Methods for enhancing engineered cell therapy in cancer treatment - Patents.com

Abstract

Methods are provided for reprogramming M2-like macrophages to M1-like macrophages, which reverse the pro-inflammatory to anti-inflammatory shift observed during the course of certain cancers, and are co-administered with one or more types of engineered cells, including, but not limited to, CAR T cells, engineered natural killer cells, engineered stem cells, etc. The compounds include immunomodulators that target cellular pattern recognition receptors and are specific to the cells of interest due to the incorporation of a targeting moiety (e.g., folate, or a functional fragment or analog thereof). Releasable and / or non-releasable linkers can be included and engineered to facilitate optimal delivery of the immunomodulator. The compounds and compositions can be used in one or more methods of cancer treatment.

Description

[Technical field]

[0001] Priority This patent application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 133,773, filed January 4, 2021, the contents of which are incorporated by reference in their entirety into this disclosure.

[0002] The present disclosure relates to methods of using one or more compounds that contain a targeting moiety and that reprogram M2-type macrophages to M1-type macrophages in combination with chimeric antigen receptor T cell and other engineered cell therapies. [Background technology]

[0003] Chimeric antigen receptor (CAR) is a recombinant receptor that performs both antigen binding and T cell activation functions, and has great potential for treating cancer due to its tumor-specific activation and killing. An exemplary second generation CAR consists of a single chain variable fragment (scFv) derived from an antibody for targeting, a CD3 zeta chain for activation, a single cytoplasmic domain of a costimulatory receptor such as CD28 or 4-1BB, and a hinge and transmembrane domain.

[0004] Although the success of CAR-T therapy in treating hematopoietic tissue cancers has been impressive, it has not been proven that CAR-T therapy can have a similar effect on patients with solid tumors. The activity and survival of CAR-T cells in the tumor microenvironment (TME) are regulated by multiple immunosuppressive cells, including tumor-associated macrophages (TAMs), myeloid-derived immunosuppressive cells (MDSCs), cancer-associated fibroblasts (CAFs), tumor-associated neutrophils (TANs), and regulatory T cells (Tregs).

[0005] One of the major challenges in killing solid tumors is often posed by TAMs, well-known immune cells in the TME. TAMs, which account for up to 50% of solid tumor mass, interact with cancer cells and other immune cells to promote tumor growth by promoting angiogenesis, immunosuppression and inflammation. To enhance the performance of CAR-T cells in solid tumors, it is essential to convert TAMs in the TME from tumor-supporting to tumor-killing.

[0006] Stem cells from different sources exhibit different abilities of proliferation, migration and differentiation, which determines their application in antitumor therapy. Various strategies have been developed for cancer treatment using stem cell-based therapy, including hematopoietic stem cell (HSC) transplantation, mesenchymal stem cell (MSC) injection for post-cancer treatment, stem cells for therapeutic carriers, generation of immune effector cells and vaccine generation. Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can be used for adoptive cell transfer technology to generate effector immune cells that are later constructed into CARs. Furthermore, ESC and iPSC can be potential sources for manufacturing anticancer vaccines. Furthermore, exosomes extracted from drug-primed MSC and neuronal stem cell (NSC) cultures can be used to target drugs to tumor sites. Stem cell-based therapy can improve the therapeutic efficacy of other therapies due to its enhanced targeting on tumors, thereby reducing off-target events.

[0007] Furthermore, cancer is often treated by chemotherapy, which utilizes highly potent drugs such as mitomycin, paclitaxel and camptothecin. In many cases, these chemotherapeutic agents show a dose-response effect, and tumor inhibition is proportional to the amount of drug administered. Thus, aggressive dosing regimens are used to treat neoplasms. However, high-dose chemotherapy is hindered by poor selectivity for cancer cells and toxicity to normal cells. Lack of tumor specificity is one of the many obstacles that conventional chemotherapy must overcome.

[0008] Despite the clear need for cancer prevention and treatment, cancer continues to be a major cause of death and / or morbidity worldwide, as there are currently no effective therapeutic options that can cure the condition. Furthermore, when drugs or other therapies are available, such treatments usually use very potent drugs that have poor selectivity for the cancer cells of interest and therefore pose the risk of systemic toxicity in the potential subject. What is needed is a treatment that is not only effective in disrupting the cycle of growth-promoting factors initiated by activated M2-type (alternatively, activated) macrophages, but can do so with very high specificity for the cancer cells in question. Summary of the Invention

[0009] In view of the above, it is an object of the present disclosure to provide materials and methods for rendering TAMs in the TME tumoricidal, which are highly specific for cancer cells in the TME. These and other objects and advantages, as well as features of the invention, will be apparent from the detailed description of the invention presented herein.

[0010] Combination cancer therapies are provided that combine the use of engineered cells (e.g., CAR T cells, stem cells, etc.) with drug compounds or compositions that include a folate receptor binding ligand and a Toll-like receptor (TLR) agonist.

[0011] In at least one exemplary embodiment, a method of treating a patient suffering from (or suffering from) cancer is provided. The method includes administering a combination cancer therapy to the patient, whereupon the patient's cancer is treated. Such combination cancer therapy can include, for example, administering a first treatment to the patient and administering a second treatment to the patient. In certain embodiments, the first treatment includes at least one small molecule drug conjugate (SMDC) including (i) a drug moiety (e.g., an immunomodulator) conjugated to (ii) a ligand (e.g., a targeting moiety, such as a folate ligand, or a functional fragment or analog thereof), wherein the ligand can be bound by a cell-surface receptor of an immunosuppressive cell or a cell-surface receptor of a cancerous cell. The first and second treatments can be administered simultaneously, sequentially, consecutively, or alternatingly.

[0012] In certain embodiments, the first treatment comprises a compound comprising a folate ligand, or a functional fragment or analog thereof, linked to a TLR agonist via a linker. In some examples, the TLR agonist can be a TLR7 agonist, a TLR8 agonist, a TLR9 agonist, or a TLR7 / 8 agonist.

[0013] The second treatment can include engineered cells or engineered cell therapy. For example, the second treatment can include chimeric antigen receptor (CAR)-expressing cytotoxic lymphocytes. The lymphocytes can be autologous, or alternatively, the lymphocytes can be heterologous. In certain embodiments, the engineered cells are engineered natural killer (NK) cells, or NK cells prepared from progenitor or stem cells. The combination can include a first amount of the first treatment and a second amount of the second treatment, which together are effective for treating cancer.

[0014] In some instances, activated M2 phenotype macrophages play a role in cancer, such as by secreting anti-inflammatory cytokines that activate fibroblasts and synthesize collagen and other extracellular matrix proteins. In certain instances, these macrophages also cause the release of problematic growth factors in subjects experiencing cancer. For example, such growth factors can promote the growth of cancerous tumors. In addition, in some instances, macrophages release (e.g., simultaneously) immunosuppressive cytokines. Thus, macrophages can play an important role in promoting the establishment and growth of cancer.

[0015] In some cases, activated macrophages derived from tissue-resident macrophages or peripheral blood monocytes induce fibroblast activation through secretion of chemokine (CC motif) ligand 18 (CCL18), transforming growth factor-β1 (TGFβ1) and / or platelet-derived growth factor (PDGF). This activation, in some cases, can promote collagen secretion by fibroblasts, which can lead to the progression of fibroblast-associated cancers. In the later stages of many cancers, activated macrophages and myofibroblasts can cross-stimulate each other, promoting the growth of cancerous tumors (e.g., by growth factors secreted by activated macrophages, anti-inflammatory responses and / or collagen formation in cancerous tumors (e.g., by downstream fibrous collagen production that can result in cancerous tumors that block drug penetration into the tumor, making treatment more difficult)).

[0016] In some embodiments, the compound represented by formula QLT is provided herein. In some embodiments, Q is a radical of a folate receptor binding ligand. In some embodiments, L is a linker. In some embodiments, T is a radical of a TLR agonist. In some embodiments, QLT is a pharmaceutically acceptable salt thereof.

[0017] In some embodiments, the linker is a non-releasable linker. In some embodiments, the non-releasable linker has the formula:

[0018] [ka] It is represented by:

[0019] In some embodiments, n is 1 to 30. In some embodiments, n is 1 to 24. In some embodiments, n is 1 to 12. In some embodiments, n is 1 to 3. In some embodiments, n is 12. In some embodiments, n is 3.

[0020] In some embodiments, w is 0 to 5. In some embodiments, w is 0 to 2. In some embodiments, w is 1.

[0021] In some embodiments, the TLR agonist of the first treatment compound has (or is represented by) the structure (or a radical thereof) of formula 2-I, or a pharma- ceutically acceptable salt of formula 2-I:

[0022] [ka] and In formula 2-I, R 1 , R 3 , R 4 and R 5 are each independently hydrogen (H), alkyl, alkoxy, alkenyl, alkynyl, alicyclic, aryl, biaryl, halo, heteroaryl, -COR 2x ,

[0023] [ka] and R 2 are H, -OH, -NH2, -NHR 2x, N3, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2,

[0024] [ka] and Y is H, -OH, -NH2, -NHR 2x , -OR 2X , -SO-R 2x , -SH, -SO3H, -N3, -CHO, -COOH, -CONH2, -COSH, -COR 2x , -SO2NH2, alkenyl, alkynyl, alkoxyl, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2,

[0025] [ka] and Where: R 2x and R 2y are each independently selected from the group consisting of H, -OH, -CH-OH, -NH, -CH-NH, -COOMe, -COOH, -CONH, -COCH, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl; R 2z are -NH2 and -NR 2q R 2q’ , -OR 2q , -SO-R 2q and -COR 2q R 2q and R 2q’ are each independently alkyl or H;

[0026] [ka] is a non-aromatic, monocyclic or bicyclic 3-10 membered N-containing heterocycle; In formula 2-I, X 1 , X 2 and X 3are each independently q or N and R q each is independently H, halogen, or optionally substituted alkyl; In formula 2-I, n is an integer of 0 to 30, and m is an integer of 0 to 4.

[0027] In certain exemplary embodiments, the first treatment compound is:

[0028] [ka] or a pharma- ceutically acceptable salt thereof.

[0029] As previously described, in some embodiments, the TLR agonist of the first treatment compound is a Toll-like receptor 7 (TLR7) agonist. In some embodiments, the radical of the TLR agonist has the structure represented by formula X:

[0030] [ka] has.

[0031] In some embodiments, R1 is -NH2 or -NH-R 1X In some embodiments, R2 is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X ,

[0032] [ka] In some embodiments, R 1X , R 2X and R 2Y is each independently selected from the group consisting of hydrogen (H), alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl.

[0033] [ka] is a 3-10 membered nitrogen (N) containing non-aromatic monocyclic or bicyclic heterocycle.

[0034] In some embodiments of formula X, R3 is -OH, -SH, -NH2, or -NH-R 1X In some embodiments of formula X, R1 is -NH2 or -NH-R 1X and R2 is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X ,

[0035] [ka] and R 1X , R 2X and R 2Y are each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl;

[0036] [ka] is a 3-10 membered N-containing non-aromatic monocyclic or bicyclic heterocycle, and R3 is -OH, -SH, -NH2 or -NH-R 1X It is.

[0037] In some embodiments, the radical of the TLR agonist has the structure represented by formula XX:

[0038] [ka] has.

[0039] In some embodiments, R1 is -NH2 or -NH-R 1XIn some embodiments, R2 is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X ,

[0040] [ka] In some embodiments, R 1X , R 2X and R 2Y is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl.

[0041] [ka] is a 3-10 membered N-containing non-aromatic monocyclic or bicyclic heterocycle. In some embodiments, X is CH, CR2 or N. In some embodiments, R1 is -NH2 or -NH-R 1X and R2 is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X ,

[0042] [ka] and R 1X , R 2X and R 2Y are each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl;

[0043] [ka] is a 3-10 membered N-containing non-aromatic monocyclic or bicyclic heterocycle, and X is CH, CR2 or N.

[0044] In some embodiments, the first compound of the first treatment comprises a linker L between the targeting moiety and the immune modulator or a pharma- ceutically acceptable salt thereof. n and further comprising a linker L n is configured to avoid release of the TLR7 agonist in free form, and n is an integer less than or equal to 50. In some embodiments, the linker L n comprises polyethylene glycol (PEG) or a PEG derivative, n is an integer selected from the range of 1 to 32, and the radical of the folate receptor binding ligand is a folate receptor β (FBβ) binding ligand.

[0045] In some embodiments, the compound of the first treatment is

[0046] [ka] The compound has a structure represented by:

[0047] In some embodiments, the compound of the first treatment is

[0048] [ka] The compound has a structure represented by:

[0049] In some embodiments, the compound of the first treatment is

[0050] [ka] The compound has a structure represented by:

[0051] In some embodiments, the compound of the first treatment is

[0052] [ka] The compound has a structure represented by:

[0053] In some embodiments, provided herein are pharmaceutical compositions comprising one or more compounds of the present disclosure, wherein the TLR7 agonist has a structure represented by formula XX:

[0054] In certain examples, provided herein are methods of treating a subject suffering from cancer, comprising contacting cells of the subject with at least one compound, including a compound described herein, wherein the immune modulator comprises an agonist of TLR7, 8, 9 or 7 / 8.

[0055] In some embodiments, a compound comprising a folate ligand, or a functional fragment or analog thereof, linked to a TLR agonist via a linker, wherein the TLR agonist has the following formula:

[0056] [ka] Provided herein are compounds having the formula:

[0057] In some embodiments, R 1 is an amine group, R 2 is a single bond, -NH-, and R 3 is H, alkyl, hydroxyl or any other substituted group thereof, X is CH, NH, oxygen (O) or sulfur (S), and the linker is R 1 、 R 2 or R 3 are bonded at

[0058] In some embodiments, there is provided a pharmaceutical composition comprising a compound of any one of the formulae provided herein, wherein the linker comprises a PEG linker or a PEG derivative linker, R 3 or R 1 、 R 2Or R 3 and a releasable linker attached at

[0059] In some embodiments, the pharma- ceutically acceptable salt is selected from hydrobromide, citrate, trifluoroacetate, ascorbate, hydrochloride, tartrate, trifluoromethanesulfonate, maleate, mesylate, formate, acetate, or fumarate.

[0060] In certain embodiments, administering the compound of the first treatment activates anti-tumor or pro-inflammatory signaling cascades in the subject.

[0061] In some embodiments, the present invention provides a method for preventing or treating cancer, comprising contacting a cell with at least one compound (e.g., any compound provided by the formula provided herein) comprising an immunomodulator or a pharma- ceutically acceptable salt thereof linked to a folate ligand, or a functional fragment or analog thereof, via a linker, wherein the immunomodulator or a pharma- ceutically acceptable salt thereof targets a pattern recognition receptor.In some embodiments, the cell comprises a cell of a subject experiencing cancer or a subject at risk of experiencing cancer, and contacting the cell with at least one compound further comprises administering or applying a therapeutically effective amount of at least one compound to the subject.In some embodiments, the subject is a patient experiencing cancer, and at least one compound is administered to the subject intravenously, intramuscularly, intraperitoneally, topically, or by inhalation.

[0062] In another embodiment, a method for treating a subject suffering from cancer includes administering a first treatment to the subject, the first treatment comprising a compound comprising a folate ligand, or a functional fragment or analog thereof, linked to a TLR agonist (e.g., an immunomodulator) via a linker (as described herein), and administering a second treatment to the subject, the second treatment comprising an engineered cell (e.g., configured to treat cancer). The TLR agonist may be an agonist of Toll-like receptor 7, 8, 9 or 7 / 8. In a further embodiment, the second treatment is a CAR T cell therapy or an engineered cell therapy, or a combination thereof. The first and second treatments may be administered simultaneously, sequentially, consecutively, or alternatingly.

[0063] In some embodiments, a method for preventing or treating a disease state is provided herein, comprising contacting a cell with at least one engineered cell configured to treat the disease state, and contacting the cell with at least one compound (e.g., any compound provided by the formula provided herein) comprising an immunomodulator or a pharma- ceutically acceptable salt thereof linked to a folate ligand, or a functional fragment or analog thereof, via a linker, wherein the immunomodulator or a pharma- ceutically acceptable salt thereof targets a pattern recognition receptor. In some embodiments, the cell comprises a cell of a subject experiencing a cancerous disease state or a subject at risk of experiencing a cancerous disease state, and contacting the cell with at least one compound further comprises administering or applying a therapeutically effective amount of at least one compound to the subject, and contacting the cell with at least one engineered cell further comprises administering or applying a therapeutically effective amount of the engineered cell to the subject. In some embodiments, the subject is a patient experiencing cancer, and the at least one compound and the at least one engineered cell are administered to the subject intravenously, intramuscularly, intraperitoneally, topically, or by inhalation. In some embodiments, the engineered cells are CAR T cells, engineered T cells, T cells prepared from progenitor or stem cells, engineered NK cells, NK cells prepared from progenitor or stem cells, engineered stem cells, or any combination of the above.

[0064] In at least one embodiment of the method of the present disclosure, administering at least one compound of the first treatment, when performed as a first treatment, reprograms the subject's M2-type macrophages to M1-type macrophages and enhances the efficacy of at least one engineered cell of the second treatment compared to the baseline efficacy of the at least one engineered cell. In certain embodiments, administering and / or contacting the cell with at least one compound comprising an immune modulator or a pharma- ceutically acceptable salt thereof activates anti-tumor cells or pro-inflammatory signaling cascades in the subject. In certain embodiments, the anti-tumor cells are T cells, engineered T cells, or T cells prepared from progenitor or stem cells (e.g., at least one engineered cell configured to treat a disease state).

[0065] In some embodiments, the method further comprises obtaining or having obtained a sample from the subject, and quantifying the expression level of one or more biomarkers in the sample. In at least one embodiment of the method of the present disclosure, the one or more biomarkers are each selected from the group consisting of CCL18, arginase 1 (Arg1), matrix metallopeptidase 9 (MMP9), metalloproteinase 3 (TIMP3), interleukin 1 beta (IL-1 beta), hydroxyproline, collagen, PDGF, TGF beta, folate receptor beta (FR beta), tumor necrosis F-alpha (TNF alpha), interferon gamma (IFN-gamma), mannose receptor (CD206), cluster of differentiation 163 (CD163), cluster of differentiation 86 (CD86), interleukin 6 (IL-6), chemokine 10 (CXCL10) and immune interferon (IFN alpha). In at least one embodiment of the method of the present disclosure, the biological sample is obtained from a volume of peripheral blood drawn from the subject. In at least one embodiment of the method of the present disclosure, the quantification step is performed using a method selected from the group consisting of qPCR, mass spectrometry, ELISA, and another modality capable of measuring or quantifying the expression of the biomarker.

[0066] In at least one embodiment of the disclosed method, the method further comprises comparing the expression level of each of the one or more biomarkers with the expression level of such biomarkers in a control, the control being a healthy individual or an individual not experiencing cancer. In at least one embodiment, the method may further comprise administering or having administered to the subject a therapeutically effective amount of an unconjugated agonist or inhibitor and the engineered cells if CCL18, Arg1, MMP9, TIMP3, IL-1β, PDGF, TGFβ, FRβ, CD206, CD163, hydroxyproline or collagen is upregulated relative to the expression level of the control, or if TNFα, IFN-γ, IL-6, CXCL10, IFNα or CD86 is downregulated or not expressed relative to the expression level of the control.

[0067] In some embodiments, the folate ligand, or a functional fragment or analog thereof, is specific for and binds to cellular FRβ.

[0068] In at least one embodiment of the methods of the disclosure, the immune modulator, or a pharma- ceutically acceptable salt thereof, comprises a Toll-like receptor (TLR) 7, 8, 9 or 7 / 8 agonist.

[0069] In at least one embodiment of the disclosed method, at least one compound (e.g., of the first treatment) has the following formula:

[0070] [ka] has.

[0071] In at least one embodiment of the methods of the disclosure, the immune modulator comprises a TLR agonist having a structure of Formula X or Formula XX, or a pharma- ceutically acceptable salt of Formula X or Formula XX:

[0072] [ka] and In formulas X and XX, R1 is -NH2 or -NH-R 1X and R2 is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X ,

[0073] [ka] and

[0074] [ka] is a 3-10 membered N-containing non-aromatic monocyclic or bicyclic heterocycle, in which R3 is -OH, -SH, -NH2 or -NH-R 1X In the formula XX, X is CH or N, and R 1X , R 2X and R 2Y is each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl.

[0075] In at least one embodiment of the disclosed method, the compound / immunomodulator comprises:

[0076] [ka] or a pharma- ceutically acceptable salt thereof, 1 , R 3 , R 4 and R 5 are each independently hydrogen (H), alkyl, alkoxy, alkenyl, alkynyl, alicyclic, aryl, biaryl, halo, heteroaryl, -COR 2x ,

[0077] [ka] and R 2 are H, -OH, -NH2, -NHR 2x , N3, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2,

[0078] [ka] and Y is H, -OH, -NH2, -NHR 2x , -OR 2X , -SO-R 2x , -SH, -SO3H, -N3, -CHO, -COOH, -CONH2, -COSH, -COR 2x , -SO2NH2, alkenyl, alkynyl, alkoxyl, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2,

[0079] [ka] and R 2x and R 2y are each independently selected from the group consisting of H, -OH, -CH-OH, -NH, -CH-NH, -COOMe, -COOH, -CONH, -COCH, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl; R 2z are -NH2 and -NR 2q R 2q’ , -OR 2q , -SO-R 2q and -COR 2q R 2q and R 2q’ are each independently alkyl or H;

[0080] [ka] is a non-aromatic, monocyclic or bicyclic 3-10 membered N-containing heterocycle, and in formula 2-I, X 1 , X 2 and X3 are each independently q or N and R q is each independently H, halogen, or optionally substituted alkyl, and in formula 2-I, n is 0-30, and m is 0-4.

[0081] In at least one embodiment of the method of the present disclosure, the subject is experiencing or at risk of experiencing cancer, and the step of administering the first treatment further comprises administering or applying to the subject a therapeutically effective amount of at least one compound. In certain embodiments, the cancer is a solid tumor cancer.

[0082] In at least one embodiment of the methods of the present disclosure, at least one compound of the first treatment is administered to the subject intravenously, intramuscularly, intraperitoneally, topically, or by inhalation.

[0083] In at least one embodiment of the methods of the present disclosure, the subject's M2-type macrophages include myeloid-derived immunosuppressive cells (MDSCs), tumor-associated macrophages (TAMs), or both MDSCs and TAMs.

[0084] In at least one embodiment of the methods of the present disclosure, at least one compound of the first treatment comprises a composition containing one or more pharma- ceutically acceptable carriers, adjuvants, diluents, excipients and / or vehicles, or combinations thereof.

[0085] In at least one embodiment of the disclosed method, the subject is a human, a mouse, or any other mammal.

[0086] In at least one embodiment of the methods of the disclosure, the immune modulator, or a pharma- ceutically acceptable salt thereof, is a TLR agonist having the formula:

[0087] [ka] Including, In the formula, R 1 is an amine group, R 2 is a single bond, -NH-, and R 3 is H, alkyl, hydroxy or any other substituted group thereof, X is CH, NH, O or S, and the linker is R 1 、 R 2 or R 3 are bonded at

[0088] In at least one embodiment of the methods of the present disclosure, the linker of at least one compound of the first treatment comprises a PEG linker or a PEG derivative linker and is a non-releasable linker.

[0089] In at least one embodiment of the method of the present disclosure, the first and second treatments are administered simultaneously, sequentially, consecutively or alternatingly.

[0090] In some embodiments, a method for preventing or treating a disease state is provided herein, comprising contacting a cell with at least one engineered cell configured to treat the disease state, and contacting the cell with at least one compound (e.g., any compound provided by the formula provided herein) comprising an immunomodulator or a pharma- ceutically acceptable salt thereof linked to a folate ligand, or a functional fragment or analog thereof, via a linker, wherein the immunomodulator or a pharma- ceutically acceptable salt thereof targets a pattern recognition receptor. In some embodiments, the cell comprises a cell of a subject experiencing a cancerous disease state or a subject at risk of experiencing a cancerous disease state, and contacting the cell with at least one compound further comprises administering or applying a therapeutically effective amount of at least one compound to the subject, and contacting the cell with at least one engineered cell further comprises administering or applying a therapeutically effective amount of the engineered cell to the subject. In some embodiments, the subject is a patient experiencing cancer, and the at least one compound and the at least one engineered cell are administered to the subject intravenously, intramuscularly, intraperitoneally, topically, or by inhalation. In some embodiments, the engineered cells are CAR T cells, engineered stem cells, or a combination of the two. In at least some embodiments, contacting the cells of the subject with at least one compound comprising an immunomodulator or a pharma- ceutically acceptable salt thereof reprograms M2-type macrophages of the subject to M1-type macrophages.

[0091] In some embodiments, the folate ligand, or a functional fragment or analog thereof, is specific for and binds to cellular FRβ.

[0092] In some embodiments, provided herein is a method of treating a subject experiencing a cancerous disease state (e.g., cancer) comprising enhancing the efficacy of one or more engineered cell therapies administered to the subject by administering a second treatment comprising one or more compounds comprising a targeting moiety (e.g., a folate ligand, or a functional fragment or analog thereof) linked via a linker to an immune modulator or a pharma- ceutically acceptable salt thereof (e.g., any of the TLR agonists of the present disclosure, including, but not limited to, TLR7, 8, 9, or 7 / 8 agonists), wherein the targeting moiety targets a pattern recognition receptor of the cell. In certain embodiments, contacting the subject's cells with one or more compounds of the second treatment reprograms the subject's M2-type macrophages to M1-type macrophages. In at least one exemplary embodiment of this method, the immune modulator or a pharma- ceutically acceptable salt thereof of the second treatment is a TLR7 agonist and the linker is a releasable linker. In at least one additional embodiment, the linker is a non-releasable linker.

[0093] In some embodiments of the method of the present disclosure, administering at least one compound of the second treatment activates anti-tumor cells or proinflammatory signaling cascades in the subject.In at least one embodiment, such anti-tumor cells are T cells, natural killer (NK) cells, engineered NK cells, or NK cells prepared from progenitor cells or stem cells.In addition or alternatively, such anti-tumor cells are macrophages.

[0094] In some embodiments, provided herein are compounds comprising a targeting moiety linked to an immune modulator, or a pharma- ceutically acceptable salt thereof, that targets a cellular pattern recognition receptor, wherein the targeting moiety comprises a folate ligand, or a functional fragment or analog thereof.

[0095] The disclosed embodiments and other features, advantages and aspects contained herein, and materials for obtaining same, will become apparent in light of the following detailed description of various exemplary embodiments of the present disclosure, which will be better understood in conjunction with the accompanying drawings. [Brief description of the drawings]

