SMART PEPTIDES AND TRANSFORMABLE NANOPARTICLES FOR CANCER IMMUNOTHERAPY

BACKGROUND OF THE INVENTION

Clinical success in cancer immunotherapy in recent years has brought great enthusiasm to our war against cancer. Immune checkpoint receptor pathway blockade monoclonal antibodies such as anti-PD-1, anti-PD-L1, and anti-CTLA-4 can reverse T effector cell (Teff) dysfunction and exhaustion, resulting in dramatic tumour shrinkage and sometimes complete remission in some patients, even with late stage metastatic diseases. However, the response rate varies greatly between tumour types: up to 40% in melanoma, 25% in non-small cell lung cancer, but <10% in most other tumour types. To date, US Food and Drug Administration (FDA) had approved seven immune checkpoint blockade monoclonal antibodies (ICB-Ab): one CTLA-4 inhibitor (ipilimumab), three PD-1 inhibitors (nivolumab, pembrolizumab, and cemiplimab), and three PD-L1 inhibitors (atezolizumab, durvalumab, and avelumab), used either alone, or in combination with other chemotherapies, against a range of tumour types.

Tumour microenvironment (TME), comprised of immune and stromal cells, vasculature, extracellular matrix, cytokines, chemokines, and growth factors, can all influence tumour response to immune checkpoint blockage (ICB) therapies. Emerging data indicates that defects in Teff cell homing to the tumour sites is a critical factor in resistance to ICB therapy. Other mechanisms of ICB resistance include the presence of immunosuppressive regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and M2 macrophages at the tumour sites. Elevated level of CCL5, CCL17, CCL22, CXCL8 and CXCL12 facilitates the recruitment of Tregs and MDSCs to the TME, resulting in a diminished ICB response. In contrast, CXCL9 and CXCL10 promote homing of cytotoxic T-cells (CTLs) to the tumour sites, boosting anti-tumour immune response; transforming growth factor beta (TGF-β) does the opposite and also upregulates Tregs. VEGF upregulates inhibitory receptors on CTLs, contributing to their exhaustion. Upregulation of other immune checkpoint receptors such as mucin domain-3 protein (TIM-3), lymphocyte-activation gene 3 (LAG-3), B and T lymphocyte attenuator (BTLA), T-cell immunoreceptor, tyrosine-based inhibition motif domain (TIGIT), and V-domain immunoglobulin-containing suppressor of T-cell activation (VISTA) has been implicated in ICB resistance. Co-expression of these checkpoint receptors can lead to T cell exhaustion. Oncogenic or tumour suppressor pathways, such as mitogen-activated protein kinase (MAPK) and PI3K-γ in the cancer cells can also influence TME by altering the immune cell compositions and cytokine profile, contributing to ICB resistance. Inhibitors against these pathways have been found to improve ICB response.

In an attempt to overcome ICB resistance, many combination therapeutic strategies have been tried preclinically and clinically. These include the addition of the following drugs to a ICB-Ab: one other ICB-Ab (antibodies against CTLA-4, PD-1, PD-L1, LAG-3 and TIM-3), chemotherapeutic agents (paclitaxel, gemcitabine and carboplatin), radiation therapy, targeted therapy (inhibitors against PI3K, VEGF, BRAF/MEK, IDO, A2AR, FGFR, EGFR, PARP and mTOR), macrophage inhibitors (inhibitors against CSF1R and ARG1), cytokine/chemokine inhibitors (inhibitors against CXCR4, CXCR2 and TGF-β), epigenetic modulators (histone deacetylase inhibitors and hypomethylating agents), immunomodulatory agents (antibodies against OX40, 41BB, GITR, CD40 and ICOS), adoptive cell transfer therapy (car T, TIL and TCR), and modulation of gut microbiome.

Advancement and optimization of nano-immunotherapy lie in the development of innovative approaches to enhance the specificity and controllability of immunotherapeutic interventions, targeting desired cell types at the TME. Advanced bionanomaterials or approaches in a more controlled manner could enhance immunotherapeutic potency by increasing the accumulation and prolonging the retention of immunomodulatory and immune cell homing agents at the TME while sparing the normal tissues and organs, thus reducing off-target adverse effects such as systemic cytokine storm. In situ assembly of nanomaterial has been demonstrated to improve the performance of bioactive molecules. One plausible explanation is that T cell targeting ligands and/or immunomodulatory agents incorporated into in situ fibrillar-transformable nanoplatform, will generate nanofibrillar networks at the TME, enhancing Teff cells homing to the tumour sites and improving immunotherapeutic efficacy, with or without additional ICB therapy.

Human epidermal growth factor receptor 2 (HER2) is overexpressed in over 20% breast cancers, and to a lesser degree in gastric cancers, colorectal cancer, ovarian cancers and bladder cancers. Unlike those cancers caused by mutated or fusion oncogenes (e.g. EGFR in lung cancers and Bcr-Abl in chronic myelocytic leukemia) which respond well to monotherapy, cancers with HER2 overexpression often require drug combinations. It is because this latter group of tumours are driven by gene amplification and massive overexpression of HER2. HER2 is a receptor tyrosine kinase that is normally activated via induced dimerization with itself or with its family members EGFR, HER3 or HER4. In HER2 positive tumours, HER2s are massively overexpressed and constitutively dimerized, leading to unrelenting activation of down-stream proliferation and survival pathways and malignant phenotype.

Because of the high expression level of HER2, trastuzumab and pertuzumab, the two anti-HER2 monoclonal antibodies are ineffective as monotherapy against these tumours. They need to be given in combinations with other HER2-targeted therapy, chemotherapy or hormonal therapy. Herein, some embodiments describe a novel HER2-mediated, peptide-based, and non-toxic transformative nano-agent that is highly efficacious as a monotherapy against HER2+ breast cancer xenograft models. This receptor-mediated transformable nanotherapy (RMTN) is comprised of peptide with unique domains that allow self-assembly to form micelles under aqueous condition and transformation into nanofibrils at the tumour site, where HER2 is encountered. The resulting nanofibrillar network effectively suppresses HER2 dimerization and downstream signaling, and facilitates tumour cell death.

Herein, smart supramolecular materials for cancer immunotherapy were constructed.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a compound of formula (I): A-B-C (I), wherein A is a hydrophobic moiety; B is a peptide, wherein the peptide forms a beta-sheet; and C is a hydrophilic targeting ligand, wherein the hydrophilic targeting ligand is a LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, folate, a LHRH peptide, a HER2 ligand, an EGFR ligand, or a toll-like receptor agonist CpG oligonucleotides.

