Bryostatin compounds for enhancement of immunotherapy

Abstract

To provide a method for selectively enhancing expression, translocation, and / or cell surface presentation of an antigen in target cells of interest so as to modulate the immunogenicity of the target cells.SOLUTION: The method comprises administering to a subject an effective amount of a bryostatin agent to modulate immunogenicity of target cells. The method includes a method of treating cancer, comprising administering to the subject an effective amount of a bryostatin agent to enhance presentation of a cell surface antigen or neoantigen on target cells of the subject, and administering to the subject a therapeutically effective amount of a therapeutic agent that specifically binds to the cell surface antigen to treat the cancer in the subject. Aspects of the method also include the use of bryostatin agents to sensitize the target cells to clearance by the subject's immune system.SELECTED DRAWING: Figure 1-1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 850,905, filed May 21, 2019, the entire contents of which are incorporated herein by reference.

[0002] Federal Research Funding Statement This invention was made with support from the United States government under contract numbers CA031845 and AI124743 awarded by the National Institutes of Health. The United States government has certain rights in this invention. [Background technology]

[0003] Introduction Bryostatin 1 has been advanced in clinical medicine for the treatment of various diseases, disorders, and conditions, including HIV / AIDS, Alzheimer's disease (AD), and cancer. Bryostatin can be isolated from the marine organism Bugula neritina (e.g., in 0.00014% yield) and can be produced in small amounts biosynthetically. Chemical synthesis of various naturally occurring bryostatin compounds and bryostatin analogs, which can provide access to sufficient material for clinical development, has been described by Wender et al. (see, e.g., WO 2018067382).

[0004] Leukemia and lymphoma are difficult-to-treat cancers, with a 5-year survival rate of approximately 60% and accounting for approximately 10% of cancer-related deaths. Novel therapies of interest in development include chimeric antigen receptor T-cell therapy (CAR-T cell therapy) and various targeted therapies, such as antibody-based therapies. CAR-T cell therapy involves the use of autologous cultured T cells, including adoptive cell transfer (ACT). In CAR-T cell therapy, T cells are extracted from the patient and genetically engineered to express a CAR against an antigen specific to a known cancer (e.g., tumor), or T cells can be genetically engineered to express a CAR in situ. Alternatively, T cells from a healthy donor are genetically engineered to express a CAR against an antigen specific to a known cancer. Autologous or allogeneic cells are expanded ex vivo to sufficient numbers and then infused back into the patient to effect antigen-specific destruction of the cancer.

[0005] HIV's ability to establish long-lived latent infections in resting CD4+ T cells leads to persistence and intermittent replenishment of the virus in patients undergoing antiretroviral therapy (ART), thereby preventing eradication of the disease. Bryostatin can activate these latently infected cells, potentially leading to their elimination through virus-mediated cytopathic effects, host immune responses, and / or therapeutic strategies targeting cells actively expressing the virus (see, e.g., Marsden et al., "In vivo activation of latent HIV with a synthetic bryostatin analog effects both latent cell 'kick' and 'kill' in a strategy for virus eradication," PLOS Pathogens 13(9): e1006575). Eliminating latently infected cells in conjunction with ART to eliminate active virus represents a strategy for treating, preventing, or eradicating the disease. Elimination of latently infected cells in conjunction with broadly neutralizing antibodies (bNAbs) to remove active virus may also be a strategy to treat, prevent, or eradicate disease (see, e.g., Borducchi et al., "Antibody and TLR7 agonist delay viral rebound in SHIV-infected monkeys," Nature (2008) Nov;563(7731): 360-364). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO 2018067382 [Non-patent literature]

[0007] [Non-Patent Document 1] Marsden et al., “In vivo activation of latent HIV with a synthetic bryostatin analog effects both latent cell “kick” and “kill” in strategy for virus eradication”, PLOS Pathogens 13(9): e1006575 [Non-patent document 2] Borducchi et al., “Antibody and TLR7 agonist delay viral rebound in SHIV-infected monkeys,” Nature (2008) Nov;563(7731): 360-364 Summary of the Invention

[0008] overview The present disclosure provides the use of bryostatin 1 and analogs based on the bryostatin backbone ("bryostatin agents") as cell-modulating agents. The bryostatin agents can be used to selectively enhance one or more of antigen expression, translocation, cell surface presentation, and surface retention in target cells of interest. Non-limiting examples of antigens in target cells of interest include protein antigens, peptide antigens, neoantigens, and antigens derived from mRNA processing in target cells. Provided herein are methods for modulating target cells in a subject. Aspects of the method include administering an effective amount of a bryostatin agent to a subject to modulate the immunogenicity of the target cells. Aspects of the method include contacting autologous or allogeneic cells with the bryostatin agent ex vivo to modulate the immunogenicity of the autologous or allogeneic cells. The method includes modulating target cells for use in immunotherapy. The method includes modulating target cells for disease treatment. Non-limiting examples of diseases treated by the method include cancer, HIV, neurological disorders, dementia, and Alzheimer's disease. Thus, the present methods include methods of treating cancer, comprising administering to a subject an effective amount of a bryostatin agent that enhances cell surface antigen or neoantigen presentation on target cells in the subject, and administering to the subject a therapeutically effective amount of a therapeutic agent that specifically binds to the cell surface antigen and treats cancer in the subject. The methods can include selectively enhancing cell surface presentation of the target antigen or neoantigen and selectively decreasing cell surface presentation of other antigens or neoantigens. Aspects of the methods also include the use of a bryostatin agent to sensitize target cells to elimination by the subject's immune system. [The present invention 1001] 1. A method of modulating target cells in a subject, comprising: below: (a) expression of the antigen in the target cell, (b) translocation of the antigen in the target cell, (c) cell surface presentation of the antigen in the target cell, and (d) cell surface retention of the antigen in the target cell. contacting the target cells with an effective amount of a bryostatin agent to selectively enhance one or more of: A method comprising: [The present invention 1002] The antigen is Protein antigens, peptide antigens, neoantigens, and antigens derived from target cell mRNA processing 1001. The method of claim 1001, wherein the method is selected from the group consisting of: [The present invention 1003] the target cells are chimeric antigen receptor (CAR)-modified T cells or chimeric antigen receptor-natural killer cells (CAR-NK cells); 1003. The method of any one of claims 1001 to 1002, wherein said step of contacting said target cells with a bryostatin agent enhances expression or cell surface presentation and persistence of said CAR. [The present invention 1004] 10. The method of claim 10, wherein said contacting step is performed ex vivo, and said target cells are removed from said subject (are autologous cells). [The present invention 1005] The method of claim 1003, wherein said contacting step is performed ex vivo and said target cells are removed from a donor (are allogeneic cells). [The present invention 1006] 1001. The method of claim 1001, wherein said target cells are selected from cancer cells, cancer stem cells, and cancer progenitor cells. [The present invention 1007] 1006. The method of claim 1006, wherein said contacting step is performed in vivo, comprising administering said bryostatin agent to a subject having cancer. [The present invention 1008] 1008. The method of any one of claims 1006 to 1007, wherein said target cells are sensitized to elimination by the subject's immune system. [The present invention 1009] administering to the subject an effective amount of a therapeutic agent capable of one or more of inhibiting proliferation of the modulated target cells or eliminating the modulated target cells. The method of the present invention 1007 further comprising: [The present invention 1010] 1001. The method of claim 1001, wherein said target cells are HIV-infected cells. [The present invention 1011] wherein the contacting step is performed in vivo and comprises administering the bryostatin agent to a subject diagnosed with or suspected of having HIV; 1001. The method of claim 1001, wherein said contacting step can have a therapeutic effect. [The present invention 1012] 1. A method of treating cancer in a subject, comprising: (a) administering to a subject an effective amount of a bryostatin agent to enhance the presentation and persistence of cell surface antigens or neoantigens on target cells in said subject; and (b) administering to the subject a therapeutically effective amount of a therapeutic agent that specifically binds to the cell surface antigen and treats cancer in the subject. A method comprising: [The present invention 1013] The method of claim 1012, wherein said subject is relapsed or refractory to targeted anti-cancer therapy. [The present invention 1014] The method of claim 1012, wherein, prior to step (a), the target cancer cells present a therapeutically ineffective level of cell surface antigens on the surface of the target cells. [The present invention 1015] The bryostatin agent is (a) expression of a cell surface antigen, (b) transfer of the expressed cell surface antigen to the surface of the target cell, and (c) retention of the cell surface antigen on the surface of the target cell. The method of the present invention 1012 enhances one or more of the following: [The present invention 1016] Any of the methods of claims 1012 to 1015, wherein the therapeutic agent is selected from chimeric antigen receptor-expressing T cells (CAR T cells), CAR-natural killer cells (CAR-NK cells), an antibody agent, an antibody-drug conjugate (ADC), and a bispecific antibody agent. [The present invention 1017] The method of any one of claims 1012 to 1016, wherein the cancer is leukemia or B-cell lymphoma. [The present invention 1018] The method of any one of claims 1012 to 1016, wherein the cancer is melanoma, prostate cancer, breast cancer, ovarian cancer, esophageal cancer, or kidney cancer. [The present invention 1019] 19. The method of any of claims 1012 to 1018, further comprising the step of determining the level or expression or presentation of a cell surface antigen in target cancer cells of a sample obtained from said subject. [The present invention 1020] further comprising administering at least one additional anti-cancer therapy to said patient; 1020. The method of any of claims 1012 to 1019, wherein said additional anti-cancer therapy is selected from radiation therapy, chemotherapy, immunotherapy, checkpoint inhibitors, surgery, and vascular-targeted therapy. [The present invention 1021] assessing one or more biomarkers in the subject's sample to assay the cancer status. Any of the methods of 1012 to 1020 of the present invention, further comprising: [Brief explanation of the drawings]

[0009] [Figure 1-1]Figure 1, panels A–D, illustrate synthetic strategies for preparing bryostatin and analogs, as well as their bound structures with protein kinase C (PKC). Panel A: Retrosynthetic analysis of the bryostatin scaffold, showing the pharmacophore elements of the C-ring subunit as the C1 carbonyl, C19 hemiketal, and C26 alcohol. C13 is highlighted (shown as a sphere) as an area of interest for analog synthesis. Panel B: Rendering of the PKC-bryostatin-membrane ternary complex. Bryostatin 1 is shown within a rectangular box. The pharmacophore elements of the C-ring subunit of the bryostatin scaffold (see Figure 1, panel A) interact directly with PKC (dark gray), while the A- and B-rings are embedded in the plasma membrane (light gray). Panel C: Representative conformers of the bryostatin scaffold bound to PKC, determined by REDOR NMR and fitted to experimentally determined intramolecular distances. Panel D: Convergent construction of the bryostatin scaffold from acid 1 and enal 2. The C13 functionality provides a versatile starting point for subsequent diversification while avoiding interference with the C-ring pharmacophore elements (shown in Figure 1, panel A). [Figure 1-2] See description of Figure 1-1. [Figure 1-3] See description of Figure 1-1. [Figure 1-4] See description of Figure 1-1. [Figure 2] Figures 2A-2B illustrate therapeutic strategies for methods of treating cancer: Figure 2A is a schematic showing the effect of cytotoxic chemotherapeutic agents, and Figure 2B is a schematic showing selective mAb or CAR T cell therapeutic agents. [Figure 3A]Figure 3, panels A–D, illustrate that CD22 site density on target cells limits CD22 CAR functionality. Panel A: Histogram of CD22 expression in CRISPR / Cas9-edited CD22-negative NALM6 B-ALL lines transduced to express various levels of CD22. NALM6 refers to the parental cell line. Production of interferon gamma (panel B) and IL-2 (panel C) by CD22 CAR-transduced T cells after coculture with NALM6 cell lines expressing various CD22 site densities. By one-way analysis of variance (ANOVA), * = p<0.05, *** = p<0.005, and **** = p<0.001. Data shown in panels B and C represent three independent experiments. Lines represent the mean ± standard error of triplicate well measurements. Panel D: Xenograft model demonstrating that CD22 CAR T cells eliminated parental NALM6 at a dose of 6 x 106 / mouse (NOD SCID gamma (NSG)) administered 3 days after leukemia injection, but failed to eradicate NALM6 expressing low CD22 site density, despite the CAR T cells initially delaying leukemia progression. Represents three independent experiments. Corresponds to Figure 4 in Fry et al., Nature Medicine 2018, 24, 20-27. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 4A] FIG. 4, panels AD, shows that bryostatin 1 (B1) and various exemplary bryostatin agents induced sustained CD22 surface expression in NALM6, JP, and 2F7 cells in vitro. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 5]Figure 5 illustrates that administering Bryostatin 1 to mice along with CD22 CAR T cell therapy enhances the durability of the response. On day 0, NSG mice were injected with 1 x 106 GPF-positive NALM6 tumor cells. On day 3, they were injected with 4 x 106 mock CAR or CD22 CAR for treatment. Mice were given Bryostatin 1 or DMSO three times a week for two weeks. Mice were imaged using IVIS™ technology and IP injection of luciferin-D. [Figure 6A] Figure 6, panel A, illustrates bryostatin-induced PKC activation, as determined by confocal microscopy to observe translocation of PKCδ-GFP to the plasma membrane. Figure 6, panels B-D, illustrate cytosolic fluorescence normalized to t = 0 (the time point immediately before compound addition to the medium) and plotted against time. Error bars have been omitted for clarity. Maximum translocation of PKCδ-GFP to the plasma membrane is reported in Table 2. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 7A] 1 depicts biological data for exemplary bryostatin agents. [Figure 7B] 1 depicts biological data for exemplary bryostatin agents. [Figure 7C] 1 depicts biological data for exemplary bryostatin agents. [Figure 7D] 1 depicts biological data for exemplary bryostatin agents. [Figure 7E] 1 depicts biological data for exemplary bryostatin agents. [Figure 7F] 1 depicts biological data for exemplary bryostatin agents. [Figure 7G] 1 depicts biological data for exemplary bryostatin agents. [Figure 7H] 1 depicts biological data for exemplary bryostatin agents. [Figure 7I] 1 depicts biological data for exemplary bryostatin agents. [Figure 7J] 1 depicts biological data for exemplary bryostatin agents. [Figure 7K] 1 depicts biological data for exemplary bryostatin agents. [Figure 7L] 1 depicts biological data for exemplary bryostatin agents. [Figure 7M] 1 depicts biological data for exemplary bryostatin agents. [Figure 7N] 1 depicts biological data for exemplary bryostatin agents. [Figure 7O] 1 depicts biological data for exemplary bryostatin agents. [Figure 7P] 1 depicts biological data for exemplary bryostatin agents. [Figure 7Q] 1 depicts biological data for exemplary bryostatin agents. [Figure 7R] 1 depicts biological data for exemplary bryostatin agents. DETAILED DESCRIPTION OF THE INVENTION

[0010] definition Before describing exemplary embodiments in detail, the following definitions are set forth to illustrate and define the meaning and scope of terms used herein.

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, for clarity and ease of reference, certain terms are defined below.

[0012] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. For example, the term "primer" refers to one or more primers, i.e., one primer and multiple primers. It should also be noted that a claim may be written to exclude any optional element. This statement therefore serves as a basis for prior use of exclusive language, such as "solely," "only," or "negative" limitations, in relation to the recitation of claim elements.

[0013] Numeric ranges are inclusive of the numbers defining the range.

[0014] "Alkyl" refers to monovalent saturated aliphatic hydrocarbyl groups having 1 to 20 carbon atoms, e.g., 1 to 10 carbon atoms, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, etc. This term includes, by way of example, straight-chain and branched hydrocarbyl groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0015] The term "substituted alkyl" refers to an alkyl group, as defined herein, where one or more carbon atoms of the alkyl chain are substituted with -O-, -N-, -S-, -S(O) n-(n is 0 to 2), -NR- (R is hydrogen or alkyl), and may be substituted with a heteroatom such as alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NR a R b where R refers to an alkyl having 1 to 5 substituents selected from the group consisting of ’ and R ” may be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, and heterocycle.

[0016] "Alkylene" refers to a straight or branched chain alkylene, and includes -O-, -NR 10 -, -NR 10 C(O)-, -C(O)NR 10 -, and the like, wherein R refers to a divalent aliphatic hydrocarbyl group having 1 to 20, and in some cases 1 to 10, or 1 to 6, or 1 to 3 carbon atoms, optionally interrupted by one or more groups selected from 10is selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl, as defined herein. This term includes, by way of example, methylene (-CH-), ethylene (-CHCH-), n-propylene (-CHCHCH-), iso-propylene (-CHCH(CH)-), (-C(CH)CHCH-), (-C(CH)CHC(O)-), (-C(CH)CHC(O)NH-), (-CH(CH)CH-), and the like.

[0017] "Substituted alkylene" refers to an alkylene group in which one to three hydrogens have been replaced with a substituent, as described for carbon in the definition of "substituted" below.

[0018] The term "alkane" refers to alkyl and alkylene groups as defined herein.

[0019] The terms “alkylaminoalkyl,” “alkylaminoalkenyl,” and “alkylaminoalkynyl” refer to R ’ NHR ” - refers to the group where R ’ is an alkyl group as defined herein, and R ” is an alkylene, alkenylene, or alkynylene group as defined herein.

[0020] The terms "alkaryl" or "aralkyl" refer to the groups -alkylene-aryl and -substituted alkylene-aryl, where alkylene, substituted alkylene, and aryl are defined herein.

[0021] "Alkoxy" refers to the group -O-alkyl, where alkyl is as defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. The term "alkoxy" also refers to the groups alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0022] The term "substituted alkoxy" refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O-, where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0023] The term "alkoxyamino" refers to the group --NH-alkoxy, where alkoxy is defined herein.

[0024] The term "haloalkoxy" refers to the group alkyl-O-, where one or more of the alkyl group's hydrogen atoms has been replaced with a halo group and includes, by way of example, groups such as trifluoromethoxy.

[0025] The term "haloalkyl" refers to a substituted alkyl group, as defined above, in which one or more of the alkyl group's hydrogen atoms has been replaced with a halo group. Examples of such groups include, but are not limited to, fluoroalkyl groups such as trifluoromethyl, difluoromethyl, trifluoroethyl, and the like.

[0026] The term "alkylalkoxy" refers to the groups -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, and substituted alkylene-O-substituted alkyl, where alkyl, substituted alkyl, alkylene, and substituted alkylene are as defined herein.

[0027] The term "alkylthioalkoxy" refers to the groups -alkylene-S-alkyl, alkylene-S-substituted alkyl, substituted alkylene-S-alkyl, and substituted alkylene-S-substituted alkyl, where alkyl, substituted alkyl, alkylene, and substituted alkylene are as defined herein.

[0028] "Alkenyl" refers to a straight-chain or branched hydrocarbyl group having 2 to 20 carbon atoms, in some cases 2 to 10 carbon atoms, e.g., 2 to 7 carbon atoms, and having at least one, and in some cases 1 to 2, sites of double bond unsaturation. The term includes, by way of example, bi-vinyl, allyl, and but-3-en-1-yl. The term includes cis and trans isomers or mixtures of these isomers.

[0029] The term "substituted alkenyl" refers to an alkenyl group, as defined herein, having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0030] "Allenyl" refers to a straight or branched chain hydrocarbyl group having 2 to 20 carbon atoms, in some cases 2 to 10 carbon atoms, e.g., 2 to 7 carbon atoms, and having a carbon atom with double bond unsaturation to each of two adjacent carbon atoms. The term includes stereoisomers or mixtures of these isomers.

[0031] The term "substituted allenyl" refers to an alkenyl group, as defined herein, having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0032] "Alkynyl" refers to a straight-chain or branched monovalent hydrocarbyl group having from 2 to 20 carbon atoms, in some cases from 2 to 10 carbon atoms, e.g., from 2 to 7 carbon atoms, and having at least one, and in some cases one or two, sites of triple bond unsaturation. Examples of such alkynyl groups include acetylenyl (-C≡CH) and propargyl (-CHC≡CH).

[0033] The term "substituted alkynyl" refers to an alkynyl group, as defined herein, having from one to five substituents, or one to three substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0034] "Alkynyloxy" refers to the group -O-alkynyl, where alkynyl is as defined herein. Alkynyloxy includes, by way of example, ethynyloxy, propynyloxy, and the like.

[0035] "Acyl" means HC(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted hetero " refers to the groups aryl-C(O)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)-, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. For example, acyl includes the "acetyl" group CHC(O)-.

[0036] "Acylamino" is -NR 20 C(O) alkyl, -NR 20 C(O) substituted alkyl, NR 20 C(O)cycloalkyl, -NR 20 C(O)-substituted cycloalkyl, -NR 20 C(O)cycloalkenyl, -NR 20 C(O)-substituted cycloalkenyl, -NR 20 C(O)alkenyl, -NR 20 C(O) substituted alkenyl, -NR 20 C(O)alkynyl, -NR 20 C(O) substituted alkynyl, -NR 20 C(O)aryl, -NR 20 C(O) substituted aryl, -NR 20 C(O)heteroaryl, -NR 20 C(O)-substituted heteroaryl, -NR 20 C(O) heterocyclic, and -NR 20 C(O)-substituted heterocyclic groups, where R 20is hydrogen or alkyl, and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0037] The term "aminocarbonyl" or "aminoacyl" refers to the group -C(O)NR 21 R 22 where R 21 and R 22 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic; and R 21 and R 22 may be joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0038] "Aminocarbonylamino" is -NR 21 C(O)NR 22 R 23 where R 21 , R 22 , and R 23 is independently selected from hydrogen, alkyl, aryl, or cycloalkyl, or two R groups join to form a heterocyclyl group.

[0039] The term "alkoxycarbonylamino" refers to the group -NRC(O)OR, where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl, where alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0040] The term "acyloxy" refers to the groups alkyl-C(O)O-, substituted alkyl-C(O)O-, cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)O-, aryl-C(O)O-, heteroaryl-C(O)O-, and heterocyclyl-C(O)O-, where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0041] "Aminosulfonyl" is -SO2NR 21 R 22 where R 21 and R 22 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic; and R 21 and R 22 may be joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0042] "Sulfonylamino" is -NR 21 SO2R 22 where R 21 and R 22is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic; and R 21 and R 22 may be joined together with the atoms bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0043] "Aryl" or "Ar" refers to a monovalent aromatic carbocyclic group of 6 to 18 carbon atoms having a single ring (as in a phenyl group) or a ring system having multiple condensed rings (examples of such aromatic ring systems include naphthyl, anthryl, and indanyl), which may or may not be aromatic, provided that the point of attachment is through an aromatic ring atom. The term includes, by way of example, phenyl and naphthyl. Unless otherwise limited by the definition of the aryl substituent, such aryl groups are optionally substituted with one to five substituents, or one to three substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl.

[0044] "Aryloxy" refers to the group -O-aryl, where aryl is as defined herein and includes, by way of example, phenoxy, naphthoxy, and the like, and includes optionally substituted aryl groups, also defined herein.

[0045] "Amino" refers to the group -NH2.

[0046] The term "substituted amino" refers to the group -NRR, where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl, provided that at least one R is not hydrogen.

[0047] The term "azido" refers to the group -N3.

[0048] "Carboxyl," "carboxy," or "carboxylate" refers to -CO2H or a salt thereof.

[0049] The terms "carboxyl ester" or "carboxy esters" or "carboxyalkyl" or "carboxylalkyl" refer to -C(O)O-alkyl, -C(O)O-substituted alkyl, -C(O)O-alkenyl, -C(O)O-substituted alkenyl, -C(O)O-alkynyl, -C(O)O-substituted alkynyl, -C(O)O-aryl, -C(O)O-substituted aryl, -C(O)O-cycloalkyl, -C(O)O-substituted cycloalkyl, -C(O)O-cycloalkenyl, -C(O)O -substituted cycloalkenyl, -C(O)O-heteroaryl, -C(O)O-substituted heteroaryl, -C(O)O-heterocyclic, and -C(O)O-substituted heterocyclic groups, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0050] "(Carboxyl ester)oxy" or "carbonate" refers to -OC(O)O-alkyl, -OC(O)O-substituted alkyl, -OC(O)O-alkenyl, -OC(O)O-substituted alkenyl, -OC(O)O-alkynyl, -OC(O)O-substituted alkynyl, -OC(O)O-aryl, -OC(O)O-substituted aryl, -OC(O)O-cycloalkyl, -OC(O)O-substituted cycloalkyl, -OC(O)O-cycloalkenyl, -OC(O)O-substituted cycloalkenyl, -OC(O)O- refers to the groups -OC(O)O-heteroaryl, -OC(O)O-substituted heteroaryl, -OC(O)O-heterocyclic, and -OC(O)O-substituted heterocyclic, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0051] "Cyano" or "nitrile" refers to the group --CN.

[0052] "Cycloalkyl" refers to cyclic alkyl groups of from 3 to 10 carbon atoms having one or more cyclic rings, including fused, bridged, and spirocyclic systems. Examples of suitable cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like. Such cycloalkyl groups include, for example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl.

[0053] The term "substituted cycloalkyl" refers to a cycloalkyl group having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0054] "Cycloalkenyl" refers to a non-aromatic cyclic alkyl group of 3 to 10 carbon atoms having mono- or polycyclic rings and having at least one double bond, and in some cases 1 to 2 double bonds.

[0055] The term "substituted cycloalkenyl" refers to a cycloalkenyl group having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0056] "Cycloalkynyl" refers to a non-aromatic cycloalkyl group of from 5 to 10 carbon atoms having mono- or polycyclic rings and having at least one triple bond.

[0057] "Cycloalkoxy" refers to -O-cycloalkyl.

[0058] "Cycloalkenyloxy" refers to -O-cycloalkenyl.

[0059] "Halo" or "halogen" refers to fluoro, chloro, bromo, and iodo.

[0060] "Hydroxy" or "hydroxyl" refers to the group --OH.

[0061] "Heteroaryl" refers to an aromatic group of 1 to 15 carbon atoms, e.g., 1 to 10 carbon atoms, and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a single ring (e.g., pyridinyl, imidazolyl, or furyl) or multiple condensed rings in a ring system (e.g., as in groups such as indolizinyl, quinolinyl, benzofuran, benzimidazolyl, or benzothienyl), where at least one ring in the ring system is aromatic and at least one ring in the ring system is aromatic, provided that the point of attachment is through an aromatic ring atom. In certain embodiments, the nitrogen and / or sulfur ring atoms of a heteroaryl group can be oxidized to provide N-oxide (N→O), sulfinyl, or sulfonyl moieties. This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise limited by the definition of the heteroaryl substituent, such heteroaryl groups are optionally substituted with from 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl, and trihalomethyl.

[0062] The term "heteroaralkyl" refers to the group -alkylene-heteroaryl, where alkylene and heteroaryl are defined herein. This term includes, by way of example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

[0063] "Heteroaryloxy" refers to -O-heteroaryl.

[0064] "Heterocycle," "heterocyclic," "heterocycloalkyl," and "heterocyclyl" refer to saturated or unsaturated groups having a single ring or multiple condensed rings, including fused, bridged, and spiro ring systems, and having 3 to 20 ring atoms, e.g., 1 to 10 heteroatoms. These ring atoms are selected from the group consisting of nitrogen, sulfur, or oxygen, where in fused ring systems, one or more of the rings can be cycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atoms of the heterocyclic group can be oxidized to provide an N-oxide, -S(O)-, or -SO2- moiety.

[0065] Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenanthroline ... Examples of suitable amines include azine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also called thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, and tetrahydrofuranyl.

[0066] Unless otherwise limited by the definition of the heterocyclic substituent, such heterocyclic groups are optionally substituted with 1 to 5, or 1 to 3, substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and fused heterocycle.

[0067] "Heterocyclyloxy" refers to the group --O-heterocyclyl.

[0068] The term "heterocyclylthio" refers to the group heterocyclic -S-.

[0069] The term "heterocyclene" refers to a diradical group formed from a heterocycle, as defined herein.

[0070] The term "hydroxyamino" refers to the group -NHOH.

[0071] "Nitro" refers to the radical -NO2.

[0072] "Oxo" refers to the atom (=O).

[0073] "Sulfonyl" refers to SO-alkyl, SO-substituted alkyl, SO-alkenyl, SO-substituted alkenyl, SO-cycloalkyl, SO-substituted cycloalkyl, SO-cycloalkenyl, SO-substituted cycloalkenyl, SO-aryl, SO-substituted aryl, SO-heteroaryl, SO-substituted heteroaryl, SO-heterocyclic, and SO-substituted heterocyclic groups, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Sulfonyl, by way of example, includes methyl-SO-, phenyl-SO-, and 4-methylphenyl-SO-.

[0074] "Sulfonyloxy" refers to the groups -OSO2-alkyl, OSO2-substituted alkyl, OSO2-alkenyl, OSO2-substituted alkenyl, OSO2-cycloalkyl, OSO2-substituted cycloalkyl, OSO2-cycloalkenyl, OSO2-substituted cycloalkenyl, OSO2-aryl, OSO2-substituted aryl, OSO2-heteroaryl, OSO2-substituted heteroaryl, OSO2-heterocyclic, and OSO2-substituted heterocyclic, where alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0075] The term "aminocarbonyloxy" refers to the group -OC(O)NRR, where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic, where alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclic are as defined herein.

[0076] "Thiol" refers to the group --SH.

[0077] The term "thioxo" or "thioketo" refers to the atom (=S).

[0078] The term "alkylthio" or "thioalkoxy" refers to the group -S-alkyl, where alkyl is as defined herein. In certain embodiments, the sulfur may be oxidized to -S(O)-. Sulfoxides can exist as one or more stereoisomers.

[0079] The term "substituted thioalkoxy" refers to the group -S-substituted alkyl.

[0080] The term "thioaryloxy" refers to the group aryl-S-, where aryl is as defined herein and includes optionally substituted aryl groups, also as defined herein.

[0081] The term "thioheteroaryloxy" refers to the group heteroaryl-S-, where heteroaryl is as defined herein and includes optionally substituted aryl groups, also as defined herein.