[0096] [Figure 1-1] FIG. 1A shows the chemical structures of exemplary compounds having a targeting moiety (a folate receptor ligand) attached to an immune modulator (a Toll-like receptor 7 (TLR7) agonist radical) via a non-releasable linker (e.g., comprising a polyethylene glycol (PEG) backbone moiety). [Figure 1-2] FIG. 1B shows the chemical structure of an exemplary compound having a targeting moiety (a folate receptor ligand) linked to an immune modulator (a TLR7 agonist radical) via a releasable linker (e.g., comprising a disulfide moiety in its backbone), and an exemplary drug release mechanism. [Figure 1-3] FIG. 1C shows chemical structures of exemplary compounds provided herein. [Diagram 2] FIG. 2 depicts an exemplary flow chart of a method of treating a subject experiencing or at risk of experiencing a fibrotic disease or cancer. [Figure 3-1]Figures 3A-3F show graphical data of various marker levels measured from human M2 macrophages when contacted with an exemplary free (non-targeted) or an exemplary targeted (e.g., via a folate receptor binding ligand) TLR7 agonist at various concentrations of each compound. The data shown in Figures 3A-3C confirm that administration of either the non-targeted or targeted TLR7 agonist successfully reprogrammed M2 macrophages into M1 macrophages (i.e., down-regulated M2 anti-inflammatory macrophages), and the data shown in Figures 3D-3F confirm that administration of the test compound up-regulated M1 macrophages. Each value represents the mean ± standard deviation for each group; #P<0.05, ##P<0.01, ###P<0.005, ####P<0.0001; treated groups vs. M2 untreated group by Dunnett's multiple comparison test. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 4-1] Figures 4A-4E and 5A-5D show representative graphical data of various marker levels measured from M2 macrophages incubated with various concentrations of exemplary free or targeted TLR7 agonists for 2 hours (Figures 4A-4E) or 46 hours (Figures 5A-5D). Figures 4A-4E and 5A-5D confirm that the M2 anti-inflammatory phenotype was down-regulated after administration of free and targeted TLR7 agonists. Each value represents the mean ± standard deviation for each group; #P<0.05, ##P<0.01, ###P<0.005, ####P<0.0001; Compound 1A and Compound 1B treated groups vs. M2 untreated group in Figures 4A-5D by Dunnett's multiple comparison test. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 5-1]Figures 4A-4E and 5A-5D show representative graphical data of various marker levels measured from M2 macrophages incubated with various concentrations of exemplary free or targeted TLR7 agonists for 2 hours (Figures 4A-4E) or 46 hours (Figures 5A-5D). Figures 4A-4E and 5A-5D confirm that the M2 anti-inflammatory phenotype was down-regulated after administration of free and targeted TLR7 agonists. Each value represents the mean ± standard deviation for each group; #P<0.05, ##P<0.01, ###P<0.005, ####P<0.0001; Compound 1A and Compound 1B treated groups vs. M2 untreated group in Figures 4A-5D by Dunnett's multiple comparison test. [Figure 5-2] Same as above. [Figure 6-1] 6A-6D show representative graphical data of various marker levels measured from M2 macrophages (i) treated with various concentrations of exemplary free and targeted TLR7 agonists for 48 hours (FIGS. 6A and 6B), or (ii) treated with various concentrations of exemplary free and targeted TLR7 agonists for 2 hours, then replaced with fresh medium and cultured for the remaining 46 hours (FIGS. 6C and 6D). Values ​​represent the mean ± standard deviation for each group; #P<0.05, ##P<0.01, ###P<0.005, ####P<0.0001; Compound 1A and Compound 1B treated groups vs. M2 untreated group by Dunnett's multiple comparison test. [Figure 6-2] Same as above. [Figure 6-3] FIG. 6E shows flow cytometry data confirming that THP-1 (a human monocytic cell line derived from an acute monocytic leukemia patient)-induced macrophages were folate receptor beta (FRβ)-positive (FRβ+). [Figure 6-4] FIG. 6F shows that exemplary targeted TLR7 agonists are stable. [Figure 7-1]FIG. 7A shows post-staining images of lungs taken from mice with bleomycin (BM)-induced experimental fibrosis and stained using anti-mouse FRβ antibody, and hematoxylin-eosin (H&E) staining was performed on days 7, 14, and 21 after BM-induced lung injury. [Figure 7-2] FIG. 7B shows quantification of FRβ staining in the panel of FIG. 7A. [Figure 7-3] 7C and 7D show FRβ immunohistochemistry (IHC) staining of human idiopathic pulmonary fibrosis (IPF) lung tissue (FIG. 7C) and healthy human lung tissue (FIG. 7D). [Figure 7-4] FIG. 7E shows images of mouse tissues / organs taken from mice with BM or (phosphate buffered saline (PBS) control) without BM-induced experimental fibrosis and imaged with a folate receptor-targeted fluorescent dye. [Figure 7-5] FIG. 7F shows fluorescence-activated cell sorter (FACS) analysis of mice with BM-induced experimental fibrosis. [Figure 7-6] Same as above. [Figure 7-7] Same as above. [Figure 8-1] FIG. 8A illustrates the treatment regimen of free and targeted TLR7 agonists in a BM model. [Figure 8-2] 8B-8G show anti-inflammatory (FIGS. 8B-8D) and pro-inflammatory (FIGS. 8E-8G) marker levels measured from mice treated with the BM model of FIG. 8A. [Figure 8-3] Same as above. [Figure 8-4] Same as above. [Figure 8-5] FIG. 8H shows cell counts in bronchoalveolar lavage fluid (BALF) from mice treated with the BM model of FIG. 8A. [Figure 9-1] 9 and 9B show survival curves (FIG. 9A) and body weight changes (FIG. 9B) of mice with pulmonary fibrosis treated with non-targeted and targeted TLR7 drugs. [Figure 9-2] Same as above. [Figure 10-1] FIG. 10A shows hydroxyproline content (μg / lung) of lung tissue as a measure of fibrosis. [Figure 10-2] 10B and 10C show the lung tissue of FIG. 9A by H&E staining (FIG. 10B) and Masson's Trichrome (collagen) staining (FIG. 10C). [Figure 10-3] Same as above. [Figure 11-1] 11A and 11B show the survival curve (FIG. 11A) and body weight change (FIG. 11B) of mice with pulmonary fibrosis treated with an exemplary targeted TLR7 agonist, with each value representing the mean ± standard deviation of each group. [Figure 11-2] Same as above. [Figure 12-1] 12A shows the dose-dependent effect of an exemplary targeted TLR7 agonist of the present disclosure on suppressing fibrosis in BM-induced mice.FIG 12A shows graphical data on the body weight of BM-induced mice over time. [Figure 12-2] FIG. 12B shows measurements of hydroxyproline content in lung tissue treated with various doses of an exemplary conjugate provided herein (eg, Compound 1B). [Figure 12-3] Figure 12C shows images of histological analysis of lung tissues by various staining. Each value represents the mean ± standard deviation of each group; *P<0.05, **P<0.005, ***<0.0005; saline vs. vehicle group, treatment vs. vehicle group by Student's t-test. [Figure 13-1] 13A-13D show various marker levels measured from M2 macrophages reprogrammed according to the methods of the present disclosure with various concentrations of an exemplary targeted TLR7 agonist for 48 hours, with each value representing the mean ± standard deviation for each group. [Figure 13-2] Same as above. [Figure 14-1]14A-14C show various marker levels measured from M2 macrophages reprogrammed according to the methods of the present disclosure with various concentrations of exemplary free and targeted TLR7 agonists. Values ​​shown in FIG. 14A-14C represent the mean ± standard deviation of each group; #P<0.05, ##P<0.005, ###P<0.0005, ####P<0.0001; Compound 3A, Compound 3B and Compound 3C treated groups vs. M2 untreated group by Dunnett's multiple comparison test. [Figure 14-2] Same as above. [Figure 14-3] Same as above. [Figure 15] FIG. 15 shows secreted chemokine (CC motif) ligand 18 (CCL18) protein levels in each group of cells from FIGS. 14A-14C following treatment with exemplary free and targeted TLR7 agonists. [Figure 16] FIG. 16 illustrates the method in the BM mouse model. [Figure 17-1] 17A and 17B show the purity of exemplary targeted TLR7 agonists provided herein. [Figure 17-2] Same as above. [Figure 18-1] 18A-18F show data from the in vivo study method of FIG. 16, including survival curves (FIG. 18A), body weight change (FIGS. 18B and 18D), concentration of cells with BALF present (FIG. 18C), and hydroxyproline concentration (μg HP / leaf) in surviving mice (FIG. 18E) and in all mice (i.e., including both surviving mice and mice that died before day 21) (FIG. 18F). [Figure 18-2] Same as above. [Figure 18-3] Same as above. [Figure 18-4] Same as above. [Figure 19-1]Figure 19: Both targeted and non-targeted TLR7 agonists reprogram human monocyte-derived anti-inflammatory macrophages to a pro-inflammatory phenotype (Figures 19A-19F). Mean ± SD. Statistical significance between groups was determined using unpaired two-tailed t-test (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). [Figure 19-2] Same as above. [Figure 19-3] Same as above. [Figure 20-1] FIG. 20A-20F show a comparison of plasma cytokine levels following treatment with Compound 1A versus Compound 1B in healthy mice. [Figure 20-2] Same as above. [Figure 20-3] Same as above. [Figure 20-4] Figure 20G shows the change in body weight after treatment of mice with exemplary compounds provided herein, including the change in body weight as a measure of systemic toxicity during alternate day dosing (n=2); Mean ± standard deviation. Statistical significance between groups was compared using unpaired two-tailed t-test (*P<0.05, **P<0.01, ***P<0.001). [Figure 21] FIG. 21 shows healthy and fibrotic lungs as described in FIG. 6 stained with 4′,6-diamidino-2-phenylindole (DAPI) (nuclei; blue), anti-F4 / 80 (macrophages; red) and anti-mannose receptor (CD206). [Figure 22] FIG. 22 shows the effect of various exemplary compounds on interleukin 6 (IL-6) expression in peripheral blood mononuclear cells. [Figure 23-1] 23A and 23B show the in vitro effects of various exemplary compounds on IL-6 and C-X-C motif chemokine 10 (CXCL-10) induction in monocyte-derived M2 macrophages over a 48 hour period. [Figure 23-2] 23C and 23D show the in vivo effects of various exemplary compounds on IL-6 and tumor necrosis factor alpha (TNF-α) production. [Figure 24]Figures 24A-24F show the expression of TLR7 in 4T1, CT26 and EMT6 cells. Cells were fixed, permeabilized and stained with anti-mouse TLR7-PE antibody. Figure 24A shows the negative control for 4T1 cells, while Figure 24B shows the negative control for CT26 cells and Figure 24C shows the negative control for EMT6 cells. Figure 24D shows the results of staining 4T1 cells with anti-mouse TLR7-PE antibody, while Figure 24E shows the results of staining CT26 cells with anti-mouse TLR7-PE antibody and Figure 24F shows the results of staining EMT6 cells with anti-mouse TLR7-PE antibody. [Figure 25-1] Figures 25A-25C are graphs of CD19 versus percent maximum (Max), showing CD19 expression in 4T1, CT26 and EMT6 cells. Figure 25A shows the overlay of stained 4T1-mCD19-F7 cells (i.e., transduced cells labeled with anti-CD19-PE) and unstained 4T1-mCD19-F7 cells, while Figure 25B shows the overlay of stained CT26-mCD19 cells (i.e., transduced cells labeled with anti-CD19-PE) and unstained CT26-mCD19 cells. [Figure 25-2] FIG. 25C shows the overlay of stained EMT6-mCD19-C10 cells (i.e., transduced cells labeled with anti-CD19-PE) and unstained EMT6-mCD19-C10 cells. [Figure 26] Figures 26A-26C are plots of anti-mouse CD19 CAR vs. SSC-A (10^3) ​​showing expression of anti-mouse CD19 scFv in transduced mouse T cells as measured by flow cytometry using anti-rat-Alexa594 antibody for staining. Figure 26A shows the results of staining non-transduced mouse T cells (negative control), while Figure 26B shows the results of staining single transduced mouse T cells and Figure 24C shows the results of staining double transduced mouse T cells. [Figure 27]Figure 27 is a graph of cells vs. % cytotoxicity against mouse CD19+ cancer cells, which shows the results of an assay to determine whether anti-mouse CD19 CAR-T cells are cytotoxic to mouse CD19+ cancer cells. [Figure 28] FIG. 28 is a graph of tumor size (mm3) versus days after the first FA-TLR7A-1A injection, showing the change in tumor size obtained with treatment with CAR-T cells alone (CAR-T), or CAR-T cells in combination with a non-releasable folate-TLR7A agonist (CAR-T+FA-TLR7A) compared to control (untreated). [Figure 29] FIG. 29 is a graph of % body weight change versus days after tumor implantation, showing the percent body weight change obtained with treatment with CAR-T cells or a combination of CAR-T cells and non-releasable folate-TLR7A agonist (CAR-T+FA-TLR7A) compared to control (untreated). [Diagram 30] Figure 30A is a graph of iNOS+ / Arginase 1+ in treatment vs. F4 / 80+, which shows the M1 / M2 (iNOS+ / Arginase-1+) macrophage ratio in tumors after treatment with CAR-T cells alone or in combination with a non-releasable folate-TLR7A agonist compared to untreated. Figure 30B is a graph of F4 / 80+% in treatment vs. tumors, which shows the percentage of total macrophages in tumors after treatment with CAR-T cells alone or in combination with a non-releasable folate-TLR7A agonist compared to untreated. [Diagram 31] FIG. 31 is a graph of %CD11b+Gr-1+ cells in tumor versus treatment, showing the percentage of total bone marrow-derived stem cells (MDSC) in tumors after treatment with CAR-T cells alone (CAR-T), or CAR-T cells in combination with a non-releasable folate-TLR7 agonist, compared to untreated. [Diagram 32]Figure 32A is a graph of % CD3+ T cells in tumor versus treatment, showing the percentage of total T cells in tumors after treatment with CAR-T cells alone (CAR-T), or CAR-T cells in combination with a non-releasable folate-TLR7A agonist (CAR-T+FA-TLR7A) compared to no treatment. Figure 32B is a graph of % CAR-T cells in tumor versus treatment, showing the percentage of CAR-T cells in tumors after treatment with CAR-T cells alone (CAR-T), or CAR-T cells in combination with a non-releasable folate-TLR7A agonist (CAR-T+FA-TLR7A) compared to no treatment. [Diagram 33] Figure 33A is a graph of %CD25+T cells in tumor versus treatment, showing the percentage of CD25+T cells in tumors after treatment with CAR-T cells alone (CAR-T), or CAR-T cells in combination with a non-releasable folate-TLR7A agonist (CAR-T+FA-TLR7A) compared to no treatment. Figure 33B is a graph of %CD25+CAR-T cells in tumor versus treatment, showing the percentage of CD25+CAR-T cells in tumors after treatment with CAR-T cells alone (CAR-T), or CAR-T cells in combination with a non-releasable folate-TLR7A agonist (CAR-T+FA-TLR7A) compared to no treatment. [Diagram 34] Figure 34A is a graph of % CD69+ T cells in tumor versus treatment, showing the percentage of CD69+ T cells in tumors after treatment with CAR-T cells alone (CAR-T), or CAR-T cells in combination with a non-releasable folate-TLR7A agonist (CAR-T+FA-TLR7A) compared to no treatment. Figure 34B is a graph of % CD69+ CAR-T cells in tumor versus treatment, showing the percentage of CD69+ CAR-T cells in tumors after treatment with CAR-T cells alone (CAR-T), or CAR-T cells in combination with a non-releasable folate-TLR7A agonist (CAR-T+FA-TLR7A) compared to no treatment. [Diagram 35] FIG. 35 shows the effect of various exemplary compounds on interleukin 6 (IL-6) expression in peripheral blood mononuclear cells. [Figure 36-1] 36A and 36B show the in vitro effects of various exemplary compounds on IL-6 and C-X-C motif chemokine 10 (CXCL-10) induction in monocyte-derived M2 macrophages over a 48 hour period. [Figure 36-2] 36C and 36D show the in vivo effects of various exemplary compounds on IL-6 and tumor necrosis factor alpha (TNF-α) production. [Figure 37] FIG. 37 shows exemplary structures of releasable (FA-PEG3-(R)TLR7-1A) and non-releasable (FA-PEG3-(NR)TLR7-1A) forms of the folate-TLR7 agonist. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0097] While the present disclosure is susceptible to various modifications and alternative forms, exemplary embodiments thereof have been shown by way of example in the drawings and are herein described in detail.

[0098] In order to promote understanding of the principles of the present disclosure, reference will now be made to the embodiments illustrated in the drawings, and specific phrases will be used to describe them. It will nevertheless be understood that the description of these embodiments is not intended to limit the scope. On the contrary, the present disclosure is intended to embrace alternatives, modifications, and equivalents that may be included within the spirit and scope of the present application as defined by the appended claims. As previously stated, the present technology may be illustrated and described in one or more preferred embodiments, but the compositions, compounds, and methods thereof may include many different configurations, forms, materials, and auxiliary materials.

[0099] All patents, patent application publications, journal articles, textbooks, and other publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All such publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0100] In the following description, numerous specific details are set forth to provide a thorough understanding of the present disclosure. It should be understood that certain examples can be practiced without some or all of these specific details, and that the present disclosure is not limited to a particular biological system, a particular cancer, or a particular organ or tissue, which may, of course, vary, but remain applicable in light of the data presented herein.

[0101] Various techniques and mechanisms of the present disclosure sometimes describe a connection or association between two components. Words such as coupled, associated, connected, connected, and similar terms with their inflected morphemes are used interchangeably unless a distinction is specified or otherwise made clear from the context. These words and expressions do not necessarily imply a direct relationship, but include a connection through an intermediate component. It should be noted that a relationship between two components does not necessarily imply a direct, unobstructed relationship, since various other components may exist between the two components of interest. Thus, a relationship does not necessarily imply a direct, unobstructed relationship, unless otherwise specified.

[0102] Moreover, wherever feasible and convenient, the same reference numbers are used in the drawings and description to refer to the same or similar parts or steps. The drawings are in simplified form and are not to scale. It is understood that the present disclosure is thus presented for illustrative purposes only, and the principles and embodiments described herein may be applied to components of compounds and / or compositions having configurations other than those specifically described herein. Indeed, it is expressly contemplated that the components of the compositions and compounds of the present disclosure may be adjusted in the promotion of their desired applications.

[0103] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art of chemistry and biology. Although any methods and materials similar or equivalent to those described herein can be used to practice or test the subject matter of this application, preferred methods and materials are described herein. Furthermore, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, if a compound / composition is substituted with "one" alkyl or aryl, the compound / composition is optionally substituted with at least one alkyl and / or at least one aryl.

[0104] When ranges relating to physical properties such as molecular weight or chemical properties such as chemical formulas are used herein, all combinations and subcombinations of ranges and specific embodiments herein are intended to be included. The term "about," when referring to a number or numerical range, means that the number or numerical range referred to is an approximation within experimental variation (or within statistical experimental error), and thus the number or numerical range may vary between 1% and 15% of the stated number or numerical range. The term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") is not intended to exclude that in certain other embodiments, for example, any compositions, compositions, methods or processes of matter described herein, the embodiments may "consist of" or "consist essentially of" the described features.

[0105] In certain embodiments, the compounds and / or compositions provided herein are also useful for preventing and / or treating cancer. In some embodiments, the compounds, compositions and methods provided herein utilize strategies to target (e.g., selectively) the innate immune system and reprogram macrophage polarization from M2 to M1, and to exploit, for example, its anti-cancer properties. In some embodiments, the compounds include Toll-like receptor (TLR) 7 and / or 8 agonists. In certain embodiments, the compounds provided herein are provided or used alone, in conjunction with a targeting agent, and / or in combination therapy with other interventions, such as, for example, engineered cell therapy, which are described in more detail below. In some embodiments, administration of the compounds provided herein enhances the efficacy / potency of a second treatment (e.g., engineered cell or engineered cell therapy) administered to the subject by reprogramming and / or activating proinflammatory signaling cascades in the subject.

[0106] In general, the methods and compounds, and combinations thereof, use at least one small molecule drug conjugate (SMDC) comprising a drug moiety (e.g., agonist) conjugated to a ligand. In certain embodiments, the ligand specifically binds to a cell-surface receptor on folate receptor beta (FRβ)-expressing myeloid cells, which in tumor-bearing mammals are primarily immunosuppressive and located almost exclusively within the tumor microenvironment (TME). Upon uptake by a target cell, the drug moiety of the SMDC can bind to a TLR, initiate signaling events, and reprogram the cell to a more immunostimulatory phenotype (e.g., M1-like). Administration of the SMDC can be further combined with administration of an engineered cell therapy (e.g., chimeric antigen receptor (CAR)-expressing cytotoxic lymphocytes, T cells prepared from progenitor cells or stem cells, etc.), resulting in enhanced efficacy of the engineered cell therapy with little or no observed off-target toxicity.

[0107] Thus, the present combinations, compounds and methods provide cancer prevention and treatment that are not only effective against solid tumors, but can selectively target agonists (i.e., immunomodulators) to receptors on tumor-associated macrophages (TAMs) and / or myeloid-derived immunosuppressive cells (MDSCs) within cancerous tumors, thus avoiding systemic and / or off-target toxicity. Furthermore, immunomodulators / TLR agonists can modify certain properties of other infiltrating immune cells, including engineered cells (e.g., CAR T cells, other engineered T cells, engineered natural killer (NK) cells, etc.) and normal T cells, thereby significantly enhancing the efficacy of the combined engineered cell therapy.

[0108] The term "off-target toxicity" refers to organ or tissue damage, or loss of body weight in a subject that is undesirable to the physician or other individual treating the subject, or any other effect on the subject that is a potential adverse indicator to the treating physician (e.g., B-cell aplasia, fever, decreased blood pressure, or pulmonary edema). The terms "treat", "treating", "treated" or "treatment" (with respect to a disease or condition) are preferably an approach for obtaining beneficial or desired results, including clinical results, and may include, but are not limited to, one or more of the following: amelioration of conditions associated with the disease, curing the disease, reducing the severity of the disease, improving the quality of life of a person suffering from the disease, prolonging survival, and / or prophylactic or preventative treatment. In particular, with reference to cancer, the terms "treat," "treating," "treated," or "treatment" can further mean the reduction in tumor size, complete or partial removal of a tumor (e.g., complete or partial response), induction of stable disease, prevention of cancer progression (e.g., progression-free survival), or any other effect on cancer that is considered by a physician to be a therapeutic, prophylactic, or preventative treatment of the cancer.

[0109] As used herein, engineered cell therapy can include various immunotherapies based on biologically engineered cells, including, but not limited to, CAR therapy. "CAR therapy" refers to cytotoxic lymphocyte cells (e.g., T cells or NK cells) or populations thereof modified by molecular biology methods to express CAR on the cell surface. CAR is a polypeptide with a predefined binding specificity for a desired target, operably linked (e.g., as separate chains linked by fusion, one or more disulfide bonds, etc.) to the intracellular portion of a cell activation domain. By bypassing MHC class I and class II restrictions, CAR-engineered lymphocyte cells of both CD8+ and CD4+ subsets can be recruited for redirected target cell recognition. While CAR T cell therapy is well known, it will be understood that CAR-based cell therapy can also be used with NK cells (e.g., CAR-NK therapy).

[0110] The CAR comprises a recognition region, which is further defined herein. In certain embodiments, the CAR can further comprise an activation signaling domain, which can be derived from, for example, the T cell CD3-zeta (CD3ζ) chain, the Fc receptor gamma signaling domain, or the Fc receptor gamma, or one or more costimulatory domains, such as CD28, CD137 (4-1BB), CD278 (ICOS), or CD134 (OX40).

[0111] Certain CARs are fusions of binding functions (e.g., as single chain variable fragments (scFv) derived from monoclonal antibodies) to CD3 zeta transmembrane domains and endodomains. Such molecules result in the transmission of zeta signals in response to recognition by the recognition receptor binding functions of their targets. However, numerous alternatives exist. As a non-limiting example, the antigen recognition domains derived from the innate T cell receptor (TCR) alpha and beta single chains can be used as binding functions. Alternatively, receptor ectodomains (e.g., CD4 ectodomains) can be used. All that is required for the binding function is that it can bind to a given target in a specific manner with high affinity.

[0112] In particular, engineered cell therapy is not limited to CAR therapy.In fact, various types of immune cells (such as T cells and NK cells) can be reprogrammed with enhanced survival and functional activity as known in the art.Engineered cell therapy using engineered T cells, T cells prepared from progenitor cells or stem cells, engineered NK cells, or NK cells prepared from progenitor cells or stem cells can also be used in the combination method provided herein.

[0113] Additionally, "specifically binds," "binds with high affinity," or "specifically" or "selectively," when referring to a ligand / receptor, recognition region / targeting moiety, nucleic acid / complementary nucleic acid, antibody / antigen, or other binding pair, refers to a binding reaction that is determinative of the presence of a protein in a heterogeneous population of proteins and other biologics. Thus, under specified conditions, a specified ligand or recognition region binds to a particular receptor (e.g., one present on the surface of a cancer cell) or targeting moiety, respectively, and does not bind in significant amounts 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 a binding compound, ligand, antibody, or binding composition derived from an antigen-binding site of an antibody of the contemplated methods binds to its target with an affinity that is often at least 25% higher, more often at least 50% higher, most often at least 100% (2-fold) higher, usually at least 10-fold higher, more usually at least 20-fold higher, and most usually at least 100-fold higher than the affinity of any other binding compound.

[0114] In typical embodiments, a molecule that specifically binds to a target has a binding affinity of at least about 10, e.g., as determined by Scatchard analysis. 6 Liter / mol (K 'O =10 ~6 M) and preferably has an affinity of at least about 10 liters / mol. It will be appreciated by those skilled in the art that some binding compounds can specifically bind to more than one target, for example, an antibody will specifically bind to its antigen, to a lectin via the antibody's oligosaccharides, and / or to an Fc receptor via the antibody's Fc region.

[0115] The combination, compound and method will now be described in detail.In order to promote understanding of the principles presented herein, reference will be made to the embodiments illustrated in the drawings, and specific terms will be used to describe them.Nevertheless, it will be understood that the description of these embodiments is not intended to limit the scope.On the contrary, the present disclosure is intended to encompass alternatives, modifications and equivalents that may be included within the spirit and scope of the present application as defined by the appended claims.

[0116] As specified above, the combinations, compounds and methods use at least one SMDC in combination with the administration of engineered cells or engineered cell therapy. In general, and without any intent of limitation, the novel compounds, compositions and methods of the present disclosure target the innate immune system of a subject and reprogram the polarization of macrophages from M2 type to M1 type in favor of the proinflammatory properties of the M1 type phenotype. For example, in at least one exemplary embodiment, such compounds and compositions include a targeting moiety that targets FRβ, such as a folate receptor binding ligand, or an analog, functional fragment, derivative or radical thereof (e.g., pteroyl amino acid), linked to an immunomodulator or a pharma-ceutically acceptable salt thereof. As described in detail below, such an embodiment takes advantage of the restricted expression of FRβ to localize a systemically administered compound directly to FRβ-expressing cells (e.g., those in cancerous tissues), such that the immunomodulator component can then convert, e.g., reprogram, activated myeloid cells (e.g., M2-like macrophages) to a proinflammatory M1 polarization. This targeted design advantageously prevents systemic activation of the immune system (ie, reduces systemic exposure to such compounds), thus avoiding toxicity.

[0117] Further exemplary embodiments may include a linker disposed between the targeting moiety and the immunomodulator. Such linkers may be releasable or non-releasable. As described herein, compounds / compositions of the present disclosure that include a releasable linker, when administered, release the targeting moiety and the immunomodulator from each other at or about the time that the immunomodulator becomes active. Additionally or alternatively, in embodiments where compounds / compositions of the present disclosure include a non-releasable linker, when administered, the targeting moiety and the immunomodulator do not release rapidly under physiological conditions. Thus, the components remain together after uptake by the target cell and / or after activation of the immunomodulator.

[0118] Primarily, there are two main immune strategies found in vertebrates: the innate immune system and the adaptive immune system. The innate or non-specific immune response is the first line of defense against non-self pathogens and consists of physical, chemical and cellular defenses. On the other hand, the adaptive immune system is initiated against pathogens that have escaped or overcome the primary innate immune defense.

[0119] Inflammatory response plays an important role in immunity.When tissue is damaged or pathogen is detected, for example, inflammatory response is initiated and immune system is mobilized.Immune cells of innate immune system (i.e., neutrophils and eosinophils) are first mobilized to the site of tissue injury or damage or the location of pathogen through blood vessel and lymphatic system, then by macrophage.

[0120] Cells of the innate immune system can express specialized pattern recognition receptors that detect and bind to specific protein sequences present on microbial pathogens or other non-self molecules. As used herein, "pattern recognition receptor" refers to and includes any immune receptor expressed on the membrane surface of white blood cells, e.g., at least macrophages, that can bind to a specific ligand that activates the receptor, ultimately leading to an innate immune response (and, in some cases, ultimately the development of antigen-specific adaptive immunity).

[0121] Two examples of classes of molecules that can bind to pattern recognition receptors include pathogen-associated molecular patterns associated with microbial pathogens, and damage-associated molecular patterns associated with host cell components released during cell damage or death. Recognition of these protein sequences by pattern recognition receptors can initiate signaling pathways that trigger the expression of certain genes whose products control innate immune responses (e.g., in some cases direct the development of antigen-specific adaptive immunity). Thus, pattern recognition receptors mediate these signaling pathways, and in some cases can be used to positively or negatively control innate and even adaptive immune responses.

[0122] Macrophages are a diverse group of leukocytes known to eliminate pathogens by phagocytosis and are broadly classified as having either an M1 or M2 phenotype, depending on the specific differentiation they undergo in response to the local tissue environment. In some instances, macrophages are polarized toward the M1 phenotype upon exposure to interferon gamma (IFN-γ), lipopolysaccharide (LPS) and / or granulocyte-macrophage colony-stimulating factor (GM-CSF). In certain instances, the M1 phenotype is characterized by the production of high levels of proinflammatory cytokines (such as interleukin 1β (IL-1β), tumor necrosis factor (TNF), interleukin 12 (IL-12), interleukin 18 (IL-18) and / or interleukin 23 (IL-23)), the ability to mediate resistance to pathogens, potent bactericidal properties, advanced production of reactive nitrogen and oxygen intermediates, and / or promotion of T helper type 1 (Th1) responses. In some instances, M1 polarization is associated with the "attack and kill" phase of the innate immune response. In certain instances, M1 polarization acts to inhibit or prevent the initial establishment of an infection and / or to eliminate damaged tissue.

[0123] In certain instances, after the innate immune system has performed this "attack and kill" phase, macrophages can reprogram themselves to become healing (i.e., M2 type) and release, for example, growth factors to promote healing. Such growth factors can include (but are not limited to) certain cytokines such as interleukin 4 (IL-4), interleukin 10 (IL-10), platelet-derived growth factor (PDGF), transforming growth factor-β1 (TGFβ), chemokine (CC motif) ligand 18 (CCL18), and / or interleukin 13 (IL-13). In certain instances, exposure to such cytokines / growth factors alternatively activates the M2 macrophage phenotype.

[0124] In contrast to M1, M2 macrophages can be associated with wound healing and tissue repair. In some cases, M2 macrophages are characterized by their involvement in tissue remodeling, immune regulation / suppression and / or tumor promotion. In certain cases, M2 macrophages produce polyamines to induce cell proliferation and / or proline to induce collagen production. While this healing response is beneficial for healthy subjects, the presence of M2 macrophages may have a significant detrimental effect on subjects suffering from cancer, by suppressing immunity and / or promoting tumor growth and fibrosis.

[0125] Chemokines and other factors can be released to promote the infiltration of immune cells into damaged tissues (e.g., innate immune response), including, for example, monocytes and macrophages that are considered to be of M2-like phenotype, releasing, for example, anti-inflammatory cytokines. In turn, chronic secretion of these cytokines activates tissue-resident and infiltrating fibroblasts / fibrocytes to become myofibroblasts, which in turn secrete collagen and other extracellular matrix proteins that can harden the surrounding tissue. In some cases, these M2 macrophages exacerbate the disease by promoting fibrosis. In some cases, growth factors and other cytokines produced by the M2 phenotype drive the growth of cancerous tumors by similar pathways.

[0126] Reprogramming M2-like macrophages into M1-like macrophages In certain cancers, macrophages can be disproportionately biased toward an anti-inflammatory (M2-like) phenotype. In certain instances, immune modulators can convert, e.g., reprogram, activated myeloid cells (e.g., M2-like macrophages and / or anti-tumor cells) to a pro-inflammatory M1 polarization (e.g., where macrophages produce little or no growth factors and / or related cytokines, and, e.g., slow down or even eliminate the progression of the disease state (i.e., cancer). In certain instances, the compositions and methods provided herein reverse the pro-inflammatory to anti-inflammatory shift observed during the developmental course of certain cancers. In some embodiments, the compositions and methods provided herein reduce the amount / expression of cancer biomarkers (e.g., those associated with anti-inflammatory activity (e.g., CCL18, hydroxyproline and collagen)) in a sample taken from an individual or subject, which is indicative of macrophage conversion to the M1 phenotype and thus initiation of anti-tumor cell activation (e.g., T cells, NK cells and / or macrophages) and pro-inflammatory signaling cascades. An "individual," "subject," or "patient," as used herein, is a mammal, preferably a human, but may be an animal.

[0127] A "marker" or "biomarker", as the term is used herein, can be described as being differentially expressed if the level of expression in a subject experiencing an active disease state is significantly different from the expression level in a sample taken from the subject, or from a healthy subject, or from a subject not experiencing the disease state. A differentially expressed marker can be overexpressed or underexpressed (in the embodiment mentioned in the immediately preceding paragraph, the biomarker is decreased or underexpressed) compared to the expression level in a normal or control sample, or the baseline of the subject. The increase or decrease or quantification of a marker in a biological sample can be determined by any of several methods known in the art for measuring the presence and / or relative abundance of a gene product or transcript. The level of a marker can be determined as an absolute value, or relative to a baseline value, and the level of the marker in the subject compared to a cutoff index. Alternatively, the relative abundance of a marker(s) can be determined compared to a control, which can be a clinically normal subject. Additionally, as used herein, the terms "gene overexpression" and "overexpression" (when used in reference to a gene) and their terminological elements have the meanings ascribed to them by those of skill in the art, which include (without limitation) the overexpression or misexpression of a wild-type gene product, which may result in a mutant phenotype and / or result in increased target protein expression.

[0128] In some embodiments, compositions and methods provided herein increase pro-inflammatory biomarkers (e.g., TNFα and IFN-γ). In some embodiments, compositions are provided that reverse the M2-like phenotype shift (e.g., resulting in the realization of effective treatment of cancer).

[0129] The administration of immune modulators is combined with the administration of engineered cells to prevent the inactivation of such engineered cells in the TME observed when using conventional techniques. In certain embodiments, the immune modulators target immunosuppressive cells (and / or cancerous cells) in tumors and deliver drug moieties to the target cells, thereby increasing the infiltration and activity of engineered cells in the TME while also avoiding systemic toxicity. The administration of immune modulators together with engineered cell therapy results in superior cytotoxicity against cancer cells in solid tumors than engineered cell therapy alone.

[0130] Thus, a combination method of treating cancer is provided. In certain embodiments, the method comprises (a) administering a first compound comprising a drug moiety (e.g., a TLR agonist) conjugated to a ligand (e.g., a targeting moiety) that can be bound by a cell-surface receptor of an immunosuppressive cell or a cell-surface receptor of a cancerous cell, and (b) an engineered cell, the combination comprising a first amount of (a) and a second amount of (b), which together are therapeutically effective for treating cancer. The various components of the embodiment of the first treatment (i.e., the immunomodulator compound) will now be described in more detail.

[0131] Immune Modulators / Drug Moieties In at least one embodiment, a drug comprising an immunomodulator is used to make the compound used in the methods described herein.As used herein, "immunomodulator" refers to any drug, warhead or other composition or compound that stimulates or otherwise affects the immune system of a subject by inducing the activation of one or more components of the immune system or by increasing their activity.For example and without limitation, an immunomodulator can include a compound or composition that targets one or more pattern recognition receptors in addition to or instead of targeting signaling pathways in immune cells.

[0132] Illustrative examples of immune modulators of the present disclosure include, but are not limited to, agonists of TLRs, stimulators of interferon genes (STING), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), absent in melanoma 2 (AIM2)-like receptors (ALRs), receptor for advanced glycation end products (RAGE), or any other pattern recognition receptors located in the endosomes or cytoplasm of cells. The immune modulators of the present disclosure may additionally or alternatively include nuclear factor kappa light chain enhancer of activated B cells (NFκβ) activators or Iκβ kinase inhibitors, which act further downstream in the pathway. Table 1 provides examples of such NFκβ activators or Iκβ kinase inhibitors that may be used as immune modulators of the present disclosure.

[0133] [Table 1-1]

[0134] [Table 1-2]

[0135] "Toll-like receptors" or "TLRs" are a class of proteins that play a role in the innate immune system and are examples of pattern recognition receptors. TLRs can be single transmembrane receptors that recognize structurally conserved molecules derived from microorganisms. TLRs can be expressed on the membrane surface of white blood cells, including, for example, dendritic cells, macrophages, natural killer cells, cells of adaptive immunity (e.g., T and B lymphocytes) and non-immune cells (epithelial cells and endothelial cells and fibroblasts). Non-limiting examples of TLRs include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 and TLR13. In some embodiments, the TLR agonists provided herein bind to one or more TLRs. In some embodiments, the TLR agonists provided herein bind to TLR7, TLR8 or TLR9. In some embodiments, the TLR agonists provided herein bind to TLR7. In some embodiments, the TLR agonists provided herein bind to TLR7 and TLR8. In some embodiments, the agonist is a ligand that binds to and activates the receptor.

[0136] In some cases, such as when the compounds provided herein are (e.g., potent) TLR-7 / 8 agonists, the unconjugated compounds provided herein are highly toxic when delivered systemically. In some cases, it is desirable to reduce and / or eliminate the systemic toxicity associated with such compounds. In some cases, the conjugate radicals of the compounds provided herein have reduced toxicity (e.g., at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, or at least 90%) compared to the free forms of such compounds. Furthermore, in some cases, the compounds provided herein (conjugates) have reduced toxicity at comparable or lower concentrations (e.g., 120% or less, 100% or less, 80% or less, 60% or less, or 40% or less of the effective dose (ED) of the free form) compared to the free forms of the compounds. 50 ) with a median concentration.

[0137] Any therapeutic agent (e.g., drug) suitable for reprogramming activated macrophages (M2-like phenotype) to M1-like phenotype can be used, and the drug moiety (or warhead) can act in the endosomes and / or cytoplasm (e.g., depending on its structure) of the cell. In at least one embodiment, the therapeutic agent comprises an immune modulator (e.g., one that positively regulates a pattern recognition receptor and / or its downstream signaling pathway (in each case, part of the innate immune system) (e.g., TLR, NLR, RLR, ALR, RAGE and / or STING agonists, etc.), and / or kinases of the Pelle / Interleukin-1 Receptor Associated Kinase (IRAK) family, such as IRAK-M inhibitors). Furthermore, in some embodiments, the therapeutic agent comprises at least one small molecule drug conjugate (SMDC) that comprises a TLR7 agonist. In other embodiments, the compounds provided herein comprise phosphoinositide 3-kinase (PI3K) kinase inhibitors or other inhibitors that negatively regulate the adaptive immune system (e.g., it may be used alone or in conjunction with an immunomodulator that targets a pattern recognition receptor). In some embodiments used for the treatment of cancer, the compositions or compounds (e.g., drug moieties) comprise (a) (i) at least one SMDC comprising an immunomodulator that targets a pattern recognition receptor and / or is an agonist of the downstream signaling pathway of the innate immune system, (ii) conjugated to a ligand that can be bound by a cell-surface receptor or cell-surface receptor (i.e., a targeting moiety described below) of an immunosuppressive cell, and (b) a CAR-expressing cytotoxic lymphocyte.