In another embodiment, the present invention provides a compound of formula (I): A-B-C (I), wherein A is a hydrophobic moiety; B is a peptide, wherein the peptide forms a beta-sheet; and C is a hydrophilic targeting ligand, wherein the hydrophilic targeting ligand is a LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, a LHRH peptide, a HER2 ligand, an EGFR ligand, or a toll-like receptor agonist CpG oligonucleotides and wherein when the hydrophobic moiety is bis-pyrene, then C is a LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, a LHRH peptide, an EGFR ligand, or a toll-like receptor agonist CpG oligonucleotides.

In another embodiment, the present invention provides a nanocarrier having an interior and an exterior, the nanocarrier comprising a plurality of compounds of the present invention, wherein each compound self-assembles in an aqueous solvent to form the nanocarrier such that a hydrophobic pocket is formed in the interior of the nanocarrier, and a hydrophilic group self-assembles on the exterior of the nanocarrier.

In another embodiment, the present invention provides a nanocarrier having an interior and an exterior, the nanocarrier comprising a plurality of a first conjugate and a second conjugate wherein the first conjugate comprises formula (I): A-B-C (I); and the second conjugate comprises formula (II): A′-B′-C′ (II) wherein: A and A′ are each independently a hydrophobic moiety; B and B′ are each independently a peptide, wherein each peptide independently forms a beta-sheet; and C and C′ are each independently a hydrophilic targeting ligands, wherein each hydrophilic targeting ligand is independently a LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, folate, a LHRH peptide, a HER2 ligand, an EGFR ligand, or a radiometal chelator; and wherein A and A′ are different hydrophobic moieties and/or C and C′ are different hydrophilic targeting ligands.

In another embodiment, the present invention provides a method of forming nanofibrils, comprising contacting a nanocarrier of the present invention with a cell surface or acellular component at a tumor microenvironment, wherein the nanocarrier undergoes in situ transformation to form fibrillary structures, thereby forming the nanofibrils.

In another embodiment, the present invention provides a method of treating a disease, comprising administering to a subject in need thereof, a therapeutically effective amount of a nanocarrier of the present invention, wherein the nanocarrier forms nanofibrils in situ after binding to a cell surface or acellular component at the tumor microenvironment, thereby treating the disease.

in another embodiment, the present invention provides a method of imaging, comprising administering to a subject to be imaged, an effective amount of a nanocarrier of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F show assembly and fibrillar-transformation of transformable peptide monomer 1 (TPM1′) BP-FFVLK-YCDGFYACYMDV. FIGS. 1A-1B show changes in UV-vis absorption (FIG. 1A) and fluorescence (FIG. 1B) of NPs1 upon gradual addition of water into DMSO solution of NPs1 with the H2O:DMSO from 0:100, 20:80, 40:60, 60:40, 80:20, 90:10, 98:2 and 99.5:0.5. Ex=380 nm. FIG. 1C TEM images of initial NPs1 and nanofibers (NFs1) transformed by NPs1 interaction with HER2 protein (Mw≈72 KDa) at different time points (0.5, 6, 24 h). The scale bar in d: 100 nm. FIGS. 1D-1F—show variation of size distribution (FIG. 1D), CD spectra (FIG. 1E) and fluorescence signal (FIG. 1F) of initial NPs1 and NFs1 at the different time points. The molar ratio of HER2 peptide/HER2 protein was approximately 1000:1.

FIGS. 2A-2H show the morphological characterizations of fibrillar-transformable NPs1 co-culture with HER2 positive cancer cells. FIGS. 2A-2C show cellular fluorescence distribution images of NPs1 interaction with SKBR-3 cells (HER2+) (FIG. 2A), BT474 cells (HER2+) (FIG. 2B) and MCF-7 cells (HER2−) (FIG. 2C) at 6 h time point. Scale bar in FIGS. 2A-2C: 50 μm. FIG. 2D shows Western blot and quantitative analysis of relative HER2 protein expression in MCF-7 cells and MCF-7/C6 cells. ***P<0.001. FIG. 2E shows cellular fluorescence distribution images of NPs1 interaction with MCF-7/C6 cells (HER2+) at the different time points (0.5, 6, 24 h). Scale bar in e: 50 μm. FIG. 2F shows fluorescence binding distribution images of the nanofibrillar network of NFs1 and HER2 antibody (29D8, rabbit, different receptor binding site with HER2 peptide of NPs1) on the cell membrane of MCF-7/C6 cells. HER2 antibody was used to label HER2 receptors. FIG. 2G shows SEM images of untreated MCF-7/C6 cells and cells treated by NPs1 for 6 h and 24 h. FIG. 2H shows TEM images of untreated MCF-7/C6 cells and cells treated by NPs1 for 24 h. The red arrow shows fibrillar network. The concentration of NPs1 was 50 μM.

FIGS. 3A-3G show the extracellular and intracellular mechanisms of fibrillar-transformable NPs interaction with MCF-7/C6 breast cancer cells. FIG. 3A shows cellular fluorescence distribution images of NPs1, NPs2 and HER2 antibody (29D8, rabbit, different receptor binding site with HER2 peptide of NPs1 and NPs2) binding HER2 receptors of MCF-7/C6 cells, respectively. HER2 antibody was used to label HER2 receptors. The concentration of NPs1 and NPs2 were 50 μM. The scale bar in a: 20 μm. FIG. 3B shows the viability of MCF-7/C6 cells incubated with NPs1-4 at the different concentration (n=3). *P<0.05, **P<0.01. FIG. 3C shows Western blot analysis of apoptosis related proteins and HER2 total protein in MCF-7/C6 cells treated by NPs1 for 24 h with different concentration. FIGS. 3D-3E show Western blot analysis of inhibition and disaggregation mechanism of HER2 protein dimer in MCF-7/C6 cells treated by NPs1 for 24 h with different concentration (FIG. 3D) and at 50 μM under different time point (FIG. 3E). FIG. 3F shows Western blot analysis of inhibition mechanism of proliferation protein in MCF-7/C6 cells treated by NPs1 at 50 μM under different time point and at 24 h under different concentration. FIG. 3G shows Western blot analysis of inhibition mechanism of proliferation protein in MCF-7/C6 cells treated by NPs1-4 and Herceptin (HP) at 36 h. The concentration of NPs1-4 were 50 μM, and the concentration of Herceptin was 15 μg/mL as a positive control group.

