Compositions and uses thereof for chimeric antigen receptor T cell therapy

Abstract

The present disclosure describes an amphiphilic ligand conjugate comprising a chimeric antigen receptor (CAR) ligand, a lipid (diacyl lipid), and a linker (hydrophilic polymer, hydrophilic amino acid, polysaccharide), and claims compositions and methods using this construct, for example, to stimulate proliferation of CAR-expressing cells. In some embodiments, the lipid inserts into the cell membrane under physiological conditions. In some embodiments, the lipid binds to albumin under physiological conditions. In some embodiments, the lipid inserts into the cell membrane under physiological conditions and binds to albumin under physiological conditions. In some embodiments, the amphiphilic ligand conjugate is transported to lymph nodes and contains a lipid that inserts into the cell membrane of resident antigen-presenting cells (APCs), thereby co-presenting the CAR-T cell ligand on the cell surface along with native cytokine / receptor costimulatory signals.

Description

[Technical field]

[0001] Related information paragraph This application claims the benefit of the priority date of U.S. Provisional Patent Application No. 62 / 560,588, filed September 19, 2017, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Dramatic advances have been made in the clinical treatment of cancer using immunotherapy. One of the most powerful treatments developed to date is adoptive cell therapy with chimeric antigen receptor T cells (CAR T cells, or CAR-T). CAR-T are autologous lymphocytes from patients transduced with a synthetic antigen receptor formed by fusing an antigen-binding domain to the CD3 signaling chain from the T cell receptor complex and a costimulatory domain from one of several well-known co-receptors that provide supportive signals during T cell activation. CAR-T cells have shown dramatic complete responses in hematological malignancies, and the FDA recently approved CAR-T therapy for the treatment of B-cell leukemia.

[0003] However, CAR-T cells are currently simply infused into patients, receive no further stimulation except through tumor cell encounters in vivo, and lack many of the important signaling cues that are normally provided to T cells to promote their full effector function. Moreover, CAR-T cells fail to functionally persist in some patients and generally respond poorly to solid tumors. Thus, agents that improve CAR-T cell therapy are needed. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure is based, at least in part, on the discovery that chimeric antigen receptor (CAR) ligands are efficiently delivered to lymph nodes by the use of amphiphile conjugates that bind human serum albumin and partition into the membrane of resident antigen presenting cells (APCs), thereby co-presenting the CAR-T cell ligand on the cell surface along with native cytokine / receptor costimulatory signals. Without being bound by theory, it is believed that these dual properties of amphiphile conjugates (i.e., lymph node targeting and membrane insertion) combine to enable a booster vaccine for CAR-T cells, which efficiently expands CAR-T cells in vivo, increasing their functionality and enhancing antitumor activity.

[0005] It has been demonstrated that amphipathic ligand conjugates containing either a tag or a tumor-associated antigen activated and induced the proliferation of T cells expressing CARs containing either a tag or a tumor-associated antigen binding domain, or both. Notably, such amphipathic ligand conjugates retained this activity in vivo, thus allowing the expansion and activation of CAR-T cells after administration to subjects. Furthermore, administration of the amphipathic ligand conjugates of the present disclosure also resulted in significantly increased CAR-T infiltration into tumors, and tumor-infiltrating CAR-T cells showed enhanced reactivity against tumor cells despite the surface expression of checkpoint inhibitors PD1 and TIM3. Treatment with the amphipathic ligand conjugates of the present disclosure in conjunction with CAR-T cell therapy significantly delayed tumor growth and extended survival.

[0006] The present disclosure is also based, at least in part, on the discovery that the amphipathic ligand conjugates described herein overcome the poor response of CAR-T cells in solid tumors. As demonstrated herein, administration of CAR-T cells expressing tumor-associated antigens, when administered in combination with an amphipathic ligand conjugate, was able to delay tumor growth of solid tumors and increase survival of tumor-bearing mice compared to controls and CAR-T cells alone.

[0007] Furthermore, the present disclosure is based at least in part on the discovery that the enhanced efficacy of CAR-T cell therapy is maintained in lymphreplete conditions in combination with the amphipathic ligand conjugates of the present disclosure. Current CAR-T cell therapy requires lymphodepletion, which is associated with severe toxicity. As shown herein, CAR-T cell therapy in combination with the amphipathic ligand conjugates of the present disclosure resulted in delayed tumor growth and increased survival of lymphodepleted tumor-bearing mice. The delayed tumor growth and increased survival were comparable to lymphodepleted mice receiving the same treatment regimen. Without wishing to be bound by theory, these results indicate that administration of the amphipathic ligand conjugates of the present disclosure can eliminate the need for lymphodepletion prior to CAR-T cell therapy, thereby mitigating toxicity in subjects.

[0008] Thus, in one aspect, the present disclosure provides an amphiphilic ligand conjugate comprising a chimeric antigen receptor (CAR) ligand and a lipid operably linked to the CAR ligand. In some aspects, the lipid inserts into the cell membrane under physiological conditions. In some aspects, the lipid binds to albumin under physiological conditions. In some aspects, the lipid inserts into the cell membrane under physiological conditions and binds to albumin under physiological conditions. In some aspects, the amphiphilic ligand conjugate is transported to lymph nodes and comprises a lipid that inserts into the cell membrane of resident antigen presenting cells (APCs), thereby co-presenting the CAR-T cell ligand on the cell surface together with native cytokine / receptor co-stimulatory signals.

[0009] In any of the above or related embodiments, the amphiphilic ligand conjugate of the present disclosure comprises a diacyl lipid. In some embodiments, the diacyl lipid comprises an acyl chain comprising 12-30 hydrocarbon units. In some embodiments, the diacyl lipid comprises an acyl chain comprising 14-25 hydrocarbon units. In some embodiments, the diacyl lipid comprises an acyl chain comprising 16-20 hydrocarbon units. In some embodiments, the diacyl lipid comprises an acyl chain comprising 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hydrocarbon units. In some embodiments, the diacyl lipid comprises an acyl chain comprising 18 hydrocarbon units.

[0010] In any of the above or related embodiments, the amphiphilic ligand conjugate comprises a CAR ligand operably linked to a lipid via a linker. In some embodiments, the linker is selected from the group consisting of a hydrophilic polymer, a series of hydrophilic amino acids, a polysaccharide, or a combination thereof. In some embodiments, the linker comprises "N" consecutive polyethylene glycol units, where N is between 25 and 50.

[0011] In another aspect, the disclosure provides an amphipathic ligand conjugate comprising a CAR ligand operably linked to a diacyl lipid via a linker, wherein the diacyl lipid comprises an acyl chain comprising 12-30 hydrocarbon units, and the linker comprises "N" consecutive polyethylene glycol units, where N is between 25 and 50.

[0012] In any of the above or related embodiments, the amphiphilic ligand conjugate of the present disclosure comprises a CAR ligand that is a tag. In some embodiments, the tag is selected from the group consisting of fluorescein isothiocyanate (FITC), streptavidin, biotin, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horseradish peroxidase, palmitoylation, nitrosylation, alkaline phosphatase, glucose oxidase, and maltose binding protein.

[0013] In other embodiments, the amphipathic ligand conjugate comprises a CAR ligand that is a tumor-associated antigen or a fragment thereof. Exemplary tumor antigens include one or more of CD19, CD20, CD22, k light chain, CD30, CD33, CD123, CD38, ROR1, ErbB2, ErbB3 / 4, EGFr vIII, carcinoembryonic antigen, EGP2, EGP40, mesothelin, TAG72, PSMA, NKG2D ligand, B7-H6, IL-13 receptor alpha 2, MUC1, MUC16, CA9, GD2, GD3, HMW-MAA, CD171, Lewis Y, G250 / CALX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSC1, folate receptor-alpha, CD44v7 / 8, 8H9, NCAM, VEGF receptor, 5T4, fetal AchR, NKG2D ligand, CD44v6, TEM1 and / or TEM8.

[0014] In another aspect, the present disclosure provides an amphipathic ligand conjugate comprising a lipid operably linked to fluorescein isothiocyanate (FITC) via a polyethylene glycol moiety. In yet another aspect, the present disclosure provides an amphipathic ligand conjugate comprising a lipid operably linked to a fragment of a tumor-associated antigen (e.g., CD19, CD20, CD22, HER2, EGFRvII) via a polyethylene glycol moiety. In some aspects, the lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and the polyethylene glycol moiety is PEG-2000.

[0015] In any of the above or related embodiments, the amphiphilic ligand conjugate of the present disclosure comprises a lipid, the lipid being 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). In some embodiments, the amphiphilic ligand conjugate of the present disclosure comprises 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) linked to a CAR ligand via PEG-2000.

[0016] In another aspect, the present disclosure provides an amphipathic ligand conjugate comprising 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) operably linked to fluorescein isothiocyanate (FITC) via a polyethylene glycol moiety. In another aspect, the present disclosure provides an amphipathic ligand conjugate comprising 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) operably linked to a fragment of a tumor-associated antigen (e.g., CD19, CD20, CD22, HER2, EGFRvII) via a polyethylene glycol moiety.

[0017] In yet another aspect, the present disclosure provides an amphipathic ligand conjugate comprising 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) operably linked to fluorescein isothiocyanate (FITC) via PEG-2000. In yet a further aspect, the present disclosure provides an amphipathic ligand conjugate comprising 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) operably linked to a fragment of a tumor-associated antigen (e.g., CD19, CD20, CD22, HER2, EGFRvII) via PEG-2000.

[0018] In any of the above or related aspects, an amphipathic ligand conjugate of the present disclosure comprises a CAR ligand that binds to a CAR, where the CAR comprises a costimulatory domain.

[0019] In any of the above or related embodiments, the amphiphilic ligand conjugate of the present disclosure comprises a CAR ligand that binds to a CAR, and the CAR comprises a bispecific binding domain. In some embodiments, the bispecific binding domain comprises a tag binding domain and a tumor-associated antigen binding domain (e.g., CD19, CD20, CD22, HER2, EGFRvII). In some embodiments, the bispecific binding domain comprises a first tumor-associated antigen binding domain (e.g., CD19, CD20, CD22, HER2, EGFRvII) and a second tumor-associated antigen binding domain (e.g., CD19, CD20, CD22, HER2, EGFRvII). In some embodiments, the bispecific binding domain comprises a tag binding domain and a tumor-associated antigen binding domain, and the CAR ligand is a tag. In some embodiments, the bispecific binding domain comprises a first tumor-associated antigen binding domain and a second tumor-associated antigen binding domain, and the CAR ligand comprises a first or second tumor-associated antigen, or a fragment thereof.

[0020] In any of the above or related embodiments, the amphipathic ligand conjugate of the present disclosure comprises a CAR ligand comprising a tag, wherein the CAR comprises a tag-binding domain. In other embodiments, the CAR ligand is a tumor-associated antigen or a fragment thereof, and the CAR comprises a tumor-associated antigen-binding domain.

[0021] In another aspect, the present disclosure provides an amphipathic ligand conjugate comprising a diacyl lipid operably linked to a tag, wherein the tag binds to a CAR comprising a tag-binding domain. In another aspect, the present disclosure provides an amphipathic ligand conjugate comprising a diacyl lipid operably linked to a tag via a polyethylene glycol moiety, wherein the tag binds to a CAR comprising a tag-binding domain.

[0022] In another aspect, the present disclosure provides an amphipathic ligand conjugate comprising a diacyl lipid operably linked to a tag, wherein the tag binds to a CAR comprising a tag-binding domain and a tumor-associated antigen-binding domain. In another aspect, the present disclosure provides an amphipathic ligand conjugate comprising a diacyl lipid operably linked to a tag via a polyethylene glycol moiety, wherein the tag binds to a CAR comprising a tag-binding domain and a tumor-associated antigen-binding domain.

[0023] In another aspect, the present disclosure provides an amphipathic ligand conjugate comprising a diacyl lipid operably linked to a tumor-associated antigen or a fragment thereof, wherein the tumor-associated antigen binds to a CAR comprising a tumor-associated antigen binding domain (e.g., CD19, CD20, CD22, HER2, EGFRvII). In another aspect, the present disclosure provides an amphipathic ligand conjugate comprising a diacyl lipid operably linked to a tumor-associated antigen or a fragment thereof via a polyethylene glycol moiety, wherein the tumor-associated antigen or a fragment thereof binds to a CAR comprising a tumor-associated antigen binding domain binding domain. In some aspects, the CAR comprises a first tumor-associated antigen binding domain and a second tumor-associated antigen binding domain, and the amphipathic ligand conjugate comprises either the first or second tumor-associated antigen.

[0024] In another aspect, the present disclosure provides a composition comprising an amphiphilic ligand conjugate described herein and a pharma- ceutically acceptable carrier.

[0025] In another aspect, the disclosure provides an immunogenic composition comprising a composition described herein and an adjuvant.

[0026] In some embodiments, the immunogenic composition comprises an adjuvant, the adjuvant being an amphipathic oligonucleotide conjugate comprising an immunostimulatory oligonucleotide conjugated to a lipid with or without a linker and optionally a polar compound. In some embodiments, the immunostimulatory oligonucleotide binds to a pattern recognition receptor. In some embodiments, the immunostimulatory oligonucleotide comprises CpG. In some embodiments, the immunostimulatory oligonucleotide is a ligand for a toll-like receptor.

[0027] In any of the above-mentioned embodiments, the amphipathic oligonucleotide conjugate comprises a linker, the linker being an oligonucleotide linker. In some embodiments, the oligonucleotide linker comprises "N" consecutive guanines, N being between 0 and 2. In some embodiments, the lipid of the amphipathic oligonucleotide conjugate is a diacyl lipid. In some embodiments, the diacyl lipid comprises an acyl chain comprising 12-30 hydrocarbon units. In some embodiments, the diacyl lipid comprises an acyl chain comprising 14-25 hydrocarbon units. In some embodiments, the diacyl lipid comprises an acyl chain comprising 16-20 hydrocarbon units. In some embodiments, the diacyl lipid comprises an acyl chain comprising 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hydrocarbon units. In some embodiments, the diacyl lipid comprises an acyl chain comprising 18 hydrocarbon units.

[0028] In other embodiments, the immunogenic composition comprises an adjuvant, and the adjuvant is cyclic di-GMP (CDG).

[0029] In another aspect, the disclosure provides a method of activating, expanding or increasing the proliferation of CAR-T cells in a subject, comprising administering to the subject an amphipathic ligand conjugate, composition or immunogenic composition described herein. In some aspects, the proliferation of CAR(-) T cells is not increased in the subject. In some aspects, the CAR comprises a tag binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tag. In some aspects, the CAR comprises a tumor-associated antigen binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tumor-associated antigen or a fragment thereof. In some aspects, the CAR comprises a tag binding domain and a tumor-associated antigen binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tag. In some aspects, the CAR comprises a first tumor-associated antigen binding domain and a second tumor-associated antigen binding domain, and the CAR ligand of the amphipathic ligand conjugate is a first or second tumor-associated antigen, or a fragment thereof.

[0030] In yet another aspect, the disclosure provides a method of reducing or decreasing tumor size or inhibiting tumor growth in a subject in need thereof, comprising administering to the subject an amphipathic ligand conjugate, composition, or immunogenic composition described herein, wherein the subject is undergoing or has undergone CAR-T cell therapy. In some aspects, the CAR comprises a tag-binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tag. In some aspects, the CAR comprises a tumor-associated antigen binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tumor-associated antigen or a fragment thereof. In some aspects, the CAR comprises a tag-binding domain and a tumor-associated antigen binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tag. In some aspects, the CAR comprises a first tumor-associated antigen binding domain and a second tumor-associated antigen binding domain, and the CAR ligand of the amphipathic ligand conjugate is a first or second tumor-associated antigen, or a fragment thereof.

[0031] In a further aspect, the present disclosure provides a method of inducing an anti-tumor response in a subject having cancer, comprising administering to the subject an amphipathic ligand conjugate, composition or immunogenic composition as described herein, wherein the subject is undergoing or has undergone CAR-T cell therapy. In some aspects, the CAR comprises a tag-binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tag. In some aspects, the CAR comprises a tumor-associated antigen binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tumor-associated antigen or a fragment thereof. In some aspects, the CAR comprises a tag-binding domain and a tumor-associated antigen binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tag. In some aspects, the CAR comprises a first tumor-associated antigen binding domain and a second tumor-associated antigen binding domain, and the CAR ligand of the amphipathic ligand conjugate is a first or second tumor-associated antigen, or a fragment thereof.

[0032] In another aspect, the present disclosure provides a method of stimulating an immune response against a target cell population or target tissue expressing an antigen in a subject, comprising administering to the subject a CAR-T cell targeted to the antigen and an amphipathic ligand conjugate, composition or immunogenic composition described herein. In some aspects, the immune response is a T cell-mediated immune response or an anti-tumor immune response. In some aspects, the target cell population or target tissue is a tumor cell or tumor tissue. In some aspects, the CAR comprises a tag-binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tag. In some aspects, the CAR comprises a tumor-associated antigen-binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tumor-associated antigen or a fragment thereof. In some aspects, the CAR comprises a tag-binding domain and a tumor-associated antigen-binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tag. In some embodiments, the CAR comprises a first tumor-associated antigen binding domain and a second tumor-associated antigen binding domain, and the CAR ligand of the amphipathic ligand conjugate is the first or second tumor-associated antigen, or a fragment thereof.

[0033] In another aspect, the present disclosure provides a method of stimulating an immune response against a target cell population or target tissue expressing an antigen in a subject, comprising administering to the subject a CAR-T cell targeted to the antigen and an amphipathic ligand conjugate, composition or immunogenic composition as described herein, wherein the target cell population or target tissue is a cell population or tissue infected with a virus. In some aspects, the virus is human immunodeficiency virus (HIV). In some aspects, the immune response is a T cell-mediated immune response. In some aspects, the CAR comprises a tag-binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tag.

[0034] In a further aspect, the present disclosure provides a method of treating a subject having a disease, disorder or condition associated with expression or increased expression of an antigen, comprising administering to the subject a CAR-T cell targeted to the antigen and an amphipathic ligand conjugate, composition or immunogenic composition described herein. In some aspects, the antigen is a viral antigen or a cancer antigen. In some aspects, the CAR comprises a tag-binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tag. In some aspects, the CAR comprises a tumor-associated antigen-binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tumor-associated antigen or a fragment thereof. In some aspects, the CAR comprises a tag-binding domain and a tumor-associated antigen-binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tag.

[0035] In any of the above-mentioned embodiments, the method comprises administering the amphipathic ligand conjugate, composition or immunogenic composition to the subject prior to receiving the CAR-T cells. In other embodiments, the method comprises administering the amphipathic ligand conjugate, composition or immunogenic composition to the subject after receiving the CAR-T cells. In another embodiment, the method comprises administering the amphipathic ligand conjugate, composition or immunogenic composition to the subject in conjunction with the co-administered CAR-T cells.

[0036] In any of the above-mentioned related aspects, the amphipathic ligand conjugate of the present disclosure is transported to lymph nodes. In some aspects, the amphipathic ligand conjugate is transported to inguinal and auxiliary lymph nodes. In some aspects, once transported to lymph nodes, the amphipathic ligand conjugate is inserted into the membrane of an antigen-presenting cell. In some aspects, the antigen-presenting cell is a medullary macrophage, a CD8+ dendritic cell, and / or a CD11b+ dendritic cell.

[0037] In any of the above-described embodiments, the CAR ligand is retained in the lymph node for at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, or at least 25 days.

[0038] In any of the above-mentioned embodiments in which the CAR ligand is a tag and the CAR comprises a tag-binding domain, the methods further comprise administering a formulation of tagged protein, where the tag-binding domain binds to the tagged protein. In some embodiments, the protein of the tagged protein is an antibody or an antigen-binding fragment thereof. In some embodiments, the tag-binding domain is an antibody or an antigen-binding fragment thereof. In some embodiments, the formulation of tagged protein is administered to the subject prior to administration of the CART cells and the amphipathic ligand conjugate, composition or immunogenic composition. In other embodiments, the formulation of tagged protein is administered to the subject in conjunction with administration of the CAR-T cells and the amphipathic ligand conjugate, composition or immunogenic composition. In yet other embodiments, the formulation of tagged protein is administered to the subject after administration of the CAR-T cells and the amphipathic ligand conjugate, composition or immunogenic composition.

[0039] In any of the above-mentioned embodiments, the CAR-T cells are administered prior to administration of the amphipathic ligand conjugate, composition or immunogenic composition. In other embodiments, the CAR-T cells are administered after administration of the amphipathic ligand conjugate, composition or immunogenic composition. In yet other embodiments, the CAR-T cells are administered in conjunction with administration of the amphipathic ligand conjugate, composition or immunogenic composition.

[0040] In any of the above aspects, the amphipathic ligand conjugate, composition or immunogenic composition described herein is administered parenterally in a non-tumor draining lymph node, parenterally in a tumor draining lymph node, or intratumorally.

[0041] In any of the above aspects, the subject has cancer. In any of the above aspects, the subject is a human.

[0042] In another aspect, the present disclosure provides a kit for treating or delaying the progression of cancer in an individual undergoing CAR-T cell therapy, comprising a container containing a composition comprising an amphipathic ligand conjugate as described herein, an optional pharma- ceutically acceptable carrier, and a package insert containing instructions for administering the composition. In some embodiments, the CAR comprises a tag binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tag. In some embodiments, the CAR comprises a tumor-associated antigen binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tumor-associated antigen or a fragment thereof. In some embodiments, the CAR comprises a tag binding domain and a tumor-associated antigen binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tag. In some embodiments, the CAR comprises a first tumor-associated antigen binding domain and a second tumor-associated antigen binding domain, and the CAR ligand of the amphipathic ligand conjugate is a first or second tumor-associated antigen, or a fragment thereof.

[0043] In yet another aspect, the present disclosure provides a kit comprising a medicament comprising a composition comprising an amphipathic ligand conjugate as described herein, optionally a pharmaceutical acceptable carrier, for treating or delaying the progression of cancer in an individual undergoing CAR-T cell therapy, and a package insert comprising instructions for administration of the medicament alone or in combination with a composition comprising an adjuvant and optionally a pharmaceutical acceptable carrier. In some embodiments, the CAR comprises a tag-binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tag. In some embodiments, the CAR comprises a tumor-associated antigen binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tumor-associated antigen or a fragment thereof. In some embodiments, the CAR comprises a tag-binding domain and a tumor-associated antigen binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tag. In some embodiments, the CAR comprises a first tumor-associated antigen binding domain and a second tumor-associated antigen binding domain, and the CAR ligand of the amphipathic ligand conjugate is a first or second tumor-associated antigen, or a fragment thereof.

[0044] In other aspects, the present disclosure provides a kit comprising a container containing a composition comprising an amphipathic ligand conjugate as described herein, an optional pharma- ceutically acceptable carrier, and a package insert containing instructions for administering the composition vaccine for activating, expanding or increasing the proliferation of CAR-T cells in an individual undergoing CAR-T cell therapy. In some aspects, the CAR comprises a tag binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tag. In some aspects, the CAR comprises a tumor-associated antigen binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tumor-associated antigen or a fragment thereof. In some aspects, the CAR comprises a tag binding domain and a tumor-associated antigen binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tag. In some aspects, the CAR comprises a first tumor-associated antigen binding domain and a second tumor-associated antigen binding domain, and the CAR ligand of the amphipathic ligand conjugate is a first or second tumor-associated antigen, or a fragment thereof.

[0045] In some aspects, the present disclosure provides a kit comprising a medicament comprising a composition comprising an amphipathic ligand conjugate as described herein, optionally a pharmaceutical acceptable carrier, for activating, expanding or increasing the proliferation of CAR-T cells in an individual undergoing CAR-T cell therapy, and a package insert comprising instructions for administration of the medicament alone or in combination with a composition comprising an adjuvant and optionally a pharmaceutical acceptable carrier. In some aspects, the CAR comprises a tag-binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tag. In some aspects, the CAR comprises a tumor-associated antigen binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tumor-associated antigen or a fragment thereof. In some aspects, the CAR comprises a tag-binding domain and a tumor-associated antigen binding domain, and the CAR ligand of the amphipathic ligand conjugate is a tag. In some aspects, the CAR comprises a first tumor-associated antigen binding domain and a second tumor-associated antigen binding domain, and the CAR ligand of the amphipathic ligand conjugate is a first or second tumor-associated antigen, or a fragment thereof.

[0046] In any of the above-mentioned embodiments, the kit includes an adjuvant and instructions for administration of the adjuvant to treat or delay the progression of cancer in an individual undergoing CAR-T cell therapy. In some embodiments, the adjuvant is an amphipathic oligonucleotide conjugate comprising an immunostimulatory oligonucleotide as described herein.

[0047] In another aspect, the disclosure provides a use of an amphipathic ligand conjugate, composition or immunogenic composition described herein to activate, expand or increase the proliferation of CAR-T cells in an individual undergoing CAR-T cell therapy.

[0048] In yet another aspect, the present disclosure provides a use of an amphipathic ligand conjugate, composition or immunogenic composition described herein for treating or delaying the progression of cancer in an individual.

[0049] In another aspect, the disclosure provides the use of an amphipathic ligand conjugate, composition or immunogenic composition described herein in the manufacture of a medicament for treating or delaying the progression of cancer in an individual.