[0082] The term "thioheterocyclooxy" refers to the group heterocyclyl-S-, where heterocyclyl is as defined herein and includes optionally substituted heterocyclyl groups, also as defined herein.

[0083] Further to the disclosure herein, the term "substituted," when used to modify a particular group or radical, can also mean that one or more hydrogen atoms of the particular group or radical have been replaced, each independently of the other, with the same or different substituents as defined below.

[0084] In addition to the groups disclosed herein for individual terms, substituents replacing one or more hydrogens on a saturated carbon atom of a particular group or radical (where any two hydrogens on a carbon are ═O, ═NR, etc.) are also included. 70 , =N-OR 70 , =N2, or =S) are -R unless otherwise specified. 60 , halo, =O, -OR 70 , -SR 70 , -NR 80 R 80 , trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R 70 , -SO2O - M + , -SO2OR 70 , -OSO2R 70 , -OSO2O - M + , -OSO2OR 70 , -P(O)(O - )2(M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )2, -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -C(O)O - M + , -C(O)OR 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OC(O)O - M + , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 CO2 - M+ , -NR 70 CO2R 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 , and -NR 70 C(NR 70 )NR 80 R 80 where R 60 is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl; and each R 70 are independently hydrogen or R 60 and each R 80 is independently R 70 or two R 80 are joined together, together with the nitrogen atom to which they are attached, to form a 5-, 6-, or 7-membered heterocycloalkyl, which may contain 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N, and S, wherein N may have substitution of -H or C1-C3 alkyl; and each M + is a positively charged counterion. + are independently, for example, alkali ions, e.g., K + , Na + , Li + ammonium ions, e.g. + N(R 60 )4; or alkaline earth ions, such as [Ca 2+ ] 0.5 , [Mg 2+ ] 0.5 , or [Ba 2+ ] 0.5(The "0.5" subscript means that one of the counterions to such divalent alkaline earth ions may be an ionized form of a compound of the present invention and the other may be a typical counterion such as chloride; alternatively, a two-ionized compound disclosed herein may serve as the counterion to such divalent alkaline earth ions, or a doubly-ionized compound of the present invention may serve as the counterion to such divalent alkaline earth ions.) Specific examples include -NR 80 R 80 is intended to include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, N-methyl-piperazin-1-yl, and N-morpholinyl.

[0085] Further to the disclosure herein, the substitution of hydrogen on an unsaturated carbon atom of a "substituted" alkene, alkyne, aryl, and heteroaryl group is represented by -R unless otherwise specified. 60 , halo, -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R 70 , -SO3 - M + , -SO3R 70 , -OSO2R 70 , -OSO3 - M + , -OSO3R 70 , -PO3 -2 (M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )2, -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -CO2 - M + , -CO2R 70 , -C(S)OR 70 , -C(O)NR80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OCO2 - M + , -OCO2R 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 CO2 - M + , -NR 70 CO2R 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 , and -NR 70 C(NR 70 )NR 80 R 80 where R 60 , R 70 , R 80 , and M + is as previously defined, except that in the case of a substituted alkene or alkyne, the substituent is -O - M + , -OR 70 , -SR 70 , or -S - M + isn't it.

[0086] In addition to the groups disclosed herein for each individual term, the substituent of a hydrogen on a nitrogen atom of a "substituted" heteroalkyl and cycloheteroalkyl group is, unless otherwise specified, -R 60 , -O - M + , -OR 70 , -SR 70 , -S - M + , -NR 80 R80 , trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R 70 , -S(O)2O - M + , -S(O)2OR 70 , -OS(O)2R 70 , -OS(O)2O - M + , -OS(O)2OR 70 , -P(O)(O - )2(M + )2, -P(O)(OR 70 )O - M + , -P(O)(OR 70 )(OR 70 ), -C(O)R 70 , -C(S)R 70 , -C(NR 70 )R 70 , -C(O)OR 70 , -C(S)OR 70 , -C(O)NR 80 R 80 , -C(NR 70 )NR 80 R 80 , -OC(O)R 70 , -OC(S)R 70 , -OC(O)OR 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 C(O)OR 70 , -NR 70 C(S)OR 70 , -NR 70 C(O)NR 80 R 80 , -NR 70 C(NR 70 )R 70 , and -NR 70 C(NR 70 )NR 80 R 80 where R 60 , R 70 , R 80 , and M +is as already defined.

[0087] Further to the disclosure herein, in certain embodiments, a substituted group has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0088] For all of the substituents defined above, it is understood that this specification is not intended to include polymers arrived at by defining further substituents on the substituent itself (e.g., substituted aryl having as a substituent a substituted aryl group which is substituted with a substituted aryl group which is further substituted with a substituted aryl group, etc.). In such cases, the maximum number of such substitutions is three. For example, serial substitutions of substituted aryl groups specifically contemplated herein are limited to substituted aryl-(substituted aryl)-substituted aryl.

[0089] Unless otherwise indicated, the naming of substituents not expressly defined herein is arrived at by naming the functionality of the terminal moiety followed by the adjacent functionality toward the point of attachment. For example, the substituent "arylalkyloxycarbonyl" refers to the group (aryl)-(alkyl)-OC(O)-.

[0090] For any group containing one or more substituents disclosed herein, it is of course understood that such group does not include sterically impractical and / or unsynthetic substitutions or substitution patterns. In addition, the present compounds include all stereochemical isomers arising from the substitution of such compounds.

[0091] The terms "synthetic equivalent" or "reactive equivalent" are well understood by those skilled in the art, particularly in the field of retrosynthesis, to refer to a compound or compounds corresponding to a given "synthon" (EJ Corey, Pure App. Chem., 1967, 14: 30-37). Any given synthon may have multiple synthetic equivalents. The term "synthon" refers to a compound containing a portion of the core building block of the target molecule to be synthesized, considered the basis of a synthetic procedure. For example, a synthon may refer to a fragment identified by retrosynthetic analysis or a synthetic building block related to a possible synthetic procedure. The term "synthetic equivalent" refers to a compound that can be used as a substitute for a target intermediate or starting material in a synthetic strategy without having to substantially change the strategy and procedure. It is understood that a synthetic equivalent can be related to a target intermediate or starting material by containing functional groups in the same arrangement as on the underlying fragment of the target scaffold of interest, or precursors or protected versions thereof. A synthetic equivalent can refer to different functional groups with the same chemical properties. A synthon can refer to a fragment resulting from retrosynthetic analysis, for example, from the dissociation of a carbon-carbon bond in a target molecule. A synthetic equivalent can refer to the actual substrate used in a synthetic procedure toward a target molecule. In some cases, the terms synthon and synthetic equivalent can refer to the same molecule. In some cases, the term synthon refers to a synthetic fragment that allows for multiple synthetic equivalents. The definition of a synthetic equivalent includes compounds in which a portion of the compound of interest that is unstable or reactive under the conditions used in the chemical reaction is protected or masked with an appropriate protecting group that can be discarded after the chemical reaction. In some cases, the definition includes compounds in which a portion of the compound of interest is protected or masked with a protecting group that is designed to be discarded during the chemical reaction to provide an unstable or reactive group in situ.

[0092] "Promoiety" refers to a form of protecting group that, when used to mask a functional group within an active agent, converts the active agent into a prodrug.

[0093] The term "pharmaceutically acceptable salt" means a salt that is acceptable for administration to a patient, such as a mammal (a salt having a counterion that has an acceptable safety profile for the mammal for a given administration regimen). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salts" refers to pharmaceutically acceptable salts of a compound, such salts derived from a variety of organic and inorganic counterions well known in the art, including, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and, if the molecule contains a basic functional group, includes salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, etc., hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid, as well as salts of organic acids, such as para-toluenesulfonic acid, methanesulfonic acid, oxalic acid, para-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid, and related inorganic and organic acids. Accordingly, such pharmaceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate (e.g., 3-hexyne-1,6-dioate), ... -dioate), benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, hippurate, gluconate, lactobionate, and the like.In certain specific embodiments, pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as fumaric acid and maleic acid.

[0094] The term "salt thereof" refers to a compound formed by replacing a proton of an acid with a cation, such as a metal cation or an organic cation. The salt is optionally a pharmaceutically acceptable salt, although salts of intermediate compounds not intended for administration to a patient need not be. By way of example, salts of the present compounds include salts in which the compound is protonated with an inorganic or organic acid to form a cation, and the conjugate base of the inorganic or organic acid is the anionic component of the salt.

[0095] "Solvate" refers to a complex formed by the combination of solvent molecules with molecules or ions of the solute. The solvent can be an organic compound, an inorganic compound, or a mixture of both. Some examples of solvents include, but are not limited to, methanol, N,N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.

[0096] "Stereoisomer" and "stereoisomers" refer to compounds that have the same connectivity of their atoms but differ in the arrangement of their atoms in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.

[0097] "Tautomers" refer to alternative forms of molecules that differ only in the electronic bonding of the atoms and / or the position of protons, e.g., enol-keto and imine-enamine tautomers, or tautomeric forms of heteroaryl groups containing the -N=C(H)-NH- ring atom configuration, e.g., pyrazole, imidazole, benzimidazole, triazole, and tetrazole. One of ordinary skill in the art will recognize that other tautomeric ring atom configurations are also possible.

[0098] It will be understood that the term "or a salt or solvate or stereoisomer thereof" includes salts, solvates, and all stereoisomeric variations, for example solvates of pharmaceutically acceptable salts of stereoisomers of the compound.

[0099] As used herein, the term "treating" or "treatment" means treating or curing a disease or condition in a patient, such as a mammal (particularly a human), and includes (a) preventing the disease or condition from occurring, such as prophylactic treatment of a subject; (b) ameliorating the disease or condition, such as eliminating or causing regression of the disease or condition in a patient; (c) inhibiting the disease or condition, for example, by slowing or halting the progression of the disease or condition in a patient; or (d) alleviating the symptoms of the disease or condition in a patient.

[0100] As used herein, the term "contacting a target cell" refers to administering a therapeutically effective amount of an agent, such as a bryostatin agent described herein, to the target cell.

[0101] The term "eliminating" or "elimination" in the context of "eliminating regulated target cells" or "elimination by the immune system of a subject" refers to eradicating the regulated cells from the subject. In some cases, elimination of the regulated cells from the subject is achieved by administering a therapeutically effective amount of a therapeutic agent. In some cases, contacting the target cells with a bryostatin agent (e.g., as described herein) is sufficient to eliminate the regulated cells from the subject. Thus, in some cases, contacting the target cells with a bryostatin agent is sufficient to provide a therapeutic effect.

[0102] The term "cell surface antigen" refers to a molecule present on the cell membrane and cell surface of a cell, which may be cell-specific depending on the cell type and can be used to characterize, identify, and target the cell. The immune system can recognize cell surface antigens. Cell surface antigens include neoantigens, i.e., newly formed antigens that have not previously been recognized by the immune system.

[0103] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to polymeric nucleotides of any length, ribonucleotides or deoxyribonucleotides. Thus, the terms include, but are not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0104] The terms "polypeptide," "peptide," and "protein," as used interchangeably herein, refer to polymeric amino acids of any length and can include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. These terms include fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences with or without an N-terminal methionine residue; immunotagged proteins; and the like.

[0105] The terms "chimeric antigen receptor" and "CAR," used interchangeably herein, refer to an artificial multimodular molecule that can induce or inhibit immune cell activation and generally, but not exclusively, includes an extracellular domain (e.g., a ligand / antigen-binding domain), a transmembrane domain, and one or more intracellular signaling domains. In some cases, the cells are T cells and are converted into CAR-T cells. In other cases, the cells are NK cells and are converted into CAR-NK cells. The term CAR is not limited to specific CAR molecules and also includes CAR variants. CAR variants include split CARs, in which the extracellular portion (e.g., a ligand-binding portion) and the intracellular portion (e.g., an intracellular signaling portion) of the CAR are present in two separate molecules. CAR variants also include ON-switch CARs, which are CARs that can be conditionally activated, including, for example, split CARs, in which the conditional heterodimerization of the two portions of the split CAR is pharmacologically controlled. CAR variants also include bispecific CARs, which include a secondary CAR-binding domain that can amplify or inhibit the activity of the primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs), which may be used, for example, as components of bispecific CAR systems, in which activation of the primary CAR is inhibited as a result of binding of the secondary CAR binding domain.CAR molecules and their derivatives (i.e., CAR variants) have been described, for example, in PCT International Application US2014 / 016527; Fedorov et al. Sci Transl Med (2013) ;5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151-5; Riddell et al. Cancer J (2014) 20(2):141-4; Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev (2014) 257(1):91-106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304, the disclosures of which are incorporated herein by reference in their entireties.

[0106] As used herein, "expressed in" may be used to describe a cellular moiety (e.g., a protein or complex thereof) or precursor thereof that is normally produced within a cell and presented on the surface of the cell as a result of translocation to the extracellular surface of the cell membrane.

[0107] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to mammals, including, but not limited to, murines (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cattle, sheep, pigs, goats), etc. In some cases, the individual is a human.

[0108] As used herein, the term "donor" refers to a mammal, including, but not limited to, murines (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cattle, sheep, pigs, goats), etc., from which target cells can be derived (e.g., allogeneic cells). In some cases, the donor is a human. In some cases, the donor is a healthy donor. In some cases, the donor is a diseased donor.

[0109] A "therapeutically effective amount" or "effective amount" refers to the amount of one agent, or the amount of two agents combined, that, when administered to a mammal or other subject for treating a disease, is sufficient to effect successful treatment for such disease. A "therapeutically effective amount" will vary depending on the agent, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0110] "Specifically bind" or "selectively bind" means that a molecule binds preferentially to a target of interest or binds to a target with higher affinity than other molecules. For example, a DNA molecule will bind to a substantially complementary sequence but not to unrelated sequences. Specific binding can refer to the non-covalent or covalent preferential binding of a molecule over other molecules or moieties in a solution or reaction mixture (e.g., an antibody that specifically binds to a particular polypeptide or epitope over other polypeptides it can bind). In some embodiments, the affinity of a molecule for another molecule to which it specifically binds is greater than or equal to 10. -5 M or less (for example, 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -13 M or less, 10 -14 M or less, 10 -15 M or less, or 10 -16 M or less) K D"Affinity" refers to the strength of binding, and increased binding affinity is characterized by a lower K D There is a correlation with.

[0111] As used herein, the terms "antibody" and "immunoglobulin" are used interchangeably and can broadly refer to whole or intact molecules or fragments thereof, as well as modified and / or conjugated antibodies or fragments thereof. Immunoglobulins can be divided into five distinct classes based on differences in the amino acid sequences of their heavy chain constant regions. Immunoglobulins within a given class all have very similar heavy chain constant regions. These differences can be detected by sequence analysis or, more commonly, by serological means (i.e., by the use of antibodies directed against such differences). The immunoglobulin classes include IgG (gamma heavy chain), IgM (mu heavy chain), IgA (alpha heavy chain), IgD (delta heavy chain), and IgE (epsilon heavy chain).

[0112] Antibodies or immunoglobulins can refer to a class of structurally related glycoproteins, consisting of two pairs of polypeptide chains: one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized; see, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain typically contains a heavy chain variable region (V H ) and heavy chain constant region (C H The heavy chain constant region is typically composed of C H 1. C H 2, and C H Each light chain typically consists of three domains: a light chain variable region (V L ) and the light chain constant region (C L The light chain constant region is typically composed of C L It consists of one domain. VH and V L The regions can be further subdivided into hypervariable regions (i.e., regions that may be hypervariable in sequence and / or in the form of structurally defined loops), also called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs).

[0113] Whole or nearly intact antibodies are generally multivalent, meaning that they can simultaneously bind to two or more molecules of an antigen, whereas antibody fragments can be monovalent. Antibodies produced by an organism as part of an immune response are generally monospecific, meaning that they generally bind to one type of antigen. Multivalent monospecific antibodies, i.e., antibodies that bind to two or more molecules of one type of antigen, can bind to one antigen epitope (e.g., monoclonal antibodies) or to multiple different antigen epitopes (e.g., polyclonal antibodies).

[0114] Multispecific (e.g., bispecific) antibodies that bind to multiple antigens can be readily produced by one of skill in the art and are therefore appropriately encompassed by the use of the term "antibody" herein. Similarly, multivalent antibody fragments can be produced, for example, by linking two monovalent antibody fragments. Thus, bivalent and / or multivalent antibody fragments can be appropriately encompassed by the use of the term "antibody," as one of skill in the art will readily recognize antibody fragments that can be linked in any convenient and appropriate combination to create multivalent monospecific or multispecific (e.g., bispecific) antibody fragments, for example, as described below.

[0115] Antibody fragments include, but are not limited to, antigen-binding fragments (Fab or F(ab), e.g., Fab' or F(ab'), (Fab)2, F(ab')2, etc.), single-chain variable fragments (scFv or Fv), "third generation" (3G) molecules, etc., which are capable of binding to an epitopic determinant. Such antibody fragments retain some ability to selectively bind to an antigen of interest, and examples of which include, but are not limited to, the following: (1) Fab, the fragment containing a monovalent antigen-binding fragment of an antibody molecule, which can be produced by digestion of a whole antibody with the enzyme papain to yield one intact light chain and a portion of one heavy chain; (2) Fab', a fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by shortening to leave one intact light chain and a portion of one heavy chain; two Fab' fragments can be obtained per antibody molecule; (3) the fragment of an antibody that can be obtained by treating the whole antibody with the enzyme pepsin without subsequent reduction, (Fab)2; (4) F(ab)2 is a dimer of two Fab' fragments held together by two disulfide bonds; (5) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; (6) Single-chain antibodies ("SCAs"), defined as genetically engineered molecules comprising a light chain variable region and a heavy chain variable region linked as a genetically fused single-chain molecule by a suitable polypeptide linker; such single-chain antibodies may be in the form of diabodies, triabodies, tetrabodies, or other multimers, which may or may not be multispecific (see, e.g., WO 94 / 07921 and WO 98 / 44001); and (7) "3G," which includes single domain (typically a variable heavy domain without a light chain) and "miniaturized" antibody molecules (typically full-size Abs or mAbs with non-essential domains removed).

[0116] As used herein, "antigen-specific T cell" and "T cell specific for an antigen" refer to a T cell that expresses on its cell surface a TCR that specifically binds to an antigen by virtue of the structure of the T cell receptor (TCR) polypeptide, such as the α and β polypeptide chains comprising the variable regions. T cells whose TCR is specific for an antigen may have undergone TCR genomic locus recombination during maturation and / or may have been genetically modified to express one or more TCR polypeptides or modified TCR-like receptors (e.g., chimeric antigen receptors).

[0117] A "disease antigen" or "disease-associated antigen" refers to an epitope (e.g., an antigenic peptide, lipid, polysaccharide, nucleic acid, etc.) that elicits an immune response, such as a T cell-mediated immune response. When the disease is a tumor, the tumor antigen or tumor-associated antigen can be an epitope expressed on the surface of a tumor cell. A tumor antigen can be unique to the tumor cell and not normally expressed on the surface of other cells in the body, particularly of the same lineage. In some cases, a tumor antigen can be an epitope normally expressed on other cells in the body but that does not elicit an immune response in a non-tumor context. A tumor antigen can have one or more epitopes that are typically expressed on normal cells during fetal development, when the immune system is immature and unable to respond. A tumor antigen can have one or more epitopes that are normally presented at very low levels on normal cells but are expressed at much higher levels on tumor cells.

[0118] Other definitions for each term may appear throughout this specification.

[0119] Detailed Description The present disclosure provides the use of bryostatin 1 and analogs based on the bryostatin backbone ("bryostatin agents") as cell-modulating agents. The bryostatin agents can be used to selectively enhance one or more of antigen expression, translocation, cell surface presentation, and cell surface retention in target cells of interest. Non-limiting examples of antigens in target cells of interest include protein antigens, peptide antigens, neoantigens, and antigens derived from mRNA processing in target cells. Provided herein are methods for modulating target cells in a subject. Aspects of the method include administering an effective amount of a bryostatin agent to a subject to modulate the immunogenicity of the target cells. Aspects of the method include contacting autologous or allogeneic cells with the bryostatin agent ex vivo to modulate the immunogenicity of the autologous or allogeneic cells. The method includes modulating target cells for use in immunotherapy. The method includes modulating target cells for disease treatment. Non-limiting examples of diseases treated by the method include cancer, HIV, neurological disorders, dementia, and Alzheimer's disease. The methods include methods for treating cancer, comprising administering to a subject an effective amount of a bryostatin agent that enhances cell surface antigen or neoantigen presentation on target cells of the subject, and administering to the subject a therapeutically effective amount of a therapeutic agent that specifically binds to the cell surface antigen and treats cancer in the subject. The methods may include selectively enhancing cell surface presentation of the target antigen or neoantigen and selectively decreasing cell surface presentation of other antigens or neoantigens. Aspects of the methods also include the use of a bryostatin agent to sensitize target cells to elimination by the subject's immune system.

[0120] Non-limiting examples of target cells modulated by the present methods may include diseased cells, infected cells, modified cells, and normal antigen-presenting cells. In some cases, the target cells are normal antigen-presenting cells that, after treatment with the present methods (e.g., as described herein), become more effective and exhibit an enhanced ability to eliminate disease or target cells. For example, the present methods can use normal antigen-presenting cells to produce mRNA proteins that induce an immune response, thus strengthening the immune system. In certain cases, non-limiting examples of target cells modulated by the present methods may include HIV-infected cells, cancer cells, chimeric antigen receptor (CAR)-modified T cells (CAR-T cells), and chimeric antigen receptor-natural killer cells (CAR-NK). The present bryostatin agents can be used in combination with chimeric antigen receptor-T cell therapy (CAR-T cell therapy) or CAR-NK cell therapy to improve patient response and prevent relapse due to low and variable antigen expression on the surface of the target cells of interest. CARs represent an emerging cancer therapy (e.g., treatment of B and T cell lymphomas) and other malignant disease treatments. CAR-T cells can include patient-derived memory CD8+ T cells (e.g., autologous cells) that have been modified to express a recombinant T cell receptor specific for a known antigen presented on the surface of a tumor of interest, for example. In this regard, T cells can be removed from a patient and modified to express a CAR against a particular antigen (e.g., on the surface of a tumor of interest), or the T cells can be modified to express a CAR in vivo. CAR-T cells can also include donor-derived memory CD8+ T cells (e.g., allogeneic cells removed from a donor) that have been modified to express a recombinant T cell receptor specific for a known antigen presented on the surface of a tumor of interest, for example. While the present disclosure is generally described in the context of using CAR-T cell therapy to treat cancer, it should be understood that it is not limited to such therapy.

[0121] Before describing the present invention in further detail, it is to be understood that the invention is not limited to the particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention is limited only by the appended claims.

[0122] Where a range of values is provided, unless the context clearly indicates otherwise, it is understood that each value between the upper and lower limit of that range, and any other value in that range or intervening value, to the nearest tenth of the lower limit, is encompassed within the invention. The upper and lower limits of such smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any explicitly stated excluded limit in the stated range. When the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0123] In the ranges described herein, some numerical values are preceded by the term "about." In this specification, the term "about" is used not only to literally support the number that follows it, but also to provide a number that is close to or approximately the number that follows it. When determining whether a number is close to or approximately a specifically stated number, the unstated close or approximately value may be a number that provides a substantial equivalent to the specifically stated number in the context in which it is stated.

[0124] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, representative exemplary methods and materials are described below.

[0125] All publications and patents cited herein are incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference to disclose and describe the methods and / or materials in connection with which such publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the stated dates of publication may differ from the actual publication dates, which may require independent confirmation.

[0126] As used herein and in the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. It is further noted that a claim may be drafted to exclude any optional element. This statement therefore serves as a basis for the use of exclusive language, such as "solely," "only," or "negative" limitations, in relation to the recitation of claim elements.

[0127] As will be apparent to those skilled in the art upon reading this disclosure, each embodiment described and illustrated herein has distinct components and features that may be readily separated from or combined with any features of the other embodiments without departing from the scope or spirit of the invention. Any described methods can be carried out in the order described, or in any other order that is logically possible.

[0128] Although apparatus and methods have been or will be described by functional descriptions for the sake of grammatical flow, the claims should in no way be construed as necessarily limited by construction of "means" or "step" limitations unless expressly recited under 35 U.S.C. §112, but rather, it should be expressly understood that under the doctrine of legal equivalents, the full meaning and scope of equivalents of the definitions provided by the claims should be given, and that if a claim is expressly recited under 35 U.S.C. §112, all legal equivalents under 35 U.S.C. §112 should be given.

[0129] method As summarized above, the methods of the present disclosure provide for the use of a bryostatin agent to selectively enhance one or more of the expression, translocation, cell surface presentation, and persistence of an antigen (e.g., as described herein) in a target cell of interest. As disclosed herein, target cells include diseased cells, infected cells, modified cells, and normal antigen-presenting cells. Provided herein are methods for modulating target autologous or allogeneic cells ex vivo. Provided herein are methods for modulating target cells in vivo. Provided herein are methods for modulating target cells in a subject. Thus, the methods comprise administering to the subject an effective amount of a bryostatin agent. In certain aspects, the methods include methods for treating cancer, comprising administering to the subject an effective amount of a bryostatin agent that enhances the presentation of a cell surface antigen or neoantigen on a target cell of the subject, and administering to the subject a therapeutically effective amount of a therapeutic agent that specifically binds to the cell surface antigen and treats the cancer in the subject.

[0130] The present disclosure provides the use of bryostatin agents to modulate the presentation and persistence of cell surface antigens. In some cases, specific cell surface antigens can be targeted for selective modulation, e.g., selective enhancement of cell surface presentation. The present disclosure provides for selective enhancement of target cell surface antigens and neoantigens over non-target antigens, e.g., cell surface antigens associated with general immune activation. Selective modulation can include enhancing the expression or presentation of target antigens. In certain cases, the antigen is selected from protein antigens, peptide antigens, neoantigens, and antigens derived from mRNA processing of target cells.

[0131] "Selectively," as used herein in the context of "selectively enhancing expression" of a protein in a target cell of interest or "selectively enhancing cell surface display" of a protein in a target cell of interest, refers to treatment of a target protein population that facilitates distinguishing between members of the population with desirable attributes (e.g., target antigens or neoantigens) and members with less desirable attributes. In other words, certain members of the protein population are preferentially enhanced (e.g., in terms of expression or surface display) in the target cell over other proteins of the population.

[0132] "Enhancing expression" or "enhancing target antigen expression" means increasing the expression of a target protein or antigen by 50% or more. In some cases, a bryostatin agent increases cell surface presentation by 55% or more, e.g., 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 100% or more, or even more. In some cases, a bryostatin agent enhances expression by 2-fold or more, e.g., 3-fold or more, 4-fold or more, 5-fold or more, or even more. "Enhancing cell surface presentation" or "enhancing target antigen presentation" means increasing the presentation of a target protein or antigen by 50% or more. In some cases, a bryostatin agent increases cell surface presentation by 55% or more, e.g., 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 100% or more, or even more. In some cases, the bryostatin agent increases cell surface presentation by 2-fold or more, e.g., 3-fold or more, 4-fold or more, 5-fold or more, or even more. In some cases, the bryostatin agent will enhance presentation and retention of more than one target antigen, e.g., 2 or more target antigens, 3 or more target antigens, 4 or more target antigens, 5 or more target antigens, or more.

[0133] "Modulating a target cell" or "modulating the immunogenicity of a target cell" is intended to include enhancing the cell surface presentation of a particular protein and enhancing the expression of a particular protein in the target cell. Modulating a target cell can also include reducing the expression or cell surface presentation of other proteins in the target cell. Modulating a target cell includes modulating cell surface antigen presentation and cellular antigen expression. In some aspects, the methods disclosed herein include modulating the immunogenicity of a target cell by selectively modulating a target antigen, e.g., by upregulating the target antigen. In some aspects, the methods include modulating the immunogenicity of a target cell by downregulating other antigens, e.g., antigens associated with general immune activation. "Immunogenicity" refers to the ability of a target cell to be more visible to a subject's immune system or the ability of a target cell to elicit an immune response by a subject.

[0134] This method can be used in combination with chimeric antigen receptor-T cell therapy (CAR-T cell therapy) and chimeric antigen receptor-natural killer cell therapy (CAR-NK) to improve patient responses and prevent relapse in patients due to low and variable antigen expression on the surface of the target cells of interest. To date, CAR-T cell therapy has been limited, in part, by the gradual and variable expression of target antigens, the induction of antigen-specific toxicity targeting normal tissues expressing the target antigen, and the development of life-threatening cytokine release syndrome due to the high potency of CAR-T cell and / or CAR-NK cell therapy. Specifically, it has been observed that high-affinity interactions of T cell receptors with large antigen loads can lead to activation-induced cell death.

[0135] Recently, preliminary phase I data from a clinical trial of CD22-targeted CAR T therapy in patients with acute lymphoblastic leukemia (ALL) showed promising results, with approximately 70% of patients achieving complete cure with a median duration of 6 months. However, despite this success, relapses were observed in patients with diminishing and variable CD22 surface expression levels. A mouse tumor xenograft model demonstrated that critical CD22 surface activity is required for anti-CD22 CAR T cell activation and tumor elimination (see, for example, Fry et al., Nat. Med., 2017, 24, 20-28).

[0136] Protein kinase C (PKC) modulators, C1 domain binders, and non-C1 domain targets that mediate bryostatin activity may be useful adjuvants for targeted cancer therapy. Among the most extensively studied PKC modulators, plant-derived phorbol esters (PE) have been shown to induce antigen presentation in a variety of cell lines. The marine macrolide bryostatin 1 (see, e.g., Pettit et al. J. Am. Chem. Soc. 1982, 104 (24), 6846-6848; and Kortmansky et al. Cancer Invest. 2003, 21 (6), 924-936) can alter the expression of surface antigens in tumors and other cell lines, making them more immunogenic and therefore more susceptible to immune elimination. Data presented herein suggest that bryostatin agents can be used in conjunction with CAR-T cell therapy to enhance CAR T cell activity by increasing the number and density of cell surface antigens on target cells.