FIG. 4A-4F show in vivo evaluation of fibrillar-transformable NPs. FIG. 4A show time-dependent ex vivo fluorescence images and FIG. 4B show quantitative analysis of tumour tissues and major organs (heart, liver, spleen, lung, kidney, intestine, muscle and skin) collected at 10, 24, 48, 72 and 168 h post-injection of NPs1. In FIG. 4B ***P<0.001, the fluorescence signal in tumour tissue at 72 h and 168 h compared with other organs displays tumour accumulation and in situ transformation of fibrillar network with long retention time; ***P<0.001, the fluorescence signal in liver and kidney at 10 h compared with that at 72 and 168 h displays that NPs1 could be removed rapidly from liver and kidney. FIG. 4C shows the fluorescence distribution images and H&E image of NPs1 in tumour tissue and normal skin tissue at 72 h post-injection (green color: BP of NPs1; blue color: DAPI; scale bar in c: 100 μm). FIG. 4D shows time-dependent ex vivo fluorescence images of tumour tissues and major organs collected at 72 h post-injection of NPs2-4. FIG. 4E shows quantitative analysis of tumour tissues and livers collected at 72 h post-injection of NPs1-4. In FIG. 4E ***P<0.001, the fluorescence signal of tumour tissue in NPs1 group compared with that in other control groups displays that fibrillar networks in NPs1 group promote long retention time in tumour site. FIG. 4F shows TEM images of distribution in tumour tissue and in situ fibrillar transformation of NPs1-4 at 72 h post-i.v. injection and untreated group. The dose of NPs1-4 were 8 mg/Kg per injection. In FIG. 4F, “C” means MCF-7/C6 cell; “N” means cell nucleus.

FIG. 5A-5K show anti-tumour activity of NPs in Balb/c nude mice bearing HER2 positive breast tumour. FIG. 5A shows schematic illustration of tumour inoculation and treatment protocol of mice. FIGS. 5B-5C show observation of the tumour inhibition effect (FIG. 5B) and weight change of mice (FIG. 5C) in subcutaneous tumour model during the 40 days of treatment (n=8 per group; the dose of NPs1-4 were 8 mg/Kg per injection). **P<0.01, ***P<0.001. FIG. 5D shows cumulative survival of different treatment groups of mice bearing MCF-7/C6 breast tumours. FIG. 5E shows schematic illustration of three times treatment protocol of mice for tumour tissue analysis. FIG. 5F shows the fluorescence distribution images in tumour tissue and H&E anti-tumour image post three times injection of NPs1 (green color: BP of NPs1; blue color: DAPI; scale bar in f: 100 μm). FIG. 5G shows representative TEM images of late membrane rapture and cell death by the nanofibrillar network after injection of NPs1 three times. The red arrow shows fibrillar network. FIG. 5H shows Ki-67 stain images of tumour tissues treated by different groups after injection three times. Scale bar in h: 25 μm. FIG. 5I shows Western blot analysis of inhibition mechanism of HER2 protein and proliferation proteins in MCF-7/C6 tumour tissues treated by different groups after injection three times. FIGS. 5J-5K shows observation of the tumour inhibition effect in subcutaneous tumour SKBR-3 (FIG. 5J) and BT474 HER2 positive breast cancer (FIG. 5K) models during the 40 days of treatment (n=8 per group; the dose of NPs1 were 8 mg/Kg per injection). ***P<0.001 compared with PBS control group.

FIG. 6 shows chemical structure and mass spectra via MALDI-TOF of transformable peptide monomer 1 BP-FFVLK-YCDGFYACYMDV.

FIG. 7 shows chemical structure and mass spectra via MALDI-TOF of transformable peptide monomer 2 BP-GGAAK-YCDGFYACYMDV.

FIG. 8 shows chemical structure and mass spectra via MALDI-TOF of transformable peptide monomer 3 BP-FFVLK-PEG.

FIG. 9 shows chemical structure and mass spectra via MALDI-TOF of transformable peptide monomer 4 BP-GGAAK-PEG.

FIG. 10 shows effect of HER2 protein/peptide ligand ratio on fibrillar transformation. TEM images and particle size measurements of NPs1 were obtained after incubation with soluble HER2 protein for 24 h in PBS solution. NPs1 concentration was maintained constant at 20 μM. The scale bar is 200 nm. The HER2 protein/peptide ligand ratio is labeled for each micrograph. Experiments were repeated three times.

FIG. 11A shows observation on the anti-tumour effect in subcutaneous SKBR-3 tumour during the 40 days of treatment (n=6 per group; the dose of NPs1-4 were 8 mg/kg per injection, q.o.d.; data are presented as the mean±s.d.). The statistical significance was calculated via one-way ANOVA with a Tukey post-hoc test. *P<0.05. FIGS. 11B-11C show body weight of mice bearing subcutaneous BT474 tumour (FIG. 11B) and SKBR-3 tumour (FIG. 11C) during the 40 days of treatment (n=6 per group; data are presented as the mean s.d.). Red arrows depict each single i.v. injection.

FIG. 12 shows nanofibrillar networks promote T cell homing and reprogram tumour microenvironment for enhanced immunotherapy. Schematic illustration of self-assembly and fibrillar transformation of TPMs, and (I), (II), (III) process in tumour tissue: in situ fibrillar transformation of NPs, LLP2A conversion from proLLP2A, followed attracting and targeting T cells, and TAMs re-education of from M2 to M1 phenotype. TPMs, NPs, NFs, M1-TAM and M2-TAM represent transformable peptide monomers, nanoparticles, nanofibrils, M1-like tumour-associated microphage and M2-like tumour-associated microphage, respectively.

FIG. 13A-13H shows assembly and fibrillar transformation of transformable peptide TPM1 (LXY30-KLVFFK(Pa)) and TPM2 (proLLP2A-KLVFFK(R848)). FIG. 13A shows schematic illustration of molecular structure and function of TPM1 and TPM2. FIG. 13B shows changes in fluorescence (FL) of T-NPs following the gradual addition of water (from 0 to 99%) to a solution of T-NPs in DMSO comprised of TPM1 and TPM2 at a 1:1 ratio; excitation wavelength, 405 nm. FIG. 13C shows TEM images of initial T-NPs and T-NPs transformed into nanofibrils (T-NFs) after interaction with soluble α₃β₁integrin protein for 24 h (H₂O to DMSO ratio of 99:1). The concentration of T-NPs used in the experiment was 20 μM. The scale bars in c are 100 nm. FIG. 13D shows variation in fluorescence signal of Pa in the fibrillar-transformation process of T-NPs to T-NFs over time. FIG. 13A E shows TEM images of initial T-NPs and T-NFs after interaction with esterase, soluble α₄β₁integrin protein or α₄β₁integrin protein plus esterase for 24 h (H₂O to DMSO ratio of 99:1). The concentration of T-NPs used in the experiment was 20 μM. The scale bars in e are 100 nm. FIGS. 13F-3G show variation in size distribution (FIG. 13F) and circular dichroism spectra (FIG. 13G) of initial T-NPs and T-NFs under different conditions. FIG. 13H shows Tte in vitro release profile of R848 from T-NFs over time. The molar ratio of α₃β₁or α₄β₁integrin protein to peptide ligand was approximately 1:1000. a.u., arbitrary units; mdeg, millidegrees.