[0050] In other aspects, the present disclosure provides a kit comprising a pharmaceutical composition comprising an amphipathic ligand conjugate as described herein, an optional pharmaceutical acceptable carrier, and a package insert comprising instructions for administering the composition for treating or delaying the progression of a viral infection in an individual undergoing CAR-T cell therapy. In some aspects, the kit comprises a formulation of a tagged protein and instructions for administering the formulation of the tagged protein, wherein the CAR comprises a tag-binding domain that binds to the tagged protein. In some aspects, the kit comprises an adjuvant and instructions for administering the adjuvant for treating or delaying the progression of a viral infection in an individual undergoing CAR-T cell therapy. In some aspects, the adjuvant is an amphipathic oligonucleotide conjugate as described herein. (Item 1) Chimeric antigen receptor (CAR) ligands; and A lipid operably linked to the CAR ligand. 2. An amphiphilic ligand conjugate comprising: (Item 2) 2. The amphiphilic ligand conjugate according to claim 1, wherein the lipid inserts into a cell membrane under physiological conditions, binds to albumin under physiological conditions, or both. (Item 3) 3. The amphiphilic ligand conjugate according to claim 1 or 2, wherein the lipid is a diacyl lipid. (Item 4) 4. The amphiphilic ligand conjugate according to claim 3, wherein the diacyl lipid comprises an acyl chain comprising 12 to 30 hydrocarbon units, 14 to 25 hydrocarbon units, 16 to 20 hydrocarbon units, or 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 hydrocarbon units. (Item 5) 5. The amphiphilic ligand conjugate according to any one of claims 1 to 4, wherein the CAR ligand is operably linked to the lipid via a linker. (Item 6) 6. The amphiphilic ligand conjugate according to claim 5, wherein the linker is selected from the group consisting of a hydrophilic polymer, a series of hydrophilic amino acids, a polysaccharide, or a combination thereof. (Item 7) 6. The amphiphilic ligand conjugate according to item 5, wherein the linker comprises "N" consecutive polyethylene glycol units, where N is between 25 and 50. (Item 8) 1. An amphipathic ligand conjugate comprising a CAR ligand operably linked to a diacyl lipid via a linker, wherein the diacyl lipid comprises an acyl chain comprising 12-30 hydrocarbon units, and the linker comprises "N" consecutive polyethylene glycol units, where N is between 25 and 50. (Item 9) 9. The amphiphilic ligand conjugate according to any one of the preceding claims, wherein the CAR ligand is a tag. (Item 10) 10. The amphiphilic ligand conjugate according to item 9, wherein the tag is selected from the group consisting of fluorescein isothiocyanate (FITC), streptavidin, biotin, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horseradish peroxidase, palmitoylation, nitrosylation, alkaline phosphatase, glucose oxidase and maltose binding protein. (Item 11) 9. The amphiphilic ligand conjugate according to any one of items 1 to 8, wherein the CAR ligand is a tumor associated antigen or a fragment thereof. (Item 12) An amphiphilic ligand conjugate comprising a lipid operably linked to fluorescein isothiocyanate (FITC) via a polyethylene glycol moiety. (Item 13) 13. The amphiphilic ligand conjugate according to any one of items 8 to 12, wherein the lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and the polyethylene glycol moiety is PEG-2000. (Item 14) 14. The amphiphilic ligand conjugate of any one of items 1 to 13, wherein the CAR ligand binds to a CAR, and the CAR comprises a costimulatory domain. (Item 15) 15. The amphiphilic ligand conjugate of item 14, wherein the CAR comprises bispecific binding domains. (Item 16) 16. The amphiphilic ligand conjugate according to item 15, wherein the bispecific binding domain comprises a tag binding domain and a tumor associated antigen binding domain or a first tumor associated antigen binding domain and a second tumor associated antigen binding domain. (Item 17) 17. The amphiphilic ligand conjugate according to claim 16, wherein the bispecific binding domain comprises a tag-binding domain and a tumor-associated antigen-binding domain, and the CAR ligand is a tag. (Item 18) 16. The amphipathic ligand conjugate of claim 15, wherein the bispecific binding domain comprises a first tumor associated antigen binding domain and a second tumor associated antigen binding domain, and the CAR ligand is the first or second tumor associated antigen, or a fragment thereof. (Item 19) 15. The amphiphilic ligand conjugate of item 14, wherein the CAR comprises a tag-binding domain and the CAR ligand is a tag. (Item 20) 15. The amphipathic ligand conjugate of item 14, wherein the CAR comprises a tumor-associated antigen binding domain and the CAR ligand is a tumor-associated antigen or a fragment thereof. (Item 21) 20. A composition comprising an amphiphilic ligand conjugate according to any one of items 1 to 19 and a pharma- ceutically acceptable carrier. (Item 22) 22. An immunogenic composition comprising the composition according to item 21 and an adjuvant. (Item 23) 23. The immunogenic composition of claim 22, wherein the adjuvant is an amphipathic oligonucleotide conjugate comprising an immunostimulatory oligonucleotide conjugated to a lipid with or without a linker and optionally a polar compound. (Item 24) 24. The immunogenic composition of claim 23, wherein the immunostimulatory oligonucleotide binds to a pattern recognition receptor. (Item 25) 25. The immunogenic composition of claim 24, wherein the immunostimulatory oligonucleotide comprises CpG. (Item 26) 24. The immunogenic composition of claim 23, wherein the immunostimulatory oligonucleotide is a ligand for a toll-like receptor. (Item 27) 27. The immunogenic composition according to any one of items 23 to 26, wherein the linker is an oligonucleotide linker. (Item 28) 28. The immunogenic composition according to item 27, wherein the oligonucleotide linker comprises "N" consecutive guanines, where N is between 0 and 2. (Item 29) 29. The immunogenic composition according to any one of items 23 to 28, wherein the lipid is a diacyl lipid. (Item 30) 30. The immunogenic composition of item 29, wherein the diacyl lipid comprises an acyl chain containing 12 to 30 carbohydrate units. (Item 31) 23. The immunogenic composition of claim 22, wherein the adjuvant is cyclic di-GMP (CDG). (Item 32) 32. A method for activating, expanding or increasing the proliferation of CAR-T cells in a subject, comprising administering to the subject an amphipathic ligand conjugate according to any one of items 1 to 20, a composition according to item 21 or an immunogenic composition according to any one of items 23 to 31. (Item 33) The method of claim 32, wherein proliferation of CAR(-) T cells is not increased in the subject. (Item 34) 32. A method of reducing or decreasing tumor size or inhibiting tumor growth in a subject in need thereof, comprising administering to the subject an amphipathic ligand conjugate according to any one of items 1 to 20, a composition according to item 21 or an immunogenic composition according to any one of items 23 to 31, wherein the subject is undergoing or has undergone CAR-T cell therapy. (Item 35) 32. A method for inducing an anti-tumor response in a subject having cancer, comprising administering to the subject an amphipathic ligand conjugate according to any one of items 1 to 20, a composition according to item 21 or an immunogenic composition according to any one of items 23 to 31, wherein the subject is undergoing or has undergone CAR-T cell therapy. (Item 36) 32. A method for stimulating an immune response against a target cell population or target tissue expressing an antigen in a subject, comprising administering to the subject CAR-T cells targeted to said antigen and an amphipathic ligand conjugate according to any one of items 1 to 20, the composition according to item 21 or the immunogenic composition according to any one of items 23 to 31. (Item 37) 37. The method of claim 36, wherein the immune response is a T cell-mediated immune response or an anti-tumor immune response. (Item 38) 38. The method of claim 36 or 37, wherein the target cell population or target tissue is a tumor cell or tumor tissue. (Item 39) 32. A method of treating a subject having a disease, disorder or condition associated with expression or increased expression of an antigen, comprising administering to the subject CAR-T cells targeted to said antigen and an amphipathic ligand conjugate according to any one of items 1 to 20, the composition according to item 21 or the immunogenic composition according to any one of items 23 to 31. (Item 40) 35. The method of any one of items 32 to 34, wherein the subject is administered the amphipathic ligand conjugate, the composition or the immunogenic composition prior to receiving CAR T cells. (Item 41) 35. The method of any one of items 32 to 34, wherein the subject is administered the amphipathic ligand conjugate, the composition or the immunogenic composition after receiving CAR-T cells. (Item 42) 35. The method of any one of items 32 to 34, wherein the amphipathic ligand conjugate, the composition or the immunogenic composition, and the CAR-T cells are administered simultaneously. (Item 43) 43. The method of any one of items 32 to 42, wherein the amphiphilic ligand conjugate is transported to a lymph node. (Item 44) 43. The method of any one of items 32 to 42, wherein the amphiphilic ligand conjugate is transported to the inguinal lymph nodes and auxiliary lymph nodes. (Item 45) 45. The method of any one of items 32 to 44, wherein the amphipathic ligand conjugate, upon transport to the lymph node, is inserted into the membrane of an antigen presenting cell. (Item 46) 46. ​​The method of claim 45, wherein the antigen-presenting cells are medullary macrophages, CD8+ dendritic cells and / or CD11b+ dendritic cells. (Item 47) 47. The method of any one of items 32 to 46, wherein the CAR ligand is retained in the lymph node for at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, or at least 25 days. (Item 48) 48. The method of any one of items 32 to 47, wherein the CAR ligand is a tag and the CAR comprises a tag-binding domain, and the method further comprises administering a formulation of a tagged protein, wherein the tag-binding domain binds to the tagged protein. (Item 49) 49. The method of claim 48, wherein the tagged protein is an antibody or an antigen-binding fragment thereof. (Item 50) 50. The method of claim 48 or 49, wherein the tag binding domain is an antibody or an antigen-binding fragment thereof. (Item 51) 51. The method of any one of items 48 to 50, wherein a formulation of the tagged protein is administered to the subject prior to administration of the CAR-T cells, and amphipathic ligand conjugate, composition or immunogenic composition. (Item 52) 51. The method of any one of items 48 to 50, wherein a formulation of the tagged protein is administered to the subject in conjunction with administration of the CAR-T cells, and amphipathic ligand conjugate, composition, or immunogenic composition. (Item 53) 51. The method of any one of items 48 to 50, wherein a formulation of the tagged protein is administered to the subject after administration of the CAR-T cells, and the amphipathic ligand conjugate, composition, or immunogenic composition. (Item 54) 54. The method of any one of items 51 to 53, wherein the CAR-T cells are administered prior to administration of the amphipathic ligand conjugate, composition or immunogenic composition. (Item 55) 54. The method of any one of items 51 to 53, wherein the CAR-T cells are administered following administration of the amphipathic ligand conjugate, composition or immunogenic composition. (Item 56) 54. The method of any one of items 51 to 53, wherein the CAR-T cells are administered in conjunction with administration of the amphipathic ligand conjugate, composition or immunogenic composition. (Item 57) 57. The method of any one of items 32 to 34 and 49 to 56, wherein the subject has cancer. (Item 58) 58. The method of any one of items 32 to 57, wherein the subject is a human. (Item 59) 21. A kit comprising a container containing a composition comprising the amphiphilic ligand conjugate according to any one of items 1 to 20, optionally a pharma- ceutically acceptable carrier, and a package insert containing instructions for administration of said composition for treating or delaying the progression of cancer in an individual undergoing CAR-T cell therapy. (Item 60) 21. A kit comprising a medicament comprising a composition comprising the amphipathic ligand conjugate according to any one of items 1 to 20, optionally a pharma- ceutically acceptable carrier, for treating or delaying the progression of cancer in an individual undergoing CAR-T cell therapy, and a package insert comprising instructions for administration of said medicament alone or in combination with a composition comprising an adjuvant and optionally a pharma- ceutically acceptable carrier. (Item 61) 21. A kit comprising a container containing a composition comprising the amphipathic ligand conjugate according to any one of items 1 to 20, optionally a pharma- ceutically acceptable carrier, and a package insert containing instructions for administration of the composition vaccine, for activating, expanding or increasing the proliferation of CAR-T cells in an individual undergoing CAR-T cell therapy. (Item 62) 21. A kit comprising a medicament comprising a composition comprising the amphipathic ligand conjugate according to any one of items 1 to 20, optionally a pharma- ceutically acceptable carrier, for activating, expanding or increasing the proliferation of CAR-T cells in an individual undergoing CAR-T cell therapy, and a package insert comprising instructions for administration of said medicament alone or in combination with a composition comprising an adjuvant and optionally a pharma- ceutically acceptable carrier. (Item 63) 62. The kit of claim 59 or claim 61, further comprising an adjuvant and instructions for administration of the adjuvant to treat or delay the progression of cancer in an individual undergoing CAR-T cell therapy. (Item 64) 64. The kit according to any one of items 60, 62 and 63, wherein the adjuvant is an amphipathic oligonucleotide conjugate comprising an immunostimulatory oligonucleotide conjugated to a lipid with or without a linker and optionally a polar compound. (Item 65) 32. Use of the amphipathic ligand conjugate according to any one of items 1 to 20, the composition according to item 21 or the immunogenic composition according to any one of items 23 to 31 for activating, expanding or increasing the proliferation of CAR-T cells in an individual undergoing CAR-T cell therapy. (Item 66) Use of the amphiphilic ligand conjugate according to any one of items 1 to 20, the composition according to item 21 or the immunogenic composition according to any one of items 23 to 31 for treating or delaying the progression of cancer in an individual. (Item 67) 32. Use of the amphiphilic ligand conjugate according to any one of items 1 to 20, the composition according to item 21 or the immunogenic composition according to any one of items 23 to 31 in the manufacture of a medicament for treating or delaying the progression of cancer in an individual. (Item 68) 59. The method of any one of items 32 to 58, comprising administering the amphipathic ligand conjugate, the composition or the immunogenic composition parenterally in a non-tumor draining lymph node, parenterally in a tumor draining lymph node or intratumorally. (Item 69) 37. The method of claim 36, wherein the target cell population or target tissue is a virally infected cell population or tissue. (Item 70) 70. The method of claim 69, wherein the virus is human immunodeficiency virus (HIV). (Item 71) The method of claim 69 or 70, wherein the immune response is a T cell-mediated immune response. (Item 72) 40. The method of claim 39, wherein the antigen is a viral antigen or a cancer antigen. (Item 73) 21. A kit comprising a medicament comprising a composition comprising the amphipathic ligand conjugate according to any one of items 1 to 20, optionally a pharma- ceutically acceptable carrier, and a package insert comprising instructions for administration of said composition for treating or delaying the progression of a viral infection in an individual undergoing CAR-T cell therapy. (Item 74) 74. The kit of claim 73, further comprising a formulation of a tagged protein and instructions for administering the formulation of the tagged protein, wherein the CAR comprises a tag binding domain that binds to the tagged protein. (Item 75) The kit of claim 73 or claim 74, further comprising an adjuvant and instructions for administration of the adjuvant to treat or delay the progression of a viral infection in an individual receiving CAR-T cell therapy. (Item 76) 76. The kit of item 75, wherein the adjuvant is an amphipathic oligonucleotide conjugate comprising an immunostimulatory oligonucleotide conjugated to a lipid with or without a linker and optionally a polar compound. [Brief description of the drawings]

[0051] [Figure 1A] Figure 1A provides a schematic diagram of amphiphilic ligand conjugates comprising a lipid tail (e.g., DSPE) conjugated via a PEG-2000 linker to a small molecule (top), a short linear peptide (middle) or a protein domain (bottom).

[0052] [Figure 1B] FIG. 1B provides a schematic diagram showing the interaction between antigen-presenting cells decorated with an amphipathic ligand conjugate containing a chimeric antigen receptor (CAR) ligand and CAR-T cells.

[0053] [Figure 2A] FIG. 2A provides a schematic of the domain structure and orientation of the transmembrane anti-FITC CAR.

[0054] [Figure 2B] FIG. 2B provides a graph of flow cytometry data showing the extent of anti-FITC CAR surface expression following retroviral transduction into primary mouse T cells.

[0055] [Figure 2C] Figure 2C provides a graph showing quantification of IFNγ produced by anti-FITC CAR-T cells after interaction with K562 cells decorated with various concentrations of DSPE-PEG-FITC as indicated. ***p<0.0001, **p<0.01, *p<0.05.

[0056] [Figure 2D]Figure 2D provides a graph showing the percentage of cell death of DSPE-PEG-FITC coated DC2.4 cells after 6 hours of co-culture with FITC-CAR-T cells at an effector to target (E:T) ratio of 10:1. ***p<0.0001, **p<0.01, *p<0.05.

[0057] [Figure 3A] FIG. 3A provides a graph showing the extent of DSPE-PEG-FITC retention (as measured by radioefficiency) in lymph nodes removed from mice after subsequent days following vaccination with various doses of DSPE-PEG-FITC or FITC alone as indicated.

[0058] [Figure 3B] FIG. 3B provides a graph showing DSPE-PEG-FITC uptake by different lymphatic populations in the draining inguinal lymph node 24 hours after subcutaneous injection.

[0059] [Figure 3C] FIG. 3C provides a graph of flow cytometry data showing uptake of various doses of DSP-PEG-FITC by three different APCs following subcutaneous injection.

[0060] [Figure 4] Figure 4 provides a graph showing the proliferation index of FITC CAR-T cells in inguinal lymph nodes primed with PBS, c-di-GMP (CDG), DSPE-PEG-FITC, or DSPE-PEG-FITC+CDG. The effect of PBS and CDG alone was evaluated 1 day after vaccination.

[0061] [Diagram 5]Figure 5 provides a graph showing DSPE-PEG-FITC display on antigen-presenting cell surface with or without CDG in lymph node cell populations. Lymph nodes were harvested 24 hours and 3 days after DSPE-PEG-FITC vaccination + / - CDG. ***p<0.0001, **p<0.01, *p<0.05.

[0062] [Figure 6] Figure 6 provides a graph showing the mean fluorescence intensity (MFI) of various costimulatory molecules on CD11c+ cells that take up DSPE-PEG-FITC with or without CDG. ***p<0.0001, **p<0.01, *p<0.05.

[0063] [Figure 7] Figure 7 provides a schematic showing the experimental timeline (top) and a graph showing the percentage of CD45.1 FITC CAR-T cells following two rounds of DSPE-PEG-FITC vaccination in lymphodepleted CD45.2 mice (bottom). ***p<0.0001, **p<0.01, *p<0.05.

[0064] [Figure 8] Figure 8 provides a schematic showing the experimental timeline (top) and a graph showing the percentage of CD45.1 FITC CAR-T cells following two rounds of DSPE-PEG-FITC vaccination in lymphoid-replete CD45.2 mice. ***p<0.0001, **p<0.01, *p<0.05.

[0065] [Figure 9] Figure 9 provides a graph showing antibody responses over time to repeated DSPE-PEG-FITC vaccinations. ***p<0.0001, **p<0.01, *p<0.05.

[0066] [Figure 10A] FIG. 10A provides a schematic showing EGFRvIII peptide conjugated to DSPE-PEG.

[0067] [Figure 10B] FIG. 10B shows surface expression of EGFRvIII CAR on mouse T cells after immunization with DSPE-PEG-EGFRvIII.

[0068] [Figure 10C] Figure 10C shows the proliferation of EGFRvIII CAR T cells in lymph nodes 48 hours after DSPE-PEG-EGFRvIII vaccination as determined by cell trace violet tracing.

[0069] [Figure 11A] Figure 11A provides a graph showing quantification of IFNγ produced by EGFRvIII CAR-T cells or control T cells following interaction with CT-2A glioma cells with or without EGFRvIII expressed on the cell surface. ***p<0.0001, **p<0.01, *p<0.05.

[0070] [Figure 11B] Figure 11B provides a graph showing the percentage of cell death of CT-2A glioma cells with wild-type EGFR or EGFRvIII after co-culture with EGFRvIII CAR-T cells or control T cells. ***p<0.0001, **p<0.01, *p<0.05.

[0071] [Figure 12] Figure 12 provides a graph showing the percentage of EGFRvIII CAR T cells in mice vaccinated with DSPE-PEG-EGFRvIII ("VAX") or a control vaccine.

[0072] [Figure 13] Figure 13 provides a graph showing cytokine (IFNγ and TNFα) secretion of circulating CAR T or non-CAR T cells (n=5) in response to EGFRvIII-expressing target cells with or without DSPE-PEG-EGFRvIII ("VAX") in vitro.

[0073] [Figure 14] Figure 14 provides a schematic showing the experimental timeline (top) and a graph showing tumor infiltration of EGFRvIII CAR-T cells, as measured by the number of CAR-T cells per mg of tumor, in mice implanted with EGFRvIII-expressing CT-2A cells and administered DSPE-PEG-EGFRvIII ("PepVIII Vax").

[0074] [Figure 15] Figure 15 provides a graph showing cytokine (IFNγ and TNFα) secretion of tumor-infiltrating CAR-T cells in response to PBS or DSPE-PEG-EGFRvIII ("VAX").

[0075] [Figure 16] Figure 16 provides graphs showing Granzyme B expression levels (left) and proliferation, as determined by Ki67 (right), of tumor-infiltrating CAR-T cells in response to PBS or DSPE-PEG-EGFRvIII ("PepVIII Vax").

[0076] [Figure 17] Figure 17 provides a graph showing expression of PD-1 and TIM3 on tumor-infiltrating EGFRvIII CAR T cells with or without DSPE-PEG-EGFRvIII ("VAX").

[0077] [Figure 18A] Figure 18A provides a graph showing tumor volume in CT-2A tumor-bearing mice treated with EGFRvIII CAR-T + / - DSPE-PEG-EGFRvIII vaccination ("VAX") under lymphodepleting conditions. ***p<0.0001, **p<0.01, *p<0.05.

[0078] [Figure 18B] FIG. 18B provides a Kaplan-Meier survival graph of the CT-2A tumor-bearing mice of FIG. 18A.

[0079] [Figure 19] FIG. 19 provides a schematic of a FITC-antigen bispecific CAR design that targets both FITC and the melanoma-associated antigen TRP1.

[0080] [Figure 20] FIG. 20 provides a graph showing FITC-TRP1 CAR expression on the surface of T cells.

[0081] [Figure 21] Figure 21 provides a graph showing IFNγ secretion of FITC-TRP1 bispecific CAR T upon co-culture with DSPE-PEG-FITC coated K562 or B16F10 cells. Monospecific FITC CAR T cells and TRP1 CAR T cells were included as controls. ***p<0.0001, **p<0.01, *p<0.05.

[0082] [Figure 22] Figure 22 provides a graph showing the percentage of cell death of TRP1-expressing target cells when co-cultured in vitro with FITC-TRP1 bispecific CAR-T or monospecific TRP1 CAR T cells. Co-culture was set up for 6 hours at an effector to target (E:T) ratio of 10:1.

[0083] [Diagram 23] FIG. 23 provides a graph showing FITC-TRP1 CAR-T proliferation in lymph nodes 48 hours after DSPE-PEG-FITC vaccination as measured by cell trace violet tracing.

[0084] [Figure 24A]Figures 24A and 24B show tumor growth (Figure 24A) and animal survival (Figure 24B) of B16F10 tumor-bearing mice treated with FITC-TRP1 bispecific CAR-T therapy alone or CAR-T plus DSPE-PEG-FITC vaccination ("VAX") with lymphodepleting preconditioning. [Figure 24B] Figures 24A and 24B show tumor growth (Figure 24A) and animal survival (Figure 24B) of B16F10 tumor-bearing mice treated with FITC-TRP1 bispecific CAR-T therapy alone or CAR-T plus DSPE-PEG-FITC vaccination ("VAX") with lymphodepleting preconditioning.

[0085] [Diagram 25] Figure 25 provides a graph showing the number of FITC-TRP1 bispecific CAR-T in the peripheral blood of mice receiving PBS or DSPE-PEG-FITC vaccinations ("VAX").

[0086] [Figure 26] Figure 26 provides a graph showing infiltration of FITC / TRP1-CAR T cells into B16F10 tumors in mice receiving PBS or DSPE-PEG-FITC vaccinations.

[0087] [Figure 27A] Figures 27A and 27B show tumor growth (Figure 27A) and animal survival (Figure 27B) of lymphoid-filled B16F10 tumor-bearing mice treated with FITC-TRP1 bispecific CAR-T therapy alone or CAR-T plus DSPE-PEG-FITC vaccination ("VAX"). [Figure 27B] Figures 27A and 27B show tumor growth (Figure 27A) and animal survival (Figure 27B) of lymphoid-filled B16F10 tumor-bearing mice treated with FITC-TRP1 bispecific CAR-T therapy alone or CAR-T plus DSPE-PEG-FITC vaccination ("VAX"). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0088] overview Various diseases are characterized by the development of progressive immunosuppression in patients. The presence of impaired immune response in patients with malignant tumors has been particularly well documented. Cancer patients and tumor-bearing mice show various altered immune functions, such as a decrease in delayed-type hypersensitivity, a decrease in lymphocyte lytic function and proliferation response. Increasing immune function in cancer patients could have beneficial effects on tumor control.

[0089] Chimeric antigen receptor (CAR) T-cell therapy has been successful in treating hematological malignancies. However, CAR-T cells cannot be functionally sustained in some patients and generally show poor response in solid tumors. Current protocols for CAR-T therapy rely on the infusion of large numbers of CAR-T cells, but these cells may die or rapidly lose functional activity against tumors. In preclinical animal models, in vivo expansion of T cells via vaccination is one of the most effective strategies to support the efficacy of T cell therapy, but traditional vaccines are known to be unable to boost CAR-T via their chimeric antigen receptors.

[0090] Based on the present disclosure, the activation and proliferation of CAR-T is enhanced using an amphipathic ligand conjugate comprising a chimeric antigen receptor ligand and a lipid. The amphipathic ligand conjugate of the present disclosure provides an answer to some of the shortcomings of the current approach for generating therapeutic CAR-T cells by stimulating transferred CAR-T cells in vivo, and can reduce the amount of infused CAR-T cells required for durable therapeutic response and alleviate the need for patient lymphodepletion. definition

[0091] Terms used in the claims and this specification are defined as set forth below, unless otherwise specified.

[0092] It should be noted that 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.

[0093] As used herein, "about" will be understood by one of ordinary skill in the art and will vary to some extent depending on the context in which it is used. If uses of the term would not be clear to persons of ordinary skill in the art given the context in which it is used, "about" will mean up to plus or minus 10% of the particular value.

[0094] As used herein, the term "adjuvant" refers to a compound that enhances or otherwise alters or modifies the immune response generated with a particular immunogen or antigen. Modification of the immune response includes enhancing or broadening the specificity of either or both antibody and cellular immune responses. Modification of the immune response can also mean decreasing or suppressing a particular antigen-specific immune response. In certain embodiments, the adjuvant is a cyclic dinucleotide. In some embodiments, the adjuvant is an immunostimulatory oligonucleotide as described herein. In some embodiments, the adjuvant is administered before, together with, or after administration of the amphipathic ligand conjugate or a composition comprising the conjugate. In some embodiments, the adjuvant is co-formulated in the same composition as the amphipathic ligand conjugate.

[0095] "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of amino acids, but function in a manner similar to naturally occurring amino acids.

[0096] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes.

[0097] An "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a given amino acid sequence (the amino acid sequence of the starting polypeptide) with a second, different, "replacement" amino acid residue. An "amino acid insertion" refers to the incorporation of at least one additional amino acid into a given amino acid sequence. An insertion usually consists of the insertion of one or two amino acid residues, although larger "peptide insertions" of the invention can be made, for example, insertions of about 3 to about 5, or even up to about 10, 15 or 20 amino acid residues. The inserted residue(s) can be naturally or non-naturally occurring, as disclosed above. An "amino acid deletion" refers to the removal of at least one amino acid residue from a given amino acid sequence.

[0098] As used herein, "amphiphile" or "amphiphilic" refers to a conjugate that includes a hydrophilic head group and a hydrophobic tail, thereby forming an amphiphilic conjugate. In some embodiments, the amphiphile conjugate includes a chimeric antigen receptor (CAR) ligand and one or more hydrophobic lipid tails, and is referred to herein as an "amphiphilic ligand conjugate." In some embodiments, the amphiphile conjugate further includes a polymer (e.g., polyethylene glycol), which is conjugated to one or more lipids or the CAR ligand.

[0099] The term "alleviating" refers to any therapeutically beneficial outcome in the treatment of a disease state, e.g., cancer, including prevention, reduction in severity or progression, remission, or cure of the disease state.

[0100] As used herein, the term "antigenic preparation" or "antigenic composition" or "immunogenic composition" refers to a preparation that induces an immune response when administered to a vertebrate, especially a mammal.

[0101] The term "antigen-presenting cell" or "APC" is a cell that presents foreign antigens complexed with MHC on its surface. T cells recognize this complex using the T cell receptor (TCR). Examples of APCs include, but are not limited to, dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), monocytes (e.g., THP-1), B lymphoblastoid cells (e.g., C1R.A2, 1518 B-LCL), and monocyte-derived dendritic cells (DCs). Some APCs internalize antigens by either phagocytosis or receptor-mediated endocytosis.

[0102] As used herein, the term "bispecific" or "bifunctional antibody" refers to an artificial hybrid antibody or fragment thereof having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments. See, for example, Songsivilai & Lachmann, (1990) Clin. Exp. Immunol. 79:315-321; Kostelny et al., (1992) J. Immunol. 148:1547-1553.

[0103] As used herein, the term "chimeric antigen receptor (CAR)" refers to an artificial transmembrane protein receptor that includes (i) an extracellular domain capable of binding at least one predetermined CAR ligand or antigen, or a predetermined CAR ligand and antigen, (ii) an intracellular segment that includes one or more cytoplasmic domains derived from a signaling protein that is different from the polypeptide from which the extracellular domain is derived, and (iii) a transmembrane domain. A "chimeric antigen receptor (CAR)" may also be referred to as a "chimeric receptor," "T-body," or "chimeric immune receptor (CIR)."

[0104] The phrase "CAR ligand", used interchangeably with "CAR antigen", refers to any natural or synthetic molecule (e.g., small molecule, protein, peptide, lipid, carbohydrate, nucleic acid), or a portion or fragment thereof, that can specifically bind to a CAR (e.g., the extracellular domain of a CAR). In some embodiments, the CAR ligand is a tumor-associated antigen or a fragment thereof. In some embodiments, the CAR ligand is a tag. One of skill in the art can determine the appropriate CAR ligand for use in a CAR-based amphipathic ligand conjugate utilized in cell therapy.

[0105] "Intracellular signaling domain" means any oligopeptide or polypeptide domain known to function to transmit a signal that causes the activation or inhibition of a biological process in a cell, e.g., activation of an immune cell, such as a T cell or an NK cell. Examples include the ILR chains, CD28 and / or CD3zeta.

[0106] As used herein, "cancer antigen" refers to (i) a tumor-specific antigen, (ii) a tumor-associated antigen, (iii) a cell expressing a tumor-specific antigen, (iv) a cell expressing a tumor-associated antigen, (v) an embryonic antigen on a tumor, (vi) an autologous tumor cell, (vii) a tumor-specific membrane antigen, (viii) a tumor-associated membrane antigen, (ix) a growth factor receptor, (x) a growth factor ligand, and (xi) any other type of antigen or antigen-presenting cell or material associated with cancer.

[0107] As used herein, "CG oligodeoxynucleotides (CG ODNs)," also known as "CpG ODNs," are short, single-stranded synthetic DNA molecules that contain a cytosine nucleotide (C) followed by a guanine nucleotide (G). In certain embodiments, the immunostimulatory oligonucleotide is a CG ODN.

[0108] As used herein, the term "costimulatory ligand" includes a molecule on an antigen-presenting cell (e.g., APC, dendritic cell, B cell, etc.) that specifically binds to a cognate co-stimulatory molecule on a T cell, thereby providing a signal that mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by the binding of the TCR / CD3 complex to a peptide-loaded MHC molecule. Costimulatory ligands can include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (rCAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to the Toll ligand receptor, and ligands that specifically bind to B7-H3. Costimulatory ligands also include antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, ligands that specifically bind to CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, 1COS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83, among others.

[0109] "Costimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.

[0110] A "costimulatory signal," as used herein, refers to a signal that combines with a primary signal, e.g., TCR / CD3 ligation, to result in T cell proliferation and / or up- or down-regulation of key molecules.

[0111] A polypeptide or amino acid sequence "derived from" a specified polypeptide or protein refers to the origin of the polypeptide. Preferably, a polypeptide or amino acid sequence derived from a particular sequence has an amino acid sequence that is essentially identical to that sequence or a portion thereof, which portion consists of at least 10-20 amino acids, preferably at least 20-30 amino acids, more preferably at least 30-50 amino acids, or is otherwise identifiable by one of skill in the art as having its origin in that sequence.

[0112] A polypeptide derived from another peptide may have one or more mutations compared to the starting polypeptide, e.g., one or more amino acid residues substituted with another amino acid residue or having one or more amino acid residues inserted or deleted.

[0113] Polypeptides may include non-naturally occurring amino acid sequences. Such variants necessarily have less than 100% sequence identity or similarity with the starting molecule. In preferred embodiments, variants have an amino acid sequence with less than about 75%-100% amino acid sequence identity or similarity, more preferably less than about 80%-100%, more preferably less than about 85%-100%, more preferably less than about 90%-100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and most preferably less than about 95%-100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide, for example, over the length of the variant molecule.

