Patent Application
20230218771
The text of the computer readable sequence listing filed herewith, titled “JHU-39025-252_SQL”, created Jul. 25, 2022, having a file size of 12,563 bytes, is hereby incorporated by reference in its entirety.
Cancer immunotherapy has shown great promise in treating patients with advanced or metastatic tumors. However, only patients with immune-stimulating tumors, which are characterized by favorable tumor-infiltrating lymphocytes (TILs), seem to achieve effective clinical responses. The tumor microenvironment (TME) is the primary location where tumor cells and the TILs interact; the responsiveness of tumors to immunotherapy therefore depends, at least in part, on the TME immunophenotype. As a consequence, promoting an immune-stimulating TME that favors TILs has been proposed to be one of the most critical methods for maximizing the potential of cancer immunotherapy.
Thus, there remains a need for compositions and methods that not only kill cancer cells, but also stimulate and maintain an anti-cancer immune response in the TME.
The present disclosure provides a composition comprising: (a) a prodrug comprising one or more cytotoxic agents conjugated to a hydrophilic moiety by a linker, and (b) one or more immunomodulators. Also provided is a hydrogel comprising the composition and methods of using the composition to kill cancer cells.
The present disclosure is predicated, at least in part, on the generation of a prodrug containing a cytotoxic agent (e.g., a chemotherapeutic agent) conjugated to a hydrophilic moiety, such as peptide or polypeptide. When complexed with an immunomodulator (e.g., a checkpoint inhibitor), the prodrug can self-assemble into hydrogels at a tumor site, followed by long-term release of the cytotoxic agent and the immunomodulator. Release of the cytotoxic agent and immunomodulator results in killing of cancer cells and elicits an immune-stimulating tumor microenvironment which sensitizes against cancer recurrence.
The disclosure demonstrates that local administration of a composition described herein can self-assemble into a nanofiber hydrogel, which induces durable T cell memory and robust systemic antitumor immunity, which may be used to prevent tumor recurrence and potential metastasis. Thus, the present inventive compositions and methods of local therapy with immunomodulators can enhance antitumor immune responses and improve the overall response rate.
In some embodiments, the compositions and methods described herein may lower the dosage of immunomodulator required for therapeutic efficacy and minimize systemic exposure. Moreover, the use of the disclosed prodrug hydrogelators to deliver an immunomodulator (e.g., an immune checkpoint inhibitor) eases concerns about any possible short- or long-term toxicities of synthetic or natural drug carriers, representing a simple yet effective means to achieve combination chemo- and immuno-therapy with translation potential. In some embodiments, the incorporation of an MMP-2 responsive substrate and an iRGD segment in the prodrug design contributes to the improved treatment efficacy and reduced side effects.
The present disclosure therefore describes a combined chemo-immunotherapy strategy on the basis of a prodrug hydrogel to boost immunity against cancer. This in situ-formed prodrug hydrogel can serve as a therapeutic reservoir for sustained intratumoral release of both a cytotoxic agent (e.g., chemotherapeutic agent) and an immunomodulator (e.g., an immune checkpoint inhibitor). The prodrug hydrogel increases the frequency of T effector cells and reduces the population of immune suppressor cells, so as to provoke a robust antitumor immune response. The long-term memory T cell and systemic immune response induced by local delivery of the prodrug composition suggests that this platform may be used to treat tumor recurrence and metastasis.
In some embodiments, the disclosure provides a composition comprising: (a) a prodrug comprising one or more cytotoxic agents conjugated to a hydrophilic moiety by a linker, and (b) one or more immunomodulators. The term “prodrug,” as used herein, refers to a biologically inactive compound which can be metabolized in a subject (e.g., a human) to produce a pharmacologically active compound (e.g., a drug). Prodrugs may be used to improve how a compound is absorbed, distributed, metabolized, and excreted by the subject.
An agent is “cytotoxic” and induces “cytotoxicity” if the agent kills or inhibits the growth of cells, particularly cancer cells. In some embodiments, for example, cytotoxicity includes preventing cancer cell division and growth, as well as reducing the size of a tumor or cancer. Cytotoxicity of cancer cells may be measured using any suitable cell viability assay known in the art, such as, for example, assays which measure cell lysis, cell membrane leakage, and apoptosis. For example, methods including but not limited to trypan blue assays, propidium iodide assays, lactate dehydrogenase (LDH) assays, tetrazolium reduction assays, resazurin reduction assays, protease marker assays, 5-bromo-2′-deoxy-uridine (BrdU) assays, and ATP detection may be used. Cell viability assay systems that are commercially available also may be used and include, for example, CELLTITER-GLO® 2.0 (Promega, Madison, Wis.), VIVAFIX™ 583/603 Cell Viability Assay (Bio-Rad, Hercules, Calif.); and CYTOTOX-FLUOR™ Cytotoxicity Assay (Promega, Madison, Wis.).
Any cytotoxic agent that kills cancer cells or inhibits cancer cell proliferation may be included in the prodrug. Examples of cytotoxic agents include, but are not limited to, alkylating agents, antibiotics, antimetabolites, free radical generators and mitotic inhibitors. It will be appreciated that cytotoxic agents may also be referred to in the art as “cytotoxic chemotherapeutics” or simply “chemotherapeutics.” Thus, in some embodiments, the cytotoxic agent employed in the disclosed prodrug comprises one or more chemotherapeutics. Examples of cytotoxic chemotherapeutics include, but are not limited to, alkylating agents, nitrogen mustard alkylating agents, nitrosourea alkylating agents, antimetabolites, purine analog antimetabolites, pyrimidine analog antimetabolites, hormonal antineoplastics, natural antineoplastics, antibiotic natural antineoplastics, and vinca alkaloid natural antineoplastics, such as carboplatin and cisplatin. More specifically, chemotherapeutic agents that may be employed in the disclosed prodrug include, for example, goserelin, leuprolide, tamoxifen, aldesleukin, tretinoin (ATRA), adriamycin, asparaginase, bleomycin, busulphan, cisplatin, carboplatin, carmustine, capecitabine, chlorambucil, cytarabine, cyclophosphamide, camptothecin (CPT), dacarbazine, dactinomycin, daunorubicin, dexrazoxane, docetaxel, doxorubicin, etoposide, floxuridine, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine, mercaptopurine, meplhalan, methotrexate, mitomycin, mitotane, mitoxantrone, nitrosurea, paclitaxel, pamidronate, pentostatin, plicamycin, procarbazine, rituximab, streptozocin, teniposide, thioguanine, thiotepa, vinblastine, vincristine, vinorelbine, taxol, transplatinum, anti-vascular endothelial growth factor compounds (“anti-VEGFs”), anti-epidermal growth factor receptor compounds (“anti-EGFRs”), 5-fluorouracil, etc. In some embodiments, the cytotoxic agent is camptothecin, paclitaxel, bumetanide, verteporfrin, or vorapaxar.
The type and number of chemotherapeutics used in the disclosed prodrug composition will depend on the standard chemotherapeutic regimen for a particular tumor type. In some embodiments, two or more different cytotoxic agents may be included in the prodrug. For example, two different chemotherapeutic agents can be included in a single prodrug. In other embodiments, three or four different cytotoxic agents may each be linked by a biodegradable linker to the hydrophilic moiety of the prodrug of the present invention.
In some embodiments, the cytotoxic agent or drug (D) acts as a hydrophobic portion of the prodrug compositions of the present invention. As used herein, the term “hydrophobic” biologically active agents or drug molecules describes a heterogeneous group of molecules that exhibit poor solubility in water but that are typically, but certainly not always, soluble in various organic solvents. Often, the terms slightly soluble (1-10 mg/ml), very slightly soluble (0.1-1 mg/ml), and practically insoluble (<0.1 mg/ml) are used to categorize such substances. Drugs such as steroids and many anticancer drugs (e.g., chemotherapeutic agents) are important classes of poorly water-soluble drugs; however, their water solubility varies over at least two orders of magnitudes. Typically, such molecules require secondary solubilizers such as carrier molecules, liposomes, polymers, or macrocyclic molecules such as cyclodextrins to help the hydrophobic drug molecules dissolve in aqueous solutions necessary for drug delivery in vivo. Other types of hydrophobic drugs show even a lower aqueous solubility of only a few ng/ml. Since insufficient solubility commonly accompanies undesired pharmacokinetic properties, the high-throughput screening of kinetic and thermodynamic solubility as well as the prediction of solubility is of major importance in discovery (lead identification and optimization) and development.
While the prodrug ideally comprises a cytotoxic agent as described above, in some embodiments the prodrug may comprise other types of biologically active agents. As used herein, the term “biologically active agent” includes any compound or molecule for treating tumor-related diseases, e.g. drugs, inhibitors, and proteins. An active agent and a biologically active agent are used interchangeably herein to refer to a chemical or biological compound that induces a desired pharmacological and/or physiological effect, wherein the effect may be prophylactic or therapeutic. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of those active agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the terms “active agent,” “pharmacologically active agent” and “drug” are used, then, it is to be understood that the invention includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs etc.
As used herein, the term “biologically active agent” can also include imaging agents for use in identifying the location of the molecules in the tissues. In accordance with an embodiment, the imaging agent is a fluorescent dye. The dyes may be emitters in the visible or near-infrared (NIR) spectrum. Known dyes useful in the present invention include carbocyanine, indocarbocyanine, oxacarbocyanine, thiocarbocyanine and merocyanine, polymethine, coumarine, rhodamine, xanthene, fluorescein, boron-dipyrromethane (BODIPY), Cy5, Cy5.5, Cy7, VivoTag-680, VivoTag-S680, VivoTag-S750, AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight547, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, and ADS832WS.
Organic dyes which are active in the NIR region are known in biomedical applications. However, there are only a few NIR dyes that are readily available due to the limitations of conventional dyes, such as poor hydrophilicity and photostability, low quantum yield, insufficient stability and low detection sensitivity in biological system, etc. Significant progress has been made on the recent development of NIR dyes (including cyanine dyes, squaraine, phthalocyanines, porphyrin derivatives and BODIPY (borondipyrromethane) analogues) with much improved chemical and photostability, high fluorescence intensity and long fluorescent life. Examples of NIR dyes include cyanine dyes (also called as polymethine cyanine dyes) are small organic molecules with two aromatic nitrogen-containing heterocycles linked by a polymethine bridge and include Cy5, Cy5.5, Cy7 and their derivatives. Squaraines (often called Squarylium dyes) consist of an oxocyclobutenolate core with aromatic or heterocyclic components at both ends of the molecules, an example is KSQ-4-H. Phthalocyanines, are two-dimensional 18n-electron aromatic porphyrin derivatives, consisting of four bridged pyrrole subunits linked together through nitrogen atoms. BODIPY (borondipyrromethane) dyes have a general structure of 4,4′-difluoro-4-bora-3a, 4a-diaza-s-indacene) and sharp fluorescence with high quantum yield and excellent thermal and photochemical stability.
