure in the literature. (Ref: DESIGN AND DEVELOPMENT OF NEUROKININ- 1 RECEPTOR-BINDING AGENT DELIVERY CONJUGATES, Application Number: PCT/US2015/44229; incorporated herein by reference.
• CA9 ligand (53.6mg) was dissolved in DMF (2-3mL) in a 50mL round bottom flask using a Teflon magnetic stir bar. Ambient air was removed using a vacuum and replaced with nitrogen gas, this was done in three cycles. The round bottom flask was kept under constant nitrogen gas. To the flask, 28.9mg of N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) was added followed by 21.6mg 1-Hydroxybenzotriazole hydrate (HOBt) and 18.9 ⁇ L of Boc- PEG2-NH2 (Sigma Aldrich).
• EDC N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride
• PBMCs Human peripheral blood mononuclear cells
• PBMCs Human peripheral blood mononuclear cells
• GE Healthcare Lifesciences Ficoll density gradient centrifugation
• T cells were then isolated from PBMCs by using an EasySepTM Human T Cell Isolation Kit (STEM CELL technologies).
• T cells were cultured in TexMACS medium (Miltenyi Biotech Inc) with 40- 100 IU/mL human IL-2 (Miltenyi Biotech), 2% human AB type serum, and 1% penicillin/streptomycin sulfate.
• Dynabeads Human T-Activator CD3/CD28 (ThermoFisher Scientific) were added to T cells at 1:1 ratio to activate T cells.
• T cells were transduced with FITC-CAR lentiviral particles in the presence of 8 pg/mL polybrine (Santa Cruiz Biotech) by spinfection at 1,200 g for 90 minutes at 22-32 °C.
• T cell mixture containing those with CAR modification (CAR-Ts) and those without CAR modification (non-transformed Ts) was cultured in the presence of activation beads for 6 days before the removal of activation beads.
• Fluorescence-Activated Cell Sorting was used to sort out CAR-T cells (GFP positive) and non-transformed T cells (GFP negative) based on their GFP expression.
• the sorted T cells were cultured for 7-15 days before injection into mice. When a T cell mixture was used, CAR-T cells and non-transformed T cells were mixed at the desired ratio before mouse injection. The data shown in Figures 2-4E was obtained with T cells prepared with these procedures.
• the resulting CAR construct (1551bp) was inserted into EcoRI/Notl cleaved lentiviral expression vector pCDH-EFl-MCS-(PGK-GFP) ( Figure 1, System Biosciences).
• the sequence of the CAR construct in lentiviral vector was confirmed by DNA sequencing.
• the CAR construct used to generate the data for the Examples has the nucleic acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2.
• An exemplary CAR nucleic acid coding sequence can comprise SEQ ID NO: 1, wherein the first ATG is the start codon.
• An exemplary CAR amino acid sequence can comprise SEQ ID NO: 2.
• An exemplary insert can comprise SEQ ID NO: 3, wherein the first GCCACC sequence can comprise a restriction enzyme cleavage site, followed by the ATG start codon.
• the amino acid sequence encoded by SEQ ID NO: 1 or 3 can comprise SEQ ID NO: 2
• lentivirus containing CAR gene for human T cell transduction [0243] To prepare lentiviral virus containing an anti-fluorescein (i.e., anti-FITC) single chain fragment variable (scFv) CAR, a HEK-293TN packaging cell line was co-transfected with the lentiviral vector encoding anti-fluorescein scFv CAR and a 2nd generation of a lentiviral packaging plasmid mix (Cellecta) or ViraPower Lentivrial Packaging Mix (ThermoFisher). After 24 and 48 hours of transfection, supernatants containing the lentivirus with the CAR gene were harvested and virus particles were concentrated by the standard polyethylene glycol virus concentration method (Clontech) for future transduction with human T cells.