[0114] In one embodiment, there is one amino acid difference between the starting polypeptide sequence and the sequence derived therefrom. Identity or similarity with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., the same residues) with the starting amino acid residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.

[0115] As used herein, the term antigen "cross-presentation" refers to the presentation of exogenous protein antigens to T cells via MHC class I and class II molecules on APCs.

[0116] As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. A CTL response is a response that is induced by CD8 + It is primarily mediated by T cells.

[0117] As used herein, the term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect.The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially stop the disease and its complications in a patient who is already suffering from the disease.The amount effective for this use depends on the severity of the disorder being treated and the general condition of the patient's own immune system.

[0118] As used herein, the term "effector cell" or "effector immune cell" refers to a cell that is involved in an immune response, e.g., in promoting an immune effector response. In some embodiments, an immune effector cell specifically recognizes an antigen. Examples of immune effector cells include, but are not limited to, natural killer (NK) cells, B cells, monocytes, macrophages, and T cells (e.g., cytotoxic T lymphocytes (CTLs)). In some embodiments, an effector cell is a T cell.

[0119] As used herein, the term "immune effector function" or "immune effector response" refers to the function or response of immune effector cells that promote an immune response against a target.

[0120] As used herein, the term "hematological cancer" includes lymphoma, leukemia, myeloma or lymphoid malignancies, as well as cancer of the spleen and lymph nodes.Exemplary lymphomas include both B-cell lymphoma (B-cell hematological cancer) and T-cell lymphoma.B-cell lymphomas include both Hodgkin's lymphoma and most non-Hodgkin's lymphomas. Non-limiting examples of B cell lymphomas include diffuse large B cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma (overlaps with chronic lymphocytic leukemia), mantle cell lymphoma (MCL), Burkitt's lymphoma, mediastinal large B cell lymphoma, Waldenstrom's macroglobulinemia, nodal marginal zone B cell lymphoma, splenic marginal zone lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis. Non-limiting examples of T cell lymphomas include extranodal T cell lymphoma, cutaneous T cell lymphoma, anaplastic large cell lymphoma, and angioimmunoblastic T cell lymphoma. Hematological malignancies also include leukemia, such as but not limited to secondary leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia and acute lymphoblastic leukemia.Hematological malignancies further include myeloma, such as but not limited to multiple myeloma and smoldering multiple myeloma.Other hematological and / or B-cell or T-cell related cancers are encompassed by the term hematological malignancies.

[0121] As used herein, "immune cells" are cells of hematopoietic origin that play a role in immune response. Immune cells include lymphocytes (e.g., B cells and T cells), natural killer cells, and myeloid cells (e.g., monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes).

[0122] As used herein, an "immunostimulatory oligonucleotide" is an oligonucleotide that can stimulate (eg, induce or enhance) an immune response.

[0123] The terms "inducing an immune response" and "enhancing an immune response" are used interchangeably and refer to the stimulation of an immune response (i.e., either passive or adaptive) to a particular antigen. When used in reference to inducing CDC or ADCC, the term "inducing" refers to the stimulation of a specific direct cell killing mechanism.

[0124] As used herein, a subject "in need of prevention," "in need of treatment," or "in need of" refers to a subject who, as determined by the judgment of an appropriate medical practitioner (e.g., in the case of a human, a physician, nurse or nurse practitioner; in the case of a non-human mammal, a veterinarian), would reasonably benefit from a given treatment (e.g., treatment with a composition comprising an amphipathic ligand conjugate).

[0125] The term "in vivo" refers to a process that occurs in a living organism.

[0126] As used herein, the terms "linked", "operably linked", "fused" or "fusion" are used interchangeably. These terms further refer to connecting two elements or components or domains together by suitable means, including chemical conjugation or recombinant DNA technology. Methods of chemical conjugation (e.g., using heterobifunctional crosslinkers) are known in the art, as are methods of recombinant DNA technology.

[0127] The term "lipid" refers to a biomolecule that is soluble in non-polar solvents and insoluble in water. Lipids are often described as hydrophobic or amphipathic molecules that allow them to form structures such as vesicles or membranes in aqueous environments. Lipids include fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids (including cholesterol), prenol lipids, saccharolipids, and polyketides. In some embodiments, lipids suitable for the amphipathic ligand conjugates of the present disclosure bind to human serum albumin under physiological conditions. In some embodiments, lipids suitable for the amphipathic ligand conjugates of the present disclosure insert into cell membranes under physiological conditions. In some embodiments, lipids bind to albumin and insert into cell membranes under physiological conditions. In some embodiments, lipids are diacyl lipids. In some embodiments, diacyl lipids contain more than 12 carbons. In some embodiments, the diacyl lipid contains at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 carbons.

[0128] The terms "mammal" or "subject" or "patient" as used herein include both humans and non-humans, including, but not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0129] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and their polymers, either in single-stranded or double-stranded form.Unless otherwise specified, this term encompasses nucleic acids that contain known analogs of natural nucleotides, which have similar binding properties as reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides.Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Cassol et al., 1992; Rossolini et al., Mol. Cell. Probes 8:91-98, 1994). For arginine and leucine, the modification at the second base can also be conservative. The term nucleic acid is used interchangeably with gene, cDNA encoded by gene, and mRNA.

[0130] The polynucleotide of the present invention may be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides may be composed of single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules that include DNA and RNA that may be single-stranded or more typically double-stranded or may be a mixture of single-stranded and double-stranded regions. Furthermore, polynucleotides may be composed of triple-stranded regions that include RNA or DNA, or both RNA and DNA. Polynucleotides may also include one or more modified bases, or DNA or RNA backbones that are modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases, such as inosine. Various modifications can be made to DNA and RNA; thus, "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms.

[0131] In some embodiments, the peptide of the present invention is encoded by a nucleotide sequence.The nucleotide sequence of the present invention can be useful for several applications, including cloning, gene therapy, protein expression and purification, mutagenesis, DNA vaccination of a host in need thereof, antibody generation for passive immunization, PCR, primer and probe generation, etc.

[0132] As used herein, "parenteral administration," "administered parenterally," and other grammatically equivalent phrases refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intranasal, intraocular, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid, and intrasternal injection and infusion.

[0133] As generally used herein, "pharmacologically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs and / or body fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0134] As used herein, the term "physiological conditions" refers to the in vivo conditions of a subject. In some embodiments, physiological conditions refer to a neutral pH (e.g., a pH between 6 and 8).

[0135] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers.

[0136] As used herein, a "small molecule" is a molecule having a molecular weight of less than about 500 Daltons.

[0137] As used herein, the term "subject" includes any human or non-human animal.For example, the methods and compositions of the present invention can be used to treat subjects with cancer or infectious diseases.The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0138] The term "sufficient amount" or "an amount sufficient to" means an amount sufficient to produce a desired effect, for example, an amount sufficient to reduce the diameter of a tumor.

[0139] The term "T cell" refers to a type of white blood cell that can be distinguished from other white blood cells by the presence of T cell receptors on the cell surface. T helper cells (also known as T HCells or CD4 + T cells) and T H 1. T H 2. T H 3. T H 17, T H 9 and T FH The subtypes include cytotoxic T cells (i.e., T C cells, CD8 + T cells, cytotoxic T lymphocytes, T-killer cells, killer T cells), memory T cells and central memory T cells (T CM cells), effector memory T cells (T EM and T EMRA cells) and resident memory T cells (T RM T cells), including the subtype regulatory T cells (also known as T reg cells or suppressor T cells) and CD4 + FOXP3 + T reg cells, CD4 + FOXP3 - T reg cells, Tr1 cells, Th3 cells and T reg There are several subsets of T cells, including but not limited to subtypes including IL-17 cells, natural killer T cells (aka NKT cells), mucosal-associated invariant T cells (MAIT), and gamma delta T cells (γδ T cells), including Vγ9 / Vδ2 T cells. Any one or more of the T cells mentioned above or not mentioned may be the target cell type for the methods of use of the present invention.

[0140] As used herein, the term "T cell activation" or "activation of T cells" refers to the cellular process in which mature T cells expressing antigen-specific T cell receptors on their surface recognize their cognate antigen and respond by entering the cell cycle, secreting cytokines or lytic enzymes, and initiating or becoming competent to perform cell-based effector functions. T cell activation requires at least two signals to be fully activated. The first occurs after engagement of the T cell antigen-specific receptor (TCR) by the antigen-major histocompatibility complex (MHC), and the second occurs by subsequent engagement of costimulatory molecules (e.g., CD28). These signals are transmitted to the nucleus and result in clonal expansion of T cells, upregulation of activation markers on the cell surface, differentiation into effector cells, induction of cytotoxicity or cytokine secretion, induction of apoptosis, or a combination thereof.

[0141] As used herein, the term "T cell-mediated response" refers to a response mediated by effector T cells (e.g., CD8 + cells) and helper T cells (e.g., CD4 + "T-cell mediated responses" refers to any response mediated by T cells, including but not limited to T cells. T cell mediated responses include, for example, T cell cytotoxicity and proliferation.

[0142] The term "T cell cytotoxicity" includes any immune response mediated by CD8+ T cell activation. Exemplary immune responses include cytokine production, CD8+ T cell proliferation, granzyme or perforin production, and clearance of infectious agents.

[0143] A "therapeutic antibody" is an antibody, an antibody fragment, or an antibody-derived construct that can bind to a cell surface antigen on a target cell and cause a therapeutic effect. Such antibodies can be chimeric, humanized, or fully human. Methods for producing such antibodies are known in the art. Such antibodies include single-chain Fc fragments of antibodies, minibodies, and diabodies. Any of the therapeutic antibodies known in the art to be useful for cancer treatment can be used in combination therapy with the compositions described herein. The therapeutic antibody can be a monoclonal or polyclonal antibody. In a preferred embodiment, the therapeutic antibody targets a cancer antigen. In some embodiments, the therapeutic antibody comprises a tag-binding domain that is recognized by an amphipathic ligand conjugate that comprises a tag.

[0144] As used herein, "therapeutic protein" refers to any polypeptide, protein, protein variant, fusion protein and / or fragments thereof that can be administered to a subject as a pharmaceutical.

[0145] The term "therapeutically effective amount" is an amount effective for alleviating symptoms of a disease. Since prevention can be considered treatment, a therapeutically effective amount can be a "prophylactically effective amount."

[0146] The terms "treat", "treating" and "treatment" as used herein refer to therapeutic or prophylactic measures as described herein. The method of "treatment" employs administration of an amphipathic ligand conjugate of the present disclosure to a subject in need of such treatment, for example, a subject undergoing CAR T cell therapy. In some embodiments, the amphipathic ligand conjugate is administered to a subject in need of an enhanced immune response against a particular antigen to prevent, cure, delay a disorder or recurring disorder, reduce the severity of a disorder or recurring disorder, or alleviate one or more symptoms of a disorder or recurring disorder, or to extend the subject's survival beyond that expected in the absence of such treatment, or to a subject who may eventually acquire such a disorder.

[0147] As used herein, "vaccine" refers to a formulation comprising the amphiphilic ligand conjugate described herein in combination with an adjuvant, in a form that can be administered to a vertebrate animal to induce a protective immune response sufficient to prevent and / or alleviate an infection or disease, and / or to reduce at least one symptom of an infection or disease, and / or to enhance the effectiveness of another dose of synthetic nanoparticles. Typically, a vaccine comprises a conventional saline or buffered aqueous medium in which a composition described herein is suspended or dissolved. In this form, the composition described herein is used to prevent, alleviate or otherwise treat an infection or disease. When introduced into a host, the vaccine elicits an immune response, including, but not limited to, the production of antibodies and / or cytokines and / or the activation of cytotoxic T cells, antigen presenting cells, helper T cells, dendritic cells and / or other cellular responses. Chimeric Antigen Receptor

[0148] In some aspects, the present disclosure provides compositions and methods for use or practiced in conjunction with chimeric antigen receptor (CAR) effector cells.

[0149] Chimeric antigen receptors (CARs) are engineered artificial transmembrane receptors that confer specificity to an immune effector cell (e.g., T cells, natural killer cells, or other immune cells) for a given ligand, resulting in activation of the effector cell upon recognition and binding to the ligand. Typically, these receptors are used to confer the antigen specificity of a monoclonal antibody to a T cell.

[0150] In some embodiments, a CAR comprises three domains: 1) an ectodomain, which typically comprises a signal peptide, a ligand or antigen recognition region (e.g., scFv) and a flexible spacer; 2) a transmembrane (TM) domain; and 3) an endodomain (also known as an "activation domain"), which typically comprises one or more intracellular signaling domains. The ectodomain of a CAR resides on the outside of the cell and is exposed to the extracellular space, thereby making it accessible for interaction with its cognate ligand. The TM domain allows the CAR to be anchored in the cell membrane of an effector cell. The third endodomain (also known as an "activation domain") assists in effector cell activation upon binding of the CAR to its specific ligand. In some embodiments, effector cell activation includes induction of cytokine and chemokine production, as well as activation of the cytolytic activity of the cell. In some embodiments, the CAR redirects cytotoxicity to tumor cells.

[0151] In some embodiments, the CAR comprises a ligand-specific or antigen-specific recognition domain (also called a binding domain) that binds to a specific target ligand or antigen. In some embodiments, the binding domain is a single chain antibody variable fragment (scFv), an anchored ligand, or an extracellular domain of a co-receptor fused to a transmembrane domain that is in turn linked to a signaling domain. In some embodiments, the signaling domain is derived from CD3ζ or FcRγ. In some embodiments, the CAR comprises one or more costimulatory domains derived from proteins such as CD28, CD137 (also known as 4-lBB), CD134 (also known as OX40), and CD278 (also known as ICOS).

[0152] Engagement of the antigen-binding domain of the CAR with its target antigen on the surface of the target cell results in clustering of the CAR and delivery of an activation stimulus to the CAR-containing cell. In some embodiments, the main feature of CARs is their ability to redirect the specificity of immune effector cells, thereby inducing the production of molecules that can mediate proliferation, cytokine production, phagocytosis, or cell death of target antigen-expressing cells in a major histocompatibility complex (MHC)-independent manner that leverages the cell-specific targeting ability of monoclonal antibodies, soluble ligands, or cell-specific co-receptors. Although scFv-based CARs engineered to contain signaling domains derived from CD3ζ or FcRγ have been shown to deliver potent signals for T cell activation and effector function, they are insufficient to initiate signals that promote T cell survival and expansion in the absence of concomitant costimulatory signals. De novo generation of CARs containing binding domains, hinge, transmembrane and signaling domains derived from CD3ζ or FcRγ together with one or more costimulatory signaling domains (e.g., intracellular costimulatory domains derived from CD28, CD137, CD134 and CD278) have been shown to more efficiently direct anti-tumor activity as well as increased cytokine secretion, lytic activity, survival and proliferation in CAR-expressing T cells in vitro, in animal models and in cancer patients (Milone et al., Molecular Therapy, 2009; 17: 1453-1464; Zhong et al., Molecular Therapy, 2010; 18: 413-420; Carpenito et al., PNAS, 2009; 106:3360-3365).

[0153] In some embodiments, chimeric antigen receptor-expressing effector cells (e.g., CAR-T cells) are cells derived from a patient with a disease or condition and genetically modified in vitro to express at least one CAR with any specificity for a ligand. These cells are stimulated or induced by specific binding of a ligand to the CAR to perform at least one effector function (e.g., induction of cytokines) useful for treating the disease or condition of the same patient. Effector cells can be T cells (e.g., cytotoxic T cells or helper T cells). Those skilled in the art should understand that other cell types (e.g., natural killer cells or stem cells) can express CARs, and that chimeric antigen receptor effector cells can include effector cells other than T cells. In some embodiments, an effector cell is a T cell (e.g., a cytotoxic T cell) that exerts its effector function (e.g., a cytotoxic T cell response) against a target cell or target cell (e.g., a cancer cell) upon contact with or proximity to the target cell (see, e.g., Chang and Chen (2017) Trends Mol Med 23(5):430-450).

[0154] Prolonged exposure of T cells to their cognate antigens can result in exhaustion of effector functions, allowing the persistence of infected or transformed cells. Recently developed strategies to stimulate or rejuvenate host effector functions using agents that induce immune checkpoint blockade have been successful in treating some cancers. Emerging evidence suggests that T cell exhaustion can also represent a significant impediment to sustained, long-lived antitumor activity by chimeric antigen receptor-expressing T cells (CAR-T cells). In some embodiments, the differentiation state of T cells retrieved from patients prior to CAR transduction and the conditioning regimen that patients undergo prior to reintroduction of CAR-T cells (e.g., the addition or elimination of alkylating agents, fludarabine, total body irradiation) can greatly affect the persistence and cytotoxic potential of CAR-T cells. In vitro culture conditions that stimulate (via anti-CD3 / CD28 or stimulator cells) and expand (via cytokines such as IL-2) T cell populations can also alter the differentiation state and effector function of CAR-T cells (Ghoneim et al., (2016) Trends in Molecular Medicine 22(12):1000-1011).

[0155] The present disclosure addresses several shortcomings of current approaches for the generation of therapeutic CAR-T cells. Existing methods of therapeutic CAR-T cell preparation often require extensive cell culture in vitro to obtain sufficient numbers of modified cells for adoptive cell transfer, during which the natural identity or differentiation state of the T cells may be altered and T cell function may be impaired. Furthermore, when a patient is in urgent need of treatment to prevent disease progression, the time required to generate sufficient amounts of CAR-T cells may not coincide with the opportunity to treat the patient, resulting in treatment failure and patient death. The compositions and methods provided by the present disclosure circumvent this obstacle and provide an expedient and more physiologically relevant therapeutic approach by stimulating the activation and proliferation of CAR-T cells in vivo. Furthermore, current CAR-T cell therapy regimens require prior lymphodepletion, which weakens the patient's health and destroys the conducive environment that may improve the efficacy of CAR-T. In some aspects, the present disclosure provides methods to stimulate adoptively transferred CAR-T cells such that they are still able to engraft, actively proliferate, and expand in vivo in the absence of lymphodepletion.

[0156] Current CAR-T cell therapy relies only on engineered costimulatory signals to maintain CAR-T effector function. The lack of other costimulatory signals and natural stimulatory environment may lead to incomplete T cell maturation and increased T cell exhaustion. In one aspect, the present disclosure provides a method and composition for mobilizing T cells into lymph nodes, which is a physiologically appropriate activation environment for immune cells, and co-administration of adjuvants to activate APCs, which provide a complete set of essential costimulatory signals for optimal CAR-T cell activation.

[0157] In some embodiments, particularly for the treatment of ALL and / or NHL, suitable CARs target CD19 or CD20. Non-limiting examples include CARs that include the following structures: (i) anti-CD19 scFv, CD8 H / TM domain, 4-1BB CS domain, and CD3ζ TCR signaling domain; (ii) anti-CD19 scFv, CD28 hinge and transmembrane domain, CD28 costimulatory domain, and CD3ζ TCR signaling domain; and (iii) anti-CD20 scFv, IgG hinge and transmembrane domain, CD28 / 4-1BB costimulatory domain, and CD3ζ TCR signaling domain. In some embodiments, CAR effector cells suitable for combination with the combinations and methods disclosed herein, including but not limited to Kymriah™ (tisagenlecleucel; Novartis; formerly CTL019) and Yescarta™ (axicabtadine cilorucel; Kite Pharma), target CD19 or CD20. Retargeted CAR T cells

[0158] In some embodiments, effector cells (e.g., T cells) engineered to express a universal immune receptor, a tag, a switch, or a CAR that binds to the Fc region on an immunoglobulin are suitable for the compositions and methods described herein.

[0159] In some embodiments, effector cells (e.g., T cells) are modified to express a universal immune receptor, i.e., UnivIR. One type of UnivIR is a biotin-binding immune receptor (BBIR) (see, e.g., U.S. Patent Publication US20140234348A1, which is incorporated herein by reference in its entirety). Other examples of methods and compositions relating to universal chimeric receptors and / or effector cells expressing universal chimeric receptors are described in International Patent Applications WO2016123122A1, WO2017143094A1, WO2013074916A1, U.S. Patent Application US20160348073A1, all of which are incorporated herein by reference in their entirety.

[0160] In some embodiments, effector cells (e.g., T cells) are engineered to express a universal modular anti-tag chimeric antigen receptor (UniCAR). This system allows for retargeting of UniCAR-grafted immune cells to multiple antigens (see, e.g., U.S. Patent Publication US20170240612A1, which is incorporated herein by reference in its entirety; Cartellieri et al., (2016) Blood Cancer Journal 6, e458, which is incorporated herein by reference in its entirety).

[0161] In some embodiments, effector cells (e.g., T cells) are engineered to express a switchable chimeric antigen receptor and a chimeric antigen receptor effector cell (CAR-EC) switch. In this system, the CAR-EC switch has a first region that is bound by the chimeric antigen receptor on the CAR-EC and a second region that binds to a cell surface molecule on the target cell, thereby stimulating an immune response from the CAR-EC that is cytotoxic to the bound target cell. In some embodiments, the CAR-EC is a T cell, and the CAR-EC switch can act as an "on switch" for CAR-EC activity. Activity can be "turned off" by reducing or ceasing administration of the switch. These CAR-EC switches can be used with the CAR-ECs disclosed herein, as well as existing CAR T cells, for the treatment of diseases or conditions, such as cancer, where the target cell is a malignant cell. Such treatments can be referred to herein as switchable immunotherapy (U.S. Patent Publication US9624276B2, which is incorporated herein by reference in its entirety).

[0162] In some embodiments, effector cells (e.g., T cells) are engineered to express a receptor that binds to the Fc portion of human immunoglobulin (e.g., CD16V-BB-ζ) (Kudo et al., (2014) Cancer Res 74(1):93-103, which is incorporated by reference in its entirety).

[0163] In some embodiments, effector cells (e.g., T cells) are engineered to express a universal immune receptor (e.g., switchable CAR, sCAR) that binds to a peptide neoepitope (PNE). In some embodiments, the peptide neoepitope (PNE) is incorporated into an antibody (antibody switch) that targets an antigen at a defined, distinct location. Thus, sCAR-T cell specificity is redirected only to PNEs that are not present in the human proteome, thus allowing for orthogonal interactions between sCAR-T cells and the antibody switch. In this way, sCAR-T cells are strictly dependent on the presence of the antibody switch to be fully activated, thus eliminating CAR T cell off-target recognition of endogenous tissues or antigens in the absence of the antibody switch (Arcangeli et al., (2016) Transl Cancer Res 5(Suppl 2):S174-S177, which is incorporated herein by reference in its entirety). Other examples of switchable CARs are provided by U.S. Patent Application US20160272718A1, which is incorporated by reference in its entirety.

[0164] As used herein, the term "tag" encompasses the universal immune receptor, tag, switch, or Fc region of an immunoglobulin. In some embodiments, the effector cell is modified to express a CAR that includes a tag-binding domain. In some embodiments, the CAR binds to fluorescein isothiocyanate (FITC), streptavidin, biotin, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horseradish peroxidase, palmitoylation, nitrosylation, alkaline phosphatase, glucose oxidase, or maltose binding protein. Anti-tag chimeric antigen receptor (AT-CAR)

[0165] There are some limitations to the generalized clinical application of CAR T cells.For example, there is no single tumor antigen that is universally expressed by all cancer types, so each scFv in the CAR needs to be engineered to have specificity for the desired tumor antigen.In addition, the tumor antigen targeted by the CAR may be downregulated or mutated in response to treatment, resulting in tumor escape.

[0166] As an alternative, universal anti-tag chimeric antigen receptor (AT-CAR) and CAR-T cells have been developed. For example, human T cells have been engineered to express anti-fluorescein isothiocyanate (FITC) CAR (called anti-FITC-CAR). This platform takes advantage of the high affinity interaction between anti-FITC scFv (on the surface of the cell) and FITC, as well as the ability to conjugate FITC molecules (or other tags) to any anti-cancer-based monoclonal antibody, such as cetuximab (anti-EGFR), rituximab (anti-CD20) and herceptin (anti-Her2).

[0167] Thus, in some embodiments, effector cells (e.g., T cells) are modified to express a universal anti-tag chimeric antigen receptor (AT-CAR), at least as described in WO2012082841 and US20160129109A1, the entirety of which is incorporated herein by reference. In such AT-CAR systems, T cells recognize and bind to tagged proteins, such as antibodies. For example, in some embodiments, AT-CAR T cells recognize tagged antibodies, such as FITC-labeled antibodies. In some embodiments, anti-tumor antigen antibodies are conjugated to a tag (e.g., FITC) and administered before, in conjunction with, or after AT-CAR therapy. Anti-tumor antigen antibodies are known to those skilled in the art.

[0168] As shown, the binding specificity of tag-binding domain depends on the identity of the tag that is conjugated to the protein that is used to bind to target cells.For example, in some embodiments of the present disclosure, the tag is FITC and the tag-binding domain is anti-FITC scFv.Alternatively, in some embodiments of the present disclosure, the tag is biotin or PE (phycoerythrin) and the tag-binding domain is anti-biotin scFv or anti-PE scFv.

[0169] In some embodiments, the protein of each preparation of tagged protein is the same or different, and the protein is an antibody or an antigen-binding fragment thereof. In some aspects, the antibody or an antigen-binding fragment thereof is cetuximab (anti-EGFR), nimotuzumab (anti-EGFR), panitumumab (anti-EGFR), rituximab (anti-CD20), omalizumab (anti-CD20), tositumomab (anti-CD20), trastuzumab (anti-Her2), gemtuzumab (anti-CD33), alemtuzumab (anti-CD52) and bevacizumab (anti-VEGF).

[0170] Thus, in some embodiments, the tagged proteins include FITC-conjugated antibodies, biotin-conjugated antibodies, PE-conjugated antibodies, histidine-conjugated antibodies and streptavidin-conjugated antibodies that bind to a TAA or TSA expressed by a target cell. For example, tagged proteins include, but are not limited to, FITC-conjugated cetuximab, FITC-conjugated rituximab, FITC-conjugated Herceptin, biotin-conjugated cetuximab, biotin-conjugated rituximab, biotin-conjugated Herceptin, PE-conjugated cetuximab, PE-conjugated rituximab, PE-conjugated Herceptin, histidine-conjugated cetuximab, histidine-conjugated rituximab, histidine-conjugated Herceptin, streptavidin-conjugated cetuximab, streptavidin-conjugated rituximab, and streptavidin-conjugated Herceptin.

[0171] In some embodiments, the AT-CARs of each population of AT-CAR-expressing T cells are the same or different, and the AT-CARs comprise a tag-binding domain, a transmembrane domain, and an activation domain. In some embodiments, the tag-binding domain is an antibody or an antigen-binding fragment thereof. In some aspects, the tag-binding domain specifically binds to FITC, biotin, PE, histidine, or streptavidin. In some embodiments, the tag-binding domain is an antigen-binding fragment, and the antigen-binding fragment is a single-chain variable fragment (scFv), such as an scFv that specifically binds to FITC, biotin, PE, histidine, or streptavidin. In some embodiments, the transmembrane domain is the hinge and transmembrane region of human CD8α chain. In some embodiments, the activation domain comprises one or more of the cytoplasmic region of CD28, CD137 (41BB), OX40, HVEM, CD3ζ, and the cytoplasmic region of FcRε.

[0172] In some embodiments, the tag of each preparation of tagged protein is the same or different, and the tag is selected from the group consisting of fluorescein isothiocyanate (FITC), streptavidin, biotin, histidine, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horseradish peroxidase, palmitoylation, nitrosylation, alkaline phosphatase, glucose oxidase, and maltose binding protein.

[0173] Tags can be conjugated to proteins using techniques such as chemical coupling and chemical cross-linkers. Alternatively, polynucleotide vectors can be prepared that encode the tagged protein as a fusion protein. Cell lines can then be engineered to express the tagged protein, which can be isolated from the culture medium, purified, and used in the methods disclosed herein.

[0174] In some embodiments, the tagged protein is administered to the subject before, in conjunction with, or after administration of AT-CAR-expressing T cells. In some embodiments, the present disclosure provides a method of treating cancer in a subject, comprising: (a) administering to a subject in need of treatment a formulation of a tagged protein, where the tagged protein binds to cancer cells in the subject, and (b) administering to the subject a therapeutically effective population of anti-tag chimeric antigen receptor (AT-CAR)-expressing T cells, where the AT-CAR-expressing T cells bind to the tagged protein and induce cancer cell death, thereby treating cancer in the subject. Tandem CAR (TanCAR) effector cells

[0175] It has been observed that using CAR approaches for cancer treatment, tumor heterogeneity and immunoediting can cause escape from CAR treatment (Grupp et al., New Eng. J. Med (2013) 368:1509-1518). As an alternative approach, bispecific CARs, known as tandem CARs or TanCARs, have been developed in an attempt to simultaneously target multiple cancer-specific markers. In TanCARs, the extracellular domain contains two antigen binding specificities in tandem, connected by a linker. Thus, both binding specificities (scFvs) are linked to a single transmembrane moiety: one scFv is adjacent to the membrane and the other scFv is in a distal position. As an exemplary TanCAR, Grada et al. (Mol Ther Nucleic Acids (2013) 2, e105) describe a TanCAR that includes a CD19-specific scFv followed by a Gly-Ser linker and a HER2-specific scFv. The HER2-scFv was in a juxtamembrane position and the CD19-scFv in a distal position. TanCAR was shown to induce distinct T cell reactivity against each of the two tumor restricted antigens.