FIG. 14 shows DLS experiment to confirm transformation of T-NPs to T-NFs. The peak at 20 nm gradually went down in the solution, while the peak around 700 nm went up.

FIG. 15A-15H shows morphological characterization of fibrillar-transformable nanoparticles after incubation with 4T1 murine breast cancer cells. FIG. 15A shows cellular fluorescence distribution images of T-NPs and UT-NPs interaction for 6 h with 4T1 cells. Scale bar is 10 μm. Experiments were repeated three times. FIG. 15B shows cellular fluorescence signal retention images of 4T1 cells after exposure to T-NPs and UT-NPs for 6 h followed by incubation with fresh medium without NPs for 18 h. Scale bar is 10 μm. Experiments were repeated three times. FIG. 15C shows representative TEM images of 4T1 cells treated with T-NPs and UT-NPs for 24 h, showing abundance of nanofibrils around cells treated with T-NPs. Scale bar is 200 nm. Experiments were repeated three times. The concentration of T-NPs was 50 μM. FIG. 15D shows cellular fluorescence distribution images of Jurkat T-lymphoma cells (GFP labeled) after incubation with esterase-treated T-NPs. Jurkat cells were used to mimic T-lymphocytes, which also express α₄β₁integrin. Scale bar is 10 μm. Experiments were repeated three times. FIG. 15E shows representative SEM images of untreated 4T1 and Jurkat cells, and cells treated with T-NPs for 6 h. Scale bar is 10 μm. Experiments were repeated three times. FIG. 15F shows experimental scheme and cellular fluorescence distribution images of T-NPs (fluorescent red), after interaction with 4T1 and GFP-labeled Jurkat cells. It shows nanofibrillar networks covering 4T1 cells, which in turn could attract and bind Jurkat malignant T-cells. Scale bar is 10 μm. Experiments were repeated three times. FIG. 5G shows representative SEM images of 4T1 and Jurkat cells after treatment with T-NPs (see FIG. 15F). Experiments were repeated three times. FIG. 15H shows representative images of M2-like murine macrophages and subsequent re-education by T-NFs, T-NFs plus esterase, or R848 at different time points. Scale bar is 20 μm. Experiments were repeated three times. Statistical significance was calculated using a two-sided unpaired t test; *P<0.05, **P<0.01, ***P<0.001.

FIG. 16A-16M shows in vivo evaluation of fibrillar-transformable nanoparticles. FIG. 16A-16B show time-dependent ex vivo fluorescence (FL) images (FIG. 16A) and quantitative analysis (FIG. 16B) of tumour tissues and major organs (heart (H), liver (Li), spleen (Sp), lung (Lu), kidney (K), intestine (I), muscle (M) and skin (Sk)) collected at 10, 24, 48, 72, 120 and 168 h post-injection of T-NPs. Data are presented as mean±s.d., n=3 independent experiments. FIG. 16C shows time-dependent ex vivo fluorescence (FL) images of tumour tissues collected at 10, 24, 48, 72, 120 and 168 h post-injection of UT-NPs. Data are presented as mean±s.d., n=3 independent experiments. FIG. 16D shows fluorescence (FL) quantification of tumour tissues collected at 10, 24, 48, 72, 120 and 168 h post-injection of T-NPs and UT-NPs. FIG. 16E shows representative TEM images of distribution in tumour tissue and in situ fibrillar transformation of T-NPs, UT-NPs and untreated control group at 72 h post-injection. “N” depicts nucleus. FIG. 16F shows fluorescence (FL) distribution images of T-NPs in tumour tissue and normal skin tissue at 72 h post-injection (red, Pa of T-NPs; blue, DAPI; scale bars, 50 μm). FIG. 16G shows R484 distribution retention in tumour tissues at different time points post injection of T-NPs and UT-NPs. Dose of R848: 0.94 mg kg⁻¹; data were mean±s.d., n=3 for each time point. FIG. 16H shows the expression of CXCL10 chemokine within the tumour tissues after 3 days of T-NPs, UT-NPs and saline treatment (n=3; data were mean±s.d.). FIG. 16I-16K show representative flow cytometric analysis images of CD45⁺CD3⁺ (FIG. 16I), CD8⁺/CD4⁺ (FIG. 16J) and CD4⁺Foxp3⁺ (FIG. 16K) T cell within the 4T1 tumours excised from mice treated with T-NPs, UT-NPs or saline control. FIG. 16L shows immunohistochemistry (IHC) of tumours excised from mice after treatment with T-NPs or UT-NPs. Representative images are shown for the IHC staining of T cells (CD8⁺, CD4⁺, Foxp3⁺) and macrophage markers (CD68, CD163). Scale bar is 100 μm. FIG. 16M shows the expression levels (qPCR assay) of IFN-γ, TGF-β, IL12, IL10, Nos2 and Arg-1 in 4T1 tumours excised from mice 15 days after treatment with T-NPs or UT-NPs (n=3; data were mean±s.d.). Statistical significance was calculated using a two-sided unpaired t test; *P<0.05, **P<0.01, ***P<0.001.

FIG. 17A-17G shows anti-tumour efficacy of fibrillar-transformable nanoparticles in Balb/c mice bearing 4T1 breast tumour. FIG. 17A shows experimental design: orthotopic tumour inoculation and treatment protocol; regimen 6 is T-NPs with all the 4 critical components. FIG. 17B-17C show Oservation of tumour inhibitory effect (FIG. 17B) and weight change (FIG. 17C) of mice bearing orthotopic 4T1 tumour over 21 d after initiation of treatment (n=8 per group). Data are presented as mean±s.d. FIG. 17D shows cumulative survival of different treatment groups of mice bearing 4T1 breast tumours. FIG. 17E shows representative flow cytometric analysis images of CD3⁺CD8⁺ T cell within the 4T1 tumours excised from treated mice on day 21. FIG. 17F shows H&E and IHC images of excised tumors. Representative images are shown for the IHC staining of Ki67, T cells (CD8, Foxp3) and macrophage markers (CD68, CD163). Scale bar is 100 μm. FIG. 17G shows the expression levels (analyzed by qPCR) of IFN-γ, TNF-α, IL12, IL6, TGF-β, IL10, Nos2 and Arg-1 in 4T1 tumours excised from mice on day 21 (data were mean±s.d.). Statistical significance was calculated using a two-sided unpaired t test; *P<0.05, **P<0.01, ***P<0.001.