Other imaging agents which may be employed in the compositions of the present invention include PET and SPECT imaging agents. The most widely used agents include branched chelating agents such as di-ethylene tri-amine penta-acetic acid (DTPA), 1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and their analogs. Chelating agents, such as di-amine dithiols, activated mercaptoacetyl-glycyl-glycyl-gylcine (MAG3), and hydrazidonicotinamide (HYNIC), are able to chelate metals like 99mTc and 186Re. Instead of using chelating agents, a prosthetic group such as N-succinimidyl-4-18F-fluorobenzoate (18F-SFB) is necessary for labeling peptides with 18F. In accordance with a preferred embodiment, the chelating agent is DOTA.
Various forms of the biologically active agents may be used. These include, without limitation, such forms as uncharged molecules, molecular complexes, salts, ethers, esters, amides, prodrug forms and the like, which are biologically activated when implanted, injected or otherwise placed into a subject.
A moiety, substance, compound, or molecule is “hydrophilic” when its interactions with water and other polar substances are more thermodynamically favorable than its interactions with oil or other hydrophobic solvents. A hydrophilic agent is typically charge-polarized and capable of hydrogen bonding, which makes it soluble not only in water but also in other polar solvents. In some cases, both hydrophilic and hydrophobic properties occur in a single molecule, and such molecules are referred to as “amphiphilic” molecules or “amphiphiles.” Examples of amphiphilic molecules include, but are not limited to, lipids that form cell membrane, soaps, alcohols, and certain amino acids.
The hydrophilic moiety may be any molecule or portion of a molecule that confers a self-assembly feature to the disclosed prodrug. Exemplary moieties include, but are not limited to, peptides, polypeptides, and oligo ethyleneoxide (OEG). Any suitable hydrophilic peptide or polypeptide may be included in the prodrug, but ideally the peptide or polypeptide comprises a biological activity such as tumor targeting, tissue penetrating, cell penetrating, apoptosis-inducing, and/or is capable of binding to known cellular epitopes, such as integrins or cancer cell receptors, and derivatives, or functional fragments or functional homolog of such peptides. The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. Oligo(ethylene glycol) (OEG) chains are hydrophilic and have been widely used as side chains of conjugated polymers. In addition to hydrophilicity, OEG chains exhibit high polarity, high flexibility, and ionic conductivity.
The term, “amino acid” includes the residues of the natural α-amino acids (e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Lys, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well as β-amino acids, synthetic and non-natural amino acids. Many types of amino acid residues are useful in the polypeptides and the invention is not limited to natural, genetically-encoded amino acids. Examples of amino acids that can be utilized in the peptides described herein can be found, for example, in Fasman, 1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc., and the reference cited therein. Another source of a wide array of amino acid residues is provided by the website of RSP Amino Acids LLC.
Reference herein to “derivatives” includes parts, fragments and portions of the hydrophilic moiety of the prodrug molecule. For peptides and polypeptides, a derivative also includes a single or multiple amino acid substitution, deletion and/or addition. Homologues include functionally, structurally or stereochemically similar peptides from the naturally occurring peptide or protein. All such homologs are contemplated by the present invention.
Analogs and mimetics include molecules which contain non-naturally occurring amino acids or which do not contain amino acids but nevertheless behave functionally the same as the peptide. Examples of non-natural amino acids and amino acid derivatives include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, omithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. Natural product screening is one useful strategy for identifying analogs and mimetics.
Exemplary hydrophilic peptides or polypeptides include, but are not limited to, RGD or RGDR (SEQ ID NO: 1), HDK, iRGD (cCRGDKGPDC) (SEQ ID NO: 2), or derivatives thereof, having z=1 to 6 repeating moieties. As used herein, the term “iRGD” refers to 9-amino acid cyclic (c) peptide (sequence: CRGDKGPDC) (SEQ ID NO: 2) molecular mimicry agent that was originally identified in an in vivo screening of phage display libraries in tumor-bearing mice. The peptide is able to home to tumor tissues, but in contrast to standard RGD (arginylglycylaspartic acid) peptides, iRGD is distributed much more extensively into extravascular tumor tissue. It was later identified that this extravasation and transport through extravascular tumor tissue was due to the bi-functional action of the molecule: after the initial RGD-mediated tumor homing, another pharmacological motif is able to manipulate tumor microenvironment, making it temporarily accessible to circulating drugs. This second step is mediated through specific secondary binding to neuropilin-1 receptor, and subsequent activation of a trans-tissue pathway, dubbed the C-end Rule (CendR) pathway. It is believed that the RGD sequence motif mediates binding to αVβ3 and αV5 integrins that are expressed on tumor endothelial cells. Second, upon αV binding, a protease cleavage event is activated, revealing the c-terminal CendR motif (R/KXXR/K) (SEQ ID NO: 3) of the peptide. The CendR motif is then able to bind to neuropilin-1, activating an endocytotic/exocytotic transport pathway. Other hydrophilic peptides which can be used in conjunction with the compositions of the present invention include tumor associated antigens, such as, for example, CEA, TAG-72, CyclinB 1, Ep-CAM, Her2/neu, CDK4, fibronectin, p53, ras, etc.
The one or more cytotoxic agents of the disclosed prodrug may be conjugated to a hydrophilic moiety (e.g., a peptide or polypeptide) by a linker. A linker is any chemical moiety that is capable of linking one compound, usually a drug, to another compound (e.g., a cell-binding agent such as an peptide ligand or antibody) in a stable, covalent manner. Linkers can be susceptible to or be substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the antibody remains active. In some embodiments, the linker can be any amino acid with a side chain having a free amino, carboxyl or disulfide group. Exemplary amino acids useful as amino acid linkers in the nanofiber hydrogels of the present invention include lysine (K), glutamic acid (E), arginine (R) and cysteine (C).
Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, and esterase labile groups. Linkers also include charged linkers, and hydrophilic forms thereof as described herein and known in the art. In some embodiments, the linker may be a cleavable linker, a non-cleavable linker, a hydrophilic linker, and a dicarboxylic acid-based linker. It is contemplated that the cytotoxic agent is covalently linked to a hydrophilic moiety via a biodegradable bond. For example, amino groups, carboxyl groups and disulfide bonds are capable of being cleaved in vitro by various chemical and biological or enzymatic processes. In some embodiments, the linker comprises at least one disulfide group (referred to as a “disulfide linker”). For example, in some embodiments, the linker can comprise a C1-C6 acyl-disulfide group. For example, the linker can be (4-(pyridin-2-yldisulfanyl)butanoate) (buSS), or (4-(pyridin-2-yldisulfanyl)ethyl carbonate) (etcSS). Other linkers that may be employed include, but are not limited to, N-succinimidyl 4-(2pyridyldithiojpentanoate (SPP); N-succinimidyl 4-(2-pyridyldithio)-2-sulfopentanoate (sulfoSPP); N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB); N-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB); N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC); N-sulfosuccinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (sulfoSMCC); N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB); and N-succinimidyl-[(Nmaleimidopropionamidoj-tetraethyleneglycol] ester (NHS-PEG4-maleimide).
In other embodiments, the linker further comprises a matrix metalloproteinase-2 (MMP-2) cleavable peptide (also referred to herein as an “MMP-2 degradable peptide”). Matrix metalloproteinases (MMPs) are major extracellular enzymes involved in cancer initiation, progression, and metastasis. MMPs are widely used as cancer biomarkers and therapeutic targets. Recently, MMPs have been investigated as robust tumor microenvironmental stimuli for ‘smart’ MMP-responsive drug delivery and tumor targeting and have shown great potential in cancer diagnosis and therapy (see, e.g., Yao et al., Trends in Pharmacological Sciences, 39(8): 766-781 (2018); doi.org/10.1016/j.tips.2018.06.003). For example, because MMPs cleave active agents from the extracellular matrix by degrading proteins, MMPs have been used to cleave prodrugs and thus release the active drug selectively in the diseased tissue overexpressing MMPs (e.g., cancers). MMP-activated peptide prodrugs have been generated, in which therapeutic drugs are attached to an MMP substrate peptide. When the peptide is cleaved, the drug becomes active. The prodrug can be orally available or parenterally administered and targeting depends only on the peptide sequence specificity. Many such prodrugs have been designed for various anti-cancer agents (see, e.g., U.S. Pat. Nos. 6,844,318, 6,855,689, and 5,659,061; and Timar et al., Cancer Chemother Pharmacol., 41(4): 292-8 (1998)). There has been a growing interest in the development of peptide-based supramolecular filaments responsive to specific MMPs due to their important biomedical applications (Yang et al., Soft Matter 2009, 5, 2546-2548; Giano et al, Biomaterials 2011, 32, 6471-6477; Galler et al., J. Am. Chem. Soc. 2010, 132, 3217-3223; and Chau et al., Biomaterials 2008, 29, 17131719).
Any suitable peptide that can be cleaved or degraded by MMP-2 may be employed in the prodrug described herein. In some embodiments the MMP-2 cleavable peptide comprises one or more of the following amino acid sequences PLGLAG (SEQ ID NO: 4), PLGVR (SEQ ID NO: 5), or GPLGIAGQ (SEQ ID NO: 6), or functional variants or derivatives thereof. Without being limited to any particular example, the composition of the present invention can be a hetero-dual drug amphiphile comprising a first cytotoxic agent and a second cytotoxic agent linked by the same or different linker, for example buSS, to the peptide portion of the prodrug.
The composition further comprises one or more immunomodulators. The terms “immune modulator,” “immune modulator protein,” and “immunomodulator,” may be used interchangeably to refer to a substance or protein that affects normal immune function of an organism. In some embodiments, an immune modulator stimulates immune functions of an organism, such as by activating, boosting, or restoring immune responses. In other embodiments, an immune modulator may exert a negative effect on immune function, such as by attenuating an existing immune response or preventing the stimulation of an immune response. Immunomodulators may be naturally occurring substances (e.g., proteins) or may be synthetically generated compounds. Examples of naturally occurring immunomodulators include, but are not limited to, cytokines, chemokines, and interleukins. Cytokines are small proteins (˜25 kDa) that are released by a variety of cell types, typically in response to an activating stimulus, and induce responses through binding to specific receptors. Examples of cytokines include, but are not limited to, interferons (i.e., IFN-α, IFN-β, IFN-γ), leukemia inhibitory factor (LIF), oncostatin M (OSM), granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), tumor necrosis factors (e.g., TNF-α), transforming growth factor (TGF)-β family members (e.g., TGF-β1 and TGF-β2). Chemokines are a class of cytokines that have chemoattractant properties, inducing cells with the appropriate receptors to migrate toward the source of the chemokine. Chemokines fall mainly into two groups: CC chemokines comprising two adjacent cysteines near the amino terminus, or CXC chemokines, in which two equivalent cysteine residues are separated by another amino acid. CC chemokines include, but are not limited to, chemokine ligands (CCL) 1 to 28, and CXC chemokines include, but are not limited to, CXC ligands (CXCL) 1 to 17. Interleukins are a structurally diverse group of cytokines which are secreted by macrophages in response to pathogens and include, for example, interleukin-1 (IL-1), IL-2, IL-6, IL-12, and IL-8. Other cytokines, chemokines, and interleukins are known in the art (see, e.g., Cameron M. J., and Kelvin D. J., Cytokines, Chemokines and Their Receptors. In: Madame Curie Bioscience Database, Austin (Tex.): Landes Bioscience; 2000-2013). In other embodiments, an immunomodulator may be synthetically or recombinantly generated. For example, an immunomodulator may be a fusion protein, a chimeric protein, or any modified version of a naturally-occurring immune modulator.