[0176] Thus, some embodiments of the present disclosure relate to tandem chimeric antigen receptors that mediate bispecific activation and targeting of T cells. Although the present disclosure refers to bispecificity for CARs, in some embodiments, CARs can target three, four or more tumor antigens. Using CAR T cells to target multiple antigens can enhance T cell activation and / or counteract tumor escape due to antigen loss. TanCARs can also target multiple expressed antigens, target various tumors using the same cell product with broad specificity, and / or provide a better toxicity profile by CARs that signal less strongly to achieve the same result due to multiple specificities.

[0177] In some embodiments, the present disclosure provides a TanCAR that comprises two targeting domains. In some embodiments, the present disclosure provides a multispecific TanCAR that comprises three or more targeting domains. In another embodiment, the present disclosure provides a first CAR and a second CAR on the cell surface, each CAR comprises an antigen binding domain, and the antigen binding domain of the first CAR binds to a first tumor antigen (e.g., CD19, CD20, CD22, HER2), and the antigen binding domain of the second CAR binds to another (different) tumor antigen. TanCAR is described in US20160303230A1 and US20170340705A1, which are incorporated herein by reference.

[0178] In some embodiments, the TanCARs of the present disclosure target two or more tumor antigens. Exemplary tumor antigens include one or more of CD19, CD20, CD22, k light chain, CD30, CD33, CD123, CD38, ROR1, ErbB2, ErbB3 / 4, EGFr vIII, carcinoembryonic antigen, EGP2, EGP40, mesothelin, TAG72, PSMA, NKG2D ligand, B7-H6, IL-13 receptor alpha 2, MUC1, MUC16, CA9, GD2, GD3, HMW-MAA, CD171, Lewis Y, G250 / CALX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSC1, folate receptor-alpha, CD44v7 / 8, 8H9, NCAM, VEGF receptor, 5T4, fetal AchR, NKG2D ligand, CD44v6, TEM1 and / or TEM8.

[0179] In some embodiments, the present disclosure provides a bispecific TanCAR that targets CD19 and another tumor antigen. In some embodiments, the present disclosure provides a bispecific TanCAR that targets CD22 and another tumor antigen. In some embodiments, the present disclosure provides a bispecific TanCAR that targets HER2 and another tumor antigen. In some embodiments, the present disclosure provides a bispecific TanCAR that targets IL13R-alpha2 and another tumor antigen. In some embodiments, the present disclosure provides a bispecific TanCAR that targets VEGF-A and another tumor antigen. In some embodiments, the present disclosure provides a bispecific TanCAR that targets Tem8 and another tumor antigen. In some embodiments, the present disclosure provides a bispecific TanCAR that targets FAP and another tumor antigen. In some embodiments, the present disclosure provides a bispecific TanCAR that targets EphA2 and another tumor antigen. In some embodiments, the present disclosure provides bispecific TanCARs that target one or more, two or more, three or more, or four or more of the following tumor antigens: CD19, CD22, HER2, IL13R-alpha2, VEGF-A, Tem8, FAP, or EphA2, and any combination thereof. In some embodiments, the present disclosure provides bispecific TanCARs that target HER2 and IL13R-alpha2. In some embodiments, the present disclosure provides bispecific TanCARs that target CD19 and CD22. Methods for generating chimeric antigen receptors and CAR effector cells

[0180] In some embodiments, the subject's effector cells (e.g., T cells) are genetically modified with a chimeric antigen receptor (Sadelain et al., Cancer Discov. 3:388-398, 2013). For example, effector cells (e.g., T cells) are provided, and a recombinant nucleic acid encoding a chimeric antigen receptor is introduced into the patient-derived effector cells (e.g., T cells) to generate a CAR cell. In some embodiments, effector cells (e.g., T cells) that are not derived from the subject are genetically modified with a chimeric antigen receptor. For example, in some embodiments, the effector cells (e.g., T cells) are allogeneic cells engineered to be used as an "off-the-shelf" adoptive cell therapy, such as Universal Chimeric Antigen Receptor T cells (UCARTs) developed by Cellectis. UCARTs are allogeneic CAR T cells engineered to be used to treat the maximum number of patients with a particular cancer type. Non-limiting examples of UCARTs being developed by Cellectis include those targeting the following tumor antigens: CD19, CD123, CD22, CS1 and CD38.

[0181] A variety of different methods known in the art can be used to introduce any of the nucleic acids or expression vectors disclosed herein into effector cells (e.g., T cells).Non-limiting examples of methods for introducing nucleic acids into effector cells (e.g., T cells) include lipofection, transfection (e.g., calcium phosphate transfection, transfection using highly branched organic compounds, transfection using cationic polymers, dendrimer-based transfection, optical transfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposomes), microinjection, electroporation, cell squeezing, sonoporation, protoplast fusion, impalefection, hydrodynamic delivery, gene gun, magnetofection, viral transfection, and nucleofection. Additionally, CRISPR / Cas9 genome editing technology known in the art can be used to introduce CAR nucleic acids into effector cells (e.g., T cells) and / or introduce other genetic modifications (e.g., those described below) into effector cells (e.g., T cells) to enhance CAR cell activity (see, e.g., US9,890,393; US9,855,297; US2017 / 0175128; US2016 / 0184362; US2016 / 0272999; WO2015 / 161276; WO2014 / 191128; CN106755088; CN106591363; CN106480097; CN106399375; CN104894068 for the use of CRISPR / Cas9 technology with respect to CAR T cells).

[0182] Provided herein are methods that can be used to generate any of the cells or compositions described herein, each of which can express a CAR (e.g., any of the CARs described herein).

[0183] A chimeric antigen receptor (CAR) comprises an antigen-binding domain, a transmembrane domain, and a cytoplasmic sequence of a CD3ζ sequence sufficient to stimulate a T cell when the antigen-binding domain binds to an antigen, and optionally a cytoplasmic signaling domain that includes a cytoplasmic sequence of one or more (e.g., two, three, or four) costimulatory proteins (e.g., one or more cytoplasmic sequences of B7-H3, BTLA, CD2, CD7, CD27, CD28, CD30, CD40, CD40L, CD80, CD160, CD244, ICOS, LAG3, LFA-1, LIGHT, NKG2C, 4-1BB, OX40, PD-1, PD-L1, TIM3, and a ligand that specifically binds to CD83) that provide costimulation of the T cell when the antigen-binding domain binds to the antigen. In some embodiments, a CAR may further comprise a linker. Non-limiting aspects and features of CARs are described below. Additional embodiments of CARs and CAR cells, including exemplary antigen binding domains, linkers, transmembrane domains, and cytoplasmic signaling domains, are described in, e.g., Kakarla et al., Cancer J. 20:151-155, 2014; Srivastava et al., Trends Immunol. 36:494-502, 2015; Nishio et al., Oncoimmunology 4(2): e988098, 2015; Ghorashian et al., Br. J. Haematol. 169:463-478, 2015; Levine, Cancer Gene Ther. 22:79-84, 2015; Jensen et al., Curr. Opin. Immunol. 33:9-15, 2015; Singh et al., Cancer Gene Ther. 22:95-100, 2015; Li et al., Zhongguo Shi Yan Xue Ye Xue Za Zhi 22:1753-1756, 2014;Gill et al., Immunol. Rev. 263:68-89, 2015;Magee et al., Discov. Med. 18:265-271, 2014;Gargett et al., Front. Pharmacol. 5:235, 2014;Yuan et al., Zhongguo Shi Yan Xue Ye Xue Za Zhi 22:1137-1141, 2014;Pedgram et al., Cancer J. 20:127-133, 2014;Eshhar et al., Cancer J. 20:123-126, 2014;Ramos et al., Cancer J. 20:112-118, 2014;Maus et al., Blood 123:2625-2635, 2014;Jena et al., Curr. Hematol. Malig. Rep. 9:50-56, 2014;Maher et al., Curr. Gene Ther. 14:35-43, 2014;Riches et al., Discov. Med. 16:295-302, 2013; Cheadle et al., Immunol. Rev. 257:83-90, 2014; Davila et al., Int. J. Hematol. 99:361-371, 2014; Xu et al., Cancer Lett. 343:172-178, 2014; Kochenderfer et al., Nat. Rev. Clin. Oncol. 10:267-276, 2013; Hosing et al., Curr. Hematol. Malig. Rep. 8:60-70, 2013; Hombach et al., Curr. Mol. Med. 13:1079-1088, 2013; Xu et al., Leuk. Lymphoma 54:255-260, 2013; Gilham et al., Trends Mol. Med. 18:377-384, 2012; Lipowska-Bhalla et al., Cancer Immunol. Immunother. 61:953-962, 2012; Chmielewski et al., Cancer Immunol. Immunother. 61:1269-1277, 2013; Jena et al., Blood 116:1035-1044, 2010;Dotti et al, Immunology Reviews 257(1): 107-126, 2013;Dai et al., Journal of the National Cancer Institute 108(7): djv439, 2016;Wang and Riviere, Molecular Therapy-Oncolytics 3: 16015, 2016; US Patent Application Publication No. 2018 / 0057609; Same as No. 2018 / 0037625; Same as No. 2017 / 0362295; Same as No. 2017 / 0137783; Same as No. 2016 / 0152723, Same as 2016 / 02 No. 06656, same as No. 2016 / 0199412, same as No. 2016 / 0208018, same as No. 2015 / 0232880, same as No. 2015 / 0225480; same as No. 2015 / 0224143; same as No. 2015 / 0224142 ;Same as No. 2015 / 0190428;Same as No. 2015 / 0196599;Same as No. 2015 / 0152181;Same as No. 2015 / 0140023;Same as No. 2015 / 0118202;Same as No. 2015 / 0110760;Same as No. 2015 / 0099299;Same as No. 2015 / 0093822;Same as No. 2015 / 0093401;Same as No. 2015 / 0051266;Same as No. 2015 / 0050729;Same as No. 2015 / 0024482;Same as No. 2015 / 002 No. 3937; same as No. 2015 / 0017141; same as No. 2015 / 0017136; same as No. 2015 / 0017120; same as No. 2014 / 0370045; same as No. 2014 / 0370017; same as No. 2014 / 0369977; same as No. 2014 / 0349402; same as No. 2014 / 0328812; same as No. 2014 / 0322275; same as No. 2014 / 0322216; same as No. 2014 / 0322212; same as No. 2014 / 0322183; same as No. 201 No. 4 / 0314795; same as No. 2014 / 0308259; same as No. 2014 / 0301993; same as No. 2014 / 0296492; same as No. 2014 / 0294784; same as No. 2014 / 0286973; same as No. 2014 / 0274909; same as No. 2014 / 0274801; same as No. 2014 / 0271635; same as No. 2014 / 0271582; ​​same as No. 2014 / 0271581; same as No. 2014 / 0271579; same as No. 2014 / 0255363;2014 / 0242701; 2014 / 0242049; 2014 / 0227272; 2014 / 0219975; 2014 / 0170114; No. 2014 / 0134720; No. 2014 / 0134142; No. 2014 / 0120622; No. 2014 / 0120136; No. 2014 / 0106449; No. 2014 / 0106449; 2014 / 0099340; 2014 / 0086828; 2014 / 0065629; 2014 / 0050708; 2 014 / 0024809; 2013 / 0344039; 2013 / 0323214; 2013 / 0315884; 2013 / 0309258; 201 3 / 0288368; 2013 / 0287752; 2013 / 0287748; 2013 / 0280221; 2013 / 0280220; 2013 / 0266551; 2013 / 0216528; 2013 / 0202622; 2013 / 0071414; 2012 / 0321667; 2012 / 0 302466; 2012 / 0301448; 2012 / 0301447; 2012 / 0060230; 2011 / 0213288; 2011 / 0158957; 2011 / 0104128; 2011 / 0038836; 2007 / 0036773; and 2004 / 0043401. Further embodiments of CARs and CAR cells, including exemplary antigen binding domains, linkers, transmembrane domains, and cytoplasmic signaling domains, are described in WO 2016 / 168595; WO 12 / 079000; WO 2015 / 0141347; WO 2015 / 0031624; WO 2015 / 0030597; WO 2014 / 0378389; WO 2014 / 0219978; WO 2014 / 02066 20;2014 / 0037628;2013 / 0274203;2013 / 0225668;2013 / 0116167;2012 / 0230962;2012 / 0213783;2012 / 0093842;2012 / 0071420;2012 / 0015888;2011 / 0268754;2010 / 0297093;2010 / 0158881;2010 / 0034834; 2010 / 0015113; 2009 / 0304657; 2004 / 0043401; 2014 / 0322253; 2015 / 0118208; 2015 / 0038684; 2014 / 0024601; 2012 / 0148552; 2011 / 0223129; 2009 / 0257994; 2008 / 0160607; 2008 / 0003683; 2013 / 0121960; 2011 / 0052554; and 2010 / 0178276. ; A. Antigen-binding Domains

[0184] The antigen-binding domain contained in a chimeric antigen receptor (CAR) can specifically bind to an antigen (e.g., a tumor-associated antigen (TAA) or an antigen not expressed on a non-cancerous cell) or a universal receptor (e.g., a tag). Non-limiting examples of antigen-binding domains include monoclonal antibodies (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgE and IgD) (e.g., fully human or chimeric (e.g., humanized) antibodies), antigen-binding fragments of antibodies (e.g., Fab, Fab' or F(ab')2 fragments) (e.g., fragments of fully human or chimeric (e.g., humanized) antibodies), diabodies, triabodies, tetrabodies, minibodies, scFv, scFv-Fc, (scFv)2, scFab, bis-scFv, hc-IgG, BiTEs, single domain antibodies (e.g., V-NAR domain or VhH domain), IgNAR, and multispecific (e.g., bispecific) antibodies. Methods for making these antigen-binding domains are known in the art.

[0185] In some embodiments, the antigen-binding domain comprises at least one (e.g., one, two, three, four, five or six) CDRs (e.g., any of the three CDRs from an immunoglobulin light chain variable domain or any of the three CDRs from an immunoglobulin heavy chain variable domain) of an antibody, such as an immunoglobulin molecule (e.g., a light or heavy chain immunoglobulin molecule) and an immunologically active (antigen-binding) fragment of an immunoglobulin molecule, capable of specifically binding to a target antigen.

[0186] In some embodiments, the antigen binding domain is a single chain antibody (e.g., a V-NAR domain or a V H In some embodiments, the antigen-binding domain is a whole antibody molecule (e.g., a human, humanized or chimeric antibody) or a multimeric antibody (e.g., a bispecific antibody).

[0187] In some embodiments, antigen-binding domains include antibody fragments and multispecific (e.g., bispecific) antibodies or antibody fragments. Examples of antibodies and antigen-binding fragments thereof include, but are not limited to, single chain Fvs (scFvs), Fab fragments, Fab' fragments, F(ab')2, disulfide-linked Fvs (sdFvs), Fvs, and fragments containing either the VL or VH domains.

[0188] Further antigen-binding domains provided herein are polyclonal, monoclonal, multispecific (multimeric, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single chain antibodies, intracellularly generated antibodies (i.e., intrabodies), and antigen-binding fragments thereof. The antibody or antigen-binding fragment thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antigen-binding domain is an IgG1 antibody or antigen-binding fragment thereof. In some examples, the antigen-binding domain is an IgG4 antibody or antigen-binding fragment thereof. In some embodiments, the antigen-binding domain is an immunoglobulin comprising a heavy chain and a light chain.

[0189] Further examples of antigen-binding domains are antigen-binding fragments of IgG (e.g., an antigen-binding fragment of IgG1, IgG2, IgG3 or IgG4) (e.g., an antigen-binding fragment of human or humanized IgG, e.g., a human or humanized IgG1, IgG2, IgG3 or IgG4), an antigen-binding fragment of IgA (e.g., an antigen-binding fragment of IgA1 or IgA2) (e.g., an antigen-binding fragment of human or humanized IgA, e.g., a human or humanized IgA1 or IgA2), an antigen-binding fragment of IgD (e.g., an antigen-binding fragment of human or humanized IgD), an antigen-binding fragment of IgE (e.g., an antigen-binding fragment of human or humanized IgE) or an antigen-binding fragment of IgM (e.g., an antigen-binding fragment of human or humanized IgM).

[0190] In some embodiments, the antigen binding domain is at about 1×10 -7 M or 1×10 -7 Less than M (e.g., about 1 × 10 -8 M or 1×10 -8 Less than M, about 5 x 10 -9 M or 5×10 -9 Less than M, about 2 x 10 -9 M or 2×10 -9 Less than M or about 1×10-9 M or 1×10 -9 Affinity (K D ) and can bind to a specific antigen (e.g., a tumor-associated antigen).

[0191] As can be understood by those skilled in the art, the selection of the antigen binding domain to be included in the CAR depends on the type and number of ligands that define the surface of the cell (e.g., cancer cell or tumor) to be targeted in the subject in need thereof, and / or on the ligand present on the amphipathic ligand conjugate. For example, in some embodiments, the antigen binding domain is selected to recognize a ligand that acts as a cell surface marker on a cancer cell or is a tumor-associated antigen (e.g., CD19, CD30, Her2 / neu, EGFR, or BCMA) or a tumor-specific antigen (TSA). In some embodiments, the antigen binding domain recognizes a ligand on the amphipathic ligand conjugate.

[0192] In some embodiments, the CAR effector cells (e.g., CAR T cells) comprise a CAR molecule that binds to a tumor antigen (e.g., comprises a tumor antigen binding domain). In some embodiments, the CAR molecule comprises an antigen binding domain that recognizes a tumor antigen of a solid tumor (e.g., breast cancer, colon cancer, etc.). In some embodiments, the CAR molecule is a tandem CAR molecule comprising at least two antigen binding domains. In some embodiments, the CAR molecule comprises an antigen binding domain that recognizes a tumor antigen of a hematological malignancy (e.g., leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute promyelocytic leukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma and marginal zone B-cell lymphoma, polycythemia vera, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, etc.).

[0193] In some embodiments, the tumor antigen is a tumor-specific antigen (TSA). A TSA is unique to tumor cells and is not present on other cells in the body. In some embodiments, the tumor antigen is a tumor-associated antigen (TAA). A TAA is not unique to tumor cells, but instead is also expressed on normal cells under conditions that cannot induce a state of immune tolerance to the antigen. Expression of an antigen on a tumor can occur under conditions that allow the immune system to respond to the antigen. In some embodiments, a TAA is expressed on normal cells during fetal development, when the immune system is immature and cannot respond, or is normally present at very low levels on normal cells, but is expressed at much higher levels on tumor cells.

[0194] In certain embodiments, tumor-associated antigens are determined by sequencing the patient's tumor cells and identifying mutated proteins found only in tumors. These antigens are called "neo-antigens." Once neo-antigens are identified, therapeutic antibodies can be produced against them and used in the methods described herein.

[0195] In some embodiments, the tumor antigen is an epithelial cancer antigen (e.g., breast, gastrointestinal, lung), prostate-specific cancer antigen (PSA) or prostate-specific membrane antigen (PSMA), bladder cancer antigen, lung (e.g., small cell lung) cancer antigen, colon cancer antigen, ovarian cancer antigen, brain cancer antigen, gastric cancer antigen, renal cell carcinoma antigen, pancreatic cancer antigen, liver cancer antigen, esophageal cancer antigen, head and neck cancer antigen, or colorectal cancer antigen. In certain embodiments, the tumor antigen is a lymphoma antigen (e.g., non-Hodgkin's lymphoma or Hodgkin's lymphoma), B-cell lymphoma cancer antigen, leukemia antigen, myeloma (e.g., multiple myeloma or plasma cell myeloma) antigen, acute lymphoblastic leukemia antigen, chronic myeloid leukemia antigen, or acute myelogenous leukemia antigen.

[0196] Tumor antigens (e.g., tumor-associated antigens (TAA) and tumor-specific antigens (TSA)) that can be targeted by CAR effector cells (e.g., CAR T cells) include 1GH-IGK, 43-9F, 5T4, 791Tgp72, acyclophilin C-related protein, alpha-fetoprotein (AFP), α-actinin-4, A3, antigen specific for the A33 antibody, ART-4, B7, Ba733, BAGE, BCR-ABL, beta-catenin, beta-HCG, BrE3-antigen, BCA225, BTAA, CA125, CA15-3 / CA 27.29 / BCAA, CA195, CA242, CA-50, CAM43, CAMEL, CAP-1, carbonic anhydrase IX, c-Met, CA19-9, CA72-4, CAM17.1, CASP-8 / m , CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD2 3, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD6 6a-e, CD67, CD68, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147, CD154 , CDC27, CDK4, CDK4m, CDKN2A, CO-029, CTLA4, CXCR4, CXCR7, CXCL12, HIF-1a, colon-specific antigen-p (CSAp), CEA (CEACAM5) , CEACAM6, c-Met, DAM, E2A-PRL, EGFR, EGFRvIII, EGP-1(TROP-2), EGP-2, ELF2-M, Ep-CAM, fibroblast growth factor (FGF), FGF -5, Flt-1, Flt-3, folate receptor, G250 antigen, Ga733VEpCAM, GAGE, gp100, GRO-β, H4-RET, HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its subunits, HER2 / neu, HMGB-1, hypoxia-inducible factor (HIF-1), HSP70-2M, HST-2, HTgp-175, Ia, IGF-1R, IFN-γ, IFN-α,IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growth factor Child-1 (IGF-1), KC4-antigen, KSA, KS-1-antigen, KS1-4, LAGE-1a, Le-Y, LDR / FUT, M344, MA-50, macrophage migration inhibitory factor (MIF), MAGE, MAGE-1, MAGE-3, MAGE-4 , MAGE-5, MAGE-6, MART-1, MART-2, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MG7-Ag, MOV18, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, M UC16, MUM-1 / 2, MUM-3, MYL-RAR, NB / 70K, Nm23H1, NuMA, NCA66, NCA95, NCA90, NY-ESO-1, p15, p16, p185erbB2, p180erbB3, PAM4 antigen, pancreatic cancer whip PD1 receptor (PD-1), PD-1 receptor ligand 1 (PD-L1), PD-1 receptor ligand 2 (PD-L2), PI5, placenta growth factor, p53, PLAGL2, Pmel17 prostatic acid phosphatase, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-1R, IL-6, IL-25, RCAS1, RS5, RAGE, RANTES, Ras, T101, SAGE, S100, survivin, survivin-2B, SDDCAG16, TA-90 / Mac2 binding protein, T These include, but are not limited to, AAL6, TAC, TAG-72, TLP, tenascin, TRAIL receptor, TRP-1, TRP-2, TSP-180, TNF-α, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigen, tyrosinase, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen, complement factors C3, C3a, C3b, C5a, C5, angiogenesis markers, bc1-2, bc1-6 and K-ras, oncogene markers and oncogene products (e.g.,See Sensi et al., Clin Cancer Res 2006, 12:5023-32; Parmiani et al., J Immunol 2007, 178:1975-79; Novellino et al. Cancer Immunol Immunother 2005, 54:187-207). ,

[0197] In some embodiments, the tumor antigen is a viral antigen derived from a virus associated with a human chronic disease or cancer (e.g., cervical cancer). For example, in some embodiments, the viral antigen is derived from Epstein-Barr virus (EBV), HPV antigens E6 and / or E7, Hepatitis C virus (HCV), Hepatitis B virus (HBV), or Cytomegalovirus (CMV).

[0198] Exemplary cancers or tumors and the specific tumor antigens associated with such tumors (although not exclusively) include acute lymphoblastic leukemia (etv6, aml1, cyclophilin b), B cell lymphoma (Ig-idiotype), glioma (E-cadherin, α-catenin, β-catenin, γ-catenin, p120ctn), bladder cancer (p21ras), biliary tract cancer (p21ras), breast cancer (MUC family, HER2 / neu, c-erbB-2), cervical cancer (p53 , p21ras), colon cancer (p21ras, HER2 / neu, c-erbB-2, MUC family), colorectal cancer (colorectal-associated antigen (CRC)-CO17-1A / GA733, APC), choriocarcinoma (CEA), epithelial cell carcinoma (cyclophilin b), gastric cancer (HER2 / neu, c-erbB-2, ga733 glycoprotein), hepatocellular carcinoma (α-fetoprotein), Hodgkin lymphoma (Imp-1, EBNA-1), lung cancer (CEA, MAGE-3, NY -ESO-1), lymphoid leukemia (cyclophilin b), melanoma (p5 protein, gp75, carcinoembryonic antigen, GM2 and GD2 gangliosides, Melan-A / MART-1, cdc27, MAGE-3, p21ras, gp100), myeloma (MUC family, p21ras), non-small cell lung cancer (HER2 / neu, c-erbB-2), nasopharyngeal carcinoma (Imp-1, EBNA-1), ovarian cancer (MUC family, HER 2 / neu, c-erbB-2), prostate cancer (prostate-specific antigen (PSA) and its antigenic epitopes PSA-1, PSA-2 and PSA-3, PSMA, HER2 / neu, c-erbB-2, ga733 glycoprotein), renal cancer (HER2 / neu, c-erbB-2), squamous cell carcinoma of the cervix and esophagus, testicular cancer (NY-ESO-1), and T-cell leukemia (HTLV-1 epitopes), as well as viral products or proteins.

[0199] In some embodiments, immune effector cells (e.g., CAR T cells) containing a CAR molecule useful in the methods disclosed herein express a CAR that contains a mesothelin-binding domain (i.e., the CAR T cells specifically recognize mesothelin). Mesothelin is a tumor antigen that is overexpressed in various cancers, including ovarian, lung, and pancreatic cancers.

[0200] In some embodiments, immune effector cells (e.g., CAR T cells) comprising a CAR molecule useful in the methods disclosed herein express a CAR comprising a CD19 binding domain. In some embodiments, immune effector cells (e.g., CAR T cells) comprising a CAR molecule useful in the methods disclosed herein express a CAR comprising a HER2 binding domain. In some embodiments, immune effector cells (e.g., CAR T cells) comprising a CAR molecule useful in the methods disclosed herein express a CAR comprising an EGFR binding domain.

[0201] In some embodiments, the CAR effector cell expressing a CAR comprising a CD19 targeting or binding domain is Kymriah™ (tisagenlecleucel; Novartis; see WO2016109410, which is incorporated by reference in its entirety) or Yescarta™ (axicabtadine ciloreucel; Kite; see US20160346326, which is incorporated by reference in its entirety). B. Linker

[0202] Provided herein are CARs that may optionally include a linker between (1) the antigen-binding domain and the transmembrane domain, and / or (2) the transmembrane domain and the cytoplasmic signaling domain. In some embodiments, the linker may be a polypeptide linker. For example, the linker may be between about 1 amino acid and about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, about 10 amino acids, about 8 amino acids, about 6 amino acids, about 4 amino acids, or about 2 amino acids; between about 2 amino acids and about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, about 10 amino acids, about 8 amino acids, about 8 amino acids, about 9 amino acids, about 10 ... amino acids, about 6 amino acids or about 4 amino acids; about 4 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, about 10 amino acids, about 8 amino acids or about 6 amino acids; about 6 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, about 10 amino acids or about 8 amino acids;about 8 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, or about 10 amino acids; about 10 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, or about 12 amino acids; about 12 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids , about 20 amino acids, about 18 amino acids, about 16 amino acids, or about 14 amino acids; about 14 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, or about 16 amino acids; about 16 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 1 00 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, or about 18 amino acids; about 18 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, or about 20 amino acids;about 20 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, or about 25 amino acids; about 25 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, or about 30 amino acids amino acids; about 30 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, or about 35 amino acids; about 35 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, or about 40 amino acids; about 40 amino acids to about 500 amino acids, about 400 amino acids about 50 to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, or about 50 amino acids; about 50 to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, or about 60 amino acids; about 60 to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 150 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, or about 70 Amino acids; about 70 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids or about 80 amino acids; about 80 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids or about 90 amino acids; about 90 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids or about 100 amino acids; about 100 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids or about 200 amino acids;It may have a length of about 200 amino acids to about 500 amino acids, about 400 amino acids, or about 300 amino acids; about 300 amino acids to about 500 amino acids, or about 400 amino acids; or about 400 amino acids to about 500 amino acids;

[0203] Further examples and embodiments of linkers are described in the references cited herein, and therefore are incorporated herein in their entireties. C. Transmembrane domain

[0204] In some embodiments, the CAR described herein also comprises a transmembrane domain.In some embodiments, the transmembrane domain naturally associates with the sequence in the cytoplasmic domain.In some embodiments, the transmembrane domain can be modified by one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid substitutions to avoid the domain binding to other transmembrane domains (e.g., transmembrane domains of the same or different surface membrane proteins) to minimize interaction with other members of the receptor complex.

[0205] In some embodiments, transmembrane domain can be derived from natural source.In some embodiments, transmembrane domain can be derived from any membrane-bound or transmembrane protein.Non-limiting examples of transmembrane domain that can be used herein can be derived from (e.g., can include at least their transmembrane sequence or part of their transmembrane sequence) from T cell receptor alpha, beta or zeta chain, CD28, CD3 epsilon, CD33, CD37, CD64, CD80, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD86, CD134, CD137 or CD154.