[0137] Accordingly, provided herein are methods of modulating target cells in a subject. The methods include: contacting a target cell with an effective amount of a bryostatin agent to selectively enhance one or more of: (a) expression of the antigen in the target cell; (b) translocation of the antigen in the target cell; (c) cell surface presentation of the antigen in the target cell; and (d) cell surface retention of the antigen in the target cell, thereby modulating the immunogenicity of the target cell. In some embodiments, the antigen is a protein antigen. In some cases, the antigen is a peptide antigen. In some cases, the antigen is a neo-antigen. In some other cases, the antigen is derived from mRNA processing in the target cell (e.g., an antigen derived from delivery and expression of mRNA).

[0138] In some embodiments of the methods of modulating a cell, the target cell is an HIV-infected cell. In certain cases, the target cell is a cell infected with latent HIV, and modulating the immunogenicity of the target cell comprises activating expression of HIV in the latent viral reservoir.

[0139] In some cases, the bryostatin agent activates HIV expression by 10% or more, e.g., 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or even more. In some cases, the bryostatin agent activates HIV expression by 2-fold or more, e.g., 3-fold or more, 4-fold or more, 5-fold or more, or even more.

[0140] In some embodiments of the method of modulating cells, the contacting step is performed in vivo and includes administering a bryostatin agent to a subject diagnosed with or suspected of having HIV. In certain cases, the method of modulating cells further includes administering to the subject a therapeutically effective amount of a therapeutic agent capable of eliminating the modulated target cells having activated HIV expression. In some cases, the therapeutic agent capable of eliminating the modulated target cells is antiretroviral therapy (ART). In some cases, the therapeutic agent capable of eliminating the modulated target cells is a broadly neutralizing antibody (bNAb). In certain embodiments, the method of modulating cells includes eliminating the modulated target cells having activated HIV expression without administering an additional therapeutic agent; for example, bryostatin itself may be capable of eliminating target cells.

[0141] In some embodiments of the cell modulating method, the target cell is a chimeric antigen receptor (CAR)-modified T cell or a CAR-NK cell, and the expression or cell surface presentation of the CAR is enhanced by contacting the target cell with a bryostatin agent. In certain cases, the CAR has affinity for a target cell surface antigen selected from a viral antigen, a bacterial antigen, a parasitic antigen, a tumor cell-associated antigen (TAA), a disease cell-associated antigen, an antigen derived from cellular mRNA processing, and any fragment thereof. In certain cases, the modified T cell is obtained from peripheral blood mononuclear cells, umbilical cord blood cells, a purified T cell population, or a T cell line.

[0142] In some embodiments of the methods of modulating cells, the contacting step is performed ex vivo and the target cells are removed from the subject to be treated (e.g., autologous cells), while in other embodiments, the contacting step is performed ex vivo and the target cells are removed from a donor (e.g., allogeneic cells).

[0143] In this method, contacting target cells ex vivo with a bryostatin agent can enhance the production of CAR-T cells and (CAR)-NK cells in a manner similar to enhancing antigen presentation in target cells. In some cases, the enhancement of the CAR is more efficient externalization of the engineered recognition fragment.

[0144] In some embodiments of the method of modulating cells, the target cells are selected from cancer cells, cancer stem cells, and cancer progenitor cells. In certain cases, the cancer cells are derived from cancers such as, but not limited to, breast cancer, prostate cancer, bladder cancer, soft tissue sarcoma, lymphoma, esophageal cancer, uterine cancer, bone cancer, adrenal cancer, lung cancer, thyroid cancer, colon cancer, glioma, liver cancer, pancreatic cancer, kidney cancer, cervical cancer, testicular cancer, head and neck cancer, ovarian cancer, neuroblastoma, and melanoma.

[0145] In some embodiments of the method of modulating cells, the contacting step is performed in vivo and includes administering a bryostatin agent to a subject with cancer. The bryostatin agent can be administered by any convenient route, such as orally, intraocularly, auricularly, subcutaneously, intravenously, intramuscularly, intradermally, intraperitoneally, and by inhalation.

[0146] In some cases, the bryostatin agent is administered subcutaneously. In some cases, the bryostatin agent is administered orally. In some cases, the bryostatin agent is administered intraocularly. In some cases, the bryostatin agent is administered intravenously. In some cases, the bryostatin agent is administered intramuscularly. In some cases, the bryostatin agent is administered intradermally. In some cases, the bryostatin agent is administered intraperitoneally. In some cases, the bryostatin agent is administered by inhalation.

[0147] In some embodiments of the methods of modulating cells, the methods sensitize the target cells to elimination by the subject's immune system. In some cases, the methods sensitize the target cells to elimination by cells of the subject's innate immune system. In some cases, the methods sensitize the target cells to elimination by cells of the subject's adaptive immune system.

[0148] Cell modulating methods induce an immune response in a subject by sensitizing target cells to elimination by the subject's immune system. In some cases, cell modulating methods do not affect the subject's own immune system cells, but can selectively enhance antigen expression or cell surface presentation for elimination by the subject's immune system.

[0149] In some embodiments of the methods of modulating cells, the subject is relapsed to immune cell mediated elimination and the bryostatin agent modulates a T cell mediated immune response against a target cell population. In some cases, the subject is refractory to immune cell mediated elimination and the bryostatin agent modulates a T cell mediated immune response against a target cell population. In some cases, the bryostatin agent modulates a T cell mediated immune response against a target cell population, such that the immune response against the target cell population is increased by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or even more, compared to an untreated subject.

[0150] The terms "relapse" or "recurrent" refer to the return of disease after recovery. The term "refractory to disease" refers to a subject's resistance or unresponsiveness to treatment for a particular disease. For example, a refractory or resistant cancer is unresponsive to first-line, and sometimes second-line, chemotherapy drugs, biological agents, and / or radiation therapy. A refractory cancer may shrink, but not to the point where treatment is determined to be effective. In most cases, the tumor either remains the same size as before treatment (stable disease) or grows (progressive disease).

[0151] In some embodiments of the methods of modulating cells, the subject is undergoing cancer immunotherapy. In certain cases, the subject is undergoing a tumor antigen peptide vaccine.

[0152] In some embodiments of the method for modulating cells, the method further comprises administering to the subject an effective amount of a therapeutic agent capable of one or more of inhibiting proliferation of the modulated target cells and eliminating the modulated target cells. In some cases, the method is used in combination with other therapies, where the combination provides an additive or synergistic benefit to the subject.

[0153] Also provided herein is a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of a bryostatin agent that enhances the presentation of a cell surface antigen or neoantigen on a target cell in the subject; and administering to the subject a therapeutically effective amount of a therapeutic agent that specifically binds to the cell surface antigen and treats cancer in the subject.

[0154] In some embodiments of the methods of treating cancer, the subject is relapsed to the targeted anti-cancer therapy. In some cases, the subject is refractory to the targeted anti-cancer therapy.

[0155] In some embodiments of the methods of treating cancer, the bryostatin agent sensitizes the target cancer cells to growth inhibition by a therapeutic agent. In some cases of the methods of treating cancer, the bryostatin agent sensitizes the target cancer cells to elimination by a therapeutic agent. In some embodiments of the methods of treating cancer, prior to administering to the subject an effective amount of a bryostatin agent, the target cancer cells present a therapeutically ineffective level of cell surface antigens on the target cell surface (e.g., a level of presentation insufficient to induce cytotoxicity with the agent).

[0156] In some embodiments of the methods of treating cancer, the bryostatin agent enhances one or more of: (a) expression of a cell surface antigen; (b) translocation of the expressed cell surface antigen to the surface of a target cell; and (c) retention of the cell surface antigen on the surface of the target cell.

[0157] In some embodiments of the methods of treating cancer, the bryostatin agent enhances (increases) cell surface presentation of a cell surface antigen by 50% or more, e.g., 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 100% or more, or even more. In some cases, cell surface presentation of the antigen is enhanced by 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, or even more.

[0158] In some embodiments of the methods of treating cancer, cell surface antigen presentation of the target cancer cells is enhanced for 2 or more days after administration of the bryostatin agent. In some cases, cell surface antigen presentation of the target cancer cells is enhanced (increased) for 10 or more hours, 20 or more hours, 30 or more hours, 1 or more days, 2 or more days, 3 or more days, 4 or more days, 5 or more days, 6 or more days, 7 or more days, 8 or more days, 9 or more days, 10 or more days, or longer.

[0159] Thus, in some methods of treating cancer, the bryostatin agent enhances (increases) retention of the cell surface antigen on the target cell surface by 50% or more, e.g., 60% or more, 70% or more, 80% or more, 90% or more, or even more. In some cases, cell surface presentation of the antigen is enhanced by 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, or even more. In some cases, the bryostatin agent enhances (increases) retention of the cell surface antigen on the target cell surface for a period of 10 hours or more, 20 hours or more, 30 hours or more, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, or longer.

[0160] In some embodiments of the methods of treating cancer, the therapeutic agent that specifically binds to a cell surface antigen is selected from chimeric antigen receptor-expressing T cells (CAR T cells), chimeric antigen receptor-expressing natural killer cells (CAR-NK cells), an antibody agent, an antibody-drug conjugate (ADC), and a bispecific antibody agent. In some cases, the therapeutic agent is a CAR T cell. In some cases, the therapeutic agent is a CAR-NK cell. In some cases, the therapeutic agent is an antibody. In some cases, the therapeutic agent is an ADC. In other cases, the therapeutic agent is a bispecific antibody agent.

[0161] In some embodiments of the method for treating cancer, administering a therapeutically effective amount of a therapeutic agent to a subject comprises administering a composition comprising a therapeutically effective amount of CAR T cells that specifically bind to a cell surface antigen presented on a target cell population to the subject. In certain cases, the bryostatin agent regulates T cell-mediated immune response to the target cell population. In certain cases, the target cell population comprises a tumor antigen selected from CD10, CD19, CD20, CD21, CD22, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD52, CD80, CD86, CD137, CDK4, CDK6, OX40, and CD340. In certain cases, the target cell population comprises a tumor antigen CD22.

[0162] In some embodiments of the methods of treating cancer, the therapeutic agent that specifically binds to a cell surface antigen is a chimeric antigen receptor-expressing T cell (CAR T cell) or a CAR-NK cell, and the CAR T cell or CAR-NK cell is effective in treating a B-cell malignancy, CLL, ALL, B-ALL, leukemia, lymphoma, or a solid tumor. In certain cases, the solid tumor is selected from breast cancer, prostate cancer, bladder cancer, soft tissue sarcoma, lymphoma, esophageal cancer, uterine cancer, bone cancer, adrenal cancer, lung cancer, thyroid cancer, colon cancer, glioma, liver cancer, pancreatic cancer, kidney cancer, cervical cancer, testicular cancer, head and neck cancer, ovarian cancer, neuroblastoma, and melanoma.

[0163] In some embodiments of the methods of treating cancer, a bryostatin agent is administered first, followed by a therapeutic agent that specifically binds to a cell surface antigen and treats cancer in a subject. In some cases, the bryostatin agent is administered first, followed by a therapeutically effective amount of CAR-T cells. In some cases, the bryostatin agent is administered first, followed by a therapeutically effective amount of CAR-NK cells. In some cases, the bryostatin agent is administered first, followed by a therapeutically effective amount of an antibody agent. In some cases, the bryostatin agent is administered first, followed by a therapeutically effective amount of an antibody-drug conjugate (ADC). In some cases, the bryostatin agent is administered first, followed by a therapeutically effective amount of a bispecific antibody agent.

[0164] In some embodiments of the methods of treating cancer, a bryostatin agent is administered simultaneously with the administration of a therapeutic agent that specifically binds to a cell surface antigen and treats cancer in a subject. In some cases, the bryostatin agent is administered simultaneously with the administration of a therapeutically effective amount of CAR-T cells. In some cases, the bryostatin agent is administered simultaneously with the administration of a therapeutically effective amount of CAR-NK cells. In some cases, the bryostatin agent is administered simultaneously with the administration of a therapeutically effective amount of an antibody agent. In some cases, the bryostatin agent is administered simultaneously with the administration of a therapeutically effective amount of an antibody-drug conjugate (ADC). In some cases, the bryostatin agent is administered simultaneously with the administration of a therapeutically effective amount of a bispecific antibody agent.

[0165] In some embodiments of the methods of treating cancer, a bryostatin agent is administered following administration of a therapeutic agent that specifically binds to a cell surface antigen and treats cancer in a subject. In some cases, a therapeutically effective amount of CAR-T cells is administered following administration of a bryostatin agent. In some cases, a therapeutically effective amount of CAR-NK cells is administered following administration of a bryostatin agent. In some cases, a therapeutically effective amount of an antibody agent is administered following administration of a bryostatin agent. In some cases, a therapeutically effective amount of an antibody-drug conjugate (ADC) is administered following administration of a bryostatin agent. In some cases, a therapeutically effective amount of a bispecific antibody agent is administered following administration of a bryostatin agent. In some cases, a bryostatin agent is administered following administration of mRNA to enhance expression, translocation, and presentation of the encoded protein.

[0166] In some embodiments of the methods for treating cancer, administering the therapeutic agent comprises administering to the subject a therapeutically effective amount of an antibody agent, ADC, or bispecific antibody agent that specifically binds to a cell surface antigen.

[0167] In some embodiments of the methods of treating cancer, the antibody agent comprises a human monoclonal antibody, or an antigen-binding portion thereof. In certain cases, the antibody agent is an antibody, including a full-length antibody of the IgG1 or IgG4 isotype.

[0168] In some embodiments of the method for treating cancer, the therapeutic agent that specifically binds to a cell surface antigen is an ADC containing a cytotoxic agent. In certain cases, the cytotoxic agent is a cytotoxin or a radioactive agent. In some cases, the cytotoxic agent is conjugated to the antibody of the ADC via a linker. In certain cases, the linker is selected from a peptidyl linker, a hydrazine linker, and a disulfide linker. In certain cases, the cytotoxic agent is selected from a calicheamicin, an auristatin, a maytansinoid, a taxol derivative, and a duocarmycin.

[0169] In some embodiments of the methods of treating cancer, the therapeutic agent that specifically binds to a cell surface antigen is an ADC selected from inotuzumab ozogamicin and gemtuzumab ozogamicin.

[0170] In some embodiments of the methods of treating cancer, the therapeutic agent that specifically binds to a cell surface antigen is a bispecific antibody agent. In particular cases, the bispecific antibody is an anti-CD20 / anti-CD22 bispecific antibody fusion protein or an anti-CD19 / anti-CD22 bispecific antibody fusion protein.

[0171] In some embodiments of the methods of treating cancer, the therapeutic agent is administered by a route selected from oral, intraocular, otic, subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal, and inhalation. In some cases, the agent is administered orally. In some cases, the agent is administered intraocularly. In some cases, the agent is administered otically. In some cases, the agent is administered subcutaneously. In some cases, the agent is administered intravenously. In some cases, the agent is administered intramuscularly. In some cases, the agent is administered intradermally. In some cases, the agent is administered intraperitoneally. In some cases, the agent is administered by inhalation.

[0172] The present method can be used to treat a variety of different cancers. For example, representative cancer conditions and cell types for which the method of the present disclosure may be useful include melanoma, myeloma, chronic lymphocytic leukemia (CLL), AIDS-related lymphoma, non-Hodgkin's lymphoma, colorectal cancer, renal cancer, prostate cancer, head, neck, stomach, esophageal, anal, or cervical cancer, ovarian cancer, breast cancer, peritoneal cancer, and non-small cell lung cancer. In one embodiment, the cancer is leukemia or B-cell lymphoma. In certain cases, the B-cell lymphoma is non-Hodgkin's lymphoma. In one embodiment, the cancer is selected from Burkitt's lymphoma and B-cell chronic lymphocytic leukemia. In certain cases, the cancer is melanoma, prostate cancer, breast cancer, ovarian cancer, esophageal cancer, or kidney cancer.

[0173] In some embodiments of the methods of treating cancer, the subject is a mammal. In some cases, the mammal is a human. In some cases, the mammal is non-human, such as a murine (e.g., rat, mouse), lagomorph (e.g., rabbit), non-human primate, dog, cat, ungulate (e.g., horse, cow, sheep, pig, goat), etc.

[0174] In some embodiments of the methods for treating cancer, the mammalian subject is relapsed or refractory to cell surface antigen-targeted therapy. In certain cases, the cell surface antigen is selected from CD10, CD19, CD20, CD21, CD22, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD52, CD80, CD86, CD137, CDK4, CDK6, OX40, and CD340.

[0175] In some embodiments of the methods of treating cancer, the method further comprises determining the level or expression or presentation of a cell surface antigen in target cancer cells of a sample obtained from the subject. In certain cases, cells can be removed from a patient and treated with a bryostatin agent (e.g., as described herein) to enhance expression or surface presentation of the target antigen, and then treated with the agent to detect the level of cancer in the patient.

[0176] In some embodiments of the methods of treating cancer, the method further comprises administering to the patient at least one additional anti-cancer therapy, wherein the additional anti-cancer therapy is selected from radiation therapy, chemotherapy, immunotherapy, checkpoint inhibitors, surgery, and vascular-targeted therapy.

[0177] In some embodiments of the methods of treating cancer, the method further comprises evaluating one or more biomarkers in the subject's sample to assay the cancer status.

[0178] In some embodiments of the methods of treating cancer, the bryostatin agent is an analog of bryostatin 1, and the tolerable dose range of the bryostatin agent is improved by 50% or more relative to bryostatin 1. In certain cases, the tolerable dose range of the bryostatin agent is improved by 50% or more, e.g., 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or even more, relative to bryostatin 1. In certain cases, the tolerable dose range of the bryostatin agent is improved by 2-fold or more, e.g., 3-fold or more, 4-fold or more, 5-fold or more, or even more, relative to bryostatin 1.

[0179] Bryostatin As used herein, the terms "bryostatin compound" and "bryostatin agent" are used interchangeably to refer to compounds having the basic bryostatin pharmacophore, which is based on the bryostatin natural product and, in some cases, has a backbone characterized by a ring containing a macrocyclic lactone with three embedded six-membered rings (e.g., tetrahydropyran rings designated as A, B, and C rings), and a configuration of carbon atoms numbered C1 through C26, as exemplified in the structure of bryostatin 1 shown below. The backbone lactone is defined by the bond between the C1 carbonyl and the oxygen of the C25 hydroxyl group. TIFF2025121975000002.tif76128

[0180] The bryostatin skeleton may contain an alkene between C16 and C17 and an exocyclic alkene at the C13 and C21 positions. The bryostatin skeleton may contain specific stereogenic arrangements, for example, at C3, C5, C7, C9, C11, C15, C19, C20, and C23, C25, and / or C26. Various substituents and derivative groups (e.g., ester or ether groups) may be included in the present bryostatin compounds (e.g., as described herein). Naturally occurring bryostatins, originally isolated from marine exocysts, comprise a family of approximately 21 known compounds. The terms "bryostatin compound" and "bryostatin agent" are intended to include both naturally occurring bryostatin compounds, such as bryostatin 1, and "bryostatin analog compounds," which include non-naturally occurring bryostatin analogs and derivative compounds of interest that retain functional groups necessary for biological activity.

[0181] The terms "bryostatin compound" and "bryostatin agent" are intended to include compounds that have the same basic bryostatin pharmacophore, i.e., three hydrogen bond donors and acceptors that provide its binding function, and a three-dimensional spatial arrangement of lipid domains that provides its binding to membranes and the functions that result therefrom. The bryostatin pharmacophore, C1 domain model, non-C1 domain target model (e.g., regulated by bryostatin or its analogs), or PKC pharmacophore model was introduced by Wender's team in 1986 (see, e.g., Wender, PNAS, 1986, 83, 4214-4218), which was extended to bryostatin in 1988 (see, e.g., Wender, PNAS, 1988, 85, 7197-7201), leading to the design of the first bryostatin analogs (see, e.g., Wender, JACS, 1998, 120, 4534-4535; and Wender, PNAS, 1998, 95, 6624-6629, the disclosures of which are incorporated herein by reference in their entireties). This pharmacophore model is described in U.S. Pat. No. 8,735,609, the disclosure of which is incorporated herein by reference in its entirety. Bryostatin compounds can be broadly described as having two major regions, referred to herein as the "recognition domain" (or pharmacophore region) and the relatively lipophilic "spacer domain" (or linker region). The recognition domain has structural features similar to those of C17 through C26 through C1, including the C ring formed in part by atoms C19 through C23 of the native bryostatin macrocycle and the lactone bond between C1 and C25. The other spacer domain connects atoms corresponding to C1 through C17 of the native bryostatin macrocycle, substantially maintaining the relative distance between the C1 and C17 atoms and the orientation of the C1C2 and C16C17 bonds.In addition to maintaining the recognition domain in an active conformation, the spacer domain provides a moiety that can be easily derivatized by any convenient synthetic methodology, thereby providing analogs with improved in vivo stability and pharmacological properties (e.g., by modulating the side effect profile) while retaining biological activity. Exemplary synthetic procedures for obtaining bryostatin 1 and bryostatin analog compounds are described in Wender et al. Science 2017, 358 (6360), 218-223 and International Publication No. PCT / US2017 / 054158, filed September 28, 2017, the disclosures of which are also incorporated herein by reference. The linker region of the bryostatin family can vary significantly to provide analogs that retain bryostatin-like pan-PKC isoform binding selectivity, binding selectivity for other protein targets with C1 domains, binding selectivity and affinity for other non-C1 domain targets, and other analogs that exhibit selectivity and affinity for only one or more PKC isoforms. Thus, a wide variety of linkers can be used to retain the binding activity of bryostatin 1 or to create complementary selectivity. Such selectivity influences the translocation of PKC and other target proteins, and the therapeutic activity of PKC, as well as off-target effects. In some cases, bryostatin compounds include a linker moiety, L, that is a linear, cyclic, or polycyclic linker moiety comprising a continuous chain of 6 to 14 chain atoms, one embodiment of which defines the shortest path from C25 through C1 to C17. The distance "d" should be about 2.5 to 5.0 angstroms (as determined experimentally, e.g., by NMR spectroscopy), preferably about 3.5 to 4.5 angstroms, and most preferably about 4.0 angstroms, e.g., about 3.92 angstroms. Thus, L may consist solely of a linear chain of atoms connecting C17 through C1 to C25, or it may contain one or more ring structures that assist in connecting C17 through C1 to C25.In certain cases, the linker region contains a lactone group (-C(=O)O-) or a lactam group (-C(=O)NH-), which connects to C25 of the recognition region by analogy with the C1 lactone moiety present in native bryostatin. In addition, the linker may contain a hydroxyl group similar to the C3 hydroxyl found in native bryostatin, thus allowing for the formation of an intramolecular hydrogen bond between the C3 hydroxyl of the linker and the C19 hydroxyl group of the recognition region (and, optionally, the oxygen of the native B ring). In some embodiments, the linker terminates in -CH(OH)CHC(=O)O- and is attached to C25 of the recognition region by an ester (or lactone, if cyclic) bond. Exemplary linker domains are shown below. TIFF2025121975000003.tif41128

[0182] For all bryostatin compounds and their synthetic precursors described herein, it is understood that a numbering scheme may be used to refer to atoms corresponding to those of the macrocyclic ring and attached substituents as described above for the basic bryostatin skeleton. Bryostatin compounds of interest include, but are not limited to, naturally occurring bryostatins, e.g., bryostatin 1, bryostatin 2, and bryostatin 3, and bryostatin analogs, e.g., those described in U.S. Pat. No. 8,735,609, U.S. Pat. No. 7,256,286, U.S. Pat. No. 8,816,122, U.S. Pat. No. 9,096,550, and International Publication No. PCT / US2017 / 054158, the disclosures of which are incorporated herein by reference.

[0183] Various novel bryostatin compounds are described herein and are obtainable by the methods disclosed in International Publication No. PCT / US2017 / 054158. In some cases, the bryostatin compounds have activity as protein kinase C modulators both in vitro and in vivo. In some cases, the bryostatin compounds have PKC isoform selectivity. In certain cases, the bryostatin compounds bind to the C1 domain of PKC. In certain cases, the bryostatin compounds have activity as modulators of signaling protein targets that contain a C1 domain. Any convenient C1 domain-containing protein can be targeted for modulation by the bryostatin compounds. Exemplary C1 domain-containing proteins of interest include, but are not limited to, PKC, PKD, chimerin, diacylglycerol kinase, Unc-13 and Munc-13, guanine nucleotide exchange factors, myotonic dystrophy kinase-related Cdc42-binding kinase, and the like. Protein targets having a "C1 domain" include AKAP13, ARAF, ARHGAP29, ARHGEF2, BRAF, CDC42BPA, CDC42BPB, CDC42BPG, CHN1, CHN2, CIT, DGKA, DGKB, DGKD, DGKE, DGKG, DGKH, DGKI, DGKK, DGKQ, DGKZ, GMIP, HMHA1, KSR1, KSR2, MYO9A, MYO9B, PDZD8, PRKCA, PRKCB1, PRKCD, and PRKCE. , PRKCG, PRKCH, PRKCI, PRKCN, PRKCQ, PRKCZ, PRKD1, PRKD2, PRKD3, RACGAP1, RAF1, RASGRP, RASGRP1, RASGRP2, RASGRP3, RASGRP4, RASSF1, RASSF5, ROCK1, ROCK2, STAC, STAC2, STAC3, TENC1, UNC13A, UNC13B, UNC13C, VAV1, VAV2, and VAV3. In certain cases, the bryostatin compounds have activity as modulators of C1 domain-free signaling protein targets.

[0184] Bryostatin compounds of interest include, but are not limited to, compounds featuring variations of the C7 ester. Any number of ester or ether substituents can be incorporated at the C7 position by using specific anhydrides in the esterification reaction, or by performing etherification using, for example, any number of alkyl bromides or other convenient etherification reagents. In some cases, the compounds have the same A-ring functionalization at the C7 and C9 positions as the target native bryostatin, such as bryostatin 1.

[0185] In some embodiments, the bryostatin compound has the formula (XXIVb): TIFF2025121975000004.tif59128, wherein W 1 is alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkyl, or substituted alkyl; If the covalent bond marked "b" is a double bond, it is Z. 2 is =CR 5 R 6 or =NR 7 and when the covalent bond designated "b" is a single bond, Z 2 HA-OR 8 or -N(R 7 )2; X 1 is H or OR 11 and; Y 1 is H or OR 12 and; R 5 , R 6 , R 7 , and R 8 are each independently H, alkyloxycarbonyl (e.g., —COMe), substituted alkyloxycarbonyl, alkyl, or substituted alkyl; R 11 is acyl, substituted acyl, alkyl, or substituted alkyl; Each R 12 are independently H, alkyl, or substituted alkyl; R 13is alkyl or substituted alkyl; R 14 and R 15 is independently an H or a pro moiety; R 16 is H, alkyl, or substituted alkyl.

[0186] In some embodiments of Formula (XXIVb), the bryostatin compound has the formula (XXIIb): TIFF2025121975000005.tif53128, wherein R 4 is alkyl or substituted alkyl; If the covalent bond marked "b" is a double bond, it is Z. 2 is =CR 5 R 6 or =NR 7 and; When the covalent bond marked "b" is a single bond, it is Z. 2 HA-OR 8 or -N(R 7 )2; R 5 , R 6 , R 7 , R 8 are each independently H, alkoxycarbonyl (e.g., —COMe), substituted alkoxycarbonyl, alkyl, or substituted alkyl; R 11 is H, acyl, substituted acyl, alkyl, or substituted alkyl; Each R 12 are independently H, alkyl, or substituted alkyl; R 13 is H, alkyl, or substituted alkyl; R 14 and R 15 is independently an H or a pro moiety; R 16 is H, alkyl, or substituted alkyl.

[0187] In some embodiments of Formula (XXIIb), the bryostatin compound has the formula (XXIIIb): I have TIFF2025121975000006.tif52128.

[0188] In some cases of formula (XXIIb) and (XXIVb), R 16 is methyl. In some cases of formulas (XXIIb), (XXIIIb), and (XXIVb), R 14 is H or a promoiety. In some cases of formulas (XXIIb), (XXIIIb), and (XXIVb), R 13 is methyl. In some cases of formula (XXIIb) and (XXIVb), R 12 is methyl. In some cases of formula (XXIIb) and (XXIVb), R 12 is H. In some cases of formulas (XXIIb), (XXIIIb), and (XXIVb), R 11 is acetyl. In some cases of formulas (XXIIb), (XXIIIb), and (XXIVb), R 11 is H. In some cases of formula (XXIIb) and (XXIIIb), R 4 is C3H7. In some cases of formulas (XXIIb), (XXIIIb), and (XXIVb), R 15 is H.

[0189] In some embodiments, the bryostatin compound has the formula (XXXI): TIFF2025121975000007.tif56128, or a solvate, hydrate, or prodrug form thereof and / or a salt thereof, wherein R 4 is alkyl or substituted alkyl; When the covalent bond marked "b" is a double bond, Z 2 is CR 5 R 6 or NR 7 and; When the covalent bond marked "b" is a single bond, Z 2 is OR 8 or N(R 7)2; R 5 , R 6 , R 7 , and R 8 are each independently H, halogen, alkyloxycarbonyl, substituted alkyloxycarbonyl, alkyl, or substituted alkyl; R 11 is acyl, substituted acyl, alkyl, or substituted alkyl; R 12 is H, alkyl, or substituted alkyl; R 13 is H, alkyl, or substituted alkyl; R 14 and R 15 is independently H, a hydroxyl protecting group, or a promoiety.

[0190] In some cases of formula (XXXI), R 4 is propyl. In some cases of formula (XXXI), R 11 is alkyl or substituted alkyl. In some cases of formula (XXXI), R 11 is acyl or substituted acyl. In some cases of formula (XXXI), R 12 is alkyl or substituted alkyl.