The immunomodulator may be an immune checkpoint regulator. Immune checkpoints are molecules on immune cells that must be activated or inhibited to stimulate immune system activity. Tumors can use such checkpoints to evade attacks by the immune system. The immune checkpoint regulator may be an antagonist of an inhibitory signal of an immune cell, also referred to as a “checkpoint inhibitor,” which blocks inhibitory checkpoints (i.e., molecules that normally inhibit immune responses). For example, the immune checkpoint regulator may be an antagonist of A2AR, BTLA, B7-H3, B7-H4, CTLA4, GALS, IDO, KIR, LAG3, PD-1, TDO, TIGIT, TIM3 and/or VISTA. Checkpoint inhibitor therapy therefore can block inhibitory checkpoints, restoring immune system function. Currently approved checkpoint inhibitors target the molecules CTLA4, PD-1, and PD-L1, and include ipilimumab (YERVOY®), nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), and durvalumab (IMFINZI®). Any suitable checkpoint inhibitor, such as those described in, e.g., Kyi, C. and M. A. Postow, Immunotherapy, 8(7): 821-37 (2016); Collin, M., Expert Opin Ther Pat., 26(5): 555-64 (2016); Pardoll, D. M., Nat Rev Cancer, 12(4): 252-6 (2012); and Gubin et al., Nature, 515(7528): 577-81 (2014)) may be included in the disclosed compositions. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody, such as an anti-CTLA-4 antibody, an anti-B7-H4 antibody, an anti-B7-H1 antibody, an anti-LAG3 antibody, an anti-CD47 antibody, or an anti-PD1 antibody. More particularly, the immune checkpoint inhibitor is an anti-CD47 antibody (also referred to herein as “aCD47”) or an anti-PD1 antibody (also referred to herein as “aPD1”). In other embodiments, the immune checkpoint regulator may be an agonist of an immune cell stimulatory receptor, such as an agonist of BAFFR, BCMA, CD27, CD28, CD40, CD122, CD137, CD226, CRTAM, GITR, HVEM, ICOS, DR3, LTBR, TACI and/or OX40.
PD-1 ligands (PD-L1 and PD-L2) expressed on the surface of tumor cells and antigen presenting cells (APCs) engage PD-1+ T cells, resulting in T cell apoptosis, anergy, and exhaustion. Blocking these interactions between PD-1 and its ligands with an anti-PD-1 antibody (aPD1) leads to restoration of T cell function and long-term antitumor immune response. Despite its promising potential, current aPD1 therapy benefits a relatively small fraction of patients, exhibiting only a 10 to 30% treatment response rate. This is partially because patients with non-immunogenic (cold) tumors, which are characterized by insufficient infiltration of tumor antigen-specific T cells (primarily CD8+ T cells) and low tumoral expression of PD-L1, respond poorly to immune checkpoint inhibitors. Furthermore, systemic administration as a conventional means results in off-target binding of the antibodies to normal tissues, compromising the efficacy of aPD1 therapy and inducing severe immune-related side effects. Consequently, to fully realize the potential of immune checkpoint inhibitors, approaches are needed to convert cold tumors to immune-stimulating tumors and to sensitize tumors to aPD1 with minimal off-target adverse events. Examples of anti-PD1 antibodies include, but are not limited to, pembrolizumab, nivolumab, cemiplimab, avelumab, durvalumab, atezolizumab, spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), toripalimab (JS 001), MPDL3280A, MEDI4736, AMP-224, AMP-514, and MSB0010718C.
CD47 is an immunoglobulin that is overexpressed on the surface of many types of cancer cells. CD47 forms a signaling complex with signal-regulatory protein a (SIRPα), enabling the escape of these cancer cells from macrophage-mediated phagocytosis. In recent years, CD47 has been shown to be highly expressed by various types of solid tumors and to be associated with poor patient prognosis in various types of cancer. A growing number of studies have since demonstrated that inhibiting the CD47-SIRPα signaling pathway promotes the adaptive immune response and enhances the phagocytosis of tumor cells by macrophages. Indeed, blocking CD47-SIRPα interaction with an anti-CD47 antibody (aCD47) can promote tumor cell phagocytosis, and at the same time, trigger antitumor T cell immune response. Exemplary anti-CD47 monoclonal antibodies include, but are not limited to, Hu5F9-G4 (Liu et al., PLoS One.; 10(9): e0137345 (2015)), AO-176 (Puro et al., Mol Cancer Ther, 19(3) 835-846 (2020); DOI: 10.1158/1535-7163.MCT-19-1079), and commercially available antibodies (e.g., Cat. No. 127519, BioLegend, Inc., San Diego, Calif.).
In other embodiments, the immunomodulator may be a small molecule. The term “small molecule,” as used herein, refers to a low molecular weight (<900 daltons) organic compound that may regulate a biological process, with a size typically on the order of 1 nm. Small molecules exhibit a variety of biological functions and may serve a variety applications, such as in cell signaling, as pharmaceuticals, and as pesticides. Examples of small molecules include amino acids, fatty acids, phenolic compounds, alkaloids, steroids, bilins, retinoids, etc.
Recent work has demonstrated that activation of the stimulator of interferon genes (STING) signal pathway vigorously stimulates innate and adaptive immune responses in the TME, which has been proposed to enhance tumor immunogenicity. It is also well-known that chemotherapeutic drugs such as camptothecin (CPT) and etoposide can trigger cell death by instigating DNA damage. It has been reported that these anticancer drugs can lead to release of fragmented DNA into the cytosol and stimulation of intrinsic STING-dependent activity, suggesting that these small molecule drugs may exert their anticancer effects partly through triggering the STING pathway and activating the immune system.
Thus, in some embodiments, the small molecule is an agonist of the stimulator of interferon genes (STING) protein. STING is a cytosolic adaptor protein that responds to endogenous and exogenous cytosolic nucleic acid ligands. Cytosolic DNA is recognized by cyclic GMP-AMP synthetase (cGAS), which catalyzes the generation of the secondary messenger cGAMP. cGAMP binds to STING and leads to the production of type I interferons (IFNs) and other cytokines. Type I IFNs selectively stimulate antigen-presenting cells (in particular dendritic cells, DCs), which in turn prime and activate tumor antigen-specific T cells. Moreover, activation of the STING pathway in tumor cells can lead to natural killer (NK) cells infiltrating into the tumor and mobilizing an antitumor response. In light of these discoveries, STING agonists, such as cGAMP and cyclic dinucleotides (CDNs), have been utilized to promote an immune-stimulating TME and augment antitumor immune responses. While promising, CDNs as a monotherapy have shown poor therapeutic benefit in preclinical trials due to its high toxicity, requiring multiple doses and combination with immune checkpoint blockade. Examples of STING agonists that may be used in the disclosed compositions include, but are not limited to, cGAMP and cyclic dinucleotides, 2′3′-cGAMP (cyclic [G(2′,5′)pA(3′,5′)p]), 2′2′-cGAMP, cyclic [G(2′,5′)pA(2′,5′)p], 3′3′-cGAMP (cyclic [G(3′,5′)pA(3′,5′)p] (cGAMP), Bis-(3′-5′)-cyclic dimeric adenosine monophosphate (c-di-AMP), Bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP), cyclic (adenine monophosphate-inosine monophosphate) (cAIMP), cyclic [I(3′,5′)pI(3′,5′)p] di-inosine monophosphate (c-di-IMP), MK-1454, dimeric amidobenzimidazole (diABZI), and 5,6-dimethylxanthenone-4-acetic acid (DMXAA).
In aqueous solution, the disclosed prodrug composition can assemble into one-dimensional nanostructures, such as filamentous nanostructures, forming a nanostructure solution. In some cases, the one-dimensional nanostructures may assemble into nanotubes (NTs). Upon local in vivo administration, the nanostructure solution can immediately form a hydrogel. Thus, the disclosure also provides a hydrogel comprising the above-described prodrug composition. The term “hydrogel,” as used herein, refers to a three-dimensional network composed of hydrophilic polymers crosslinked either through covalent bonds or via physical intramolecular or intermolecular interactions. Hydrogels can absorb large amounts of water or biological fluids (up to several thousand percent), and swell readily without dissolving. The high hydrophilicity of hydrogels is primarily due to the presence of hydrophilic moieties such as carboxyl, amide, amino, and hydroxyl groups distributed along the backbone of polymeric chains. In the swollen state, hydrogels are soft and rubbery, closely resembling living tissues. Many hydrogels, such as chitosan and alginate-based hydrogels, exhibit desirable biocompatibility (see, e.g., El-Sherbiny, I. M., and Yacoub, M. H. Global Cardiology Science & Practice, 2013(3): 316-342 (2013); and Kyung et al, J. Appl. Polym. Sci., 83: 128-136 (2002)). Since their discovery more than 50 years ago, hydrogels have been employed in a variety of applications including, for example, drug delivery, wound healing, ophthalmic materials, and tissue engineering (see, e.g., El-Serbiny and Yacoub, supra; Hoffman, A. S., Ann. NY Acad. Sci., 944: 62-73 (2001); and Peppas et al., Eur. J. Pharm. Biopharm., 50: 27-46 (2000)).
Hydrogels typically reach their equilibrium swelling when a balance occurs between osmotic driving forces, which encourage the entrance of water or biological fluids into the hydrophilic hydrogel matrix, and the cohesive forces exerted by the polymer strands within the hydrogel. These cohesive forces resist the hydrogel expansion and the extent of these forces depends particularly on the hydrogel crosslinking density. Generally, the more hydrophilic the polymer forming the hydrogel, the higher the total water amount absorbed by the hydrogel. Likewise, the higher the crosslinking extent of a particular hydrogel, the lower the extent of the gel swelling. Hydrogels in their dried forms are referred to in the art as “xerogels,” while dry porous hydrogels resulting from the use of drying techniques (e.g., freeze-drying or solvent extraction) are referred to in the art as “aerogels” (see, e.g., Guenet, J. M., Thermoreversible gelation of polymers and biopolymers; Academic Press, New York (1992), p. 89).