[0206] In some embodiments, the transmembrane domain may be synthetic. For example, in some embodiments where the transmembrane domain is derived from a synthetic source, the transmembrane domain may comprise (e.g., may predominantly comprise) hydrophobic residues (e.g., leucine and valine). In some embodiments, the synthetic transmembrane domain comprises at least one (e.g., at least two, at least three, at least four, at least five, or at least six) triplet of phenylalanine, tryptophan, and valine at the end of the synthetic transmembrane domain. In some embodiments, the transmembrane domain of the CAR may comprise a CD8 hinge domain.

[0207] Further specific examples of transmembrane domains are described in the references cited herein. D. Cytoplasmic domain

[0208] Also provided herein are CAR molecules that contain a cytoplasmic signaling domain that contains a cytoplasmic sequence of CD3zeta sufficient to stimulate T cells when the antigen-binding domain binds to an antigen, and optionally one or more cytoplasmic sequences of costimulatory proteins that provide costimulation of T cells (e.g., one or more cytoplasmic sequences of any of CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3, NKG2C, B7-H3, a ligand that specifically binds to CD83, and ITAM sequences). Stimulation of the CAR immune effector cell can result in activation of one or more anti-cancer activities of the CAR immune effector cell. For example, in some embodiments, stimulation of the CAR immune effector cell can result in an increase in the cytolytic or helper activity of the CAR immune effector cell, including secretion of cytokines. In some embodiments, the entire intracellular signaling domain of the costimulatory protein is included in the cytoplasmic signaling domain. In some embodiments, the cytoplasmic signaling domain includes a shortened part of the intracellular signaling domain of the costimulatory protein (e.g., a shortened part of the intracellular signaling domain that transmits effector function signals in CAR immune effector cells). Non-limiting examples of intracellular signaling domains that can be included in the cytoplasmic signaling domain include the cytoplasmic sequences of T cell receptor (TCR) and co-receptor, which work together to initiate signaling after antigen receptor engagement, and any variant of these sequences that includes at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) substitution and has the same or nearly the same functional ability.

[0209] In some embodiments, the cytoplasmic signaling domain may comprise two distinct classes of cytoplasmic signaling sequences: signaling sequences that initiate antigen-dependent activation via the TCR (primary cytoplasmic signaling sequences) (e.g., CD3ζ cytoplasmic signaling sequences) and the cytoplasmic sequences of one or more costimulatory proteins that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences).

[0210] In some embodiments, the cytoplasmic domain of the CAR can be designed to include a CD3ζ signaling domain, either by itself or in combination with any other desired cytoplasmic signaling sequence(s) useful in the context of the CAR. In some examples, the cytoplasmic domain of the CAR can include a CD3ζ chain portion and a costimulatory cytoplasmic signaling sequence. A costimulatory cytoplasmic signaling sequence refers to a portion of the CAR that includes a cytoplasmic signaling sequence of a costimulatory protein (e.g., CD27, CD28, 4-IBB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83).

[0211] In some embodiments, the cytoplasmic signaling sequences in the cytoplasmic signaling domain of the CAR are positioned in random order.In some embodiments, the cytoplasmic signaling sequences in the cytoplasmic signaling domain of the CAR are linked to each other in a specific order.In some embodiments, a linker (e.g., any of the linkers described herein) can be used to form a link between different cytoplasmic signaling sequences.

[0212] In some embodiments, the cytoplasmic signaling domain is designed to include the cytoplasmic signaling sequence of CD3ζ and the cytoplasmic signaling sequence of the costimulatory protein CD28. In some embodiments, the cytoplasmic signaling domain is designed to include the cytoplasmic signaling sequence of CD3ζ and the cytoplasmic signaling sequence of the costimulatory protein 4-IBB. In some embodiments, the cytoplasmic signaling domain is designed to include the cytoplasmic signaling sequence of CD3ζ and the cytoplasmic signaling sequences of the costimulatory proteins CD28 and 4-1BB. In some embodiments, the cytoplasmic signaling domain does not include the cytoplasmic signaling sequence of 4-1BB. Further engineering of CAR T cells

[0213] In another embodiment, the therapeutic efficacy of CAR effector cells (e.g., CAR T cells) is enhanced by disruption of methylcytosine dioxygenase genes (e.g., Tetl, Tet2, Tet3), which results in increased proliferation, modulation of effector cytokine production, and reduced total levels of 5-hydroxymethylcytosine, which is associated with degranulation, thereby increasing the proliferation and / or function of CAR effector cells (e.g., CAR T cells), as described in PCT Publication WO2017 / 049166. Thus, effector cells (e.g., T cells) can be engineered to express a CAR, and expression and / or function of Tetl, Tet2 and / or Tet3 in said effector cells (e.g., T cells) is reduced or eliminated.

[0214] In another embodiment, the therapeutic efficacy of a CAR effector cell (e.g., a CAR T cell) is enhanced by using an effector cell (e.g., a T cell) that constitutively expresses the CAR (referred to as a nonconditional CAR) and conditionally expresses another agent useful for treating cancer, as described in PCT Publication WO2016 / 126608 and US Publication No. 2018 / 0044424. In such an embodiment, the conditionally expressed agent is expressed upon activation of the effector cell (e.g., a T cell), e.g., upon binding of the nonconditional CAR to its target. In one embodiment, the conditionally expressed agent is a CAR (referred to herein as a conditional CAR). In another embodiment, the conditionally expressed agent inhibits a checkpoint inhibitor of the immune response. In another embodiment, the conditionally expressed agent improves or enhances the efficacy of the CAR, which may include a cytokine.

[0215] In another embodiment, the therapeutic efficacy of the CAR T cells is enhanced by modifying the CAR T cells with a nucleic acid capable of altering (e.g., down-modulating) the expression of an endogenous gene selected from the group consisting of TCR alpha chain, TCR beta chain, beta-2 microglobulin, an HLA molecule, CTLA-4, PD1, and FAS, as described in PCT Publication WO2016 / 069282 and US Publication No. 2017 / 0335331.

[0216] In another embodiment, the therapeutic efficacy of CAR T cells is enhanced by co-expressing the CAR and one or more enhancers of T cell priming ("ETPs") in T cells, as described in PCT Publication WO2015 / 112626 and US Publication No. 2016 / 0340406. The addition of ETP components to CAR T cells confers enhanced "professional" antigen-presenting cell (APC) function. In an embodiment, the CAR and one or more ETPs are transiently co-expressed in T cells. Thus, the engineered T cells are safe (given the transient nature of CAR / ETP expression) and induce long-lasting immunity via APC function.

[0217] In another embodiment, the therapeutic efficacy of CAR T cells is enhanced by co-expressing a CAR and an inhibitory membrane protein (IMP) containing a binding (or dimerization) domain in T cells, as described in PCT Publication WO2016 / 055551 and US Publication No. 2017 / 0292118. Both the CAR and the IMP are rendered responsive to soluble compounds, particularly via a second binding domain contained within the CAR, thereby allowing co-localization of the inhibitory signaling domain of the IMP with the signaling domain of the CAR, which has the effect of reducing CAR activation by dimerization or ligand recognition. The inhibitory signaling domain is preferably programmed death-1 (PD-1), which attenuates T cell receptor (TCR)-mediated activation of IL-2 production and T cell proliferation.

[0218] In another embodiment, the therapeutic efficacy of CAR T cells is enhanced using a system in which controlled variations in the conformation of the extracellular portion of the CAR, including the antigen binding domain, are obtained upon the addition of small molecules, as described in PCT Publication WO2017 / 032777. This integrated system switches the interaction between the antigen and the antigen binding domain between on / off states. By being able to control the conformation of the extracellular portion of the CAR, the downstream function of the CAR T cells, such as cytotoxicity, can be directly modulated. Thus, a CAR can be characterized in that it comprises: a) at least one external domain comprising i) an extracellular antigen binding domain; and ii) a switch domain comprising at least a first multimerization ligand binding domain and a second multimerization ligand binding domain capable of binding to a given multivalent ligand to form a multimer comprising the two binding domains and the multivalent ligand to which they can bind; b) at least one transmembrane domain; and c) at least one internal domain comprising a signaling domain and optionally a costimulatory domain; where the switch domain is located between the extracellular antigen binding domain and the transmembrane domain. Amphiphilic conjugates A. Overview

[0219] Amphiphile vaccine technology has been developed that involves linking an adjuvant or antigen (e.g., peptide) to a lipophilic polymer tail that promotes localization of the vaccine to lymph nodes (Liu et al. (2014) Nature 507:519-522). Such amphiphile-antigens (e.g., amph-peptides) can also insert into cell membranes (see, e.g., Liu et al. (2011) Angewandte Chemie-Intl. Ed. 50:7052-7055). Thus, the present disclosure provides amphiphilic conjugates comprising a CAR ligand for use in stimulating, expanding, and activating CAR effector cells (e.g., CAR-T cells).

[0220] In some embodiments, the amphiphilic conjugates of the present disclosure are used in chimeric antigen receptor (CAR)-expressing cell therapy (e.g., CAR-T cell therapy). In some embodiments, the amphiphilic conjugates of the present disclosure stimulate a specific immune response against a specific target, such as a tumor-associated antigen. In some embodiments, the amphiphilic conjugates of the present disclosure stimulate the proliferation of CAR-expressing cells (e.g., CAR-T cells) in vivo. In some embodiments, the amphiphilic conjugates of the present disclosure comprise a CAR ligand, herein referred to as an amphiphilic ligand conjugate. In some embodiments, the amphiphilic conjugates comprise an immunostimulatory oligonucleotide, herein referred to as an amphiphilic oligonucleotide conjugate.

[0221] As shown in FIG. 1A, a variety of amphipathic ligand conjugate structures are disclosed in which a lipophilic moiety, or "lipid tail" (e.g., DSPE), is linked (e.g., covalently linked) to a CAR ligand via a linker (e.g., PEG-2000). The modularity of this design allows a variety of ligands, including but not limited to small molecules (e.g., FITC), short peptides (e.g., linear peptides that provide epitopes specific to CAR), or modular protein domains (e.g., folded polypeptides or polypeptide fragments that provide conformational epitopes specific to CAR), to be linked (e.g., covalently) to the lipid to produce amphipathic ligand conjugates with tailored specificity.

[0222] Without being bound by theory, it is believed that the amphipathic ligand conjugates of the present disclosure are primarily delivered to lymph nodes, where the lipid tail moiety is inserted into the membrane of antigen-presenting cells (APCs), resulting in the decoration of the APCs with the CAR ligand (FIG. 1B). The embedded CAR ligand serves as a specific target for the CAR expressed on the surface of CAR-expressing cells (e.g., CAR T cells) (which are administered prior to, subsequent to, or co-administered with the amphipathic ligand conjugates of the present disclosure), resulting in the recruitment of the CAR-expressing cells to the CAR-ligand-decorated APCs. The interaction of the CAR with the embedded CAR ligand provides a stimulatory signal via the CAR, while the APCs further present other naturally occurring co-stimulatory signals, resulting in optimal CAR-expressing cell activation, long-term survival, and efficient memory formation. B. Lipid Conjugates

[0223] In certain embodiments, lipid conjugates (e.g., amphiphilic conjugates) described in US2013 / 0295129, which is incorporated herein by reference, are used in the methods disclosed herein. In some embodiments, the lipid conjugates include a hydrophobic tail that inserts into the cell membrane. In some embodiments, the lipid conjugates include an albumin-binding lipid to efficiently target the conjugate to lymph nodes in vivo. In some embodiments, the lipid conjugates include an albumin-binding lipid that includes a hydrophobic tail that inserts into the cell membrane and efficiently targets the conjugate to lymph nodes in vivo. In some embodiments, the lipid conjugates bind to endogenous albumin targeting them to lymphatic vessels and draining lymph nodes, where they accumulate due to sorting of albumin by antigen-presenting cells. In some embodiments, the lipid conjugates include an antigenic peptide or a molecular adjuvant, thereby inducing or enhancing a robust immune response. In some embodiments, the lipid conjugate comprises a CAR ligand, thereby inducing or enhancing the expansion, proliferation and / or activation of a CAR-expressing cell (e.g., a CAR effector cell, e.g., a CAR-T cell). A lipid conjugate comprising a CAR ligand is referred to as an "amphiphilic ligand conjugate," as defined above.

[0224] In some embodiments, the lipid conjugate that is efficiently targeted to lymph nodes is called a "lymph node targeting conjugate". In some embodiments, the lymph node targeting conjugate comprises a highly lipophilic albumin binding domain (e.g., albumin binding lipid), and a cargo, e.g., a CAR ligand or molecular adjuvant. In some embodiments, the lymph node targeting conjugate comprises three domains: a highly lipophilic albumin binding domain (e.g., albumin binding lipid), a cargo, e.g., a CAR ligand or molecular adjuvant, and a polar block linker that promotes the solubility of the conjugate and reduces the ability of the lipid to insert into the cell plasma membrane. Thus, in certain embodiments, the general structure of the conjugate is LPC, where "L" is an albumin binding lipid, "P" is a polar block, and "C" is a cargo, e.g., a CAR ligand or molecular adjuvant. In some embodiments, the cargo itself can also function as a polar block domain, and a separate polar block domain is not required. Thus, in certain embodiments, the conjugate has only two domains: the albumin-binding lipid and the cargo.

[0225] In some embodiments, the cargo of the conjugate is a CAR ligand, thereby resulting in an amphipathic ligand conjugate. In some embodiments, the amphipathic ligand conjugate is administered or formulated with an adjuvant, which is a molecular adjuvant, for example, an immunostimulatory oligonucleotide, or an amphipathic ligand that contains a peptide antigen as cargo. (i) Lipids

[0226] In some embodiments, the lipid component of the amphipathic conjugate comprises a hydrophobic tail. In some embodiments, the hydrophobic tail inserts into a cell membrane. In some embodiments, the lipid is linear, branched, or cyclic. In some embodiments, the lipid is greater than 12 carbons in length. In some embodiments, the lipid is 13 carbons in length. In some embodiments, the lipid is 14 carbons in length. In some embodiments, the lipid is 15 carbons in length. In some embodiments, the lipid is 16 carbons in length. In some embodiments, the lipid is 17 carbons in length. In some embodiments, the lipid is 18 carbons in length. In some embodiments, the lipid is 19 carbons in length. In some embodiments, the lipid is 20 carbons in length. In some embodiments, the lipid is 21 carbons in length. In some embodiments, the lipid is 22 carbons in length. In some embodiments, the lipid is 23 carbons in length. In some embodiments, the lipid is 24 carbons in length. In some embodiments, the lipid is 25 carbons in length. In some embodiments, the lipid is 26 carbons in length. In some embodiments, the lipid is 27 carbons in length. In some embodiments, the lipid is 28 carbons long. In some embodiments, the lipid is 29 carbons long. In some embodiments, the lipid is 30 carbons long. In some embodiments, the lipid is at least 17-18 carbons long, but may be shorter if it exhibits good albumin binding and adequate targeting to lymph nodes.

[0227] Lymph node targeting conjugates include amphipathic ligand conjugates and amphipathic oligonucleotide conjugates that can be transported from the site of delivery to lymph nodes via lymph.In certain embodiments, activity is based in part on the ability of the conjugate to associate with albumin in the blood of a subject.Thus, lymph node targeting conjugates typically include lipids that can bind to albumin under physiological conditions.Appropriate lipids for targeting lymph nodes can be selected based on the ability of lipids or lipid conjugates that include lipids to bind to albumin.Appropriate methods for testing the ability of lipids or lipid conjugates to bind to albumin are known in the art.

[0228] For example, in certain embodiments, a plurality of lipid conjugates can spontaneously form micelles in an aqueous solution. The micelles are incubated with a solution containing albumin, such as albumin or fetal bovine serum (FBS). The sample can be analyzed, for example, by ELISA, size exclusion chromatography, or other methods, to determine whether binding has occurred. The lipid conjugate can be selected as a lymph node targeting conjugate if, as discussed above, in the presence of a solution containing albumin, such as albumin or fetal bovine serum (FBS), the micelles dissociate and the lipid conjugate binds to albumin.

[0229] Examples of preferred lipids for use in lymph node-targeting lipid conjugates include, but are not limited to, straight-chain unsaturated and saturated fatty acids, branched-chain saturated and unsaturated fatty acids, and fatty acid derivatives, such as fatty acid esters, fatty acid amides and fatty acid thioesters, diacyl lipids, cholesterol, cholesterol derivatives, and steroid acids, such as bile acids, lipid A, or combinations thereof, having an aliphatic tail of 8-30 carbons. In some embodiments, the lipid is saturated. In some embodiments, the lipid comprises at least one lipid tail comprising 8-30, 12-30, 15-25, or 16-20 carbons.

[0230] In certain embodiments, the lipid is a diacyl lipid or a double-chain lipid. In some embodiments, the tail in the diacyl lipid contains about 8 to about 30 carbons and can be saturated, unsaturated, or a combination thereof. In some embodiments, the diacyl lipid is saturated. In some embodiments, the diacyl lipid is saturated and each tail contains about 8 to about 30 carbons. In some embodiments, the diacyl lipid is saturated and each tail contains 12 carbons. In some embodiments, the diacyl lipid is saturated and each tail contains 13 carbons. In some embodiments, the diacyl lipid is saturated and each tail contains 14 carbons. In some embodiments, the diacyl lipid is saturated and each tail contains 15 carbons. In some embodiments, the diacyl lipid is saturated and each tail contains 16 carbons. In some embodiments, the diacyl lipid is saturated and each tail contains 17 carbons. In some embodiments, the diacyl lipid is saturated and each tail contains 18 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 19 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 20 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 21 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 22 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 23 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 24 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 25 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 26 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 27 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 28 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 29 carbons. In some embodiments, the diacyl lipids are saturated and each tail comprises 30 carbons. The tails can be coupled to the head group via an ester bond linkage, an amide bond linkage, a thioester bond linkage, or a combination thereof.In certain embodiments, the diacyl lipid is a phosphate lipid, a glycolipid, a sphingolipid, or a combination thereof.

[0231] In some embodiments, the lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). In some embodiments, the diacyl lipid is synthesized as described in US9,107,904, the entirety of which is incorporated herein by reference. In some embodiments, the diacyl lipid is synthesized as provided below: [ka] #1 Stearoyl Chloride

[0232] Preferably, the lymph node targeting conjugate comprises a lipid of 8 or more carbon units in length. Increasing the number of lipid units is believed to reduce lipid insertion into the plasma membrane of the cell, thereby allowing the lipid conjugate to freely bind albumin and traffic to the lymph nodes.

[0233] For example, in some embodiments, the lipid may be a diacyl lipid composed of two C18 hydrocarbon tails. In certain embodiments, the lipid for use in preparing lymph node-targeting lipid conjugates is not a single chain hydrocarbon (e.g., C18). (ii) Molecular adjuvants

[0234] In certain embodiments, an amphipathic oligonucleotide conjugate is used in conjunction with an amphipathic ligand conjugate. The oligonucleotide conjugate typically comprises an immunostimulatory oligonucleotide.

[0235] In certain embodiments, immunostimulatory oligonucleotides can function as ligands for pattern recognition receptors (PRRs).Examples of PRRs include the Toll-like family of signaling molecules, which play a role in the initiation of innate immune response and also affect the later, more antigen-specific adaptive immune response.Thus, oligonucleotides can function as ligands for Toll-like family of signaling molecules, such as Toll-like receptor 9 (TLR9).

[0236] For example, unmethylated CpG sites can be detected by TLR9 on plasmacytoid dendritic cells and B cells in humans (Zaida, et al., Infection and Immunity, 76(5):2123-2129, (2008)). Thus, the sequence of an oligonucleotide can contain one or more unmethylated cytosine-guanine (CG or CpG, used interchangeably) dinucleotide motifs. "p" refers to the phosphodiester backbone of DNA, as discussed in more detail below, and some oligonucleotides containing CG can have modified backbones, such as phosphorothioate (PS) backbones.

[0237] In certain embodiments, the immunostimulatory oligonucleotide may contain more than one CG dinucleotide, either closely spaced or separated by intervening nucleotide(s). The CpG motif(s) may be internal to the oligonucleotide sequence. Numerous nucleotide sequences stimulate TLR9 with variations in the number and position of the CG dinucleotide(s) and the exact base sequence adjacent to the CG dimer.

[0238] Typically, CG ODNs are classified based on their sequence, secondary structure, and effect on human peripheral blood mononuclear cells (PBMCs). The five classes are class A (D type), class B (K type), class C, class P, and class S (Vollmer, J & Krieg, AM, Advanced drug delivery reviews 61(3): 195-204 (2009), incorporated herein by reference). CG ODNs can stimulate the production of type I interferons (e.g., IFNα) and induce the maturation of dendritic cells (DCs). Some classes of ODNs are also strong activators of natural killer (NK) cells via indirect cytokine signaling. Some classes are potent stimulators of human B cell and monocyte maturation (Weiner, GL, PNAS USA 94(20): 10833-7 (1997); Dalpke, AH, Immunology 106(1): 102-12 (2002); Hartmann, G, J of Immun. 164(3):1617-2 (2000), each of which is incorporated herein by reference).

[0239] According to some embodiments, lipophilic-CpG oligonucleotide conjugates are used to enhance immune responses to antigens. Lipophilic-CpG oligonucleotides are represented by the following formula, where "L" is a lipophilic compound, e.g., a diacyl lipid, and "G n " is a guanine repeat linker, and "n" indicates 1, 2, 3, 4 or 5. 5'-LG n TCCATGACGTTCCTGACGTT-3'

[0240] Other PRR Toll-like receptors include TLR3 and TLR7, which can recognize double-stranded RNA, single-stranded and short double-stranded RNA, respectively, and retinoic acid-inducible gene I (RIG-I)-like receptor, i.e., RIG-I, and melanoma differentiation-associated gene 5 (MDA5), which is best known as an RNA-sensing receptor in the cytosol. Thus, in certain embodiments, the oligonucleotide comprises a functional ligand for TLR3, TLR7 or RIG-I-like receptor, or a combination thereof.

[0241] Examples of immunostimulatory oligonucleotides and methods for making them are known in the art. See, e.g., Bodera, P. Recent Pat Inflamm Allergy Drug Discov. 5(1):87-93 (2011), incorporated herein by reference.

[0242] In certain embodiments, the oligonucleotide cargo comprises two or more immunostimulatory sequences.

[0243] Oligonucleotides can be between 2 and 100 nucleotide bases in length, including, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 95, 98, 100 or more nucleotide bases in length.

[0244] The 3' or 5' end of the oligonucleotide may be conjugated to a polar block or lipid. In certain embodiments, the 5' end of the oligonucleotide is linked to a polar block or lipid.

[0245] Oligonucleotides can be DNA or RNA nucleotides that typically contain a heterocyclic base (nucleobase), a sugar moiety attached to the heterocyclic base, and a phosphate moiety that esterifies the hydroxyl functional group of the sugar moiety. The major naturally occurring nucleotides contain uracil, thymine, cytosine, adenine, and guanine as heterocyclic bases, and ribose or deoxyribose sugars linked by phosphodiester bonds. In certain embodiments, oligonucleotides are composed of nucleotide analogs that have been chemically modified to improve stability, half-life, or specificity or affinity for target receptors compared to their DNA or RNA counterparts. Chemical modifications include chemical modifications of the nucleobase, sugar moiety, nucleotide linkage, or combinations thereof. As used herein, "modified nucleotide" or "chemically modified nucleotide" defines a nucleotide that has one or more chemical modifications of the heterocyclic base, sugar moiety, or phosphate moiety components. In certain embodiments, the charge of the modified nucleotide is reduced compared to a DNA or RNA oligonucleotide of the same nucleobase sequence. For example, an oligonucleotide can have a low negative charge, no charge, or a positive charge.

[0246] Typically, nucleoside analogs support bases that can hydrogen bond with standard polynucleotide bases by Watson-Crick base pairing, where the analog backbone presents the bases in a manner that allows such hydrogen bonding in a sequence-specific manner between the oligonucleotide analog molecule and the bases in a standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA).In certain embodiments, the analog has a substantially uncharged phosphorus-containing backbone. (iii) chimeric antigen receptor ligand

[0247] In some embodiments, the CAR ligand of the amphiphilic ligand conjugate is an antigenic protein or polypeptide, such as a tumor-associated antigen or a portion thereof. In some embodiments, the CAR ligand is a small molecule, a peptide or a protein domain, or a fragment thereof. In some embodiments, the ligand binds to a CAR on a CAR-expressing cell (e.g., a CAR-T cell). Thus, the methods and compositions described herein utilize an amphiphilic ligand conjugate that is complementary to a CAR-expressing cell (e.g., a CAR-T cell). In some embodiments, the CAR ligand binds to any one of the above CARs.

[0248] In some embodiments, the peptide is between 2 and 100 amino acids, including, for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids. In some embodiments, the peptide is greater than 50 amino acids. In some embodiments, the peptide is >100 amino acids.

[0249] In some embodiments, the protein / peptide is linear, branched or cyclic. In some embodiments, the peptide comprises D-amino acids, L-amino acids, or a combination thereof. In some embodiments, the peptide or protein is conjugated to a polar block or lipid at the N-terminus or C-terminus of the peptide or protein.

[0250] In some embodiments, the protein or polypeptide can be any protein or peptide that can induce or enhance the immune system's ability to develop antibody and T cell responses against this protein or peptide.Cancer antigens are typically antigens that are preferentially expressed by cancer cells (i.e., they are expressed at higher levels in cancer cells than on non-cancerous cells), and in some cases, are expressed only by cancer cells.Cancer antigens can be expressed within or on the surface of cancer cells. The cancer antigen can be, but is not limited to, CD19, TRP-1, TRP-2, MART-1 / Melan-A, gp100, adenosine deaminase binding protein (ADAbp), FAP, cyclophilin b, colorectal-related antigen (CRC) - C017-1A / GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostate-specific antigen (PSA), PSA-1, PSA-2, PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor / CD3-zeta chain, and CD20.Cancer antigens are MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE -A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C 1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05), GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p The antigen may be selected from the group consisting of 21ras, RCAS1, alpha-fetoprotein, E-cadherin, alpha-catenin, beta-catenin, gamma-catenin, p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig-idiotype, p15, gp75, GM2 ganglioside, GD2 ganglioside, human papillomavirus protein, tumor antigens of the Smad family, lmp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, CD20, or c-erbB-2.

[0251] In some embodiments, the methods and compositions of the present disclosure are used in combination with Kymriah™ (tisagenlecleucel; Novartis) suspension for intravenous infusion, formerly CTL019. For example, in one embodiment, the compositions of the present disclosure include an amphipathic ligand conjugate in which the CAR ligand is CD19 or an antigenic portion thereof. Such compositions can be administered to a subject in combination with CD19-specific CAR-T cells (e.g., a population of CD19-specific CAR-T cells), cancer, such as Kymriah™ (tisagenlecleucel; Novartis) for the treatment of B-cell acute lymphoblastic leukemia (ALL).

[0252] Suitable antigens are known in the art and available from commercial government and scientific sources. In certain embodiments, the antigen is a whole inactivated or irradiated tumor cell. The antigen can be a purified or partially purified polypeptide derived from a tumor. The antigen can be a recombinant polypeptide produced by expressing DNA encoding the polypeptide antigen in a heterologous expression system. The antigen can be DNA encoding all or part of an antigenic protein. The DNA can be in the form of vector DNA, e.g., plasmid DNA.

[0253] In certain embodiments, antigens can be provided as single antigens or in combination. Antigens can also be provided as complex mixtures of polypeptides or nucleic acids.

[0254] In some embodiments, the CAR ligand of the amphiphilic ligand conjugate is a tag that binds to the CAR, which comprises the tag-binding domain described above. In some embodiments, the tag is fluorescein isothiocyanate (FITC), streptavidin, biotin, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horseradish peroxidase, palmitoylation, nitrosylation, alkaline phosphatase, glucose oxidase, or maltose binding protein.

[0255] In some embodiments, the CAR comprises a tumor antigen binding domain, and the CAR ligand is a tumor antigen or a fragment thereof. In some embodiments, the CAR comprises a tag binding domain (e.g., AT-CAR), and the CAR ligand is a tag. In some embodiments, the CAR is a tandem CAR, and the CAR ligand binds to at least one of the antigen binding domains present on the tandem CAR. In some embodiments, the CAR is bispecific, comprises a tumor antigen binding domain and a tag binding domain, and the CAR ligand is a tag. In some embodiments, the CAR is bispecific, comprises a tumor antigen binding domain and a tag binding domain, and the CAR ligand is a tumor antigen or a fragment thereof. In some embodiments, the CAR comprises a first tumor associated antigen binding domain and a second tumor associated antigen binding domain, and the CAR ligand is a first or second tumor associated antigen. (iv) Polar block / linker

[0256] In order for the conjugate to be efficiently transported to lymph nodes, the conjugate should remain soluble. Thus, in some embodiments, a polar block linker is included between the cargo and the lipid to increase the solubility of the conjugate. The polar block reduces or prevents the ability of the lipid to insert into the plasma membrane of cells, for example, cells in tissues adjacent to the injection site. The polar block can also reduce or prevent the ability of the cargo, for example, a synthetic oligonucleotide comprising a PS backbone, to non-specifically associate with extracellular matrix proteins at the site of administration. In some embodiments, the polar block increases the solubility of the conjugate without preventing its ability to bind to albumin. This combination of features is believed to allow the conjugate to bind to albumin present in serum or interstitial fluid and remain in circulation until the albumin is transported to and retained in lymph nodes. In some embodiments, the cargo functions as a polar block, and therefore a separate polar block is not required.