[0138] In one embodiment, a combination therapy and / or method for the treatment of cancer is provided, the combination therapy comprising the use and / or administration of (a) at least one SMDC comprising (i) an immunomodulator that targets a pattern recognition receptor and / or is an agonist of a downstream signaling pathway of the innate immune system, (ii) conjugated to a cell-surface receptor of an immunosuppressive cell or a ligand capable of binding a cell-surface receptor (i.e., a targeting moiety described below), and (b) at least one engineered cell (or a composition comprising one or more engineered cells).Embodiments of the combination therapy / method for treating cancer may utilize a TRL7 agonist, TLR8, TLR9 agonist or TLR7 / 8 agonist used in combination with either CAR-T, or CAR-NK cells, stem cells, or other engineered cells, or combinations thereof. In one embodiment of the combination therapy, TRL7 agonist, TLR8, TLR9 agonist or TLR7 / 8 agonist is used with engineered cells to treat cancer. In another embodiment, the combination therapy includes SMDCs used in combination with CAR T cells, stem cells, other engineered cells, or any combination of the above. In another embodiment of the combination therapy to treat cancer, TRL7 agonist, TLR8, TLR9 agonist or TLR7 / 8 agonist is used with CAR T cells. In a further embodiment of the combination therapy to treat cancer, folate-TRL7 agonist, folate-TLR8, folate-TLR9 agonist or folate-TLR7 / 8 agonist is used in combination with CAR T cells to treat cancer.

[0139] In certain embodiments, the therapeutic / drug moiety of the disclosed compounds is conjugated to a targeting moiety (or radical thereof) that targets a cellular pattern recognition receptor via a linker. The linker may be releasable or non-releasable, as described in more detail herein.

[0140] In at least one exemplary embodiment, the targeting moiety comprises a folate ligand, or a functional fragment or analog thereof. "Folate" refers to folate receptor binding molecules, including, for example, folic acid and analogs and derivatives of folic acid, such as, but not limited to, folinic acid, pteroylpolyglutamic acid, pteroyl-D-glutamic acid, and folate receptor binding pteridines (such as tetrahydropterin, dihydrofolate, tetrahydrofolate, and their deaza and dideaza analogs).

[0141] The terms "deaza" and "dideaza" analog refer to art-recognized analogs that have carbon atoms substituted for one or two nitrogen atoms in the natural folic acid structure, or in its analog or derivative.For example, deaza analogs can include 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 tetrahydropterin, dihydrofolate and tetrahydrofolate).Dideaza analogs include, for example, 1,5-dideaza, 5,10-dideaza, 8,10-dideaza and 5,8-dideaza analogs of folate. Other folates useful as complexing ligands in the context of the present disclosure include the folate receptor binding analogs pemetrexed, proguanil, pyrimethamine, trimethoprim, pralatrexate, raltitrexed, aminopterin, amethopterin (also known as methotrexate), N 10 -Methylfolate, 2-deamino-hydroxyfolate (dydroxyfolate), deaza analogues (such as 1-deazamethopterin or 3-deazamethopterin), and 3',5'-dichloro-4-amino-4-deoxy-N 10 -Methylpteroylglutamic acid (dichloromethotrexate).

[0142] Folic acid and the above analogs and / or derivatives are also referred to as "a folate", "the folate" or "folates", reflecting their ability to bind to folate receptors. As described herein, such molecules, when conjugated with exogenous molecules, are effective in enhancing transmembrane transport, such as folate-mediated endocytosis. The above may be used for the folate receptor binding ligands described herein. As used herein, the term "ligand" refers to a molecule, ion or atom that is bound to a central atom or ion of a compound (e.g., a drug).

[0143] Certain embodiments of the novel compounds of the present disclosure are now presented. It will be understood by those skilled in the art that the compounds of the present disclosure may exhibit polymorphism. In fact, the compounds of the present disclosure may include any of the racemic, optically active, polymorphic or stereoisomers of the compounds described herein that exhibit the useful properties described, or mixtures thereof, and methods for preparing optically active forms (e.g., by resolving the racemic form by recrystallization techniques, synthesis from optically active starting materials, chiral synthesis, or chromatographic separation using chiral stationary phases) and determining antitumor activity using standard tests described herein or other similar tests known in the art are well known in the art. Furthermore, unless expressly stated otherwise, the structures depicted herein are also intended to include all stereochemical forms of the structures, i.e., the right-handed (R) and left-handed (S) configurations of each asymmetric center. Thus, single stereochemical isomers of the compositions, as well as enantiomeric and diastereomeric mixtures, are within the scope of the present disclosure.

[0144] Specific values ​​listed herein for radicals, substituents, and ranges are for illustrative purposes only, unless otherwise specified. Such examples are not exclusive of other stated values ​​or other values ​​within the ranges defined for radicals and substituents. For example, (C1-C6) alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl. (C1-C3) alkyl can be iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl. (C1-C3) alkoxy can be methoxy, ethoxy, or propoxy; and (C2-C6) alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy.

[0145] Furthermore, when a moiety is substituted with an R substituent or substituted group, the group may be referred to as "R-substituted." When a moiety is specifically described as being R-substituted or generally comprising a substituted group, the moiety is substituted with at least one R substituent, each of which is optionally different. It will be understood that the substituted group (or R substituent) may comprise any molecule or combination of molecules, provided that the inclusion does not substantially affect the overall structure and shape of the compound, and does not alter any hydrogen bonds essential to the underlying compound achieving its intended purpose (e.g., binding to a targeted pattern recognition receptor).

[0146] When substituents are designated by a conventional chemical formula written from left to right, those substituents equally encompass the chemically identical substituents that would occur if the structure were written from right to left; for example, -CHO- is equivalent to -OCH-.

[0147] In certain embodiments, the immune modulator / drug moiety group of the compounds provided herein comprises a TLR agonist and has a structure represented by formula X or XX, or a pharma- ceutically acceptable salt of formula X or XX:

[0148] [ka] and In formulas X and XX: R1 is -NH2 or -NH-R 1X and R2 is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X ,

[0149] [ka] and

[0150] [ka] is a 3-10 membered N-containing non-aromatic monocyclic or bicyclic heterocycle; In formula X, R3 is -OH, -SH, -NH2 or -NH-R 1X and In the formula XX, X is CH, CR2 or N; R 1X , R 2X and R 2Y is each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl.

[0151] In some embodiments, the immune modulator (e.g., TLR7 agonist) group of the compounds provided herein is a radical having a structure of formula XX, or more particularly, formula XX′:

[0152] [ka] and During the ceremony, R 1B is -NH2 or -NH-R 1X and R 2B is hydrogen (H), alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X ,

[0153] [ka] and R 1X , R 2X and R 2Y are each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl;

[0154] [ka] is a 3-10 membered N-containing non-aromatic monocyclic or bicyclic heterocycle; X is CH or nitrogen (N).

[0155] Alkyl, alkoxy, and the like, as used herein, represent straight chain (i.e., unbranched) or branched chain, or combinations thereof, which may be fully saturated, mono- or polyunsaturated, and can include divalent and polyvalent radicals having the number of carbon atoms specified (i.e., C1-C6). 10means 1 to 10 carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs, and isomers such as, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Unsaturated alkyl groups are those having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl-2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers. An alkoxy is an alkyl that is attached to the remainder of the molecule via an oxygen linker (-O-). In some embodiments, alkoxy refers to a radical that is attached through an oxygen atom of the formula -O-alkyl.

[0156] In general, the term "acyl" or "acyl substituent" refers to a group derived by removal of one or more hydroxyl groups from an oxoacid, including inorganic acids, and contains a double-bonded oxygen atom and an alkyl group. Furthermore, when referring to individual radicals such as "propyl," only straight-chain radicals are encompassed; branched-chain isomers such as "isopropyl" are specifically mentioned.

[0157] In some embodiments, the TLR7 agonist has the formula X, and the TLR7 agonist is linked via a linker to R. 1A , R 1B , R 3A or R 3B and the TLR7 agonist has the formula XX', the TLR7 agonist is conjugated to the targeting moiety via a linker, 1A , R 1B , R 3A or R 3B is conjugated to a targeting moiety at one of

[0158] As used herein, the term "linker" includes a chain of atoms adapted with a biofunctional group to form a chemical bond with A, B, or S, connecting two or more functional moieties of a molecule to form a compound of the present disclosure. By way of example, the chain of atoms may be selected from carbon (C), 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 link various functional capabilities of a compound, such as a folate and a drug. The linker may include a wide range of linking groups, such as ranging from about 2 to about 100 atoms in a continuous backbone, and may include releasable or non-releasable linkers, as described in more detail below. In some embodiments, the immune modulator (e.g., TLR7 agonist) group of the compounds provided herein includes a radical having a structure of formula XXX, more specifically formula XXX':

[0159] [ka] and During the ceremony, R 1C is -NH2 or -NH-R 1X and R 2C is a bond, NH, -NR 1X or CH2, Where applicable,

[0160] [ka] is a 3-10 membered N-containing non-aromatic monocyclic or bicyclic heterocycle; X A is CH2, NH2 or -NH-R 1X and R 1X are each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl; The TLR7 agonist is linked to the R 1C , R 2C or R3B is conjugated to a targeting moiety.

[0161] In some embodiments, the compounds may include a linker ("L" or "L") between or otherwise connecting the targeting moiety and the immunomodulator. n In some embodiments, the linker L n is configured to avoid release of the immune modulator, and n is an integer less than or equal to 50. In some embodiments, the linker L n comprises a polyethylene glycol (PEG) linker or a PEG derivative linker, n is an integer selected from the range of 1 to 32, and the targeting moiety is specific for the folate receptor β. In some embodiments, n is 1 to 50, 1 to 10, 2 to 8, or 2 to 4.

[0162] In some embodiments, L is a hydrolyzable linker. In some embodiments, L is a non-hydrolyzable linker. In some embodiments, L is an optionally substituted heteroalkyl.

[0163] The term "alkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from alkyl, exemplified by, but not limited to, -CH2CH2CH2CH2-. Typically, an alkyl (or alkylene) group has from 1 to 24 carbon atoms. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.

[0164] The term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a stable linear or branched chain, or combination thereof, consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom O, N, P, S, and Si may be located at any of the interior positions of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH2=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3 and -CN. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3.

[0165] Similarly, the term "heteroalkylene" by itself or as part of another substituent refers (unless otherwise stated) to a divalent radical derived from heteroalkyl, exemplified by, but not limited to, -CH2-CH2-S-CH2-CH2 and -CH2-S-CH2-CH2-NH-CH2. In the case of heteroalkylene groups, heteroatoms can also occupy either or both of the terminal chains (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Furthermore, in the case of alkylene and heteroakylene linking groups, the orientation of these linking groups is not implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)2R'- represents both -C(O)2R'- and -R'C(O)2-. As noted above, heteroalkyl groups, as used herein, include groups that are attached to the remainder of the molecule via a heteroatom, such as -C(O)R', -C(O)NR', ​​-NR'R", -OR', -SR' and / or -SOR'. When "heteroalkyl" is recited followed by a recitation of a specific heteroalkyl group, such as -NR'R", it is understood that the terms heteroalkyl and -NR'R" are not unnecessary or mutually exclusive. Rather, the specific heteroalkyl groups are recited for added clarity. Thus, the term "heteroalkyl" should not be construed herein as excluding specific heteroalkyl groups, such as -NR'R".

[0166] In some embodiments, L is a substituted heteroalkyl containing at least one substituent selected from the group consisting of alkyl, hydroxyl, oxo, PEG, carboxylate, and halo. "Halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

[0167] In some embodiments, L comprises a spacer (e.g., as described elsewhere herein). In some embodiments, the spacer comprises a peptidoglycan or a sugar.

[0168] In some embodiments, L is a heteroalkyl substituted with at least one disulfide bond in its backbone, hi some embodiments, L is a peptide having at least one disulfide bond in its backbone.

[0169] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, a polypeptide, or a fragment of a polypeptide, peptide or fusion polypeptide. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0170] In some embodiments, L is -CONH-CH(COOH)-CH-SS-CH-CR a R b -O-CO-, -CONH-CH(COOH)CR a R b -O-CO-, -C(O)NHCH(COOH)(CH2)2-CONH-CH(COOH)CR a R b -O-CO- or -C(O)NHCH(COOH)(CH2)2-CONH-CH(COOH)-CH2-SS-CH2-CR a R b -O-CO-, R a and R b is independently H, alkyl, or heteroalkyl (e.g., PEG).

[0171] In some embodiments, L is

[0172] [ka] wherein n and m are each independently 0 to 10.

[0173] In some embodiments, L is

[0174] [ka] The structure includes wherein n is 1 to 32. In at least one exemplary embodiment, n is 1 to 30 and w is 0 to 5.

[0175] In some embodiments, L is

[0176] [ka] The structure includes In the formula, n is 1 to 30, and w is 0 to 5.

[0177] In some embodiments, the compound has the formula:

[0178] [ka] The compound has a structure represented by:

[0179] In some embodiments, the compound has the formula:

[0180] [ka] The compound has a structure represented by:

[0181] In some embodiments, the compound has the formula:

[0182] [ka] The compound has a structure represented by:

[0183] In some embodiments, the compound has the formula:

[0184] [ka] The compound has a structure represented by:

[0185] In some embodiments, the compound has the formula:

[0186] [ka] The compound has a structure represented by:

[0187] In certain embodiments, a compound comprising a targeting moiety comprising a folate ligand, or a functional fragment or analog thereof, linked via a linker to an immune modulator comprising a TLR agonist, wherein the TLR agonist has the structure of formula XXX-I below:

[0188] [ka] Provided herein is a compound having the formula: In the formula, R 1 is an amine group, R 2 is a single bond, -NH-, and R 3 is H, alkyl, hydroxy or any other substituted group thereof, X is CH, NH, O or S, and the linker is R 1 、 R 2 or R 3 Additionally or alternatively, R1 is -NH2 or -NH-R 1X R2 may be H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X ,

[0189] [ka] R may be 1X , R 2X and R 2Yeach may be independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl;

[0190] [ka] may be a 3-10 membered N-containing non-aromatic monocyclic or bicyclic heterocycle and / or X may be CH, CR2 or N.

[0191] In certain embodiments, a compound (e.g., of a first treatment) comprises a targeting moiety comprising a folate ligand, or a functional fragment or analog thereof, linked via a linker to a drug / immunomodulator comprising a TLR agonist, wherein the TLR agonist has the structure of formula XXX:

[0192] [ka] Provided herein is a compound having the formula: During the ceremony, R 1 is an amine group, R 2 is a bond (e.g., a single bond), -NH-, -NR 1X or CH2, If applicable,

[0193] [ka] is a 3-10 membered N-containing non-aromatic monocyclic or bicyclic heterocycle; X is CH2, NH, NH2, O, S, -NH-R 1X and R 1X are each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl; TLR7 agonists are either "L" or "L n " through a linker such as R1 , R 2 or R 3 The linker L is conjugated to a targeting moiety at one of the n can be configured to avoid release of the compound, and n can be an integer less than or equal to 50. The linker Ln can comprise a PEG linker or a PEG derivative linker, and n can be an integer selected from the range of 1 to 32, and the targeting moiety can be specific for the folate receptor β. Thus, n can be 1 to 50, 1 to 32, 1 to 10, 2 to 8, or 2 to 4.

[0194] L can be a hydrolyzable linker. Alternatively, L can be a non-hydrolyzable linker. L can also be an optionally substituted heteroalkyl.

[0195] Additionally or alternatively, R 3 is independently selected from the group consisting of H, alkyl, hydroxyl or any other substituted groups thereof. Additionally or alternatively, R is -NH or -NH-R 1X R2 may be H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X ,

[0196] [ka] R may be 1X , R 2X and R 2Y may each be independently selected from the group consisting of H, alkyl, alkenyl and alkynyl, alicylclic, aryl, biaryl and heteroaryl;

[0197] [ka] is a 3-10 membered N-containing non-aromatic monocyclic or bicyclic heterocycle, and / or X is CH2, NH, NH2, O, S, -NH-R 1X or X may be CH, CR2 or N.

[0198] In some embodiments, a pharmaceutical composition comprising any of the formulas or compounds provided herein, wherein the linker comprises PEG or a PEG derivative, and in some examples, R 3 or R 1 、 R 2 Or R 3 and a releasable linker attached at

[0199] In some embodiments, the pharma- ceutically acceptable salt is selected from hydrobromide, citrate, trifluoroacetate, ascorbate, hydrochloride, tartrate, trifluoromethanesulfonate, maleate, mesylate, formate, acetate, or fumarate.

[0200] In some embodiments, the compounds include TLR agonists (e.g., radicals thereof), and include, for example and without limitation, TLR3 agonists, TLR7 agonists, TLR7 / 8 agonists, TLR8 agonists, or TLR9 agonists (e.g., all of which bind to Toll-like receptors present in the endosomes of cells). For example and without limitation, in at least one exemplary embodiment, the immune modulator drug / compound may be selected from the compounds listed in Table 2 below.

[0201] [Table 2-1]

[0202] [Table 2-2]

[0203] [Table 2-3]

[0204] [Table 2-4]

[0205] [Table 2-5]

[0206] In some embodiments, the compounds provided herein are compounds (or radicals) of formula I (e.g., TLR7 agonists):

[0207] [ka] or a pharma- ceutically acceptable salt thereof. 1 is an amine. In some embodiments, R 2 is a (e.g., single) bond and / or an amine (e.g., —NH—). In certain embodiments, R 3 is H, alkyl, hydroxy, or any other substituted or suitable substituent thereof (e.g., as described herein). In some embodiments, X is CH, NH, O, or S. In some embodiments, where the compounds provided herein comprise a radical of formula I, the targeting moiety is R 1 , R 2 and / or R 3 In certain embodiments, the linker is conjugated or attached (e.g., via a linker and / or directly) to the radical of formula I at any suitable position, such as at or via R 1 , R 2 or R 3 are bonded at

[0208] In at least one exemplary embodiment, the compounds described herein include compounds (or radicals) of formula Ia (e.g., TLR7 agonists):

[0209] [ka] or a pharma- ceutically acceptable salt thereof. In some embodiments, X is CH or N. In some embodiments, R is -NH or -NH-R 1X In some embodiments, R2 is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X ,

[0210] [ka] In certain embodiments, R 1X , R 2X and R 2Y is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl.

[0211] [ka] is a 3-10 membered N-containing non-aromatic monocyclic or bicyclic heterocycle. In some embodiments, where the compounds provided herein contain a radical of formula Ia, the targeting moiety is R 1 , R 2 and / or R 3 is conjugated or attached (e.g., via a linker and / or directly) to the radical of formula Ia at any suitable position, such as at or via:

[0212] In some embodiments, the compounds provided herein are compounds (or radicals) of formula II (e.g., TLR7 agonists):

[0213] [ka] or a pharma- ceutically acceptable salt thereof. 1 is an amine. In some embodiments, R 2 is a (e.g., single) bond or -NH-. In some embodiments, R 3 is H, an alkyl, a hydroxy group, or any other substituent such as those described herein. In some embodiments, X is CH, NH, O, or S. In some embodiments, where the compounds provided herein comprise a radical of formula II, the targeting moiety is R 1 , R 2 and / or R 3 The radical of formula II is conjugated or attached (e.g., via a linker and / or directly) to the radical of formula II at any suitable position, such as at or via:

[0214] In other embodiments, compounds of the present disclosure include TLR agonists of formula III (e.g., or a radical thereof) or a pharma- ceutically acceptable salt thereof:

[0215] [ka] and In the formula, R 1 is an amine group, R 3 is a hydroxy group. Additionally, if desired, a targeting moiety (e.g., or a radical thereof) or other ligand may be added to R 1 or R 3wherein the TLR agonist of formula III (e.g., or a radical thereof) is a TLR7 agonist that is at least 10 times (10x) more potent than a conventionally available TLR7 agonist.

[0216] In some embodiments, the TLR7 agonist has formula IV (e.g., or a radical thereof), or a pharma- ceutically acceptable salt thereof:

[0217] [ka] is provided herein, In the formula, R 1 is an amine group, R 2 is a single bond, -NH-.

[0218] In certain embodiments, the TLR agonist of the compounds provided herein (e.g., immune modulator and / or first therapy) has the structure of formula (2-I), a radical thereof, or a pharma- ceutically acceptable salt of formula (2-I):

[0219] [ka] and In formula (2-I), R 1 , R 3 , R 4 and R 5 are each independently hydrogen (H), alkyl, alkoxy, alkenyl, alkynyl, alicyclic, aryl, biaryl, halo, heteroaryl, -COR 2x ,

[0220] [ka] and R 2 are H, -OH, -NH2, -NHR 2x, N3, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2,

[0221] [ka] and Y is H, -OH, -NH2, -NHR 2x , -OR 2X , -SO-R 2x , -SH, -SO3H, -N3, -CHO, -COOH, -CONH2, -COSH, -COR 2x , -SO2NH2, alkenyl, alkynyl, alkoxyl, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2,

[0222] [ka] where: R 2x and R 2y are each independently selected from the group consisting of H, -OH, -CH-OH, -NH, -CH-NH, -COOMe, -COOH, -CONH, -COCH, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl; R 2z are -NH2 and -NR 2q R 2q’ , -OR 2q , -SO-R 2q and -COR 2q R 2q and R 2q’ are each independently alkyl or H;

[0223] [ka] is a non-aromatic, monocyclic or bicyclic 3-10 membered N-containing heterocycle; In formula (2-I), X 1 , X 2 and X 3 are each independentlyq or N and R q each is independently H, halogen, or optionally substituted alkyl; In formula (2-I), n is an integer of 0 to 30, and m is an integer of 0 to 4.

[0224] In certain embodiments, the TLR agonist of the compound has the structure of formula (2-IA) (or a radical or a pharma- ceutically acceptable salt thereof):

[0225] [ka] (In the formula, R 1 is an optionally substituted C3-C8 alkyl (e.g., acyclic or cyclic) (e.g., optionally substituted with one or more substituents, each of which is independently halogen, alkyl, heteroalkyl, alkoxy, or cycloalkyl); R 2 H, -OR z , -SO2N(R z )2, -NR 2x R 2y or N3, Y is H, -OR z , -NR 2x R 2y , -SR z , -SOR z , -SO3R z , -N3, -COR z , -COOR z , -CON(R z )2, -COSR z , -SO2N(R z )2 or -CON(R z )2, R 2x and R 2y are each independently hydrogen, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z)2, or optionally substituted alkyl (e.g., optionally substituted with one or more substituents, each of which is independently oxo, halogen, alkyl, heteroalkyl, alkoxy, or cycloalkyl), and R z are each independently hydrogen, halogen or optionally substituted alkyl, or R 2x and R 2y taken together form an optionally substituted heterocycloalkyl (e.g., the optionally substituted heterocycloalkyl is a monocyclic or bicyclic heterocycloalkyl and / or the optionally substituted heterocycloalkyl is a 3-10 membered heterocycloalkyl); R 3 are each independently halogen, -N3, -CN, -NO2, -COR z , -COOR z , -CON(R z )2, -COSR z , -SO2N(R z )2, -CON(R z 2) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, amino, hydroxy, or thiol, wherein the alkyl, alkoxy, heteroalkyl, cycloalkyl, or heterocycloalkyl is optionally substituted; R 4 and R 5 are each independently alkyl, alkoxy, halogen, or cycloalkyl, wherein alkyl, alkoxy, and cycloalkyl are optionally substituted; n is 1 to 6, and m is 0 to 4. or a pharma- ceutically acceptable salt thereof.

[0226] One embodiment provides a compound having the structure of Formula (2-I) or (2-IA), or a pharma- ceutically acceptable salt thereof, where n is 1 to 30. In one embodiment, n is 1 to 6. In another embodiment, n is 1 to 3. In another embodiment, n is 1 or 2. In another embodiment, n is 0. In another embodiment, n is 1. In another embodiment, n is 1 and Y is -OH. In another embodiment, n is 1 and Y is -NH2.

[0227] In one embodiment, the compound is represented by the structure of TLR7 (TLR7)-1 (Compound 1A). In one embodiment, the compound is represented by the structure of TLR7-1 (Compound 2A). In one embodiment, the compound is represented by the structure of TLR7-1 (Compound 3A). The structures of such compounds are illustrated in FIG. 1C.

[0228] One embodiment provides a compound having the structure of formula (2-I) or (2-IA), or a pharma- ceutically acceptable salt thereof, wherein Y is -OH, OCH, -NH, -NHNH, -NHCONH, -SH, -SONH, -N, -COOH, -COCH, -COOCH, or -CONH.

[0229] One embodiment provides a compound having the structure of formula (2-I) or (2-IA), or a pharma- ceutically acceptable salt thereof, wherein Y is H, —NH, —NHR 2x , -OR 2X , -SO-R 2x , -SH, -SO3H, -N3, -CHO, -COOH, -CONH2, -COSH, -COR 2x , -SO2NH2, alkenyl, alkynyl, alkoxyl, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2,

[0230] [ka] It is.

[0231] One embodiment provides a compound having the structure of formula (2-I) or (2-IA), or a pharma- ceutically acceptable salt thereof, wherein Y is OH.

[0232] One embodiment provides a compound having the structure of formula (2-I) or (2-IA), or a pharma- ceutically acceptable salt thereof, wherein Y is NH2.

[0233] One embodiment provides a compound having the structure of formula (2-I) or (2-IA), or a pharma- ceutically acceptable salt thereof, wherein n is 1 and Y is OH.

[0234] One embodiment provides a compound having the structure of formula (2-I) or (2-IA), or a pharma- ceutically acceptable salt thereof, wherein n is 1 and Y is NH2.

[0235] One embodiment provides a compound having the structure of formula (2-I) or (2-IA), or a pharma- ceutically acceptable salt thereof, wherein n is 0 and Y is NH2.

[0236] One embodiment provides a compound comprising the structure of formula (2-I) or (2-IA), or a pharma- ceutically acceptable salt thereof, wherein R 1 is optionally substituted alkyl. In one embodiment, R 1 is an optionally substituted C-C alkyl. In another embodiment, R 1 is an optionally substituted acyclic C-C alkyl. In another embodiment, R 1 is butyl.

[0237] One embodiment provides a compound comprising the structure of formula (2-I) or (2-IA), or a pharma- ceutically acceptable salt thereof, wherein R 2 -NR 2x R 2y In one embodiment, R 2 is NH2.

[0238] One embodiment provides a compound comprising the structure of formula (2-I) or (2-IA), or a pharma- ceutically acceptable salt thereof, wherein R 3 is H.

[0239] One embodiment provides a compound comprising the structure of formula (2-I) or (2-IA), or a pharma- ceutically acceptable salt thereof, wherein R 4 is alkyl. In one embodiment, R 4 is methyl.

[0240] One embodiment provides a compound comprising the structure of formula (2-I) or (2-IA), or a pharma- ceutically acceptable salt thereof, wherein R 5 is alkyl. In one embodiment, R 5 is methyl.

[0241] One embodiment provides a compound comprising the structure of formula (2-I) or (2-IA), or a pharma- ceutically acceptable salt thereof, wherein R 4 and R 5 Each is alkyl. In one embodiment, R 4 and R 5 are each methyl.

[0242] One embodiment provides a compound comprising the structure of formula (2-I) or (2-IA), or a pharma- ceutically acceptable salt thereof, wherein m is 0. In another embodiment, m is 1. In another embodiment, m is 2. In another embodiment, m is 3. In another embodiment, m is 4.

[0243] One embodiment provides a compound comprising the structure of formula (2-I) or a pharma- ceutically acceptable salt thereof, wherein X 1 , X 2 and X 3 are each N. In one embodiment, X 1 is N. In another embodiment, X 2 is N. In another embodiment, X 3 is N.

[0244] One embodiment provides a compound comprising the structure of formula (2-I) or a pharma- ceutically acceptable salt thereof, with the proviso that compounds in which n is 0 are excluded.

[0245] One embodiment provides a compound comprising the structure of formula (2-I) or a pharma- ceutically acceptable salt thereof, with the proviso that compounds in which n is 0 and Y is OH are excluded.

[0246] One embodiment provides a compound comprising the structure of formula (2-I) or a pharma- ceutically acceptable salt thereof, wherein n is 0, Y is OH, and R 1 is butyl, and R 2 is NH2 and R 3 is H and R 4 and R 5 However, it is provided that compounds in which each is methyl are excluded.

[0247] One embodiment provides a compound comprising the structure of formula (2-I) or a pharma- ceutically acceptable salt thereof, with the proviso that the compound TLR7-1 is excluded.

[0248] In some embodiments, the compound has any one or more of the formulas:

[0249] [ka] or a pharma- ceutically acceptable salt thereof.

[0250] In some embodiments, the compound has any one or more of the formulas:

[0251] [ka] or a pharma- ceutically acceptable salt thereof.

[0252] In some embodiments, the compound has any one or more of the formulas:

[0253] [ka] or a pharma- ceutically acceptable salt thereof.

[0254] In some embodiments, administration of a compound provided herein (e.g., to an individual) converts macrophages in cancerous tissues from an M2-like phenotype to a proinflammatory M1-like phenotype. In some embodiments, a decrease in collagen synthesis stimulating cytokines (i.e., CCL18, PDGF, and IL-1β) occurs following administration of a compound provided herein and a concomitant increase in collagen production inhibiting cytokines (e.g., IFN-γ). Notably, in at least one embodiment, the cytokine profile following administration of a compound provided herein is consistent with reprogramming from an M2-like phenotype to an M1-like phenotype. As used herein, a "profile" or "assay" refers to a set of one or more markers and their presence, absence, and / or relative level or abundance (compared to one or more controls). For example, a cytokine profile is a set of data of the presence, absence, relative level, or abundance of a cytokine present in a sample. A genomic or nucleic acid profile is a set of data of the presence, absence, relative level, or abundance of expressed nucleic acids (e.g., transcripts, mRNAs, etc.). The profile may alternatively be referred to as an expression profile.

[0255] In some embodiments, the net result of reprogramming is an increase in alveolar air sacs, a decrease in extracellular matrix deposition, and a decrease in hydroxyproline / collagen biosynthesis, effectively reversing the disease (see, e.g., Example 4).

[0256] Although specific drugs and formulas are described herein, it should be understood that any compound (e.g., drug) useful for reprogramming activated myeloid cells to a proinflammatory M1-like phenotype can be used in the novel compounds and methods thereof (e.g., any compound (e.g., drug) capable of binding to a pattern recognition receptor and inhibiting at least a portion of the innate immune system response downstream thereof). In some embodiments, the analogs and / or derivatives, compounds described herein can be used for targeting the compounds provided herein.

[0257] In addition, more than one compound / conjugate can be administered, and in some examples, the compound can include different drugs. For example, the different drugs can be selected from TLR7 agonists and TLR9 agonists. In yet another embodiment, one or more compounds can be administered in a composition with one or more conjugated and / or unconjugated drugs (e.g., the conjugated embodiments described below). In some embodiments, any of the compounds and drugs described herein can be used according to the methods described herein, and in some examples, depending on the desired application, can be combined with other drugs that reduce or inhibit bone marrow-derived immunosuppressive cells (e.g., in conjunction with cancer treatment), downregulate the production of growth factors (e.g., pirfenidone), directly modify fibroblasts by inhibiting mammalian target of rapamycin complex 1 (mTORC1) signaling (e.g., CZ415), and / or any other pro-inflammatory and / or anti-cancer drugs and therapies.

[0258] As used herein, "downregulation" and its terminology elements (e.g., "downregulation" or "downregulated") may be used interchangeably and refer to a decrease in the level of a marker, such as a gene, nucleic acid, metabolite, transcript, protein or polypeptide.Similarly, "upregulation" and its terminology elements (e.g., "upregulation" or "upregulated") may also be used interchangeably and refer to an increase in the level of a marker, such as a gene, nucleic acid, metabolite, transcript, protein or polypeptide.Similarly, a pathway, such as a signal transduction or metabolic pathway, may be upregulated or downregulated.

[0259] Targeting part In some cases, the toxicity associated with systemic administration of at least conventional drugs identified herein has hindered their practical use in the treatment of cancer.For example, TLR agonists may not be tolerated by individuals, and in some cases, may cause subject death (e.g., when administered systemically by conventional modalities).In some embodiments, the compounds provided herein, such as those having formula I and / or II, are significantly more potent than conventional drugs that can be used with the compounds of the present disclosure, and in some cases, the mechanism of avoiding systemic toxicity is favorable.

[0260] In certain embodiments, provided herein are therapeutic agents (e.g., drugs (already described)) conjugated to a targeting moiety. In some embodiments, the targeting moiety comprises a ligand or other atom or molecule that targets (e.g., with high specificity) a particular region or tissue of an individual, and in certain examples may comprise, for example, a hormone, an antibody and / or a vitamin. As described in more detail below, in at least one embodiment, the targeting moiety comprises a molecule that has (e.g., high) affinity for FRβ. In some examples, the targeting moiety has specific affinity for any receptor that is unique to cancer cells or tissues.