Hydrogels can be classified based on a variety of characteristics, such as, for example origin, durability, response to stimuli, charge, structure, and composition. With respect to origin, hydrogels can be classified as natural, synthetic or semi-synthetic. Most synthetic hydrogels are synthesized by traditional polymerization of vinyl or vinyl-activated monomers. The equilibrium swelling values of these synthetic hydrogels vary widely according to the hydrophilicity of the monomers and the crosslinking density. Natural hydrogels typically are made of natural polymers including, for example, polynucleotides, polypeptides, and polysaccharides that can be obtained from a variety of sources (e.g., collagen from mammals and chitosan from shellfish exoskeletons). With respect to durability, hydrogels can be classified as durable (such as most polyacrylate-based hydrogels) or biodegradable (such as polysaccharide-based hydrogels), depending on their stability characteristics in a physiological environment. Biodegradable hydrogels have recently been developed in which degradable polymers inside the hydrogel matrices undergo chain scission to form oligomers of low molecular weight. The resulting oligomers are either eliminated by the organism or undergo further degradation. Such biodegradable hydrogels can be used in both biomedical and non-biomedical applications (see e.g., Zhu, W. and Ding, J., J. Appl Polym Sci., 99: 2375 (2006)). With respect to response to environmental stimuli, “smart” hydrogels have been developed that exhibit changes in swelling behavior, network structure, and/or mechanical characteristics in response to various environmental stimuli such as pH, temperature, light, ionic strength or electric field (see, e.g., Gutowska et al., J Control Release, 22: 95-104 (1992); Ferreira et al, Int J Pharm., 794: 169-180 (2000); and D'Emanuele, A. and Staniforth, J. N., Pharm Res., 8: 913-918 (1991)). These changes typically disappear upon removal of the stimulus and the hydrogels are restored to their original state in a reversible manner.
Supramolecular hydrogels are formed by the physical crosslinking of filamentous assemblies, derived of peptide amphiphile building units. Such supramolecular hydrogels employ responsive sol-gel phase transitioning, permitting their direct injection into the target sites with minimal non-surgical invasiveness. Recently, direct conjugation of therapeutic agents onto the peptides has been shown to convert the drug into an effective hydrogelator (e.g., a liquid vehicle which becomes a gel upon contact with body tissues or fluids), capable of creating a “self-delivery” hydrogel. In addition, the ability of hydrogels to respond the tumor microenvironment allows for a better controlled release kinetics and improved therapeutic efficacy.
Starting materials and reagents that may be used to prepare prodrug hydrogel compositions of the present invention are either available from commercial suppliers such as Aldrich Chemical Company (Milwaukee, Wis.), Bachem (Torrance, Calif.), Sigma (St. Louis, Mo.), or are prepared by methods well known to those of ordinary skill in the art (see, e.g., Fieser and Fieser's Reagents for Organic Synthesis, vols. 1-17, John Wiley and Sons, New York, N.Y., 1991; Rodd's Chemistry of Carbon Compounds, vols. 1-5 and supplements, Elsevier Science Publishers, 1989; Organic Reactions, vols. 1-40, John Wiley and Sons, New York, N.Y., 1991; March J; Advanced Organic Chemistry, 4th ed. John Wiley and Sons, New York, N.Y., 1992; and Larock: Comprehensive Organic Transformations, VCH Publishers, 1989). In most instances, amino acids and their esters or amides, and protected amino acids, are widely commercially available; and the preparation of modified amino acids and their amides or esters are extensively described in the chemical and biochemical literature and thus well-known to persons of ordinary skill in the art.
General procedures for preparing prodrug hydrogel compositions of the present invention involve initially attaching a carboxyl-terminal protected amino acid to a resin. After attachment, the resin is filtered, washed, and the protecting group on the alpha-amino group of the carboxyl-terminal amino acid is removed. The removal of this protecting group must take place, of course, without breaking the bond between that amino acid and the resin. The next amino, and if necessary, side chain protected amino acid, is then coupled to the free amino group of the amino acid on the resin. This coupling takes place by the formation of an amide bond between the free carboxyl group of the second amino acid and the amino group of the first amino acid attached to the resin. This sequence of events is repeated with successive amino acids until all amino acids are attached to the resin. Finally, the protected peptide is cleaved from the resin and the protecting groups removed to reveal the desired peptide. The cleavage techniques used to separate the peptide from the resin and to remove the protecting groups depend upon the selection of resin and protecting groups and are known to those familiar with the art of peptide synthesis.
In some embodiments, a prodrug hydrogel composition as described herein may have the following formula:
In other embodiments, a prodrug hydrogel composition as described herein may have the following formula:
In other embodiments, a prodrug hydrogel composition as described herein may have the following formula:
The disclosure also provides method of killing cancer cells which comprises contacting cancer cells with the above-described composition, wherein the prodrug spontaneously assembles into a hydrogel and the cytotoxic agent and immunomodulator are released from the composition, thereby killing the cancer cells.
The term “tumor,” as used herein, refers to an abnormal mass of tissue that results when cells divide more than they should or do not die when they should. In the context of the present disclosure, the term tumor may refer to tumor cells and tumor-associated stromal cells. Tumors may be benign and non-cancerous if they do not invade nearby tissue or spread to other parts of the organism. In contrast, the terms “malignant tumor,” “cancer,” and “cancer cells” may be used interchangeably herein and refer to a tumor comprising cells that divide uncontrollably and can invade nearby tissues. Cancer cells also can spread or “metastasize” to other parts of the body through the blood and lymph systems. The disclosed method ideally induces cytotoxicity in malignant tumor cells or cancer cells. The malignant tumor cells or cancer cells may be from a carcinoma (cancer arising from epithelial cells), a sarcoma (cancer arising from bone and soft tissues), a lymphoma (cancer arising from lymphocytes), a blood cancer (e.g., myeloma or leukemia), a melanoma, or brain and spinal cord tumors. The malignant tumor or cancer cells can be located in the oral cavity (e.g., the tongue and tissues of the mouth) and pharynx, the digestive system, the respiratory system, bones and joints (e.g., bony metastases), soft tissue, the skin (e.g., melanoma), breast, the genital system, the urinary system, the eye and orbit, the brain and nervous system (e.g., glioma or glioblastoma), or the endocrine system (e.g., thyroid) and is not necessarily the primary tumor. More particularly, cancers of the digestive system can affect the esophagus, stomach, small intestine, colon, rectum, anus, liver, gall bladder, and pancreas. Cancers of the respiratory system can affect the larynx, lung, and bronchus and include, for example, non-small cell lung carcinoma. Cancers of the reproductive system can affect the uterine cervix, uterine corpus, ovaries, vulva, vagina, prostate, testis, and penis. Cancers of the urinary system can affect the urinary bladder, kidney, renal pelvis, and ureter. Cancer cells also can be associated with lymphoma (e.g., Hodgkin's disease and Non-Hodgkin's lymphoma), multiple myeloma, or leukemia (e.g., acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, and the like). In some embodiments, the cancer is glioblastoma or colorectal cancer.
Ideally, the disclosed method promotes inhibition of cancer cell proliferation, the eradication of cancer cells, and/or a reduction in the size of at least one tumor such that a mammal (e.g., a human) is treated for cancer. By “treatment of cancer” is meant alleviation of cancer in whole or in part. In one embodiment, the disclosed method reduces the size of a tumor at least about 20% (e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%). Ideally, the cancer is completely eliminated, i.e., the cancer cells are killed.
The cancer cells may be contacted with the composition in vitro or in vivo. The term “in vivo” refers to a method that is conducted within living organisms in their normal, intact state, while an “in vitro” method is conducted using components of an organism that have been isolated from its usual biological context. When the cell is contacted with the composition in vitro, the cell may be any suitable prokaryotic or eukaryotic cell. When the cell is contacted with the composition in vivo, the composition may be administered to an animal, such as a mammal, particularly a human, using standard administration techniques and routes. Suitable administration routes include, but are not limited to, oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. The composition preferably is suitable for parenteral administration. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In other embodiments, the composition may be administered to a mammal using systemic delivery by intravenous, intramuscular, intraperitoneal, or subcutaneous injection.
In an exemplary embodiment, the compositions of the present invention can be used before or after surgical resection of a tumor in a subject. In some embodiments, the compositions are applied to the tissue margins and surrounding tissues after removal of the tumor. The tissues are then surgically closed. As used herein, the term “application” refers to the local in situ administration of the compositions of the present invention to the site of interest.
Without being held to any particular mechanism of action, the compositions of the present invention allow for the sustained release of biologically active agents (e.g., cytotoxic agents) and immunomodulators directly into the tumor site and surrounding tissues, as well as being administered post-operatively to enhance the effectiveness of the surgical treatment by local chemotherapeutic action on any remaining tumor cells which evaded surgical resection. The cytotoxic agent and immunomodulator will be released from the hydrogel through dissolution and through the biodegradation of the hydrogel and the bonds between the cytotoxic agent, linker, and hydrophilic moiety, to allow diffusion of the cytotoxic agent and one or more immunomodulators to come into contact with the surrounding tissues.
The hydrogel may be applied or administered to a subject by other means. It is contemplated that the prodrug hydrogels of the present invention can be made in gel, or liquid form, and then applied to the tissues of interest by spraying, swabbing, injection, or otherwise applying the compositions directly to the tissues or tumor site.
An advantage of the compositions and methods described herein is the fact that the use of local administration allows for high concentrations of the cytotoxic agent and one or more immunomodulators at the site of the tumor without having systemic effects in the subject.
Another advantage of the compositions and methods described herein is the ability to provide chemotherapy in a sustained release formulation, in parts of the body where there would otherwise be limited access of the biologically active agent to the site of interest. For example, the brain is well known for the blood-brain barrier preventing hydrophobic and polar molecules from entering the brain tissues. Systemic doses of chemotherapeutic agents do not typically cross the barrier without other measures or formulations which can cause systemic toxicities. However, application of the compositions of the present invention directly into the brain after tumor resection avoids this common problem.
The dose of the prodrug hydrogel compositions of the present invention also will be determined by the existence, nature, and extent of any adverse side effects that might accompany the administration of a particular composition. Preferably, the composition comprises an “effective amount” of the prodrug and the immunomodulator. As used herein, the term “effective amount” refers to the amount of a therapy (e.g., a prophylactic or therapeutic agent), which is sufficient to reduce the severity and/or duration of a disease, ameliorate one or more symptoms thereof, prevent the advancement of a disease or cause regression of a disease, or which is sufficient to result in the prevention of the development, recurrence, onset, or progression of a disease or one or more symptoms thereof, or enhance or improve the prophylactic and/or therapeutic effect(s) of another therapy (e.g., another therapeutic agent) useful for treating a disease, such as cancer.
Typically, an attending physician will decide the dosage of the pharmaceutical composition with which to treat each individual subject, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, compound to be administered, route of administration, and the severity of the condition being treated. By way of example, and not intending to limit the invention, the dose of the pharmaceutical compositions of the present invention can be about 0.001 to about 1000 mg/kg body weight of the subject being treated, from about 0.01 to about 100 mg/kg body weight, from about 0.1 mg/kg to about 10 mg/kg, and from about 0.5 mg to about 5 mg/kg body weight. In another embodiment, the dose of the pharmaceutical compositions of the present invention can be at a concentration from about 1 nM to about 10,000 nM, preferably from about 10 nM to about 5,000 nM, more preferably from about 100 nM to about 500 nM.