• an anti-fluorescein i.e., anti-FITC
• scFv single chain fragment variable
• T cells were isolated from human peripheral blood mononuclear cells (PBMC) by Ficoll density gradient centrifugation (GE Healthcare Lifesciences). After washing away remaining Ficoll solution, T cells were isolated by using an EasySepTM Human T Cell Isolation Kit (STEM CELL technologies). Purified T cells were cultured in TexMACSTM medium (Miltenyi Biotech Inc) with 1% penicillin and streptomycin sulfate in the presence of human IL-2 (100 IU/mL, Miltenyi Biotech Inc). T cells were cultured at density of lxl 0 6 cells/mL in multi-well plates. T cells were split and re-feed every 2-3 days.
• Isolated T cells were activated with Dynabeads coupled with anti-CD3/CD28 antibodies (Life Technologies) for 12-24 hours in the presence of human IL-2 (100 IU/mL), then transduced with lentivirus encoding an anti-fluorescein CAR gene. Cells were harvested after 72 hours and the expression of CAR on transduced T cells was identified by measuring GFP fluorescent cells using flow cytometry.
• exhausted CAR-T cells (10 4 /well) were generated by their continuous transfer (every 12 hours) to fresh MDA-MB-231 cells (10 4 /well) in presence of a FITC-folate adaptor compound while monitoring for appearance of exhaustion markers (PD-1 + Tim3 + LAG3 + ) and loss of CAR-T cell cytotoxicity.
• a FITC-folate adaptor compound monitoring for appearance of exhaustion markers (PD-1 + Tim3 + LAG3 + ) and loss of CAR-T cell cytotoxicity.
• the abilities of both nontargeted and FITC-targeted TLR7 agonists to reverse the CAR-T cell exhaustion were then documented.
• anti-FITC CAR-T cells (10 4 /well) were co cultured with MDA-MB-231 (10 4 /well) at 1:1 ratio in a 96 well plate, while fresh MDA-MB-231 cell (10 4 /well) were added every 12 h to the wells two times to exhaust the FITC-CAR-T cells.
• MDA-MB-231 10 4 /well
• mcherry+ MDA-MB-231; 10 4 /well
• the number of live mcherry+ (MDA-MB-231) cells were then counted by Incucyte S3 every 4 hours.
• the killing efficacy was calculated by dividing the numbers of cells at 16 h/numbers of cell at 8h and multiplying xl00%.
• mice For the in vivo model, more particularly, 8-10-week-old NSG mice (strain No. 005557) from Jackson Lab were used. All of the NSG mice were maintained on a folic acid-deficient diet (TD.95247, Envigo) in order to reduce the level of folic acid in mice to physiological levels found in humans. NSG mice were then implanted with 1.5 ⁇ 10 6 KB cells into the flank. Once tumors reached around 30-50 mm 3 , all the mice were divided into two groups: a non-CAR-T cell treatment group and a CAR-T cell treatment group. The CAR-T cell treatment group received 1 x 10 7 anti-FITC-CAR-T cells by i.v. injection.
• TD.95247 folic acid-deficient diet
• the CAR-T cell treatment group mice were given 500 nmol/kg of FITC-folic acid adaptor compound.
• the mice in the TLR7 treatment group received 10 nmol/mouse of FITC-TLR7 two times per week.
• Control groups received an equal volume of 100 pi of saline vehicle two times/week. All of the CAR-T cell treatment group mice continued to be given 500 nmol/kg of FITC-folic acid once a week. Tumor volume was measured unblinded with a caliper and was calculated using the formula (a ⁇ b 2 )/2 (a being the largest and b being the smallest diameter of the tumor).
• FIG. 2B shows the results of an assay where MDA-MB-231 cells were co-cultured with anti- FITC CAR-T cells and FITC-folic acid adaptor compound, and fresh MDA-MB-231 cells were added to the co-culture every 12 hours 3 times consecutively.
• the results in Figure 2B shows that these CAR-T cells became exhausted after stimulation 2 times by MDA-MB-231 cells in vitro, indicated by the decreased killing efficacy, and increased exhaustion markers expression (PD- 1+Tim3+Lag3+).
• FIG. 4 shows the rejuvenation effect of the FITC-TLR7 agonist conjugate in the KB xenograft model, as indicated by the decreased tumor size (Figure 4A) and decreased exhaustion marker expression (PD-1+Tim3+) ( Figures 4B and 4C).
• Figures 4D and 4E shows the change in the percentage of CAR T cells and change in the mice body weight during treatment.

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