[0261] In some instances, FRβ is significantly upregulated in activated myeloid cells (e.g., mainly activated monocytes and M2-like macrophages), and all data recorded to date support that FRβ is induced only in cells of myeloid origin after exposure to anti-inflammatory or pro-inflammatory stimuli. Folate receptors can be upregulated (e.g., more than 90%) in non-mucinous ovarian cancer. In certain instances, folate receptors are present in kidney cancer, brain cancer, lung cancer and breast cancer. For example, there are some cancers that do not express a sufficient number of folate receptors themselves to achieve the desired specificity, but cancerous tumors express, for example, myeloid-derived immune suppressor cells (MDSCs), which express FRβ and can be targeted, for example, by the targeting moieties provided herein. In some embodiments, folate receptors are substantially absent (e.g., present at extremely low levels) in healthy (non-myeloid) tissues (e.g., not involving lung, liver, spleen, heart, brain, muscle, intestine, pancreas, bladder, etc.). In some cases, even the resting tissue-dwelling macrophages present in large amounts throughout the body are mainly FRβ negative.In some cases, the uptake of folate-targeted imaging agents is in, for example, inflamed tissue, malignant lesions and kidney.In some cases, only subjects without cancer retain folate-targeted drugs in kidney and inflammation sites.In some cases, the difference in folate receptor expression provides a mechanism for selectively targeting cancer cells.

[0262] In some embodiments, the compounds and methods provided herein take advantage of the restricted expression of FRβ to target / localize potent compounds (e.g., conjugates or drugs) administered systemically to cancerous tissues. In some examples, the compounds provided herein are delivered directly to FRβ-expressing cells, for example, which can advantageously prevent systemic activation of the immune system and avoid (e.g., at least some of) the toxicity that has previously prevented the systemic use of non-targeted compounds (e.g., drugs) described herein. In some embodiments, the methods described herein are used to treat cancer, for example, regardless of whether the cancer expresses a folate receptor. In some embodiments, folic acid and other folate receptor binding ligands (or radicals thereof), such as folate, are used as targeting moieties, for example, because they have affinity for FRβ.

[0263] Folic acid is a member of the B family of vitamins and may play an essential role in cell survival, for example, by participating in the biosynthesis of nucleic acids and amino acids. Folic acid can enhance the specificity of conjugated immunomodulator drugs by targeting activated myeloid cells, and the specificity of conjugated anticancer drugs by targeting folate receptor-positive cancer cells. In some examples, compounds are provided herein that include a folate ligand (or a radical thereof) or a functional fragment or analog thereof, and an immunomodulator (e.g., a TLR7, TLR8, TLR7 / 8, TLR9, or TLR3 agonist) as a targeting moiety. In some examples, TLR7, TLR8, TLR7 / 8, TLR9, and TLR3 are present in endosomes. In some embodiments, the compound or a radical thereof binds to a TLR. In some embodiments, the TLR is TLR7.

[0264] Pyrido[2,3-d]pyrimidine analog ligands (e.g., or radicals thereof), functional fragments or analogs thereof, or any other molecules, fragments, or atoms with affinity (and, for example, without limitation, high specificity) for FRβ may alternatively be used as targeting moieties (or radicals thereof). For example, such folate analog molecules can have a relative affinity for binding to FRβ of about 0.01 or greater compared to folic acid at temperatures of about 20° C. / 25° C. / 30° C. / physiological conditions. Similarly, galectin-3 ligands, translocator protein (TSPO) ligands, and any other ligands or targeting moieties with high specific affinity for cancerous cells or tissues may be used.

[0265] Specific examples of suitable targeting moieties (or radicals thereof) are now provided. However, it will be understood that the targeting moieties (or radicals thereof) of the present disclosure may include any ligand (or radicals thereof) useful for targeting FRβ and are not limited to the structures specified herein. The ligand (or radicals thereof) is capable of binding to FRβ.

[0266] In at least one embodiment, the compounds provided herein have the structure of Formula V, or a functional fragment or analog thereof:

[0267] [ka] and a targeting moiety (or radical thereof) having the formula: During the ceremony, X1, X2, X3, X4, X5, X6, X7, X8 and X9 are each independently N, NH, CH, CH2, O or S; Y is C, CH, CH2, N, NH, O or S; Z is glutamic acid, valine or a substrate; R1 and R2 are each independently NH2, OH, SH, CH3, or H; R3 is "hours" or alkyl; m and n are each independently 0, 1, or a value between 0 and 1;

[0268] [ka] represents either a single bond or a double bond C=C.

[0269] In a further embodiment, as a non-limiting example, the targeting moiety of formula V (or a radical thereof) has the structure of VI (or a functional fragment or analog thereof):

[0270] [ka] having During the ceremony, X1, X2, X3, X5, X6, X7, X8 and X9 are each independently N, NH, CH, CH2, O or S; Y is C, CH, CH2, N, NH, O or S; Z is glutamic acid, valine or a substrate; R1 and R2 are each independently NH2, OH, SH, CH3, or H; R3 is "hours" or alkyl; m and n are each independently 0, 1, or a value between 0 and 1;

[0271] [ka] represents either a single bond or a double bond C=C.

[0272] Another particular targeting moiety (or radical thereof) of formula V (or functional fragment or analog thereof) has the structure of formula VII:

[0273] [ka] having During the ceremony, X1, X2, X3, X4, X5, X6, X7, X8 and X9 are each independently N, NH, CH, CH2, O or S; Y is C, CH, CH2, N, NH, O or S; Z is glutamic acid, valine or a substrate; R1 and R2 are each independently NH2, OH, SH, CH3, or H; R3 is "hours" or alkyl; m and n are each independently 0, 1, or a value between 0 and 1;

[0274] [ka] represents either a single bond or a double bond C=C.

[0275] In some embodiments, the targeting moiety of formula VI (or a radical thereof) has the structure of formula VIII:

[0276] [ka] having During the ceremony, X1, X2, X3, X5, X6, X7, X8 and X9 are each independently N, NH, CH, CH2, O or S; Y is C, CH, CH2, N, NH, O or S; Z is glutamic acid, valine or a substrate; R1 and R2 are each independently NH2, OH, SH, CH3, or H; R3 is "hours" or alkyl; m is 0, 1, or between 0 and 1;

[0277] [ka] represents either a single bond or a double bond C=C.

[0278] In some embodiments, the targeting moiety of formula VI (or a radical thereof) has the structure of formula IX:

[0279] [ka] having During the ceremony, X1, X2, X3, X5, X6, X7, X8 and X9 are each independently N, NH, CH, CH2, O or S; Y is C, CH, CH2, N, NH, O or S; Z is glutamic acid, valine or a substrate; R1 and R2 are each independently NH2, OH, SH, CH3, or H; R3 is "hours" or alkyl; m is 0, 1, or between 0 and 1;

[0280] [ka] represents either a single bond or a double bond C=C.

[0281] In some embodiments, the targeting moiety of formula VII (or a radical thereof) has the structure of formula X or XI:

[0282] [ka] having During the ceremony, X1, X2, X3, X4, X5, X6, X7, X8 and X9 are each independently N, NH, CH, CH2, O or S; Y is C, CH, CH2, N, NH, O or S; Z is glutamic acid, valine or a substrate; R1 and R2 are each independently NH2, OH, SH, CH3, or H; R3 is "hours" or alkyl; m is 0, 1, or between 0 and 1;

[0283] [ka] is a single or double bond C=C, or

[0284] [ka] represents one of the following: During the ceremony, X1, X2, X3, X4, X5, X6, X7, X8 and X9 are each independently N, NH, CH, CH2, O or S; Y is C, CH, CH2, N, NH, O or S; Z is glutamic acid, valine or a substrate; R1 and R2 are each independently NH2, OH, SH, CH3, or H; R3 is "hours" or alkyl; m is 0, 1, or between 0 and 1;

[0285] [ka] represents either a single bond or a double bond C=C.

[0286] Chemical structures and spectroscopic data for some additional embodiments of targeting moieties (eg, or radicals thereof) of the present disclosure are provided in Tables 3, 4, 5, and 6 below.

[0287] Table 3 provides non-limiting examples of additional embodiments of targeting moieties (eg, or radicals thereof) having the structure of Formula VIII.

[0288] [Table 3-1]

[0289] [Table 3-2]

[0290] [Table 3-3]

[0291] Table 4 provides non-limiting examples of additional embodiments of targeting moieties (eg, or radicals thereof) having the structure of Formula IX.

[0292] [Table 4-1]

[0293] [Table 4-2]

[0294] Table 5 provides non-limiting examples of additional embodiments of targeting moieties having the structure of Formula X.

[0295] [Table 5]

[0296] As noted above, instead of folate, the targeting moiety (e.g., radicals thereof) may be one or more non-classical anti-folate analogs, such as, for example, a pyrido[2,3-d]pyrimidine or similar analog (or radicals thereof) having a formula (e.g., radicals of this formula) set forth in Table 6 below (or analogs or functional fragments thereof).

[0297] [Table 6-1]

[0298] [Table 6-2]

[0299] [Table 6-3]

[0300] [Table 6-4]

[0301] [Table 6-5]

[0302] In some examples, the compounds presented herein include a drug (e.g., a radical thereof) (e.g., an immunomodulator) that is conjugated to a targeting moiety (e.g., a radical thereof). The immunomodulator (e.g., a radical thereof) may be conjugated to the targeting moiety (e.g., a radical thereof) directly or via a linker (e.g., optionally including a spacer). FIG. 1A shows at least one embodiment of compound 100. Here, compound 100 includes an immunomodulator (or a drug or radical thereof) 102, e.g., having formula I, 3 is a hydroxy group. The immune modulator (e.g., a radical thereof) 102 is conjugated to a targeting moiety (e.g., a radical thereof) 104 via a linker 106, where the targeting moiety (e.g., a radical thereof) 104 is a folate (e.g., non-releasable) and the linker 106 is a PEG linker repeated n times, where n is between 1 and 32.

[0303] In at least one embodiment, and without limitation, compound 100 may be represented by the formula: QLT, where Q is a radical of a folate receptor binding ligand / targeting moiety 104, L is a linker 106, and T is a radical of a TLR agonist / immunomodulator 102. The linker L may include any of the linker formulas presented herein.

[0304] Similarly, Figure IB depicts at least one embodiment of compound 150. Compound 150 has an immune modulator / drug (e.g., radical thereof) 152, which is a TLR7 agonist (e.g., radical thereof), e.g., having formula III, conjugated to a targeting moiety (e.g., radical thereof) 154 via a (e.g., releasable) linker 156.

[0305] Linker Linker (L or L n ) may be releasable or non-releasable. In some cases, the target of a compound containing a non-releasable linker is, for example, an endosome (e.g., of a cell of interest), while the target of a releasable linker is, in some cases, an endosome, cytoplasm, or both (e.g., of a cell of interest).

[0306] In at least one exemplary embodiment, the linker L n is disposed between the targeting moiety (e.g., a radical thereof) and the immunomodulator or a pharma- ceutically acceptable salt thereof, and a linker L or L n is configured to avoid release of the TLR7 agonist in free form, and n is an integer less than or equal to 50. Additionally or alternatively, the compound may be linked to a linker L comprising a PEG or PEG derivative. n where n can be an integer selected from the range of 1 to 32, and the targeting moiety (e.g., a radical thereof) can include a radical of a folate receptor binding ligand, including a FRβ binding ligand.

[0307] The term "releasable" in the context of a linker refers to 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, reducing agent labile, pH labile, acid labile, base labile, oxidatively unstable, metabolically unstable, biochemically unstable, enzyme labile, or p-aminobenzyl-based polyvalent releasable bond. It is understood that the physiological conditions that result in bond cleavage do not necessarily include biological or metabolic processes, but may instead include standard chemical reactions, such as, for example, hydrolysis reactions at physiological pH, or as a result of compartmentalization into organelles such as endosomes that have a lower pH than the pH of the cytoplasm. The cleavable bond can link two adjacent atoms in the releasable linker, and / or can link other linker moieties or targeting moieties and / or drugs described herein, for example, at one or both ends of the releasable linker. In some examples, the releasable linker is cleaved into two or more fragments. In some instances, the releasable linker is separated from the targeting moiety, hi some embodiments, the targeting moiety and the immunomodulator are released from each other such that the immunomodulator is in an activated state.

[0308] In contrast, the term "non-releasable" in the context of a linker refers to a linker that includes at least one bond that is not easily or rapidly cleaved under physiological conditions. In some embodiments, a non-releasable linker includes a backbone that is stable under physiological conditions (e.g., the backbone is not susceptible to hydrolysis (e.g., hydrolysis by water or enzymatic hydrolysis)). In some embodiments, a composition provided herein that includes a non-releasable linker does not release any component of the composition (e.g., a targeting ligand (e.g., a fully amorphous (FA) ligand) or an immune modulator (e.g., a TLR7 agonist)). In some embodiments, a non-releasable linker does not have a disulfide bond (e.g., SS) or an ester in the backbone. In some embodiments, the composition includes a targeting moiety and an immune modulator linked by a backbone that is substantially stable during the entire circulation of the composition (e.g., during endocytosis into the endosomes of a target cell). In some embodiments, compositions comprising non-releasable linkers are particularly beneficial when the immunomodulator targets TLRs, NOD-like receptors, and / or other pattern recognition receptors present in the endosomes of cells. Non-releasable linkers may include amides, esters, ethers, amines, and / or thioethers (e.g., thio-maleimides). Although specific examples are provided herein, it will be understood that any molecule may be used for a non-releasable linker, provided that at least one bond is formed that is not easily or rapidly cleaved under physiological conditions.

[0309] Perhaps more particularly, a non-releasable linker includes a linker that undergoes less than ten percent (10%) (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.1%, less than 0.01% or less than 0.001%) hydrolysis at neutral pH, for example, in water (e.g., a buffer (e.g., a phosphate buffer) solution) within a period of time (e.g., 24 hours). In some embodiments, when a non-releasable linker is used, less than about ten percent (10%), and preferably less than five percent (5%) of the administered conjugate compound releases free drug or does not release any free drug (e.g., in the systemic circulation before being taken up by the target cell / tissue). In some embodiments, less than five percent (5%) of the free drug is released from the conjugate within one hour of administration while the compound is in the systemic circulation.

[0310] In some embodiments, the targeting moiety is not cleaved from the drug / immunomodulator to make the compound therapeutically effective in vivo. In some embodiments, this is advantageous, for example, because a negligible amount (if any) of the drug (e.g., immunomodulator, e.g., TLR7 agonist) is released (e.g., systemically) prior to targeted delivery of the compound, allowing the use of targeted compositions that contain a potent drug (e.g., TLR7 agonist). In some embodiments, controlling the release profile of the active component is a challenging aspect of preparing an effective pharmaceutical composition. In some embodiments, compositions that include a non-releasable linker provided herein avoid the difficulty of preparing an effective pharmaceutical composition (e.g., by eliminating the need for time control of release). In some embodiments, the immunomodulator or warhead of the compounds provided herein becomes active upon binding (e.g., conjugated to a targeting conjugate). In some embodiments, while the warhead / immunomodulator is active, the non-releasable linker and targeting moiety prevent release (e.g., by the subject's body) of toxic cytokines that activate the immune system (such as, for example, interleukin 6 (IL-6)) (e.g., because the compound is specifically targeted (e.g., using folate or an analog thereof)). In certain instances, for example, even when the warhead / immunomodulator of the compound is activated when linked to a non-releasable linker, the immunomodulator is unable to access the appropriate (e.g., targeted) receptor in the endosome of a cell until the compound binds to the targeted receptor (e.g., a folate receptor).

[0311] As a non-limiting example, linker 106 in Figure 1A is a non-releasable PEG linker, while linker 156 in Figure 1B is a self-immolative, releasable linker (e.g., comprising a disulfide bond (e.g., S-S)). For example, the scheme shown in Figure 1B illustrates the self-immolative cascade of compound 150 upon cleavage from targeting moiety 154.

[0312] In some embodiments, the linker 156 is configured such that the drug is cleaved from the targeting moiety 154 only after sufficient time has passed for the compound to circulate in the subject's systemic circulation following administration (e.g., be cleared from non-target tissues and be captured and internalized by target cells and / or receptors). In some embodiments, the release period will vary (e.g., from subject to subject (e.g., based on a variety of factors)). In some embodiments, the releasable linker can be engineered to not cleave / release for at least up to 24 hours after administration, or even over a period of one week. In some embodiments, the compound can safely pass through the subject's system and any amounts not captured by target cells (e.g., cells expressing FRβ) can be excreted prior to release / activation, thus preventing toxicity (e.g., because the immunomodulator is not active when attached to the releasable linker).

[0313] Both releasable and non-releasable linkers can be engineered to optimize biodistribution, bioavailability and PK / PD (e.g., of the compounds) and / or to increase uptake (e.g., of the compounds) into target tissues according to methods generally known in the art or hereafter developed methods, such as by PEGylation, etc. In some embodiments, the linker is configured to avoid significant release of pharmacoactive amounts of the drug into the circulation before being captured by cells (e.g., cells of interest (e.g., macrophages in fibrotic or cancerous tissue to be treated)).

[0314] In some embodiments, conjugates that include a releasable linker may be designed to diffuse across the membrane of an endosome, for example, into the cytoplasm of a target cell. The releasable linker may be designed such that the immunomodulator is not released until the compound reaches the cytoplasm.

[0315] Conjugates provided herein may include a releasable linker (e.g., to facilitate release of the immunomodulator in the cytoplasm, e.g., where the immunomodulator comprises an activator of PI3K kinase, IRAK, or 1-kappa-β (Iκβ) kinase (e.g., using prostratin, etc.) or nuclear factor kappa-light-chain enhancer of activated B cells (NF-κβ) (see, e.g., Table 1) or a myeloid differentiation primary response 88 (MyD88) agonist). In some embodiments, the releasable linker prevents release of the immunomodulator until, for example, after the targeting moiety binds to an appropriate target (e.g., macrophage folate receptor), is internalized into the endosome of the target cell, and / or diffuses into the cytoplasm (e.g., where the desired pattern recognition receptor is located). In some embodiments, the releasable linker releases the immunomodulator within the endosome.

[0316] In some embodiments, the linkers provided herein may include one or more spacers (e.g., to facilitate a specific release time, promote increased uptake into target tissues, and / or optimize the biodistribution, bioavailability, and / or PK / PD of the compounds provided herein). The spacers may include one or more of alkyl chains, PEG, peptides, sugars, peptidoglycans, clickable linkers (e.g., triazoles), rigid linkers such as polyproline and polypiperidine, and the like.

[0317] In some embodiments, PEG 12significantly reduces, if not completely avoids, non-specific uptake of the compounds provided herein (e.g., in non-target organs (e.g., in the liver and / or kidneys of a subject following administration). In some embodiments, the compounds avoid delivery to the liver and kidneys. In some embodiments, the targeting moiety (its free form, its radical, or a conjugate thereof) does not bind to uptake receptors on the surface of non-target cells (e.g., provided that the organs are not target sites, thus irritation of immune complexes in those organs can be avoided, which is highly beneficial in clinical situations).

[0318] In some embodiments, the conjugates provided herein that include a non-releasable linker reduce or eliminate the toxicity of the component released from the conjugate in its free form (e.g., the free form of the compound and / or ligand provided herein).At least one embodiment of the present disclosure provides a compound having the structure of formula (2-II), (2-IIA), (2-III) or (2-IIIA) as set forth below, or a pharma- ceutically acceptable salt thereof, wherein L is a cleavable linker.

[0319] In at least one embodiment, the linker comprises a hydrophilic spacer. In some embodiments, the compound has the structure of formula XII (e.g., a substructure of the TLR7 agonist of formula III conjugated to folate via a releasable linker containing a first hydrophilic spacer):

[0320] [ka]

[0321] In some embodiments, the compound has the structure of Formula XIII (e.g., the structure of a TLR7 agonist of Formula III conjugated (covalently) to a folate via a non-releasable linker that includes a second hydrophilic spacer):

[0322] [ka]

[0323] Specific examples of exemplary conjugated compounds are provided herein.

[0324] In some embodiments, the compounds provided herein comprise a radical of a targeting moiety conjugated with a radical of an immunomodulator or a pharma- ceutically acceptable salt thereof, so that the immunomodulator (or its radical) or its pharma-ceutically acceptable salt remains pharma- ceutically active when conjugated.The targeting moiety may comprise any of the targeting moieties described herein, and in at least one embodiment, comprises a folate ligand, any other folate receptor binding molecule (or, for example, any functional fragment or analogue described above) or a pyrido[2,3-d]pyrimidine analogue.In some embodiments, the targeting moiety (or its conjugate or radical) is specific for FRβ.

[0325] In some embodiments, the compounds provided herein include one or more linkers, and the radical of the targeting moiety is conjugated to the radical of the immunomodulator via one or more linkers. For example, when the immunomodulator or a pharma- ceutically acceptable salt thereof has formula I or II, the radical of the immunomodulator is conjugated via a linker or directly to R 1 , R 2 or R 3 Similarly, when the immunomodulator or a pharma- ceutically acceptable salt thereof has formula III, the radical of the immunomodulator may be conjugated to the radical of the targeting moiety through a linker or directly through R 1 or R 3Alternatively, when the immunomodulator or a pharma- ceutically acceptable salt thereof has formula IV, the radical of the immunomodulator may be conjugated to the radical of the targeting moiety via a linker or directly, R 1 or R 2 The linker may be conjugated to the radical of the targeting moiety in one of the following: As described herein, the linker may be releasable or non-releasable.

[0326] In some embodiments, one or more linkers of the compounds provided herein may comprise PEG, PEG derivatives, or any other linkers known in the art or subsequently developed that can achieve the objectives described herein. In some embodiments, the linker may be repeated n times, where n is a positive integer. For example, and without limitation, n may be any integer selected from the ranges of 1-16, 1-32, 1-64, or 1-96. The number of repeats in the linker (i.e., n) may be selected to achieve the desired functionality, size, and / or potency of the compound, and / or in view of the desired application. In some embodiments, one or more of the linkers comprise one or more spacers (e.g., which may also be used to specifically design the characteristics of the compound).

[0327] In some embodiments, the linker is a hydrolyzable linker. In some embodiments, the linker is a non-hydrolyzable linker. In some embodiments, the linker is an optionally substituted heteroalkyl. In some embodiments, the linker is a substituted heteroalkyl containing at least one substituent selected from the group consisting of alkyl, hydroxyl, oxo, PEG, carboxylate and halo. In some embodiments, the linker comprises a spacer (e.g., as described elsewhere herein).

[0328] In some embodiments, the linker is a heteroalkyl substituted with at least one disulfide bond in its backbone, hi some embodiments, the linker is a peptide having at least one disulfide bond in its backbone.

[0329] In some embodiments, the linker is -CONH-CH(COOH)-CH-SS-CH-CR a R b -O-CO-, -CONH-CH(COOH)CR a R b -O-CO-, -C(O)NHCH(COOH)(CH2)2-CONH-CH(COOH)CR a R b -O-CO- or -C(O)NHCH(COOH)(CH2)2-CONH-CH(COOH)-CH2-SS-CH2-CR a R b -O-CO-, R a and R b is independently H, alkyl, or heteroalkyl (e.g., PEG).

[0330] In some embodiments, the linker has the following structure:

[0331] [ka] Including, n and m each independently represent an integer of 0 to 10.

[0332] In some embodiments, the linker has the following structure:

[0333] [ka] Including, n and m each independently represent an integer of 0 to 10.

[0334] In some embodiments, the linker has the following structure:

[0335] [ka] Including, In the formula, n is 1 to 32.

[0336] In some embodiments, the linker has the following structure:

[0337] [ka] Including, In the formula, n is 1 to 16.

[0338] Conjugates As stated above, the present disclosure further relates to compounds provided herein (e.g., radicals thereof) conjugated directly or via a linker to a targeting moiety that targets a cellular pattern recognition receptor (e.g., a TLR7 and / or 8 (TLR7 / 8) agonist as described above). In some embodiments, the targeting ligand comprises a folate ligand, or a functional fragment or analog thereof, e.g., a pteroyl amino acid. In some embodiments, the linker is non-releasable. In some embodiments, the conjugate provides a targeting molecule with a non-releasable linker, thereby reducing the systemic exposure of the TLR7 / 8 agonist. In some embodiments, the conjugate provides a targeting molecule with a non-releasable linker, thereby reducing the systemic adverse effects of the TLR7 / 8 agonist.

[0339] It is understood that any combination of compound radicals (e.g., compound radicals in any one of Tables 1 or 2), linkers (e.g., as provided herein), and ligand radicals (e.g., ligand radicals in any one of Tables 3-6) can be combined to form a conjugate provided herein. In some embodiments, the compound radical or ligand radical is a carbon atom or a heteroatom (e.g., O, S, N, etc.). In some embodiments, the compound radical is C or O. In some embodiments, the ligand radical is C or O. In some embodiments, the attachment point of the compound and the ligand (e.g., via the linker) depends on the configuration of the radicals. In some embodiments, the linker includes a spacer (e.g., as described elsewhere herein). It is also understood that any conjugate provided herein can be synthesized in a similar manner to that presented in the examples.

[0340] Non-limiting examples of conjugates provided herein are presented in Table 7.

[0341] [Table 7-1]

[0342] [Table 7-2]

[0343] [Table 7-3]

[0344] Non-limiting examples of conjugates provided herein are presented in Table 8.

[0345] [Table 8-1]

[0346] [Table 8-2]

[0347] [Table 8-3]

[0348] [Table 8-4]

[0349] [Table 8-5]

[0350] In some examples, a conjugated compound provided herein (e.g., of a first treatment) has the structure of Formula XIV (or a functional fragment or analog thereof, including a TLR7 agonist of Formula III conjugated to folate, e.g., via a releasable linker):

[0351] [ka]

[0352] In another embodiment, the conjugated compounds provided herein have the structure of Formula XV (or a functional fragment or analog thereof (e.g., compound 3B), including a TLR7 agonist of Formula II conjugated to a folate, e.g., via a releasable linker):

[0353] [ka]

[0354] In yet another embodiment, the conjugated compounds provided herein have the structure of Formula XVI (or a functional fragment or analog thereof, including a TLR7 agonist of Formula II conjugated to a folate, e.g., via a non-releasable linker comprising three PEGs (e.g., Compound 3D):

[0355] [ka]

[0356] In yet another embodiment, the conjugated compounds provided herein have the structure of Formula XVII (or a functional fragment or analog thereof, including a TLR7 agonist of Formula II conjugated to a folate, e.g., via a non-releasable linker comprising 12 PEG):

[0357] [ka]

[0358] Further embodiments of the conjugated compounds provided herein have the structure of Formula XVIII (or a functional fragment or analog thereof (e.g., compound 3D'), including the TLR7 agonist of Formula II conjugated to folate, e.g., via a non-releasable linker comprising 16 PEG):

[0359] [ka]

[0360] Further embodiments of the conjugated compounds provided herein have the structure of Formula XIX (or a functional fragment or analog thereof, including, for example, a TLR7 agonist of Formula III conjugated to folate (Compound 1B):

[0361] [ka]

[0362] In some embodiments, TLR-7 / 8 agonists that are conjugated with folate provide specificity for diseased cell types. In one embodiment, a folate-TLR7 / 8 agonist conjugate can be delivered (e.g., specifically) to endosomes of FRβ+macrophages, for example, while limiting exposure of the system to the TLR-7 / 8 agonist.

[0363] Additional Embodiments As described herein, the compound (e.g., of the first treatment) comprises an immunomodulator conjugated (via a linker or directly) to a targeting moiety. In certain exemplary embodiments, the compound (e.g., of the first treatment) has the structure of formula (2-II):

[0364] [ka] TLR agonist having or a pharma- ceutically acceptable salt thereof, wherein: R 1 , R 3 , R 4 , R 5 each independently represents H, alkyl, alkoxy, alkenyl, alkynyl, alicyclic, aryl, biaryl, halo, heteroaryl, -COR 2x ,

[0365] [ka] and R 2 are H, -OH, -NH2, -NHR 2x , N3, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2,

[0366] [ka] and Z is a group of the formula GL-, GO-, GLO-, GLO-alkyl-, GLS-, G-SO2-NH-, GL-NR a R b -, GLS(O) x -Alkyl-, GL-CO-, GL-aryl-, GL-NH-CO-NH-, GL-NH-O-, GL-NH-NH-, GL-NH-CS-NH, GLC(O)-alkyl-, GL-SO2-,

[0367] [ka] Based on L is a linker and G is a folate receptor binding ligand; R a and R b are each independently H, halo, hydroxy, alkoxy, aryl, amino, acyl, or C(O)R c and R c is alkyl, aryl, oxy or alkoxy, x is 0 to 3, R 2x and R 2y are each independently selected from the group consisting of H, -OH, -CH2-OH, -NH2, -CH2-NH2, -COOMe, -COOH, -CONH2, -COCH3, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl; R 2z are -NH2 and -NR 2q R 2q’ , -OR 2q , -SO-R 2q and -COR 2q are independently selected from the group consisting of R 2q and R 2q’ are each independently alkyl or H;

[0368] [ka] is a 3-10 membered N-containing non-aromatic monocyclic or bicyclic heterocycle; In formula 2-II, X 1 , X 2 and X 3 CR q or N and R q each is independently hydrogen, halogen, or optionally substituted alkyl; In formula 2-II, n is an integer of 0 to 30, and m is an integer of 0 to 4.

[0369] One embodiment is a compound represented by the structure of formula (2-IIA):

[0370] [ka] (In the formula, R 1 is optionally substituted alkyl (e.g., acyclic or cyclic) (e.g., optionally substituted with one or more substituents, each of which is independently halogen, alkyl, heteroalkyl, alkoxy, or cycloalkyl); R 2 H, -OR z , -SO2N(R z )2, -NR 2x R 2y or N3, R 2x and R 2y are each independently hydrogen, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or optionally substituted alkyl (e.g., optionally substituted with one or more substituents, each of which is independently oxo, halogen, alkyl, heteroalkyl, alkoxy, or cycloalkyl), and R z are each independently hydrogen, halogen or optionally substituted alkyl; R2x and R 2y taken together form an optionally substituted heterocycloalkyl (e.g., the optionally substituted heterocycloalkyl is a monocyclic or bicyclic heterocycloalkyl and / or the optionally substituted heterocycloalkyl is a 3-10 membered heterocycloalkyl); R 3 each is independently halogen, -N3, -CN, -NO2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, aryl, heteroaryl, heterocycloalkyl, amino, hydroxy, carbonyl, or thiol, wherein alkyl, alkoxy, heteroalkyl, cycloalkyl, or heterocycloalkyl is optionally substituted; R 4 and R 5 are each independently alkyl, alkoxy, halogen, or cycloalkyl, wherein alkyl, alkoxy, and cycloalkyl are optionally substituted; X 1 , X 2 and X 3 are each independently q or N and R q each is independently hydrogen, halogen, or optionally substituted alkyl; Z is LG, L is a linker, and G is a folate receptor binding ligand; n is 1 to 6, and m is 0 to 4. or a pharma- ceutically acceptable salt thereof.

[0371] One embodiment provides a compound having the structure of Formula (2-II) or (2-IIA), or a pharma- ceutically acceptable salt thereof, wherein n is 1 to 30. In one embodiment, n is 1 to 6. In another embodiment, n is 1 to 3. In another embodiment, n is 1 or 2. In another embodiment, n is 0. In another embodiment, n is 1.

[0372] One embodiment is R1 In one embodiment, R is an optionally substituted alkyl group. 1 is an optionally substituted C-C alkyl. In another embodiment, R 1 is an optionally substituted acyclic C-C alkyl. In another embodiment, R 1 is butyl.

[0373] One embodiment is R 2 But -NR 2x R 2y In one embodiment, R is a compound having the structure of Formula (2-II) or (2-IIA), or a pharma- ceutically acceptable salt thereof. 2 is NH2.

[0374] One embodiment is R 3 is H, or a pharma- ceutically acceptable salt thereof.

[0375] One embodiment is R 4 In one embodiment, R is an alkyl group. 4 is methyl.

[0376] One embodiment is R 5 In one embodiment, R is an alkyl group. 5 is methyl.

[0377] One embodiment is R 4 and R 5 In one embodiment, R is an alkyl group, R is an alkyl group, and R is an alkyl group. 4 and R 5 are each methyl.

[0378] One embodiment provides a compound having the structure of Formula (2-II) or (2-IIA), or a pharma- ceutically acceptable salt thereof, wherein m is 0. In another embodiment, m is 1. In another embodiment, m is 2. In another embodiment, m is 3. In another embodiment, m is 4.

[0379] In one embodiment, X 1 , X 2 and X 3 and n is an integer from 1 to 3, and n is an integer from 1 to 3. In one embodiment, X is an integer from 1 to 3, or n is an integer from 1 to 3. 1 is N. In another embodiment, X 2 is N. In another embodiment, X 3 is N.