[0191] In some cases of formula (XXXI), the covalent bond designated "b" is a double bond, and Z 2 is NR 7 and R 7 is H, alkyloxycarbonyl, substituted alkyloxycarbonyl, alkyl, or substituted alkyl. In certain cases, Z 2 is CFC0R', where R' is alkyl (e.g., methyl). In some cases of formula (XXXI), the covalent bond designated "b" is a single bond and Z 2 is OR 8 or N(R 7 )2 and R 7 and R 8are each independently H, alkyloxycarbonyl (e.g., —COMe), substituted alkyloxycarbonyl, alkyl, or substituted alkyl.

[0192] In some cases, R 13 is an alkyl containing at least two carbons or one substituted alkyl. In some cases of formula (XXXI), R 4 is a substituted alkyl. In some cases of formula (XXXI), R 14 is the pro part.

[0193] It is understood that all bryostatin analog compounds (e.g., as described herein) may be modified by a subsequent C-H oxidation reaction (e.g., near the end of a synthetic sequence) to include aliphatic (sp3-hybridized) carbons in the backbone (C1-C26) that are substituted with alkyl, substituted alkyl, alkoxy, substituted alkoxy, amino, substituted amino, azide, or halogen (e.g., F, Cl). In some cases, the method further comprises incorporating a substituent of interest at one of the C1-C26 positions of the backbone by a subsequent C-H oxidation reaction.

[0194] Bryostatin compounds of interest include, but are not limited to, those featuring variations of the C19 hemiketal. Any number of ketals can be incorporated, for example, by utilizing any convenient alcohol solvent.

[0195] Bryostatin compounds of interest include, but are not limited to, those featuring variations at the C13 position. Variability at the C13 position can be easily achieved, for example, by olefination or imine formation. In some cases, the C13 ketone can be reduced to an alcohol, followed by acylation or etherification. In certain cases, the C13 ketone can be modified by reductive amination using any convenient amino reactant. In some cases, the E isomer of the C13 enoate can be obtained. In certain cases, the C13 ketone is modified to form a spirocyclic ring. In certain cases, the C13 ketone is modified to form a phosphate, a hetero group, or an organoselenium group.

[0196] In some embodiments, the bryostatin compound has the formula (XXXII): It is an analogue of natural bryostatin with TIFF2025121975000008.tif55128.

[0197] Flexibility at the C13 position can also be achieved by modifying the C13 alkene. In some cases, the C13 alkene is substituted, for example, with an alkyl or halogen group. In some cases, the C13 alkene can be reduced and optionally further substituted, for example, with an alkyl, alkoxy, halogen group, etc. In some cases, the alkene at the C13 position can be modified to form a carbocycle or heterocycle, for example, by epoxidation, cyclopropanation, aziridine formation, thiirane formation, cycloadduct formation, or spirocyclization. In some cases, carbocycles are formed with three or more carbons, for example, four or more carbons, for example, five or more carbons, for example, six or more carbons, or more. In some cases, three-membered heterocycles are formed. In some cases, larger heterocycles are formed, for example, four-membered, five-membered, six-membered, or even larger heterocycles.

[0198] In some embodiments, the bryostatin compound has the formula (XXXIA)-(XXXIC): TIFF2025121975000009.tif123137, wherein: R 4 is alkyl or substituted alkyl; If the covalent bond marked "b" is a double bond, it is Z. 2 is =CR 5 R 6 or =NR 7 and; When the covalent bond marked "b" is a single bond, it is Z. 2 HA-OR 8 or -N(R 7 )2; R 5 , R 6 , R 7 , and R 8 are each independently H, alkyloxycarbonyl (e.g., —COMe), substituted alkyloxycarbonyl, alkyl, or substituted alkyl; R 11 is H, acyl, substituted acyl, alkyl, or substituted alkyl; R 12 is H, alkyl, or substituted alkyl; R 13 is H, alkyl, or substituted alkyl; R 14 is the H, or pro part.

[0199] In some cases of any of formulas (XXXIA) to (XXXIC), R 4 is selected from propyl, butyl, pentyl, hexyl, heptyl, or octyl. In some cases of formula (XXXIA), R 4 is propyl. In some cases of formula (XXXIB), R 4 is pentyl. In some cases of formula (XXXIC), R 4 is heptyl. In some cases of any of formulas (XXXIA) to (XXXIC), R 11 is alkyl or substituted alkyl. In some cases of any of formulas (XXXIA) to (XXXIC), R11 is acyl or substituted acyl. In some cases of formulae (XXXIA) to (XXXIC), R 12 is alkyl or substituted alkyl. In some cases, R 12 is hydrogen. In some cases of any of formulas (XXXIA) to (XXXIC), R 13 is hydrogen. In some cases of any of formulas (XXXIA) to (XXXIC), R 13 is alkyl (e.g., methyl). In some cases of formulae (XXXIA) to (XXXIC), R 14 is hydrogen.

[0200] In some cases of any of formulas (XXXIA)-(XXXIC), the covalent bond designated "b" is a double bond, and Z 2 is CR 5 R 6 and R 5 , R 6 are each independently H, alkyloxycarbonyl, or substituted alkyloxycarbonyl. 5 is H and R 6 is a substituted alkyloxycarbonyl. In some cases, R 5 is H and R 6 is alkyloxycarbonyl (e.g., -CO2Me or CO2Et). In some cases, R 5 and R 6 are both H. In some cases of any one of formulas (XXXIA) through (XXXIC), the covalent bond designated "b" is a single bond, and Z 2 is OR 8 and R 8 is selected from H, alkyloxycarbonyl (e.g., —COMe), and substituted alkyloxycarbonyl. In some cases, R 8 is a substituted alkyloxycarbonyl. In some cases, R 8 is alkyloxycarbonyl (e.g., -COMe). In some cases, R 8 is H.

[0201] In some embodiments, the bryostatin compound is an analog of a naturally occurring bryostatin described by any of the formulas (XXXIA): In some cases, the structure of formula (XXXIA) is described by any of the following structures: TIFF2025121975000010.tif211147TIFF2025121975000011.tif157141TIFF2025121975000012.tif150159

[0202] In some embodiments, the bryostatin compound is an analog of a naturally occurring bryostatin described by any of formulas (XXXIB): In some cases, the structure of formula (XXXIB) is described by any of the following structures: TIFF2025121975000013.tif68146

[0203] In some embodiments, the bryostatin compound is an analog of a naturally occurring bryostatin described by any of the formulas (XXXIC). In some cases, the structure of formula (XXXIC) is described by any of the following structures: TIFF2025121975000014.tif66148

[0204] In some embodiments, the bryostatin compound has the formula (XXXIII): TIFF2025121975000015.tif57128, wherein W 1 is alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, allenyl, substituted allenyl, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteoraryl, heteroalkyl, substituted heteroalkyl, heterocyclic, substituted heterocyclic, or carbon chains containing rings and substituted rings that contain oxygen or nitrogen atoms and / or cyclalkyl, cycloalkenyl, etc. (e.g., PEG or modified PEG groups); X 1 is H or OR11 and; X 2 and X 3 is independently selected from H, halogen, alkyl, substituted alkyl, alkoxy, amine, substituted amine, amide, substituted amide, acyl, hydroxyl, heteroalkyl, heteroaryl, substituted heteroalkyl, substituted heteroaryl, phosphate, organoselenium, thio, substituted thio; or X 2 and X 3 and combine to form a carbocyclic or heterocyclic ring, such as a cyclopropane, an epoxide, an aziridine, a thiirane, a 4-membered spiro ring, a 5-membered spiro ring, or a 6-membered spiro ring; Y 1 is H or OR 12 and; Each R 12 are independently H, alkyl, or substituted alkyl; R 13 is H, alkyl, or substituted alkyl; R 16 is H, alkyl, or substituted alkyl; R 14 and R 15 is independently H, a hydroxyl protecting group, or a promoiety; or a solvate, hydrate, or prodrug form thereof and / or a salt thereof.

[0205] In certain embodiments of the bryostatin analog of formula (XXXIII), X 2 and X 3 are both hydrogen. In certain cases, X 2 or X 3 One of the X is hydroxyl. 2 and X 3 are both hydroxyls.

[0206] In some embodiments of the bryostatin analog of Formula (XXXIII), the compound is described by any of the following structures: TIFF2025121975000016.tif66135

[0207] Bryostatin compounds of interest include, but are not limited to, those that feature variations at the C12 or C14 position, or at both the C12 and C14 positions. Variability in the B ring can be achieved by modifying the C12 or C14 carbon, or both the C12 and C14 carbons, of any structure and formula disclosed herein. In some cases, the C12 carbon is alkylated. In some cases, the C14 carbon is alkylated. In some cases, the C12 carbon is substituted with a halogen. In some cases, the C14 carbon is substituted with a halogen. In some cases, the C12 or C14 position is independently substituted with a group selected from the group consisting of substituted alkyl, alkoxy, amine, substituted amine, amide, substituted amide, acyl, hydroxyl, heteroalkyl, heteroaryl, substituted heteroalkyl, substituted heteroaryl, phosphate, phosphoryl, sulfate, sulfonyl, organoselenium, thio, and substituted thio.

[0208] In some embodiments, the bryostatin compound has the formula (XXXIV): TIFF2025121975000017.tif60128, wherein W 1 is alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, allenyl, substituted allenyl, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heteroalkyl, substituted heteroalkyl, heterocyclic, substituted heterocyclic, or carbon chains containing oxygen or nitrogen atoms and / or rings and substituted rings containing cycloalkyl, cycloalkenyl, etc. (e.g., PEG or modified PEG groups); If the covalent bond marked "b" is a double bond, it is Z. 2 is CR 5 R 6 or NR 7 and; When the covalent bond marked "b" is a single bond, it is Z. 2 is OR 8, phosphate, phosphoryl, thio group, sulfate, sulfonyl, organoselenium group, or N(R 7 )2; R 5 , R 6 , R 7 , and R 8 are each independently H, halogen, alkyloxycarbonyl, substituted alkyloxycarbonyl, alkyl, or substituted alkyl; X 1 is H or OR 11 and; X 4 and X 5 is independently selected from H, halogen, alkyl, substituted alkyl, alkoxy, amine, substituted amine, amide, substituted amide, acyl, hydroxyl, heteroalkyl, heteroaryl, substituted heteroalkyl, substituted heteroaryl, phosphate, organoselenium, thio, and substituted thio; Y 1 is H or OR 12 and; Each R 12 are independently H, alkyl, or substituted alkyl; R 13 is H, alkyl, or substituted alkyl; R 16 is H, alkyl, or substituted alkyl; R 14 and R 15 is independently H, a hydroxyl protecting group or a promoiety; or a solvate, hydrate, or prodrug form thereof and / or a salt thereof.

[0209] Interesting bryostatin compounds include, but are not limited to, those that feature variations at the C26 position. By modifying the synthesis method of the southern hemisphere fragment, variations can be introduced into the C26 alcohol. In some cases of the present bryostatin compounds, a hydroxy group at the C26 position is necessary for compound activity. In some cases, variations at the C26 position provide a prodrug form of the interested bryostatin compound, where the prodrug form can be converted to a free C26 hydroxyl group in vivo.

[0210] In some embodiments, the compounds are provided in prodrug form. "Prodrug" refers to a derivative of an active agent that requires conversion in the body to release the active agent. In certain embodiments, the conversion is enzymatic. Prodrugs are often, but not necessarily, pharmacologically inactive until converted to an active agent. "Promoiety" refers to a form of protecting group that, when used to mask a functional group in an active agent, converts the active agent into a prodrug. In some cases, the promoiety is attached to the drug by a bond that is cleaved in vivo by enzymatic or non-enzymatic means. Any convenient prodrug form of the compounds can be prepared, for example, according to the strategies and methods described in Rautio et al. ("Prodrugs: design and clinical applications", Nature Reviews Drug Discovery 7, 255-270 (February 2008)).

[0211] Prodrugs of bryostatin compounds include C26-specific bryostatin analogs. Methods for the synthesis of southern hemisphere fragments can provide for derivatization of the C26 alcohol with, for example, an ester. In some cases, the introduction of an ester group at the C26 position can inactivate the resulting bryostatin derivative. In certain embodiments, the C26 ester group can be cleaved, for example, chemically (e.g., at a specific pH, by photochemical means) or biologically (e.g., by the action of endogenous esterases, reduction of a gamma or epsilon disulfide bond, promotion of intramolecular transthioesterification, and release of a free hydroxyl group) to release a bryostatin compound with a free C26 hydroxyl group. This prodrug strategy allows bryostatin compounds of interest to be easily modified to improve their pharmacological properties, such as their PK (pharmacokinetics) and ADME (absorption, distribution, metabolism, and excretion) properties, while maintaining bryostatin activity, allowing for sustained release as a free drug following administration of the compound.

[0212] Cleavable linkages may include groups that can be cleaved hydrolytically, enzymatically, or otherwise in vivo. Inert groups may range from alkyl groups (e.g., selected to provide a specific cleavage rate) to oligopeptides or lipids (e.g., to enhance cellular uptake). Modifications may include, but are not limited to, esters, carbonates, carbamates, and ethers, all of which may contain alkyl, alkenyl, alkynyl, amines, hydroxyl, guanidinium, carbocycles, and heterocycles.

[0213] In some embodiments, the compound of formula (XXXV): Bryostatin 1 is modified to form the structure shown in TIFF2025121975000018.tif58128.

[0214] In the above formula, R 17is selected from esters, carbonates, carbamates, and ethers, all of which may be optionally substituted with one or more groups selected from alkyls, alkenyls, alkynyls, amines, hydroxyls, disulfides, guanidinium, carbocycles, and heterocycles. In some cases, the acetate group at C7 is replaced with an H atom, an ester, carbonate, carbamate, or ether, all of which may be optionally substituted with one or more groups selected from alkyls, alkenyls, alkynyls, amines, hydroxyls, disulfides, guanidinium, carbocycles, and heterocycles.

[0215] Bryostatin compounds of interest include, but are not limited to, those featuring variations at the C26 methyl position. The method for preparing the southern hemisphere fragment may be adapted to prepare analogs with any convenient C26 substituent at the C26 methyl position.

[0216] Bryostatin compounds of interest include, but are not limited to, those featuring variations at the C20 ester position. Any convenient ester group can be incorporated onto the C20 hydroxyl group (e.g., as described herein). In some cases, the ester group is an alkyne containing a precursor of the octadienoate group, such as the octadienoate present at the C20 ester position of bryostatin 1. By modifying the Southern Hemisphere synthesis, analogs can be prepared by esterifying the C20 alcohol moiety with a variety of ester groups. It is understood that in some cases, all of the ester groups described herein can be considered the corresponding acyl or substituted acyl substituents of the C20 hydroxyl. In some cases, the ester group is an alkyl or substituted alkyl ester. In some cases, the ester group is an aryl or substituted aryl ester. In some cases, the ester group is an alkenyl or substituted alkenyl. In some cases, the ester group is an alkynyl or substituted alkynyl ester.

[0217] Bryostatin compounds of interest include, but are not limited to, those featuring variations at the C21 enoate ester position. Any convenient ester group (e.g., as described herein) can be incorporated onto the C20 hydroxyl group.

[0218] Aspects of the present disclosure include bryostatin compounds, their salts (e.g., pharmaceutically acceptable salts), and / or solvates, hydrates, and / or prodrug forms thereof. Additionally, for any compounds described herein having one or more chiral centers, unless the absolute stereochemistry is specified, it is understood that each center may independently be of the R or S configuration, or a mixture thereof. It will be understood that the present disclosure is intended to encompass all variations of salts, solvates, hydrates, prodrugs, and stereoisomers.

[0219] In some embodiments, the bryostatin compounds, or prodrug forms thereof, are provided in the form of pharmaceutically acceptable salts. Compounds containing amine, imine, or nitrogen-containing groups may be basic in nature and therefore capable of reacting with any number of inorganic and organic acids to form pharmaceutically acceptable acid addition salts.

[0220] In some embodiments, the compound, its prodrug, stereoisomer, or salt is provided in the form of a solvate (e.g., hydrate). As used herein, the term "solvate" refers to a complex or aggregate formed by one or more solute molecules, such as a prodrug or a pharmaceutically acceptable salt thereof, with one or more solvent molecules. Such solvates are typically crystalline solids having a substantially fixed molar ratio of solute to solvent. Representative solvents include, for example, water, methanol, ethanol, isopropanol, acetic acid, etc. When the solvent is water, the solvate formed is a hydrate.

[0221] cell surface antigen As used herein, cell surface antigens are intended to include neoantigens and antigens derived from delivery or expression of mRNA. In certain cases, the cell surface antigen is endogenous. In certain cases, the cell surface antigen is an endogenous neoantigen. In certain cases, the cell surface antigen is derived from administration of mRNA and is expressed to produce a protein (e.g., corresponding to the mRNA message) that produces the antigen. In this regard, any protein can be expressed using mRNA, and externalization of that protein can create a novel antigen. The protein produced can be any protein that produces an antigen, and the bryostatin agent can act to enhance the translocation and presentation of the expressed antigen to the cell surface and prolong its persistence on the cell surface.

[0222] In some embodiments, the therapeutic agent is one of the following: TIFF2025121975000019.tif204166TIFF2025121975000020.tif67166.

[0223] Enhanced CAR-T cell therapy methods The present disclosure contemplates a method for modulating a T cell-mediated immune response against a target cell population in a subject using CAR-T cell therapy and a bryostatin agent. The present disclosure also contemplates a method for modulating an NK cell-mediated immune response against a target cell population in a subject using CAR-NK cell therapy and a bryostatin agent. Certain embodiments contemplate a method for modulating a T cell-mediated or NK cell-mediated immune response against a target cell population in a subject, the method comprising: (a) introducing into the subject a plurality of therapeutically effective cells genetically modified to express a chimeric antigen receptor, the chimeric antigen receptor comprising at least one antigen-specific targeting region capable of binding to the target cell population, wherein the chimeric antigen receptor targeting region is capable of inducing activation-induced cell death upon binding to the target cell population; and (b) administering to the subject a therapeutically effective amount of a bryostatin agent sufficient to prevent or limit the activation-induced cell death. In certain embodiments, the CAR comprises an antigen-binding domain that specifically recognizes a CD22 target cell population. In certain embodiments of the present disclosure, the bryostatin agent enhances the function of activated memory CD8+ T cells. In other embodiments, the dosage of the bryostatin agent is sufficient to enhance cytotoxic function. In certain cases, the dosage of the bryostatin agent is sufficient to enhance the activity of CAR T cells by increasing the number of cell surface antigens on target cells.

[0224] Embodiments are contemplated in which the bryostatin agent is administered prior to, simultaneously with, or subsequent to the administration of the therapeutically effective plurality of cells. In certain embodiments of the present disclosure, the bryostatin agent is administered subcutaneously.

[0225] In certain cases, the bryostatin agent is contacted with a plurality of cells ex vivo prior to administration to the subject. In certain embodiments, the cells contacted with the bryostatin agent ex vivo are removed from the subject to be treated (e.g., autologous cells). In other embodiments, the cells contacted with the bryostatin agent ex vivo are removed from a donor (e.g., allogeneic cells).

[0226] The chimeric antigen receptor of the present disclosure can comprise two or more polypeptide chains. In some embodiments, the receptor is a T cell receptor (TCR). In some embodiments, the TCR comprises one or more CD3 polypeptides, e.g., one or more CD3ζ polypeptides. In some embodiments, the cell surface receptor is a TCR comprising a protease cleavage site located between the variable region of the alpha chain (αv) and the constant region of the alpha chain (αc); between the constant region of the alpha chain (αc) and the transmembrane region of the alpha chain (αt); between the variable region of the beta chain (βv) and the constant region of the beta chain (βc); between the constant region of the beta chain (βc) and the transmembrane region of the beta chain (βt); between the transmembrane domain of the CD3ζ polypeptide and the cytoplasmic domain of the CD3ζ polypeptide, if a CD3ζ polypeptide is present; or any combination thereof, if the TCR has two or more protease cleavage sites. In some cases, one or more protease cleavage sites (and optionally one or more corresponding proteases) may be located (1) between the variable region of the alpha chain (αv) and the constant region of the alpha chain (αc); (2) between the constant region of the alpha chain (αc) and the transmembrane region of the alpha chain (αt); (3) between the variable region of the beta chain (βv) and the constant region of the beta chain (βc); (4) between the constant region of the beta chain (βc) and the transmembrane region of the beta chain (βt); and (5) between the transmembrane region of CD3ζ and the cytoplasmic domain of CD3ζ. In some embodiments, the TCRs of the present disclosure comprise a cleavage site and protease (in cis configuration), e.g., present within a linker. In some embodiments, when the cell surface receptor is a TCR, the protease is provided in trans, i.e., the protease is not part of the polypeptide chain containing the cleavage site. In some embodiments, when the protease is provided in trans, the protease is tethered to another chain of the TCR. For example, if the cleavage site is located in the α chain, the protease is provided on the β chain, or vice versa. Also, by way of example, the cleavage site may be located in one of the CD3 chains (epsilon, gamma, delta, or zeta) and the protease may be provided in another CD3 chain.

[0227] The extracellular binding domain of the modified receptor (e.g., CAR or modified TCR) of the present disclosure can specifically bind to an antigen, e.g., a cell surface antigen, e.g., an antigen on the surface of a cancer cell or an MHC molecule-associated antigenic peptide. The extracellular binding domain can be, for example, about 10 5 M -1 Affinity or K a (i.e., the equilibrium association constant of a particular binding interaction, in units of 1 / M). In certain embodiments, the extracellular binding domain "specifically binds" to an antigen if it binds to or binds to the antigen with a constant of about 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 , or 10 13 M -1 Higher than or equal to K a "High affinity" binding is defined as binding to an antigen with a binding affinity of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , at least 10 13 M -1 , or higher K a Alternatively, affinity can be expressed as the equilibrium dissociation constant (K) of a particular binding interaction, in units of M. D ) (for example, 10 -5 M~10 -13 In some embodiments, specific binding can be defined as a binding activity between the extracellular binding domain and the target protein (e.g., a nucleotide sequence of about 10 M or lower). -5Lower than or equal to M, approximately 10 -6 Lower than or equal to M, approximately 10 -7 Lower than or equal to M, approximately 10 -8 Lower than or equal to M, approximately 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M or lower K D The binding affinity of the extracellular binding domain to the target antigen can be readily determined by conventional techniques, such as competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, using surface plasmon resonance (SPR) (e.g., BIAcore 2000 machine, according to the general procedures outlined by the manufacturer), radioimmunoassay, etc.

[0228] The extracellular binding domain binds to a target antigen of interest, e.g., a specific antigen on the surface of a target cell, and can comprise or consist of an antibody (e.g., a single chain antibody such as an scFv), a receptor (e.g., a variable lymphocyte receptor), a receptor fragment (e.g., an Fc receptor fragment), a ligand, a cytokine, a DARPin, an adnectin, a nanobody, and a peptide.

[0229] In some embodiments, the extracellular binding domain of the CAR includes a single-chain antibody, non-limiting examples of which include a single-chain variable fragment (scFv). The single-chain antibody can be a monoclonal single-chain antibody (e.g., as described herein), a chimeric single-chain antibody, a humanized single-chain antibody, a fully human single-chain antibody, etc. Suitable extracellular binding domains include those described in Labanieh et al. (2018) Nature Biomedical Engineering 2:377-391, the entire disclosure of which is incorporated herein by reference for all purposes. In some embodiments, the extracellular binding domain of the CAR is an antibody approved by the U.S. Food and Drug Administration and / or the European Medicines Agency (EMA) for use as a therapeutic antibody (e.g., to induce antibody-dependent cellular cytotoxicity (ADCC) of specific disease-associated cells in patients), or a fragment thereof (e.g., a single-chain version of such an antibody, e.g., an scFv version of the antibody) that retains the ability to bind to the target molecule.

[0230] In another aspect, the extracellular binding domain of the CAR specifically binds to a molecule on the surface of a target cell. The target cell can be any cell type of interest. For example, the target cell can be a genetically and / or phenotypically normal cell. In other embodiments, the target cell can be a genetically and / or phenotypically abnormal cell. Abnormal cells of interest include, but are not limited to, cancer cells, cells of the tumor microenvironment (e.g., tumor stromal cells), such as cancer-associated fibroblasts (CAFs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), tumor endothelial cells (TECs), and the like. See, for example, Labanieh et al. (2018) Nature Biomedical Engineering 2:377-391. "Cancer cell" refers to a cell that exhibits a neoplastic cell phenotype, which may be characterized, for example, by one or more of aberrant cell growth, aberrant cell proliferation, loss of density-dependent growth inhibition, the potential for anchorage-independent growth, the ability to promote tumor growth and / or development in immunocompromised non-human animal models, and / or any suitable indicator of cellular transformation. "Cancer cell" is used interchangeably herein with "tumor cell," "malignant cell," or "cancerous cell," and includes cancer cells of solid tumors, semi-solid tumors, hematological malignancies (e.g., leukemia cells, lymphoma cells, myeloma cells, etc.), primary tumors, metastatic tumors, etc.

[0231] In certain embodiments, the CAR-expressing T cells or NK cells are effective in treating B-cell malignancies, CLL, ALL, B-ALL, leukemia, lymphoma, or solid tumors. In some cases, the solid tumor is selected from breast cancer, prostate cancer, bladder cancer, soft tissue sarcoma, lymphoma, esophageal cancer, uterine cancer, bone cancer, adrenal cancer, lung cancer, thyroid cancer, colon cancer, glioma, liver cancer, pancreatic cancer, kidney cancer, cervical cancer, testicular cancer, head and neck cancer, ovarian cancer, neuroblastoma, and melanoma.

[0232] A CAR can comprise an antigen-binding domain (e.g., an antibody such as an scFv), a transmembrane domain, and an intracellular signaling domain.

[0233] In some embodiments, a CAR comprises one or more linker sequences between various domains. A "variable region linking sequence" is an amino acid sequence that connects the heavy chain variable region to the light chain variable region and provides a spacer function compatible with the interaction of these two sub-binding domains, so that the resulting polypeptide retains the same specific binding affinity for the same target molecule as an antibody comprising the same light and heavy chain variable regions. In certain aspects, a linker separates one or more heavy or light chain variable domains, hinge domains, transmembrane domains, costimulatory domains, and / or primary signaling domains. In certain embodiments, a CAR comprises one, two, three, four, five, or more linkers. In certain embodiments, the linker is about 1 to about 25 amino acids in length, about 5 to about 20 amino acids in length, or about 10 to about 20 amino acids in length, or any amino acid length therebetween. In some embodiments, the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids in length.

[0234] In some embodiments, the binding domain of the CAR is followed by one or more spacer domains, which distance the antigen-binding domain from the effector cell surface (e.g., the surface of a T cell expressing the CAR) to allow for proper cell-cell contact, antigen binding, and / or activation. The spacer domain (and any other spacer domains, linkers, etc. described herein) may be derived from natural, synthetic, semi-synthetic, or recombinant sources. In certain embodiments, the spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domain may comprise the amino acid sequence of a hinge region of a native immunoglobulin or a modified immunoglobulin hinge region. In one embodiment, the spacer domain comprises the CH2 and / or CH3 of IgG1, IgG4, or IgD. Exemplary spacer domains suitable for use in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8α and CD4, which may be wild-type hinge regions derived from such molecules or variants thereof. In certain aspects, the hinge domain comprises a CD8α hinge region. In some embodiments, the hinge is a PD-1 hinge or a CD152 hinge.

[0235] The "transmembrane domain" (TM domain) is the portion of a CAR that fuses the extracellular binding moiety with the intracellular signaling domain and anchors the CAR to the cell membrane of a cell (e.g., an immune effector cell). The Tm domain can be derived from natural, synthetic, semi-synthetic, or recombinant sources. In some embodiments, the Tm domain is derived from (e.g., comprising at least a transmembrane region or functional portion thereof) the alpha or beta chain of the T cell receptor, CD35, CD3ζ, CD3γ, CD3δ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD-1.

[0236] In one embodiment, the CAR comprises a Tm domain derived from CD8α. In certain aspects, the CAR comprises a Tm domain derived from CD8α and a short oligo- or polypeptide linker, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, that connects the Tm domain of the CAR to the intracellular signaling domain. For example, a glycine-serine linker can be used as such a linker.

[0237] The "intracellular signaling" domain of a CAR refers to the portion of the CAR that is involved in transmitting a signal from the binding of the CAR to a target molecule / antigen to the inside of an immune effector cell, which signaling induces an effector cell function, such as activation, cytokine production, proliferation, and / or cytotoxic activity, such as the release of a cytotoxic factor into the target cell to which the CAR is bound, or other cellular response elicited by the binding of the target molecule / antigen to the extracellular CAR domain. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transmits an effector function signal and directs a cell to perform a particular function. When a truncated portion of the intracellular signaling domain is used, it can be used in place of the full-length intracellular signaling domain, so long as such truncated portion transmits the effector function signal. The term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain that is sufficient to transmit the effector function signal.

[0238] To fully activate a T cell, signals generated through the T cell receptor (TCR) alone are insufficient; a secondary or costimulatory signal is also required. Thus, T cell activation is mediated by two distinct classes of intracellular signaling domains: primary signaling domains, which initiate antigen-dependent primary activation by the TCR (e.g., the TCR / CD3 complex), and costimulatory signaling domains, which act antigen-independently to provide a secondary or costimulatory signal. Thus, a CAR of the present disclosure can comprise an intracellular signaling domain that includes one or more "co-stimulatory signaling domains" and a "primary signaling domain."

[0239] The primary signaling domain controls the primary activation of the TCR complex by stimulation or inhibition. Primary signaling domains that act by stimulation can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif (or "ITAM"). Non-limiting examples of primary signaling domains containing ITAMs suitable for use in the CARs of the present disclosure include those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79α, CD79β, and CD66δ. In certain embodiments, the CAR comprises one CD3ζ primary signaling domain and one or more costimulatory signaling domains. The intracellular primary signaling domain and the costimulatory signaling domain are operably linked to the carboxyl terminus of the transmembrane domain.