In some embodiments, the method induces a memory T cell response against a cancer in the subject. The term “memory T cell,” as used herein, can be defined as a CD8+ T cell that has responded to a cognate antigen and persists long-term. Compared to naïve cells of the same antigen-specificity, memory T cells persist in greater numbers; can populate peripheral organs; are poised to immediately proliferate, execute cytotoxic functions, and secrete effector cytokines upon antigenic re-encounter; and exist in different metabolic, transcriptional, and epigenetic states (Homann et al., Nat Med., 7: 913-9. doi: 10.1038/90950 (2001); Masopust et al., J Immunol., 172: 4875-82. doi: 10.4049/jimmuno1.172.8.4875 (2004); DiSpirito J R, Shen H, Cell Res., 20: 13-23. doi: 10.1038/cr.2009.140 (2010); Veiga-Fernandes H, Rocha B., Nat Immunol. 5: 31-7. doi: 10.1038/ni1015 (2004); and Lalvani et al., J Exp Med., 186: 859-65 (1997)). As such, hosts possessing memory T cells are often better protected against solid tumors and infection with intracellular bacteria, viruses, and protozoan parasites than their naïve counterparts (Martin, M. D., Badovinac, V. P., Frontiers in Immunology, 9: 2692 (2018)). Two major subsets of memory T cells have been identified: CD62Llo/CCR7lo effector memory T cells (Tem) and CD62Lhi/CCR7hi central memory T cells (Tcm). Expression of CCR7 and CD62L on Tcm cells facilitates homing to secondary lymphoid organs, while Tem cells are more cytolytic and express integrins and chemokine receptors necessary for localization to inflamed tissues (Sallusto et al., Nature, 401: 708-12. doi: 10.1038/44385 (1999)).
The disclosed method can be performed in combination with other therapeutic methods to achieve a desired biological effect in a patient. Ideally, the disclosed method may include, or be performed in conjunction with, one or more cancer treatments. The choice of cancer treatment used in combination with the disclosed method will depend on a variety of factors, including the cancer/tumor type, stage and/or grade of the tumor or cancer, the patient's age, etc. Suitable cancer treatments that may be employed include, but are not limited, surgery, chemotherapy, radiation therapy, immunotherapy, hormone therapy, and stem cell transplantation.
The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
The following methods and materials were employed in Examples 1-16.
The objective of this study was to develop the in situ-formed, prodrug hydrogels for local delivery of aPD1 and CPT for enhanced cancer chemo-immunotherapy. The antitumor efficacy was evaluated in GL-261 and CT 26 tumor models. Mice were randomized to different treatment groups on the basis of tumor size and body weight. Animals from varying groups were imaged to assess tumor progression, tracked to create survival curves, and re-challenged with tumors to assess immune memory. To assess the phenotype of the TME, mice were euthanized at various time points. Sample sizes were selected on based on previous experimental experience. Body weight and tumor size were measured every two days, and mice were euthanized when tumor volume exceeded 1 cm3 or when body weight loss exceeded 20%. Blinding was not performed. All experiments were repeated at least three times.
Rink Amide MBHA Resin and amino acids were obtained from AAPPTEC (Louisville, Ky.). Anti-PD1 antibody (cat. no. 135234) was purchased from BioLegend. Camptothecin (CPT) was purchased from Ava Chem Scientific (San Antonio, Tex.). Human MMP2 recombinant protein was obtained from Thermo Fisher. Cy3 NHS-ester and Cy5 NHS-ester were purchased from Lumiprobe. CDA (2′3′-c-di-AM(PS)2 (Rp,Rp), cat. no. tlrl-nacda2r) was purchased from InvivoGen. FITC labeled-c-di-AMP (cat. no. F011) and Cy7-labeled cGAMP (cat. no. C240) were purchased from Biolog Life Science Institute. All other chemicals were purchased from Sigma-Aldrich and were used without any further purification.
GL-261-luc brain cancer cell line, 4T1-luc breast cancer cell line, and CT 26 colon cancer cell line were generously donated by M. Lim, J. Hanes, and J. Fu at The Johns Hopkins University School of Medicine, respectively. GL-261-luc and 4T1-luc cells were grown in DMEM (Gibco, Invitrogen) supplemented with 10% FBS, 100 Units/mL penicillin G sodium, 100 μg/mL streptomycin sulfate, and 100 μg/ml of G418 (Invitrogen). CT 26 cells were grown in RPMI 1640 (Gibco, Invitrogen) supplemented with 10% FBS, 100 Units/mL penicillin G sodium, and 100 μg/mL streptomycin sulfate.
8-10 weeks old Female C57BL/6, BALB/c mice (Charles River) and C56BL/6J-Tmem173gt/J STING-deficient mice (STINGgt/gt, Jackson Laboratory) were kept under a 12 h light/dark cycle at the Animal Care Facility with food and water ad libitum. Animal experiments were performed in accordance with the animal protocol approved by the Animal Care and Welfare Committee at The Johns Hopkins University.
Synthesis of diCPT-iRGD
The peptide C2K-cyl[CRGDRGPDC] (SEQ ID NO: 7) was synthesized using an AAPPTEC Focus XC synthesizer via the standard Fmoc-solid phase technique. The peptide was confirmed by MALDI-TOF MS. CPT-etcSS-Pyr was synthesized using the same method as reported previously. Then, C2K-cyl[CRGDRGPDC] (10 mg, 7.2 μmol) was dissolved in an N2-purged DMSO solution of CPT-etcSS-Pyr (16 mg, 28.8 μmol) and allowed to react overnight. The solution was purified by RP-HPLC. Product fractions were combined and immediately lyophilized to give diCPT-iRGD as a white to yellow powder. The product identity was confirmed by ESI MS.
Synthesis of diCPT-PLGLAG-iRGD.
The peptide C2K-GPLGLAG-cyl[CRGDRGPDC] (SEQ ID NO: 8) was synthesized using an AAPPTEC Focus XC synthesizer via the standard Fmoc-solid phase technique. The purity and identity of the peptide were confirmed by HPLC and MALDI-TOF MS, respectively. CPT-etcSS-Pyr was synthesized using the same method as previously reported (Chem. Commun. 50, 6039-6042 (2014)). Then, C2K-GPLGLAG-cyl[CRGDRGPDC] (100 mg, 51 μmol) was dissolved in 5 ml N2-purged DMSO solution of CPT-etcSS-Pyr (115 mg, 204 μmol) and allowed to react overnight. The solution was purified by RP-HPLC and the product was lyophilized to obtain diCPT-PLGLAG-iRGD. The identity of diCPT-PLGLAG-iRGD was confirmed by ESI MS.
Self-Assembly of diCPT-PLGLAG-iRGD.
For preparation of self-assembled diCPT-PLGLAG-iRGD nanotubes (P-NT), diCPT-PLGLAG-iRGD conjugate was directly dissolved in deionized water at 500 μM and aged for 24 h at room temperature. The structure was then characterized by transmission electron microscopy (TEM, Technai12 TWIN).
For preparation of self-assembled NTs, the diCPT-iRGD conjugate was directly dissolved in deionized water at 500 μM and aged for 24 h at room temperature. After 24 hours, adding cyclic-di-AMP molecule into nanotube solution. The negative charged cyclic-di-AMP condensed onto positive charged nanotube surface based on electrostatic interactions. The gelation was triggered to occur in physiological conditions, where biomolecules and counter ions screen charges and promote nanotube-nanotube interactions to percolate into a network. The structure was then imaged by transmission electron microscopy (TEM, Tecnai12 TWIN). CDA-NT was prepared by adding 20 μg of CDA to the diCPT-iRGD NT solution (CPT, 100 μg in 25 μl). After vortexing, the mixtures were incubated at 37° C. for 30 min. The zeta potential and morphology of CDA-NT were determined using a Zetasizer and TEM, respectively. For in vitro release study and confocal imaging, CDA was labeled with FITC. For in vivo imaging, Cy7-CDA was used.
Solution-to-hydrogel transition experiments were performed by adding 20 μl of 10×PBS to 180 μl of 7.2 mM P-NT solution. The sol-gel transition behavior was tested by the inverted-vial method. For preparation of aPD1 loaded hydrogel (P-NT-aPD1), 50 μg of aPD1 was added to the P-NT solution (150 μg of CPT). After vortexing, the mixture was incubated at 37° C. for 30 min. 10×PBS was then added to the P-NT-aPD1 solution to induce the formation of a hydrogel. Distribution of aPD1 in hydrogel was further characterized using confocal microscopy (Zeiss LSM 510). For in vitro release studies and confocal imaging, aPD1 was labeled with Cy3. For in vivo imaging, Cy5.5 labeled aPD1 was used. For the diCPT-iRGD NT or CDA-NT solutions, solution-to-gelation transition experiments were performed by adding 20 μl of 10×PBS to 180 μl of 5.75 mM diCPT-iRGD NT or CDA-NT solutions. The sol-gel transition behavior was tested by the inverted-vial method. Encapsulation and distribution of CDA (labeled with FITC) in hydrogel was further characterized by a confocal microscope (Zeiss LSM 510).
Briefly, 20 mM glutathione (GSH) and 400 μM diCPT-PLGLAG-iRGD or diCPT-iRGD stock solutions were prepared in water or PBS (pH 7.4). Equal volumes of GSH and diCPT-PLGLAG-iRGD or diCPT-iRGD solutions were then mixed to reach a final GSH concentration of 10 mM and the mixtures were incubated at 37° C. Samples were then taken at predetermined time points (0, 0.5, 1, 2, 4, 8, 12 and 24 h) and quantified using HPLC.
4 μg/ml of MMP-2 recombinant protein and 400 μM of diCPT-PLGLAG-iRGD stock solutions were prepared in water. Equal volumes of MMP-2 and diCPT-PLGLAG-iRGD solutions were then mixed and incubated at 37° C. for the desired period of time (0, 0.5, 1, 2, 4, 8, 12 and 24 h). Samples were then quantified using HPLC.
GL-261 cells (2.5×103 cells/well) were incubated in a 96-well plate overnight. The cells were then treated with varying concentrations of CPT or diCPT-PLGLAG-iRGD or diCPT-iRGD solutions, with untreated cells used as a control. After 48 h incubation, cell viability was tested using an MTT or WST-1 assay. The percentage of cell viability was calculated as follows: Cell Viability (%)=A570sample/A570control×100%. IC50 values of the drug formulations were analyzed using Graph Pad Prism 5.
GL-261 cells were seeded at a density of 1×104 cells/well into agarose coated 48-well plates. After the culture plates were incubated at 37° C. for 7 days, the spheroids were treated with free CPT, NT and P-NT at the CPT concentration of 5 μM. The tumor spheroids were photographed every two days. Growth inhibition was evaluated by measuring the size of the tumor spheroids. Volume of the tumor spheroids was calculated using the equation V=(π×dmax×dmin)/6, with dmax dmin representing the maximum and minimum diameter of the spheroid, respectively.
aPD1 and CPT Release from P-NT-aPD1 Hydrogel
The in vitro hydrogel (P-NT-aPD1) release study was performed at 37° C. in the presence or absence of 2 μg/ml of MMP-2. The quantity of aPD1 (labeled with Cy3) released was determined using a Fluorescence spectrophotometer whereas CPT release was quantified using HPLC.