[0380] One embodiment provides a compound having the structure of formula (2-II) or (2-IIA), or a pharma- ceutically acceptable salt thereof, the compound having the structure:

[0381] [ka] It is represented by:

[0382] In certain embodiments, the compound is a conjugated compound comprising a TLR agonist (e.g., an immunomodulator) having the structure of formula (2-II) or a pharma- ceutically acceptable salt thereof, which has the structure:

[0383] [ka] It is represented by:

[0384] One embodiment is a structure (or radical) of formula (2-III):

[0385] [ka] a TLR agonist expressed by and pharma- ceutically acceptable salts thereof, wherein: R 1 , R 3 , R 4 and R 5 each independently represents H, alkyl, alkoxy, alkenyl, alkynyl, alicyclic, aryl, biaryl, halo, heteroaryl, -COR 2x ,

[0386] [ka] and R 2x and R 2y are each independently selected from the group consisting of H, -OH, -CH-OH, -NH, -CH-NH, -COOMe, -COOH, -CONH, -COCH, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl; R 2z are -NH2 and -NR 2x R 2y , -OR 2x , -SO-R 2x and -COR 2x are independently selected from the group consisting of Z is a group of the formula GL-, GL-CO-, GLC(O)-alkyl-, L is a linker, and G is a folate receptor binding ligand; X 1 , X 2 and X 3 are CR q or N and R q each is independently hydrogen, halogen, or optionally substituted alkyl; In formula 2-III, n is an integer of 0 to 30, and m is an integer of 0 to 4.

[0387] One embodiment is a TLR agonist represented by the structure (or radical) of formula (2-IIIA):

[0388] [ka] (In the formula, R 1 is optionally substituted alkyl (e.g., acyclic or cyclic) (e.g., optionally substituted with one or more substituents, each of which is independently halogen, alkyl, heteroalkyl, alkoxy, or cycloalkyl); Y is H, -OR z , -NR 2x R 2y , -SR z , -SOR z , -SO3R z , -N3, -COR z , -COOR z , -CONR z 2. -COSR z , -SO2N(R z )2 or -CON(R z )2, where: R 2x and R 2y are each independently hydrogen, -N(R z )2, -CON(R z )2, -C(R z )2-N(R z )2, -CS-N(R z )2, or optionally substituted alkyl (e.g., optionally substituted with one or more substituents, each of which is independently oxo, halogen, alkyl, heteroalkyl, alkoxy, or cycloalkyl), and R z are each independently hydrogen, halogen or optionally substituted alkyl; R 2x and R 2y taken together form an optionally substituted heterocycloalkyl (e.g., the optionally substituted heterocycloalkyl is a monocyclic or bicyclic heterocycloalkyl and / or the optionally substituted heterocycloalkyl is a 3-10 membered heterocycloalkyl); R 3each is independently halogen, -N3, -CN, -NO2, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, aryl, heteroaryl, heterocycloalkyl, amino, hydroxy, carbonyl, or thiol, wherein alkyl, alkoxy, heteroalkyl, cycloalkyl, or heterocycloalkyl is optionally substituted; R 4 and R 5 are each independently alkyl, alkoxy, halogen, or cycloalkyl, wherein the alkyl, alkoxy, or cycloalkyl is optionally substituted; X 1 , X 2 and X 3 are each independently q or N and R q each is independently hydrogen, halogen, or optionally substituted alkyl; Z is LG, L is a linker, and G is a folate receptor binding ligand; n is 1 to 6, and m is 0 to 4. or a pharma- ceutically acceptable salt thereof.

[0389] One embodiment provides a compound having the structure of Formula (2-III) or (2-IIIA), or a pharma- ceutically acceptable salt thereof, wherein n is 1 to 30. In one embodiment, n is 1 to 6. In another embodiment, n is 1 to 3. In another embodiment, n is 1 or 2. In another embodiment, n is 0. In another embodiment, n is 1. In another embodiment, n is 1 and Y is OH. In another embodiment, n is 1 and Y is NH2.

[0390] One embodiment provides a compound having the structure of formula (2-III) or (2-IIIA), or a pharma- ceutically acceptable salt thereof, wherein Y is OH.

[0391] One embodiment provides a compound having the structure of formula (2-III) or (2-IIIA), or a pharma- ceutically acceptable salt thereof, wherein Y is NH2.

[0392] One embodiment provides a compound having the structure of formula (2-III) or (2-IIIA), or a pharma- ceutically acceptable salt thereof, wherein n is 1 and Y is OH.

[0393] One embodiment provides a compound having the structure of formula (2-III) or (2-IIIA), or a pharma- ceutically acceptable salt thereof, wherein n is 1 and Y is NH2.

[0394] One embodiment provides a compound having the structure of formula (2-III) or (2-IIIA), or a pharma- ceutically acceptable salt thereof, wherein n is 0 and Y is NH2.

[0395] One embodiment is R 1 In one embodiment, R is an optionally substituted alkyl. 1 is an optionally substituted C-C alkyl. In another embodiment, R 1 is an optionally substituted acyclic C-C alkyl. In another embodiment, R 1 is butyl.

[0396] One embodiment is R 3 is H, or a pharma- ceutically acceptable salt thereof.

[0397] One embodiment is R 4 In one embodiment, R is an alkyl group. 4 is methyl.

[0398] One embodiment is R 5In one embodiment, R is an alkyl group. 5 is methyl.

[0399] One embodiment is R 4 and R 5 In one embodiment, R is an alkyl group, and R is an alkyl group. 4 and R 5 are each methyl.

[0400] One embodiment provides a compound having the structure of Formula (2-III) or (2-IIIA), or a pharma- ceutically acceptable salt thereof, wherein m is 0. In another embodiment, m is 1. In another embodiment, m is 2. In another embodiment, m is 3. In another embodiment, m is 4.

[0401] In one embodiment, X 1 , X 2 and X 3 In one embodiment, the compound has the structure of formula (2-III) or (2-IIIA), or a pharma- ceutically acceptable salt thereof, wherein each X is N. 1 is N. In another embodiment, X 2 is N. In another embodiment, X 3 is N.

[0402] In some embodiments, the compound has the structure:

[0403] [ka] or a pharma- ceutically acceptable salt thereof.

[0404] In some embodiments, the compound has the structure:

[0405] [ka] or a pharma- ceutically acceptable salt thereof.

[0406] As previously described, the compounds of the present disclosure, including the TLR-7 / 8 conjugates provided herein, can be conjugated to targeting moieties via linkers.Any of the linkers provided herein can be used with the TLR7 / 8 agonists provided herein.For example, and without limitation, in some embodiments, the conjugates provided herein that include non-releasable linkers reduce or eliminate the toxicity of the components released from the conjugate in their free form (e.g., the free form of the compounds and / or ligands provided herein).

[0407] At least one embodiment of the present disclosure provides a compound having the structure of formula (2-II), (2-IIA), (2-III) or (2-IIIA), or a pharma- ceutically acceptable salt thereof, wherein L is a cleavable linker.

[0408] In some embodiments, one or more linkers of the compounds provided herein may include PEG, PEG derivatives, or any other linkers known in the art or subsequently developed that can achieve the objectives described herein. In some embodiments, the linker may be repeated n times, where n is a positive integer. For example, and without limitation, n may be any integer selected from the ranges of 1-16, 1-32, 1-64, or 1-96. The number of repeats in the linker may be selected to achieve the desired functionality, size, and / or potency of the compound, and / or the desired application. In some embodiments, one or more of the linkers include one or more spacers (e.g., this may also be used to specifically design the characteristics of the compound).

[0409] In some embodiments, the linker is a hydrolyzable linker. In some embodiments, the linker is a non-hydrolyzable linker. In some embodiments, the linker is an optionally substituted heteroalkyl. In some embodiments, the linker is a substituted heteroalkyl containing at least one substituent selected from the group consisting of alkyl, hydroxyl, oxo, PEG, carboxylate and halo. In some embodiments, the linker comprises a spacer (e.g., as described elsewhere herein).

[0410] At least one embodiment provides a compound having the structure of formula (2-II), (2-IIA), (2-III) or (2-IIIA), or a pharma- ceutically acceptable salt thereof, wherein L is a hydrolyzable linker (e.g., an amide, ester, ether, or sulfonamide).

[0411] In another embodiment, L is an optionally substituted heteroalkyl. In some embodiments, the heteroalkyl is unsubstituted. In other embodiments, the heteroaryl is substituted with at least one substituent selected from the group consisting of alkyl, hydroxyl, acyl, PEG, carboxylate, and halo. In another embodiment, L is a heteroalkyl substituted with at least one disulfide bond in its backbone.

[0412] In another embodiment, L is a peptide or peptidoglycan having at least one disulfide bond in its backbone.

[0413] In another embodiment, L is a cleavable linker that can be cleaved by enzymatic reaction, reactive oxygen species (ROS), or reducing conditions.

[0414] In some embodiments, L is a group of the formula: -NH-CH-CR 6 R 7 -SS-CH2-CH2-O-CO-, R 6 and R 7are each independently H, alkyl, or heteroalkyl.

[0415] In some embodiments, L has the formula:

[0416] [ka] is or contains the group In the formula, p is 0 to 30, and d is 1 to 40. R 8 and R 9 are each independently H, alkyl, cyclic, aryl, or heteroalkyl.

[0417] One embodiment provides a compound having the structure of formula (2-II), (2-IIA), (2-III) or (2-IIIA), or a pharma- ceutically acceptable salt thereof, wherein L is a non-cleavable linker.

[0418] One embodiment provides a compound having the structure of formula (2-II), (2-IIA), (2-III) or (2-IIIA), or a pharma- ceutically acceptable salt thereof, wherein L is a non-hydrolyzable linker.

[0419] In some embodiments, L is selected from the group consisting of alkylene, heteroalkylene, -O-alkynylene, alkenylene, acyl, aryl, heteroaryl, amide, oxime, ether, ester, triazole, PEG, and carboxylate.

[0420] In one embodiment, L is an alkyl ether. In another embodiment, L is an amide. In another embodiment, L is a peptide or peptidoglycan. In another embodiment, L is an amino acid. In another embodiment, L is PEG (e.g., -OCH2-CH2-O-). In another embodiment, L is a polysaccharide. In another embodiment, L has the structure:

[0421] [ka] is represented by w is an integer from 0 to 5, and p is an integer from 1 to 30.

[0422] In one embodiment, L is selected from the following list:

[0423] [ka] (wherein n is 0 to 30).

[0424] Folate Receptor Binding Ligands One embodiment provides a compound having structural formula (2-II), (2-IIA), (2-III) or (2-IIIA), or a pharma- ceutically acceptable salt thereof, in which G is a folate receptor binding ligand. In one embodiment, G is or is derived from folate, folic acid, or a functional fragment or derivative thereof. In one embodiment, G is a folate or folic acid derivative. In another embodiment, G is pteroic acid or a pteroyl derivative.

[0425] One embodiment is where G is a group of formula (2-IV) or this group:

[0426] [ka] Including, Each R is independently

[0427] [ka] and R is a natural or unnatural amino acid, or a derivative or fragment thereof, or a pharma- ceutically acceptable salt thereof.

[0428] In one embodiment, G is a group of formula (2-V) or a group thereof:

[0429] [ka] The present invention provides a compound having the structure of formula (2-II), (2-IIA), (2-III) or (2-IIIA), or a pharma- ceutically acceptable salt thereof, comprising:

[0430] One embodiment is where G is a group of formula (2-VI) or this group:

[0431] [ka] or a pharma- ceutically acceptable salt thereof, wherein R is an integer from 1 to 3;

[0432] One embodiment has the structure:

[0433] [ka]

[0434] [ka]

[0435] [ka]

[0436] [ka] or a pharma- ceutically acceptable salt thereof.

[0437] One embodiment has one of the following structures:

[0438] [ka]

[0439] [ka]

[0440] [ka]

[0441] [ka] or a pharma- ceutically acceptable salt thereof.

[0442] The compounds described herein can be prepared by conventional methods of organic synthesis practiced by those skilled in the art. The general reaction sequences outlined below represent general methods useful for preparing the compounds of the present disclosure and are not intended to be limiting in scope or utility.

[0443] The description of the compound of the present invention is limited by the principles of chemical bond formation known to those skilled in the art.Therefore, when a group can be substituted by one or more of several substituents, such substitutions are selected to conform to the principles of chemical bond formation to give a compound that is not inherently unstable and / or is known to those skilled in the art to be likely to be unstable under ambient conditions such as aqueous, neutral and some known physiological conditions.For example, heterocycloalkyl or heteroaryl is bonded to the rest of the molecule through ring heteroatom according to the principles of chemical bond formation known to those skilled in the art, thereby avoiding the compound being inherently unstable.

[0444] The term "identical" or % "identity" in the context of two or more polypeptide sequences refers to two or more sequences or subsequences that are identical or have a specified percentage of identical peptides (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned over a comparison window or maximum match over a specified region, such as a target end, a folate end, a linker or a warhead). Such sequences are then said to be "substantially identical". In other words, the identity exists over one or more regions throughout the sequence, so long as the general shape and structure of the molecules, as well as hydrogen bonds, are maintained to be substantially compatible with the target binding site and function as an agonist thereto, if appropriate.

[0445] The compounds described herein can be administered in unit dosage forms and / or compositions that contain one or more pharma- ceutically acceptable carriers, adjuvants, diluents, excipients and / or vehicles, and combinations thereof.As used herein, the term "administering" and its terminology generally refer to any and all means of introducing the compounds described herein into host subjects, including, but not limited to, oral, intravenous, intramuscular, subcutaneous, transdermal, inhalation, buccal, ocular, sublingual, vaginal, rectal, and similar routes of administration.

[0446] The administration of the compounds of the present disclosure as salts may be appropriate.The examples of acceptable salts include, but are not limited to, alkali metal (e.g., sodium, potassium or lithium) or alkaline earth metal (e.g., calcium) salts.However, when administered to the subject to be treated, generally any non-toxic and effective salt is acceptable.Similarly, "pharmaceutical acceptable salt" refers to the salt with counter ion that may be used in pharmaceutical products. Such salts can include, but are not limited to, (1) acid addition salts that 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, or organic acids such as acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or (2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion, or coordinates to an organic base such as ethanolamine, diethanolamine, triethanolamine, trimethamine, N-methylglucamine, etc. Pharmaceutically acceptable salts are well known to those of skill in the art, and any such pharma-ceutically acceptable salt may be contemplated in connection with the embodiments described herein.

[0447] Acceptable salts can be obtained using standard procedures known in the art, including but not limited to reacting sufficiently acidic compounds with a suitable base to provide a physiologically acceptable anion. Suitable acid addition salts are formed from acids which form non-toxic salts. Illustrative, though non-limiting, examples include acetate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hybenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, saccharate, stearate, succinate, tartrate, tosylate, and trifluoroacetate. Suitable base salts of the compounds described herein are formed from bases that form non-toxic salts.Non-limiting illustrative examples include arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.Half-salts of acids and bases, such as hemisulfate and hemicalcium salts, can also be formed.

[0448] As used herein, the term "composition" generally refers to any product that contains more than one component, including the compounds described herein. It should be understood that the compositions described herein may be prepared from the isolated compounds described herein, or from the salts, solutions, hydrates, solvates and other forms of the compounds described herein. It is understood that certain functional groups, such as hydroxy, amino and similar groups, may form complexes with water and / or various solvents in various physical forms of the compounds. It should also be understood that the compositions may be prepared from various amorphous, non-amorphous, partially crystalline, crystalline and / or other morphological forms of the compounds described herein, and that the compositions may be prepared from various hydrates and / or solvates of the compounds described herein. Thus, a pharmaceutical composition that lists the compounds described herein includes each of the compounds described herein, or any combination thereof, or its individual forms, various morphological forms, and / or solvates or hydrate forms.

[0449] The compounds of the present disclosure can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in various forms that are compatible with the selected route of administration.For example, the pharmaceutical compositions can be formulated for and administered via oral or parenteral, intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intraventricular, intraurethral, ​​intrasternal, intracranial, intratumoral, intramuscular, topical, inhalation and / or subcutaneous routes.Indeed, in at least one embodiment, the compounds and / or compositions described herein can be administered directly into the bloodstream, into muscle, or into an internal organ.

[0450] For example, in at least one embodiment, the compounds may be administered systemically (e.g., orally) in combination with a pharma- ceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The percentage of the compositions and preparations may vary and may be between about 1 and about 99% by weight of the active ingredient, as well as binders, excipients, disintegrants, lubricants, and / or sweeteners, as known in the art. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0451] Preparation of parenteral compounds / compositions under sterile conditions, for example by lyophilization, can be easily carried out using standard pharmaceutical techniques well known to those skilled in the art. In at least one embodiment, the solubility of the compounds used in the preparation of parenteral compositions can be increased by the use of suitable formulation techniques, such as the incorporation of solubility enhancers.

[0452] As stated above, the compounds / compositions of the present disclosure may also be administered by infusion or injection (e.g., using needle (including microneedle) injectors and / or needle-free injectors). Solutions of the active compositions may be aqueous, optionally mixed with non-toxic surfactants, and / or may contain carriers or excipients such as salts, carbohydrates, and buffering agents (preferably pH 3-9), although for some applications, they may be more suitably formulated as sterile non-aqueous solutions or as dry forms used in conjunction with suitable vehicles such as sterile, pyrogen-free water or phosphate buffered saline (PBS). For example, dispersions may be prepared in glycerol, liquid PEG, triacetin, and mixtures thereof, as well as oils. Under ordinary conditions of storage and use, these preparations may further contain a preservative to prevent the growth of microorganisms.

[0453] Pharmaceutical dosage forms suitable for injection or injectable can include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient, which are optionally encapsulated in liposomes and are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions. In all cases, the final 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, including, for example and without limitation, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid PEG, etc.), vegetable oils, non-toxic glyceryl esters, and / or suitable mixtures thereof. In at least one embodiment, the proper fluidity can be maintained by the formation of liposomes, by maintaining 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 parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In certain cases, it is 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 inclusion of agents formulated to delay absorption, for example, aluminum monostearate and gelatin.

[0454] Sterile injectable solutions can be prepared by mixing active compound and / or one or more of the other components described above in the composition in the required amount of suitable solvent, and then filter sterilization.For the preparation of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying technique and freeze-drying technique, which obtains the powder of active ingredient and any additional desired components present in the solution previously sterile filtered.

[0455] For topical administration, it may be desirable to administer the compound to the skin as a composition or formulation in combination with a dermatologically acceptable carrier, which may be solid or liquid.For example, in certain embodiments, the solid carrier may comprise finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, etc.Similarly, useful liquid carriers may comprise water, alcohol or glycol, or water-alcohol / glycol blends, in which the compound may be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.Additionally or alternatively, adjuvants such as fragrances and antimicrobial agents may be added to optimize the properties for a given use.The resulting liquid composition may be applied from absorbent pads used to impregnate bandages and / or other dressings, may be sprayed onto the target area using pump-type or aerosol sprayers, or may simply be applied directly onto the desired area of ​​the subject.

[0456] Thickening agents such as synthetic polymers, fatty acids, salts and esters of fatty acids, fatty alcohols, modified cellulose, or modified inorganic materials may also be used with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like for application directly to the skin of a subject.

[0457] As used herein, the term "therapeutically effective", "therapeutically effective dose", "therapeutically effective amount", "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of a compound that, when administered once or throughout the course of a treatment cycle (unless otherwise specifically stated), affects the health, welfare or mortality of a subject (and, for example, but not limited to, delays the onset of symptoms associated with cancer and / or reduces the severity of one or more of the symptoms). Useful dosages of the compounds of the present disclosure can be determined by comparing their in vitro and in vivo activities in animal models. Methods for extrapolating effective dosages in mice and other animals to human subjects are known in the art. In practice, dosages of the compounds can vary considerably depending on the condition of the host subject, the cancer being treated, how the pathology is progressing, the route of administration of the compound, and tissue distribution, and the possible simultaneous use of other therapeutic treatments (such as radiation therapy, or additional drugs in combination therapy). The amount of the composition required for use in treatment (e.g., a therapeutically or prophylactically effective amount or dose) will vary not only depending on the particular application, but also on the selected salt (if applicable) and the characteristics of the subject (e.g., age, condition, sex, body surface area and / or weight of the subject, tolerance to the drug), and will ultimately be at the discretion of the attending physician, clinician, or otherwise. A therapeutically or prophylactically effective amount, or dose, can be in the range of, for example, about 0.05 mg / kg to about 30.0 mg / kg of patient body weight, or about 0.01 mg / kg to about 5.0 mg / kg of patient body weight, including but not limited to, 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, and 5.0 mg / kg (all of which are per kg of patient body weight).The total therapeutically or prophylactically effective amount of the compound may be administered in single or divided doses and, at the physician's discretion, may fall outside of the typical ranges given herein.

[0458] In another embodiment, the compound is administered at about 0.5 g / m 2 ~about 500mg / m 2 , about 0.5g / m 2 ~about 300mg / m 2 or about 100 g / m 2 ~about 200mg / m 2 In another embodiment, the amount is about 0.5 mg / m 2 ~about 500mg / m 2 , about 0.5mg / m 2 ~about 300mg / m 2 , about 0.5mg / m 2 ~about 200mg / m 2 , about 0.5mg / m 2 ~about 100mg / m 2 , about 0.5mg / m 2 ~about 50mg / m 2 , about 0.5mg / m 2 ~about 600mg / m 2 , about 0.5mg / m 2 ~about 6.0mg / m 2 , about 0.5mg / m 2 ~about 4.0mg / m 2 or about 0.5 mg / m 2 ~about 2.0mg / m 2 The total amount may be administered in a single dose or in divided doses and may be outside the typical ranges given herein, at the physician's discretion. These amounts are based on m of body surface area.

[0459] In some embodiments, in connection with measuring the expression of certain biomarkers and / or analyzing cytokine levels in samples from subjects, the important aspect of the present disclosure is not the particular method used to detect the marker or set of markers, but which markers are used to detect. There are many methods that can be used to detect the expression, quantification or profile of one or more biomarkers. Once the marker or set of markers to be detected or quantified is identified, any of several techniques (now known or hereafter developed) may be used under the provision of appropriate reagents. Once presented with the identified biomarker or biomarkers, the skilled artisan will be able to select an appropriate assay (e.g., PCR-based or microassay-based assay for nucleic acid markers, enzyme-linked immunosorbent assay (ELISA), protein or antibody microarray, or similar immunological assay, etc.) to carry out the methods disclosed herein.

[0460] Engineered Cells and Engineered Cell Therapies As set forth above, in certain embodiments, the method includes administering to the subject a first treatment and a second treatment. Various components of the second treatment embodiment (i.e., the engineered cells or engineered cell-based therapy or composition) are now described in more detail.

[0461] In addition to administering a compound (e.g., a first treatment), the method can include administering a second treatment comprising engineered cells and / or engineered cell compositions. Such engineered cells can be cytotoxic lymphocytes, such as cytotoxic T cells, NK cells, lymphokine-activated killer (LAK) cells, or a combination of two or more of the above. A variety of engineered cell therapies are now known in the art, and it will be understood that in certain embodiments, the second treatment can include any currently known, or later discovered, engineered cell, or cell therapy currently useful for treating or preventing cancer.

[0462] In certain embodiments, the engineered cells are NK cells prepared from progenitor or stem cells. In certain embodiments, the engineered cells are T cells prepared from progenitor or stem cells.

[0463] In at least one embodiment, the T lymphocytes (e.g., cytotoxic T lymphocytes) are engineered to express a CAR. In at least one embodiment, the NK cells are engineered to express a CAR.

[0464] CAR is a fusion protein that includes a recognition domain, a costimulatory domain and an activation signaling domain. In certain embodiments, CAR binds with high specificity to cell-surface antigens of immunosuppressive cells or cancerous cells.

[0465] In certain embodiments, the recognition region of the CAR can be an scFv, Fab fragment, etc., of an antibody that specifically (e.g., with high specificity) binds to a cell-surface antigen (e.g., cluster of differentiation 19 (CD19)). When the recognition region of the CAR comprises an scFv region, the scFv region can be prepared from (i) an antibody known in the art that binds to a targeting moiety, (ii) an antibody prepared de novo using at least one targeting moiety, such as a hapten, and (iii) a sequence variant derived from the scFv region of such an antibody, e.g., the scFv region has 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 scFv region from which they are derived.

[0466] "Percent (%) sequence identity" with respect to a reference to a polypeptide sequence is defined as the percentage of amino acid or nucleic acid residues in a candidate sequence that are identical to the residues in the reference sequence, respectively, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and any conservative substitutions are not considered as part of the sequence identity. Alignment to determine percent sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software. For example, the determination of percent identity or similarity between sequences can be performed, for example, by using the GAP program (Genetics Computer Group, software; currently available from Accelrys online), and alignment can be performed, for example, using the ClustalW algorithm (VNTI software, InforMax Inc). In addition, sequence databases can be searched using the nucleic acid or amino acid sequence of interest. Algorithms for database searching are usually based on the BLAST software (Altschul et al., 1990), although one skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm needed to achieve maximal alignment over the full length of the sequences being compared. In some embodiments, the percent identity can be determined along the full length of the nucleic acid or amino acid sequence.

[0467] The costimulatory domain of the CAR can act to enhance the proliferation and survival of cytotoxic lymphocytes when the CAR is bound to a targeting moiety. In certain embodiments, the costimulatory domain of the CAR can be CD28 (cluster of differentiation 28), CD137 (cluster of differentiation 137; 4-1BB), CD134 (cluster of differentiation 134; OX40), CD278 (cluster of differentiation 278; ICOS), CD2 (cluster of differentiation 2), CD27 (cluster of differentiation 27), CD40L (cluster of differentiation 2; CD154), DAP10, NKG2D, signaling lymphocyte activation molecule (SLAM)-related receptor family (such as 2B4), TLR, or a combination thereof. Those skilled in the art will understand that sequence variants of these costimulatory domains can be used without adversely affecting the present invention, where the variants have the same or similar activity as the domains when they are modeled. In various embodiments, 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.

[0468] In certain embodiments, the activation signaling domain generates a lymphocyte activation signal upon binding to the targeting portion of the CAR. Suitable activation signaling domains can be, but are not limited to, T cell CD3 chain, CD3 delta receptor protein, mbl receptor protein, B29 receptor protein, or Fc receptor gamma. Those skilled in the art will appreciate that sequence variants of these activation signaling domains can be used, where the variants have the same or similar activity as the domain when they are modeled. In various embodiments, 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.

[0469] The construct encoding the CAR 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), both of which are incorporated herein by reference in their entirety (collectively, "Protocols").

[0470] As non-limiting examples, plasmid or viral expression vectors (e.g., lentiviral vectors, retroviral vectors, Sleeping Beauty and PiggyBac (transposon / transposase systems, including non-viral mediated CAR gene delivery systems)) can be prepared that encode a fusion protein comprising a recognition region, one or more costimulatory domains and an activation signaling domain, linked in frame and in a 5' to 3' direction.

[0471] Other configurations are also permissible and include a recognition domain, an activating signaling domain and one or more costimulatory domains.

[0472] The term "vector" refers to any nucleic acid that functions to carry, harbor, or express a nucleic acid of interest. Nucleic acid vectors can have dedicated functions such as expression, packaging, pseudotyping, or transduction. Vectors can also have operational functions, such as when adapted for use as cloning or shuttle vectors. The structure of a vector can include any desired form that is feasible to produce and desirable for a particular use. These can include, for example, circular forms, such as plasmids and phagemids, as well as linear or branched forms. Nucleic acid vectors can be composed of, for example, DNA or RNA, and contain nucleotide derivatives, analogs, or mimetics, partially or completely. Such vectors can be obtained from natural sources, can be produced recombinantly, or can be chemically synthesized.

[0473] The positioning of the recognition region in the fusion protein is generally such that display of a region outside the cell is achieved. If desired, the CAR can also include additional elements such as a signal peptide (e.g., CD8α signal peptide) to ensure proper transport of the fusion protein to the cell surface, a transmembrane domain to ensure that the fusion protein remains an integral membrane protein (e.g., CD8α transmembrane domain, CD28 transmembrane domain, or CD3ζ transmembrane domain), and a hinge domain (e.g., CD8α hinge) that provides flexibility to the recognition region and allows for strong binding to the targeting moiety.

[0474] Cytotoxic lymphocytes (e.g., cytotoxic T lymphocytes or NK cells) can be engineered to express a CAR construct by transfecting a population of lymphocytes with an expression vector encoding the CAR construct. Suitable methods for preparing a transduced population of lymphocytes expressing a selected CAR construct are well known to those of skill in the art.

[0475] In one embodiment, the cells used in the methods described herein can be autologous cells, although heterologous cells can also be used if the patient being treated has received high doses of chemotherapy or radiation treatments that destroy the patient's immune system. In one embodiment, allogeneic cells can be used.

[0476] Lymphocytes can be obtained from a subject by means well known in the art.For example, T cells (e.g., cytotoxic T cells) can be obtained by collecting peripheral blood from a subject, subjecting the blood to Ficoll density gradient centrifugation, and then isolating a T cell population from the peripheral blood using a negative T cell isolation kit (such as EasySep™ T cell isolation kit).

[0477] In certain embodiments, the cell population need not be pure and may contain multiple types of cells, such as T cells, monocytes, macrophages, NK cells, and B cells. Furthermore, in at least one embodiment, the harvested population may comprise at least about 90% of a selected cell type, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of a selected cell type.

[0478] Generally, after obtaining the cells to be engineered, the cells are cultured under conditions that promote activation of the cells. In at least one embodiment, the culture conditions are such that the cells can be administered to a subject without concern for reactivity to components of the culture medium. For example, the culture conditions may be free of bovine serum products, such as bovine serum albumin. In one aspect, activation can be achieved by introducing a known activator, such as an anti-CD3 antibody, into the culture medium in the case of cytotoxic T cells. Other suitable activators are generally known and include, for example, anti-CD28 antibodies. The population of cells can be cultured under conditions that promote activation, for example, for about 1 to about 4 days. The appropriate level of activation can be determined by cell type, size, proliferation rate, or activation markers, as determined by flow cytometry.

[0479] In at least one embodiment, the population of cells is cultured under conditions that promote activation, and then the cells are transfected with an expression vector encoding a CAR. In various embodiments, vectors and transfection methods suitable for use are known in the art. After transfection, the cells can be administered immediately to the patient, or the cells can be cultured for a period of time that allows the cells to be recovered from transfection, such as 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 for about 5 to about 12 days, about 6 to about 13 days, about 7 to about 14 days, or about 8 to about 15 days. In one aspect, suitable culture conditions can be similar to the conditions under which the cells are cultured to be activated, either with or without the agent used to promote activation.

[0480] Thus, as described above, the methods of treatment described herein can further include the steps of 1) obtaining a population of autologous or heterologous cytotoxic cells (e.g., cytotoxic T lymphocytes, NK cells, etc.), 2) culturing the cells under conditions that promote activation of the cells, and 3) transfecting the cells with an expression vector encoding a CAR to form a CAR-expressing cell.

[0481] Alternatively, the methods of treatment described herein may further comprise the step of preparing T cells or NK cells from progenitor or stem cells as known in the art.

[0482] Once the cells are transfected (if appropriate) and activated, a composition containing the engineered cells can be prepared and administered to a subject. In at least one embodiment, the engineered cells can be cultured using a culture medium that is free of any animal products, such as bovine serum. In another embodiment, tissue culture conditions commonly used by those skilled in the art can be used to avoid contamination with bacteria, fungi and mycoplasma. In certain embodiments, the cells are pelleted, washed and resuspended in a pharma- ceutically acceptable carrier or diluent prior to administration to a patient.

[0483] Exemplary compositions comprising engineered cells include compositions comprising cells in sterile 290 mOsm saline in insoluble cryomedia (containing Plasma-Lyte A, dextrose, sodium chloride injection, human serum albumin and DMSO), in 0.9% NaCl with 2% human serum albumin, or any other sterile 290 mOsm insoluble material. In certain embodiments, depending on what the culture medium is, the engineered cells can be administered in the culture medium as a composition or can be concentrated and resuspended in the culture medium prior to administration. In various embodiments, the engineered cell composition can be administered to the subject by any suitable means, such as parenteral administration, e.g., intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, or intrathecal.

[0484] In one aspect, the total number of engineered cells and the concentration of cells in the composition administered to a patient will vary depending on several factors, including the type of lymphocytes used (e.g., cytotoxic T lymphocytes), the binding specificity of the CAR (if applicable), which cancer, the location of the cancer in the patient, the means used to administer the composition to the patient, and the health, age, and weight of the patient being treated. In various embodiments, suitable compositions comprising engineered cells include those having volumes of about 0.1 ml to about 200 ml and about 0.1 ml to about 125 ml.

[0485] Similarly, methods of treating a patient for cancer are provided, comprising administering to the patient any of the compounds described above, and administering to the patient any of the engineered cell compositions or engineered cell therapies described above, such that the patient is treated for cancer.

[0486] In the methods described herein, the cancer can be further imaged before administering the compound or its pharma- ceutically acceptable salt, or the engineered cell composition (e.g., CAR-expressing cytotoxic lymphocyte composition or CAR-NK cell composition) to the subject. The cancer can also or alternatively be imaged during or after administration to diagnose metastasis and, for example, the effectiveness of treatment. For example, imaging can be performed by positron emission tomography (PET) imaging, magnetic resonance imaging (MRI) or single photon emission computed tomography (SPECT) / computed tomography (CT) imaging. The imaging method can be any suitable imaging method known in the art.