[0240] In some embodiments, CARs comprise one or more costimulatory signaling domains to enhance the efficacy and expansion of CAR-expressing T cells. As used herein, the term "co-stimulatory signaling domain" or "co-stimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule or an active fragment thereof. Examples of costimulatory molecules suitable for use in CARs contemplated in certain embodiments include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and ZAP70. In some embodiments, the CAR comprises one or more costimulatory signaling domains selected from the group consisting of 4-1BB, CD28, CD137, and CD134, and a CD3ζ primary signaling domain.

[0241] In certain aspects, the CAR of the present disclosure comprises an antigen-binding portion (e.g., a single-chain antibody such as an scFv) that binds to an antigen of interest; a transmembrane domain derived from a polypeptide selected from the group consisting of CD4, CD8α, CD154, and PD-1; one or more intracellular costimulatory signaling domains derived from a polypeptide selected from the group consisting of 4-1BB, CD28, CD134, and CD137; and an intracellular signaling domain derived from a polypeptide selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79α, CD79β, and CD66δ. Such CARs may further comprise a spacer domain, such as a CD8 alpha hinge, between the antigen-binding portion and the transmembrane domain.

[0242] Enhanced Targeted Anticancer Methods The present disclosure contemplates methods for enhancing targeted anti-cancer therapy by utilizing a bryostatin agent to modulate target cancer cells and selectively enhance antigen expression or cell surface presentation on the cancer cells. Certain embodiments contemplate methods for treating cancer in a subject, the method comprising: (a) introducing into the subject a therapeutically effective amount of a bryostatin agent (e.g., as described herein) that enhances cell surface antigen or neo-antigen presentation on the target cancer cells; and (b) administering to the subject a therapeutically effective amount of a therapeutic agent that specifically binds to the cell surface antigen and treats the subject's cancer. In certain embodiments, the subject is relapsed or refractory to targeted anti-cancer therapy.

[0243] In certain aspects of the present disclosure, target cancer cells present a therapeutically ineffective level of cell surface antigens on the target cell surface prior to treatment with a bryostatin agent. The bryostatin agent can enhance the cell surface antigens presented on the cell surface so that they are presented at a therapeutically effective level. In some cases, the bryostatin agent enhances the expression of the cell surface antigen. In some cases, the bryostatin agent enhances the translocation of the expressed cell surface antigen to the target cell surface. In some cases, the bryostatin agent enhances the retention of the cell surface antigen on the target cell surface. Thus, the dose of the bryostatin agent is sufficient to enhance the cytotoxic function of the surface antigen.

[0244] In some embodiments of the methods of treating cancer, the bryostatin agent enhances cell surface presentation of the cell surface antigen by 50% or more. In some cases, the bryostatin agent enhances cell surface presentation by 55% or more, e.g., 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 100% or more, or even more. In some cases, the bryostatin agent enhances cell surface presentation by 2-fold or more, e.g., 3-fold or more, 4-fold or more, 5-fold or more, or even even more.

[0245] In some embodiments of the methods of treating cancer, persistence of cell surface antigens on target cancer cells is enhanced for two or more days after administration of the bryostatin agent. In certain cases, cell surface antigen presentation on target cancer cells is enhanced for three or more days, e.g., four or more days, five or more days, six or more days, seven or more days, eight or more days, nine or more days, ten or more days, or longer, after administration of the bryostatin agent.

[0246] Embodiments are contemplated in which the bryostatin agent is administered prior to, simultaneously with, or subsequent to the administration of a therapeutically effective amount of a therapeutic agent that specifically binds to a cell surface antigen to treat cancer in a subject. In certain embodiments of the present disclosure, the bryostatin agent is administered subcutaneously.

[0247] In some embodiments, when the target cell is a cancer cell, the molecule on the surface of the cancer cell to which the antigen-binding portion of the therapeutic agent binds is a tumor-associated cell surface molecule or a tumor-specific cell surface molecule. By "tumor-associated cell surface molecule" is meant a cell surface molecule that is expressed on malignant cells but with limited expression on cells of normal tissue, that is expressed at a much higher density on malignant cells than on normal cells, or that is developmentally expressed.

[0248] When the target cell is a cancer cell, the cancer cell may express a tumor-associated or tumor-specific molecule on its surface to which the antigen-binding portion of the therapeutic agent binds. In certain embodiments, the target cancer cell contains a tumor antigen selected from CD10, CD19, CD20, CD21, CD22, CD30, CD34, CD40, CD52, CD80, CD86, and CD340. In certain embodiments, such tumor-associated or tumor-specific molecules are selected from the group consisting of HER2, B7-H3 (CD276), CD19, CD20, GD2, CD22, CD30, CD33, CD56, CD66 / CEACAM5, CD70, CD74, CD79b, CD123, CD133CD138, CD171, nectin-4, mesothelin, transmembrane glycoprotein NMB (GPNMB), prostate-specific membrane antigen (PSMA), SLC44A4, CA6, tyrosine-protein kinase Met (c-Met), epidermal growth factor receptor variant III (EGFRvIII), mucin 1 (MUC1), ephrin type A receptor 2 (EphA2), glypican 2 (GPC2), The antigen is selected from glypican 3 (GPC3), fms-like tyrosine kinase 3 (FLT3), folate receptor alpha (FRα), IL-13 receptor alpha 2 (IL13Rα2), fibroblast activation protein (FAP), receptor tyrosine kinase-like orphan receptor 1 (ROR1), B-cell maturation antigen (BCMA), delta-like 3 (DLL3), kappa light chain, vascular endothelial growth factor receptor 2 (VEGFR2), trophoblast glycoprotein (TPBG), anaplastic lymphoma kinase (ALK), CA-IX, integrins, C-X-C chemokine receptor type 4 (CXCR4), neuropilin-1 (NRP1), matriptase, and other tumor-associated or tumor-specific molecules of interest.

[0249] In certain embodiments, the therapeutic agent that specifically binds to a cell surface antigen to treat cancer in a subject is an antibody agent. Antibodies that may be used as inhibitors in connection with the present disclosure include, but are not limited to, monoclonal antibodies, polyclonal antibodies, bispecific antibodies, Fab antibody fragments, F(ab)2 antibody fragments, Fv antibody fragments (e.g., V H or V L), single-chain Fv antibody fragments, and dsFv antibody fragments. Furthermore, antibody molecules can be fully human, humanized, or chimeric. Antibodies that can be used in connection with the present disclosure can include any antibody variable region linked to any immunoglobulin constant region, mature or native. Minor variations in the amino acid sequence of an antibody or immunoglobulin molecule are encompassed by the present disclosure, provided that such variations maintain 75% or more, e.g., 80% or more, 90% or more, 95% or more, or 99% or more of the sequence. Specifically, conservative amino acid substitutions are contemplated. Conservative substitutions are those that occur within a family of amino acids whose side chains are related to each other. Whether an amino acid change results in a functional peptide can be determined by assaying the specific activity of the polypeptide derivative.

[0250] An "antibody fragment" comprises a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from multiple antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with one antigen-binding site, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking antigen.

[0251] An "Fv" is the minimum antibody fragment containing a complete antigen-recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain tightly bound by noncovalent bonds. The three CDRs from each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. The six CDRs collectively confer antigen-binding specificity to the antibody. However, although a single variable domain (or half of an Fv, containing only three antigen-specific CDRs) can recognize and bind antigen, it does so with lower affinity than the entire binding site.

[0252] The "Fab" fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab' fragments in which the cysteine residue(s) of the constant domains bear a free thiol group are referred to herein as Fab'-SH. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0253] The "light chains" of antibodies (immunoglobulins) of any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domain. Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

[0254] "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0255] Thus, antibodies that can be used in connection with the present disclosure can include monoclonal antibodies, polyclonal antibodies, bispecific antibodies, Fab antibody fragments, F(ab)2 antibody fragments, Fv antibody fragments (e.g., VH or VL), single-chain Fv antibody fragments, and dsFv antibody fragments. Furthermore, antibody molecules can be fully human, humanized, or chimeric. In some embodiments, the antibody molecule is a monoclonal fully human antibody.

[0256] The antibodies that can be used in connection with the present disclosure can include any antibody variable region linked to any immunoglobulin constant region, whether mature or unprocessed. When the light chain variable region is linked to a constant region, it can be a kappa chain constant region. When the heavy chain variable region is linked to a constant region, it can be a human gamma 1, gamma 2, gamma 3, or gamma 4 constant region, more preferably gamma 1, gamma 2, or gamma 4, more preferably gamma 1 or gamma 4.

[0257] Minor variations in the amino acid sequence of an antibody or immunoglobulin molecule are encompassed within the present disclosure, provided that such variations maintain at least 75% or more of the sequence, e.g., at least 80% or more, 90% or more, 95% or more, or 99% or more. Specifically, conservative amino acid substitutions are contemplated. Conservative substitutions are those that occur within a family of amino acids whose side chains are related to each other. Whether an amino acid change results in a functional peptide can be determined by assaying the specific activity of the polypeptide derivative. Fragments (or analogs) of antibody or immunoglobulin molecules can be readily prepared by those skilled in the art. Preferred amino- and carboxy-termini of fragments or analogs are near the boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data to public or private sequence databases. Preferably, computer-based comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known. Sequence motifs and structural conformations can be used to define structural and functional domains of the present invention.

[0258] Non-limiting examples of antibodies with which the present disclosure may be used include adecatumumab, ascrinvacumab, cizutumumab, conatumumab, daratumumab, drozitumab, durigotumab, durvalumab, dusigitumab, enfortumab, enoticumab, figitumumab, ganitumab, glenbatumumab, intetumumab, ipilimumab, iratumumab, icrucumab, lexatumumab, lucatumumab, mapatumumab, naruna Nesvacumab, ofatumumab, olaratumumab, panitumumab, patritumumab, pritumumab, radletumumab, ramucirumab, rilotumumab, lobatumumab, seribantumab, tarextumab, teprotumumab, tobetumumab, vantictumab, besencumab, votumumab, zalutumumab, framvotumab, altumomab, anatumomab, arcitumomab, bectumomab, blinatumomab, detumomab, ibritumomab, minletumomab, mitumomab, Moxetumomab, Naptumomab, Nofetumomab, Pemtumomab, Pintumomab, Racotumomab, Satumomab, Solitomab, Taplitumomab, Tenatumomab, Tositumomab, Tremelimumab, Abagovomab, Igovomab, Oregovomab, Capromab, Edrecolomab, Nacolomab, Amatuximab, Bavituximab, Brentuximab, Cetuximab, Derlotuximab, Dinutuximab, Ensituximab, Futuximab , Girentuximab, Indatuximab, Isatuximab, Margetuximab, Rituximab, Siltuximab, Ublituximab, Ecromeximab, Abituzumab, Alemtuzumab, Bevacizumab, Bivatuzumab, Brontuzumab, Cantuzumab, Cantuzumab, Sitanuzumab, Crivatuzumab, Dacetuzumab, Demicizumab, Dalotuzumab,Denintuzumab, Elotuzumab, Emactuzumab, Emibetuzumab, Enoblitzumab, Etracizumab, Farletuzumab, Ficlatuzumab, Gemtuzumab, Imgatuzumab, Inotuzumab, Labetuzumab, Rifatuzumab, Lintuzumab, Lorvotuzumab, Lumuletuzumab, Matuzumab, Milatuzumab, Nimotuzumab, Obinutuzumab, Ocaratuzumab, Otlertuzumab, Onartuzumab, Oportuzumab, Parasatuzumab, Pertuzumab, Pinatuzumab, Polatuzumab, Sibrotuzumab, Simtuzumab, Takatuzumab, Tigatuzumab, Examples of such agents include rastuzumab, tucotuzumab, vandortuzumab, vanucizumab, veltuzumab, borsetuzumab, sofituzumab, catumaxomab, ertumaxomab, depatuxizumab, ontuxizumab, brontuzumab, tamtuvetmab, or antigen-binding variants thereof. As used herein, the term "variant" refers to an antibody that binds to a particular cognate antigen (e.g., HER2 for trastuzumab), but has fewer or more amino acids than the parent antibody, has one or more amino acid substitutions relative to the parent antibody, is a single-chain variant of the parent antibody (e.g., an scFv variant), or any combination thereof.

[0259] In certain embodiments, the therapeutic agent that specifically binds to a cell surface antigen and treats cancer in a subject is an antibody (e.g., as described herein) conjugated to a cytotoxic agent. In certain embodiments, the cytotoxic agent is a cytotoxin or a radioactive agent. In certain cases, the cytotoxic agent is selected from calicheamicin, auristatin, maytansinoid, taxol derivatives, and duocarmycin.

[0260] In certain cases, the cytotoxic agent is a chemotherapeutic agent. Specific chemotherapeutic agents of interest include, but are not limited to, gemcitabine, docetaxel, bleomycin, erlotinib, gefitinib, lapatinib, imatinib, dasatinib, nilotinib, bosutinib, crizotinib, ceritinib, trametinib, bevacizumab, sunitinib, sorafenib, trastuzumab, ado-trastuzumab emtansine, rituximab, ipilimumab, rapamycin, temsirolimus, everolimus, methotrexate, doxorubicin, Abraxane, Forfirinox, cisplatin, carboplatin, 5-fluorouracil, Teysumo, paclitaxel, prednisone, levothyroxine, pemetrexed, navitoclax, and ABT-199. Peptide compounds can also be used.Interesting cancer chemotherapeutic agents include but are not limited to dolastatin and its active analogue and derivative; and auristatin and its active analogue and derivative (for example, monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF) etc.).For example, see WO 96 / 33212, WO 96 / 14856 and US 6,323,315. Suitable cancer chemotherapeutic agents also include maytansinoids and their active analogs and derivatives (see, e.g., EP 1391213; and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623); duocarmycins and their active analogs and derivatives (including, e.g., synthetic analogs, KW-2189 and CB1-TM1); benzodiazepines and their active analogs and derivatives (e.g., pyrrolobenzodiazepines (PBDs)); and calicheamicins and their active analogs and derivatives (e.g., antibody-drug conjugates with calicheamicin include gemtuzumab ozogamicin and inotuzumab ozogamicin). In some embodiments, the ADC is selected from inotuzumab ozogamicin and gemtuzumab ozogamicin.

[0261] In certain embodiments of the ADC, the cytotoxic agent is conjugated to the antibody via a linker. Any convenient linking group can be utilized in the ADC. The terms "linker," "link," and "linking group" are used interchangeably and refer to a linking moiety that covalently connects two or more compounds (e.g., an antibody and a cytotoxic agent of interest). In some cases, the linker is bivalent. In certain cases, the linker is a branched or trivalent linking group. In some cases, the linker has a linear or branched backbone of 200 atoms or less (e.g., 100 atoms or less, 80 atoms or less, 60 atoms or less, 50 atoms or less, 40 atoms or less, 30 atoms or less, or 20 atoms or less) in length. The linking moiety can be a covalent bond connecting two groups, or a straight or branched chain 1 to 200 atoms long, e.g., about 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 100, 150, or 200 carbon atoms long, where the linker can be straight, branched, cyclic, or single atom. In certain cases, 1, 2, 3, 4, or 5 or more carbon atoms in the linker backbone can be optionally substituted with sulfur, nitrogen, or oxygen heteroatoms. In certain cases, when the linker contains a PEG group, every third atom in that segment of the linker backbone is substituted with oxygen. The bond between the atoms in the backbone can be saturated or unsaturated, and typically there is only one, no more than two, or no more than three unsaturated bonds in the linker backbone. The linker can include one or more substituents, e.g., alkyl, aryl, or alkenyl groups. Linkers can include, but are not limited to, oligo(ethylene glycol), ether, thioether, disulfide, amide, carbonate, carbamate, tertiary amine, alkyl (which can be linear or branched), e.g., methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), etc. The linker backbone can include a cyclic group, e.g., an aryl, heterocycle, or cycloalkyl group, where two or more atoms, e.g., 2, 3, or 4 atoms, of the cyclic group are included in the backbone. The linker can be cleavable or non-cleavable. The linker can be a peptide, e.g., a linked sequence of residues. The linker can be a hydrazine linker.The linker can be a disulfide linker.

[0262] In certain embodiments, the therapeutic agent that specifically binds to a cell surface antigen and treats cancer in a subject is a bispecific antibody. In certain cases, the bispecific antibody is an anti-CD20 / anti-CD22 bispecific antibody fusion protein. In certain cases, the bispecific antibody is an anti-CD19 / anti-CD22 bispecific antibody fusion protein.

[0263] In certain embodiments of the enhanced targeted anti-cancer method, the subject's innate immune system effectively acts as a therapeutic agent to treat the subject's cancer. In certain cases of the enhanced targeted anti-cancer method, the subject's adaptive immune system effectively acts as a therapeutic agent to treat the subject's cancer. In this regard, certain embodiments contemplate a method of treating cancer in a subject, the method comprising: (a) introducing into the subject a therapeutically effective amount of a bryostatin agent (e.g., as described herein) that enhances the presentation of cell surface antigens or neoantigens of target cancer cells; and (b) treating the subject's cancer by elimination of the enhanced cell surface antigens or neoantigens by the subject's immune system.

[0264] composition Aspects of the invention also include compositions, eg, compositions that include a bryostatin agent of interest.

[0265] The compositions discussed herein can be formulated using any convenient excipients, reagents, and methods. The compositions are provided as formulations with pharmaceutically acceptable excipients. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be detailed here. Pharmaceutically acceptable excipients are described, for example, in A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AH Kibbe et al., eds., 3 rd and is widely described in various publications, including the American Pharmaceutical Assoc.

[0266] Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers, or diluents, are readily available to the public. Additionally, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like, are readily available to the public.

[0267] In some embodiments, the bryostatin agent is formulated in an aqueous buffer. Suitable aqueous buffers include, but are not limited to, acetate, succinate, citrate, and phosphate buffers in various strengths ranging from 5 mM to 100 mM. In some embodiments, the aqueous buffer contains an agent that provides an isotonic solution. Such agents include, but are not limited to, sodium chloride; and sugars such as mannitol, dextrose, and sucrose. In some embodiments, the aqueous buffer further contains a non-ionic surfactant such as polysorbate 20 or 80. Optionally, the formulation may further contain a preservative. Suitable preservatives include, but are not limited to, benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, and the like. In most cases, the formulation is stored at about 4°C. The formulation may be lyophilized, in which case the formulation generally contains a cryoprotectant such as sucrose, trehalose, lactose, maltose, or mannitol. Lyophilized formulations can be stored for long periods at ambient temperatures. In some embodiments, the compounds are formulated for sustained release.

[0268] In some embodiments, the bryostatin agent and a second therapeutic agent that specifically binds to a cell surface antigen (e.g., as described herein), such as a chimeric antigen receptor-expressing T cell (CAR T cell), an antibody agent, an antibody-drug conjugate (ADC), and a bispecific antibody agent, etc. (e.g., as described herein), are administered to an individual as a formulation (e.g., the same or separate agents) with a pharmaceutically acceptable excipient. In some embodiments, the formulation is administered to a patient in conjunction with at least one additional anti-cancer therapy, wherein the additional anti-cancer therapy is selected from radiation therapy, chemotherapy, immunotherapy, checkpoint inhibitors, surgery, and vascular-targeted therapy. In certain embodiments, the checkpoint inhibitor is selected from a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) inhibitor, a programmed death 1 (PD-1) inhibitor, or a PD-L1 inhibitor.

[0269] In another aspect, a pharmaceutical composition is provided that comprises or consists essentially of a bryostatin agent, or a pharmaceutically acceptable salt, isomer, tautomer, or prodrug thereof, and further comprises one or more additional anticancer agents of interest. Any convenient anticancer agent can be utilized in the method together with the compound. The compound can be administered in unit dosage form and can be prepared by any method known in the art. Such methods include combining the compound with a pharmaceutically acceptable carrier or diluent, which constitutes one or more accessory ingredients. Pharmaceutically acceptable carriers are selected based on the chosen route of administration and standard pharmaceutical practice. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and not toxic to the subject. The carrier can be solid or liquid, and the type is generally selected based on the type of administration being used.

[0270] Examples of suitable solid carriers include lactose, sucrose, gelatin, agar, and bulk powder. Examples of suitable liquid carriers include water, pharmaceutically acceptable oils and fats, alcohols or other organic solvents, such as esters, emulsions, syrups or elixirs, suspensions, solutions and / or suspensions, and solutions and / or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules. Such liquid carriers may contain, for example, suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, thickeners, and solubilizers. Preferred carriers are edible oils, such as corn oil or canola oil. Polyethylene glycol, such as PEG, is also a good carrier.

[0271] Any drug delivery device or system that provides the dosage regimen of the present disclosure can be used. A wide variety of delivery devices and systems are known to those skilled in the art.

[0272] Although not necessary, the compounds and agents described herein can optionally be targeted to cancer sites using any known targeting means.The compounds of the present disclosure can be formulated with a variety of compounds that have been demonstrated to target compounds to cancer sites.The terms "target to cancer sites" and "cancer targeting" refer to targeting compounds to cancer sites, and therefore refer to at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% or more of the compounds administered to a subject entering cancer sites.

[0273] Dosage and Administration In some embodiments, a "therapeutically effective amount" is an amount of a bryostatin agent that, when administered to an individual in one or more doses in monotherapy or combination therapy, is effective to enhance expression of a protein in target cells in the subject by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to expression of the protein in target cells not treated with the bryostatin agent. In other embodiments, a "therapeutically effective amount" is an amount of a bryostatin agent that, when administered to an individual in one or more doses in monotherapy or combination therapy, is effective to enhance surface presentation of a protein in target cells in the subject by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to surface presentation of the protein in target cells not treated with the bryostatin agent.

[0274] In some embodiments, an effective amount of a bryostatin agent is from about 50 ng / ml to about 50 μg / ml (e.g., from about 50 ng / ml to about 40 μg / ml, from about 30 ng / ml to about 20 μg / ml, from about 50 ng / ml to about 10 μg / ml, from about 50 ng / ml to about 1 μg / ml, from about 50 ng / ml to about 800 ng / ml, from about 50 ng / ml to about 700 ng / ml, from about 50 ng / ml to about 600 ng / ml, from about 50 ng / ml to about 500 ng / ml, from about 50 ng / ml to about 400 ng / ml, from about 60 ng / ml to about 400 ng / ml, from about 70 ng / ml to about 300 ng / ml, from about 60 ng / ml to about 100 ng / ml, from about 65 ng / ml to about 85 ng / ml, about 70 ng / ml to about 90 ng / ml, about 200 ng / ml to about 900 ng / ml, about 200 ng / ml to about 800 ng / ml, about 200 ng / ml to about 700 ng / ml, about 200 ng / ml to about 600 ng / ml, about 200 ng / ml to about 500 ng / ml, about 200 ng / ml to about 400 ng / ml, or about 200 ng / ml to about 300 ng / ml).

[0275] In some embodiments, an effective amount of a bryostatin agent is from about 10 pg to about 100 mg, e.g., from about 10 pg to about 50 pg, from about 50 pg to about 150 pg, from about 150 pg to about 250 pg, from about 250 pg to about 500 pg, from about 500 pg to about 750 pg, from about 750 pg to about 1 ng, from about 1 ng to about 10 ng, from about 10 ng to about 50 ng, from about 50 ng to about 150 ng, from about 150 ng to about 250 ng, from about 250 ng to about 500 ng, from about 500 ng to about 750 ng, from about 750 ng to about 1 μg, from about 1 μg to about 10 μg, from about 10 μg to about 50 μg, from about 50 μg to about 150 μg, from about 150 μg to about 250 The amount is in the range of about 250 μg to about 500 μg, about 500 μg to about 750 μg, about 750 μg to about 1 mg, about 1 mg to about 50 mg, about 1 mg to about 100 mg, or about 50 mg to about 100 mg. This amount may be a single dose or a total daily amount. The total daily amount may be in the range of 10 pg to 100 mg, or 100 mg to about 500 mg, or 500 mg to about 1000 mg.

[0276] In some embodiments, a single dose of the bryostatin agent is administered. In other embodiments, multiple doses are administered. When multiple doses are administered over a period of time, the compound may be administered twice daily (bid), daily (qd), every other day (qod), every other day, three times per week (tiw), or twice per week (biw) over a period of time. For example, the compound may be administered bid, qd, qod, tiw, or biw for one day to about two years or more. For example, the bryostatin agent may be administered at any of the above frequencies for one week, two weeks, one month, two months, six months, one year, or two years or more, depending on various factors. In some embodiments, the compound may be administered orally, intraocularly, otically, subcutaneously, intravenously, intramuscularly, intradermally, intraperitoneally, and by inhalation, among other routes of administration. In some embodiments, the compound may be administered in multiple courses, allowing for "drug holidays" that may range from one to seven days.

[0277] In certain embodiments, the dose of a bryostatin agent is a prodrug of a bryostatin agent, eg, as described herein.

[0278] kit Kits containing the bryostatin agents of the present disclosure are also provided. The kits of the present disclosure may include one or more doses of a bryostatin agent and, optionally, one or more doses of one or more additional therapeutic agents. In some embodiments, the kits include one or more doses of a bryostatin agent (e.g., as described herein); and one or more doses of a therapeutic agent (e.g., as described herein) that specifically binds to a cell surface antigen in a therapeutically effective amount. The formulation may be conveniently provided in unit dosage form. Such kits include a container containing the formulation, e.g., a unit dose, as well as an information package insert describing the use of the formulation in the methods of the present invention, e.g., instructions for using the unit dose to treat a cellular condition associated with pathogen angiogenesis. The term kit refers to packaged active agent(s). In some embodiments, the system or kit includes a dose of the compound (e.g., as described herein) and a dose of a second active agent (e.g., as described herein) in amounts effective to treat an angiogenesis-related disease or condition (e.g., as described herein) in a subject.

[0279] In addition to the above-mentioned components, the kit may further include instructions for using the components of the kit, e.g., to practice the subject methods. The instructions are typically recorded on a suitable recording medium. For example, the instructions may be printed on paper, plastic, or other substrate. Thus, the instructions may be present in the kit as a package insert, on the labeling of the container of the kit or its components (i.e., in association with packaging or subpackaging), or otherwise. In other embodiments, the instructions may be present as an electronic storage data file on a suitable computer-readable storage medium, e.g., CD-ROM, DVD-ROM, Blu-ray, diskette, hard disk drive (HDD), portable flash drive, or the like. In still other embodiments, the actual instructions are not present in the kit, but rather means for obtaining the instructions from a remote source, e.g., via the Internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0280] In some embodiments, the kit comprises a first dose of the pharmaceutical composition comprising a bryostatin agent and a second dose of the pharmaceutical composition comprising a therapeutic agent of interest.

[0281] Purpose The method is used to selectively enhance the expression or cell surface presentation of a protein in a target cell of interest to modulate the activity of the target cell. The method can be used in a variety of applications where modulation of a cell of interest is desirable, including therapeutic, diagnostic, and research applications.

[0282] The method can be used to treat diseases for which there is no effective treatment, including HIV / AIDS and the eradication of cancer. The method can also be used to sensitize target cells of interest to elimination by cells of the innate or adaptive immune system. The method can also be used to treat subjects who are relapsed or refractory to targeted anti-cancer therapy.

[0283] The method may be used for diagnostic purposes, involving the evaluation of one or more biomarkers in a subject's sample to assay for a disease (eg, cancer) state.

[0284] The following examples are offered by way of illustration and not by way of limitation. [Example]

[0285] The following examples are presented so as to fully disclose and describe to those of ordinary skill in the art how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent all or the only experiments performed by the inventors. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be allowed for. Unless otherwise specified, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0286] General methods in molecular and cellular biochemistry are covered in Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harvard Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons Reagents, cloning vectors, cells, and kits for the methods referred to in or related to this disclosure are available from commercial suppliers such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., and from repositories such as Addgene, Inc., American Type Culture Collection (ATCC), for example.

[0287] Introduction The development of targeted biologics and cell therapies for cancer treatment, including monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), bispecific antibodies (biAbs), and chimeric antigen receptor (CAR) T cells, is transforming oncology. By targeting tumor-specific cell surface antigens and neoantigens, these therapies offer distinct advantages over traditional treatment options by efficiently and selectively eliminating malignant cells while avoiding the systemic toxicity associated with cytotoxic chemotherapy. While mAbs, ADCs, biAbs, and CAR T therapies rely on diverse mechanisms of action and exploit different host biology systems to eliminate tumors, they all fundamentally share a common requirement: sufficient and sustained target antigen presentation. While these recent advances in cancer immunology hold promise, tumor escape and acquired resistance due to reduced surface expression of target antigens limit the efficacy and scope of these approaches. Indeed, poor durability of responses and patient relapse associated with variable and reduced antigen expression have been observed across several indications. See, e.g., Lim et al. Cell 2017, 168(4), 724-740; June et al. Science 2018, 359 (6382), 1361-1365; Sharma et al. Cell 2017, 168 (4), 707-723; Loganzo et al. Mol. Cancer Ther. 2016, 15 (12), 2825-2834; Fry et al. Nat. Med. 2017; and Majzner et al. Cancer Discov. 2018, 8 (10), 1219-1226. Identifying adjuvants that enhance and sustain surface expression of target antigens could broadly address this problem and improve the efficacy of targeted biologics and cell therapies, as these adjuvants could be used in combination with these therapies to increase both the number of patients who respond and the duration of the response.