Hydrogel Release Studies for CDA and diCPT-iRGD
The in vitro release study was performed at 37° C. in PBS or in 10% FBS. The released CDA and diCPT-iRGD were determined using HPLC. To evaluate the in vivo release of CDA and CPT, Cy7-labeled free CDA or CDA-NT solution was injected into the tumors. At predetermined time points, fluorescence images were recorded by an IVIS Spectrum imaging system (Perkin Elmer). Mice were sacrificed to measure the amount of CPT in the tumors by HPLC. Harvested tumors were also dissected and snap-frozen in optimal cutting temperature compound (Thermo Fisher Scientific). Micrometer sections were cut using a cryotome and stained with DAPI. The slides were then imaged using a confocal microscope (Zeiss LSM 510).
100 μl of 7.2 mM P-NT solution or diCPT-iRGD NT solution (5.75 mM, 100 μL) was injected into the back of C57BL/6 mice. The mice were then sacrificed at predetermined time points (1 hour, 5, 15, 30 and 45 days). The remaining hydrogel in each mouse was photographed and the amount of drug that persisted within the hydrogel was determined using HPLC.
In Vivo Drug Release from P-NT-aPD1 Prodrug Hydrogel
To evaluate the in vivo release of aPD1 and CPT, free (CPT+aPD1) or P-NT-aPD1 solution was injected into the tumors. Fluorescence imaging of Cy5.5-aPD1 was monitored by an IVIS Spectrum imaging system (Perkin Elmer). At predetermined time points, mice were sacrificed and major organs and tumors were collected and imaged. Bioluminescence images were analyzed using Living Image software (Perkin Elmer). The amount of CPT in the tumors was measured by HPLC. Harvested tumors were also snap-frozen, cut into micrometer sections, and stained with DAPI. The slides were then imaged using a confocal microscope (Zeiss LSM 510).
For the subcutaneous GL-261-luc tumor model, 2.5×106 cells were inoculated on the right flanks of female C57BL/6 mice. After ten days (tumor volume had reached 100-150 mm3), mice were randomly assigned to five groups (n=5−10). Then the mice were intratumorally (it.) injected with saline (30 μl on days 10, 17 and 24), free (CPT+aPD1) ((50 μg CPT+50 μg aPD1)/30 μl on days 10, 17 and 24), diCPT-PLGLAG-iRGD NT solution (P-NT, 150 μg CPT/30 μl single injection on day 10), aPD1 loaded diC12-PLGLAG-iRGD NF (aPD1(L), 50 μg aPD1/30 μl single injection on day 10), or P-NT-aPD1 ((150 μg CPT+50 μg aPD1)/30 μl single injection on day 10). For re-challenge studies, mice with long term survival from all treatment groups were inoculated with 2.5×106 GL-261-luc cells on their opposite flank to develop new tumors.
To assess the systemic anti-tumor effects of P-NT-aPD1, mice were implanted with GL-261-luc cells in both the right back and left brain. Briefly, 2.5×106 cells were inoculated on the right flanks of female C57BL/6 mice. The orthotopic glioblastoma tumors were established by implanting 5×104 GL-261-luc cells into the left frontal lobe. The injection site was 2.5 mm lateral, 0.5 mm anterior from the bregma and 2.5 mm deep from the outer border of the cranium. Mice were imaged using an IVIS System (Perkin Elmer) to evaluate tumor growth. The tumor take rate was 100%. After six days, tumors on the right flank were treated with P-NT-aPD1 hydrogel (n=10), while orthotopic gliomas were designated as ‘distant tumors’ and did not receive any treatment. Control mice (n=5) were treated with Saline.
The tumor size and body weight were measured every 2 days. The tumor volume was calculated by the formula: V=(major axis)×(minor axis)2/2. The mice were also monitored using an IVIS Spectrum Imaging System (Perkin Elmer). Mice were euthanized when tumor volume exceeded 1000 mm3 or when body weight loss surpassed 20%.
In Vivo Tumor Models and Treatment for CDA and diCPT-iRGD.
To prepare tumor models, 2.5×106 GL-261-luc cells were planted on the right flank of female C57BL/6 mice or STINGgt/gt mice and 5×105 CT 26 cells were inoculated on the right flank of female BALB/C mice. After the tumor volume reached ˜100-150 mm3 (10 days for GL-261 bearing mice, 8 days for CT 26 bearing mice), tumor-bearing mice were randomly assigned to five groups (n=5-10). Then, mice were intratumorally (it) injected with either saline, free CPT+CDA, CPT NT (NT), CDA loaded diC8-iRGD NT (CDA-L), or CDA-NT (20 μg CDA per mouse, 100 μg CPT per mouse, 25 μL), as appropriate. For the metastatic breast cancer model, 2×106 4 T1-luc cells were inoculated orthotopically into the mammary gland of female BALB/C mice to generate primary tumor. After six days, tumor-bearing mice were randomly assigned to five groups and intratumorally injected with either saline, free CPT+CDA, NT, CDA-L, or CDA-NT (20 μg CDA per mouse; 100 μg CPT per mouse, 25 μL), as appropriate. (n=6 for saline treated group, n=8 for other groups). The tumor size and body weight were measured every 3 days. The tumor volume was calculated by the formula V=(major axis)×(minor axis)2/2. The tumor was also monitored using an IVIS Spectrum Imaging System (Perkin Elmer). Mice were euthanized when the tumor volume exceeded 1 cm3 for GL-261 tumor model and 1.5 cm3 for 4T1 tumor model or when body weight loss exceeded 20%. To detect the lung metastasis of 4T1 tumor-bearing mice, mice were sacrificed on day 28. The tumors and lungs were excised, photographed, fixed and sectioned at 10 μm thickness and subjected to hematoxylin and eosin (H&E). The stained sections were imaged by microscope.
For rechallenge studies, mice with long-term survival from all groups were inoculated with 2.5×106 GL-261-luc cells or 5×105 CT 26 cells on the opposite flank to develop new tumors. The tumor burden was observed using an IVIS Spectrum Imaging System.
Tumors were minced and digested with 1 mg/mL Collagenase (Type IV, Gibco) digestion buffer. Suspensions were filtered through 70 μM cell strainers. For surface staining, cells were stained with antibodies for 30 min at 4° C. For Foxp3 staining, cells were permeabilized with Fix/Perm buffer (eBioscience) for 30 min, washed, and stained with anti-Foxp3 antibody for 30 min at 4° C. Flow cytometry was then performed using a FACSCanto II instrument (BD Biosciences). Data were analyzed with FlowJo software (Tree Star). Antibodies against CD45 (30-F11), CD4 (RM4-5 or GK1.5), CD8 (53-6.7 or 53-5.8), CD3 (17A2), CD44 (IM7), CD62L (MEL-14), Foxp3 (FJK-16s or MF-14), CDiib (ICRF44), Gr-1 (RB6-8C5), PD-1 (29F.1A12), PD-L1 (10F.9G2) NK1.1(PK136), IFN-γ(XMG1.2), KLRG1(14C2A07), MHCII(10.36), CD103(2E7) and CD69(H1.2F3) were purchased from Biolegend or eBioscience.
Tumor growth in mice was observed using bioluminescence imaging. Ten minutes after intraperitoneal injection of D-luciferin (150 μg/g), mice were anesthetized and imaged using an IVIS Spectrum Imaging System (Perkin Elmer). Bioluminescence images were analyzed using Living Image software and fluorescence intensity was quantified as the average radiance (photons s−1 cm sr−1).
To deplete NK cells, CD4+ T cells and CD8+ T cells, tumor-bearing mice were intraperitoneal injected with anti-NK1.1 (clone PK136, BioXCell), anti-CD4 (clone GK1.5, BioXCell), anti-CD8-α (clone 2.43, BioXCell) or isotype control antibody (RatIgG1, BioXcell) at an initial dose of 400 mg 1 day before treatment, followed by 200 mg every 3 days. Depletions of CD8+ T cells, CD4+ T cells and NK cells were confirmed by flow cytometric analysis of PBMCs.
Splenocytes of 4T1-tumor bearing mice were assayed for tumor peptide-specific CD8+ T cell response. 10 days after intratumoral injection of saline, free CPT+CDA, NT, CDA-L, or CDA-NT, spleens were harvested, dissociated and treated with ACK Lysis buffer to lyse red blood cells. 1×108 splenocytes were stimulated 6 hours with 1 mM AH1 peptide (SPSYVYHQF, gp70 423-431 (SEQ ID NO: 9)). Cells were then stained for intracellular IFNγ and measured on FACSCanto II instrument (BD Biosciences).
Tumor tissue was harvested and then homogenized in cold PBS buffer in the presence of digestive enzymes, forming single cell suspensions. The tumoral levels of IFN-α, IFNβ and CXCL10 were measured with ELISA kits (Invitrogen) according to the manufacturer's instructions.
Statistical analysis was performed using GraphPad Prism software 5. Data are presented as means±SD. The two-tailed unpaired t-test was used to determine statistical significance between two treatment groups. Survival was plotted using Kaplan-Meier curve and assessed by log-rank (Mantel-Cox) test. *P≤0.05, **P≤0.01, ***P≤0.001.
This example describes characterization of a self-assembling prodrug hydrogel.
The amphiphilic prodrug, diCPT-PLGLAG-iRGD, was first synthesized by conjugating a hydrophilic iRGD (a cyclic peptide known to facilitate tumor tissue penetration of anticancer agents) to two hydrophobic CPT molecules through a matrix metalloproteinase 2 (MMP-2) responsive linker (PLGLAG (SEQ ID NO: 4) peptide) (
To determine the release of free CPT from the diCPT-PLGLAG-iRGD prodrug, 10 mM glutathione (GSH) was used to mimic intracellular reductive conditions. GSH is a cancer-relevant reducing agent that breaks the etcSS disulfide linker to release the parent CPT (
The PLGLAG peptide was used as a bioresponsive linker in some of the CPT prodrug embodiments because it is cleavable by MMP-2, an enzyme known to be overexpressed in the tumor extracellular matrix. MMP-2 was selected as the cleavage enzyme due to its association with the progression of malignant tumors and high expression in various tumor types including GL-261 brain tumors (
This example demonstrates that the supramolecular hydrogel scaffold formed from the self-assembling prodrug hydrogels extends local retention and release of aPD1 antibody.
The in vivo formation of gels after injection and its subsequent degradation were then evaluated in C57BL/6 mice. After subcutaneous injection into the backs of mice, a solution containing 7.2 mM diCPT-PLGLAG-iRGD was observed to undergo a sol-gel transition within 5 minutes (
This example demonstrates that a P-NT-aPD1 prodrug hydrogel elicits a robust antitumor immunity.
To assess the immune response of each treatment, all mice were sacrificed at day 25 post tumor implantation, and tumor infiltrating lymphocytes and tumor cells were then analyzed using flow cytometry (
This example demonstrates that the P-NT-aPD1 prodrug hydrogel elicits complete regression of established GL-261 brain tumors.