[0487] The cancer can be any cancer. "Cancer" has its plain and unambiguous meaning when read in the context of this specification and can include, but is not limited to, a group of diseases involving abnormal cell growth that has the potential to invade or spread (i.e., metastasize) to other parts of the body. Examples include, but are not limited to, brain, thyroid, lung, pancreas, kidney, stomach, gastrointestinal stromal, endometrial, breast, cervical, ovarian, colon, prostate cancer, leukemia, lymphoma, other blood-related cancers, or head and neck cancer. In certain embodiments, the cancer to be treated is a tumor. In certain embodiments, the cancer is malignant.

[0488] In some aspects of these embodiments, the cancer is a folate receptor-expressing cancer, including but not limited to, a folate receptor alpha-expressing cancer. In other embodiments, the cancer is a folate receptor beta-expressing cancer.

[0489] In the compounds, compositions and methods, all embodiments of compounds (including, but not limited to, drug moieties or pharma- ceutically acceptable salts thereof, and / or ligand / targeting moieties thereof), engineered cells and / or engineered cell compositions and vector compositions are applicable, including, but not limited to, linker embodiments.

[0490] Methods for treating and preventing cancer In addition to the compounds, engineered cells, engineered cell compositions and therapeutic methods described herein, methods of achieving cancer treatment and / or preventing cancer are also provided. It will be understood that unless expressly specified otherwise, the term "compound" as used in connection with the description of the methods can encompass any of the compounds and / or conjugates described herein.

[0491] In certain embodiments, provided herein is a method of treating a subject suffering from cancer, comprising administering to the subject a first treatment comprising any of the compounds provided herein, or a pharma- ceutically acceptable salt thereof, or a (e.g., pharmaceutical) composition comprising any of the compounds provided herein, and administering to the subject a second treatment comprising engineered cells. The compound of the first treatment (or a pharma- ceutically acceptable salt thereof) can comprise a compound comprising a folate ligand, or a functional fragment or analog thereof, linked to a TLR agonist via a linker. In certain embodiments, the compound of the first treatment comprises a compound comprising any one of the structures of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula VII, Formula VIII, Formula IX, Formula X, Formula XI, Formula XII, Formula XIII, Formula XIV, Formula XV, Formula XVI, Formula XVII, Formula XVIII, Formula XX, Formula XXX, Formula 2-I, Formula 2-II, Formula 2-III, Formula 2-IV, Formula 2-V, or Formula 2-VI. In some embodiments the immune modulator comprises an agonist of TLR7, 8, 9 or 7 / 8.

[0492] In certain embodiments of the method, administering the compound of the first treatment activates anti-tumor cells or proinflammatory signaling cascades in the subject. The anti-tumor cells can be T cells, engineered T cells and / or T cells prepared from progenitor or stem cells. In certain embodiments, the anti-tumor cells are NK cells, engineered NK cells, or NK cells prepared from progenitor or stem cells. In certain embodiments, the anti-tumor cells are macrophages.

[0493] The second treatment can include CAR T cell therapy, CAR-NK cell therapy, or engineered stem cell therapy. The first and second treatments can be administered simultaneously, sequentially, consecutively, or alternatingly.

[0494] In certain embodiments, the TLR agonist of the first treatment compound has the structure of formula 2-I (or a radical thereof), or a pharma- ceutically acceptable salt of formula 2-I:

[0495] [ka] and In formula 2-I, R 1 , R 3 , R 4 and R 5 are each independently hydrogen (H), alkyl, alkoxy, alkenyl, alkynyl, alicyclic, aryl, biaryl, halo, heteroaryl, -COR 2x ,

[0496] [ka] and R 2 are H, -OH, -NH2, -NHR 2x , N3, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2,

[0497] [ka] and Y is H, -OH, -NH2, -NHR 2x , -OR 2X , -SO-R 2x , -SH, -SO3H, -N3, -CHO, -COOH, -CONH2, -COSH, -COR 2x , -SO2NH2, alkenyl, alkynyl, alkoxyl, -NH-CH2-NH2, -CONH2, -SO2NH2, -NH-CS-NH2,

[0498] [ka] and Where: R 2x and R 2y are each independently selected from the group consisting of H, -OH, -CH-OH, -NH, -CH-NH, -COOMe, -COOH, -CONH, -COCH, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl; R 2z are -NH2 and -NR 2q R 2q’ , -OR 2q , -SO-R 2q and -COR 2q R 2q and R 2q’ are each independently alkyl or H;

[0499] [ka] is a non-aromatic, monocyclic or bicyclic 3-10 membered N-containing heterocycle; In formula 2-I, X 1 , X 2 and X 3 are each independently q or N and R q each is independently H, halogen, or optionally substituted alkyl; In formula 2-I, n is an integer of 0 to 30, and m is an integer of 0 to 4.

[0500] In certain embodiments, the compound of the first treatment is:

[0501] [ka] or a pharma- ceutically acceptable salt thereof.

[0502] In some embodiments, the compound of the first treatment has the structure of the following formula:

[0503] [ka] or a pharma- ceutically acceptable salt thereof.

[0504] In some embodiments of the methods provided herein, the immune modulator comprises a TLR agonist and has a structure of formula X or XX (or is a radical of formula X or XX), or a pharma- ceutically acceptable salt of formula X or XX:

[0505] [ka] and In formulas X and XX: R1 is -NH2 or -NH-R 1X and R2 is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R 2X , -OR 2X , -SR 2X ,

[0506] [ka] and

[0507] [ka] is a 3-10 membered N-containing non-aromatic monocyclic or bicyclic heterocycle; In formula X, R3 is -OH, -SH, -NH2 or -NH-R 1X and In the formula XX, X is CH, CR2 or N; R 1X , R 2X and R 2Y is each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl. In certain embodiments, compounds 1, 2, and 3 each comprise the structure of formula X:

[0508] In certain embodiments, administering the first treatment further comprises administering or applying to the subject a therapeutically effective amount of a compound of the first treatment. The compound of the first treatment may be administered to the subject, for example, intravenously, intramuscularly, intraperitoneally, topically, or by inhalation.

[0509] In certain embodiments, the TLR agonist of the first treatment compound has the structure of (or is a radical of) the following formula, or a pharma- ceutically acceptable salt thereof:

[0510] [ka] and During the ceremony, R 1 is an amine group, R 2 is a single bond, -NH-, R 3 is H, alkyl, hydroxyl or any other substituted radical thereof; X is CH, NH, O or S; The linker is R 1 , R 2 or R 3 are bonded at

[0511] In some embodiments of the method, the linker of the first treatment compound comprises a PEG linker or a PEG derivative linker. In certain embodiments, the pharma- ceutically acceptable salt of the method is selected from hydrobromide, citrate, trifluoroacetate, ascorbate, hydrochloride, tartrate, trifluoromethanesulfonate, maleate, mesylate, formate, acetate, or fumarate.

[0512] Methods of preventing or treating a disease state are also provided. Such methods can include contacting a cell with at least one engineered cell configured to treat a disease state, and contacting the cell with at least one compound comprising an immunomodulator or a pharma- ceutically acceptable salt thereof linked to a folate ligand, or a functional fragment or analog thereof, via a linker, where the immunomodulator or a pharma- ceutically acceptable salt thereof targets a pattern recognition receptor. The at least one engineered cell can be any of the engineered cells, therapies, or compositions described herein. The at least one compound comprising an immunomodulator or a pharma- ceutically acceptable salt thereof can be any of the compounds described herein. In certain embodiments, the at least one compound comprising an immunomodulator or a pharma- ceutically acceptable salt thereof comprises a TLR agonist having the structure of Formula 2-I (or a radical thereof) above or a pharma- ceutically acceptable salt of Formula 2-I above, or having the structure of Formula X or XX above (or being a radical or a pharma- ceutically acceptable salt of Formula X or XX). In certain embodiments, the at least one compound comprises an immunomodulator and has the following structure:

[0513] [ka] or a pharma- ceutically acceptable salt thereof.

[0514] In some embodiments, the cells comprise cells of a subject experiencing or at risk of experiencing cancer or a cancerous disease state, and contacting the cells with at least one compound further comprises administering or applying a therapeutically effective amount of at least one compound to the subject. In some embodiments, the at least one compound is administered to the subject intravenously, intramuscularly, intraperitoneally, topically, or by inhalation.

[0515] In some embodiments, the method includes obtaining or having obtained a sample from the subject; quantifying an expression level of one or more biomarkers in the sample, wherein each of the one or more biomarkers is selected from the group consisting of CCL18, arginase 1 (Arg1), matrix metallopeptidase 9 (MMP9), metalloproteinase 3 (TIMP3), IL-1β, hydroxyproline, collagen, PDGF, TGFβ, FRβ, TNFα, IFN-γ, anti-mannose receptor (CD206), cluster of differentiation 86 (CD86), cluster of differentiation 163 (CD163), IL-6, chemokine 10 (CXCL10), immune interferon (IFNα). The method further comprises the steps of: selecting, comparing the expression level of each of the one or more biomarkers in the sample to the expression level of such biomarkers in a control, and administering or having administered to the subject a therapeutically effective amount of an unconjugated agonist or inhibitor if CCL18, Arg1, MMP9, TIMP3, IL-1β, PDGF, TGFβ, FRβ, CD206, CD163, hydroxyproline or collagen is upregulated relative to the expression level of the control, or if TNFα, IFN-γ, IL-6, CXCL10, IFNα or CD86 is downregulated or not expressed relative to the expression level of the control. In some embodiments, the folate ligand, or functional fragment or analog thereof, is specific for and binds to FRβ in a cell.

[0516] Methods of treating a subject (e.g., suffering from cancer) are also provided. In certain embodiments, such methods include administering to the subject engineered cells and administering to the subject a compound comprising a folate ligand, or a functional fragment or analog thereof, conjugated to a TLR agonist via a linker. The compound comprising a folate ligand, or a functional fragment or analog thereof, can be any of the compounds described herein that comprise a folate ligand, or a functional fragment or analog thereof. In certain embodiments, the TLR agonist has the structure of formula 2-I (or a radical thereof) above or a pharma- ceutically acceptable salt of formula 2-I, or has the structure of formula X or XX above (or is a radical or a pharma- ceutically acceptable salt of formula X or XX). In certain embodiments, the compound TLR agonist has the structure of (or is a radical of) the following formula, or a pharma- ceutically acceptable salt thereof:

[0517] [ka] and During the ceremony, R 1 is an amine group, R 2 is a single bond, -NH-, R 3 is H, alkyl, hydroxyl or any other substituted radical thereof; X is CH, NH, O or S; The linker is R 1 , R 2 or R 3 are bonded at

[0518] In certain embodiments, the linker comprises a PEG linker or a PEG derivative linker, R 3 or R 1 、 R 2 Or R 3or a releasable linker attached at

[0519] In at least one embodiment, a method for treating and / or preventing cancer is provided. The method includes administering to a subject a therapeutically effective amount of one or more compounds comprising a targeting moiety (such as a folate receptor binding ligand) conjugated (via a linker or otherwise) to a drug to reprogram M2-like macrophages in a cancerous tissue or organ to an M1-like phenotype. For example, the drug may be a Toll-like receptor agonist (e.g., having formula I, III, 2-I or IV), or any other molecule or compound effective for reprogramming macrophages from an M2 phenotype to an M1 phenotype, conjugated to folate. In at least one embodiment, the drug may be selected from a TLR3 agonist, a TLR7 agonist, a TLR7 / 8 agonist, a TLR8 agonist and a TLR9 agonist. In some embodiments, the drug can reprogram M2-like macrophages to an M1 phenotype, thereby reducing anti-inflammatory cytokine and growth factor production. For example, in at least one embodiment, such reprogramming of M2-like macrophages to an M1 phenotype results in activation of pro-inflammatory signaling cascades within anti-tumor cells and / or the TME.

[0520] Also provided is a method for preventing or treating cancer, which comprises contacting a cell (e.g., a cancer cell) with at least one of CAR-expressing cytotoxic lymphocytes and / or other engineered cells, and contacting the cell with at least one compound comprising an immunomodulator or a pharmaceutically acceptable salt thereof linked to a folate ligand, or a functional fragment or analog thereof, via a linker, wherein the immunomodulator or a pharmaceutically acceptable salt thereof targets a pattern recognition receptor. Such at least one compound comprising an immunomodulator can comprise any of the compounds described herein. In certain embodiments, at least one compound is administered to a subject intravenously, intramuscularly, intraperitoneally, topically, or by inhalation.

[0521] Contacting cells with at least one compound immunomodulator or a pharma- ceutically acceptable salt thereof can, in certain embodiments, reprogram the subject's M2-type macrophages to M1-type macrophages (i.e., a pro-inflammatory phenotype).

[0522] In certain embodiments, the immunomodulator or its pharmaceutically acceptable salt is a TLR7, 8, 9 or 7 / 8 agonist.For example, the immunomodulator or its pharmaceutically acceptable salt can be a TLR7 agonist and the linker can be a releasable linker.In other embodiments, the linker is a non-releasable linker.

[0523] The step of administering or applying a therapeutically effective amount of at least one compound to the subject and contacting the cells with at least one of the CAR-expressing cytotoxic lymphocytes and / or otherwise engineered cells / lymphocytes can further include administering or applying a therapeutically effective amount of the CAR-expressing cytotoxic lymphocytes and / or otherwise engineered cells / lymphocytes (e.g., T cells or NK cells derived from stem or progenitor cells) to the subject.

[0524] In at least one embodiment, a method of treating a subject suffering from or at risk of experiencing a disease state is provided, the disease state comprising cancer, the method comprising contacting a cell of the subject with at least one compound. The at least one compound may comprise any of the compounds of the present disclosure, and in at least one exemplary embodiment, comprises a targeting moiety specific for FRβ. In some examples, the contacting of the cell can be achieved by administering to the subject at least one compound intravenously, intramuscularly, intraperitoneally, topically, orally or by inhalation, or by any of the other administration modalities described herein. Additionally or alternatively, the at least one compound may comprise a composition containing one or more pharma- ceutically acceptable carriers, adjuvants, diluents, excipients and / or vehicles, or combinations thereof. The dosage of the at least one compound administered may be appropriately modified by the clinician. However, the at least one compound is preferably administered in a therapeutically or prophylactically effective amount, and in at least one embodiment, the dosage ranges between 1 nmol / kg of subject body weight and 50 nmol / kg of subject body weight.

[0525] Referring now to FIG. 2, a flow chart depicting a method 1900 for treating cancer is shown using one or more of the compounds of the present disclosure. In at least one case, the method 1900 includes a step of contacting (administering) a cell of a subject with at least one compound including an immunomodulator (or a pharma- ceutically acceptable salt thereof), such as and without limitation, a TLR7 agonist, linked to a folate ligand, or a functional fragment or analog thereof, via a linker (step 1902). In at least one exemplary embodiment, the immunomodulator or a pharma- ceutically acceptable salt thereof targets a pattern recognition receptor. The cell may include, for example, a cell of a subject experiencing or at risk of experiencing a cancerous disease state, and the at least one compound may include any of the compounds provided herein.

[0526] In at least one embodiment, contacting the cells with at least one compound 1902 further comprises administering or applying a therapeutically effective amount of the at least one compound to a subject. Additionally or alternatively, the at least one compound may comprise a composition containing one or more pharma- ceutically acceptable carriers, adjuvants, diluents, excipients and / or vehicles, or combinations thereof.

[0527] The subject can be a mouse, a human, or any other mammal.

[0528] In addition to step 1902, method 1900 may optionally include steps 1904-1910. In step 1904, a biological sample is obtained from a subject, and in step 1906, the level of expression of one or more biomarkers in the sample is quantified. For example, the sample may be obtained from a volume of peripheral blood drawn from the subject.

[0529] The quantification step 1906 can be performed using any suitable method known in the art and can include, for example, qPCR, mass spectrometry, ELISA and / or any other modality capable of measuring / quantifying biomarker expression. In at least one exemplary embodiment, the one or more biomarkers are selected from the group consisting of CCL18, Arg1, MMP9, TIMP3, IL-1β, PDGF, TGFβ, FRβ, hydroxyproline, collagen, TNFα, IFN-γ, CD206, CD163, IL-6, CXCL10, IFNα and CD86.

[0530] In step 1908, the level of expression of each of the one or more biomarkers in the sample is compared to the level of expression of such biomarkers in a control. The control can be a healthy individual, or simply an individual not experiencing the disease condition under discussion. In at least one embodiment, a clinical difference between the expression level of one or more biomarkers in the sample and the expression level of the relevant biomarker in the control can indicate that the subject is afflicted with the disease condition under discussion. For example and without limitation, the comparison step 1908 indicates that the expression of one or more of the biomarkers CCL18, Arg1, CD163, MMP9, TIMP3, IL-1β, PDGF, TGFβ, FRβ, hydroxyproline, collagen and / or CD206 (i.e., "cancer biomarkers") is upregulated compared to the control, indicating a subject experiencing an anti-inflammatory immune response linked to an M2-like macrophage phenotype. Thus, in at least one embodiment, such results indicate a need to administer one or more compounds of the present disclosure to reprogram such M2-like macrophages to an M1 phenotype and one or more anti-tumor cell activation and / or pro-inflammatory signaling cascades.

[0531] In contrast, if comparison step 1908 indicates that expression of the above-mentioned biomarkers is downregulated compared to the control, or expression of one or more of TNFα, IFN-γ and / or CD86 ("pro-inflammatory biomarkers") is upregulated compared to the control, in certain embodiments this indicates either that the subject has responded positively to a previously administered compound (if applicable) and / or that the subject is experiencing a pro-inflammatory immune response linked to the M1 phenotype.

[0532] Optionally, in step 1910, an alternative treatment may be performed if the expression of one or more of the cancer biomarkers in the sample is upregulated relative to the respective expression levels in the control, or if the expression of one or more of the proinflammatory biomarkers is downregulated in the sample relative to the respective expression levels in the control. In at least one embodiment, the alternative treatment may include administering a therapeutically effective amount of a derivative of the at least one compound previously administered in step 1902, the derivative comprising at least one compound previously administered that has been modified with respect to the use of either a different targeting moiety, a different linker size, and / or a different immune modulator in an attempt to better optimize the effectiveness of the at least one compound for the subject. Additionally or alternatively, other treatments may be used, including those conventionally known for treating the fibrotic disease under discussion. Steps 1904-1910 may be included and / or repeated as necessary or desired to meet established criteria and / or to confirm that the active ingredient is effective in improving the symptoms of the cancer disease state.

[0533] As mentioned above, the methods of the present disclosure may be used to treat and / or prevent cancer (whether folate receptor positive or folate receptor negative). For example, in certain instances, such methods include administering to a host subject a therapeutically and / or prophylactically effective amount of one or more compounds, the one or more compounds comprising a targeting moiety attached to a drug (by a linker or other method) to reprogram M2-like macrophages in cancerous and / or tumor cells to an M1-like phenotype, such as, for example, a target TLR-7 agonist. If the cancer is folate receptor negative, such administration may further act to reduce or inhibit MDSCs present in such tissues / tumors. Additional drugs may also be administered in conjunction with such methods, including, for example, PI3K inhibitors, signal transducer and activator of transcription 6 (STAT6) inhibitors, mitogen-activated protein kinase (MAPK) inhibitors, inducible nitric oxide synthase (iNOS) inhibitors, and anti-inflammatory drugs (e.g., methotrexate). In at least one embodiment, the drug may inactivate MDSCs.

[0534] The disclosed compounds and compositions may be used alone or in combination with administering an engineered cell therapy. For example, in addition to step 1902, method 1900 may further include administering a CAR-T cell or another type of engineered cell therapy to the subject.

[0535] Such combination therapy methods of the present disclosure can be performed using any engineered cells suitable for treating cancer and can include the use of more than one of these types of agents. In certain embodiments, the engineered cells used in this combination therapy are CAR T cells, and may also (or alternatively) include engineered stem cells and other cells.

[0536] The engineered cells used in combination with the inventive conjugate compounds or compositions of the present disclosure can be either CAR T cells, stem cells or other engineered cells or their combinations.Various compatible cell therapies (also called cellular immunotherapy) are known in the art for use in treating cancer, and T cell immunotherapy in particular has been the subject of much interest.Some non-limiting examples of such therapies include engineered T cell receptor (TCR) therapy, CAR T cell therapy and natural killer (NK) cell therapy.

[0537] In certain embodiments, any one or more engineered cell therapies can be combined with administration of an immune modulator (or a pharma- ceutically acceptable salt thereof) linked via a linker to a folate ligand, or a functional fragment or analog thereof, and at least one compound, including, for example, but not limited to, a TLR7 agonist, for use in the methods of the present disclosure. In certain embodiments, engineered cell therapies co-administered with the targeting TLR-7, TLR-7 / 8, TLR-8 and / or TLR-9 agonists of the present disclosure include CAR T cell therapies disclosed herein.

[0538] The activity and survival of CAR-T cells in the tumor microenvironment (TME) are regulated by multiple immune suppressive cells, including tumor-associated macrophages (TAMs), myeloid-derived immune suppressive cells (MDSCs), cancer-associated fibroblasts (CAFs), tumor-associated neutrophils (TANs) and regulatory T cells (Tregs). TAMs present a particular challenge to the killing of solid tumors. TAMs often account for up to 50% of the mass of solid tumors, interacting with cancer cells and other immune cells and promoting tumor growth by promoting angiogenesis, immune suppression and inflammation. The inventive method can modify the TME itself, thereby increasing the therapeutic efficacy and potency of CAR T cells (and other engineered cells), especially in solid tumors.

[0539] In certain embodiments, as the present disclosure supports, the treatment with the novel folate-targeting TLR agonist, such as TLR7 or TLR7 / 8 agonist, reverses the immunosuppressive environment in cancerous tumor tissue by reprogramming M2-type TAM and MDSC into M1-type proinflammatory antitumor macrophages.When engineered cells are administered in conjunction with such therapy, the resulting altered TME can enhance the efficacy and effectiveness of such engineered cell-based immunotherapy.Thus, in certain embodiments, the use of the co-administration method of the present disclosure can eliminate or improve cancer (even solid tumors) without the need for additional interventions such as surgery, chemotherapy and / or radiation therapy.

[0540] In certain approaches, administration of both the conjugated agonist compounds of the present disclosure and the engineered cell therapy results in more than additive inhibition of cancer growth.

[0541] Thus, when multiple therapeutic agents and / or therapies are co-administered, dosages may be adjusted as recognized in the relevant art. "Co-administration" and combination therapy are not limited to simultaneous administration, but rather also include treatment regimens in which a targeted TLR-7, TLR-8, TLR-9 and / or TLR-7 / 8 agonist is administered to a subject at least once (for example) during the course of treatment that includes administering a cell-based therapy to the subject.

[0542] When the method of combination therapy includes administering more than one treatment to a subject, it should be understood that the order, timing, number, concentrations and amounts of administration are limited only by the medical requirements and limitations of the treatment (i.e., two treatments can be administered to a subject, for example, simultaneously, sequentially, sequentially, alternating, or according to any other regimen).

[0543] Chemical Examples Example A: Synthesis of Compound 1A Compound 1A was synthesized according to Scheme 1 below and as reported by Nikunj M. Shukla, Cole A. Mutz, Subbalakshmi S. Malladi, Hemamli J. Warshakoon, Rajalakshmi Balakrishna, and Sunil A. David, "Regioisomerism-dependent TLR7 agonism and antagonism in an imidazoquinoline; Structure-Activity Relationships in Human Toll-Like Receptor 7-Active Imidazoquinoline Analogues," J Med Chem. 2012 Feb 9; 55(3): 1106-1116.

[0544] [ka]

[0545] Step 1: Synthesis of 1-amino-2-methylpropan-2-ol (compound) 2,2-Dimethyloxirane (0.1 g, 1.388 mmol) was added dropwise to 20 mL of ice-cold solution of ammonium hydroxide. The reaction mixture was stirred at room temperature for 12 h. The solvent was removed under vacuum and the residue was dissolved in methanol. Di-tert-butyl dicarbonate (0.75 g, 3.47 mmol) was added to the reaction mixture and stirred for 4 h. The mixture was purified using column chromatography (24% ethyl acetate (EtOAc) / hexane) to give tert-butyl 2-hydroxy-2-methylpropylcarbamate. Pure tert-butyl 2-hydroxy-2-methylpropylcarbamate was dissolved in 5 mL of trifluoroacetic acid and stirred for 35 min. The solvent was removed under reduced pressure to give 1-amino-2-methylpropan-2-ol as the trifluoroacetate salt 1'. 1H NMR 500 MHz (500 MHz, CDC13, δ (ppm)): δ 8.62 (s, 2H), 3.02 (d, 2H), 2.06-2.04 (m, 2H), 1.37-1.34 (s, 6H).

[0546] Step 2: Synthesis of 2-methyl-1-(3-nitroquinolin-4-ylamino)propan-2-ol (compound 2) To a solution of 4-chloro-3-nitroquinoline (compound 1) (250 mg, 1.2 mmol) and Et3N (0.5 ml, 3 mmol) in a 4:1 mixture of toluene and 2-propanol, the trifluoroacetate salt of 1-amino-2-methylpropan-2-ol (compound) (450 mg, 2.4 mmol) was added. The mixture was heated to 70° C. for 30 min until a solid began to precipitate. The reaction mixture was then cooled, filtered, and washed with toluene / 2-propanol (7:3), ether, and cold water. The residue was dried at 80° C. to give 2-methyl-1-(3-nitroquinolin-4-ylamino)propan-2-ol (compound 2). Liquid chromatography-mass spectrometry (LCMS) analysis: [M+H] + m / z=261.

[0547] Step 3: Synthesis of 1-(3-aminoquinolin-4-ylamino)-2-methylpropan-2-ol (compound 3) 2-Methyl-1-(3-nitroquinolin-4-ylamino)propan-2-ol (compound 2) (450 mg, 1.72 mmol) was dissolved in methanol and hydrogenated with Pd / C as catalyst using a hydrogen balloon for 4 hours. The solution was then filtered using Celite, followed by solvent evaporation under reduced pressure to give 1-(3-aminoquinolin-4-ylamino)-2-ethylpropan-2-ol (compound 3). LCMS: [M+H] + m / z = 231. H NMR 500 MHz (CDC13, δ (ppm)): δ 8.12 (s, 1H), 7.61-7.58 (m, 1H), 7.48-7.40 (m, 2H), 4.90 (s, 2H), 3.47 (2H), 1.35-1.21 (s, 6H).

[0548] Step 4: Synthesis of 1-(4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol (compound 5, TLR7A) To a solution of compound 3 (100 mg, 0.43 mmol) in anhydrous THF was added triethylamine (66 mg, 0.65 mmol) and valeryl chloride (62 mg, 0.52 mmol). The reaction mixture was then stirred for 6-8 hours and then the solvent was removed under vacuum. The residue was dissolved in EtOAc, washed with water and brine, and then dried over Na2SO4 to give the intermediate amide compound. This was dissolved in methanol (MeOH), then calcium oxide was added and heated in a microwave at 110 °C for 1 hour. The solvent was then removed and the residue was purified using column chromatography (9% MeOH / dichloromethane) to give compound 4 (58 mg). To a solution of compound 4 in a solvent mixture of MeOH:dichloromethane:chloroform (0.1:1:1), 3-chloroperbenzoic acid (84 mg, 0.49 mmol) was added and the solution was refluxed at 45-50 °C for 40 minutes. The solvent was then removed and the residue was purified using column chromatography (20% MeOH / dichloromethane) to give the oxide derivative (55 mg). This was then dissolved in anhydrous dichloromethane, then benzoyl isocyanate (39 mg, 0.26 mmol) was added and heated at 45° C. for 15 min. The solvent was then removed under vacuum and the residue was dissolved in anhydrous MeOH, then excess sodium methoxide was added. The reaction mixture was then heated at 80° C. for 1 h. The solvent was removed under vacuum and the residue was purified using column chromatography (11% MeOH / dichloromethane) to give compound 5. LCMS: [M+H] + m / z = 312. H NMR 500 MHz (CDC13, δ (ppm)): δ 8.16-8.15 (d, 1H), 7.77-7.46 (d, 1H), 7.46-7.43 (m, 1H), 7.33-7.26 (m, 1H), 3.00-2.97 (m, 2H), 1.84-1.78 (m, 2H), 1.47-1.41 (m, 2H), 1.36 (s, 6H), 0.98-0.95 (m, 3H).

[0549] Example B: Synthesis of Compound 1B Compound 1A can then be used to synthesize compound 1B according to Scheme 2 below.

[0550] [ka]

[0551] Compound 1A, the folate and the linker are either commercially available or can be prepared according to methods known to those skilled in the art.

[0552] To a solution of compound 5 (33 mg, 0.106 mmol) and dimethylaminopyridine (39 mg, 0.319 mmol) in 4 mL of methylene chloride under nitrogen atmosphere, heterobifunctional linker 7 (88 mg, 0.213 mmol) was added at room temperature, and the mixture was stirred at reflux temperature for 7 h, at which point thin layer chromatography (TLC) analysis of the mixture indicated >80% conversion. The mixture was concentrated and purified by column chromatography using 10% acetonitrile in methylene chloride as the eluent. The pure product compound 9 was obtained as a light yellow solid. To a solution of drug-linker intermediate compound 9 (1.0 equiv.-1.5 equiv.) in DMSO containing dimethylaminopyridine (1 equiv.) was added a solution of compound 8 (1 equiv.) in dimethylsulfoxide (DMSO) in three portions at 20 min intervals. After stirring at room temperature under argon for 1-2 hours, LCMS analysis of the mixture indicated the formation of the desired folate-drug conjugate (compound 10) as the major product. The mixture was purified by preparative high performance liquid chromatography (HPLC). LCMS: [M+H] + m / z = 959. 1H NMR (500 MHz, DMSO-d6) δ 8.58 (s, 1H), 8.49 (d, J = 8.8 Hz, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.83 - 7.74 (m, 1H), 7.54 (d, J = 8.0 Hz, 2H), 7.48 (t, J = 7.6 Hz, 1H), 7.41 (s, 1H), 7.06 (s, 1H), 6.81 (d, J = 6.2 Hz, 1H), 6.61 (d, J = 8.3 Hz, 2H), 6.27 (s, 1H), 4.43 (d, J = 5.9 Hz, 2H), 4.28 (t, J = 6.6 Hz, 2H), 4.00 (d, J = 25.7 Hz, 3H), 3.03 (t, J = 7.5 Hz, 2H), 2.97 (dd, J = 13.0, 6.5 Hz, 1H), 2.09 (s, 2H), 1.81 (s, 7H), 1.40 (q, J = 7.4 Hz, 2H), 1.22 (s, 2H), 1.13 (s, 2H), 0.91 (t, J = 7.4 Hz, 3H).

[0553] Example C: Synthesis of Compound 2A Compound 2A can be synthesized according to Scheme 3 and Scheme 4.

[0554] [ka]

[0555] [ka]

[0556] Cysteine-loaded Wang's resin (11) was first deprotected using 20% ​​piperidine in dimethylformamide (DMF). The free amine was treated with Fmoc-Glu(OtBu)-COOH in the presence of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), N,N-diisopropylethylamine (DIPEA) and DMF. The coupled product was deprotected using 20% ​​piperidine in DMF and treated with pteroic acid in the presence of PyBop, DIPEA and DMF to generate compound 12. The trifluoroacetyl group was deprotected with 50% ammonia-DMF solution. Finally, the resin was cleaved using a cocktail solution of trifluoroacetic acid:triisopropylsilane:water:tris(2-carboxyethyl)phosphine and purified using HPLC to give folate-cysteine ​​(13) as a yellow solid.

[0557] Compound 14 was first treated with a heterobifunctional linker reagent (15) to give the folate-cystine disulfide intermediate (16), which was then reacted with folate-cysteine ​​(13) in DMSO and purified using HPLC to produce compound 17 (e.g., compound 2A). All compounds were characterized by LCMS using ammonium bicarbonate and acetonitrile as the buffer system. The observed mass from LCMS for compound 2A was [M+H]+=1082.2.

[0558] Example D: Synthesis of TLR7 agonist TLR7-1A Solvents, reagents and starting materials were purchased from commercial suppliers and used as received unless otherwise noted. All reactions were carried out at room temperature unless otherwise noted. Starting materials were either purchased from commercial suppliers or synthesized according to methods described herein or using literature procedures.