[0288] Bryostatin 1, a marine macrolide and potent PKC modulator (see, e.g., Pettit et al. J. Am. Chem. Soc. 1982, 104 (24), 6846-6848; and Kortmansky et al. Cancer Invest. 2003, 21 (6), 924-936), can alter the expression of surface antigens of tumors and other cell lines, making them more immunogenic and therefore more susceptible to immune elimination. Several preclinical and clinical studies have reported that bryostatin 1 can modify the immune phenotype and increase the immunogenicity of cancer cells in acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin's lymphoma (NHL) (e.g., Hammond et al. J. Immunother. 2005, 28 (1), 28-39; Katib et al. Hematol. 1993, 21 (1), 61-64; Varterasian et al. J. Clin. Oncol. 1998, 16 (1), 56-62; Al Katib et al. J. Immunother. 1993, 14, 33-42; Varterasian et al. Clin. Cancer Res. 2000, 6 (3), 825-828; and Shaha et al. Clin. Exp. Immunol. 2009, 158 (2), 186-198). It has also been demonstrated that designed bryostatin analogs (bryology or "bryostatin agents"), like bryostatin 1, can increase cell surface antigen presentation in chronic lymphocytic leukemia (CLL) cells (Hammond et al. J. Immunother. 2005, 28 (1), 28-39; and Katib et al. J. Immunother. 1993, 14, 33-42).

[0289] Furthermore, bryostatin 1 and some of its analogs have been found to be able to induce the cell surface expression of CD69, an activation marker, on latently HIV-infected CD4+ T cells, and thus may serve as preclinical leads for a "kick-and-kill" approach toward HIV eradication (DeChristopher et al. Nat. Chem. 2012, 4 (9), 705-710; Marsden et al. PLOS Pathog. 2017, 13 (9), e1006575; and Albert et al. Sci. Rep. 2017, 7 (1), 7456).

[0290] Among antigen-targeting strategies under clinical evaluation, CAR T cell therapy has recently emerged as a highly effective treatment for B-cell malignancies. Nevertheless, antigen loss has been observed as a major driver of resistance and patient relapse. The inventors have discovered that the use of bryostatin 1 and exemplary analogs in combination with anti-CD22 CAR T therapy can ensure that sufficient levels of CD22 surface expression are maintained to effectively eliminate malignant cells, thereby improving patient outcomes.

[0291] The majority of research in this area has focused on just a few natural products, more specifically bryostatin 1. As evidenced by studies on taxanes and avermectins, recent synthetic precursors and derivatives of natural products, so-called "close" analogs, often offer superior clinical performance (Omura et al. Nat. Rev. Microbiol. 2004, 2 (12), 984-989; and Pazdur et al. Cancer Tret. Rev. 1993, 19 (4), 351-386). However, in the case of bryostatin 1, the utilization of such analogs has been hampered by a lack of available materials needed to generate synthetic derivatives, compounded by the inherent difficulties associated with modifying the highly complex bryostatin scaffold. The initial "select" bryostatin provided material for clinical use, but the living supply of bryostatin, which produced only 18 grams from 14 tons of bryozoans (0.00014% yield), is now nearing exhaustion (Schaufelberger et al. J. Nat. Prod. 1991, 54 (5), 1265-1270). Because the availability of natural bryostatin is scarce and unreliable, recollection from this marine source would raise environmental concerns (Keough, J. Biol. Bull. 1989, 177 (2), 277-286). Aquaculture ("tank" and "open ocean") and engineered biosynthesis have been explored, but the former has encountered problems with capitalization, and the latter has encountered difficulties culturing the mutualistic bacteria required for bryostatin 1 production (Mendola, D. Biomol. Eng. 2003, 20 (4-6), 441-458). We recently reported a solution to this problem: a practical chemical synthesis of bryostatin 1, which for the first time allows sustainable access to gram-scale quantities of this natural product on demand, thus ensuring its continued evaluation (Wender et al. Science 2017, 358 (6360), 218-223).Furthermore, this synthetic approach can also serve as a platform for developing bryostatin analogs and exploring structure-activity relationships at various positions around the bryostatin macrocyclic sclerosis, thereby enabling the design and synthesis of next-generation bryostatin-inspired small molecules for human disease research and treatment. Given that bryostatin 1 has been studied in preclinical and clinical trials for numerous indications, including the treatment of Alzheimer's disease, HIV / AIDS eradication, multiple sclerosis, Niemann-Pick disease, fragile X, and enhanced immunotherapy, and the involvement of various PKC isoforms in many of these indications, it is suggested that various analogs may exhibit superior activity across a range of indications due to their distinct selectivities and tolerability.

[0292] Example 1: Design of bryostatin analogs Disclosed herein is the design, synthesis, and evaluation of exemplary bryostatin analogs. The design strategy focused on chemical modifications to the bryostatin scaffold that are not expected to significantly affect the compound's affinity for PKC, but which can affect PKC function and, optionally, can be used to tailor formulation and ADME (absorption, distribution, metabolism, and excretion) characteristics (Figure 1, Panel A). Eighteen analogs were prepared.

[0293] Figure 1. (Panel A) Retrosynthetic analysis of the bryostatin scaffold, with pharmacophore elements of the C-ring subunit shown as the C1 carbonyl, C19 hemiketal, and C26 alcohol. C13 is highlighted (depicted as a sphere) as an area of interest for analog synthesis. (Panel B) Rendering of the PKC-bryostatin-membrane ternary complex. Bryostatin 1 is shown within a rectangular box. Pharmacophore elements of the C-ring subunit of the bryostatin scaffold (see Figure 1, Panel A) interact directly with PKC (dark gray), while the A- and B-rings are embedded in the plasma membrane (light gray). See ACS Cent. Sci. 2018, 4, 89-96. (Panel C) Representative conformers of the bryostatin scaffold bound to PKC, determined by REDOR NMR, fitted to experimentally determined intramolecular distances. See ACS Cent. Sci. 2018, 4, 89-96. (Panel D) Convergent construction of the bryostatin scaffold from acid 1 and enal 2. The C13 functionality provides a universal starting point for subsequent diversification while avoiding interference with C-ring pharmacophore elements (as described above for Figure 1, Panel A).

[0294] These compounds were designed to retain the pharmacophore functional groups proposed for PKC binding in our original pharmacophore model. Consistent with that model, most of the exemplary analogs disclosed herein exhibit single-digit nanomolar affinities for representative PKC isoforms, comparable to that of bryostatin 1 (Wender et al. Proc. Natl. Acad. Sci. USA 1986, 83 (12), 4214-4218; and Wender et al. Proc. Natl. Acad. Sci. USA 1988, 85 (19), 7197-7201). In contrast, regardless of cell-free binding affinity data, diverse activity profiles were observed in functional assays measuring PKC activation in live cells, indicating that each modification can indeed induce distinct biological functions. Importantly, the ability of exemplary analogs to increase CD22 surface expression in an in vitro ALL model was also investigated, with implications for antigen-targeted therapy. Several exemplary analogs were found to exhibit activity similar to bryostatin 1, suggesting that these compounds may be more accessible, effective, and better tolerated adjuvants for cancer immunotherapy and other indications.

[0295] The design strategy for exemplary bryostatin analogs was guided by our previously proposed bryostatin pharmacophore model (Wender et al. Proc. Natl. Acad. Sci. USA 1988, 85 (19), 7197-7201) and further enhanced by molecular dynamics simulations (Ryckbosch et al. Nat. Commun. 2017, 8 (1), 6) and REDOR NMR studies (Yang et al. ACS Cent. Sci. 2018, 4 (1), 89-96) of bryostatin 1 and labeled analogs bound to PKC in membrane microenvironments. PKCs are a family of seven homologous signaling kinases classified as conventional (α, β, γ) or novel (δ, ε, η, θ) based on their subdomain structure (Newton, ACJ Biol. Chem. 1995, 270 (48), 28495-28498). Individual PKC isoforms, isoform combinations, and mutant isoforms are involved in several disease pathologies (Newton, AC AJP Endocrinol. Metab. 2010, 298 (3), E395-E402; and Newton, AC Semin. Cancer Biol. 2018, 48, 18-26). PKC maturation and activation are governed by a tightly orchestrated sequence of phosphorylation, conformational changes, and eventual translocation to the plasma membrane, where it triggers the phosphorylation of numerous downstream signaling proteins (Newton, ACJ Biol. Chem. 1995, 270 (48), 28495-28498; and Newton, AC Chem. Rev. 2001, 101 (8), 2353-2364). A hallmark of ligand-induced PKC activation is the formation of a ternary complex between the plasma membrane, the ligand, and PKC (Figure 1, Panel B). In this context, the bryostatin scaffold can be viewed as two subunits with distinct but interdependent functions.Our own computational investigations into the similarities between a number of PKC modulators reveal that hydrogen-bonding functional groups on or proximal to the C-ring subunit of the bryostatin backbone, specifically the C1 carbonyl, C26 hydroxyl, and C19 hemiketal, are spatially pre-assembled by the A- and B-rings of the North subunit into a binding conformation similar to that of the endogenous PKC ligand, DAG (Figure 1, panels A and C) (Wender et al. Proc. Natl. Acad. Sci. USA 1988, 85 (19), 7197-7201; Ryckbosch et al. Nat. Commun. 2017, 8 (1), 6; Yang et al. ACS Cent. Sci. 2018, 4 (1), 89-96; and Wender et al. Proc. Natl. Acad. Sci. USA 1998, 95 (12), 6624-6629).Indeed, key studies have shown that modification or deletion of the C1, C26, and C19 functional groups reduces or eliminates PKC binding, while alterations to the A and B rings generally have little or no effect on binding, suggesting that modifications to these subunits of exemplary bryostatin analogs may alter their function and biodistribution (Wender et al. Proc. Natl. Acad. Sci. USA 1998, 95 (12), 6624-6629; Wender et al. Proc. Natl. Acad. Sci. USA 1988, 85 (19), 7197-7201; Wender et al. In Natural Products in Medicinal Chemistry; Wiley-VCH, 2014; pp 473-544; Wender et al. J. Am. Chem. Soc. 2008, 130 (21), 6658-6659; Wender et al. Proc. Natl. Acad. Sci. USA 2011, 108 (17), 6721-6726; and DeChristopher et al. Nat. Chem. 2012, 4 (9), 705-710).

[0296] In addition to spatially restricting the pharmacophore elements of the bryostatin C-ring subunit into a productive PKC-binding conformation, recent long-time scale (400-500 μs) molecular dynamics simulations suggest that the North subunit also influences the efficiency of PKC translocation, as well as the depth and orientation of the ligand-PKC complex in the membrane (Wender et al. Proc. Natl. Acad. Sci. USA 1988, 85 (19), 7197-7201; Ryckbosch et al. Nat. Commun. 2017, 8 (1), 6; Yang et al. ACS Cent. Sci. 2018, 4 (1), 89-96; and Wender et al. Proc. Natl. Acad. Sci. USA 1998, 95 (12), 6624-6629). Specifically, interactions of oxygenated A and B rings with water molecules at the membrane-cytosol interface are predicted to cause the bryostatin PKC complex to adopt a shallow, tilted orientation relative to the membrane (Figure 1, panel B) (Ryckbosch et al. Nat. Commun. 2017, 8 (1), 6; Yang et al. ACS Cent. Sci. 2018, 4 (1), 89-96). This orientation was found to be unique to the bryostatin 1-PKC complex and not observed when modeling other potent PKC ligands, such as phorbol dibutyrate (PDBu) and prostratin, explaining their competitive ability for PKC binding but often contrasting downstream activities. In another study, a combination of REDOR NMR and molecular dynamics simulations identified a distribution of PKC-bound bryostatin conformers (Figure 1, Panel C), a feature unique to the bryostatin scaffold that was not observed in phorbol ester derivatives (Yang et al. ACS Cent. Sci. 2018, 4 (1), 89-96).Taken together, these studies suggest that the multiple PKC-binding conformers accessible to the bryostatin scaffold can generate unique orientations of the activated PKC-ligand complex and influence the interaction of PKC with downstream effector proteins, thus explaining bryostatin's unique biological activity. This suggests that modifications to the A- and B-rings of the bryostatin scaffold can affect compound function by influencing the conformational energy landscape of the bryostatin scaffold and, in turn, the orientation of the activated PKC signaling complex. Using this paradigm, we designed a series of compounds that retained pharmacophore elements in the bryostatin C-ring but contained a series of systematic modifications at C13 in the bryostatin B-ring. This site was chosen because computational studies suggested that modifications at this position would preserve PKC-binding affinity but could affect function. By exploiting the unique reactivity of this position found in later synthetic intermediates, we were able to generate exemplary bryostatin analogs with diverse chemical functionality at this position, allowing us to explore how different substitution patterns in the northern hemisphere can affect compound function by influencing membrane binding, trafficking, and downstream signaling outcomes.

[0297] Example 2: Synthesis of Bryostatin Analogs We previously reported a convergent, short-step synthesis of bryostatin 1, enabling the construction of the diversifiable bryostatin macrocycle (exo-olefin 3, Figure 1, Panel D) from two building blocks of approximately equal complexity in the longest linear sequence of 15 steps, a total of 25 steps (Figure 1, Panel D). See, e.g., Wender et al. Science 2017, 358 (6360), 218-223; and International Publication No. PCT / US2017 / 054158, filed September 28, 2017, the disclosures of which are incorporated herein by reference. Referring to Figure 1, Panel D, Yamaguchi esterification coupled carboxylic acid 1 and enal 2 in 82% yield. Subsequent Prins macrocyclization formed the bryostatin B ring, completing the macrocycle closure. Importantly, the Prins cyclization reaction yielded exo-olefin 3, which was expected to serve as a chemical handle for further diversification of this advanced intermediate. The exo-olefin 3 could be selectively modified to directly incorporate new B-ring functionalities or converted to the corresponding ketone 4 via a stoichiometric ozonolysis procedure developed by our team. These two functional groups confer largely orthogonal chemical reactivity to the other functional groups decorating the complex and delicate bryostatin scaffold. Therefore, we sought to use these groups as a starting point to explore how modifications to the B-ring of bryostatin could affect the compound's function.

[0298] Early exploration of the C13 chemical space sought to exploit the unique reactivity of the C13 exo-olefin to generate a series of exemplary structural analogs. Previous synthetic work on the bryostatin scaffold demonstrated that this olefin is likely the most reactive of the four pi-systems found in this key intermediate (Wender et al. Science 2017, 358 (6360), 218-223). An exemplary synthetic scheme for the chemoselective functionalization of the C13 exo-olefin is shown below (Scheme 1). TIFF2025121975000021.tif102164 Scheme 1. Chemoselective functionalization of C13 exo-olefins

[0299] Referring to Scheme 1, dihydroxylation of SUW200 cleanly afforded vicinal diol 5 in the presence of C16 / C17 alkene, C20 alkynoate, and C21 dienoate. In contrast, epoxidation of exo-olefin 3 and bryostatin 1 with DMDO and mCPBA proved difficult to control but, interestingly, offered different chemoselectivities. Attempts to reduce the C13 olefin using H2 and catalytic Pd / C were also difficult to control and often yielded a mixture of reduced products. Nevertheless, we were able to achieve a clean reduction of the C13 exo-olefin and C20 alkynoate without reducing either the C21 enoate or the C16 / 17 olefin, yielding compound 5, which was subsequently deprotected to give SUW226. Alternatively, olefin 3 could be directly deprotected to give SUW200 as previously described (Wender et al. Science 2017, 358 (6360), 218-223).

[0300] Efforts to modify the C13 olefin continued with a focus on the modification of C13 ketone 4. This key intermediate provided an ideal diversification point because the C13 ketone exhibits orthogonal chemical reactivity relative to the other functional groups found in this series of key intermediates, thereby circumventing the problems arising from selective modification of the C13 exo-olefin of 3 in the presence of several other pi systems. An exemplary synthetic scheme for the chemoselective functionalization of C13 ketone 4 is shown below (Scheme 2). TIFF2025121975000022.tif76168 Scheme 2. Horner-Wadsworth-Emmons olefination of C13 ketones to give C13 enoates

[0301] Referring to Scheme 2, the synthesis began with the synthesis of a series of alkyl enoates at the C13 position. A series of compounds bearing various ester groups was generated from ketone 4 to explore how the size of the substituent and the geometry of the enoate affect the compound's functionality. Horner-Wadsworth-Emmons (HWE) olefination of ketone 4 using the corresponding HWE reagent, prepared by direct coupling of the desired alcohol with diethylphosphonoacetic acid, afforded the ester derivatives. Unlike the use of chiral HWE reagents to control the double bond geometry reported in our synthesis of bryostatin 1, we chose to perform these reactions using a simple, nonstereoselective HWE reagent to obtain both the (E) and (Z) isomers of the olefination product 6. Subsequent global deprotection and HPLC separation of the enoate diastereomers afforded exemplary analogs.

[0302] In addition to the unsaturated esters derived from the ketones, it was hoped that incorporating an alcohol at C13 would provide a convenient functional handle for subsequent diversification via esterification and provide an opportunity to explore how hydridization at C13 might affect functionality. This was readily achieved by reduction of ketone 4 with borohydride. Obtaining the C13 alcohol, a number of ester groups could be incorporated via standard esterification procedures (Scheme 3), yielding a structurally diverse set of compounds including C13 alcohols, aliphatic esters, indoyl esters, phenyl carbamates, and both cationic and anionic moieties. TIFF2025121975000023.tif98165 Scheme 3. Reduction and esterification of C13 ketones to give C13 esters. EDC = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0303] Synthesis procedure The compounds can be synthesized using any convenient method. Methods that can be used to prepare the compounds of the present disclosure include exemplary synthetic methods described in Wender et al. Science 2017, 358 (6360), 218-223 and International Publication No. PCT / US2017 / 054158, filed September 28, 2017. Many general references are also available that provide widely known chemical synthesis schemes and conditions useful for synthesizing the compounds of the present disclosure (see, for example, Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978). Each reaction can be analyzed by thin layer chromatography (TLC), LC / MS, and LC / MS and 1 The reaction products can be observed by characterizing them by H NMR. Intermediates and final products can be purified by silica gel chromatography or HPLC.

[0304] Unless otherwise noted, all reactions were carried out in oven-dried or flame-dried glassware under a nitrogen or argon atmosphere. Unless otherwise noted, reaction products were concentrated under reduced pressure on a rotary evaporator. Commercially available reagents were used as received or purified using the methods described herein. Dichloromethane, diethyl ether, dimethylformamide, pentane, tetrahydrofuran, and toluene were passed through an alumina drying column (Solv-Tek Inc.) using nitrogen pressure; ethyl acetate, hexane, and petroleum ether were obtained from Fisher Scientific. Analytical thin-layer chromatography (TLC) was performed on 250 μm silica gel 60G plates using fluorescent indicator F254 (EMD Millipore). Plates were visualized under UV light and by treatment with p-anisaldehyde, ammonium cerium molybdate, or potassium permanganate followed by slow heating for color development. Flash column chromatography was performed using silica gel (230–400 mesh, grade 60, particle size 40–63 μm) purchased from Fischer Scientific. pH 7-buffered silica gel was prepared by adding 10 wt% phosphate buffer, pH 7, to silica and rotating for approximately 12 hours. NMR spectra were acquired on a Varian INOVA 600, Varian INOVA 500, or Varian 400 magnetic resonance spectrometer. 1 H chemical shifts are reported relative to the residual solvent peaks (CHCl3 = 7.26 ppm, C6H6 = 7.16 ppm) as follows: chemical shift (δ), multiplicity (app = apparent, b = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, or a combination thereof), coupling constant (Hz), and integral. 13C chemical shifts are reported relative to the residual solvent peaks (CHCl3 = 77.16 ppm, C6H6 = 128.06 ppm). Infrared spectra were obtained on a Nicolet iS 5 FT-IR spectrometer (Thermo Fisher). Optical rotations were obtained on a P-2000 digital polarimeter (Jasco). High-resolution mass spectra (HRMS) were obtained at the Vincent Coates Foundation Mass Spectrometry Laboratory at Stanford University.

[0305] Synthesis procedure for SUW200 TIFF2025121975000024.tif50128 Compound 3 (15 mg, 0.0124 mmol, 1 equiv.) and 3:1 THF / HO (1 mL) were added to a 15 mL polypropylene Falcon tube equipped with a magnetic stir bar. The Falcon tube was transferred to a 4 °C cold room. HF-pyridine (0.32 mL) was added (final concentration: approximately 0.01 M). After 96 h, the reaction mixture was allowed to warm to room temperature. After an additional 64 h (approximately 6.5 days total), the reaction mixture was quenched by slowly pouring the reaction mixture into a separatory funnel containing saturated aqueous NaHCO (20 mL) and EtOAc (20 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (4 x 20 mL). The combined organic layers were washed with 0.5 M HCl (10 mL) to remove pyridine, and the aqueous layer was back-extracted with EtOAc (2 x 20 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. Purification was carried out by silica gel flash column chromatography (25-65% EtOAc / Hex) to give SUW200 (6.6 mg, 63% yield) as a white solid. The purity of the compound was established by TLC (1 spot) analysis. TLC R f = 0.56 (60% EtOAc / Hex, UV active, dark purple spot with p-anisaldehyde); TIFF2025121975000025.tif121166

[0306] Synthesis procedure for SUW203 To a TIFF2025121975000026.tif561291 drum vial was added SUW200 (6.6 mg, 0.0078 mmol, 1 equiv.) in acetone (0.27 mL). NMO (1.4 mg, 0.0117 mmol, 1.5 equiv.) and OsO4 (30 μL of a 0.01 M aqueous solution, 0.00031 mmol, 0.04 equiv.) were added sequentially, and the resulting mixture was stirred for 24 h. The reaction mixture was diluted with EtOAc and sat. Na2SO3 and extracted with EtOAc (2 x). The combined organic layers were dried over Na2SO4 and concentrated to a white paste. Purification by flash chromatography (pipette column, 5-10% MeOH / DCM) afforded the diol SUW203 as a white powder (4 mg, 60% yield). TLC: R f = 0.1 (80% EtOAc / hexane, UV active, purple spot with p-anisaldehyde) TIFF2025121975000027.tif77166

[0307] Synthesis procedure for SUW226 TIFF2025121975000028.tif48162 Hydrogenation To an 8-dram vial equipped with a magnetic stir bar was added Prince product SI-1 (Science, 2017, 358, 218-223) (50 mg), EtOAc (500 μL), and palladium on carbon (50 mg). The vial was placed under a hydrogen atmosphere (balloon). After 2 hours, TLC analysis indicated complete conversion of the starting material. The reaction mixture was filtered through a pipette containing Celite and directly concentrated. Purification was carried out by silica gel flash column chromatography (25% EtOAc / pentane) to yield 40 mg of material.

[0308] Total deprotectionTo a cooled (0 °C) solution of this material (40 mg) in 1:1 THF / pyridine (800 μL) was added 70% HF-pyridine (400 μL) to give a 1:2:2 HF-pyridine / THF / pyridine solution. The reaction mixture was allowed to warm to room temperature. After 36 h, HO (400 μL) was added, and the reaction mixture was heated to 40 °C. After 3 h at 40 °C, the reaction mixture was cooled to 0 °C, diluted with EtOAc (10 mL), and quenched slowly by the dropwise addition of saturated aqueous NaHCO3 until no more bubbles were observed. The layers were separated, and the aqueous layer was extracted with 80% EtOAc / Hex (5 x 10 mL). The combined organic layers were dried over NaSO4, filtered, and concentrated. Purification was carried out by silica gel flash column chromatography (33–67% EtOAc / Hex) to give a mixture of SUW226 and its C9-fluoride (24 mg, 62% yield over two steps). Next, 15 mg of this diastereomeric mixture was subjected to RP-HPLC (70-100% MeCN / HO) to obtain a pure sample of SUW226 and its C9-fluoride. The stereochemistry of C13 was assigned from 2D-ROESY data (C 13 CH3 and C 11 Between H and C 13 CH3 and C 15 (A cross peak was observed between TLC R and H.) f = 0.31 (50% EtOAc / Hex, UV active, dark purple spot with p-anisaldehyde); TIFF2025121975000029.tif107166

[0309] Steps to obtain SUW201 SUW201 is the C13 (E)-enoate isomer of bryostatin 1. HWE olefination produces an approximately 10:1 mixture of C13 isomers, allowing separation of SUW201 from bryostatin 1 by RP-HPLC. See Science, 2017, 358, 218-223 for synthesis and purification conditions. TIFF2025121975000030.tif121170

[0310] Synthesis of modified C13 enoates: HWE olefination of C13 ketones Preparation of HWE reagent from diethylphosphonoacetate: TIFF2025121975000031.tif17128 Chemicals: Diethylphosphonoacetic acid (Aldrich, used without purification) DMAP (4-dimethylaminopyridine) (Aldrich): recrystallized from hexane EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (Chem-Impex): used without purification To a flame-dried 8-dram vial equipped with a magnetic stir bar was added diethylphosphonoacetic acid (1.0 equiv.) in DCM (approximately 0.2 M). The corresponding alcohol (1.1 equiv.) was added, followed by DMAP (0.5 equiv.) in one portion. EDCI (2.0 equiv.) was then added in one portion, and the reaction was stirred at room temperature for 30 min, at which point TLC indicated complete conversion of the starting material. The reaction mixture was directly diluted with water and extracted 3 x EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated. The crude residue was purified by silica gel flash chromatography (80-90% EtOAc in hexanes) to give the desired phosphonate, which was used in the subsequent HWE olefination. Compound purity was established by TLC (single spot) analysis. Characterization data were consistent with literature values reported by Lloyd et al. (allyl phosphonate, Organic and Biomolecular Chemistry 2016, 14, 8971-8988.) and O'Leary et al. (benzyl phosphonate, JACS 2001, 123, 11519-33).

[0311] It should be noted that the exact amount of phosphonate prepared varied, but the procedure was generally carried out on an approximately 200 mg scale.

[0312] HWE olefination of C13 ketones: TIFF2025121975000032.tif53143

[0313] Chemicals: NaHMDS (1 M sodium hexamethyldisilazide in THF, Aldrich): used without purification Triethylphosphonic acid (Aldrich): used without purification Alternative phosphonates prepared by the above procedure To a flame-dried 8-dram vial equipped with a magnetic stir bar was added the corresponding phosphonate in THF (azeotrope in benzene x 2). This solution was cooled to 0 °C, and NaHMDS was added dropwise. The reaction mixture was allowed to stir at 0 °C for 30 min, at which point it turned bright yellow. Separately, to a flame-dried 8-dram vial equipped with a magnetic stir bar was added the ketone 4 in THF (azeotrope in benzene x 2). This solution was cooled to -78 °C, and an aliquot of the deprotonated phosphonate was added dropwise as a THF solution. The reaction mixture was allowed to stir at -78 °C for 5 min, at which point it was transferred to a cold room and stirred at 4 °C for approximately 2 h, at which point TLC indicated complete consumption of the starting material. The reaction mixture was then pipetted into sat. aq. NH4Cl and extracted 3 x Et2O. The combined organic layers were dried over Na2SO4, filtered, and concentrated. The crude residue was purified by silica gel flash chromatography (15-25% EtOAc in hexanes; TLC R f Purification by HCl (0.52 in 60% EtOAc / hexanes, purple spot with p-anisaldehyde) gave the desired C13 enoate as a mixture of (Z) and (E) isomers that could not be separated on silica gel. This mixture of geometric isomers was carried on directly to the next full deprotection.

[0314] Synthesis of modified C13 enoates: total deprotection TIFF2025121975000033.tif49163 Chemicals: 70% HF-pyridine (Sigma-Aldrich): used without purification Pyridine (Sigma-Aldrich): distilled from CaH2 before use To a 15 mL polypropylene Falcon tube equipped with a magnetic stir bar was added enoate 7 in THF. This solution was cooled to 0 °C, and pyridine was added dropwise. HF-pyridine was then added dropwise to give a 0.0075 M solution of enoate 7 in a 1:2:2 mixture of HF-pyridine / THF / pyridine, and the resulting reaction mixture was transferred directly to a 40 °C oil bath. The reaction mixture was allowed to stir for 20 h, at which point HO (equivalent to the HF-pyridine) was added dropwise. The reaction mixture was allowed to stir for an additional 2 h at 40 °C. The reaction mixture was then poured directly into the aqueous layer of a pre-cooled EtOAc / sat. aq. NaHCO mixture. The aqueous layer was extracted 3 x EtOAc, and the combined organic layers were washed with 1 M HCl followed by brine. The combined organic layers were then dried over NaSO, filtered, and concentrated. The product was purified by silica gel flash chromatography (10-35-45-50-60% EtOAc in hexanes) to give a mixture of C13 geometric isomers, which were then separated by HPLC.

[0315] Preparative HPLC The approximately 1:1 Z:E mixture of C13 enoate isomers was further purified by preparative HPLC using a Shimadzu LC-20AP Prominence preparative liquid chromatograph with detector settings at 254 and 280 nm. Separation was performed using a Restek Ultra C18 column (5 μm particle size, 250 mm x 10 mm). The mobile phase was a gradient elution (flow rate 5 mL / min) from 75% MeCN / HO to 100% MeCN / HO for 30 min, followed by 100% MeCN for 10 min. The sample was dissolved in 1:1 MeCN / MeOH and loaded. Two fractions were collected, affording diastereomerically pure samples of the (Z)- and (E)-enoate isomers as fluffy white powders (Fraction 1: (Z) enoate, Fraction 2: (E) enoate). The stereochemistry of the C13 enoate of SUW218 was confirmed by ROESY NMR (described further below). Furthermore, the HPLC retention time and relative C30 1H chemical shifts matched the patterns observed for the C13 (Z)- and (E)-methyl enoates of bryostatin 1 / SUW201 (Science 2017, 358, 218-223).