To evaluate the synergistic antitumor effects of the P-NT-aPD1 hydrogel, a subcutaneous GL-261 brain tumor model was used (
This example demonstrates that the P-NT-aPD1 prodrug hydrogel induces durable immune response.
To assess whether P-NT-aPD1 therapy could induce a memory response, mice that displayed long-term survival from all previous treatment groups were re-challenged with GL-261 cells on the opposite flank on day 100, with naive mice used as controls (
This example demonstrates that systemic antitumor immunity was induced by localized P-NT-aPD1 therapy.
Localized P-NT-aPD1 therapy was tested to determine whether it induces systemic antitumor immunity against GL-261 tumors. An orthotopic glioblastoma tumor model was established in the left cortical surface to mimic tumor metastasis. On day 6, the primary tumors were locally treated with P-NT-aPD1 prodrug hydrogels (
This example describes the efficacy of the P-NT-aPD1 prodrug hydrogel in a colon cancer model.
To ascertain the broad application potential of the P-NT-aPD1 prodrug hydrogel, CT 26 colon cancer bearing BALB/c mice were evaluated. When tumor volume reached 100-150 mm3 at day 8, a solution containing P-NT and aPD1 was injected into the mice to from P-NT-aPD1 hydrogel in situ (
This example describes the design of an alternative in situ formed chemo-immunotherapeutic hydrogel.
Simplicity and efficacy are the two primary considerations in the design of the self-assembling CPT drug amphiphile. Two CPT molecules were chemically conjugated to iRGD, a short peptide known to facilitate tumor tissue penetration, through a reducible disulfanyl-ethyl carbonate (etcSS) linker to form an amphiphilic molecular construct (diCPT-iRGD) (
To investigate the gelation and drug release behavior of the CDA containing hydrogel, a therapeutic dose of CDA (20 μg) was added into a 25 μl aqueous solution of diCPT-iRGD (100 μg equivalent of CPT). After mixing, the reduced Zeta potential suggests that the negatively charged CDAs were effectively condensed on the surface of these positively charged NTs (
This example demonstrates that a biodegradable NT hydrogel enables local retention and sustainable release of STING agonists in vivo.
Next, the in vivo gelation and biodegradation of the diCPT-iRGD hydrogel was evaluated by subcutaneous injection of the diCPT-iRGD NT solution into the back of C57BL/6 mice. One hour after injection, the mouse was sacrificed and it was observed that the injected NT solution had translated into a well-defined, spherical-shaped hydrogel. This in situ formed NT hydrogel showed gradual biodegradation over a period of 45 days (
To further assess the potential of this CDA-NT hydrogel as a drug depot, the intratumoral therapeutic release behavior was investigated on GL-261 tumor-bearing mice with a Cy7-labeled CDA. The fluorescent CDA was first mixed with a NT aqueous solution, and the mixture was injected into the tumors, with free Cy7-labeled CDA solution (not loaded with NT) as control. The CDA signal was detected using fluorescence IVIS imaging (
This example demonstrates that CDA-NT hydrogel elicits regression of established tumors through STING-dependent immune stimulation.
Having confirmed that the release of therapeutic agents could be sustained locally in vivo, it was decided to evaluate the synergistic antitumor effects of the CDA-NT hydrogel on subcutaneous GL-261 brain tumor model. The CDA-NT solutions were injected into the tumor when the tumor volume reached ˜100-150 mm3 at day 10 post-tumor inoculation (
To dissect the changes in immune cell population within the TME after different treatments, the residual tumors were collected and analyzed using flow cytometry at day 3 and day 10 post-treatment. These results suggest that the diC8-iRGD “drug-empty” hydrogel (E-Gel) had no effects on TILs. During the priming phase, the ratio of innate immune cells, particularly activated (CD69+ or KLRG1+) NK cells, was significantly increased after CPT+CDA, NT, CDA(L) and CDA-NT treatment (
To determine whether the observed anti-tumor efficacy of CDA-NT was STING-dependent, experiments were performed on GL-261 tumor-bearing STING-deficient (STINGgt/gt) mice. Analysis of tumor tissues removed from STINGgt/gt mice revealed lack of NK cells and CD8+ T cells accumulation in tumors after each treatment with the NT, CDA(L) and CDA-NT (
This example demonstrates that local delivery of CDA-NT induces durable immune responses.
To examine whether the local delivery of the CDA-NT hydrogel also induces a memory response, mice with long-term survival from all groups were rechallenged on day 70 to see if new tumors can be developed on the opposite flank, and naive mice were challenged as control (
This example demonstrates that the CDA-NT hydrogel promotes regression in other tumor models.
To further demonstrate the broad application of the proposed CDA-NT chemo-immunotherapy hydrogel, CT 26 colon cancer-bearing BALB/c mice were evaluated. When the tumor volume reached ˜100-150 mm3 at day 8 post-tumor inoculation, the in situ formed gels were injected into the mice (
Furthermore, the efficacy of CDA-NT was evaluated in a low-immunogenic 4T1 breast cancer model, where 4T1 breast cancer cells were implanted into the mammary gland of female BALB/c mice. After 6 days, tumors were treated with different therapeutic agents (
Glioblastoma multiforme (GBM) is the most aggressive neoplasm, with extremely high patient morbidity and mortality. Current standards of care for GBM focus on maximal safe surgical resection, implantation of GLIADEL® wafers (the only FDA approved carmustine implant) in the resection site adjuvant radiotherapy and/or oral chemotherapy. Despite an initial modest therapeutic effect, infiltrating cancerous cells remain in the surrounding brain parenchyma following resection, leading to tumor recurrence and thereby limiting the 5-year survival rate to below 5%. While recent progress in immunotherapy has drastically improved clinical outcomes for patients with a few advanced cancers, brain tumors continue to be a conspicuous exception to this trend, largely due to the unique immunosuppressive tumor environment in brain and insufficient infiltration of T cells into GBM. Even the most prominent immunotherapy approaches such as checkpoint inhibition, PD-1 and CTLA-4 blockade, and CAR-T cell therapies have shown limited benefit in GBM patients to date. Given that tumor-associated microglia/macrophages (TAMs) constitute 30-50% of brain tumor mass, macrophage-directed immunotherapy may improve the treatment of GBM.
CD47 blockade has demonstrated potential in clinical trials 23, but it requires multiple large doses and combination with chemotherapy or irradiation for an efficacious treatment of malignant brain tumors 13, 14, 22, 24. Moreover, systemic administration of CD47 antagonists causes severe side effects such as anemia and thrombocytopenia and its efficacy is compromised due to aCD47 sequestration by red blood cells 25-27. As such, approaches to enhance aCD47 immune activation and to avoid off-target effects are needed for CD47-blockade-based immunotherapy in GBM patients.
Paclitaxel (PTX) is the most successful drug in cancer chemotherapy and has been approved for the use against a wide variety of cancers including GBM. Recently, there has been mounting evidence to support that PTX can trigger infiltration of TAMs and induce enrichment of CD47 on cancer cells. These findings suggest that PTX has the ability to boost the immune response in a manner synergistic with CD47 blockade immunotherapy. It has been shown that direct linkage of a chemotherapeutic agent onto a biologically active β-sheet forming peptide transforms the drug into a supramolecular hydrogelator. The rapid solution-to-hydrogel phase transition of such self-assembling prodrug systems under physiological conditions allows for their deposition and retention in the resection cavity immediately after surgical removal of GBM. Based on these observations, it was reasoned that such PTX-containing in situ formed hydrogels can be exploited for localized delivery of immunotherapeutic agents, enabling a sustained, directed release of the combination therapy into the surrounding brain tissues to eradicate residual tumor cells.
In this context, a PTX-bearing supramolecular hydrogel was developed for site-specific delivery of aCD47 and PTX. The experiments described below reveal that the aCD47-loaded PTX prodrug hydrogelator forms a well-defined hydrogel upon infusion into the resection cavity. The hydrogel serves as a drug depot for localized, sustained release of both PTX and aCD47, eliciting an immune-stimulating tumor microenvironment (TME) and inducing macrophages phagocytosis of cancer cells.
The following methods and materials were employed in Examples 17-22.
All amino acids and Rink Amide MBHA Resin were purchased from AAPPTEC (Louisville, Ky.). Anti-CD47 antibody (cat. no. 127519) was obtained from BioLegend. All other reagents and solvents were purchased from Sigma-Aldrich. GL-261-luc cells were obtained from M. Lim at The Johns Hopkins University. Cells were cultured in DMEM (Gibco, Invitrogen) supplemented with 100 μg/ml of G418 (Invitrogen). 8-10-week female C57BL/6 mice were purchased from Charles River. Animal experiments were carried out following the animal protocol approved by the Animal Care and Welfare Committee at The Johns Hopkins University.
Through the standard Fmoc-solid phase technique, an AAPPTEC Focus XC synthesizer was used to synthesize the peptide C2K-cyl[CRGDRGPDC]. RP-HPLC and MALDI-TOF MS were used to purify and analyze the peptide, respectively. Paclitaxel (PTX) was chemically modified to PTX-buss-Pyr as previously reported. Then, PTX-buss-Pyr and purified peptide were mixed at a molar ratio of 1:1.5 (PTX-buss-Pyr/peptide) in 5 ml N2-purged DMSO and allowed to react for 3 days. The crude reaction solution was purified by RP-HPLC, then the product was confirmed by ESI MS and lyophilized to obtain PTX-iRGD as a white powder.
Preparation of aCD47/PF Hydrogel
PF hydrogel formation was tested by adding 15 μl of 10×PBS to 150 μl of 17.5 mM PF solution. For preparation of aCD47 loaded PF hydrogel (aCD47/PF), aCD47 (50 μg) was added to the PF solution (200 μg of PTX). After vortexing, 10×PBS was added to the mixture to induce the formation of aCD47/PF hydrogel. The inverted-vial method was then performed to test sol-to-gel transition. aCD47 dispersion in the hydrogel was further characterized using confocal microscopy (Zeiss LSM 510). For in vivo application, aCD47 and PF mixture solution was loaded into a syringe and directly injected into the target sites to form a gel in situ.
100 μl of 17.5 mM PF solution was subcutaneously injected into C57BL/6 mice. At predetermined time points, the mice were sacrificed and the remaining hydrogel in each mouse was photographed. Hydrogel weights were determined by detecting the amount of PTX-iRGD within the hydrogel.