[0559] The synthesis of the TLR7 agonist TLR7-1A is depicted in Scheme 5:

[0560] [ka]

[0561] Example E: Synthesis of TLR7 agonist TLR7-1B The synthesis of the TLR7 agonist TLR7-1B is depicted in Scheme 6:

[0562] [ka]

[0563] Example F: Synthesis of TLR7 agonist TLR7-1C The synthesis of the TLR7 agonist TLR7-1C is depicted in Scheme 7:

[0564] [ka]

[0565] Example G: Synthesis of releasable TLR7-folate conjugates The synthesis of a releasable TLR7-folate conjugate is depicted in Scheme 8:

[0566] [ka]

[0567] Example H: Synthesis of non-releasable TLR7 folate conjugates The synthesis of a non-releasable TLR7 folate conjugate is depicted in Scheme 9:

[0568] [ka]

[0569] Example I: Synthesis of non-releasable TLR7 folate conjugates The synthesis of a non-releasable TLR7 folate conjugate (e.g., conjugate 1) is depicted in Scheme 10 (e.g., comprising compound 1):

[0570] [ka]

[0571] Example J: Synthesis of a non-releasable TLR7 folate conjugate The synthesis of non-releasable TLR7 folate conjugates is depicted in Schemes 11 and 12:

[0572] [ka]

[0573] [ka]

[0574] In light of the state of the art and in particular the above schemes, the present disclosure presents sufficient detail to enable one of ordinary skill in the art to utilize the concepts described herein to synthesize all of the other compounds of the present disclosure.

[0575] In describing representative embodiments, the disclosure may present a method and / or process as a particular order of steps. To the extent that the method or process does not depend on the particular order of steps described herein, the method or process should not be limited to the particular order of steps described. As one of ordinary skill in the art will recognize, other orders of steps may be possible. Thus, the particular order of steps disclosed herein should not be interpreted as a limitation on the claims. Furthermore, claims directed to methods and / or processes should not be limited to performing those steps in the order described, as one of ordinary skill in the art can readily recognize that the order may vary and still remain within the spirit and scope of the disclosure. EXAMPLES

[0576] Human monocytic THP-1 cells were obtained from the American Type Culture Collection and cultured in folate-deficient RPMI 1640 medium (Invitrogen, Carlsbad, CA) containing 10% heat-inactivated fetal bovine serum and 1% penicillin / streptomycin (Invitrogen, Carlsbad, CA). THP-1 cells were initially chosen as a model system because this human monocytic cell line is known to acquire an M2-like phenotype and produce significant amounts of anti-inflammatory cytokines upon stimulation with IL-4, IL-6, and IL-13.

[0577] IFN-γ, IL-4, interleukin-6 (IL-6) and interleukin-13 (IL-13) were obtained from Biolegend. Phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), all other reagents and solvents were purchased from Sigma.

[0578] [Example 1] Differentiation and polarization of THP-1 cells into M2-like macrophages in vitro THP-1 cells were seeded in 96-well plates at a density of 60,000 cells / well. Cells were incubated with 200 nM PMA for 48 hours, then differentiated into non-polarized macrophages by incubation in fresh RPMI medium for 24 hours. The resulting macrophages were polarized to an M2-like phenotype by incubation with 20 ng / ml IL-4, 20 ng / ml IL-13, and 5 ng / ml IL-6 for 3 days, then reprogrammed with various concentrations of Compound 1A and Compound 1B for 48 hours, and collected for genetic analysis by quantitative polymerase chain reaction (qPCR). Cultures were maintained at 37°C in a humidified 5% CO2 incubator.

[0579] To evaluate whether a potent TLR7 agonist (e.g., compound 1A; e.g., Formula III) can reprogram anti-inflammatory (M2) macrophages to a less cancerous phenotype, IL-4, IL-6 and IL-13 stimulated THP-1 cells were incubated with various concentrations of non-targeted compound 1A and the mRNA levels of several cancer markers, namely CCL18, CD206, IL-1β and PDGFα and β, were examined.

[0580] As shown in Figures 3A-3C, incubation with compound 1A for 48 hours induced a decrease in the expression of CCL18, CD206, and IL-1β, suggesting that the TLR7 agonist can indeed promote the shift of these anti-inflammatory (M2) polarized THP-1 cells to a pro-inflammatory (M1) phenotype. Furthermore, when the expression of TNFα, a marker of the pro-inflammatory phenotype, was examined, an increase in its expression was observed (Figure 3D), confirming that a shift of THP-1 from pro-inflammatory to anti-inflammatory properties had occurred.

[0581] [Example 2] Assessment of macrophage reprogramming To confirm that a folate-conjugated TLR7 agonist could induce the same reprogramming of THP-1 observed in Example 1, compound 1B was prepared, in which a releasable linker linking folate to compound 1A was constructed via a disulfide self-immolative bond such that compound 1A could be released into the reducing environment of the intracellular endosome following internalization of compound 1B.

[0582] Various concentrations of either Compound 1A or Compound 1B were incubated with the polarized THP-1 macrophages for the indicated times, after which culture medium was collected to analyze secreted cytokines and harvest cells for qPCR analysis.

[0583] Total RNA was prepared using the Quick-RNA™ MicroPrep kit (Zymo Research, Irvine, CA) according to the manufacturer's recommended protocol at 1x10 5 ~2x10 5 Macrophages were isolated from 1000 cells / mL of 10 ... IRAK-4 was used as an indicator of TLR7 stimulation. To control for the specificity of the amplified products, melting curve analysis was performed. No amplification of non-specific products was observed in any of the reactions. For each marker, each sample was analyzed independently in triplicate.

[0584] The above study was repeated (see Figures 3A-3F, grey bars) and the same qualitative changes were observed, only the magnitude of effect of compound 1B was somewhat reduced. This reduction in potency was expected since the non-targeted TLR7 agonist immediately enters cultured cells, whereas its folate-targeted counterpart is designed to enter cells only after folate receptor-binding and receptor-mediated endocytosis.

[0585] Figures 4A-4E and 5A-5D show graphical data representing various marker levels measured from M2 macrophage-induced THP-1 cells that were subsequently incubated with various concentrations of Compound 1B or Compound 1A for 2 hours, washed with PBS, and, for the data shown in Figures 5A-5D, re-incubated for 46 hours (for the data shown in Figures 4A-4E, cells were collected immediately after the first 2 hours of incubation). Both data sets were collected for genetic analysis of cells by qPCR. Figures 4A-4C show CCL18 mRNA levels (Figures 4A and 5A), CD206 mRNA levels (Figures 4B and 5B), IL-1β mRNA levels (Figures 4C and 5C), and PDGFβ mRNA levels (Figure 4E). These data support the downregulation of the M2 anti-inflammatory phenotype following administration of the test compounds. In particular, compound 1B down-regulated macrophage cancer / M2 markers more than compound 1A. Furthermore, FIG. 4D shows CD86mRNA levels and FIG. 5D shows TNFα levels, and this data supports that M1-like phenotype was up-regulated after administration of test compound. PDGFα data is not shown because no significant difference was observed after treatment during collection.

[0586] Because low molecular weight, water-soluble drugs such as Compound 1A and Compound 1B are often cleared from the body within 2 hours of injection, a more physiologically relevant in vitro model of in vivo drug exposure involves incubating cells with the drug for only 2 hours and then testing the efficacy of the drug after an additional 46 hours of incubation in the absence of the drug. As shown in Figures 4A-4E, when THP-1 cells were incubated with the TLR7 agonist for 2 hours before replacing the drug-containing medium with drug-free medium, Compound 1B was observed to have superior potency compared to Compound 1A, especially in the case of TNFα induction, where the folate-targeted conjugate dramatically improved. This is likely because the folate-targeted TLR7 agonist was captured by folate receptor-positive cells, while Compound 1A was not retained by the same cells.

[0587] These data support that compound 1B should be more effective at reprogramming anti-inflammatory macrophages in vivo, with the added advantage that folate-conjugated drugs (e.g., compound 1B) should also cause less systemic toxicity because they recruit to FRβ-expressing macrophages and are unable to enter folate receptor-negative cells that predominate throughout the body (e.g., compound 1B is designed to be impermeable to folate receptor-negative cells).

[0588] Figures 6A-6D show representative graphical data of various marker levels measured from M2-induced THP-1 macrophages treated with various concentrations of drugs for 48 hours (Figures 6A and 6B) or treated with various concentrations of drugs for 2 hours, then replaced with fresh medium and cultured for the remaining 46 hours (Figures 6C and 6D). In both cases, cell supernatants were collected and secreted CCL18 protein and IL-1β were detected by ELISA. The data confirm that administration of TLR7 compounds or folate-targeted TLR7 compounds down-regulates the secretion of CCL18 and IL-1β in the low concentration range (0.1-10 nM).

[0589] Furthermore, to confirm that the above mRNA analysis accurately reflects the levels of anti-inflammatory cytokines produced by IL-4, IL-6 and IL-13 stimulated THP-1 cells, the concentrations of CCL18 and IL-1β polypeptides in the supernatants of THP-1 cells were quantified by ELISA assay. As shown in Figures 6A and 6B, both compound 1A and compound 1B induced a decrease in CCL18 and IL-1β when continuously incubated with the agonists for 48 hours, but compound 1B was still found to be superior when drug exposure was limited to only 2 hours (see Figures 6C and 6D).

[0590] [Example 3] Characterization of FRβ expression by flow cytometry Fluorescence-activated cell sorter (FACS) analysis was performed to measure FRβ expression on THP-1-derived macrophages. Cells were detached using Accutase® Cell Detachment Solution (Biolegend, San Diego, CA; #423201) and gently lifted using a cell scraper. Cells were washed with PBS and non-specific binding was blocked by incubating with Fc receptor blocking solution (Biolegend, San Diego, CA; #422301) for 10 minutes at room temperature. Biotinylated anti-human FRβ monoclonal antibody (m909) was then added and cells were incubated for an additional 30 minutes on ice before being washed in staining buffer (PBS supplemented with 2% FBS). The cells were then incubated on ice for 20 minutes in fluorescein-labeled streptavidin (BD Biosciences, Franklin Lakes, NJ; #554060), washed twice in PBS, stained with 7AAD (a viability stain) for 15 minutes, and analyzed by flow cytometry using BD Accuri C6 software (BD Biosciences, Franklin Lakes, NJ). Figure 6E shows the flow cytometry data, confirming that THP-1 macrophages were FRβ+ and therefore suitable for in vitro studies of compound 1B, as well as other studies described herein.

[0591] Figure 6F confirms that compound 1B remained stable during the incubation period in culture medium at 37° C. Indeed, compound 1B maintained its original structure after 48 hours of incubation.

[0592] [Example 4] Bleomycin-induced pulmonary fibrosis and anti-inflammatory macrophage reprogramming in vivo Studies were also conducted to determine whether macrophages in pulmonary fibrosis could be specifically targeted in vivo by folate-linked drugs. After testing multiple protocols for inducing pulmonary fibrosis in mice, a protocol was selected in which 0.75 mg / kg bleomycin (BM) was instilled into the lungs of C57BL / 6 mice through a tracheal incision, and the mice were allowed to progress to the inflammatory and fibrotic stages of fibrosis before treatment was initiated. (The BM model is widely considered useful in that it allows mechanistic studies related to fibrogenesis in an in vivo context.)

[0593] As shown in Figures 7A-7D, mice treated using this protocol typically exhibit fibrosis by day 7 after BM treatment, and this nascent fibrosis develops into severe fibrosis by day 14. Pathology then continues to progress for an additional 2-5 days before beginning to spontaneously resolve by day 21.

[0594] More specifically, 8-week-old male C57BL6 mice (average weight 22-25 g) from Charles River were housed under pathogen-free conditions at room temperature (22°C) under a 12-h light-dark cycle. Mice were fed a folate-deficient diet (Envigo Teklad Global rat food pellets) for 1 week prior to infusion of BM or PBS. Fresh water and folate-deficient diet were available ad libitum. All animal procedures conformed to National Institute of Health guidelines and were approved by the Purdue Animal Care and Use Approval Committee.

[0595] Following this, mice were anesthetized with ketamine / xylazine and the neck of the mouse was shaved using hair remover lotion and then disinfected with 70% alcohol. A small incision was made in the neck to expose the trachea. Mice were placed at a 75 degree angle and a 1 cc syringe with a 26G needle was used to inject 100 μL of sterile PBS or BM (Cayman Chemicals, Ann Arbor, MI; #13877) dissolved in PBS (0.75 mg / kg) into the trachea. Body weight was monitored every other day throughout the experiment.

[0596] To assess whether anti-inflammatory lung macrophages in these mice could be specifically targeted by folate-linked drugs, 10 days after infusion, 10 nmol (for in vivo imaging) or 100 nmol (for in vivo labeling) of a folate-linked near-infrared fluorescent dye (OTL38) was injected into the tail vein of BM-treated mice with or without a 200-fold excess of the FA glucosamine (a competitor of OTL38) and dye uptake in major organs was assessed.

[0597] After 2 hours, the mice were sacrificed using CO2 asphyxiation, and an incision was immediately made in the skin from the abdomen to the neck to expose the lungs and trachea. A small incision was then made in the upper part of the trachea to insert a blunt 22-gauge needle, and a nylon suture was tied around the trachea to close the trachea around the needle. The trachea (including the inserted needle), lungs, and heart were then removed en bloc by carefully cutting the connective tissue under the lungs, and the bronchus of the left lung was clipped using a Dieffenbach receptacle clip. The right lung was inject-and-aspirate with PBS three times using a 1 ml syringe, and the collected lavage fluid was stored on ice.

[0598] Bronchoalveolar lavage fluid (BALF) was then analyzed to determine how the targeted TLR7 agonist was working. BALF samples were centrifuged at 1500 rpm for 5 min at 4°C, and an aliquot of the supernatant was taken and stored at -80°C for cytokine / chemokine analysis. The cell pellet was resuspended and cultured in pre-warmed RPMI1640 medium for 2 h, then washed 3x with pre-warmed PBS before being collected for qPCR assay. The right lung was then tied with a nylon thread and used for subsequent analysis of hydroxyproline content. The left lung was inflated with 1 ml of PBS using the inserted syringe and transferred to 10% formalin solution for subsequent histological analysis.

[0599] The right lung lobes harvested above were weighed, placed in pressure-resistant vials (Supelco Inc., Bellefonte, PA; #27003) and hydrolyzed with 6N HCl (10 ml / g, v / w) in a sand bath at 120° C. for 3.5 hours. The hydrolyzed solution was cooled at 4° C. for 15 minutes, transferred to a 1.5 ml Eppendorf tube, and centrifuged at 12,000 rcf for 15 minutes at 4° C. The supernatant was carefully collected and aliquoted for hydroxyproline (HYP) analysis.

[0600] For subsequent HYP analysis, 10 μl of the samples were transferred to a 96-well plate and neutralized with 10 μl of 5.3 M sodium hydroxide solution. Isopropanol (40 μl) was then added to each well, followed by 20 μl of oxidation buffer, and the mixture was incubated for 5 min at room temperature on a shaker. Analysis reagent (260 μl) was added, and the plate was incubated for 30 s at room temperature on a shaker and immediately incubated at 60° C. for 25 min. 560 The absorbance was measured within 15 min at 37 °C for 10 min. All reagents were prepared according to previously reported protocols.

[0601] For histological analysis of lung sections, fixed lungs (see above) were embedded in paraffin, sectioned, and stained with hematoxylin-eosin (H&E), Masson's trichrome, or F3 (anti-mouse FRβ antibody). Tissue sections were examined in a blinded fashion by a qualified pathologist. 6 More than 10 cells were quantified per section using an Aperio-Image Scope (Leica Biosystems, Wetzlar, Del.).

[0602] CCL18 and IL-1β were quantified in the supernatants of induced THP-1 cells using the human DuoSet ELISA Development System (R&D Systems Europe, Abingdon, UK; #DY394-05) and IL-1 beta Human ELISA Kit (Thermo Fisher Scientific, Waltham, MA; #BMS224-2) as described by the manufacturer. BALF samples were analyzed for mouse IFN-γ using ELISA MAX™ Deluxe (Biolegend, San Diego, CA; #430804).

[0603] Finally, for in vivo folate imaging studies, major organs (heart, lungs, spleen, liver, small intestine, large intestine, and kidneys) were excised and imaged using an AMI live imager (Spectral Instruments Imaging, Tucson, AZ). For in vivo folate receptor labeling studies, lungs were collected immediately after euthanasia and digested with a lung dissociation kit (Miltenyi Biotec, Bergisch Gladbach, DE; #130-098-427) as described by the gentleMACS Octo Dissociator with Heathers (Miltenyi Biotec, Bergisch Gladbach, DE; #130-096-427) and filtered through a 70 μm cell strainer (Miltenyi Biotec, Bergisch Gladbach, DE; #130-098-462). Red blood cells were removed from harvested cells in the filtrate by ammonium sulfate lysis, washed 2x with cold PBS, and labeled with antibodies against desired macrophage markers (FITC-CD11b, Biolegend, San Diego, CA; #101205; FE-F4 / 80, Biolegend, San Diego, CA; #123109) for 30 min on ice. Labeled macrophages were then washed twice in PBS, stained with 7AAD (a viability stain) for 15 min, and analyzed by flow cytometry using BD Accuri C6 software (BD Biosciences, San Jose, CA).

[0604] As shown in Figure 7A (top panel), untreated lungs (PBS control row) and BM-treated lungs at day 7 show similar dense alveoli interconnected by minimal extracellular matrix. In contrast, at day 14 after BM instillation, the size and frequency of air pouches is significantly reduced and the density of extracellular matrix is ​​visibly increased, suggesting the development of severe fibrosis in treated mice. By day 21, pathology in this model had already begun to resolve spontaneously, and many mice had finally recovered from BM-induced trauma by day 35.

[0605] Evidence of the development of inflammation by day 7 was seen in the infiltration of FRβ-expressing macrophages, which were almost completely absent from healthy lungs but continued to accumulate in BM-exposed lungs over 14 days (see lower panel of Fig. 7A and quantification in Fig. 7B). Furthermore, staining with F3 showed significant expression of FRβ in IPF lungs (prominent in the interstitial space) as previously reported in the literature (Fig. 7A). Expression of FRβ was restricted to inflamed lungs (either IPF patients or BM-induced PF, but not healthy lungs). Furthermore, FRβ-expressing macrophages were observed in the lungs of mice at day 7 after administration of BM, with maximum expression at day 14 (Fig. 7B). These results were supported by previously reported expression of FRβ in activated macrophages in inflamed lungs.

[0606] That these FRβ-expressing macrophages could then be targeted by a molecule linked to folate was demonstrated by the accumulation of the folate-targeted fluorescent dye, OTL38, in the lungs of BM-treated mice, but not in healthy mice, following tail vein injection. As shown in Figure 7B, OTL38 fluorescence was only observed in the kidneys (i.e., its primary site of excretion) of healthy mice, with little or no uptake in other tissues.

[0607] Figures 7C and 7D show FRβ IHC staining of human IPF lung tissue (Figure 7C) and healthy human lung tissue (Figure 7D). Eight-week-old C57BL / 6 male mice were fed a folate-free diet for 1 week followed by BM or PBS infusion, and 10 days after infusion, mice were injected into the tail vein with 10 nmol (for in vivo imaging) or 100 nmol (for in vivo labeling) OTL38 with or without a 200-fold excess of FA glucosamine. After 2 hours, mice were sacrificed and analyzed. For in vivo folate imaging studies, major organs (heart, lung, spleen, liver, small intestine, large intestine, and kidney) were excised and imaged using an AMI live imager (Spectral Instruments Imaging, Tucson, AZ). For in vivo folate receptor labeling studies, mouse lungs were collected immediately after euthanasia, digested, and then labeled with antibodies to the desired macrophage markers (FITC-CD11b, PE-F4 / 80) and 7AAD (live / dead stain) and analyzed by flow cytometry.

[0608] FIG. 7E shows images of various mouse tissues / organs taken from mice with (BM) or without BM-induced experimental fibrosis (PBS control) and imaged with OTL38, a folate receptor-targeted fluorescent dye, 10 nmol OTL38 was injected into the tail vein of healthy (row a) or BM-treated (rows b and c) mice 10 days after induction of fibrosis in the absence (b) or presence (c) of 200-fold excess of folate-targeted glucosamine (a competitive reagent for FRβ that blocks OTL38 binding) and euthanized 2 hours later for tissue dissection and fluorescent imaging, confirming that the inventive FA-targeted conjugates of the present disclosure exhibit FRβ-specific binding without being taken up by other healthy tissues.

[0609] Tail vein injection of OTL38 into BM-treated mice not only resulted in the above fluorescence in the kidney, but also resulted in significant accumulation in the fibrotic lung (see FIG. 7E). This pulmonary uptake was primarily mediated by folate receptors, as can be demonstrated by the fact that pulmonary accumulation was almost quantitatively blocked when BM-treated mice were co-injected with 200-fold excess of folate-glucosamine, i.e., a competitive inhibitor of FRβ binding (see FIG. 7E). These data demonstrate that folate-targeting molecules selectively bind to folate receptor-expressing cells in fibrotic tissues without accumulating to any significant extent in other tissues of the body. In other words, FRβ-expressing macrophages can indeed be targeted by folate-linked molecules and, in clinical applications, localize almost exclusively to fibrotic tissues. Thus, when targeting moieties are used in the compounds of the present disclosure, there will be minimal of any TLR7 agonist that is not captured by the targeted fibrotic (or cancerous) tissue.

[0610] Next, to determine which cell types capture the folate-dye conjugate in the lungs of BM-treated mice, lungs from the above animals were digested with collagenase and examined by flow cytometry for cell-specific dye uptake. Figure 7F shows data from FACS analysis obtained from in vivo labeling of such mice undergoing BM-induced experimental fibrosis, injected into the tail vein with PBS (column 1) or 100 nmol OTL38 in the absence (column 2) or presence (column 3) of a 200-fold excess of folate-targeted [glucosamine]. As shown in Figure 7F, macrophage-like cells did not show any fluorescence when isolated from BM-treated mice not injected with OTL38 (see column 1). In contrast, approximately 22% of macrophage-like cells from fibrotic mice injected with OLT38 showed significant folate-targeted dye retention (column 2), supporting that OTL38 targets FRβ-positive macrophages in inflamed lungs. Indeed, dye uptake is specifically mediated by folate receptors, as demonstrated by the observation that simultaneous tail vein injection of 200-fold excess folate-glucosamine blocked virtually all folate-dye retention, demonstrating that dye accumulation requires unoccupied folate receptors. Importantly, this conclusion is further supported by data showing that FRβ expression is virtually undetectable in untreated lungs (see FIG. 7A), but increases dramatically in BM-treated lungs during the development of fibrosis (see FIG. 7A-7D). FRβ expression is also predominantly expressed in the lungs of human IPF patients.

[0611] [Example 5] Having established the ability of the conjugated drug to target FRβ-expressing fibrotic macrophages, we next examined whether a folate-targeted TLR7 agonist could suppress signs and symptoms of fibrosis in BM-treated mice. In this case, BM-treated mice were intravenously infused with either vehicle (3% DMSO in PBS) or compound 1B every other day starting on day 10 (see Figure 8A). Compound 1A could not be evaluated in vivo as well, since the TLR7-54 agonist caused rapid weight loss and subsequent death (see Figures 9A and 9B). In BM-induced experimental pulmonary fibrosis in mice, inflammation is known to continue for approximately 9-10 days after BM introduction. In this model, the switch from inflammation to fibrosis occurs approximately on days 9-14, so cancer markers begin to appear approximately on day 10, with dosing starting on day 10 (Figure 8A).

[0612] Two doses were administered every other day until day 21. Each daily dose was separated by 6 hours to prevent any "tolerance" to the TLR agonist. Mice were then sacrificed on day 21 and lungs were immediately excised for bronchoalveolar lavage followed by immunohistochemistry and quantification of collagen and hydroxyproline.

[0613] Figures 8B-8G show representative graphical data of various marker levels measured from mice treated with the BM model of Figure 8A, where BALF was collected on day 21, centrifuged at 4°C, the resulting pellet was resuspended in medium, plated in 96-well plates, cultured for 2 hours, washed three times with pre-warmed PBS, and cells were collected for qPCR. These data show that Arg1 (Figure 8B), MMP9 (Figure 8C), and TIMP3 (Figure 8D) (e.g., cancer markers) were all downregulated. Both CD86 (Figure 8E) and IFN-γ (Figure 8F) (e.g., pro-inflammatory markers) were upregulated. In addition, the negative regulator of TLR7 signaling IRAK-4 was upregulated (Figure 8G), as was the number of BALF cells present (Figure 8H). Indeed, the total number of mouse BALF cells was dose-dependently reduced after treatment with different doses of Compound 1B. Each value shown in Figures 8B to 8G represents the mean ± standard deviation for each group; * P < 0.05, ** P<0.005, *** <0.0005; for saline vs. vehicle, Compound 1A and Compound 1B treatment vs. vehicle, calculated by Student's t-test, except for measurements of BALF cell counts and protein concentrations, and for Compound 1B treatment and vehicle, calculated by Dunnett's multiple comparison test; vehicle=3% DMSO in PBS.

[0614] As shown in Figures 8B-8D, qPCR analysis of cancer markers in the macrophage subpopulation of bronchoalveolar lavage cells revealed that tissue inhibitors of Arg1, MMP9, and TIMP3 were all elevated in BM-induced mice compared to control mice. More importantly, parallel studies demonstrated that treatment of BM-induced mice with compound 1B suppressed all of the same cancer markers and led to levels of fibrotic markers similar to those observed in healthy mice. Consistent with these data, quantification of proinflammatory markers revealed that both CD86 transcript abundance (qPCR) and IFN-γ concentrations (ELISA of lavage fluids) were elevated following treatment with compound 1B (see Figures 7E and 7F). Taken together with the observed upregulation of IRAK-4 (i.e., a marker of TLR activation; results shown in Figure 7G) and the dose-dependent reduction in the total number of BALF cells present following treatment with different doses of compound 1B (Figure 7H), these data demonstrate that administration of a folate-targeted TLR7 agonist can reprogram macrophages from an anti-inflammatory M2-like to a pro-inflammatory M1-like phenotype in vivo in the lungs of BM-treated mice.

[0615] [Example 6] Additional studies were performed to determine whether the above reprogramming of fibrotic lung macrophages actually resulted in amelioration of fibrotic conditions in fibrotic mice. Lung tissue from these mice was embedded in paraffin, sectioned, and stained with H&E and Masson's Trichrome to assess tissue density and extracellular collagen deposition, respectively.

[0616] Figures 9A and 9B show the survival curve (Figure 9A) and body weight change (Figure 9B) of mice with experimental pulmonary fibrosis treated with non-targeted and targeted TLR7 agonists. This data supports that administration of the compounds of the present disclosure (here, for example, compound 1B) improves the survival rate of BM-treated mice without causing significant weight loss. Each value represents the mean ± standard deviation for each group.

[0617] FIG. 10A shows hydroxyproline content (μg / lung) of lung tissue using collagen deposition as a measure of fibrosis. Tissues are shown at day 21 for each of the following: healthy control (saline) (●), disease control (vehicle) (■), treatment with free drug TLR7 agonist (compound 1A) (▼), and treatment with folate-targeted TLR7 agonist (compound 1B) (▲). BM-derived mice treated with 10 nmol of either compound 1B (▲) or compound 1A (▼) showed a significant reduction in total hydroxyproline content per lung compared to vehicle control (■). Each value shown in FIG. 9A represents the mean ± standard deviation for each group; * P < 0.05, ** P<0.005, *** <0.0005; saline vs. vehicle, Compound 1A and Compound 1B treated vs. vehicle by Student's t-test.

[0618] 10B and 10C show staining images of the lung tissue depicted in FIG. 10A by H&E staining (FIG. 10B) and Masson's Trichrome (collagen) staining (FIG. 10C).

[0619] As shown in the H&E staining in the panel of FIG. 10B, healthy lungs contain a large amount of air sacs surrounded by a thin reticular membrane. In contrast, BM-induced lungs show far fewer alveoli with prominent deposition of extracellular matrix where air sacs once existed. Most importantly, BM-injected mice treated with compound 1B beginning on day 10 showed lung architecture similar to that of healthy mice (FIG. 10B), suggesting that targeting of compound 1A to fibrotic lung macrophages is effective in suppressing key features of pulmonary fibrosis. Such prevention of fibrosis indeed involves blockade of collagen deposition, as evidenced by Masson's trichrome staining of parallel lunch sections (FIG. 10C), where collagen staining is strongly suppressed in mice injected with compound 1B via the tail vein (FIG. 10B). Thus, these data confirm that at least IPF mice treated with Compound 1B (▲) demonstrated suppression of IPF pathology (eg, fibrosis).

[0620] Finally, to confirm that compound 1B did indeed affect collagen production in vivo, hydroxyproline (the main component of collagen) was quantified in the total hydrolysate of diseased lung. More specifically, lung tissue from the above mice was perfused with PBS and hydrolyzed with acid to analyze hydroxyproline content. As shown in Figure 10A, induction of fibrosis induces a large increase in hydroxyproline content, which is suppressed by treatment with compound 1B. Thus, this data supports that treatment with the targeted TLR7 agonist compound of the present disclosure reduces collagen deposition in vivo, thereby reducing (and even abrogating) fibrosis.

[0621] In summary, the overall survival of mice injected with the optimized BM dose (0.75 mg / kg) was significantly improved by treatment with compound 1B, whereas compound 1A had no survival benefit, except for a significant weight loss (>25%, FIG. 7). Although the free drug performed well in reducing hydroxyproline content, the poor survival observed could be attributed to overall toxicity (i.e., weight loss, see FIG. 7B). This was not surprising, since systemic administration of TLR7 agonists is known to cause toxicity.

[0622] [Example 7] Since the use of non-targeted TLR7 agonists to treat IPF (or other fibrotic diseases) is hindered by their systemic activation of the immune system and resulting toxicity, we evaluated whether any obvious toxicity could be associated with systemic administration of compound 1B in mice. For this purpose, BM-derived mice were treated with 0, 1, 3 or 10 nmol of compound 1B every other day starting on day 10, and body weight, lung hydroxyproline content and histological analysis were performed on day 21. Unlike conventional systemic administration, targeted drugs not only improved survival rates but also reduced weight loss, which is the basis of the importance of targeted approaches (Figures 11A and 11B).

[0623] Figure 12 shows data on the dose-dependent effect of folate-targeted TLR7 agonists on suppressing fibrosis in BM-induced mice using collagen deposition as a measure of fibrosis. Data are presented for healthy controls (PBS, ●), BM-induced mice with treatment vehicle (■), 1 nmol Compound 1B (○), 3 nmol Compound 1B (□) or 10 nmol Compound 1B (▲), subsection A shows graphical data on the body weight of BM-induced mice over time, subsection B shows measurements of hydroxyproline content of lung tissue (μg / lung) treated with different doses (10 nmol, 3 nmol or 1 nmol Compound 1B), and subsection C shows images of histological analysis of right lung tissue by H&E and trichrome staining.

[0624] As can be seen in Figure 11B and subsection A of Figure 12, no difference in weight loss was observed between mice treated with 0, 1, 3 or 10 nmol of Compound 1B, suggesting that repeated administration of the compound did not cause net toxicity. Nevertheless, these treatments still had the expected effect on pulmonary fibrosis, as can be seen from a comparison of the hydroxyproline content of the various lung hydrolysates, with the order of efficacy being 10 nmol / mouse>3 nmol / mouse>1 nmol / mouse>0 nmol / mouse (subsections B and C of Figure 12). More importantly, detailed analysis of lung histology demonstrated that increasing doses of Compound 1B improved lung histology, suggesting that the tissues with the highest concentration of TLR7 agonist were indeed those with the most normal microscopic morphology. Taken together, these data support that targeting TLR7 agonist FRβ+ macrophages in fibrotic tissues can effectively prevent fibrosis without the systemic activation of the immune system that would otherwise limit the use of TLR7 agonists in humans.

[0625] Finally, to determine whether this proinflammatory effect could be achieved using lower end doses, therapeutic studies were undertaken with the two lower end doses (3 nmol / kg and 1 nmol / kg) (Figures 9 and 10). Interestingly, the 10 nmol dose provided the best survival rate, although the lower doses showed a significant reduction in hydroxyproline content and collagen deposition levels.

[0626] [Example 8] To demonstrate that embodiments of compounds of the present disclosure other than Compound 1A and Compound 1B perform similarly upon application, other representative embodiments of the compounds were tested in in vitro studies.

[0627] Figures 13A-13D show representative graphical data of various marker levels measured from human THP-1 cells induced to M2 macrophages with 20 ng / mL IL-4, 20 ng / mL IL-13, and 5 ng / mL IL-6. The cells were then reprogrammed with various concentrations (nM) of a TLR7 agonist having formula IV (e.g., compound 2A) for 48 hours and collected for genetic analysis by qPCR. The mRNA levels of the following markers relative to the expression levels of the M2-like macrophage control: CCL18 mRNA levels (Figure 12A), IL-1β mRNA levels (Figure 13B), and TNFα levels (Figure 13C), and Figure 13D shows the results of protein analysis after harvesting the cell supernatant. Secreted CCL18 protein was detected by ELISA.

[0628] In Figures 13A-13D, agonist compounds of the present disclosure having formula IV (eg, compound 2A) were evaluated for their ability to reprogram M2-like macrophages to M1-like macrophages.

[0629] Primarily, human monocytic (THP-1) cells were induced to an M2-like phenotype using methods and materials previously described. In particular, THP-1 cells were seeded in 96-well plates at a density of 60,000 cells / well. Cells were differentiated into non-polarized macrophages by incubation with 200 nM PMA for 48 hours, followed by incubation in fresh RPMI medium for 24 hours. The resulting macrophages were polarized to an M2-like phenotype by incubation with 20 ng / ml IL-4, 20 ng / ml IL-13, and 5 ng / ml IL-6 for 48 hours. Cultures were maintained at 37°C in a humidified 5% CO2 incubator.