[0316] Characterization data for SUW217-SUW220, SUW229, and SUW230 are provided below. Characterization data for SUW217: TLC R f = 0.31 (purple spot in 60% EtOAc / pentane, p-anisaldehyde); TIFF2025121975000034.tif107166

[0317] Characterization data for SUW218: TLC R f = 0.31 (purple spot in 60% EtOAc / pentane, p-anisaldehyde); TIFF2025121975000035.tif107170

[0318] Characterization data for SUW219: TLC:R f= 0.32 (50% EtOAc / pentane, UV-active, purple spot by p-anisaldehyde staining); TIFF2025121975000036.tif128170

[0319] Characterization data for SUW220: TLC:R f = 0.38 (50% EtOAc / pentane, UV-active, purple spot by p-anisaldehyde staining); TIFF2025121975000037.tif115170

[0320] Characterization data for SUW229: TLC R f = 0.38 (60% EtOAc / hexane, purple spot with p-anisaldehyde); TIFF2025121975000038.tif92170

[0321] Characterization data for SUW230: TLC R f = 0.46 (60% EtOAc / hexane, purple spot with p-anisaldehyde); TIFF2025121975000039.tif85170

[0322] Synthesis procedure for SUW204 TIFF2025121975000040.tif56157 reductionTo a cooled (-20 °C) solution of SI-2 (14 mg, 0.012 mmol, 1 equiv) in MeOH (1.15 mL, 0.01 M) was added sodium borohydride (0.5 mg, 0.013 mmol, 1.15 equiv). After 2 h, TLC analysis indicated complete conversion of the starting material. The yellow reaction mixture was quenched at -20 °C by the dropwise addition of saturated aqueous NH4Cl (2 mL). The layers were separated, and the aqueous layer was extracted with 50% EtOAc / Hex (5 x 3 mL). The combined organic layers were dried over NaSO4, filtered, and concentrated. Purification was performed by flash column chromatography on a 1.7 x 6 cm silica gel column eluting with 15–25% EtOAc / Hex, collecting 4 mL fractions. Fractions (Frxns) #20-26 gave 11.6 mg (83% yield) of SI-3, and fractions #27-31 gave 2 mg (14% yield) of its C13 diastereomer (structure not shown). After C13 acetylation gave SUW206 and SUW207, respectively, the relative stereochemistry of C13 was assigned. TLC of SI-3:R f = 0.44 (30% EtOAc / Hex, UV active); TLC of C13 diastereomer of SI-3: R f = 0.63(30% EtOAc / Hex, UV activity).

[0323] Total deprotectionTo a cooled (0 °C) solution of SI-3 (11.6 mg, 0.01 mmol, 1 equiv) in 3:1 THF / HO (1 mL, 0.01 M) was added 70% HF-pyridine (300 μL). The reaction mixture was allowed to warm to room temperature. After 48 h, TLC analysis indicated incomplete deprotection, so an additional 70% HF-pyridine (150 μL; 450 μL total) was added. After an additional 24 h (total reaction time 72 h), the reaction mixture was cooled to 0 °C, diluted with 50% EtOAc / Hex (5 mL), and slowly quenched by the dropwise addition of saturated aqueous NaHCO3 until no more bubbles were observed. The layers were separated, and the aqueous layer was extracted with 80% EtOAc / Hex (5 x 5 mL). The combined organic layers were dried over NaSO4, filtered, and concentrated. Silica gel flash column chromatography (25% EtOAc / Hex for silanol removal, followed by 50–100% EtOAc / Hex) followed by RP-HPLC (70–100% MeCN / HO) afforded SUW204 (4.8 mg, 59% yield). The same reduction / deprotection sequence was repeated with dienoate 4 to afford SI-4 and finally SUW205.

[0324] Characterization data for SUW204: TLC R f = 0.27 (100% EtOAc, no UV activity, CAM stain); TIFF2025121975000041.tif85166

[0325] Synthetic procedures for SUW206 and SUW207 TIFF2025121975000042.tif48156 Acylation To a solution of alcohol SI-4 (19 mg, ca. 4:1 dr with C13, 0.016 mmol, 1 equiv.) in CHCl (156 μL, 0.1 M) were added DMAP (1 crystal) and acetic anhydride (2 drops) successively. After 3 h, TLC analysis indicated complete conversion of the starting material. The reaction mixture was directly purified by silica gel flash column chromatography (20-30% EtOAc / Hex) to afford C13-OAc (quant., ca. 4:1 dr with C13).

[0326] Total deprotection To a cooled (0 °C) solution of this ester (19 mg, ca. 4:1 dr with C13, 0.016 mmol, 1 equiv.) in 1:1 THF / pyridine was added 70% HF-pyridine (ca. 0.0075 M solution in 1:2:2 HF-pyr / THF / pyridine). The reaction mixture was allowed to warm to room temperature. After 20 h, HO (equivalent to the HF-pyridine) was added and the reaction mixture was heated to 40 °C. After 3 h, the reaction mixture was cooled to 0 °C, diluted with EtOAc, and quenched slowly by dropwise addition of saturated aqueous NaHCO until no more bubbles were observed. The layers were separated, and the aqueous layer was extracted with 50% EtOAc / Hex. The combined organic layers were dried over NaSO, filtered, and concentrated. Purification by silica gel flash column chromatography (20% EtOAc / Hex for silanol removal, followed by 50–80% EtOAc / Hex) followed by RP-HPLC (70–100% MeCN / HO) afforded SUW206 (6.6 mg, 47% yield) and SUW207 (1.4 mg, 10% yield). The relative stereochemistry at C13 was based on numerous literature examples demonstrating that the axial proton is upfield relative to the equatorial proton. For example, in SUW206, the axial proton was observed at 4.97 ppm, whereas in SUW207, the equatorial proton was observed at 5.13 ppm.

[0327] Characterization data for SUW206 (major C13 diastereomer): TLC R f = 0.58 (75% EtOAc / Hex, UV active, dark blue spot with p-anisaldehyde); TIFF2025121975000043.tif85166

[0328] Characterization data for SUW207 (minor C13 diastereomer): TLC R f = 0.43 (75% EtOAc / Hex, UV active, dark blue spot with p-anisaldehyde); TIFF2025121975000044.tif78170

[0329] Synthesis procedure for SUW208 TIFF2025121975000045.tif57128 References : AB Smith et al., Design, Synthesis, and Evaluation of Carbamate-Substituted Analogues of (+)-Discodermolide. Org. Lett. 2005, 7, 311-314.

[0330] Acylation To a solution of SI-4 (15 mg, 0.012 mmol, 1 equiv) in 2:1 CHCl / pyridine (1.23 mL, 0.01 M) was added phenyl isocyanate (40 uL, 0.369 mmol, 30 equiv). After 48 h, the reaction mixture was quenched by the dropwise addition of saturated aqueous NHCl (2 mL). The layers were separated, and the aqueous layer was extracted with 80% EtOAc / Hex (3 x 5 mL). The combined organic layers were dried over NaSO, filtered, and concentrated. Purification was carried out by silica gel flash column chromatography (20% EtOAc / Hex) to give the C13 carbamate (assuming quantitative yield). LRMS: C 74 H 105 NNaO 17 Si2[M+Na] + Theoretical value: 1358.7; Measured value 1359.0 (TOF ESI+).

[0331] Total deprotectionTo a cooled (0 °C) solution of this carbamate (assumed 0.012 mmol, 1 equiv) in 1:1 THF / pyridine (1.31 mL) was added 70% HF-pyridine (328 uL) to give a 0.0075 M solution of 1:2:2 HF-pyr / THF / pyridine. The reaction mixture was allowed to warm to room temperature. After 20 h, HO (328 uL) was added and the reaction mixture was heated to 40 °C. After 2 h, the reaction mixture was cooled to 0 °C, diluted with 50% EtOAc / Hex (5 mL), and quenched slowly by the dropwise addition of saturated aqueous NaHCO3 until no more bubbles were observed. The layers were separated, and the aqueous layer was extracted with 80% EtOAc / Hex (5 x 5 mL). The combined organic layers were dried over NaSO4, filtered, and concentrated. Purification by silica gel flash column chromatography (25–50% EtOAc / Hex) followed by RP-HPLC (60–100% MeCN / HO) afforded SUW208 (6.6 mg, 55% yield over two steps).

[0332] Characterization data for SUW208: TLC R f = 0.36 (60% EtOAc / Hex, UV activity, CAM staining); TIFF2025121975000046.tif100170

[0333] Synthesis procedure for SUW209 TIFF2025121975000047.tif60128 Acylation To a solution of alcohol SI-4 (10 mg, 0.0082 mmol, 1 equiv.) in CHCl (1 mL), 1-adamantaneacetic acid (8 mg, 0.041 mmol, 5 equiv.), EDC-HCl (7.9 mg, 0.041 mmol, 5 equiv.), and DMAP (1 crystal) were added sequentially. After 20 h, TLC analysis indicated complete conversion of the starting material. The reaction mixture was directly purified by silica gel flash column chromatography (10% EtOAc / Hex) to afford the C-adamantyl ester in quantitative yield.

[0334] Total deprotectionTo a cooled (0°C) solution of this ester (1 equiv) in 1:1 THF / pyridine (1.5 mL) was added 70% HF-pyridine (375 uL) to give a 1:2:2 HF-pyr / THF / pyridine solution. The reaction mixture was allowed to warm to room temperature. After 24 h, HO (300 uL) was added and the reaction mixture was heated to 30°C. After 4 h, the reaction mixture was cooled to 0°C, diluted with EtOAc (10 mL), and quenched slowly by the dropwise addition of saturated aqueous NaHCO3 until no more bubbles were observed. The layers were separated, and the aqueous layer was extracted with 80% EtOAc / Hex (5 x 10 mL). The combined organic layers were dried over NaSO4, filtered, and concentrated. Purification by silica gel flash column chromatography (40–100% EtOAc / Hex) followed by RP-HPLC (70–100% MeCN / HO) afforded SUW209 (4.5 mg, 54% yield).

[0335] Characterization data for SUW209: TLC R f = 0.33 (50% EtOAc / Hex, UV active, dark blue spot with p-anisaldehyde); TIFF2025121975000048.tif85170

[0336] Synthesis procedure for SUW210 TIFF2025121975000049.tif62133 Acylation To a solution of alcohol SI-4 (10 mg, 0.008 mmol, 1 equiv.) in CHCl (0.5 mL), indole-3-propionic acid (8 mg, 0.041 mmol, 5 equiv.), EDC-HCl (8 mg, 0.0041 mmol, 5 equiv.), and DMAP (1 crystal) were added sequentially. After 24 h, the reaction mixture was directly purified by silica gel flash column chromatography (20% EtOAc / Hex) to afford the C13-indole ester (assuming quantitative yield).

[0337] Total deprotectionTo a cooled (0 °C) solution of this ester (assumed 0.008 mmol, 1 equiv) in 1:1 THF / pyridine (400 µL) was added 70% HF-pyridine (200 µL) to give a 1:2:2 HF-pyr / THF / pyridine solution. The reaction mixture was allowed to warm to room temperature. After 36 h, HO (200 µL) was added and the reaction mixture was heated to 40 °C. After 4 h, the reaction mixture was cooled to 0 °C, diluted with EtOAc (10 mL), and quenched slowly by the dropwise addition of saturated aqueous NaHCO until no more bubbles were observed. The layers were separated, and the aqueous layer was extracted with 80% EtOAc / Hex (5 x 10 mL). The combined organic layers were dried over NaSO, filtered, and concentrated. Purification was carried out by silica gel flash column chromatography (50–80% EtOAc / Hex) followed by RP-HPLC (60–100% MeCN / HO) to give SUW210 (1.6 mg, 19% over two steps).

[0338] Characterization data for SUW210: TLC R f = 0.53 (75% EtOAc / Hex, UV active, purple spot with p-anisaldehyde); TIFF2025121975000050.tif100170

[0339] Synthesis procedure for SUW211 TIFF2025121975000051.tif42167 Acylation To a vial containing SI-4 (16 mg, 0.016 mmol, 1 equiv.) in DCM (0.5 mL) was added dimethylglycine (8.4 mg, 0.082 mmol, 5 equiv.), EDCI (16 mg, 0.016 mmol, 5 equiv.), and DMAP (10 mg, 0.082 mmol, 5 equiv.). After stirring at room temperature for 16 h, the mixture was partitioned between DCM and saturated NaHCO. After extraction with DCM (2 x), the combined organics were dried over NaSO, filtered, and concentrated. Flash chromatography (30–40% EtOAc / hexanes) afforded glycinate SI-5 (10 mg, 58% yield, quant. brsm) as a white residue, which was used in the subsequent reaction.

[0340] Deprotection In a polypropylene vial, glycinate SI-5 was dissolved in 1:1 THF:pyridine (0.6 mL). HF:pyridine (0.2 mL) was added, and the reaction mixture was stirred at 40 °C for 20 h. Water (0.2 mL) was then added, and the resulting mixture was stirred at the same temperature for 2.5 h. The reaction mixture was quenched with sat. NaHCO3, extracted with EtOAc (2 x), and the combined organics were dried over Na2SO4. Flash chromatography (5-10% MeOH / DCM) afforded SUW211 (6 mg, 83% yield) as a white residue.

[0341] Characterization data for SUW211: TLC R f = 0.5 (10% MeOH / DCM, UV-active, purple spot with p-anisaldehyde); TIFF2025121975000052.tif99166

[0342] Synthesis procedure for SUW212 TIFF2025121975000053.tif49170 Acylation To a vial charged with SI-4 (11 mg, 0.011 mmol, 1 equiv.) in DCM (0.5 mL) was added succinic anhydride (3.4 mg, 0.034 mmol, 3 equiv.) and DMAP (4.1 mg, 0.034 mmol, 3 equiv.). After stirring for 16 h, the reaction mixture was partitioned between DCM and saturated NH4Cl. After extraction with DCM (2 x), the combined organics were dried over Na2SO4, filtered, and concentrated. Flash chromatography (50% EtOAc / hexanes) afforded succinate SI-6 (8 mg) as a white residue.

[0343] DeprotectionIn a polypropylene tube, succinate SI-6 was dissolved in 1:1 THF:pyridine (0.48 mL). HF-pyridine (0.12 mL) was added by running it down the side of the tube. The reaction mixture was heated in an oil bath at 40 °C for 20 h, at which point HO (0.1 mL) was added. After stirring at 40 °C for an additional 2.5 h, the reaction mixture was partitioned between HO and EtOAc. After extraction with EtOAc (3 ×), the combined organics were concentrated and purified by flash chromatography (10% MeOH / DCM) to give SUW212 (2.7 mg, 26% over two steps).

[0344] Characterization data for SUW212: TLC R f = 0.6 (10% MeOH / DCM, UV-active, purple spot with p-anisaldehyde); TIFF2025121975000054.tif100166

[0345] Example 3: PKC binding assay Having obtained a panel of compounds with diverse functional groups at C13, we began to explore how these modifications affect biological function. Since binding to PKC is a prerequisite for PKC pathway engagement, we first investigated the effect of tritium-labeled phorbol dibutyrate ([ 3 Compounds were evaluated for PKC affinity in a cell-free competitive binding assay using [H]-PDBu. Assays were performed using representative members of the classical and novel PKC families, PKCα (classical) and PKCδ (novel). Compounds were subsequently assayed for PKC activation in live cells using an isoform translocation assay. Translocation to the plasma membrane is a hallmark of PKC activation; therefore, optical observation of the subcellular localization of a PKCδ-GFP fusion protein by confocal microscopy can be used as an assay to determine whether a compound can enter cells and engage its PKC isoform target (Wender et al. Proc. Natl. Acad. Sci. USA 2011, 108 (17), 6721-6726).

[0346] PKC binding assay protocol The protein kinase C (PKC) affinity of bryostatin 1 and bryostatin analogs was determined as described below. 3 H-phorbol-12,13-dibutyrate( 3 H-PDBu) The procedure involves determining bound radioligand by glass fiber filtration.

[0347] Preparation of PKC binding assay buffer To a 50 mL polypropylene tube, Tris-HCl (pH 7.4, 1 M, 1 mL), KCl (1 M, 2 mL), CaCl (0.1 M, 30 μL), and bovine serum albumin (BSA, 40 mg, Sigma-Aldrich) were added. This mixture was diluted to 20 mL with deionized HO and gently mixed. This buffer was stored on ice until use. The final concentrations of these components are listed in Table 1.

[0348] Table 1. Composition of PKC binding assay buffer TIFF2025121975000055.tif46156

[0349] Preparation of phosphatidylserine (PS) vesicles For each duplicate assay, 3.5 mg of phosphatidylserine (Avanti Polar Lipids, porcine, 25 mg / mL CHCl3 solution) was concentrated by removing chloroform under a stream of nitrogen, followed by vacuum. Solid PS as vesicles was suspended in freshly prepared PKC binding assay buffer (3.5 mL) by six 30-second sonications with a 30-second pause between each sonication (Branson Sonifier 250, power = 2, 50% duty cycle). The resulting cloudy mixture (1 mg / mL) was stored on ice until use.

[0350] Preparation of PKC isoform solutions Assay PKC was prepared by dissolving a 4 μg aliquot of the indicated recombinant human PKC isoform (Invitrogen) in 11.6 mL of PKC binding assay buffer (enough for two assays). The diluted PKC was stored on ice and used immediately.

[0351] 3 Preparation of H-PDBu solution 3 H-PDBu (American Radiolabeled Chemicals, Inc.; 1 mCi / mL acetone solution; specific radioactivity: 20 μCi / mmol) was diluted 10-fold with DMSO. The resulting 500 nM stock solution was further diluted to 30 nM with DMSO.

[0352] Preparation of analog compound dilutions Compound dilutions were prepared as 3- or 4-fold serial dilutions from a selected "high" concentration. Seven concentrations were used to define inhibition curves for each analog compound (i.e., for SUW200, the analog concentrations used were 3000 nM, 750 nM, 188 nM, 46.9 nM, 11.7 nM, 2.93 nM, and 0.73 nM).

[0353] "Master Mix" Solution In a polypropylene tube, add 3.3 mL of 1 mg / mL PS vesicle solution, 11 mL of PKC isoform solution, and 1.1 mL of 30 nM 3 The H-PDBu solution was added, and the resulting solution was vortexed to mix and stored on ice.

[0354] PKC binding assay protocol material: Glass fiber filters (Whatman GF / B) were prepared by soaking them in a solution of aqueous polyethyleneimine (10% by volume, 18 mL) in deionized water (600 mL) for at least 1 hour. During the incubation and the rest of the assay, 500 mL of 20 mM Tris, pH 7.4 "rinse buffer" was chilled on ice.

[0355] Triplicate data points were obtained for each analog concentration. For each data point, 280 μL of the "master mix" solution at the indicated concentration and 20 μL of the analog compound were added to a polypropylene tube. Nonspecific PDBu was measured by replacing the analog compound with unlabeled PDBu (20 μL of a 75 μM stock solution, assay concentration: 5 μM). 3 H-PDBu binding was assessed in triplicate. By replacing the analogue compound with 20 μL DMSO, up to 3 H-PDBu binding was assessed in triplicate. Each solution was vortexed to mix, incubated at 37°C for 10 minutes, and then incubated on ice for at least 30 minutes before filtration. The assay contents of each polypropylene tube were vacuum filtered through polyethyleneimine-soaked filters using a Brandel Harvester, washed with rinse buffer (3X), and dried first in vacuo for 5 minutes, followed by drying at ambient conditions for ≥2 hours. The resulting filters, containing a circular perforation at each data point, were removed with tweezers and placed in scintillation vials. Scintillation vials were filled with Bio-Safe II scintillation fluid (5 mL), and radioactivity was measured in a Beckman LS 6000SC scintillation counter. Counts per minute (cpm) were averaged for each triplicate dilution. Data were plotted (cpm vs. log(concentration)) using GraphPad Software's Prism® software, and IC values were calculated using the program's built-in one-site competitive least-squares regression function. 50 It was decided that. i The value is given by the formula: K i = IC 50 / (1 + ([ 3 H-PDBu] / K d ) was calculated. 3 H-PDBu K d was measured separately in a saturation binding experiment under the same conditions.

[0356] Protocol for PKCδ-GFP translocation assay cell culture Chinese hamster ovary factor K1 (CHO-k1, ATCC) cells were cultured in F-12 Kaighn's medium (Hyclone, supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin, hereafter referred to as F-12 + / +) in an incubator (5% CO2) at 37°C. Cell cultures were maintained by subculture at a ratio of 1:3 (every 2–3 days) when the cells reached approximately 75–100% confluence, as follows:

[0357] The medium was aspirated (taking care not to disturb the adherent cells), and the cells were removed from the culture flask (T75, Falcon) using 3 mL of 0.25% trypsin-EDTA (Gibco). 1 mL of the cell suspension was then added to 9 mL of fresh F-12 + / +, and the sample was subcultured until confluence was reached (approximately 2–3 days).

[0358] Cell plating Confluent CHO-k1 cell cultures were detached from T75 flasks using 3 mL of 0.25% trypsin-EDTA. Cells were counted using a Countess II Automated Cell Counter (Fisher). The cell suspension was diluted to 240,000 cells / mL with fresh F-12 + / +, and 2.5 mL of this diluted stock solution was added to one well of a 6-well plate. Cells were then cultured for 24 hours.

[0359] Transfection Cells were transfected using Lipofectamine 2000 reagent (Invitrogen) or DA 13:11 at a charge ratio of 10:1 as previously described by McKinlay et al. (PNAS 2017, 114, E448-E456).

[0360] Lipofectamine 2000 The F-12 + / + solution was aspirated, and the cells were washed with F-12 - / -. Next, 2 mL of fresh F-12 - / - solution was added to each well, taking care not to disturb the adherent cells. For each well of CHO-k1 cells, 12.5 μL of Lipofectamine 2000 reagent (Invitrogen) was added to 250 μL of Opti-MEM reduced serum medium (Invitrogen) in a polypropylene tube and incubated at room temperature for 20 minutes. For each well, 4 μg of PKCδ-GFP pDNA and 250 μL of Opti-MEM reduced serum medium were added to a separate polypropylene tube. 250 μL of Lipofectamine 2000 suspension was added to this DNA suspension, and the solution was incubated at room temperature for 30 minutes. 500 μL of the Lipofectamine / DNA suspension was added to each well of a 6-well plate. The cells were then incubated at 37°C (5% CO2) for approximately 24 hours.

[0361] DA 13:11 The F-12 + / + solution was aspirated, and the cells were washed with F-12 − / −. Next, 2.4 mL of fresh F-12 − / − was added to each well, taking care not to disturb the adherent cells. A 4 μg aliquot of PKCδ-GFP pDNA was added to PBS (pH 5.5, final volume 100 μL). Next, 5.6 μL of DA 13:11 (2 mM stock solution in DMSO) was added to this DNA solution, and the mixture was gently mixed (by flicking) for 20 seconds, at which point it was added directly to each well of a 6-well plate. The cells were then incubated at 37°C (5% CO2) for approximately 24 hours.

[0362] Plating onto chambered coverglass slides After incubation, the medium was aspirated, and the cells were washed with PBS (2.0 mL) and trypsinized (500 μL). The cell suspension was then diluted with 2.0 mL of F-12 + / +. 200 μL aliquots were added to three wells of a Lab-Tek II 4-well chambered coverglass slide (Fisher) to create a total of four slides, each with three wells of cells. The cell suspension was then directly diluted with an additional 600 μL of F-12 + / +. The resulting samples were incubated for approximately 24 hours before imaging.

[0363] Dosing and Data Acquisition Fluorescence images were acquired using a Leica SP8 White Light Confocal microscope and the Leica AF software package. Prior to analysis, the medium was aspirated and replaced with 800 μL of PBS (Hyclone, Ca) supplemented with glucose (10 mM). 2+ Mg 2+ A single compound (with or without HCl) was added to each well of a chambered coverglass slide. Bryostatin and bryostatin analogs were diluted to the appropriate concentration in 200 μL of 10 mM glucose in PBS. Cells were imaged at predetermined locations in each well using adaptive focus control, and data were recorded in triplicate. Cells were imaged at 30-second intervals for 20–40 minutes after compound addition (set at t = 0). Data were recorded at room temperature. Images were exported in .lif file format, and fluorescence intensity was analyzed using FIJI (NIH) software. To observe migration, a small cytosolic region of interest was selected in each cell. After background subtraction and normalization, fluorescence intensity values were plotted against time. Graphed data represent the average of a minimum number of replicates.

[0364] This battery of biological assays provided important insights into the effect of B-ring substitutions on compound function. The data are summarized in Figure 6, panels A-B, and Table 2.

[0365] TIFF2025121975000056.tif158166

[0366] As can be seen from Table 2, the PKC binding affinities of the compounds were [ 3 The compounds were assessed in a competitive binding assay with [H]-phorbol dibutyrate. In vitro cellular import and binding of compounds to PKC isoforms in live cells were determined by observing translocation of PKC-GFP fusion proteins from the cytosol to the plasma membrane. Representative images are shown in Figure 6, panel A. * indicates the minimum effective concentration required to induce membrane translocation of PKC-GFP. **At 1 nM, relative to DMSO control. *** indicates a very brief transition observed (see Figure 6, panel D). ND = undetermined.

[0367] Figure 6, panel A. Bryolog-induced PKC activation as determined by confocal microscopy of PKCδ-GFP translocation to the plasma membrane. Figure 6, panels B-D. Cytosolic fluorescence was normalized to t = 0 (the time immediately before compound addition to the medium) and plotted against time. Error bars have been omitted for clarity. Maximum translocation of PKCδ-GFP to the plasma membrane is reported in Table 2.

[0368] Our goal was to determine whether cell-free PKC affinity correlates with intracellular PKC translocation and how binding and translocation affect downstream functions (e.g., CD22 induction). As expected, nearly all compounds designed using our pharmacophore model retained strong PKC binding affinity (<10 nm), comparable to that reported for bryostatin 1 (Table 2). However, certain C13 functional groups reduced PKC binding affinity. For example, charged substituents at C13 (SUW211, SUW212) reduced affinity for PKC by approximately 20- to 100-fold (Table 2), consistent with the less efficient partitioning of these groups in phosphatidylserine (PS) vesicle complexes (the membrane surrogate used in the cell-free binding assay). Furthermore, replacement of the C13 methyl-(Z)-enoate with benzyl-(Z)-enoate resulted in an approximately 30-fold decrease in potency. This same decrease in binding affinity was not observed with C13 esters bearing larger, relatively hydrophobic substituents (SUW209, SUW210, Table 2) and more conformationally flexible linkers. With these exceptions, the majority of the compounds tested exhibited single-digit nanomolar binding affinities for PKC, further demonstrating the predictive value of our proposed pharmacophore model and allowing these compounds to be advanced in vitro in PKC translocation assays.

[0369] In its inactive state, PKC resides in the cytosol. However, upon binding to its endogenous ligand, DAG, or exogenous small molecule ligands, such as phorbol esters and bryostatin, the resulting PKC-ligand complex resides in the inner leaflet of the plasma membrane (Newton, AC AJP Endocrinol. Metab. 2010, 298 (3), E395-E402). This translocation of PKC is a prerequisite for PKC activation and is therefore the most downstream activity. Both experimental studies and molecular dynamics simulations suggest that PKC-ligand complexes can adopt multiple binding states, likely influencing differential binding of scaffolding proteins and phosphorylation of downstream effector proteins, thereby resulting in diverse signaling outcomes (Newton et al. Crit. Rev. Biochem. Mol. Biol. 2018, 53 (2), 208-230; Ryckbosch et al. Nat. Commun. 2017, 8 (1), 6; Newton, ACJ Biol. Chem. 1995, 270 (48), 28495-28498; and Newton, AC Chem. Rev. 2001, 101 (8), 2353-2364). Due to the dynamic nature of this PKC signaling synapse, PKC can transmit a variety of signals with diverse biological implications (Newton, AC AJP Endocrinol. Metab. 2010, 298 (3), E395-E402). Therefore, our goal was to develop compounds with modifications to the B ring of the bryostatin scaffold that would elicit distinct signaling outcomes by establishing different interactions between the active signaling complex and the cell membrane. Monitoring the membrane translocation of PKCδ-GFP fusions in live cells is a convenient in vitro assay of compound function and cell permeability, allowing for the real-time determination of qualitative differences in precise compound activity (Wender et al. Proc. Natl. Acad. Sci. USA 2011, 108 (17), 6721-6726).Because of our design strategy for exemplary bryostatin analogs (see above), we were able to investigate how substituents at this position affect the kinetics of PKC activation.

[0370] Our studies demonstrate that the thermodynamics of PKC binding and the kinetics of ligand cellular import and translocation are often uncoupled, providing insights for isoform-selective signaling regulation. As a positive control, bryostatin 1, a single-nanomolar binder of all PKC isoforms, translocated approximately 70% of cytosolic PKCδ-GFP to the plasma membrane within 5 minutes at a concentration of 200 nM (Table 2; Figure 6, Panel C). Introducing hydrophilic substituents at C13 (SUW203 and SUW204) resulted in compounds that bound both conventional and novel PKC isoforms with single-nanomolar affinity, but these analogs behaved differently from bryostatin 1 in translocation assays (Figure 6, Panels B-C). At 200 nM, SUW204 translocated approximately 25% of cytosolic PKC to the plasma membrane in 20 minutes, whereas SUW203 required a concentration of 1000 nM to translocate approximately 50% of cytosolic PKC. Similarly, compounds with a charged substituent at C13 (SUW211, SUW212) did not achieve sustained PKC translocation even at concentrations up to 1000 nM (Table 2; Figure 6, panel D). This indicates that interactions between the membrane and functional groups on the bryostatin B ring are important for compound function, and that PKC binding is necessary but not sufficient for compound activity in vitro. Compounds with hydrophilic or charged modifications at C13 likely cannot embed effectively in the plasma membrane, thereby exhibiting attenuated PKC function.