[0257] The length and composition of polar block can be adjusted based on selected lipid and cargo.For example, for oligonucleotide conjugates, the oligonucleotide itself, for example, the oligonucleotide that is 10, 15, 20 or more nucleotides long, can be polar enough to ensure the solubility of the conjugate.Therefore, in certain embodiments, no additional polar block linker is required.However, depending on the amino acid sequence, some lipidated peptides can be essentially insoluble.In these cases, it may be desirable to include a polar block that mimics the effect of polar oligonucleotides.

[0258] In some embodiments, polar blocks are used as part of any of the lipid conjugates suitable for use in the methods disclosed herein, such as amphipathic oligonucleotide conjugates and amphipathic ligand conjugates, that reduce cell membrane insertion / preferential partitioning into albumin. In some embodiments, suitable polar blocks include, but are not limited to, oligonucleotides, such as those discussed above, hydrophilic polymers including, but not limited to, poly(ethylene glycol) (MW: 500 Da to 20,000 Da), polyacrylamide (MW: 500 Da to 20,000 Da), polyacrylic acid; a series of hydrophilic amino acids, such as serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or combinations thereof; polysaccharides including, but not limited to, dextran (MW: 1,000 Da to 2,000,000 Da), or combinations thereof.

[0259] In some embodiments, the polar block provides solubility to the entire lipid conjugate based on the molecular weight of the polar block, whether a separate component or the cargo itself. For example, in some embodiments, a polar block having a molecular weight of 2,000 Da is sufficient to make the lipid conjugate soluble for albumin binding. In some embodiments, the polar block has a molecular weight of about 300 to about 20,000 Da. In some embodiments, the polar block has a molecular weight of about 1,000 to about 15,000 Da. In some embodiments, the polar block has a molecular weight of about 1,500 to about 10,000 Da. In some embodiments, the polar block has a molecular weight of about 2,000 to about 5,000 Da. In some embodiments, the polar block has a molecular weight of about 1,000 to about 2,500 Da. In some embodiments, the polar block has a molecular weight of about 1,000 to about 3,000 Da. In some embodiments, the polar block has a molecular weight of about 1,000 to about 3,500 Da. In some embodiments, the polar block has a molecular weight of about 1,000 to about 4,000 Da. In some embodiments, the polar block has a molecular weight of about 1,000 to about 5,000 Da. In some embodiments, the polar block has a molecular weight of about 5,000 to about 10,000 Da. In some embodiments, the polar block has a molecular weight of about 15,000 to about 20,000 Da.

[0260] In some embodiments, hydrophobic lipid and linker / cargo are covalently linked.In some embodiments, the covalent bond is a non-cleavable linkage or a cleavable linkage.In some embodiments, the non-cleavable linkage comprises an amide bond or a phosphate bond, and the cleavable linkage comprises a disulfide bond, an acid-cleavable linkage, an ester bond, an anhydride bond, a biodegradable bond or an enzyme-cleavable linkage. Ethylene glycol linker

[0261] In certain embodiments, the polar block is one or more ethylene glycol (EG) units, more preferably two or more EG units (i.e., polyethylene glycol (PEG)). For example, in certain embodiments, the lipid conjugate comprises a cargo (i.e., a CAR ligand or molecular adjuvant) and a hydrophobic lipid linked by a polyethylene glycol (PEG) molecule or a derivative or analog thereof.

[0262] In certain embodiments, the lipid conjugate suitable for use in the methods disclosed herein comprises a CAR ligand linked to PEG, which is then linked to hydrophobic lipid or lipid-Gn-ON conjugate, either covalently or via the formation of a protein-oligoconjugate that hybridizes to oligomyelin.The exact number of EG units depends on the lipid and cargo, but typically, the polar block can have between about 1 and about 100, between about 20 and about 80, between about 30 and about 70, or between about 40 and about 60 EG units.In certain embodiments, the polar block has between about 45 and 55 EG units.For example, in certain embodiments, the polar block has 48 EG units.

[0263] In some embodiments, the PEG molecule has a molecular weight of about 300 to 20,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 1,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 1,500 daltons. In some embodiments, the PEG molecule has a molecular weight of about 2,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 2,500 daltons. In some embodiments, the PEG molecule has a molecular weight of about 3,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 3,500 daltons. In some embodiments, the PEG molecule has a molecular weight of about 4,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 5,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 6,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 7,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 8,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 9,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 10,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 11,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 12,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 13,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 14,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 15,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 16,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 17,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 18,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 19,000 daltons. In some embodiments, the PEG molecule has a molecular weight of about 20,000 daltons. b. Oligonucleotide linker

[0264] As discussed above, in certain embodiments, the polar block is an oligonucleotide. The polar block linker can have any sequence, for example, the sequence of the oligonucleotide can be a random sequence or a sequence specifically selected for its molecular or biochemical properties (e.g., highly polar). In certain embodiments, the polar block linker comprises one or more consecutive series of adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analogs thereof. In certain embodiments, the polar block linker consists of a series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analogs thereof.

[0265] In certain embodiments, the linker is one or more guanines, for example, between 1 and 10 guanines. Varying the number of guanines between the cargo, such as a CpG oligonucleotide, and the lipid tail has been found to control micelle stability in the presence of serum proteins. Thus, the number of guanines in the linker can be selected based on the desired affinity of the conjugate for serum proteins, such as albumin. When the cargo is a CpG immunostimulatory oligonucleotide and the lipid tail is a diacyl lipid, the number of guanines affects the ability of micelles formed in aqueous solution to dissociate in the presence of serum: non-stabilized micelles (lipo-G0T 10 In the presence of guanine, 20% of the micelles (lipo-G2T8-CG) remained intact, whereas the remaining 80% were destroyed and bound to FBS components. In the presence of guanine, the percentage of intact micelles increased from 36% (lipo-G2T8-CG) to 73% (lipo-G4T6-CG) and finally reached 90% (lipo-G6T4-CG). By increasing the number of guanines to 8 (lipo-G8T2-CG) and 10 (lipo-G 10 Increasing the amount of glycerol in the micelles to T0-CG did not further enhance micelle stability.

[0266] Thus, in certain embodiments, linkers in lymph node-targeting conjugates suitable for use in the methods disclosed herein may contain 0, 1, or 2 guanines. As discussed in more detail below, linkers containing 3 or more consecutive guanines may be used to form micelle-stabilized conjugates with properties suitable for use in the methods disclosed herein. C. Immunogenic composition

[0267] The lipid conjugate suitable for use in the methods disclosed herein can be used as a component in immunogenic compositions or vaccines.Typically, the immunogenic compositions disclosed herein include adjuvant, antigen, or a combination thereof.The combination of adjuvant and antigen can be called a vaccine.When administered to a subject in combination, the adjuvant and antigen can be administered in separate pharmaceutical compositions, or they can be administered together in the same pharmaceutical composition.When administered in combination, the adjuvant can be a lipid conjugate, the antigen can be a lipid conjugate, or both the adjuvant and antigen can be lipid conjugates.

[0268] In some embodiments, immunogenic compositions suitable for use in the methods disclosed herein comprise an amphipathic ligand conjugate administered alone or in combination with an adjuvant. In some embodiments, adjuvants include, without limitation, alum (e.g., aluminum hydroxide, aluminum phosphate); saponins purified from the bark of the Q. saponaria tree, such as QS21 (a glycolipid that elutes in the 21st peak in HPLC fractionation; Antigenics, Inc., Worcester, Mass.); poly[di(carboxylatophenoxy)phosphazene] (PCPP polymer; Virus Research Institute, USA), Flt3 ligand, Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.), ISCOMS (mixed saponins, immunostimulatory complexes that contain lipids and form virus-sized particles with pores that can retain antigens; CSL, Melbourne, Australia), Pam3Cys, SB-AS4 (a SmithKline product that contains alum and MPL). Beecham Adjuvant System #4; SBB, Belgium), micelle-forming non-ionic block copolymers such as CRL 1005 (which contain linear chains of hydrophobic polyoxypropylene flanked by chains of polyoxyethylene, Vaxcel, Inc., Norcross, Ga.), and Montanide IMS (e.g., IMS 1312, a water-based nanoparticle combined with a soluble immunostimulant, Seppic).

[0269] In some embodiments, the adjuvant is a TLR ligand, such as those discussed above. In some embodiments, the adjuvant acting through TLR3 includes, without limitation, double-stranded RNA. In some embodiments, the adjuvant acting through TLR4 includes, without limitation, derivatives of lipopolysaccharide, such as monophosphoryl lipid A (MPLA; Ribi ImmunoChem Research, Inc., Hamilton, Mont.) and muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland). In some embodiments, the adjuvant acting through TLR5 includes, without limitation, flagellin. In some embodiments, adjuvants acting through TLR7 and / or TLR8 include single-stranded RNA, oligoribonucleotides (ORNs), synthetic low molecular weight compounds, such as imidazoquinoline amines (e.g., imiquimod (R-837), resiquimod (R-848)). In some embodiments, adjuvants acting through TLR9 include DNA of viral or bacterial origin, or synthetic oligodeoxynucleotides (ODNs), such as CpG ODNs. In some embodiments, another class of adjuvants are phosphorothioate-containing molecules, such as phosphorothioate nucleotide analogs and nucleic acids that contain phosphorothioate backbone linkages.

[0270] In some embodiments, the adjuvant is selected from oil emulsions (e.g., Freund's adjuvant); saponin preparations; virosomes and virus-like particles; bacterial and microbial derivatives; immunostimulatory oligonucleotides; ADP-ribosylating toxins and detoxified derivatives; alum; BCG; mineral-containing compositions (e.g., mineral salts, such as aluminum and calcium salts, hydroxides, phosphates, sulfates, etc.); bioadhesives and / or mucoadhesives; microparticles; liposomes; polyoxyethylene ether and polyoxyethylene ester preparations; polyphosphazenes; muramyl peptides; imidazoquinolone compounds; and surfactants (e.g., lysolecithin, Pluronic® polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).

[0271] In some embodiments, the adjuvant is selected from an immunomodulator, such as a cytokine, an interleukin (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), an interferon (e.g., interferon-γ), macrophage colony-stimulating factor, and tumor necrosis factor.

[0272] In some embodiments, the adjuvant is an amphipathic oligonucleotide conjugate comprising an immunostimulatory oligonucleotide, as described above.

[0273] In some embodiments, the adjuvant is a STING (STimulator of Interferon Genes) agonist. The STING signaling pathway in immune cells is a central mediator of innate immune response, and when stimulated, it induces the expression of various interferons, cytokines and T cell recruiting factors that amplify and strengthen immune activity. Recent studies, such as those described in Dubensky, T., et al., Therapeutic Advances in Vaccines, Vol. 1(4): 131-143 (2013); and Hanson, M., et al., The Journal of Clinical Investigation, Vol. 125 (6): 2532-2546 (2015), which are incorporated herein by reference, have shown that STING agonists are effective adjuvants and efficiently induce immune response.

[0274] In some embodiments, STING agonist is cyclic dinucleotide.In certain embodiments, cyclic dinucleotide includes but is not limited to cdAMP, cdGMP, cdIMP, c-AMP-GMP, c-AMP-IMP and c-GMP-IMP, and their analogs, including but not limited to phosphorothioate analogs.In some embodiments, the cyclic dinucleotide suitable for use in the present disclosure is described in some detail in, for example, U.S. Patent Nos. 7,709,458 and 7,592,326; WO2007 / 054279; US2014 / 0205653; and Yan et al. Bioorg. Med. Chem Lett. 18: 5631 (2008), each of which is incorporated herein by reference.

[0275] In certain embodiments, STING agonist is chemically synthesized.In certain embodiments, STING agonist is a naturally occurring cyclic dinucleotide analogue.Suitable for use in the present disclosure, including cyclic dinucleotide analogue, STING agonist is provided in US Patent No. 7,709,458 and US Patent No. 7,592,326; and US2014 / 0205653. Methods for Producing Polypeptides

[0276] In some embodiments, the polypeptides (e.g., tumor-associated antigens) described herein for use in amphiphilic conjugates are produced in transformed host cells using recombinant DNA technology. For this purpose, recombinant DNA molecules encoding peptides are prepared. Methods for preparing such DNA molecules are well known in the art. For example, peptide-encoding sequences can be excised from DNA using appropriate restriction enzymes. Alternatively, DNA molecules can be synthesized using chemical synthesis techniques such as phosphoramidate method. A combination of these techniques can also be used.

[0277] The method of making polypeptide also includes a vector capable of expressing peptide in a suitable host.The vector includes the DNA molecule that codes for peptide, which is operatively linked to a suitable expression control sequence.The method of influencing this operative linkage either before or after the DNA molecule is inserted into the vector is well known.Expression control sequences include promoters, activators, enhancers, operators, ribosomal nuclease domains, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved in the control of transcription or translation.

[0278] The resulting vector, having the DNA molecule thereon, is used to transform an appropriate host, which can be carried out using methods well known in the art.

[0279] Any of a large number of available and well-known host cells may be suitable for use in the methods disclosed herein. The selection of a particular host depends on several factors recognized in the art. These include, for example, compatibility with the selected expression vector, toxicity of the peptide encoded by the DNA molecule, transformation rate, ease of peptide recovery, expression characteristics, biosafety and cost. These factors should be balanced with the understanding that not all hosts may be equally effective for the expression of a particular DNA sequence. Within these general guidelines, useful microbial hosts include bacteria (e.g., E. coli species), yeast (e.g., Saccharomyces species) and other fungi, insects, plants, mammalian (including human) cells in culture, or other hosts known in the art.

[0280] The transformed host is then cultured and purified. The host cells can be cultured under conventional fermentation conditions such that the desired compound is expressed. Such fermentation conditions are well known in the art. Finally, the peptide is purified from the culture by methods well known in the art.

[0281] Compounds can also be made by synthetic methods. For example, solid phase synthesis techniques can be used. Suitable techniques are well known in the art and include those described in Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.);Merrifield (1963), J. Am. Chem. Soc. 85: 2149;Davis et al. (1985), Biochem. Intl. 10: 394-414;Stewart and Young (1969), Solid Phase Peptide Synthesis;U.S. Pat. No. 3,941,763;Finn et al. (1976), The Proteins (3rd ed.) 2: 105-253;and Erickson et al. (1976), The Proteins (3rd ed.) 2: 257-527. Solid phase synthesis is the preferred technique for making individual peptides because it is the most cost-effective method for making small peptides. Compounds containing derivatized peptides or containing non-peptide groups can be synthesized by well-known organic chemistry techniques.

[0282] Other methods are those of molecular expression / synthesis, which are commonly known to those skilled in the art.

[0283] The nucleic acid molecules may be contained within a vector, for example, capable of directing their expression in a cell transduced with the vector. Thus, in addition to the polypeptide variants, expression vectors that contain nucleic acid molecules encoding the variants, and cells transfected with these vectors, are within certain embodiments.

[0284] Suitable vectors for use include T7-based vectors for use in bacteria (see, e.g., Rosenberg et al., Gene 56: 125, 1987), pMSXND expression vectors for use in mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988), and baculovirus-derived vectors for use in insect cells (e.g., expression vector pBacPAKS from Clontech, Palo Alto, Calif.). The nucleic acid insert encoding the polypeptide of interest in such vectors can be operably linked to a promoter selected, for example, based on the cell type in which expression is desired. For example, the T7 promoter can be used in bacteria, the polyhedrin promoter can be used in insect cells, and the cytomegalovirus or metallothionein promoter can be used in mammalian cells. Also, in the case of higher eukaryotes, tissue-specific and cell type-specific promoters are widely available. These promoters are so named for their ability to direct the expression of a nucleic acid molecule in a given tissue or cell type within the body. Those of skill in the art are well aware of the numerous promoters and other regulatory elements that can be used to direct the expression of a nucleic acid.

[0285] In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, vectors can contain an origin of replication and other genes that encode selectable markers. For example, neomycin resistance (neomycin resistance). r The ) gene confers G418 resistance to cells in which it is expressed, thus allowing phenotypic selection of transfected cells. One of skill in the art can readily determine whether a given regulatory element or selectable marker is appropriate for use in a particular experimental setting.

[0286] Viral vectors suitable for use include, for example, retroviral vectors, adenoviral and adeno-associated vectors, herpes virus vectors, simian virus 40 (SV40) vectors, and bovine papilloma virus vectors (see, e.g., Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, NY).

[0287] Prokaryotic or eukaryotic cells that contain and express a nucleic acid molecule encoding a polypeptide variant are also suitable for use. The cell is a transfected cell, i.e., a cell into which a nucleic acid molecule, e.g., a nucleic acid molecule encoding a variant polypeptide, has been introduced by recombinant DNA techniques. The progeny of such cells are also considered suitable for use in the methods disclosed herein.

[0288] The exact components of the expression system are not critical. For example, the polypeptide variant can be produced in a prokaryotic host, such as bacteria E. coli, or in a eukaryotic host, such as insect cells (e.g., Sf21 cells) or mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, Va.). When selecting an expression system, it is only important that the components are compatible with each other. Those skilled in the art can make such a decision. Furthermore, those skilled in the art can consult Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, New York, NY, 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987) if guidance is needed in selecting an expression system.

[0289] The expressed polypeptides can be purified from the expression system using routine biochemical procedures and used as described herein, e.g., conjugated to lipids. Pharmaceutical Compositions and Modes of Administration

[0290] In some embodiments, the amphipathic ligand conjugate and the CAR-expressing cell (e.g., CAR T cell) are administered together (simultaneously or sequentially). In some embodiments, the amphipathic ligand conjugate and the adjuvant (e.g., amphipathic oligonucleotide conjugate) are administered together (simultaneously or sequentially). In some embodiments, the amphipathic ligand conjugate, the adjuvant (e.g., amphipathic oligonucleotide conjugate) and the CAR-expressing cell (e.g., CAR T cell) are administered together (simultaneously or sequentially). In some embodiments, the amphipathic ligand conjugate and the CAR-expressing cell (e.g., CAR T cell) are administered separately. In some embodiments, the amphipathic ligand conjugate and the adjuvant (e.g., amphipathic oligonucleotide conjugate) are administered separately. In some embodiments, the amphipathic ligand conjugate, the adjuvant (e.g., amphipathic oligonucleotide conjugate) and the CAR-expressing cell (e.g., CAR T cell) are administered separately.

[0291] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an amphipathic ligand conjugate together with a pharma- ceutically acceptable excipient, carrier, solubilizer, emulsifier, preservative and / or adjuvant. In some embodiments, the adjuvant is an amphipathic oligonucleotide conjugate. In some embodiments, the adjuvant is a STING agonist (e.g., CDG). In some embodiments, the adjuvant is formulated in a separate pharmaceutical composition.

[0292] In some embodiments, acceptable formulation materials are preferably non-toxic to recipients at the dosages and concentrations used. In certain embodiments, the formulation material(s) are for sc and / or IV administration. In some embodiments, pharmaceutical compositions include formulation materials to modify, maintain or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or permeation of the composition.In some embodiments, suitable formulation materials include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants, flavoring agents, and diluting agents. agent); emulsifier; hydrophilic polymer (e.g., polyvinylpyrrolidone); low molecular weight polypeptide; salt-forming counterion (e.g., sodium); preservative (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvent (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohol (e.g., mannitol or sorbitol); suspending agent; surfactant or wetting agent (e.g., Pluronic® , PEG, sorbitan esters, polysorbates, e.g., polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal; stability enhancers (e.g., sucrose or sorbitol); tonicity enhancers (e.g., alkali metal halides, preferably sodium chloride or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants (Remington's Pharmaceutical Sciences, 18th Edition, AR Gennaro, ed., Mack Publishing Company (1995)).In certain embodiments, the formulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and / or 10 mM NAOAC, pH 5.2, 9% sucrose. In some embodiments, the optimal pharmaceutical composition is determined by those skilled in the art, depending, for example, on the intended route of administration, delivery format, and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. In some embodiments, such compositions can affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the amphiphilic conjugate.

[0293] In some embodiments, the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, in some embodiments, a suitable vehicle or carrier is water for injection, physiological saline solution, or artificial cerebrospinal fluid, optionally supplemented with other materials common in compositions for parenteral administration. In some embodiments, the saline solution comprises isotonic phosphate buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In some embodiments, the pharmaceutical composition comprises a Tris buffer of about pH 7.0-8.5, or an acetate buffer of about pH 4.0-5.5, which may further comprise sorbitol or a suitable substitute thereof. In some embodiments, the composition comprising the amphiphilic conjugate may be prepared for storage by mixing the selected composition having the desired degree of purity in the form of a lyophilized cake or aqueous solution with optional formulating agents (Remington's Pharmaceutical Sciences, supra). Additionally, in some embodiments, the composition comprising the amphiphilic conjugate may be formulated as a lyophilizate using suitable additives such as sucrose.

[0294] In some embodiments, the pharmaceutical composition may be selected for parenteral delivery. In some embodiments, the composition may be selected for inhalation or for delivery via the digestive tract, such as for oral delivery. The preparation of such pharma- ceutically acceptable compositions is within the skill of one of ordinary skill in the art.

[0295] In some embodiments, the formulation components are present in concentrations that are acceptable for the site of administration. In some embodiments, a buffer is used to maintain the composition at physiological pH or slightly lower, typically within a pH range of about 5 to about 8.

[0296] In some embodiments, when parenteral administration is intended, the therapeutic composition may be in the form of a pyrogen-free parenterally acceptable aqueous solution containing the amphiphilic conjugate in a pharma- ceutically acceptable vehicle. In some embodiments, the vehicle for parenteral injection is sterile distilled water in which the amphiphilic conjugate is formulated as a sterile isotonic solution that is appropriately preserved. In some embodiments, the preparation may include formulation of the desired molecule with agents such as injectable microspheres, bio-erodible particles, polymeric compounds (e.g., polylactic acid or polyglycolic acid), beads or liposomes that can provide controlled or sustained release of the product that can then be delivered via depot injection. In some embodiments, hyaluronic acid may also be used, which may have the effect of promoting sustained duration in the circulation. In some embodiments, an implantable drug delivery device may be used to introduce the desired molecule.

[0297] In some embodiments, the pharmaceutical composition may be formulated for inhalation. In some embodiments, the amphiphilic conjugate may be formulated as a dry powder for inhalation. In some embodiments, the inhalation solution containing the amphiphilic conjugate may be formulated with a propellant for aerosol delivery. In some embodiments, the solution may be nebulized. Pulmonary administration is further described in PCT Application No. PCT / US94 / 001875, which describes pulmonary delivery of chemically modified proteins.

[0298] In some embodiments, it is contemplated that the formulation may be administered orally. In some embodiments, the amphiphilic conjugate administered in this manner may be formulated with or without carriers customarily used in the preparation of solid dosage forms such as tablets and capsules. In some embodiments, the capsule may be designed to release the active portion of the formulation at the time in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. In some embodiments, at least one additional agent may be included to promote the absorption of the amphiphilic conjugate. In certain embodiments, excipients, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binders may also be used.

[0299] In some embodiments, pharmaceutical compositions may contain an effective amount of amphiphilic conjugate in a mixture with non-toxic additives suitable for tablet manufacture. In some embodiments, solutions may be prepared in unit dose form by dissolving tablets in sterile water or another suitable vehicle. In some embodiments, suitable additives include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose or calcium phosphate; or binders such as starch, gelatin or gum arabic; or lubricating agents such as magnesium stearate, stearic acid or talc.

[0300] Further pharmaceutical compositions, including formulations that include amphiphilic conjugates in sustained or controlled delivery formulations, will be apparent to those skilled in the art.In some embodiments, the techniques for formulating various other sustained or controlled delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art.See, for example, PCT Application No. PCT / US93 / 00829, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions.In some embodiments, sustained release preparations can include semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP 058,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981) and Langer, Chem. Tech., 12:98- 105 (1982)), ethylene vinyl acetate (Langer et al., supra), or poly-D(-)-3-hydroxybutyrate (EP 133,988). In some embodiments, sustained release compositions may also include liposomes, which may be prepared by any of several methods known in the art. See, e.g., Eppstein et al, Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.

[0301] In some embodiments, the pharmaceutical composition used for in vivo administration is sterile. In some embodiments, sterility is achieved by filtration through a sterile filtration membrane. In certain embodiments, the composition is lyophilized, sterilization using this method is carried out either before or after lyophilization and reconstitution. In some embodiments, the composition for parenteral administration is stored in lyophilized form or in solution. In some embodiments, the parenteral composition is placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0302] In some embodiments, once the pharmaceutical compositions have been formulated, they are stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In some embodiments, such formulations are stored either in a ready-to-use form or in a form that is reconstituted prior to administration (e.g., lyophilized form).

[0303] In some embodiments, a kit is provided for producing single-dose administration units.In some embodiments, the kit can include both a first container with dry protein and a second container with aqueous formulation.In some embodiments, the kit includes single and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes).

[0304] In some embodiments, the effective amount of pharmaceutical compositions containing amphiphilic conjugates used therapeutically depends, for example, on the therapeutic situation and purpose.Therefore, those skilled in the art will understand that the dosage level suitable for treatment according to a certain embodiment will vary depending in part on the molecule to be delivered, the indication for which the amphiphilic conjugate is used, the route of administration, and the size (weight, body surface or organ size) and / or condition (age and general health) of the patient.In some embodiments, clinicians can titrate the dosage and modify the route of administration to obtain optimal therapeutic effect.

[0305] In some embodiments, the frequency of dosing takes into account the pharmacokinetic parameters of the amphiphilic conjugate in the formulation used. In some embodiments, the clinician administers the composition until a dosage is reached that achieves the desired effect. Thus, in some embodiments, the composition can be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as continuous infusion via an implanted device or catheter. Further refinement of the appropriate dosage is routinely performed by those skilled in the art and is within the scope of routine work performed by those skilled in the art. In some embodiments, the appropriate dosage can be confirmed through the use of appropriate dose-response data.

[0306] In some embodiments, the route of administration of the pharmaceutical composition is according to known methods, for example, via injection by oral, intravenous, intraperitoneal, intracerebral (intracemdrome), intraventricular, intramuscular, subcutaneous, intraocular, intraarterial, intraportal or intralesional route; by sustained release system or implantation device.In certain embodiments, the composition can be administered by bolus injection or continuously by infusion or by implantation device.In certain embodiments, the individual components of the combination therapy can be administered by different routes.

[0307] In some embodiments, the composition may be administered locally via implantation of a membrane, sponge or another suitable material on which the desired molecule is absorbed or encapsulated. In some embodiments in which an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration. In some embodiments, it may be desirable to use pharmaceutical compositions comprising amphiphilic conjugates in an ex vivo manner. In such an example, cells, tissues, and / or organs removed from a patient are exposed to pharmaceutical compositions comprising amphiphilic conjugates, and then the cells, tissues, and / or organs are subsequently implanted back into the patient.

[0308] In some embodiments, the amphiphilic conjugates may be delivered by implanting certain cells that have been genetically engineered using methods such as those described herein to express and secrete the conjugates. In some embodiments, such cells may be animal or human cells and may be autologous, heterologous or xenogeneic. In some embodiments, the cells may be immortalized. In some embodiments, to reduce the chance of an immunological response, the cells may be encapsulated to avoid infiltration of surrounding tissues. In some embodiments, the encapsulation material is typically a biocompatible semipermeable polymeric enclosure or membrane that allows release of the protein product(s) but prevents destruction of the cells by the patient's immune system or by other harmful factors from the surrounding tissues. How to use

[0309] In some embodiments, the present disclosure provides a method of expanding or activating CAR effector cells (e.g., CAR-T cells) in vivo in a subject, the method comprising administering a composition comprising an amphiphilic lipid conjugate described herein.

[0310] In some embodiments, the present disclosure provides a method of stimulating proliferation of CAR effector cells (e.g., CAR-T cells) in vivo in a subject, the method comprising administering a composition comprising an amphiphilic lipid conjugate described herein.

[0311] Methods for determining cell expansion, activation and proliferation are known to those skilled in the art. For example, the number of cells in a particular location (e.g., lymph node, blood, tumor) can be determined by isolating the cells and analyzing them via flow cytometry. In some embodiments, the cells are stained with an appropriate marker, such as an activation marker (e.g., CD80, CD86, 41BBL, ICOSL or OX40L) and / or a proliferation marker (e.g., Ki67). In some embodiments, the number of cells is measured by introducing a dye (e.g., crystal violet) into the cells and measuring the dilution of the dye over time, where the dilution indicates cell proliferation.

[0312] In some embodiments, the disclosure provides a method for treating a subject having a disease, disorder, or condition associated with expression or increased expression of an antigen, comprising administering to the subject a CAR effector cell (e.g., a CAR-T cell) targeted to the antigen and an amphiphilic lipid conjugate.

[0313] In some embodiments, the subject is administered CAR effector cells (e.g., CAR-T cells) prior to receiving the amphiphilic lipid conjugate. In some embodiments, the subject is administered CAR effector cells (e.g., CAR-T cells) after receiving the amphiphilic lipid conjugate. In some embodiments, the subject is administered CAR effector cells (e.g., CAR-T cells) and the amphiphilic lipid conjugate, either sequentially or simultaneously.