Drug Release from aCD47/PF Hydrogel
The in vitro release study was performed at 37° C. in PBS with 10% FBS. The released PTX-iRGD was quantified by HPLC, whereas released aCD47 (labeled with Cy3) was analyzed using a fluorescence spectrophotometer. To evaluate the in vivo release of aCD47, free aCD47 or aCD47/PF solutions were injected into the subcutaneous GL-261-luc tumors, which were generated by inoculating 2.5×106 cells on the right flanks of female C57BL/6 mice for ten days. At predetermined time points, fluorescence imaging of Cy5.5-aCD47 was completed using an IVIS Spectrum imaging system (Perkin Elmer). Harvested tumors were also cut into sections, stained with DAPI, and imaged using a confocal microscope (Zeiss LSM 510). Furthermore, to evaluate the release of aCD47 in the brain, the mice were intracranially implanted with aCD47/PF hydrogel. Briefly, mice were anesthetized and stereotactically injected with 10 μl of aCD47/PF solution using a 25 μl Hamilton syringe fitted with a 26 G needle. The injection coordinates were 2.5 mm lateral, 0.5 mm anterior to bregma, and 2.5 mm deep from the outer border of the cranium. At predetermined time points, brains were taken out and fluorescence imaging of Cy5.5-aCD47 was completed using an IVIS Spectrum imaging system (Perkin Elmer).
An orthotopic glioblastoma tumor model was developed by intracranial implant of GL-261 glioma cells. Mice were anesthetized by intraperitoneal injection of ketamine/xylazine. Once prepped for surgery, the mice were positioned on a stereotactic frame and a 5 mm long midline scalp incision was made. A burr hole was drilled at the right cranial hemisphere, 2.5 mm lateral and 0.5 mm anterior to bregma. Then, 1×105 GL-261-luc cells were implanted at a depth of 2.5 mm from the dura. The skin was closed with tissue glue. On day 6 after inoculation, brain tumor-bearing mice were randomly assigned to five groups (n=8). The cranium was re-opened and 10 μL of treatment solutions were injected in the previous burr hole using a 25 μL Hamilton syringe fitted with a 26 G needle. The treatment groups were as follows: Saline, drug free DOCA-iRGD filament solution (EF), PTX-iRGD filament solution (PF), aCD47 loaded DOCA-iRGD filament solution (aCD47/EF), or aCD47 loaded PTX-iRGD filament solution (aCD47/PF). Administered drug doses were 50 μg of aCD47 and 150 μg of PTX per mouse. Body weights and behavior of the mice were monitored daily post-operation and treatment. Mice were euthanized when body weight loss surpassed 20%.
At day 8 post-tumor implantation, well-established tumors had formed and the tumor resection was performed using method similar to those previously reported 44, 55. Briefly, mice were anesthetized, then immobilized on a stereotactic frame and a midline incision was made in the skin above the cranium to expose the previous burr hole. Under a dissecting surgical microscope, the brain tumor was surgically resected using a biopsy punch. Tumor tissue and blood were aspirated using a vacuum pump. Then, 10 μL of formulation solution was injected into the resection cavity using a 25 μL Hamilton syringe fitted with a 26 G needle. Finally, the wound was closed with tissue glue. The treatment groups were as follows (n=8 for each group): Saline, EF, PF, aCD47/EF, or aCD47/PF. The dosages of administered drugs were 50 μg of aCD47 and 150 μg of PTX per mouse.
Mice were considered long-term survivors if no tumor was detected 80 days post-tumor implantation. Long-term survivors from all treatment groups were rechallenged at day 80 with 2×105 GL-261-luc cells in the contralateral hemisphere to develop new tumors. Naïve mice were intracranially implanted as controls. Tumor growth was checked weekly using IVIS imaging. Body weight and behavior of the mice were also monitored post-surgical resection and treatment. Mice were euthanized when body weight loss surpassed 20%.
Tumor growth in the brain was monitored by bioluminescence imaging using an IVIS Spectrum Imaging System. Bioluminescence images were analyzed using Living Image software (Perkin Elmer). The location and size of brain tumors were also tracked by magnetic resonance imaging (MRI) using a horizontal bore 4.7 T Biospec animal imager (Bruker Biospin). T2-weighted images were acquired in the horizontal plane at predetermined time points. Additionally, to examine the pattern of tumor growth, mice were sacrificed at different days and specimens of the brain were harvested, fixed and stained with haematoxylin and eosin (H&E). The H&E brain slides were then imaged using an optical microscope.
10-week-old healthy female C57BL/6 mice were anesthetized, a burr hole was made as described above, then 10 μL of aCD47/EF solution was injected in the burr hole using a 25 μL Hamilton syringe fitted with a 26 G needle. At predetermined time points, blood samples were harvested and complete blood cell count and serum biochemistry were determined. After 1 month, mice were sacrificed, the brain and other major organs were harvested, fixed, and stained with H&E. The slides were then imaged using an optical microscope.
Tumors collected from mice were digested with 1 mg/mL Collagenase digestion buffer (Type IV, Gibco) to get single cell suspensions. Cells were then stained with fluorescence-labelled antibodies following the manufacturer's instructions. Flow cytometry was performed using a FACSCanto II instrument (BD Biosciences) and analyzed by FlowJo software (Tree Star). Antibodies against CD45 (30-F11), CD11b (ICRF44), F4/80 (BM8), CD3 (17A2), CD4 (RM4-5), CD8 (53-6.7), Foxp3 (FJK-16s), Gr-1 (RB6-8C5), CD80 (16-10A1), CD86 (GL-1), CD103 (2E7), CD44 (IM7), and CD62L (MEL-14) were purchased from Biolegend or eBioscience. To detect the expression of CD47 on the tumor cells, cells were stained with FITC-labelled anti-CD47 antibody (miap301) and measured using flow cytometer.
Blood samples were collected from mice on day 7 post-treatment. Serum levels of IL-6, IL-12, IFN-γ, and TNF-α were measured using ELISA kits according to the manufacturer's instructions.
All results are presented as means±SD. The two-tailed unpaired t-test was used to determine statistical significance between two treatment groups and ANOVA was used for multiple comparisons. Survival was plotted using a Kaplan-Meier curve and assessed by a log-rank (Mantel-Cox) test. Statistical analysis was performed using GraphPad Prism software 5. *P≤0.05, **P≤0.01, ***P≤0.001.
This example describes the characterization of aCD47/PF supramolecular filament hydrogel.
To construct a bio-responsive hydrogel, a PTX prodrug hydrogelator (PTX-iRGD) was synthesized by chemically conjugating a PTX molecule to the iRGD peptide through a reducible 4-(pyridin-2-yl-disulfanyl)butyrate (buSS) linker using a previously reported method 50 (
The release of PTX-iRGD and aCD47 from aCD47/PF hydrogel was then investigated in PBS at 37° C. PTX-iRGD exhibited a linear release profile from aCD47/PF hydrogel, with ˜35% PTX-iRGD released within 25 days. Approximately 30% of the encapsulated aCD47 was released over the first 5 days, followed by a gradually reduced release rate, with ˜60% of aCD47 released over 25 days (
This example demonstrates that in situ formed hydrogel enhanced local retention of aCD47 in vivo and reduced side effects.
In vivo gelation was evaluated by subcutaneous injection of PF solution into mice. Injected PF transformed from a solution to a well-defined hydrogel within ten minutes (
Since the PF hydrogel can concentrate aCD47 at the desired site, it was hypothesized that localized aCD47/PF would reduce off-target toxicity and immune-related adverse events caused by conventional systemic administration of aCD47. To test this hypothesis, healthy mice were intracranially injected with aCD47/PF solution. Ex vivo imaging demonstrated that aCD47 retained in the injection site in the brain and the in situ formed hydrogel significantly prevented leakage of the aCD47 cargo (
This example demonstrates that aCD47/PF elicits regression of orthotopic GBM.
To evaluate therapeutic efficacy and investigate potential antitumor immune responses elicited by aCD47/PF hydrogel, an orthotopic GL-261 brain tumor model in mice was first utilized. For comparison, DOCA-iRGD was designed as a PTX-empty hydrogel (defined as EF gel) for the local delivery of aCD47 (aCD47/EF). The hydrogelators EF, PF, aCD47/EF, and aCD47/PF were directly injected into established brain tumors in mice. Tumor burden was monitored using bioluminescence signals and T2 weighted magnetic resonance imaging (MRI) (
Furthermore, residual tumors were collected, and the related immune cells were analyzed on day 10 post-treatment. All the therapeutic-containing hydrogels induced marked infiltration of CD45+ cells in tumor tissues. The percentage of macrophages was significantly increased after PF treatment and aCD47 blockade (
This example demonstrates that PF hydrogel elicits an immune-stimulating tumor microenvironment.
To test whether PF has the potential to boost the local immune response in a manner synergistic with CD47 blockade immunotherapy, the related immune cells and tumor cells were investigated after treatment of PF hydrogel. As shown in
The example demonstrates that aCD47/PF prevents orthotopic GBM recurrence after surgery.
Given that surgical resection is the current standard of care for GBM, a brain tumor resection model was developed to further test the aCD47/PF hydrogel as potential GBM therapy. At day 8 after tumor cell inoculation, the tumor developed to adequate size with small tumor islands around the primary tumor mass evident by MRI (
To test the inhibition of tumor recurrence by aCD47/PF hydrogel, aCD47/PF solution was intra-operatively infused into the resection cavity (
This example demonstrates that aCD47/PF elicits durable antitumor immune response.
Mice that survived following an initial treatment, called long-term survivors, were rechallenged with GL-261 cells to assess whether localized aCD47/PF elicited a memory immune response (
Examples 17-22 describe the development of a self-assembling prodrug hydrogel system to deliver both PTX and aCD47 following GBM resection. It was demonstrated that a well-defined aCD47/PF hydrogel was formed rapidly after deposition of aCD47-containing PF solution into the resection cavity. This in situ formed filaments hydrogel seamlessly filled the cavity left by GBM resection, and served as a reservoir for long-term, localized release of both PTX and aCD47 to residual tumor tissue. These in vivo results revealed that PF hydrogel elicited an immune-stimulating TME with enhanced infiltration of TAMs, DCs, and CD8+ T cells and enrichment of CD47 on cancer cells. PTX worked synergistically with the locally released aCD47 to promote phagocytosis of tumor cells by macrophages and also trigger robust T cell mediated antitumor immune response. As such, this chemo-immunotherapy aCD47/PF hydrogel significantly suppressed tumor recurrence following post-surgical removal of GBM, and demonstrated a striking 100% survival rate. Moreover, the local administration of the hydrogel generated robust effector T cell memory, thereby also preventing tumor recurrence. It is noteworthy that this self-supporting hydrogel contains only the PTX prodrug, thus avoiding any potential toxicities from additional excipient materials. More importantly, the in situ formed hydrogel increases the therapeutic concentration at the target site while preventing leakage of the drugs into blood stream and major organs, so as to reduce off-target side effects. Collectively, these results suggest that site-specific aCD47/PF hydrogel injection following surgical resection may be a clinically-relevant chemo-immunotherapy strategy for the treatment of recurrent GBM.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
Science 359, 1350-+(2018).
The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
This application is a national phase application under 35 U.S.C. § 371 of PCT International Application No. PCT/US2021/016597, filed Feb. 4, 2021, which claims priority to U.S. Provisional Patent Application No. 62/969,883, filed on Feb. 4, 2020, which is hereby incorporated by reference for all purposes as if fully set forth herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/016597 | 2/4/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62969883 | Feb 2020 | US |