FIG. 18A-18L shows anti-tumour efficacy of fibrillar-transformable nanoparticles plus anti-PD-1 therapy in mice bearing 4T1 breast tumour or Lewis lung tumour. FIG. 18A shows experimental design: orthotopic tumour inoculation and treatment protocol (4 treatment arms; regimen 4, 5 and 6 are the same as those shown in FIG. 4a). FIG. 18B shows tumor response in mice bearing orthotopic 4T1 tumour over 21 d of treatment (n=8 per group). Data are presented as mean±s.d. ***P<0.001. FIG. 18C shows cumulative survival of the four treatment groups. FIG. 18D shows experimental design: Mice previously treated with T-NPs (regimen 6) plus anti-PD-1 Ab were rechallenged with re-inoculation of cancer cells on day 90, followed by three q.o.d i.p. doses of anti-PD-1 Ab. FIG. 18E shows no anti-tumor immune memory effect was observed in same age naïve mice. FIG. 18F shows anti-tumor immune memory effect was observed in mice previously treated with T-NPs and anti-PD-1 Ab. FIG. 18G shows cumulative survival of naïve mice and previously T-NPs plus anti-PD-1 treated mice. FIG. 18H-18I show IFN-γ (FIG. 18H) and TNF-α (FIG. 18I) level in mouse sera 6 days after mice were rechallenged with 4T1 tumor cells and a day after the last dose of anti-PD-1 Ab. FIG. 18J-18K shows Observation of tumour inhibitory effect (FIG. 18J) and weight change (FIG. 18K) of mice bearing subcutaneous murine Lewis lung tumour over 21 d after initiation of treatment (n=8 per group); Treatment protocol followed experiment design in FIG. 18A, 5 cycles (i.v. regimen 4-6 and i.p. anti-PD-1. Data are presented as mean±s.d. FIG. 18L shows cumulative survival of different treatment groups of mice bearing murine Lewis lung tumours. Statistical significance was calculated using a two-sided unpaired t test; *P<0.05, **P<0.01, ***P<0.001.

FIG. 19A shows structure of CPTNPs (BP-k-l-v-f-f-k-(r)₈) where Green—Bispyrene. Blue—hydrophobic bonding motif. Red—Cell-penetrating peptide. FIG. 19B shows GG-CPTNP (BP-k-l-v-g-g-k-(r)₈) with similar coloration to A where the duel phenylalanine motif is replaced with a duel glycine motif. FIG. 19C shows DLS of CPTNPs (FF) and GG-CPTNPs (GG) in various pH. FIG. 19D shows fluorescence of CPTNP nanoparticles and CPTNP monomers where the AIEE effect of BP may be observed. FIG. 19E shows Zeta potential of FF and GG CPTNPs measured at 50 μM. (a:b, p<0.0005) FIG. 19F shows TEM images of CPTNPs in various specified environments. Scale bar is 100 μm in each image.

FIG. 20 shows Chemical structure and mass spectra via MALDI-TOF of transformable peptide monomer (TPM) 1 LXY30-KLVFFK(Pa), 2 proLLP2A-KLVFFK(R848), 3 LXY30-KAAGGK(Pa), 4 proLLP2A-KAAGGK(R848). Experiments were repeated three times.

FIG. 21A shows TEM images and size distribution of NPsTPM1, NPsTPM1 and T-NPs at the H2O and DMSO ratio of 99:1. Experiments were repeated three times. FIG. 21B shows the critical aggregation concentration (CAC) of T-NPs was measured by using pyrene as a probe. Experiments were repeated three times. FIG. 21C shows nanoparticle stability of T-NPs in serum and protease (PBS solution of pH 7.4 with/without 10% FBS and protease) at 37° C. was measured by dynamic light scattering. Data are presented as the mean±s.d., n=3 independent experiments. FIG. 21D shows TEM images of freshly prepared T-NPs and T-NPs after 24 h in PBS solution. Experiments were repeated three times. FIG. 21E show Tte CAC of T-NFs was measured by using pyrene as a probe. Experiments were repeated three times. The scale bar in all TEM images is 100 nm. The concentration of T-NPs used in FIGS. 21A, 21C, and 21D was 20 μM.

FIG. 22 shows TEM images of initial UT-NPs and UT-NPs interaction with α₃β₁integrin protein for 24 h. The molar ratio of α₃β₁integrin protein/peptide ligand was approximately 1:1000. The scale bar is 100 nm. The concentration used in the experiment was 20 μM. Experiments were repeated three times.

FIG. 23 shows biotinylated LXY30 peptide (blue curve) and negative control (red curve) incubation with 4T1 cells were analyzed with flow cytometry. Experiments were repeated three times. 3×105 cells incubated with 1 μM biotinylated LXY30 for 30 min on ice, after washing with PBS followed by incubation with 1:500 streptavidin-PE (1 mg/mL) for 30 min, then run with flow cytometry.

FIG. 24 shows viability of 4T1 cells after incubation with T-NPs and UT-NPs at different concentrations for 48 h. Data are presented as mean±s.d., n=3 independent experiments.

FIG. 25 shows blood test parameters in terms of red blood cells (RBC), white blood cells (WBC), platelets, hemoglobin, lymphocyte and total protein of healthy Balb/c mice, after 8 q.o.d. intravenous injections of T-NPs and UT-NPs (13 mg/kg per injection). Data are presented as the mean±s.d., n=3 independent experiments.

FIG. 26 shows blood test parameters in terms of liver function creatinine, alanine transaminase, aspartate transaminase, albumin, alkaline phosphatase, total bilirubin of healthy Balb/c mice after 8 q.o.d. intravenous injection of T-NPs and UT-NPs (13 mg/kg per injection). Data are presented as the mean±s.d., n=3 independent experiments.

FIG. 27 shows in vivo blood pharmacokinetics and parameter of T-NPs and UT-NPs (Data are presented as the mean±s.d., n=3 independent experiments). The C-max, AUC and T1/2 (hours) were calculated by Kinetica 5.0.

DETAILED DESCRIPTION OF THE INVENTION
I. General

The present invention provides compounds comprising a hydrophobic moiety, a beta-sheet peptide, and a hydrophilic targeting ligand, which can form nanocarriers. The nanocarriers can comprise a plurality of one conjugate or two different conjugates. The nanocarriers can transform in situ to form nanofibrils for treatment of diseases and imaging.

II. Definitions

Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present invention. For purposes of the present invention, the following terms are defined.

“A,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the agent” includes reference to one or more agents known to those skilled in the art, and so forth.

“Hydrophobic moiety” refers to the part of the compound which is substantially insoluble in water. For example, when a plurality of compounds are present which comprise a hydrophobic and hydrophilic moiety, the hydrophobic moiety will orient themselves in such a way as to avoid and minimize interaction with water molecules. Hydrophobicity of a moiety can be determined by one of ordinary skill in the art by using the octanol-water reference system to measure the logarithm of the partition coefficient (log P value). Log P values greater than 0 indicate the compound is hydrophobic, with greater values indicating greater hydrophobicity.