[0314] In some embodiments, the CAR comprises a tag-binding domain, the method disclosed herein further comprises administering a formulation of tagged protein, wherein the tag-binding domain binds to the tagged protein. In some embodiments, the protein of the tagged protein is an antibody or an antigen-binding fragment. In some embodiments, the tag-binding domain is an antibody or an antigen-binding fragment thereof. In some embodiments, the formulation of tagged protein is administered to the subject prior to administration of the CAR effector cells (e.g., CAR T cells) and the amphipathic ligand conjugate. In some embodiments, the formulation of tagged protein is administered to the subject in conjunction with (simultaneously or sequentially with) the CAR effector cells (e.g., CAR T cells) and the amphipathic ligand conjugate. In some embodiments, the formulation of tagged protein is administered to the subject after administration of the CAR effector cells (e.g., CAR T cells) and the amphipathic ligand conjugate. Cancer and Cancer Immunotherapy

[0315] In some embodiments, the amphiphilic ligand conjugates described herein are useful for treating disorders associated with abnormal apoptotic or differentiation processes, such as cell proliferation disorders (e.g., hyperproliferative disorders) or cell differentiation disorders, e.g., cancer. Non-limiting examples of cancers suitable for treatment by the methods of the invention are described below.

[0316] Examples of cell proliferation and / or differentiation disorders include cancer (e.g., carcinoma, sarcoma, metastatic disorders, or hematopoietic neoplastic disorders, e.g., leukemia). Metastatic tumors can arise from a number of primary tumor types, including, but not limited to, those of the prostate, colon, lung, breast, and liver. Thus, the compositions used herein that include amphiphilic ligand conjugates can be administered to patients with cancer.

[0317] As used herein, we may use the terms "cancer" (or "cancerous"), "hyperproliferative" and "neoplastic" to refer to cells capable of autonomous growth (i.e., an abnormal state or condition characterized by rapidly proliferating cell growth). Hyperproliferative and neoplastic disease states may be classified as pathological (i.e., characterizing or constituting a disease state) or non-pathological (i.e., as a deviation from normal that is not associated with a disease state). These terms are meant to include all types of cancerous growth or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues or organs, regardless of histopathological type or stage of invasiveness. "Pathological hyperproliferative" cells occur in disease states characterized by malignant tumor growth. Examples of non-pathological hyperproliferative cells include the proliferation of cells associated with wound repair.

[0318] The term "cancer" or "neoplasm" is used to refer to malignant tumors of various organ systems, including those affecting the lung, breast, thyroid, lymphatic glands and lymphatic tissues, gastrointestinal organs and genitourinary tract, as well as malignant tumors, e.g., adenocarcinomas, which are generally considered to include most colon cancers, renal cell carcinoma, prostate cancer and / or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.

[0319] The term "cancer" is recognized in the art and refers to malignant tumors of epithelial or endocrine tissues, including respiratory cancer, gastrointestinal cancer, genitourinary system cancer, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, and melanoma. The amphiphilic ligand conjugates can be used to treat patients with, suspected of having, or who may be at high risk of developing any type of cancer, including renal cancer or melanoma, or any viral disease. Exemplary cancers include those formed from tissues of the cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also includes carcinosarcoma, which includes malignant tumors composed of carcinomatous and sarcomatous tissues. "Adenocarcinoma" refers to cancers derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0320] Further examples of proliferation disorders include hematopoietic neoplastic disorders. As used herein, the term "hematopoietic neoplastic disorders" includes diseases involving hyperplastic / neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or their precursor cells. Preferably, these diseases arise from poorly differentiated acute leukemias (e.g., erythroblastic leukemia and acute megakaryoblastic leukemia). Further exemplary myeloid disorders include, but are not limited to, acute promyelocytic leukemia (APML), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) (reviewed in Vaickus, L. (1991) Crit. Rev. in Oncol. / Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), including B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL), and Waldenstrom's macroglobulinemia (WM). Further forms of malignant lymphoma include, but are not limited to, non-Hodgkin's lymphoma and its variants, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease, and Reed-Sternberg disease.

[0321] It will be understood by those skilled in the art that the amount of amphiphilic conjugate sufficient to reduce tumor growth and size, i.e., a therapeutically effective amount, will vary not only with the particular compound or composition selected, but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will ultimately be at the discretion of the patient's physician or pharmacist. The length of time that the compounds used in the present methods are given will vary from individual to individual.

[0322] In some embodiments, the present disclosure provides a method of reducing or decreasing tumor size or inhibiting tumor growth in a subject in need thereof, comprising administering to the subject an amphiphilic lipid conjugate described herein, wherein the subject is undergoing or has undergone CAR effector cell therapy (e.g., CAR-T cell therapy). In some embodiments, the present disclosure provides a method for inducing an anti-tumor response in a subject having cancer, comprising administering to the subject an amphiphilic lipid conjugate described herein, wherein the subject is undergoing or has undergone CAR effector cell therapy (e.g., CAR-T cell therapy).

[0323] In some embodiments, the present disclosure provides a method for stimulating an immune response against a target cell population or target tissue expressing an antigen in a subject, comprising administering an effector CAR cell (e.g., CAR-T cell) targeted to the antigen and an amphiphilic lipid conjugate. In some embodiments, the immune response is a T cell-mediated immune response. In some embodiments, the immune response is an anti-tumor immune response. In some embodiments, the target cell population or target tissue is a tumor cell or tumor tissue.

[0324] It will be understood by those skilled in the art that references herein to treatment extend to prophylaxis and treatment of the cancers and conditions noted. infectious disease

[0325] In some embodiments, the amphiphilic lipid conjugates disclosed herein are useful for treating acute or chronic infectious diseases.Since viral infections are primarily eliminated by T cells, the increase in T cell activity is therapeutically useful in situations where more rapid or complete clearance of infectious viral pathogens is beneficial for animal or human subjects. Recently, CAR-T cell therapy has been investigated for its utility in treating viral infections such as human immunodeficiency virus (HIV), as described in PCT Publication No. WO2015 / 077789;Hale et al., (2017) Engineering HIV-Resistant, Anti-HIV Chimeric Antigen Receptor T Cells. Molecular Therapy, Vol. 25(3): 570-579;Liu et al., (2016). ABSTRACT. Journal of Virology, 90(21), 9712-9724;Liu et al., (2015). ABSTRACT. Journal of Virology, 89(13), 6685-6694;Sahu et al., (2013). Virology, 446(1-2), 268-275.

[0326] Thus, in some embodiments, the amphiphilic ligand conjugate is administered to treat local or systemic viral infections, including but not limited to immunodeficiencies (e.g., HIV), papilloma (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza (e.g., human influenza virus A), and cold (e.g., human rhinovirus) viral infections. In some embodiments, pharmaceutical formulations comprising the amphiphilic ligand conjugate are administered topically to treat viral skin diseases, such as herpes lesions or shingles, or genital warts. In some embodiments, the amphiphilic ligand conjugate is administered to treat systemic viral diseases, including but not limited to AIDS, influenza, cold, or encephalitis.

[0327] In some embodiments, the disclosure provides a method for increasing proliferation of CAR effector cells (e.g., CAR-T cells) in vivo in a subject having a viral infection, comprising administering a composition comprising an amphipathic ligand conjugate, wherein the CAR comprises a viral peptide binding domain (e.g., an HIV Env binding domain) and the amphipathic ligand conjugate comprises a viral peptide (e.g., HIV Env).

[0328] In some embodiments, the disclosure provides a method for expanding CAR effector cells (e.g., CAR-T cells) in vivo in a subject having a viral infection, comprising administering a composition comprising an amphipathic ligand conjugate, wherein the CAR comprises a viral peptide binding domain (e.g., an HIV Env binding domain) and the amphipathic ligand conjugate comprises a viral peptide (e.g., HIV Env).

[0329] In some embodiments, the disclosure provides a method of reducing a viral infection in a subject in need thereof, comprising administering to the subject an amphiphilic lipid conjugate as described herein, wherein the subject is undergoing or has undergone CAR effector cell therapy (e.g., CAR-T cell therapy). In some embodiments, the disclosure provides a method for inducing an antiviral response in a subject having cancer, comprising administering to the subject an amphiphilic lipid conjugate as described herein, wherein the subject is undergoing or has undergone CAR effector cell therapy (e.g., CAR-T cell therapy).

[0330] It will be understood by those skilled in the art that references herein to treatment extend to prophylaxis and treatment of the infections and conditions noted. kit

[0331] Provided herein is a kit that includes at least the amphipathic ligand conjugate described herein and instructions for use.In some embodiments, the kit includes the amphipathic ligand conjugate, one or more controls, and various buffers, reagents, enzymes and other standard components well known in the art in a suitable container.In some embodiments, the kit further includes an adjuvant (e.g., an amphipathic oligonucleotide conjugate or a STING agonist (e.g., CDG)).Thus, in some embodiments, the amphipathic ligand conjugate and the adjuvant are in the same vial.In some embodiments, the amphipathic ligand conjugate and the adjuvant are in separate vials.

[0332] In some embodiments, the container is at least one vial, well, test tube, flask, bottle, syringe or other container means into which the amphiphilic ligand conjugate can be placed and in some cases appropriately aliquoted. If additional components are provided, the kit may include additional containers into which the compounds can be placed. The kit may also include a means for containing the amphiphilic ligand conjugate for commercial sale, and any other reagent containers that are sealed. Such containers may include injection-molded or blow-molded plastic containers into which the desired vials are held. The container and / or kit may include a label with instructions and / or warnings for use.

[0333] In some embodiments, the present disclosure provides a kit comprising a container containing a composition comprising an amphipathic ligand conjugate as described herein, an optional pharma- ceutically acceptable carrier, and a package insert containing instructions for administering the composition for treating or delaying the progression of cancer in an individual undergoing CAR-T cell therapy. In some embodiments, the kit further comprises an adjuvant and instructions for administering the adjuvant for treating or delaying the progression of cancer in an individual undergoing CAR-T cell therapy. In some embodiments, the adjuvant is an amphipathic oligonucleotide conjugate as described herein. In some embodiments, the adjuvant is a STING agonist. In some embodiments, the adjuvant is CDG.

[0334] In some embodiments, the present disclosure provides a kit comprising a medicament comprising a composition comprising an amphipathic ligand conjugate as described herein, optionally a pharma- ceutical acceptable carrier, for treating or delaying the progression of cancer in an individual undergoing CAR-T cell therapy, and a package insert containing instructions for administration of the medicament alone or in combination with a composition comprising an adjuvant and optionally a pharma- ceutical acceptable carrier.

[0335] In some embodiments, the present disclosure provides a kit comprising a container containing a composition comprising an amphipathic ligand conjugate as described herein, an optional pharma- ceutically acceptable carrier, and a package insert containing instructions for administering the composition vaccine for expanding CAR-T cells in an individual undergoing CAR-T cell therapy. In some embodiments, the kit further comprises an adjuvant and instructions for administering the adjuvant for expanding CAR-T cells in an individual undergoing CAR-T cell therapy. In some embodiments, the adjuvant is an amphipathic oligonucleotide conjugate as described herein. In some embodiments, the adjuvant is a STING agonist. In some embodiments, the adjuvant is CDG.

[0336] In some embodiments, the present disclosure provides a kit comprising a medicament comprising a composition comprising an amphipathic ligand conjugate as described herein, an optional pharmaceutical acceptable carrier, and a package insert comprising instructions for administration of the medicament alone or in combination with a composition comprising an adjuvant and an optional pharmaceutical acceptable carrier for expanding CAR-T cells in an individual undergoing CAR-T cell therapy.In some embodiments, the adjuvant is an amphipathic oligonucleotide conjugate as described herein.In some embodiments, the adjuvant is a STING agonist.In some embodiments, the adjuvant is CDG.

[0337] In some embodiments, the present disclosure provides a kit comprising a container containing a composition comprising an amphipathic ligand conjugate as described herein, an optional pharma- ceutically acceptable carrier, and a package insert containing instructions for administering the composition for increasing the proliferation of CAR-T cells in an individual undergoing CAR T cell therapy. In some aspects, the kit further comprises an adjuvant and instructions for administering the adjuvant for increasing the proliferation of CAR-T cells in an individual undergoing CAR T cell therapy. In some embodiments, the adjuvant is an amphipathic oligonucleotide conjugate as described herein. In some embodiments, the adjuvant is a STING agonist. In some embodiments, the adjuvant is CDG.

[0338] In some embodiments, the present disclosure provides a kit comprising a medicament comprising a composition comprising an amphipathic ligand conjugate as described herein, optionally a pharmaceutical acceptable carrier, for increasing the proliferation of CAR-T cells in an individual undergoing CAR-T cell therapy, and a package insert comprising instructions for administering the medicament alone or in combination with a composition comprising an adjuvant and optionally a pharmaceutical acceptable carrier. In some embodiments, the adjuvant is an amphipathic oligonucleotide conjugate as described herein. In some embodiments, the adjuvant is a STING agonist. In some embodiments, the adjuvant is CDG.

[0339] In some embodiments, any of the kits described herein further comprise a CAR-T cell comprising a CAR that binds to a CAR ligand present in the amphipathic ligand conjugate. Other embodiments of the present disclosure

[0340] Throughout this section, the term embodiment will be abbreviated as "E" followed by an ordinal number. For example, E1 is equivalent to embodiment 1.

[0341] E1. A method of expanding chimeric antigen receptor (CAR) T cells or increasing CAR T cell proliferation in a subject in vivo, comprising administering a composition in an amount sufficient to expand CAR T cells in the subject, the composition comprising an amphipathic ligand conjugate comprising a lipid, a CAR ligand, and optionally a linker.

[0342] E2. The method of embodiment 1, wherein the amphiphilic ligand conjugate binds to albumin under physiological conditions.

[0343] The method of embodiment 2, wherein proliferation of CAR(-) T cells is not increased in the subject.

[0344] E4. A method of reducing or decreasing tumor size or inhibiting tumor growth in a subject in need thereof, comprising administering to the subject a composition, wherein the subject is undergoing or has undergone chimeric antigen receptor (CAR) T-cell therapy, and the composition comprises an amphipathic ligand conjugate comprising a lipid, a CAR ligand, and optionally a linker.

[0345] E5. A method of inducing an anti-tumor response in a subject having cancer, comprising administering to the subject a composition, wherein the subject is undergoing or has undergone chimeric antigen receptor (CAR) T-cell therapy, and wherein the composition comprises an amphipathic ligand conjugate comprising a lipid, a CAR ligand, and optionally a linker.

[0346] E6. A method of stimulating an immune response in a subject against a target cell population or target tissue expressing an antigen, comprising administering to the subject chimeric antigen receptor (CAR) T cells targeted to the antigen and a composition, wherein the composition comprises an amphipathic ligand conjugate comprising a lipid, a CAR ligand and optionally a linker.

[0347] E7. The method of embodiment 6, wherein the immune response is a T cell-mediated immune response or an anti-tumor immune response.

[0348] E8. The method of embodiment 6 or 7, wherein the target cell population or target tissue is a tumor cell or tumor tissue.

[0349] E9. A method of treating a subject having a disease, disorder or condition associated with expression or increased expression of an antigen, comprising administering to the subject chimeric antigen receptor (CAR) T cells targeted to the antigen and a composition, wherein the composition comprises an amphipathic ligand conjugate comprising a lipid, a CAR ligand and optionally a linker.

[0350] The method of any one of embodiments 1 to 3, wherein the subject is administered the composition prior to receiving the CAR T cells.

[0351] The method of any one of embodiments 1 to 3, wherein the subject is administered the composition after receiving the CAR T cells.

[0352] E12. The method of any one of embodiments 1 to 3, wherein the composition and the CAR T cells are administered simultaneously.

[0353] E13. The method of any one of the above embodiments, wherein the CAR T cell comprises one costimulatory domain.

[0354] The method of embodiment 13, wherein one costimulatory domain is CD28 or 4-1BB.

[0355] E15. The method of any one of embodiments 1 to 14, wherein the amphiphilic ligand conjugate is transported to a lymph node.

[0356] E16. The method of any one of embodiments 1 to 14, wherein the amphiphilic ligand conjugate is transported to the inguinal and accessory lymph nodes.

[0357] E17. The method of any one of embodiments 1 to 16, wherein the amphipathic ligand conjugate, upon transport to the lymph node, is inserted into the membrane of an antigen-presenting cell.

[0358] E18. The method of embodiment 17, wherein the antigen-presenting cells are medullary macrophages, CD8+ dendritic cells and / or CDllb+ dendritic cells.

[0359] E19. The method according to any one of embodiments 1 to 18, wherein the CAR ligand is retained in the lymph node for at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days or at least 25 days.

[0360] E20. The method of any one of embodiments 1 to 19, wherein the composition further comprises an adjuvant.

[0361] E21. The method of embodiment 20, wherein the adjuvant is an amphipathic oligonucleotide conjugate comprising an immunostimulatory oligonucleotide conjugated to a lipid with or without a linker and optionally a polar compound.

[0362] E22. The method of embodiment 21, wherein the immunostimulatory oligonucleotide binds to a pattern recognition receptor.

[0363] E23. The method of embodiment 22, wherein the immunostimulatory oligonucleotide comprises CpG.

[0364] E24. The method of embodiment 21, wherein the immunostimulatory oligonucleotide is a ligand for a toll-like receptor.

[0365] E25. The method of any one of embodiments 1-20, wherein the linker is selected from the group consisting of a hydrophilic polymer, a series of hydrophilic amino acids, a polysaccharide, or a combination thereof.

[0366] The method of any one of embodiments 1 to 20, wherein the linker comprises "N" consecutive polyethylene glycol units, where N is between 25 and 50.

[0367] E27. The method of any one of embodiments 1 to 26, wherein the lipid is a diacyl lipid.

[0368] E28. The method of any one of embodiments 21 to 24, wherein the linker is an oligonucleotide linker.

[0369] E29. The method of embodiment 28, wherein the oligonucleotide linker comprises "N" consecutive guanines, where N is between 0 and 2.

[0370] E30. The method of any one of embodiments 21-24 and 28-29, wherein the lipid is a diacyl lipid.

[0371] The method of any one of embodiments 1 to 30, wherein the CAR ligand is a tumor-associated antigen and the CAR comprises a tumor-associated antigen binding domain.

[0372] The method of any one of embodiments 1 to 30, wherein the CAR ligand is a tag and the CAR comprises a tag-binding domain.

[0373] E33. The method of embodiment 32, wherein the tag is selected from the group consisting of fluorescein isothiocyanate (FITC), streptavidin, biotin, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horseradish peroxidase, palmitoylation, nitrosylation, alkaline phosphatase, glucose oxidase and maltose binding protein.

[0374] E34. The method of embodiment 32 or 33, further comprising administering a formulation of the tagged protein, wherein the tag-binding domain binds to the tagged protein.

[0375] E35. The method of embodiment 34, wherein the protein of the tagged protein is an antibody or an antigen-binding fragment thereof.

[0376] E36. The method of embodiment 34 or 35, wherein the tag binding domain is an antibody or an antigen-binding fragment thereof.

[0377] E37. The method of any one of embodiments 34 to 36, wherein a formulation of the tagged protein is administered to the subject prior to administration of the composition comprising the CAR T cells and the amphipathic ligand conjugate.

[0378] E38. The method of any one of embodiments 34 to 36, wherein a formulation of the tagged protein is administered to the subject in conjunction with administration of a composition comprising CAR T cells and an amphipathic ligand conjugate.

[0379] E39. The method of any one of embodiments 34 to 36, wherein a formulation of the tagged protein is administered to the subject after administration of the composition comprising the CAR T cells and the amphipathic ligand conjugate.

[0380] The method of any one of embodiments 37 to 39, wherein the CAR T cells are administered prior to administration of the composition comprising the amphipathic ligand conjugate.

[0381] The method of any one of embodiments 37 to 39, wherein the CAR T cells are administered after administration of a composition comprising an amphipathic ligand conjugate.

[0382] The method of any one of embodiments 37 to 39, wherein the CAR T cells are administered in conjunction with administration of a composition comprising an amphipathic ligand conjugate.

[0383] E43. The method of any one of embodiments 1-3 and 6-42, wherein the subject has cancer.

[0384] E44. The method of any one of embodiments 1 to 43, wherein the subject is a human.

[0385] E45. A composition comprising an amphipathic ligand conjugate and a pharma- ceutically acceptable carrier, wherein the amphipathic ligand conjugate comprises a chimeric antigen receptor (CAR) ligand, a lipid and optionally a linker.

[0386] E46. The composition of embodiment 45, wherein the linker is selected from the group consisting of a hydrophilic polymer, a series of hydrophilic amino acids, a polysaccharide, or a combination thereof.

[0387] E47. The composition of embodiment 45, wherein the linker comprises "N" consecutive polyethylene glycol units, where N is between 25 and 50.

[0388] E48. The composition of any one of embodiments 45 to 47, wherein the lipid is a diacyl lipid.

[0389] E49. The composition of any one of embodiments 45 to 48, wherein the CAR ligand is a tag.

[0390] E50. The composition of embodiment 49, wherein the tag is selected from the group consisting of fluorescein isothiocyanate (FITC), streptavidin, biotin, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horseradish peroxidase, palmitoylation, nitrosylation, alkaline phosphatase, glucose oxidase and maltose binding protein.

[0391] E51. An immunogenic composition comprising a composition according to any one of embodiments 45 to 50 and an adjuvant.

[0392] E52. The immunogenic composition of embodiment 51, wherein the adjuvant is an amphipathic oligonucleotide conjugate comprising an immunostimulatory oligonucleotide conjugated to a lipid with or without a linker and optionally a polar compound.

[0393] E53. The immunogenic composition of embodiment 52, wherein the immunostimulatory oligonucleotide binds to a pattern recognition receptor.

[0394] E54. The immunogenic composition of embodiment 53, wherein the immunostimulatory oligonucleotide comprises CpG.

[0395] E55. The immunogenic composition of embodiment 52, wherein the immunostimulatory oligonucleotide is a ligand for a toll-like receptor.

[0396] E56. The immunogenic composition of any one of embodiments 52 to 55, wherein the lipid is a diacyl lipid.

[0397] E57. The immunogenic composition of any one of embodiments 52 to 56, wherein the linker is an oligonucleotide linker.

[0398] E58. The immunogenic composition of embodiment 57, wherein the oligonucleotide linker comprises "N" consecutive guanines, where N is between 0 and 2.

[0399] E59. A kit comprising a container containing a composition comprising an amphipathic ligand conjugate, optionally a pharma- ceutically acceptable carrier, and a package insert containing instructions for administration of the composition for treating or delaying the progression of cancer in an individual undergoing CAR T cell therapy, wherein the amphipathic ligand conjugate comprises a lipid, a CAR ligand and optionally a linker.

[0400] E60. The kit of embodiment 59, further comprising an adjuvant and instructions for administration of the adjuvant to treat or delay the progression of cancer in an individual receiving chimeric antigen receptor (CAR) T cell therapy.

[0401] E61. The kit of embodiment 60, wherein the adjuvant is an amphipathic oligonucleotide conjugate comprising an immunostimulatory oligonucleotide conjugated to a lipid with or without a linker and optionally a polar compound.

[0402] E62. A kit comprising a medicament comprising a composition comprising an amphipathic ligand conjugate, optionally a pharma- ceutically acceptable carrier, for treating or delaying the progression of cancer in an individual undergoing chimeric antigen receptor (CAR) T-cell therapy, and a package insert comprising instructions for administration of the medicament alone or in combination with a composition comprising an adjuvant and optionally a pharma- ceutically acceptable carrier, wherein the amphipathic ligand conjugate comprises a lipid, a CAR ligand and optionally a linker.

[0403] E63. A kit comprising a container containing a composition comprising an amphipathic ligand conjugate, optionally a pharma- ceutically acceptable carrier, and a package insert containing instructions for administration of the composition vaccine for expanding CAR T cells in an individual undergoing CAR T cell therapy, wherein the amphipathic ligand conjugate comprises a lipid, a CAR ligand and optionally a linker.

[0404] E64. The kit of embodiment 63, further comprising an adjuvant and instructions for administration of the adjuvant, for expanding chimeric antigen receptor (CAR) T cell therapy in an individual receiving CAR T cell therapy.

[0405] E65. The kit of embodiment 64, wherein the adjuvant is an amphipathic oligonucleotide conjugate comprising an immunostimulatory oligonucleotide conjugated to a lipid with or without a linker and optionally a polar compound.

[0406] E66. A kit comprising a medicament comprising a composition comprising an amphipathic ligand conjugate, optionally a pharma- ceutical acceptable carrier, for expanding CAR T cells in an individual undergoing CAR T cell therapy, and a package insert comprising instructions for administration of the medicament alone or in combination with a composition comprising an adjuvant and optionally a pharma-ceutical acceptable carrier, wherein the amphipathic ligand conjugate comprises a lipid, a CAR ligand and optionally a linker.

[0407] E67. A kit comprising a container containing a composition comprising an amphipathic ligand conjugate, optionally a pharma- ceutically acceptable carrier, and a package insert containing instructions for administration of the composition for increasing proliferation of CAR T cells in an individual undergoing CAR T cell therapy, wherein the amphipathic ligand conjugate comprises a lipid, a CAR ligand and optionally a linker.

[0408] E68. The kit of embodiment 67, further comprising an adjuvant and instructions for administration of the adjuvant to increase proliferation of chimeric antigen receptor (CAR) T cells in an individual receiving CAR T cell therapy.

[0409] E69. The kit of embodiment 66 or 68, wherein the adjuvant is an amphipathic oligonucleotide conjugate comprising an immunostimulatory oligonucleotide conjugated to a lipid with or without a linker and optionally a polar compound.

[0410] E70. A kit comprising a medicament comprising a composition comprising an amphipathic ligand conjugate, optionally a pharma- ceutical acceptable carrier, for increasing the proliferation of CAR T cells in an individual undergoing CAR T cell therapy, and a package insert comprising instructions for administration of the medicament alone or in combination with a composition comprising an adjuvant and optionally a pharma-ceutical acceptable carrier, wherein the amphipathic ligand conjugate comprises a lipid, a CAR ligand and optionally a linker.

[0411] E71. Use of a composition according to any one of embodiments 45 to 50, an immunogenic composition according to any one of embodiments 51 to 58, or a kit according to any one of embodiments 59 to 70 for use in expanding CAR T cells in vivo in a subject.

[0412] E72. Use of a composition according to any one of embodiments 45 to 50, an immunogenic composition according to any one of embodiments 51 to 58, or a kit according to any one of embodiments 59 to 70 for use in increasing the proliferation of CAR T cells in vivo in a subject.

[0413] E73. Use of a composition according to any one of embodiments 45 to 50, an immunogenic composition according to any one of embodiments 51 to 58, or a kit according to any one of embodiments 59 to 70 for use in treating or delaying the progression of cancer in an individual.

[0414] E74. Use of a composition according to any one of embodiments 45 to 50 in the manufacture of a medicament for treating or delaying the progression of cancer in an individual, wherein the medicament comprises the composition and optionally a pharma- ceutically acceptable carrier.

[0415] E75. A composition comprising an amphipathic ligand conjugate, the amphipathic ligand conjugate comprising a lipid conjugated to fluorescein isothiocyanate (FITC) via a polyethylene glycol moiety.

[0416] E76. The composition of embodiment 75, wherein the lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and the polyethylene glycol moiety is PEG-2000.

[0417] E77. An immunogenic composition comprising an amphipathic ligand conjugate and an adjuvant, wherein the amphipathic ligand conjugate comprises a lipid, a CAR ligand and optionally a linker, and the adjuvant is an amphipathic oligonucleotide conjugate comprising a linker and an immunostimulatory oligonucleotide conjugated to a lipid with or without an optionally polar compound.

[0418] E78. An immunogenic composition comprising an amphipathic ligand conjugate and an adjuvant, wherein the amphipathic ligand conjugate comprises a lipid, a CAR ligand and optionally a linker, wherein the CAR ligand is a tag, and the adjuvant is an amphipathic oligonucleotide conjugate comprising a linker and an immunostimulatory oligonucleotide conjugated to a lipid with or without an optionally polar compound.

[0419] E79. The method of any one of embodiments 4 to 44, wherein the amphiphilic ligand conjugate binds to albumin under physiological conditions.

[0420] E80. The method of any one of embodiments 21-24 and 27-44, wherein the amphipathic oligonucleotide conjugate binds to albumin under physiological conditions.

[0421] E81. The method of any one of embodiments 1 to 44, comprising administering the composition comprising the amphiphilic ligand conjugate parenterally in a non-tumor draining lymph node, parenterally in a tumor draining lymph node, or intratumorally.

[0422] E82. The method of embodiment 6, wherein the target cell population or target tissue is a population of cells or tissue infected with a virus.

[0423] E83. The method of embodiment 82, wherein the virus is human immunodeficiency virus (HIV).

[0424] E84. The method of embodiment 82 or 83, wherein the immune response is a T cell-mediated immune response.

[0425] E85. The method of embodiment 9, wherein the antigen is a viral antigen or a cancer antigen.

[0426] E86. A kit comprising a container containing a composition comprising an amphipathic ligand conjugate, optionally a pharma- ceutically acceptable carrier, and a package insert containing instructions for administration of the composition for treating or delaying the progression of a viral infection in an individual undergoing CAR T cell therapy, wherein the amphipathic ligand comprises a lipid, a CAR ligand and optionally a linker.

[0427] The kit of embodiment 86, further comprising an adjuvant and instructions for administration of the adjuvant to treat or delay the progression of a viral infection in an individual receiving E87.CAR T cell therapy.