[0630] To evaluate whether compound 2A could reprogram anti-inflammatory macrophages to a less fibrotic phenotype, IL-4, IL-6 and IL-13 stimulated THP-1 cells were incubated with various concentrations of compound 2A and the mRNA levels of several cancer markers, namely CCL18, IL-1β and TNFα, were examined using qPCR and ELISA.

[0631] As shown in Figures 13A and 13B, incubation with compound 2A (free drug) for 48 hours induced a decrease in the expression of CCL18 and IL-1β, suggesting that TLR7 agonists can indeed promote the shift of these anti-inflammatory (M2) polarized THP-1 cells to a less fibrotic phenotype (note that Figure 13B shows a bell-shaped curve showing compound 2A having an inhibitory response at low concentrations and a stimulatory response at high concentrations, which is a typical response curve for certain drugs). Furthermore, the expression of TNFα (a marker of a proinflammatory phenotype) was examined and an increase in its expression was observed (Figure 13C), confirming that a THP-1 shift from proinflammatory to proinflammatory properties had occurred.

[0632] In addition to unconjugated TLR7 agonists, conjugated compounds of the present disclosure were also evaluated. Human THP-1 cells were induced into macrophages with an M2-like phenotype by methods described herein (e.g., using 20 ng / mL IL-4, 20 ng / mL IL-13, 5 ng / mL IL-6) and then reprogrammed with various concentrations (nM) of various compounds of the present disclosure: unconjugated (free drug) TLR7 agonist compounds having Formula I and / or II (data collectively shown as Compound 3A), a folate-conjugated TLR7 agonist compound (with a releasable linker) having Formula XV (e.g., Compound 3B), a folate-conjugated TLR7 agonist compound (with a non-releasable linker) having Formula XVII (e.g., Compound 3C), and a folate-conjugated TLR7 agonist compound (with a non-releasable linker) having Formula XVI (e.g., Compound 3D) for 2 hours. Cells were then harvested for gene analysis by qPCR to analyze the relative expression levels of CCL18 (Figure 14A), CD206 (Figure 14B) and IL-1β (Figure 14C).

[0633] The expression levels of various cancer (M2 phenotype) markers CCL18, IL-1β and CD206 markers were quantified. As shown in Figures 14A-14C, the expression levels of each of these cancer markers were decreased after administration of each of Compound 3B, Compound 3D and Compound 3C, and Compounds 3D and 3C (both non-releasable linkers) were the most effective compared to the other compounds.

[0634] Figure 15 shows secreted CCL18 protein levels in each of the groups of THP-1 cells of Figures 14A-14C following treatment with Compound 3A, Compound 3B, Compound 3C, or Compound 3D. Compound 3A and folate-targeted TLR7 compounds (e.g., Compound 3B, Compound 3C, and Compound 3D) down-regulated CCL18 secretion in the low concentration range (0.1-10 nM).

[0635] Furthermore, cell supernatants were collected and secreted CCL18 protein was detected by ELISA. Figure 15 confirms that compound 3A (free drug) and folate-targeted compounds (compounds 3B, 3C and 3D) all downregulated CCL18 secretion in the low concentration range (0.1-10 nM), further supporting that, similar to the examples described in connection with compounds 1A and 1B, these compounds can also similarly reprogram M2-like anti-inflammatory macrophages to M1-like pro-inflammatory macrophages by a similar mechanism.

[0636] [Example 9] The above study was repeated (see Figures 3A-3F, grey bars), and the same qualitative changes were observed, only the magnitude of effect of compound 1B was somewhat reduced. This reduction in potency was expected, since non-targeted TLR7 agonists immediately enter cultured cells, whereas their folate-targeted counterparts are designed to enter cells only after folate receptor-binding and receptor-mediated endocytosis. Because low molecular weight, water-soluble drugs such as compounds 1A and 1B are often cleared from the body within 2 hours of injection, a more physiologically relevant in vitro model of in vivo drug exposure involves incubating cells with the drug for only 2 hours, and then testing the efficacy of the drug after an additional 46 hours of incubation in the absence of the drug. As shown in Figures 4A-4E, when THP-1 cells were incubated with TLR7 agonists for 2 hours before replacing the drug-containing medium with drug-free medium, compound 1B was observed to have superior potency compared to compound 1A, especially in the case of TNFα induction, where the folate-targeted conjugate was dramatically improved, likely because the folate-targeted TLR7 agonist was captured by folate receptor positive cells, whereas compound 1A was not retained by the same cells.

[0637] These data support that compound 1B should be more effective at reprogramming anti-inflammatory macrophages in vivo, with the added advantage that folate-conjugated drugs (e.g., compound 1B) should also cause less systemic toxicity since they recruit to FRβ-expressing macrophages and are unable to enter the folate receptor negative cells that predominate throughout the body (e.g., compound 1B is designed to be impermeable to folate receptor negative cells).

[0638] Furthermore, to confirm that the above mRNA analysis accurately reflects the levels of anti-inflammatory cytokines produced by IL-4, IL-6 and IL-13 stimulated THP-1 cells, the concentrations of CCL18 and IL-1β polypeptides in the supernatants of THP-1 cells were quantified by ELISA assay. As shown in Figures 6A and 6B, both compound 1A and compound 1B induced a decrease in CCL18 and IL-1β when continuously incubated with the agonists for 48 hours, but compound 1B was still found to be superior when drug exposure was limited to only 2 hours (see Figures 6C and 6D).

[0639] [Example 10] Figure 16 illustrates an in vivo study of at least one embodiment of a compound of the present disclosure in a BM mouse model, the compound having formula XVII (e.g., compound 3C). Figures 17A and 17B are LC-MS spectra of compound 3C, confirming the high purity of the conjugate, with no free drug detected.

[0640] 18A-18F show results from control mice of the in vivo study method of FIG. 16, including survival curves (FIG. 18A), body weight change (FIGS. 18B and 18D), concentration of cells bearing BALF (FIG. 17C), and hydroxyproline concentration (μg HP / leaf) in surviving mice (FIG. 18E) and in total mice (e.g., including both surviving mice and mice that died before day 21) (FIG. 18F). A 10 nmol dose of a compound having formula XVII (e.g., compound 3C) improved survival of control mice while simultaneously decreasing HP and number of BALF cells. Similarly, a 3 nmol dose showed no measurable benefit to control mice.

[0641] [Example 11] M2-induced human monocyte-derived macrophages were treated with 100 nM of Compound 1A or Compound 1B in the presence or absence of FA-glucosamine (competition) for either 48 consecutive hours or for the first 2 hours, then for 46 hours in the absence of drug (2+46 hours). As shown in Figure 19, the mRNA levels of cancer markers Arg1 (Figure 19A), CD206 (Figure 19B) and CD163 (Figure 19C), and the protein levels (n=3, technical replicates) of secreted profibrotic CCL18 (Figure 19D) and proinflammatory cytokines CXCL10 (Figure 19E) and IL-6 (Figure 19F) were then determined. Both changes in the set of cytokines were inhibited by blocking unoccupied folate receptors with excess FA-glucosamine (2+46 hours, competition). This data confirms that compound 1B binds to the folate receptor, since downregulation of the biomarker was blocked by excess FA-glucosamine (a competitor).

[0642] [Example 12] Healthy mice were injected into the tail vein with 10 nmol of Compound 1A (circles) or Compound 1B (squares), and peripheral blood was collected at the indicated time points after drug injection (Figures 20A-C). Plasma IL-6 (Figure 20A), IFNα (Figure 20B), and TNFα (Figure 20C) were measured (n=3). (Figures 20D-F). The effect of drug concentration on plasma levels of IL-6 (Figure 20D), IFNα (Figure 20E), and TNFα (Figure 20F) was determined at 1.5 hours, 1 hour, or 1 hour after treatment, respectively (n=2) (Figure 20G). Compound 1A stimulates systemic cytokine release in healthy mice, whereas Compound 1B does not. Moreover, Compound 1B stimulates less inflammatory cytokine release than half the dose of Compound 1A. These data suggest that TLR7 agonists, when targeted to pulmonary macrophages by folate receptor-targeting ligands, could be safely used to reprogram macrophages in fibrotic lungs to a proinflammatory state.

[0643] [Example 13] Sections from the same healthy and fibrotic lungs described in Figure 6 were stained with DAPI (nuclei; blue), anti-F4 / 80 (macrophages; red) and anti-CD206 (M2 macrophage marker; green) and images were acquired using a Leica Versa8 hole-slide scanner as described in Methods (n=2). Scale bar, 100 μm. Differences between treatment groups indicate that Compound 1B produces a robust proinflammatory response in vivo.

[0644] Although various embodiments of the compounds, compositions and methods have been described in considerable detail herein, the embodiments are merely presented by way of non-limiting examples. Numerous variations and modifications of the embodiments described herein will be apparent to those skilled in the art in light of this disclosure. Thus, it will be understood by those skilled in the art that various changes and modifications may be made and equivalents may be substituted for the elements without departing from the scope of this disclosure. Indeed, this disclosure is not intended to be exhaustive or unduly limiting. The scope of this disclosure should be defined by the appended claims and their equivalents.

[0645] [Example 14] This example demonstrates that 4T1 and CT26 cells do not express TLR7.

[0646] 4T1 (mouse mammary adenocarcinoma cell line derived from BALB / c mouse), CT26 (mouse colorectal carcinoma cell line derived from BALB / c mouse) and EMT6 cells (experimental breast tumor 6) were fixed, permeabilized and stained with anti-mouse TLR7-PE antibody. The results are shown in Figures 22A-22F, showing the expression of TLR7 in 4T1, CT26 and EMT6 cells. Figure 22A shows the negative control for 4T1 cells, while Figure 22B shows the negative control for CT26 cells and Figure 22C shows the negative control for EMT6 cells. Figure 22D shows the results of staining 4T1 cells with anti-mouse TLR7-PE antibody, while Figure 22E shows the results of staining CT26 cells with anti-mouse TLR7-PE antibody and Figure 22F shows the results of staining EMT6 cells with anti-mouse TLR7-PE antibody. As shown in the figure, TLR7 expression was not significantly detected in 4T1, CT26 or EMT6 cells.

[0647] [Example 15] This example describes the generation of CD19-expressing mouse cancer cells.

[0648] 4T1, CT26 and EMT6 cells were transduced to express mouse CD19 and green fluorescent protein (GFP). Cells were sorted by GFP levels and selected to obtain single cell clones (4T1-mCD19, CT26-mCD19 and EMT6-mCD19). Cells were then stained with anti-mouse CD19-PE antibody. The results are shown in Figures 23A-23C, which are graphs of CD19 vs. Maximum Ratio (Max). Figure 23A shows the overlay of stained (anti-CD19-PE) and unstained 4T1-mCD19-F7 cells, while Figure 23B shows the overlay of stained (anti-CD19-PE) and unstained CT26-mCD19 cells, and Figure 23C shows the overlay of stained (anti-CD19-PE) and unstained EMT6-mCD19-C10 cells. As shown in the figure, 4T1-mCD19 cells, CT26-mCD19 cells, and EMT6-mCD19 cells all expressed mouse CD19 + It is.

[0649] [Example 16] This example describes the generation of anti-mouse CD19 chimeric antigen receptor (CAR)-T cells.

[0650] To target mouse CD19-positive cancer cells, mouse T cells were transduced to express anti-mouse CD19 CAR. Mouse T cells isolated from mouse spleens were activated with anti-CD3 / CD28 conjugated beads for 24 hours. The activated T cells were then transferred to RetroNectin-coated (Takara Bio USA, Inc., Mountain View, CA, USA) plates for transduction. To improve the expression of anti-mouse CD19 CAR on mouse T cells, a second transduction was performed 1 day after the first transduction. Since the anti-mouse CD19 scFv (single-chain variable fragment) was derived from an antibody produced by rats, an anti-rat IgG antibody conjugated with Alexa Fluor594 was used to stain transduced and non-transduced mouse T cells. Figures 24A-24C show the anti-mouse CD19 CAR versus SSC-A (10 ^324A and 24B show plots of mouse CD19 scFv expression in transduced mouse T cells as measured by flow cytometry using anti-rat-Alexa594 antibody for staining. FIG. 24A shows the results of staining non-transduced mouse T cells (negative control), while FIG. 24B shows the results of staining once-transduced mouse T cells, and FIG. 24C shows the results of staining twice-transduced mouse T cells. By the second transduction, approximately 20% of the T cells are CAR+.

[0651] [Example 17] This example describes the validation of the activity of anti-mouse CD19 CAR-T cells.

[0652] Anti-mouse CD19 CAR-T cells were co-cultured overnight with 4T1 cells expressing mouse CD19 (4T1-mCD19), CT26 cells expressing mouse CD19 (CT26-mCD19) or EMT6 cells expressing mouse CD19 (EMT6-mCD19) in a 96-well plate. The same number of target cells (4T1-mCD19, CT26-mCD19 or EMT6-mCD19) without CAR-T cells were used as spontaneous controls. The next day, the suspended cells and the supernatant were first removed from each well. Then, the attached cells (viable target cells) from each well were harvested after trypsinization and counted by flow cytometry. Anti-mouse CD19 CAR-T cells killed 94.8% of 4T1-mCD19 cells, 95.5% of CT26-mCD19 cells, and 98.6% of EMT6-mCD19 cells.

[0653] [Example 18] This example describes the evaluation of the anti-tumor activity of anti-mouse CD19 CAR-T cells in combination with a folate-TLR7 agonist in a mouse model.

[0654] 4T1-mCD19 cells (5 × 10 4) was subcutaneously injected into Balb / c mice. The mice were then divided into three groups. Group 1 was treated with phosphate buffered saline (PBS; no treatment), while Group 2 was treated with CAR-T cells alone, and Group 3 was treated with a combination of CAR-T cells and folate-TLR7 agonist. From day 6 after tumor implantation until the tumor size reached approximately 50 mm3, mice in Group 3 were injected with 3 nmol of non-releasable folate-TLR7 agonist via the tail vein five times per week. On day 6 after tumor implantation, tumor-bearing mice were subjected to 4 Gy total body irradiation (TBI) for lymphatic depletion. The next day, freshly prepared anti-mouse CD19 CAR-T cells (day 3 after transduction) were injected into mice in Groups 2 and 3.

[0655] The result was mouse CD19 + Shown in Figure 27 is a graph of cells vs. % cytotoxicity against cancer cells, which shows that anti-mouse CD19 CAR-T cells inhibited the expression of mouse CD19 + The results of an assay to determine whether CAR-T cells are cytotoxic to cancer cells are shown. Anti-mouse CD19 CAR-T cells bind to mouse CD19 + They induced more than 90% cytotoxicity against cancer cells (4T1-mCD19, CT26-mCD19, and EMT6-mCD19), whereas the same number of non-transduced T cells induced only 5.3% cytotoxicity.

[0656] Additional results were obtained by comparing days after the first FA-TLR7A-1A injection with tumor size (mm 3 ), which shows the change in tumor size obtained with treatment with CAR-T cells alone (CAR-T), or with CAR-T cells in combination with a non-releasable folate-TLR7A agonist (CAR-T+FA-TLR7A) compared to control (phosphate buffered saline; untreated).

[0657] To examine the tumor microenvironment in the three treatment groups, all tumors were digested into single cells and stained with antibodies for flow cytometric analysis.

[0658] Only mice treated with CAR-T cells had higher levels of T cell and macrophage infiltration in the tumors compared to PBS-treated (untreated) mice. However, mice treated with CAR-T cells in combination with folate-TLR7 agonist had even higher levels of T cell and macrophage infiltration in the tumors. Furthermore, there were more M1 macrophages, activated T cells, and activated CAR-T cells in tumors from mice treated with the combination therapy than in tumors from mice treated with CAR-T cells alone.

[0659] These results are shown in Figures 29-34B. Figure 29 is a graph of % body weight change versus days after tumor implantation, showing the percent body weight change obtained with treatment with CAR-T cells or the combination of CAR-T cells and non-releasable folate-TLR7A agonist (CAR-T+FA-TLR7A) compared to control (untreated).

[0660] FIG. 30A shows treatment vs. iNOS in F4 / 80+ + / Arginase 1 + 3 is a graph of M1 / M2 (iNOS) in tumors after treatment with CAR-T cells alone or in combination with CAR-T cells and a non-releasable folate-TLR7 agonist compared to untreated. + / Arginase-1 + ) macrophage ratio.

[0661] FIG. 30B shows total macrophages (F4 / 80) in the treatment versus tumor + ) which shows the percentage of total macrophages in tumors after treatment with CAR-T cells alone or the combination of CAR-T cells and non-releasable folate-TLR7A agonist compared to untreated.

[0662] FIG. 31 shows the effect of total myeloid-derived immune suppressor cells (MDSCs; CD11b + Gr-1 +) which shows the percentage of MDSCs in tumors after treatment with CAR-T cells alone (CAR-T) or the combination of CAR-T cells and non-releasable folate-TLR7 agonist compared to untreated.

[0663] FIG. 32A shows the %CD3 in tumors versus treatment. + Graph of T cells, CD3+ T cell counts in tumors after treatment with CAR-T cells alone (CAR-T) or CAR-T cells in combination with a non-releasable folate-TLR7A agonist (CAR-T+FA-TLR7A) compared to untreated. + The percentage of T cells is shown.

[0664] FIG. 32 is a graph of % CAR-T cells in tumor versus treatment, which shows the percentage of CAR-T cells in the tumor after treatment with CAR-T cells alone (CAR-T), or CAR-T cells in combination with a non-releasable folate-TLR7A agonist (CAR-T+FA-TLR7A).

[0665] FIG. 33A shows the % CD3 in tumors versus treatment. + CD25 + Graph of T cells, CD25+ TLR7A expression in tumors after treatment with CAR-T cells alone (CAR-T) or CAR-T cells in combination with a non-releasable folate-TLR7A agonist (CAR-T+FA-TLR7A) compared to untreated. + The percentage of T cells is shown.

[0666] FIG. 33B shows %CD25 in tumors versus treatment. + Graph of CAR-T cells showing CD25 expression in tumors after treatment with CAR-T cells alone (CAR-T) or CAR-T cells in combination with a non-releasable folate-TLR7A agonist (CAR-T+FA-TLR7A) compared to untreated. + The percentage of CAR-T cells is shown.

[0667] FIG. 34A shows the %CD3 in tumors versus treatment. + CD69+ Graph of T cells, CD69 T cell counts in tumors after treatment with CAR-T cells alone (CAR-T) or CAR-T cells in combination with a non-releasable folate-TLR7A agonist (CAR-T+FA-TLR7A) compared to untreated. + The percentage of T cells is shown.

[0668] FIG. 34B shows %CD69 in tumors versus treatment. + Graph of CAR-T cells showing CD69 expression in tumors after treatment with CAR-T cells alone (CAR-T) or CAR-T cells in combination with a non-releasable folate-TLR7A agonist (CAR-T+FA-TLR7A) compared to untreated. + The percentage of CAR-T cells is shown.

[0669] [Example 19] To evaluate the efficacy of TLR-7 agonists, compound 1 (TLR7-1A), compound 2 (TLR7-1B) and compound 3 (TLR7-1C) were treated with peripheral blood mononuclear cells (PBMCs) for 24 hours. TLR7-1 was used as a control. The supernatant of the cell culture was isolated and tested for IL-6 using enzyme-linked immunosorbent assay (ELISA) (Figure 35). As shown in Figure 33, TLR7 agonists caused an increase in the expression of IL-6 in PBMCs.

[0670] When these compounds were treated with human primary monocyte-derived M2 macrophages for 48 hours, they induced IL-6 and CXCL-10 more effectively than the parent compound TLR7-1 (Figures 36A and 36B). Compounds 1, 2, and 3 polarize M2 macrophages to M1 macrophages, as shown by the increase in M1 markers IL-6 (Figure 34A) and CXCL10 (Figure 36B).

[0671] [Example 20] Healthy mice were injected with 10 nmol of Compound A (TLR-1) or Compound 1 (TLR-1A) into the tail vein, and peripheral blood was collected at the indicated time points after drug injection (Figures 36C and 36D). The effects of the drugs on plasma levels of IL-6 (Figure 36C) and TNFα (Figure 36D) were determined 1 or 1.5 hours after treatment. Both compounds stimulated systemic cytokine release in healthy mice.

[0672] Furthermore, many of the examples provided herein use mouse models, but those skilled in the art will recognize that gene expression patterns in mouse models show an exceptionally significant correlation with gene expression patterns in human conditions, and that many pathways are commonly regulated by multiple conditions in humans and mice.Therefore, gene expression patterns and disease progression in mouse models closely reproduce those in human conditions, particularly with respect to inflammatory diseases and cancer, thus supporting the examples described herein to correlate with human data, specific conditions and applications.

[0673] Accordingly, the description and appended claims are intended to cover all modifications and variations that are apparent to one of ordinary skill in the art based on this disclosure. [Explanation of symbols]

[0674] 100 compounds 102 Immune Modulators 104 Targeting part 106 Linker 150 compounds 152 Immune Modulators 154 Targeting part 156 Linker 1900 Methods for Treating Cancer Contacting the cell with at least one compound. 1904 Step of obtaining a biological sample 1906 Quantifying the level of expression of a biomarker 1908. Comparing the level of expression of each of one or more biomarkers in the sample with the level of expression of such biomarkers in a control. 1910 Steps for Alternative Treatments

Claims

1. 1. A combination for use in treating cancer in a subject, comprising: a first drug comprising a compound comprising a folate ligand, or a functional fragment or analog thereof, linked to a Toll-like receptor (TLR) agonist via a linker; a second drug comprising the engineered cells; A combination comprising:

2. The combination of claim 1 , wherein the TLR agonist comprises a TLR 7, 8, 9 or 7 / 8 agonist.

3. 3. The combination of claim 1 or 2, wherein the second drug further comprises a CAR T cell or an engineered stem cell.

4. The TLR agonist of the compound of the first drug has the structure of Formula 2-I (or a radical thereof), or a pharma- ceutically acceptable salt of Formula 2-I: 【Chemistry 1】 In formula 2-I, R １ , R ３ , R ４ and R ５ are each independently hydrogen (H), alkyl, alkoxyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, halo, heteroaryl, -COR ２ｘ , 【Chemistry 2】 and R ２ は、H、-OH、-NH ２ -NHR ２ｘ 、N ３ -NH-CH ２ -NH ２ 、-CONH ２ 、-SO ２ NH ２ -NH-CS-NH ２ 、 【Chemistry 3】 and Y is H, -OH, -NH ２ , -NHR ２ｘ , -O-R ２Ｘ , -SO-R ２ｘ , -SH, -SO ３ H, -N ３ , -CHO, -COOH, -CONH ２ , -COSH, -COR ２ｘ , -SO ２ N.H. ２ , alkenyl, alkynyl, alkoxyl, -NH-CH ２ -NH ２ , -CONH ２ , -SO ２ N.H. ２ , -NH-CS-NH ２ , 【Chemistry 4】 and Where: R ２ｘ and R ２ｙ are H, -OH, and -CH ２ -OH, -NH ２ , -CH ２ -NH ２ , -COOMe, -COOH, -CONH ２ , -COCH ３ , alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, and heteroaryl; R ２ｚ are each -NH ２ , -NR ２ｑ R ２ｑ’ , -O-R ２ｑ , -SO-R ２ｑ and -COR ２ｑ R is independently selected from the group consisting of ２ｑ and R ２ｑ’ are each independently alkyl or H; 【Chemistry 5】 is a non-aromatic, monocyclic or bicyclic 3-10 membered N-containing heterocycle; In formula 2-I, X １ , X ２ and X ３ are each independently ｑ or N, R ｑ each is independently H, halogen, or optionally substituted alkyl; In formula 2-I, n is 0 to 30, and m is 0 to 4.

2. The combination of claim 1.

5. The compound of the first drug is 【Chemistry 6】 or a pharma- ceutically acceptable salt thereof.

6. The compound of the first drug has the structure of the following formula: 【Chemistry 7】 2. The combination of claim 1.

7. The TLR agonist has a structure of formula X or XX (or a radical of formula X or XX), or a pharma- ceutically acceptable salt of formula X or XX: 【Chemistry 8】 In formulas X and XX: R １ is -NH ２ Or -NH-R １Ｘ and R ２ is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R ２Ｘ , -O-R ２Ｘ , -S-R ２Ｘ , 【Chemistry 9】 and 【Chemistry 10】 is a 3-10 membered N-containing non-aromatic monocyclic or bicyclic heterocycle; In formula X, R ３ is -OH, -SH, -NH ２ Or -NH-R １Ｘ and In the formula XX, X is CH or N; R １Ｘ , R ２Ｘ and R ２Ｙ are each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl; A combination according to any one of claims 1 or 2.

8. The combination of claim 1, wherein the first drug comprises a therapeutically effective amount of the folate ligand, or a functional fragment or analog thereof, linked to a TLR agonist via a linker.

9. 9. The combination of claim 8, wherein the first drug is prepared to be administered to the subject intravenously, intramuscularly, intraperitoneally, topically, or by inhalation.

10. The TLR agonist having a structure (or a radical thereof) of the following formula: 【Chemistry 11】 During the ceremony, R １ is an amine group, R ２ is a single bond, -NH-, R ３ is H, an alkyl, a hydroxy group or any other substituted group thereof; X is CH ２ , NH, O or S; The linker is R １ , R ２ Or R ３ are bonded at 10. A combination according to any one of claims 1, 2, 8 or 9.

11. The linker comprises a polyethylene glycol (PEG) linker or a PEG derivative linker; and / or 10. A combination according to any one of claims 1, 2, 8 or 9, wherein the pharma- ceutically acceptable salt is selected from hydrobromide, citrate, trifluoroacetate, ascorbate, hydrochloride, tartrate, trifluoromethanesulfonate, maleate, mesylate, formate, acetate or fumarate.

12. The combination of any one of claims 1, 2, 6, 8 or 9, wherein the first drug activates an anti-tumor cell or pro-inflammatory signaling cascade in the subject to which the first drug is administered in combination with the second drug.

13. The combination of claim 12, wherein the anti-tumor cells are T cells, engineered T cells, T cells prepared from progenitor or stem cells, natural killer (NK) cells, engineered NK cells, NK cells prepared from progenitor or stem cells, or macrophages.

14. 1. A combination for use in the prevention or treatment of a disease condition, comprising at least one engineered cell configured to treat said disease condition; at least one compound comprising an immune modulator or a pharma- ceutically acceptable salt thereof linked via a linker to a folate ligand, or a functional fragment or analog thereof, wherein the immune modulator or the pharma- ceutically acceptable salt thereof targets a pattern recognition receptor; A combination comprising:

15. The at least one compound comprising an immune modulator or a pharma- ceutically acceptable salt thereof comprises a Toll-like receptor (TLR) agonist having the structure of Formula 2-I (or a radical thereof) or a pharma- ceutically acceptable salt of Formula 2-I, 【Chemistry 12】 In formula 2-I, R １ , R ３ , R ４ and R ５ are each independently hydrogen (H), alkyl, alkoxyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, halo, heteroaryl, -COR ２ｘ , 【Chemistry 13】 and R ２ は、H、-OH、-NH ２ -NHR ２ｘ 、N ３ -NH-CH ２ -NH ２ 、-CONH ２ 、-SO ２ NH ２ -NH-CS-NH ２ 、 【Chemistry 14】 and Y is H, -OH, -NH ２ , -NHR ２ｘ , -O-R ２Ｘ , -SO-R ２ｘ , -SH, -SO ３ H, -N ３ , -CHO, -COOH, -CONH ２ , -COSH, -COR ２ｘ , -SO ２ N.H. ２ , alkenyl, alkynyl, alkoxyl, -NH-CH ２ -NH ２ , -CONH ２ , -SO ２ N.H. ２ , -NH-CS-NH ２ , 【Chemistry 15】 where: R ２ｘ and R ２ｙ are H, -OH, and -CH ２ -OH, -NH ２ , -CH ２ -NH ２ , -COOMe, -COOH, -CONH ２ , -COCH ３ , alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, and heteroaryl; R ２ｚ are each -NH ２ , -NR ２ｑ R ２ｑ’ , -O-R ２ｑ , -SO-R ２ｑ and -COR ２ｑ R is independently selected from the group consisting of ２ｑ and R ２ｑ’ are each independently alkyl or H; 【Chemistry 16】 is a non-aromatic, monocyclic or bicyclic 3-10 membered N-containing heterocycle; In formula 2-I, X １ , X ２ and X ３ are each independently ｑ or N, R ｑ each is independently H, halogen, or optionally substituted alkyl; In formula 2-I, n is 0 to 30, and m is 0 to 4.

15. The combination of claim 14.

16. The at least one compound comprising an immune modulator comprises: 【Chemistry 17】 or a pharma- ceutically acceptable salt thereof.

17. The immune modulator comprises a TLR agonist having a structure of formula X or XX (or a radical of formula X or XX), or a pharma- ceutically acceptable salt of formula X or XX: 【Chemistry 18】 In formulas X and XX: R １ is -NH ２ Or -NH-R １Ｘ and R ２ is H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, heteroaryl, -NH-R ２Ｘ , -O-R ２Ｘ , -S-R ２Ｘ , 【Chemistry 19】 and 【Chemistry 20】 is a 3-10 membered N-containing non-aromatic monocyclic or bicyclic heterocycle; In formula X, R ３ is -OH, -SH, -NH ２ Or -NH-R １Ｘ and In the formula XX, X is CH or N; R １Ｘ , R ２Ｘ and R ２Ｙ are each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, and heteroaryl; 15. The combination of claim 14.

18. 18. The combination of any one of claims 14, 15 or 17, wherein the first drug is prepared to be administered to the subject intravenously, intramuscularly, intraperitoneally, topically, or by inhalation.

19. 18. A combination according to any one of claims 14, 15 or 17, wherein the folate ligand, or a functional fragment or analogue thereof, is specific for the folate receptor beta and binds to the folate receptor beta of the cells.

20. The combination of claim 1, wherein the engineered cells are chimeric antigen receptor (CAR)-expressing cytotoxic lymphocytes.

21. The TLR agonist of the compound has the structure of Formula 2-I (or a radical thereof) or a pharma- ceutically acceptable salt of Formula 2-I: 【Chemistry 21】 In formula 2-I, R １ , R ３ , R ４ and R ５ are each independently hydrogen (H), alkyl, alkoxyl, alkenyl, alkynyl, alicyclic, aryl, biaryl, halo, heteroaryl, -COR ２ｘ , 【Chemical 22】 and R ２ は、H、-OH、-NH ２ -NHR ２ｘ 、N ３ -NH-CH ２ -NH ２ 、-CONH ２ 、-SO ２ NH ２ -NH-CS-NH ２ 、 【Chemistry 23】 and Y is H, -OH, -NH ２ , -NHR ２ｘ , -O-R ２Ｘ , -SO-R ２ｘ , -SH, -SO ３ H, -N ３ , -CHO, -COOH, -CONH ２ , -COSH, -COR ２ｘ , -SO ２ N.H. ２ , alkenyl, alkynyl, alkoxyl, -NH-CH ２ -NH ２ , -CONH ２ , -SO ２ N.H. ２ , -NH-CS-NH ２ , 【Chemistry 24】 where: R ２ｘ and R ２ｙ are H, -OH, and -CH ２ -OH, -NH ２ , -CH ２ -NH ２ , -COOMe, -COOH, -CONH ２ , -COCH ３ , alkyl, alkenyl, alkynyl, cycloaliphatic, aryl, biaryl, and heteroaryl; R ２ｚ are each -NH ２ , -NR ２ｑ R ２ｑ’ , -O-R ２ｑ , -SO-R ２ｑ and -COR ２ｑ R is independently selected from the group consisting of ２ｑ and R ２ｑ’ are each independently alkyl or H; 【Chemistry 25】 is a non-aromatic, monocyclic or bicyclic 3-10 membered N-containing heterocycle; In formula 2-I, X １ , X ２ and X ３ are each independently ｑ or N, R ｑ each is independently H, halogen, or optionally substituted alkyl; In formula 2-I, n is 0 to 30, and m is 0 to 4.

21. The combination of claim 20.

22. The compound is 【Chemistry 26】 or a pharma- ceutically acceptable salt thereof.

23. The TLR agonist has the structure of the following formula (or a radical thereof), or a pharma- ceutically acceptable salt thereof: 【Chemistry 27】 During the ceremony, R １ is an amine group, R ２ is a single bond, -NH-, R ３ is H, an alkyl, a hydroxy group or any other substituted group thereof; X is CH ２ , NH, O or S; The linker is R １ , R ２ Or R ３ are bonded at 21. The combination of claim 20.

24. The linker comprises a polyethylene glycol (PEG) linker or a PEG derivative linker, R ３ or R １ 、 R ２ Or R ３ 24. A combination according to claim 21 or 23, wherein the linker is either

25. 2. The combination of claim 1, wherein the immune modulator or a pharma- ceutically acceptable salt thereof is a TLR7 agonist and the linker is a releasable linker.

26. The combination of claim 1 , wherein the linker is a non-releasable linker.

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