“Peptide” refers to a compound comprising two or more amino acids covalently linked by peptide bonds. As used herein, the term includes amino acid chains of any length, including full-length proteins.

“Beta-sheet”, also known as beta-pleated sheet, refers to the secondary structure in proteins and comprises beta strands stabilized by hydrogen bonds. Beta-strands can stack parallel or antiparallel to each other to form beta-sheets.

“Beta-sheet peptide domain” refers to a domain within a protein structure comprising beta-sheets.

“Beta-amyloid peptide” refers to peptides that form amyloid plaques in the brain. The formation of amyloid plaques in the brain is found in subjects with Alzheimer's disease.

“Hydrophilic targeting ligand” refers to a portion of the compound that can target cell surface receptors, cell surface proteins, or extracellular components and are hydrophilic. Hydrophilicity can be determined by measuring the log P value of a compound, wherein values less than 0 indicate hydrophilicity. Lower values indicate higher hydrophilicity. Targeting ligands can be used to target transmembrane receptors such as, but not limited to integrins and epidermal growth factor receptors, to delivery compounds, drugs, or components of interest to the cell or extracellular environment. Hydrophilic targeting ligands can include, but are not limited to peptides.

“Prodrug” refers to a compound that is biologically inactive, which becomes biologically active after being metabolized in situ. The prodrug can be metabolized by spontaneous reactions or enzymes within a mammal, resulting in an active compound. Functional groups useful in prodrugs include, but are not limited to esters, amides, carbamates, oximes, imines, ethers, phosphates, or beta-amino-ketones.

“LLP2A”, “LXY30”, and “LXW64” refer to compounds that can bind to an integrin protein. The structures of the three individual compounds are known by one of skill in the art.

“DUPA” refers to a glutamate urea compound and can be used to deliver cytotoxic drugs to prostate cancer cells. DUPA, 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid, has the following structure:

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“LHRH peptide” refers to luteinizing hormone releasing hormone peptide, and is commercially available. LHRH peptide can be used to target ovarian and prostate cancer cells.

“HER2 ligand” refers to a ligand that can bind to the HER2 protein. Examples include, but are not limited to anti-HER2 monoclonal antibodies, such as, but not limited to trastuzumab and pertuzumab and the EGFR ligands listed below.

“EGFR ligand” refers to a ligand that can bind to the EGFR protein. Examples include, but are not limited to EGF, TGF-alpha, HB-EGF, amhiregulin, betacellulin, epigen, epiregulin, neuregulin 1, neuregulin 2, neuregulin 3, and neuregulin 4.

“Toll-like receptor agonist” refers to a compound that binds to the toll-like receptor on cells, which plays a key role in the immune system. Binding to the receptor can activate the receptor to produce a biological response. An example of a toll-like receptor agonist includes, but is not limited to CpG oligonucleotides.

“CpG oligonucleotides”, also known as CpG ODN, refer to cytosine-guanosine dinucleotide motifs. The two nucleotides can be linked by a phosphodiester linker, or a modified phosphorothioate linker.

“Dye” or “fluorescent dye” refers to a chemical molecule which emits lights, commonly in the 300-700 nm range, after excitation of the chemical molecule. Upon absorption of transferred light energy (e.g., photon), a dye molecule goes into an excited state. As the molecule exits the excited state, it emits the light energy in the form of lower energy photon (e.g., emits fluorescence) and returns the dye molecule to its ground state. A dye can be a natural chemical compound or a synthetic chemical compound. Dyes include, but are not limited to cyanines, porphyrins, and bis-pyrenes.

“Porphyrin” refers to any compound, with the following porphin core:

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wherein the porphin core can be substituted or unsubstituted.

“Bis-pyrene” refers to a compound which comprises two pyrene subunits covalently linked to each other. The two pyrene subunits can be linked directly or through a linker. The linker can be any linker known to one of skill in the art, such as but not limited to, alkylenes, alkenylenes, alkynylenes, aryls, heteroaryls, aryl ketones, ketones, amines, amides, and ureas, wherein the linker can be substituted.

“Radiometal chelator” refers to a polydentate ligand binding to a single central metal atom or ion. The metal atom or ion can be a radioactive isotope of the metal. Radiometal chelators include, but are not limited to Gd(III) chelators, DOTA chelator and NOTA chelator. Gd(III) chelators include, but are not limited to gadopentetic acid, gadoteric acid, gadodiamide, gadobenic acid, gadoteridol, gadoversetamide, and gadobutrol.

“Cyanine” or “cyanine dye” refers to a synthetic dye family belonging to a polymethine group. Cyanines can be used as fluorescent dyes for biomedical imaging. Cyanines can be streptocyanines (also known as open chain cyanines), hemicyanines, and closed chain cyanines. Closed chain cyanines have nitrogens which are each independently part of a heteroaromatic moiety.

“Drug” refers to an agent capable of treating and/or ameliorating a condition or disease. A drug may be a hydrophobic drug, which is any drug that repels water. Hydrophobic drugs useful in the present invention include, but are not limited to, deoxycholic acid, taxanes, doxorubicin, etoposide, irinotecan, SN-38, cyclosporin A, podophyllotoxin, Carmustine, Amphotericin, Ixabepilone, Patupilone (epothelone class), rapamycin and platinum drugs. Other drugs includes non-steroidal anti-inflammatory drugs, and vinca alkaloids such as vinblastine and vincristine. The drugs of the present invention also include prodrug forms. One of skill in the art will appreciate that other drugs are useful in the present invention.

“Chemotherapeutic agent” refers to chemical drugs that can be used in the treatment of diseases such as, but not limited to, cancers, tumors and neoplasms. In some embodiments, a chemotherapeutic agent can be in the form of a prodrug which can be activated to a cytotoxic form. Chemotherapeutic agents commonly known by one of ordinary skill in the art can be used in the present invention. Chemotherapeutic agents include, but are not limited to resiquimod, gardiquimod, and imiquimod.

“Immunomodulatory agent” refers to a type of drug which can modify immune responses by stimulating or suppressing the immune system. Immunomodulatory agents include, but are not limited to resiquimod, gardiquimod, and imiquimod.

“Anti-HER2 rhumAb 4D5” refers to a type of HER2 antibody, and is also known as trastuzumab. Trastuzumab is commonly used to treat breast and stomach cancer and is commercially available. Trastuzumab comprises at least 50% peptide sequence identity of SEQ ID NO: 4. The peptide sequence of trastuzumab is described in “Rationally designed anti-HER2/neu peptide mimetic disables P185HER2/neu tyrosine kinases in vitro and in vivo” (Park et al. Nat Biotechnol. 2000 February; 18(2):194-8.)

“CDR-H3 loop” refers to a region inside a HER2 antibody involved with antigen binding.