[0428] E88. The kit of embodiment 87, wherein the adjuvant is an amphipathic oligonucleotide conjugate comprising an immunostimulatory oligonucleotide conjugated to a lipid with or without a linker and optionally a polar compound.

[0429] E89. The kit of any one of embodiments 59-70 and 86-88, wherein the amphiphilic ligand conjugate comprises a linker selected from the group consisting of a hydrophilic polymer, a series of hydrophilic amino acids, a polysaccharide, or a combination thereof.

[0430] E90. The kit of any one of embodiments 59-70 and 86-88, wherein the amphiphilic ligand conjugate comprises a linker comprising "N" consecutive polyethylene glycol units, wherein N is between 25 and 50.

[0431] E91. The kit of any one of embodiments 59-70 and 86-90, wherein the lipid is a diacyl lipid.

[0432] E92. The kit of any one of embodiments 61, 65, 69 or 88, wherein the amphipathic oligonucleotide conjugate comprises an oligonucleotide linker.

[0433] E93. The kit of embodiment 92, wherein the oligonucleotide linker comprises "N" consecutive guanines, where N is between 0 and 2.

[0434] E94. The kit of any one of embodiments 59 to 70 and 89 to 93, wherein the CAR ligand is a tumor-associated antigen and the CAR comprises a tumor-associated antigen binding domain.

[0435] E95. The kit of any one of embodiments 59 to 70 and 89 to 93, wherein the CAR ligand is a tag and the CAR comprises a tag-binding domain.

[0436] E96. The kit of embodiment 95, wherein the tag is selected from the group consisting of fluorescein isothiocyanate (FITC), streptavidin, biotin, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horseradish peroxidase, palmitoylation, nitrosylation, alkaline phosphatase, glucose oxidase and maltose binding protein.

[0437] E97. The kit of embodiment 95 or 96, wherein the kit further comprises a formulation of the tagged protein and instructions for administering the formulation of the tagged protein, and wherein the tag-binding domain binds to the tagged protein.

[0438] E98. The kit of embodiment 97, wherein the protein of the tagged protein is an antibody or an antigen-binding fragment thereof.

[0439] E99. The immunogenic composition of embodiment 77 or 78, wherein the amphiphilic ligand conjugate comprises a linker selected from the group consisting of a hydrophilic polymer, a series of hydrophilic amino acids, a polysaccharide, or a combination thereof.

[0440] E100. The immunogenic composition of embodiment 77 or 78, wherein the amphiphilic ligand conjugate comprises a linker comprising "N" consecutive polyethylene glycol units, wherein N is between 25 and 50.

[0441] E101. The immunogenic composition of embodiment 77, 78, 99 or 100, wherein the lipid is a diacyl lipid.

[0442] E102. The immunogenic composition of embodiment 77 or 99 to 101, wherein the CAR ligand is a tumor-associated antigen or a viral antigen.

[0443] E103. The immunogenic composition of embodiments 77, 78 or 99 to 102, wherein the amphipathic oligonucleotide conjugate comprises an oligonucleotide linker.

[0444] E104. The immunogenic composition of embodiment 103, wherein the oligonucleotide linker comprises "N" consecutive guanines, where N is between 0 and 2.

[0445] E105. The immunogenic composition of any one of embodiments 78, 99 to 101 and 103 to 104, wherein the tag is selected from the group consisting of fluorescein isothiocyanate (FITC), streptavidin, biotin, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horseradish peroxidase, palmitoylation, nitrosylation, alkaline phosphatase, glucose oxidase and maltose binding protein.

[0446] The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. EXAMPLES

[0447] Below are examples of specific embodiments for carrying out the methods described herein.These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should naturally be allowed for. Example 1 Generation of DSPE-PEG-FITC and DSPE-PEG-peptide / protein ligands

[0448] Due to poor persistence of CAR-T cells in some patient populations and the inability of CAR-T therapy to induce optimal responses in solid tumors, we hypothesized that stronger CAR-T cell expansion and enhanced functionality could be achieved by stimulation via the CAR itself. To achieve this, albumin-conjugated phospholipid polymers were utilized as previously described (Liu, H., Moynihan, KD, Zheng, Y., Szeto, GL, Li, AV, Huang, B., Irvine, DJ (2014). Structure-based programming of lymph-node targeting in molecular vaccines. Nature, 507(7493), 519-522.). Specifically, small molecule, peptide or protein ligands against CAR are attached to the polymer-lipid tail as shown in Figure 1A to form an amphiphile vaccine.

[0449] First, a retargetable CAR was used, whose chimeric antigen receptor recognizes the small molecule fluorescein (FITC), which is targeted to tumors via FITC-conjugated antitumor antibodies (Ma, JS, Kim, JY, Kazane, SA, Choi, SH, Yun, HY, Kim, MS, Cao, Y. (2016). Versatile strategy for controlling the specificity and activity of engineered T cells. Proc Natl Acad Sci USA, 113(4), E450-458). The cognate ligand is FITC-poly(ethylene glycol (PEG)-DSPE ("DSPE-PEG-FITC"). Figure 1B provides a schematic showing the stimulation of CAR T cells by antigen-presenting cells coated with the corresponding amphiphile vaccine.

[0450] To generate the DSPE-PEG-FITC vaccine, PE (phosphoethanolamine) lipid (e.g., DSPE) was dissolved in 500 μL of CHCl3 and 500 μL of DMF, and 3 equivalents of triethylamine and 1.2 equivalents of fluorescein-PEG were added. 2000 -NHS (Creative PEG Works Inc.) was added and the reaction mixture was stirred overnight. The amphiphilic fluorescein PEG amphiphile was purified by reversed-phase HPLC using a C4 column (BioBasic-4, 200 mm × 4.6 mm, Thermo Scientific) with 100 mM triethylamine-acetate buffer (TEAA, pH 7.5)-methanol (0–30 min, 10–100%) as eluent. The final product was dissolved in HO and analyzed by UV-Vis spectroscopy (fluorescein, extinction coefficient is 70,000 M at 490 nm and pH 9). -1 cm -1 ) and characterized by MALDI-TOF mass spectrometry. To generate DSPE-PEG-peptide / protein ligands, N-terminal cysteine ​​modified peptide or protein ligands were dissolved in DMF and 2 equivalents of maleimide-PEG were added.2000 -DSPE (Laysan Bio, Inc.) and the mixture was stirred at 25°C for 24 hours. Bioconjugation was determined to be essentially complete by HPLC analysis. The peptide amphiphiles were characterized by MALDI-TOF mass spectrometry. The peptide conjugates were then diluted in 10x ddH2O, lyophilized to a powder, redissolved in H2O, and stored at -80°C. Example 2 In vitro activation of anti-FITC CAR-T cells with DSPE-PEG-FITC coated cells

[0451] To determine the effect of amphiphilic ligand conjugates on chimeric antigen receptor (CAR) T cells, in vitro stimulation of CAR-T cells was evaluated after co-culture with antigen-presenting cells (APCs) that provided the amphiphilic ligand conjugates. Specifically, model CAR-T cells expressing anti-FITC CAR were generated by retroviral transduction into primary mouse T cells of a DNA vector containing an anti-FITC (fluorescein) scFV (4m5.3) coding region fused in frame to a Myc epitope tag coding region and to a CAR coding region containing a CD8 transmembrane domain, a CD28 signaling domain, and a CD3z signaling domain. The domain structure and orientation of the Myc-tagged anti-FITC CAR are shown in Figure 2A. Surface expression of the Myc-tagged anti-FITC CAR in primary mouse T cells was quantified by incubating the transduced cells with a fluorescently labeled anti-Myc antibody and quantifying fluorescent cells by flow cytometry (Figure 2B).

[0452] Next, model target cells K562 cells were tested for efficient membrane insertion of amphiphilic ligand conjugates containing a lipophilic moiety (i.e., DSPE) covalently linked to FITC via a PEG-2000 linker. At low doses (i.e., 25 nM) of DSPE-PEG-FITC, increasing serum concentrations almost completely abolished surface insertion. However, at high doses (500 nM), DSPE-PEG-FITC retained high levels of cell surface decoration (data not shown).

[0453] To mimic antigen-presenting cells in lymph nodes, dendritic cells (DC2.4) were decorated with increasing concentrations of DSPE-PEG-FITC and then co-cultured with anti-FITC CAR T cells for 0, 48 and 96 hours. The ability of FITC-decorated DC2.4 cells to stimulate anti-FITC CAR T cells was monitored by IFNγ secretion by CAR-T cells. Although most FITC molecules appeared to be internalized within 24 hours, strong induction of IFNγ by CAR-T cells was observed at 0 and 48 hours, which then faded at 96 hours (data not shown), and dose-dependent activation was observed (Figure 2C). Furthermore, when FITC-decorated DC2.4 cells were co-cultured with FITC-CAR-T cells at an effector-to-target (E:T) ratio of 10:1 for 6 h, DC2.4 cells were killed when FITC-CAR-T cells were administered DSPE-PEG-FITC (Figure 2D). Furthermore, as previously reported (Ma et al., 2016), co-culture of FITC-CAR T cells with CD19+ target cells in the presence of FITC-conjugated anti-CD19 antibody, but not control antibody, resulted in robust CAR-T activation as determined by IFNγ secretion (data not shown). Overall, these results indicate that amphipathic ligand conjugates are capable of activating CAR-T cells. Example 3 DSPE-PEG-FITC transport to lymph nodes (LN), retention and uptake by APCs

[0454] Based on the results of Example 2, we next determined whether the amphiphilic ligand conjugate DSPE-PEG-FITC could coat antigen-presenting cells in lymph nodes (LNs) to prime FITC-CAR-T cells in vivo. To evaluate DSPE-PEG-FITC transport to lymph nodes and retention and uptake by APCs, C57BL / 6 mice were given various doses of DSPE-PEG-FITC. Specifically, inguinal LNs, auxiliary LNs and iliac (lilac) LNs were harvested 24 hours after administration of 2 nmol, 5 nmol or 10 nmol doses of DSPE-PEG-FITC into the tail vein of mice. Free FITC was used as a control. Mice were sacrificed and LNs were removed at different time points for IVIS imaging (excitation 465 nm, emission 520 nm) to monitor LN retention of FITC signal. The most efficient drainage was into the inguinal LNs, followed by drainage into auxiliary LNs (data not shown). At high doses, DSPE-PEG-FITC was also observed to flow into the iliac LNs.

[0455] FITC signal was nearly lost after 4 days at the lowest dose (2 nmol), whereas signal was retained for more than 21 days at the higher dose (10 nmol) of DSPE-PEG-FITC (Figure 3A). Free FITC signal was lost at 24 hours (Figure 3A). Flow cytometric analysis of LN cells revealed substantial uptake of DSPE-PEG-FITC in CD8+ and CD11b+ dendritic cells (DCs) and macrophages, with minimal accumulation in T or B cells (Figures 3B and 3C). Confocal imaging of LNs showed that DSPE-PEG-ITC initially accumulated in interfollicular regions after 1 day, but distributed over time onto CD11c+DCs in the T cell region, and sorted FITC+CD11c+ cells from these LNs stained brightly with anti-FITC antibodies (data not shown).

[0456] Overall, these results indicate that the amphipathic ligand conjugates are expressed on antigen-presenting cells in lymph nodes. Example 4 DSPE-PEG-FITC retained in LNs robustly stimulates CAR T cell proliferation

[0457] To assess whether DSPE-PEG-FITC accumulating on lymph node antigen presenting cells results in priming of CAR T cells and how long this stimulatory effect lasts, mice were administered PBS, c-di-GMP (25ug), DSPE-PEG-FITC (10nmol), or DSPE-PEG-FITC (10nmol) + c-di-GMP (25ug) in wild type C57Bl / 6 mice on day 1. After various time points, 2x10 6 of CTV-labeled CAR-T cells were transferred into each mouse via tail vein injection. CAR-T cells were titrated to a mixture of CAR+ and CAR- cells at a 1:1 ratio. After an additional 48 hours, mice were sacrificed and LNs were removed for FACS analysis. As demonstrated in the representative results in Figure 7B, FITC-CAR-T was efficiently stimulated in lymph nodes 48 hours after adoptive transfer up to 7 days after vaccination, and co-administration of cyclic-di-GMP (CDG, a STING agonist), a strong T cell promoting adjuvant, significantly prolonged DSPE-PEG-FITC stimulation up to 14 days (Figure 7B). Minimal proliferation of CAR-T cells was observed in control mice receiving PBS or adjuvant alone. These results demonstrate the ability of amphiphilic ligand conjugates to induce CAR-T cell proliferation in vivo.

[0458] Furthermore, CDG co-administration significantly increased the duration and reachability of DSPE-PEG-FITC on multiple APC cell surfaces, including macrophages and CD11c+CD11b+DCs (Figure 5). Furthermore, CDG co-administration increased the expression levels of several costimulatory molecules, namely CD80, CD86, 41BBL, ICOSL and OX40L, compared to DSPE-PEG-FITC alone (Figure 6). Expression was measured 24 hours and 3 days after vaccination. Example 5 Effect of DSPE-PEG-FITC on long-term CAR-T cell expansion

[0459] To follow the effect of DSPE-PEG-FITC on the long-term in vivo expansion of CAR-T cells, we utilized a CD45.1 / CD45.2 congenic transplantation model. Specifically, lymphodepleted CD45.2 recipient mice were injected with various doses of CD45.1 donor FITC CAR-T cells (0.25 × 10 6 ;0.05×10 6 ;0.01×10 6 ) was given to mice. 24 hours later, mice were given PBS or vaccination with 10 nmol DSPE-PEG-FITC with or without 25ug CDG. Figure 7 provides a timeline of the experiment. The percentage of circulating CAR-T cells was determined by FACS analysis of peripheral blood collected 7 and 14 days after vaccination. CAR T cells were defined as the CD3+CD8+ / Myc tag+ population.

[0460] Dramatic longitudinal CD45.1 CAR-T expansion was observed after vaccination with DSPE-PEG-FITC alone or in combination with CDG. Specifically, 7 days after the first vaccination, 0.25 × 10 6 The group comprised >70% of peripheral CD8+ T cells and had a 0.05 × 10 6 The group accounted for >50%, which is 10 × 10 6 The second boost increased the number of CAR-T cells in mice to 0.01 × 10, which was significantly greater than that in mice transferred with ex vivo expanded CAR-T cells (Figure 7).6 The group also reached 50% by day 14.

[0461] Furthermore, the efficacy of DSPE-PEG-FITC was evaluated in lymphreplete mice. Lymphodepletion regimens enhance the efficacy of adoptive cell therapy but are associated with severe toxicity. Given the strong CAR-T boost by DSPE-PEG-FITC in lymphodepleted settings, we next examined whether DSPE-PEF-FITC could expand CAR-T cells to significant levels in lymphoreplete mice. Specifically, as shown in Figure 8, multiple doses of CD45.1 FITC CAR-T cells were transferred into lymphoreplete CD45.2 recipient mice, followed by the same vaccination scheme and subsequent analysis as above. The results are also shown in Figure 8, which shows that 10x10 6 Control mice that received only CAR-T had a circulating CD8+ T cell population of approximately 5%, compared with 0.25 × 10 6 Mice receiving 100 CAR-Ts plus DSPE-PEG-FITC reached approximately 10% by day 14, and approximately 20% reached 1 × 10 6 The results show that the FITC-CART-T group achieved a 100% T cell response in the 100% T cell group. One concern was that repeated vaccination could induce antibodies against FITC when conjugated to DSPE-PEG, thus blocking its stimulation to CAR in lymph nodes. However, no antibody response was observed when FITC was conjugated to DSPE-PEG or the carrier protein OVA (Figure 9), because DSPE-PEG did not provide a source of T cell help.

[0462] Overall, these results demonstrated that the DSPE-PEG-FITC vaccine in combination with an adjuvant (i.e., CDG) acted as a potent CAR-T booster vaccine in vivo. Example 6 Efficacy of amphiphile vaccines bearing tumor-specific antigens

[0463] We next evaluated whether the same booster vaccine concept described in the above examples could be used for a bona fide tumor antigen-specific CAR. Specifically, we utilized the murine EGFRvIII-specific CAR 139scFv, which recognizes a short linear epitope derived from EGFRvIII (Sampson, et al. (2014). EGFRvIII mCAR-modified T-cell therapy cures mice with established intracerebral glioma and generates host immunity against tumor-antigen loss. Clin Cancer Res, 20(4), 972-984). Mouse T cells were transduced with this CAR and amphiphile-EGFRvIII peptide vaccine molecules were synthesized by the following method: c-terminal cysteine ​​modified EGFRvIII peptide dissolved in dimethylformamide (DMSO) was mixed with 2.5 equivalents of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG2k) and 1 equivalent of tris(2-carboxyethyl)phosphine hydrochloride and a catalytic amount of triethylamine. The mixture was stirred at room temperature for 24 hours and subsequently purified by HPLC and dissolved in H2O. A schematic diagram of the DSPE-PEG-EGFRvIII amphiphile vaccine is shown in Figure 10A, and Figure 10B shows the expression of anti-EGFRvIII CAR on T cells.

[0464] Similar to DSPE-PEG-FITC, DSPE-PEG-EGFRvIII inserted into cell membranes in vitro, and DSPE-PEG-EGFRvIII-coated cells stimulated EGFRvIII-CAR-T cells (data not shown). 6Immunization of mice with 10ug DSPE-PEG-EGFRvIII and adjuvant (25ug cyclic-di-GMP) 24 hours after intravenous injection of cell trace violet (CTV)-labeled EGFRvIII-CAR-T cells induced extensive CAR-T cell proliferation in vivo in the draining inguinal lymph nodes 48 hours later (Figure 10C). To test the therapeutic impact of vaccine boosting, mouse CT-2A glioma cells were transduced with EGFRvIII and co-cultured with EGFRvIII-CAR-T cells. CAR-T cells secreted IFNγ in the presence of EGFRvIII-expressing CT-2A glioma cells (Figure 11A). Furthermore, in vitro co-culture of CT-2A glioma cells expressing wild-type EGFR or EGFRvIII with EGFRvIII-CAR-T at a ratio of 1:10 for 6 hours resulted in efficient killing of EGFRvIII-expressing CT-2A glioma cells by EGFRvIII-CAR-T cells, but not wild-type EGFR-expressing CT-2A glioma cells (Figure 11B).

[0465] To further explore the efficacy of the DSPE-PEG-EGFRvIII amphiphile vaccine, an in vivo model was utilized. Specifically, wild-type CD45.2 C57Bl / 6 mice were inoculated with 4 × 10 6 On day 7, CT-2A-mEGFRvIII tumor-bearing mice were sublethally irradiated and subsequently injected with different doses of EGFRvIII CAR-T cells generated from CD45.1 mice, followed by either 10ug DSPE-PEG-EGFRvIII plus 25ug CDG or no 10×10 6 In the group that received 100 CAR-T cells, circulating CAR-T cells accounted for approximately 40% of peripheral blood CD8+ T cells (Figure 12). Mice that received lower cell numbers had minimal circulating CAR-T cells, but dramatic EGFRvIII CAR-T expansion was achieved in the group that received DSPE-PEG-EGFRvIII plus CDG (Figure 12).

[0466] To evaluate the impact of amphiphilic ligand conjugates on EGFRvIII CAR T function, intracellular cytokine staining (ICS) was performed by using peripheral blood collected 7 days after vaccination. Peripheral blood mononuclear cells (PBMCs) were mixed with EGFRvIII-expressing CT-2A cells at a 1:1 ratio in a 96-well plate for 6 hours in the presence of 1× golgiplug. The cells were then surface stained, fixed, permeabilized, and then further stained with anti-IFNγ and anti-TNFα antibodies to evaluate the cytokine production of vaccine-boosted or non-boosted EGFRvIII CAR T cells in response to target cells. DSPE-PEG-EGFRvIII-boosted EGFRvIII CAR-T cells had significantly enhanced functionality, and the majority of circulating CAR-Ts responded to target tumor cells (Figure 13). Furthermore, significantly increased CAR-T infiltration into tumors in the DSPE-PEG-EGFRvIII+CDG boost group was observed 7 days after vaccination, as determined by FACS analysis of the number of CAR-T cells per mg of tumor (Figure 14). Furthermore, tumor-infiltrating CAR-T cells showed enhanced reactivity to tumor cells 7 days after vaccination. Specifically, Figure 15 shows that the level of cytokine secretion by tumor-infiltrating CAR-T cells was enhanced in the presence of DSPE-PEG-EGFRvIII+CDG compared to PBS, while Figure 16 shows that the level of granzyme B, an indicator of cytotoxicity, was increased in tumor-infiltrating CAR-T cells, and Ki67, an indicator of proliferation, was also increased. Interestingly, this enhanced reactivity occurred despite the surface expression of PD1 and TIM3 (Figure 17). Animals that received both CAR-T and DSPE-PEG-EGFRvIII+CDG had significantly delayed tumor growth (Figure 18A) and prolonged survival (Figure 18B). Notably, similar to mice vaccinated with DSPE-PEG-FITC, no antibody responses were elicited against EGFRvIII after four rounds of weekly vaccinations and only minor weight loss was observed after each vaccination, indicating that toxicity was at a manageable level (data not shown). Example 7 Design and efficacy of bispecific CAR T cells vaccinated with DSPE-PEG-FITC

[0467] While the use of surrogate peptide ligands for CAR T cells is effective, some CARs recognize three-dimensional structural epitopes (De Oliveira, et al. (2013). A CD19 / Fc fusion protein for detection of anti-CD19 chimeric antigen receptors. J Transl Med, 11, 23. doi:10.1186 / 1479-5876-11-23), for which it may be difficult or impossible to identify a simple surrogate ligand. To eliminate such limitations and provide a means to boost any CAR regardless of the nature of its binding domain or its specificity, a tandem scFv-based bispecific CAR was designed. Specifically, the anti-FITC scFV 4m5.3 was appended to the N-terminal extracellular domain of a tumor-targeting CAR via a (G4S)4 peptide linker immediately following the N-terminal signal peptide (Figure 19). To evaluate the feasibility of this approach, we utilized a bispecific mouse CAR that targets both FITC and the melanoma-associated antigen TRP1, which is well expressed in primary mouse T cells. Figure 20 shows the expression of the bispecific CAR on T cells. To confirm the response specificity of this bispecific CAR, FITC / TRP1-CAR T cells were co-cultured in vitro with either DSPE-PEG-FITC-coated target cells or TRP1-expressing B16F10 cells at an effector:target ratio of 10:1 for 6 hours, and FITC / TRP1-CAR T responded specifically and potently to both antigens, as indicated by IFNγ secretion (Figure 21). Furthermore, FITC / TRP1-CAR T cells were equivalent to monospecific TRP1-CAR T cells in upregulating TRP1 as determined by co-culture of cells at an effector-to-target (E:T) ratio of 10:1 for 6 hours. +Killed target cells (Figure 22). In vivo, DSPE-PEG-FITC vaccination robustly stimulated FITC / TRP1 bispecific CAR-T proliferation as determined by cell trace violet transport 48 hours post-vaccination (Figure 23).

[0468] To evaluate the therapeutic potential of bispecific CAR-T in combination with DSPE-PEG-FITC plus CDG, wild-type C57Bl / 6 mice were injected with 5 × 10 5 1000×1000 B16F10 tumor cells were implanted in the mice. Five days later, tumor-bearing animals were given a lymphodepletion treatment with 500 cGy of radiation, followed the next day by 10×10 6 FITC / TRP1 CAR-T cells were given intravenous injections of 10 nmol of DSPE-PEG-FITC+25 ug of CDG. While CAR-T cells alone had little effect on tumor growth compared to control T cells, mice given both CAR-T and vaccine (10 nmol of DSPE-PEG-FITC+25 ug of CDG) showed dramatically delayed tumor growth (Figure 24A) and significantly prolonged survival (Figure 24B). This was consistent with increased circulating CAR-T levels (Figure 25) and enhanced tumor infiltration (Figure 26). Weight loss was also under control in the vaccinated animal group (data not shown).

[0469] Motivated by the robust CAR-T expansion and functional enhancement by DSPE-PEG-FITC+CDG boost, we evaluated the therapeutic efficacy of CAR-T plus DSPE-PEG-FITC+CDG in tumor-bearing mice without lymphodepleted preconditioning. To facilitate in vivo tracking, CD45.1 FITC / TRP1 CAR-T cells were transferred into B16F10 melanoma-bearing CD45.2 recipient mice following a similar vaccination scheme. Mice receiving CAR-T alone had tumor growth almost indistinguishable from mice receiving control T cells, whereas combined treatment with both CAR-T and DSPE-PEG-FITC+CDG vaccination significantly delayed tumor growth and increased animal survival (Figures 27A and 27B), with minimal impact on animal weight (data not shown).

[0470] DSPE-PEG-FITC is preferentially transported to lymph nodes, where it accumulates and is taken up by APCs present in lymph nodes, but a small percentage of amphiphiles may leak into peripheral blood and insert into bystander cells, making them de novo CAR-T targets. To analyze such unintended toxicity caused by amphiphiles that have avoided lymphatic drainage, DSPE-PEG-FITC was intravenously injected into NSG mice with severely defective lymphatics to stimulate FITC CAR-T cells. Nevertheless, stimulation of FITC CAR-T proliferation was negligible (data not shown).

[0471] Overall, these results demonstrated that CAR-T cell therapy in conjunction with the use of an amphiphile vaccine is effective in solid tumors. Equivalent

[0472] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. 1. A composition for use in activating, expanding or increasing the proliferation of CAR-T cells in a subject, comprising an amphipathic ligand conjugate comprising a chimeric antigen receptor (CAR) ligand operably linked to a lipid, said lipid being capable of binding to albumin under physiological conditions and inserting itself into a cell membrane under physiological conditions, said lipid being 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and said CAR ligand binding to and activating a CAR on said CAR-T cell.

2. 1. A composition for use in reducing or decreasing tumor size or inhibiting tumor growth in a subject or inducing an anti-tumor response in a subject having cancer, the composition comprising an amphipathic ligand conjugate comprising a chimeric antigen receptor (CAR) ligand operably linked to a lipid, said subject is undergoing or has undergone CAR-T cell therapy, said lipid binds to albumin under physiological conditions and is capable of inserting itself into a cell membrane under physiological conditions, said lipid being 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and said CAR ligand binds to and activates a CAR on said CAR-T cell.

3. 1. A composition for use in a method of stimulating an immune response against a target cell population or target tissue expressing an antigen in a subject, the composition comprising an amphipathic ligand conjugate comprising a chimeric antigen receptor (CAR) ligand operably linked to a lipid, the subject being administered CAR-T cells targeted to the antigen and the composition, the lipid being capable of binding to albumin under physiological conditions and inserting itself into a cell membrane under physiological conditions, the lipid being 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), the CAR ligand binding to and activating a CAR on the CAR-T cells, and optionally the target cell population or target tissue is a population of cells or tissue infected with a virus.

4. 1. A composition for use in a method of treating a subject having a disease, disorder or condition associated with expression or increased expression of an antigen, comprising an amphipathic ligand conjugate comprising a chimeric antigen receptor (CAR) ligand operably linked to a lipid, wherein the subject is administered CAR-T cells targeted to the antigen and the composition, wherein the lipid binds to albumin under physiological conditions and is capable of inserting itself into a cell membrane under physiological conditions, wherein the lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), wherein the CAR ligand binds to and activates a CAR on the CAR-T cells, and optionally the antigen is a viral antigen or a cancer antigen.

5. The composition for use according to any one of claims 1 to 4, wherein the CAR ligand is operably linked to the lipid via a linker.

6. (i) the linker is selected from the group consisting of a hydrophilic polymer, a series of hydrophilic amino acids, a polysaccharide, or a combination thereof; or (ii) the linker comprises "N" consecutive polyethylene glycol units, where N is between 25 and 50; A composition for use according to claim 5.

7. The CAR ligand (i) a tag, optionally selected from the group consisting of fluorescein isothiocyanate (FITC), streptavidin, biotin, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horseradish peroxidase, palmitoyl, nitrosyl, alkaline phosphatase, glucose oxidase and maltose binding protein; or (ii) a tumor-associated antigen or a fragment thereof The composition for use according to any one of claims 1 to 6, wherein

8. 2. The composition for use of claim 1, wherein the CAR ligand is fluorescein isothiocyanate (FITC), and the CAR ligand is operably linked to the lipid via a polyethylene glycol moiety.

9. The composition for use according to claim 8, wherein the polyethylene glycol moiety is PEG-2000.

10. 10. The composition for use according to any one of claims 1 to 9, wherein the CAR ligand binds to a CAR, the CAR comprising a costimulatory domain, optionally the CAR comprising a bispecific binding domain, optionally the bispecific binding domain comprising a tag binding domain and a tumour associated antigen binding domain, or a first tumour associated antigen binding domain and a second tumour associated antigen binding domain.

11. The composition for use according to any one of claims 1 to 10, further comprising a pharma- ceutically acceptable carrier.

12. 12. The composition for use of claim 11, further comprising an adjuvant, optionally wherein the adjuvant is an amphipathic oligonucleotide conjugate comprising an immunostimulatory oligonucleotide conjugated to a lipid with or without a linker, optionally wherein the immunostimulatory oligonucleotide comprises CpG or is a ligand for a toll-like receptor.

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