[0371] In addition to exploring the effect of the hydrophilic functional group at C13, we also investigated a series of C13 esters with different sizes and hydrophobicities and structural motifs (aryl, heteroaryl, alkyl, and adamantyl), all of which could be efficiently derived from the corresponding C13 alcohol (see, for example, Scheme 3). While we found that diverse functional groups at this position are tolerated in terms of PKC binding affinity, our analog library exhibited widely different profiles in functional assays. In contrast to the free alcohol SUW204, capping the C13 hydroxyl group with a smaller hydrophobic substituent yielded compounds that were comparable in potency to bryostatin 1 with respect to PKC translocation (Figure 6, panel D). The two diastereomers of the C13 acetate (SUW206 and SUW207) exhibited nearly identical behavior with each other and with bryostatin 1. Although the C13 phenyl carbamate SUW208 effectively translocated PKC to the plasma membrane at 200 nM, the translocation time, approximately 15 min, was longer than that of other analogs active in the same assay (Figure 6, panel D), indicating that small changes in size and / or polarity at this site can affect the kinetics and efficacy of ligand-mediated PKC activation and signaling and / or cellular import.

[0372] To complement the synthesis of compounds with relatively similar size requirements for C13, we also incorporated larger substituents at this position, bearing significantly different functional groups than those found in natural products. The C13 adamantyl ester SUW209 and the C13 indoyl ester SUW210 were designed to enhance the potential for membrane interaction through increased localized hydrophobicity and the ability to capture cation-π contacts with cationic lipid head groups on the inner leaflet of the plasma membrane, respectively. We hypothesized that these two membrane-binding modes could differentially bias the orientation of PKC-ligand complexes in the membrane, thereby affecting downstream signaling outcomes. While both compounds are high-affinity PKC binders (Table 2), they were inactive in a PKC translocation assay at 200 nM, requiring 1000 nM to translocate approximately 70% of cytosolic PKC to the plasma membrane in approximately 5 minutes (Figure 6, panel D).

[0373] Next, we investigated how the C13 functional group might affect bulk compound properties and the correlation between compound hydrophobicity (measured by cLogP) and activity (PKC translocation at 200 nM). Plotting cLogP against the percentage of membrane-bound PKCδ-GFP at 200 nM (Figures 7A and 7B) revealed an effective window of cLogP values required for efficient translocation. Compounds with cLogP values between 1.00 and 4.00 were active at 200 nM, suggesting that lipophilicity should be effectively balanced when considering modifications to the B ring of the bryostatin scaffold. It is also noteworthy that various PKC activation kinetics were observed among compounds within the effective lipophilicity range. While most compounds exhibited logarithmic activation curves, select compounds exhibited either a delayed activation pattern (SUW208, Figure 6, Panel D) or more sustained linear activation kinetics (SUW218 and SUW229, Figure 6, Panel C). These observations regarding the kinetics of PKC-ligand binding and the intracellular distribution of the complexes can be rationalized by the timing of ligand entry into cells, with more polar compounds entering cells at a slower rate and limiting their ability to form stable complexes with the plasma membrane, and by the subsequent ligand-regulated partitioning of the PKC-ligand complex between the plasma membrane and the cytosol. This is potentially important because it is well known that the kinetics of PKC activation can have profound consequences for downstream signaling outcomes (Alfonso, SI et al., Sci. Signal. 9, ra47, https: / / doi.org / 10.1126 / scisignal.aaf6209 (2016); Newton, AC, AJP Endocrinol. Metab. 298, E395-E402 (2010)).

[0374] With the exception of the benzylenoates SUW219 and SUW220, C13 alkylenoates in both (Z) and (E) geometries were well tolerated. Interestingly, the benzylenoate SUW219 bound to PKC with approximately two-fold reduced affinity, suggesting that the (Z) olefin geometry may orient the C13 substituent in a way that influences the conformation of the southern hemisphere pharmacophore element. However, the smaller linear alkylenoates were generally strong binders and active ligands of PKC in vitro (Table 2; Figure 6, panel C). The C13 allylenoate is of strong synthetic interest, as olefin cross-metathesis at this position could render this monosubstituted alkene accessible for the final step in bryostatin diversification (see, for example, Scheme 2).

[0375] Example 4: CD22 surface expression assay Finally, the inventors also determined the effects of compounds on CD22 expression in vitro in NALM6 cells, an ALL cell line previously studied in the context of CD22-targeted CAR T cell therapy (Fry et al. Nat. Med. 2018, 24 (1), 20-28; and Ramakrishna et al. Blood 2017, 130 (Suppl 1)). Following the establishment of the generally high affinity of the exemplary bryostatin analogs, as well as their PKC translocation kinetics and extent of dynamics, the inventors sought to evaluate these exemplary analogs in assays relevant to clinical use in enhancing targeted cancer immunotherapy using bryostatin and its analogs as adjuvants. While treatment of ALL patients with CD22-targeted CAR T cell therapy has been reported, patients who fail this treatment are thought to have lower surface densities of CD22. Fry et al. clearly demonstrated that a critical threshold of CD22 surface density is required for in vitro activation of anti-CD22 CAR T cells (Nguyen, S. et al., J. Clin. Oncol. 34, 10536-10536 (2016)) and tumor elimination in a mouse tumor xenograft model (Fry et al. Nat. Med. 2017). We developed an in vitro model of bryostatin-induced increased CD22 surface expression in ALL using NALM6 cells to investigate bryostatin 1 and bryostatin analogs as adjuvant leads for CD22-targeting CAR T therapy.

[0376] CD22 surface expression protocol cell culture NALM6 and clone G5 cells (ATCC) were cultured in RPMI-1640 (Hyclone, + L-glutamine, + 10 mM HEPES, 10% fetal bovine serum, 1% penicillin / streptomycin, hereafter referred to as RPMI-1640 unless otherwise specified) in an incubator (5% CO2) at 37°C. Cell cultures were prepared according to the manufacturer's instructions at a density of 4 x 10 5 ~3 x 106 cells / mL.

[0377] Plating and administration The cell suspension from a confluent T75 flask (Fisher) was transferred to a 15 mL Falcon tube and centrifuged at 1100 rpm for 7 minutes. The supernatant was aspirated, and the cell pellet was resuspended in approximately 5 mL of fresh RPMI-1640. Cells were counted using a Countess II Automated Cell Counter (Fisher). An aliquot of the cell suspension was diluted with additional RPMI-1640 to a concentration of 1 x 10 cells. 6 A 5.2 mL stock solution of 100 cells / mL was prepared. 199 μL of this stock solution was added to 24 wells of a 96-well plate (enough for triplicate measurements of eight different experimental conditions). DMSO, bryostatin 1, and bryostatin analogs were dosed in triplicate. Each experiment included DMSO (negative control), 10 nM bryostatin 1 (positive control), and an untreated sample (negative control). 1 μL of DMSO or the appropriate compound stock solution in DMSO was added to each well. Cells were incubated for 24 hours, at which point the cell suspension was transferred to a 1.5 mL Eppendorf tube and added to 1.0 mL of PBS (Hyclone, Ca). 2+ Mg 2+ The samples were centrifuged at 2000 rpm for 5 minutes at room temperature. The supernatant was aspirated, and the cell pellet was resuspended in 400-600 μL of RPMI-1640. 2 x 10 5 ~3 x 10 6 cells / mL of cells were subcultured in 24-well plates for an additional 24 h to 7 days, at which point CD22 surface expression was assayed by flow cytometry.

[0378] Flow cytometry Cells from one well were counted using a Countess II Automated Cell Counter (Fisher). Approximately 200,000 to 300,000 cells from each well were added to a 1.5 mL Eppendorf tube containing PBS (final volume: approximately 1.2 mL). The cell suspension was centrifuged at 1500 rpm at 4°C for 7 minutes. The supernatant was aspirated, and the cell pellet was resuspended in 99 μL of pre-chilled FACS buffer (0.5% w / v BSA in PBS). 1 μL of PE mouse anti-human CD22 (5 μL / 1 x 10 6 Test cell line (BD Biosciences, catalog number 562859) was added, and the solution was incubated at 4°C for 30–45 minutes. The sample was then further diluted with 1.0 mL of FACS buffer and centrifuged at 1500 rpm for 7 minutes at 4°C. The supernatant was aspirated, and the cell pellet was resuspended in 200 μL of FACS buffer. The resulting suspension was transferred to a FACS tube (Fisher, catalog number 352058). Cells were stained with DAPI and analyzed for CD22 surface expression using a FACScan Analyzer at the Stanford Shared FACS Facility. Data analysis was performed using FlowJo and Microsoft Excel.

[0379] We sought to use this assay to determine whether Bryostatin 1 and bryostatin analogs could achieve sufficient upregulation of CD22 surface expression necessary for CD22-targeted CAR T cell-mediated tumor elimination. NALM6 cells were incubated with Bryostatin 1 and bryostatin analogs for 24 hours, at which point the test compounds were washed out of the medium and the cells were analyzed for CD22 surface expression by flow cytometry. We found that Bryostatin 1 induced a >2-fold increase in CD22 surface density (Figure 4, panels A-B). Interestingly, the bryostatin-driven increase in CD22 surface expression persisted for up to 7 days after treatment (Figure 4, panel A), suggesting that sequential administration of Bryostatin 1 followed by infusion of anti-CD22 CAR T may contribute to the CD22 surface expression that may be contributing to patient relapse. loIt has been suggested that this could be a viable strategy for eliminating tumor cells (Fry, TJ et al., Nat. Med. 24, 20-28 (2018)).

[0380] Importantly, while the C13-modified bryostatin analogs displayed a range of activity in the CD22 induction assay, select compounds were highly potent and comparable to bryostatin 1 in our assay (Figure 4, Panel B). Surprisingly, we observed a significant effect of the C13 enoate geometry on compound function for both the C13 methyl and allylic enoates (Figure 4, Panel B). The (Z)-enoate was significantly more active than the corresponding (E)-enoate, further suggesting that modifications to the B-ring of the bryostatin scaffold can modulate PKC signaling kinetics. As previously mentioned, the best-performing analog, SUW229, exhibited a distinct PKC activation profile from bryostatin 1, suggesting that distinct PKC activation kinetics may influence biologically important downstream signaling outcomes.

[0381] Protocol for CD22 surface expression on JB and 2F7 cells Culture conditions AIDS-NHL cell lines were incubated in IF10 medium (IMDM medium (Life Technologies) containing 10% fetal bovine serum (FBS, Omega Scientific), 100 units / mL penicillin, and 100 μg / mL streptomycin (Invitrogen)) at 37°C in 5% CO2.

[0382] Activation procedure for AIDS-NHL cell lines AIDS-NHL cells were cultured in round-bottom 96-well tissue culture plates at a cell density of 200,000 cells / well in 200 μL of IF10 medium containing equimolar concentrations of bryostatin 1, SUW201, or SUW229 as indicated. Cells were exposed to compounds for 24 or 48 hours and then stained for flow cytometric analysis of receptor levels.

[0383] Flow cytometry Cells in each well were washed with phosphate-buffered saline (PBS) containing 2% FBS, centrifuged at 233 x g for 9 minutes, and resuspended in 50 μL of a 1:1 dilution of PBS:human AB serum (Sigma). Cells were stained with anti-human CD22 (Biolegend, clone S-HCL-1, 363506) and incubated at 4°C for 25 minutes, then washed and fixed with 2% paraformaldehyde. Stained samples were stored at 4°C. Flow cytometry samples were analyzed using a FACSCelesta (BD Biosciences) flow cytometer, and data were analyzed using FlowJo software (version 10).

[0384] To determine whether the results observed with NALM6 cells (e.g., as outlined above) apply to other cell lines using this assay, the AIDS-related lymphoma cell lines JP and 2F7 cells were incubated with synthetic bryostatin 1, SUW201, and SEQ229 (Figure 4, panels C-D). JB is an Epstein-Barr virus (EBV)-negative AIDS-lymphoma cell line originally grown from a bone marrow sample removed from an HIV+ individual and harboring the Burkitt lymphoma translocation (Moses, A. V. et al., Nat. Med. 3, 1242-1249 (1997)). 2F7 is an AIDS-associated non-Hodgkin's lymphoma cell line of the Burkitt subtype that is EBV-positive (Widney, D.P. et al. Levels of Murine, but Not Human, CXCL13 Are Greatly Elevated in NOD-SCID Mice Bearing the AIDS-Associated Burkitt Lymphoma Cell Line, 2F7. PLoS One 8, e72414, https: / / doi.org / 10.1371 / journal.pone.0072414 (2013)). Burkitt lymphoma is one of the most common subtypes of AIDS non-Hodgkin's lymphoma, and like Hodgkin's lymphoma, AIDS patients are at significantly higher risk than the general population due to impaired cell-mediated immunity (Guech-Ongey, M. et al., Blood 116, 5600-5604 (2010)). Importantly, synthetic bryostatin 1 and analogs SUW201 and SUW229 upregulated CD22 surface expression approximately twofold in each cell line tested ( Fig. 4 , panels C–D), further highlighting the potential for PKC modulators to be used generally to enhance CD22-targeted cancer immunotherapy.

[0385] Figure 4, Panels A-D: Bryostatin-promoted CD22 cell surface expression. Panel A illustrates that synthetic bryostatin 1 promotes increased CD22 surface expression. NALM6 cells were incubated with 10 nM bryostatin 1 for 24 hours. The compound was washed out, and the cells were subcultured for the indicated times. CD22 surface expression was then assayed by flow cytometry (n = 3 biological replicates; data presented as mean ± SE). Panel B: NALM6 cells were incubated with 1 nM (left bar) or 10 nM (right bar) of compound for 24 hours. The compound was washed out, and the cells were subcultured for 24 hours. CD22 surface expression was then assayed by flow cytometry (n = 3 biological replicates; data presented as mean ± SE). Panel C: JB cells were incubated with 1 nM (left bar) or 10 nM (right bar) of compound for 48 hours. CD22 surface expression was then assayed by flow cytometry (n = 6 biological replicates; data presented as mean ± SE). Panel D: 2F7 cells were incubated with 1 nM (left bar) or 10 nM (right bar) of compound for 48 hours. CD22 surface expression was then assayed by flow cytometry (n = 6 biological replicates; data presented as mean ± SE).

[0386] This is a novel collection of bryostatin analogs, some with similar binding affinity, translocation, and CD activation activity to bryostatin, while others have similarly effective affinity but some variation in selectivity, are superior CD activators, and are better tolerated in animal studies.

[0387] summary The results presented herein emphasize the importance of design and chemical synthesis in natural product-inspired drug discovery. The efficient synthesis of bryostatin 1, a compound recently considered too complex to produce by practical methods, also allows access to its immediate precursors and derivatives. Most exemplary analogs exhibit bryostatin-like PKC affinity. Other protein targets with C1 domains similar to PKC (e.g., as described herein) may also be potential mediators of bryostatin drug activity. However, some exhibit different affinities. Some of these exemplary analogs are more amenable to synthesis, a factor that may influence candidate selection for clinical medicine. More importantly, some analogs may be comparable or even superior to bryostatin in migration assays, and selected analogs may match or even exceed the CD22 induction exhibited by bryostatin 1. Importantly, some analogs are better tolerated in animal studies. Importantly, these studies also demonstrate that bryostatin can be modified in specific regions, and that such changes can result in distinct migration and CD22 induction effects while preserving affinity.

[0388] While the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of this invention that certain changes and modifications can be made thereto without departing from the spirit and scope of the appended claims.

[0389] Accordingly, the foregoing merely illustrates the principles of the present invention. Those skilled in the art will recognize that various arrangements, not expressly described or shown herein, may be devised that embody the principles of the present invention and are within its spirit and scope. Furthermore, the primary intent of all examples and conditional language described herein is to aid the reader in understanding the principles of the present invention and the concepts the inventors have contributed to furthering the art, and should not be construed as limiting the specifically described examples and conditions. Furthermore, all statements herein that describe principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents. Furthermore, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Furthermore, none of the disclosures herein are intended to be general, whether or not such disclosure is expressly recited in the claims.

[0390] Accordingly, the scope of the present invention is not limited to the exemplary embodiments shown and described herein. The spirit and scope of the present invention are embodied in the appended claims. In the claims, 35 USC §112(f) or 35 USC §112(6) is expressly defined to be invoked with respect to a claim limitation only if the literal words "means for" or "step for" appear at the beginning of such claim limitation; if such literal words are not used in a claim limitation, 35 USC §112(f) or 35 USC §112(6) is not invoked.

[0391] Notwithstanding any appended claims, the present disclosure described herein is also described by the following clauses.

[0392] Item 1. A method for regulating target cells in a subject, the method comprising: (a) expression of the antigen in the target cell, (b) translocation of the antigen in the target cell, (c) cell surface presentation of the antigen in the target cell, and (d) cell surface retention of the antigen in the target cell. contacting the target cells with an effective amount of a bryostatin agent to selectively enhance one or more of: Includes.

[0393] Item 2. The antigen is Protein antigens, peptide antigens, neoantigens, and antigens derived from target cell mRNA processing The method according to item 1, wherein the method is selected from the group consisting of:

[0394] Item 3. The target cells are chimeric antigen receptor (CAR)-modified T cells or chimeric antigen receptor-natural killer cells (CAR-NK cells), The method of paragraph 1 or 2, wherein said step of contacting said target cell with a bryostatin agent enhances expression or cell surface presentation and persistence of said CAR.

[0395] Item 4. The CAR is: a target cell surface antigen selected from a viral antigen, a bacterial antigen, a parasitic antigen, a tumor cell-associated antigen (TAA), an antigen associated with a diseased cell, and an antigen derived from mRNA processing of said cell, and any fragment thereof; The method of item 3, wherein the antibody has affinity for

[0396] Paragraph 5. The method of Paragraph 4, wherein the modified T cells are obtained from peripheral blood mononuclear cells, umbilical cord blood cells, a purified T cell population, or a T cell line.

[0397] Item 6. The method according to any one of Items 3 to 5, wherein the contacting step is carried out ex vivo, and the target cells are removed from the subject (they are autologous cells).

[0398] Item 7. The method according to any one of Items 3 to 5, wherein the contacting step is carried out ex vivo, and the target cells are removed from a donor (they are allogenic cells).

[0399] Item 8. The method according to Item 1, wherein the target cells are selected from cancer cells, cancer stem cells, and cancer progenitor cells.

[0400] Paragraph 9. The method of Paragraph 8, wherein said contacting is performed in vivo and comprises administering said bryostatin agent to a subject having cancer.

[0401] Item 10. The method of Item 8 or 9, wherein the target cells are sensitized to elimination by the subject's immune system.

[0402] Clause 11. The method of clause 8, wherein the subject is relapsed or refractory to immune cell mediated elimination and the bryostatin agent modulates a T cell mediated immune response against the target cell population.

[0403] Item 12. The method according to any one of Items 9 to 11, wherein the subject is undergoing cancer immunotherapy.

[0404] Item 13. Administering to the subject an effective amount of a therapeutic agent capable of one or more of inhibiting the proliferation of the regulated target cells or eliminating the regulated target cells. Item 10. The method of item 9, further comprising:

[0405] Item 14. The method according to Item 1, wherein the target cells are HIV-infected cells.

[0406] Item 15. The method according to Item 14, wherein the target cell is a cell infected with latent HIV, and modulating the immunogenicity of the target cell comprises activating expression of HIV.

[0407] Item 16. The contacting step is performed in vivo and comprises administering the bryostatin agent to a subject diagnosed with or suspected of having HIV; Item 1, wherein the contacting step can have a therapeutic effect.

[0408] Item 17. The method of Item 15, further comprising administering to the subject a therapeutically effective amount of a therapeutic substance capable of eliminating the regulated target cells having activated HIV expression.

[0409] Item 18. A method for treating cancer in a subject, the method comprising: (a) administering to a subject an effective amount of a bryostatin agent to enhance the presentation and persistence of cell surface antigens or neoantigens on target cells in the subject; and (b) administering to the subject a therapeutically effective amount of a therapeutic agent that specifically binds to the cell surface antigen and treats cancer in the subject. Includes.

[0410] Item 19. The method according to Item 18, wherein the subject is relapsed or refractory to targeted anti-cancer therapy.

[0411] Item 20. The method of Item 18, wherein the bryostatin agent sensitizes the target cancer cells to growth inhibition by the therapeutic agent.

[0412] Clause 21. The method of clause 18, wherein the bryostatin agent sensitizes the target cancer cells to elimination by the therapeutic agent.

[0413] Item 22. The method according to Item 18, wherein, prior to step (a), the target cancer cells present a therapeutically ineffective level of cell surface antigens on the surface of the target cells.

[0414] Item 23. The bryostatin agent is (a) expression of a cell surface antigen, (b) transfer of the expressed cell surface antigen to the surface of the target cell, and (c) retention of the cell surface antigen on the surface of the target cell. Item 19. The method of item 18, wherein one or more of the following are enhanced:

[0415] Item 24. The method of Item 18, wherein the bryostatin agent enhances cell surface presentation of the cell surface antigen by 50% or more.

[0416] Item 25. The method of Items 23 or 24, wherein cell surface antigen presentation of the target cancer cells is enhanced for two or more days after administration of the bryostatin agent.

[0417] Item 26. The method according to any one of Items 18 to 25, wherein the therapeutic agent is selected from chimeric antigen receptor-expressing T cells (CAR T cells), CAR-natural killer cells (CAR-NK cells), an antibody agent, an antibody-drug conjugate (ADC), and a bispecific antibody agent.

[0418] Clause 27. The method of clause 26, wherein step (b) comprises administering to the subject a composition comprising a therapeutically effective amount of CAR T cells or CAR-NK cells that specifically bind to a cell surface antigen presented on the target cell population.

[0419] Clause 28. The method of Clause 27, wherein said bryostatin agent modulates a T cell-mediated or NK cell-mediated immune response against said target cell population.

[0420] Paragraph 29. The method of any one of paragraphs 27 to 28, wherein the target cell population comprises a tumor antigen selected from CD10, CD19, CD20, CD21, CD22, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD52, CD80, CD86, CD137, CDK4, CDK6, OX40, and CD340.

[0421] Item 30. The method according to any one of Items 27 to 29, wherein the chimeric antigen receptor-expressing T cells or NK cells are effective in treating a B-cell malignant disease, CLL, ALL, B-ALL, leukemia, lymphoma, or solid tumor.

[0422] Item 31. The method according to Item 30, wherein the solid tumor is selected from breast cancer, prostate cancer, bladder cancer, soft tissue sarcoma, lymphoma, esophageal cancer, uterine cancer, bone cancer, adrenal cancer, lung cancer, thyroid cancer, colon cancer, glioma, liver cancer, pancreatic cancer, kidney cancer, cervical cancer, testicular cancer, head and neck cancer, ovarian cancer, neuroblastoma, and melanoma.

[0423] Item 32. The method of any one of Items 27 to 31, wherein the administration of the bryostatin agent precedes the administration of the therapeutically effective amount of CAR-T cells or CAR-NK cells.

[0424] Item 33. The method of any one of Items 27 to 31, wherein the administration of the bryostatin agent is simultaneous with the administration of the therapeutically effective amount of CAR-T cells or CAR-NK cells.

[0425] Item 34. The method of any one of Items 27 to 31, wherein the administration of the bryostatin agent is after the administration of the therapeutically effective amount of CAR-T cells or CAR-NK cells.

[0426] Item 35. The method according to Item 26, wherein step (b) comprises administering to the subject a therapeutically effective amount of an antibody agent, ADC, or bispecific antibody agent that specifically binds to the cell surface antigen.

[0427] Item 36. The method of Item 35, wherein the antibody agent comprises a human monoclonal antibody, or an antigen-binding portion thereof.

[0428] Item 37. The method according to Item 35, wherein the antibody agent is an antibody comprising a full-length antibody of the IgG1 isotype or IgG4 isotype.

[0429] Item 38. The method according to Item 35, wherein the agent is an ADC comprising a cytotoxic agent.

[0430] Item 39. The method of Item 38, wherein the cytotoxic agent is a cytotoxin or a radioactive agent.

[0431] Clause 40. The method of clause 39, wherein the cytotoxic agent is conjugated to the antibody of the ADC via a linker.

[0432] Item 41. The method according to Item 40, wherein the linker is selected from a peptidyl linker, a hydrazine linker, and a disulfide linker.

[0433] Item 42. The method of any one of Items 38 to 41, wherein the cytotoxic agent is selected from calicheamicin, auristatin, maytansinoid, taxol derivatives, and duocarmycins.

[0434] Item 43. The method according to Item 35, wherein the ADC is selected from inotuzumab ozogamicin and gemtuzumab ozogamicin.

[0435] Item 44. The method according to Item 35, wherein the agent is a bispecific antibody agent.

[0436] Item 45. The method of Item 44, wherein the bispecific antibody is an anti-CD20 / anti-CD22 bispecific antibody fusion protein or an anti-CD19 / anti-CD22 bispecific antibody fusion protein.

[0437] Item 46. The method according to any one of Items 35 to 45, wherein the agent is administered by a route selected from oral, intraocular, aural, subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal, and inhalation.

[0438] Item 47. The method according to any one of Items 18 to 46, wherein the cancer is leukemia or B-cell lymphoma.

[0439] Item 48. The method according to Item 47, wherein the B-cell lymphoma is non-Hodgkin's lymphoma.

[0440] Clause 49. The method of clause 47, wherein the cancer is selected from Burkitt's lymphoma and B-cell chronic lymphocytic leukemia.

[0441] Item 50. The method according to any one of Items 18 to 49, wherein the cancer is melanoma, prostate cancer, breast cancer, ovarian cancer, esophageal cancer, or kidney cancer.

[0442] Item 51. The method according to any one of Items 18 to 50, wherein the subject is a mammal.

[0443] Item 52. The method of Item 51, wherein the subject is relapsed or refractory to cell surface antigen targeting therapy.

[0444] Paragraph 53. The method of Paragraph 52, wherein the cell surface antigen is selected from CD10, CD19, CD20, CD21, CD22, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD52, CD80, CD86, CD137, CDK4, CDK6, OX40, and CD340.

[0445] Item 54. The method of any one of Items 18 to 53, further comprising determining the level, expression, or presentation of a cell surface antigen in target cancer cells of a sample obtained from the subject.

[0446] Item 55. The method further comprises administering at least one additional anti-cancer therapy to the patient, 55. The method of any one of items 18 to 54, wherein the additional anti-cancer therapy is selected from radiation therapy, chemotherapy, immunotherapy, checkpoint inhibitors, surgery, and vascular-targeting therapy.

[0447] 56. Evaluating one or more biomarkers in the subject's sample to assay the cancer status. Item 56. The method according to any one of items 18 to 55, further comprising:

Claims

1. 1. A method of modulating target cells in a subject, comprising: below: (a) expression of the antigen in the target cell; (b) translocation of the antigen in the target cell; (c) cell surface presentation of the antigen in the target cell; and (d) cell surface retention of the antigen in the target cell. contacting the target cells with an effective amount of a bryostatin agent to selectively enhance one or more of: A method comprising:

2. The antigen is Protein antigens, peptide antigens, neoantigens, and antigens derived from target cell mRNA processing 2. The method of claim 1, wherein the compound is selected from the group consisting of:

3. the target cells are chimeric antigen receptor (CAR)-modified T cells or chimeric antigen receptor-natural killer cells (CAR-NK cells); 3. The method of claim 1 or 2, wherein said step of contacting said target cells with a bryostatin agent enhances expression or cell surface presentation and persistence of said CAR.

4. The method of claim 3, wherein the contacting step is performed ex vivo and the target cells are removed from the subject (are autologous cells).

5. The method of claim 3, wherein the contacting step is performed ex vivo and the target cells are removed from a donor (are allogeneic cells).

6. The method of claim 1, wherein the target cells are selected from cancer cells, cancer stem cells, and cancer progenitor cells.

7. 7. The method of claim 6, wherein said contacting step is performed in vivo and comprises administering said bryostatin agent to a subject having cancer.

8. 8. The method of claim 6 or 7, wherein the target cells are sensitized to elimination by the subject's immune system.

9. administering to the subject an effective amount of a therapeutic agent capable of one or more of inhibiting proliferation of the modulated target cells or eliminating the modulated target cells.

8. The method of claim 7, further comprising:

10. The method of claim 1, wherein the target cell is an HIV-infected cell.

11. wherein the contacting step is performed in vivo and comprises administering the bryostatin agent to a subject diagnosed with or suspected of having HIV; 10. The method of claim 1, wherein the contacting step can have a therapeutic effect.

12. 1. A method of treating cancer in a subject, comprising: (a) administering to a subject an effective amount of a bryostatin agent to enhance the presentation and persistence of cell surface antigens or neoantigens on target cells in the subject; and (b) administering to the subject a therapeutically effective amount of a therapeutic agent that specifically binds to the cell surface antigen and treats cancer in the subject. A method comprising:

13. 13. The method of claim 12, wherein the subject is relapsed or refractory to a targeted anti-cancer therapy.

14. The method of claim 12, wherein prior to step (a), the target cancer cells present a therapeutically ineffective level of cell surface antigens on the target cell surface.

15. The bryostatin agent is (a) expression of a cell surface antigen, (b) transfer of the expressed cell surface antigen to the surface of the target cell, and (c) retention of the cell surface antigen on the surface of the target cell.

13. The method of claim 12, wherein one or more of:

16. 16. The method of any one of claims 12 to 15, wherein the therapeutic agent is selected from chimeric antigen receptor-expressing T cells (CAR T cells), CAR-natural killer cells (CAR-NK cells), an antibody agent, an antibody-drug conjugate (ADC), and a bispecific antibody agent.

17. 17. The method of any one of claims 12 to 16, wherein the cancer is leukemia or B-cell lymphoma.

18. 17. The method of any one of claims 12 to 16, wherein the cancer is melanoma, prostate cancer, breast cancer, ovarian cancer, esophageal cancer, or renal cancer.

19. The method of any one of claims 12 to 18, further comprising determining the level or expression or presentation of a cell surface antigen in target cancer cells of a sample obtained from said subject.

20. further comprising administering at least one additional anti-cancer therapy to said patient; 20. The method of any one of claims 12 to 19, wherein the additional anti-cancer therapy is selected from radiation therapy, chemotherapy, immunotherapy, checkpoint inhibitors, surgery, and vascular-targeted therapy.

21. assessing one or more biomarkers in the subject's sample to assay the cancer status.

21. The method of any one of claims 12 to 20, further comprising:

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