“Nanocarrier” or “nanoparticle” refers to a micelle resulting from aggregation of the compounds of the invention. The nanocarrier of the present invention can have a hydrophobic core and a hydrophilic exterior.

“Nanofibrils” refer to tubular, rod-like fibrils which have a diameter ranging from tens to hundreds of nanometers. Nanofibrils can have high length-to-diameter ratios. Nanofibrils of the present invention can be formed by an in situ transformation of the nanoparticles after binding at the targeted site.

“Fibrillary structures” refer to linear, rod-like fibrils with diameters on the order of nanometers to micrometers and have a high length-to-diameter ratio. Fibrillary structures may include biopolymers. Fibrillary structures include, but are not limited to, nanofibrils and microfibrils.

“Cell surface” refers to the plasma membrane, which separates the extracellular space from the interior of the cell. The cell surface comprises the lipid bilayer, proteins, and carbohydrates.

“Acellular component” refers to the extracellular environment of a cell and includes, but is not limited to the extracellular matrix, extracellular vesicles, and cytokines surround a cell. The extracellular matrix comprises collagens, fibronectin, and other matrix proteins. Ligands and compounds can interact with an acellular component of cancerous cells to affect the growth of cancer cells.

“Tumor microenvironment” refers to tumor cells and the acellular environment surrounding it, including, but not limited to the extracellular matrix, signaling molecules, immune cells, stromal cells, vasculature, blood vessels, cytokines, chemokines, growth factors, and fibroblasts. Tumors can interact with the surround cells in the microenvironment through the lymphatic and circulatory systems to affect the growth and evolution of cancer cells.

“Treat”, “treating” and “treatment” refers to any indicia of success in the treatment or amelioration of an injury, pathology, condition, or symptom (e.g., pain), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the symptom, injury, pathology or condition more tolerable to the patient; decreasing the frequency or duration of the symptom or condition; or, in some situations, preventing the onset of the symptom. The treatment or amelioration of symptoms can be based on any objective or subjective parameter; including, e.g., the result of a physical examination.

“Administering” refers to oral administration, administration as a suppository, topical contact, parenteral, intravenous, intraperitoneal, intramuscular, intralesional, intranasal or subcutaneous administration, intrathecal administration, or the implantation of a slow-release device e.g., a mini-osmotic pump, to the subject.

“Subject” refers to animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In certain embodiments, the subject is a human.

“Therapeutically effective amount” or “therapeutically sufficient amount” or “effective or sufficient amount” refers to a dose that produces therapeutic effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). In sensitized cells, the therapeutically effective dose can often be lower than the conventional therapeutically effective dose for non-sensitized cells.

“Cancer” refers to diseases with abnormal cell growth and divides without control. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. The term is also intended to include any disease of an organ or tissue characterized by poorly controlled or uncontrolled multiplication of normal or abnormal cells in that tissue and its effect on the body as a whole.

“Imaging” refers to using a device outside of the subject to determine the location of an imaging agent, such as a compound of the present invention. Examples of imaging tools include, but are not limited to, fluorescence microscopy, positron emission tomography (PET), magnetic resonance imaging (MRI), ultrasound, single photon emission computed tomography (SPECT) and x-ray computed tomography (CT). The positron emission tomography detects radiation from the emission of positrons by an imaging agent.

III. Compounds

In some embodiments, the present invention provides a compound of formula (I) wherein A is bis-pyrene; B is a peptide, wherein the peptide forms a beta-sheet; and C is a HER2 ligand.

In some embodiments, the present invention provides a compound of formula (I): A-B-C (I), wherein A is a hydrophobic moiety; B is a peptide, wherein the peptide forms a beta-sheet; and C is a hydrophilic targeting ligand, wherein the hydrophilic targeting ligand is a LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, folate, a LHRH peptide, a HER2 ligand, an EGFR ligand, or a toll-like receptor agonist CpG oligonucleotides and wherein when the hydrophobic moiety is bis-pyrene, then C is a LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, folate, a LHRH peptide, an EGFR ligand, or a toll-like receptor agonist CpG oligonucleotides.

In some embodiments, the present invention provides a compound of formula (I): A-B-C (I), wherein A is a hydrophobic moiety; B is a peptide, wherein the peptide forms a beta-sheet; and C is a hydrophilic targeting ligand, wherein the hydrophilic targeting ligand is a LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, a LHRH peptide, a HER2 ligand, an EGFR ligand, or a toll-like receptor agonist CpG oligonucleotides and wherein when the hydrophobic moiety is bis-pyrene, then C is a LLP2A prodrug, LLP2A, LXY30, LXW64, DUPA, a LHRH peptide, an EGFR ligand, or a toll-like receptor agonist CpG oligonucleotides.

Hydrophobic moieties useful in the present invention includes any suitable hydrophobic moiety known by one of skill in the art. Hydrophobicity and hydrophilicity are commonly measured by the log P values of the compounds using the octane-water reference system. Values lower than 0 indicate hydrophilicity whereas values higher than 0 indicate hydrophobicity. Hydrophobic moieties useful in the present invention includes moieties with log P values of at least 1. In some embodiments, hydrophobic moieties useful in the present invention have a log P value of at least 1.5. In some embodiments, hydrophobic moieties useful in the present invention have a log P value of 1.5-15. Hydrophobic moieties include, but are not limited to cholesterol, vitamin D, vitamin D derivatives, vitamin E, vitamin E derivatives, dyes, drugs, and radiometal chelators. In some embodiments, the hydrophobic moiety is cholesterol, vitamin D, vitamin D derivatives, vitamin E, vitamin E derivatives, a dye, or a drug. In some embodiments, the hydrophobic moiety is cholesterol, vitamin D, vitamin E, a dye, or a drug. In some embodiments, the hydrophobic moiety is cholesterol, vitamin D, or vitamin E. In some embodiments, the hydrophobic moiety is a dye or drug.

Dyes useful in the present invention include any dye described in, but not limited to, Johnson, I., Histochemical Journal, 20:123-140 (1998), and The Molecular Probes® Handbook, 11^thEdition, ed. Johnson and Spence, Life Technologies, Carlsbad, Calif., 2010. The dyes can be fluorescent dyes, triarylmethane dyes, cyanine dyes, benzylidene imidazolinone dyes, indigo dyes, bis-pyrenes and porphyrins. In some embodiments, the hydrophobic moiety is a dye. In some embodiments, the hydrophobic moiety is a fluorescent dye, porphyrin, or bis-pyrene. In some embodiments, the hydrophobic moiety is a cyanine dye, porphyrin, or bis-pyrene.

Drugs useful in the present invention include chemotherapeutic agents and immunomodulatory agents. For example, the drugs can be, but are not limited to, deoxycholic acid, or the salt form deoxycholate, pembrolizumab, nivolumab, cemiplimab, a taxane (e.g., paclitaxel, docetaxel, cabazitaxel, Baccatin III, 10-deacetylbaccatin, Hongdoushan A, Hongdoushan B, or Hongdoushan C), doxorubicin, etoposide, irinotecan, SN-38, cyclosporin A, podophyllotoxin, Carmustine, Amphotericin, Ixabepilone, Patupilone (epothelone class), rapamycin and platinum drugs. Other drugs include non-steroidal anti-inflammatory drugs, and vinca alkaloids such as vinblastine and vincristine. In some embodiments, the drug is paclitaxel, resiquimod, gardiquimod, or deoxycholate.

In some embodiments, the hydrophobic moiety is a chemotherapeutic agent, a fluorescent dye, an immunomodulatory agent, a toll-like receptor agonist, a small molecule agonist of stimulator of interferon gene (STING), porphyrin, deoxycholate, cholesterol, vitamin D, or vitamin E. In some embodiments, the hydrophobic moiety is a chemotherapeutic agent, a fluorescent dye, an immunomodulatory agent, a small molecule agonist of stimulator of interferon gene (STING), porphyrin, cholesterol, vitamin D, or vitamin E. In some embodiments, the hydrophobic moiety is a chemotherapeutic agent, a fluorescent dye, an immunomodulatory agent, a small molecule agonist of stimulator of interferon gene (STING), porphyrin or deoxycholate. In some embodiments, the hydrophobic moiety is a chemotherapeutic agent, a fluorescent dye, an immunomodulatory agent, porphyrin or deoxycholate. In some embodiments, the hydrophobic moiety is paclitaxel, bis-pyrene, cyanine dye, resiquimod, gardiquimod, amidobenzimidazole, porphyrin, or deoxycholate. In some embodiments, the hydrophobic moiety is paclitaxel, bis-pyrene, cyanine dye, resiquimod, gardiquimod, porphyrin, or deoxycholate. In some embodiments, the hydrophobic moiety is resiquimod or porphyrin.

Porphyrins useful in the present invention include any porphyrin known by one of skill in the art. In some embodiments, the porphyrin is a substituted or unsubstituted porphin, protoporphyrin IX, octaethylporphyrin, tetraphenyl porphyrin, pyropheophorbide-a, pheophorbide, chlorin e6, purpurin or purpurinimide. In some embodiments, the porphyrin is pyropheophorbide-a, pheophorbide, chlorin e6, purpurin or purpurinimide. In some embodiments, the porphyrin is pheophorbide-a. In some embodiments, the porphyrin has the following structure:

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In some embodiments, the hydrophobic moiety is bis-pyrene. Bis-pyrenes useful in the present invention include any bis-pyrene known by one of skill in the art. In some embodiments, the bis-pyrene comprises the following moieties:

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In some embodiments, the bis-pyrene comprises the following:

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In some embodiments, the bis-pyrene has the following structure:

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The peptides useful in the present invention can be any suitable peptide, and have any suitable peptide sequence length known by one of skill in the art. In some embodiments, the peptide is a peptide sequence 5-50 amino acids in length. In some embodiments, the peptide is a peptide sequence 5-40 amino acids in length. In some embodiments, the peptide is a peptide sequence 5-30 amino acids in length. In some embodiments, the peptide is a peptide sequence 5-25 amino acids in length. In some embodiments, the peptide is a peptide sequence 5-20 amino acids in length. In some embodiments, the peptide is a peptide sequence 5-15 amino acids in length. In some embodiments, the peptide is a peptide sequence about 5-10 amino acids in length.

Adjacent beta-strand peptides form hydrogen bonds between each strand resulting in beta sheet peptides. The beta-sheet peptide sequences useful in the present invention can be any suitable peptide sequence known by one of skill in the art. For example, commonly known beta-sheet peptides are described in “Branched KLVFF tetramers strongly potentiate inhibition of beta-amyloid aggregation” (Chafekar et al., Chembiochem. 2007 Oct. 15; 8(15):1857-64). In some embodiments, the peptide comprises a peptide sequence from a beta-sheet peptide domain of green fluorescent protein, interleukins, immunoglobulins, or beta-amyloid peptide. In some embodiments, the peptide comprises a peptide sequence from a beta-sheet peptide domain of a beta-amyloid peptide. In some embodiments, the beta-amyloid peptide is beta-amyloid 40 or beta-amyloid 42. In some embodiments, the beta-amyloid peptide is beta-amyloid 40.

In some embodiments, the peptide comprises at least 40% sequence identity to SEQ ID NO:1. In some embodiments, the peptide comprises at least 50% sequence identity to SEQ ID NO:1. In some embodiments, the peptide comprises at least 60% sequence identity to SEQ ID NO:1. In some embodiments, the peptide comprises at least 80% sequence identity to SEQ ID NO:1. In some embodiments, the peptide comprises SEQ ID NO:1.

In some embodiments, the peptide comprises at least 40% sequence identity to SEQ ID NO:2. In some embodiments, the peptide comprises at least 50% sequence identity to SEQ ID NO:2. In some embodiments, the peptide comprises at least 60% sequence identity to SEQ ID NO:2. In some embodiments, the peptide comprises at least 80% sequence identity to SEQ ID NO:2. In some embodiments, the peptide comprises SEQ ID NO:2.

In some embodiments, the peptide comprises at least 40% sequence identity to SEQ ID NO:3. In some embodiments, the peptide comprises at least 50% sequence identity to SEQ ID NO:3. In some embodiments, the peptide comprises at least 60% sequence identity to SEQ ID NO:3. In some embodiments, the peptide comprises at least 80% sequence identity to SEQ ID NO:3. In some embodiments, the peptide comprises SEQ ID NO:3.

Hydrophilic targeting ligands useful in the present invention can target receptors on the cell surface, or the acellular component of the tumor microenvironment. Hydrophilicity and hydrophobicity are commonly measured by the log P values of the compounds using the octane-water reference system. Values lower than 0 indicate hydrophilicity whereas values higher than 0 indicate hydrophobicity. In some embodiments, the hydrophilic targeting ligand includes peptides which target cell surface receptors or acellular components in the tumor microenvironment, which includes, but is not limited to immune cells such as macrophages, T cells, and B cells. In some embodiments, the hydrophilic targeting ligand targets cell surface receptors such as, but not limited to, integrins and epidermal growth factor receptors. In some embodiments, the hydrophilic targeting ligand targets integrins, epidermal growth factors, and toll-like receptors.

In some embodiments, the hydrophilic targeting ligand is a HER2 ligand, a prodrug for a HER2 ligand, a receptor tyrosine-protein kinase-targeting ligand, an integrin-targeting ligand, epidermal growth factor receptor-targeting ligand, ovarian cancer cell-targeting ligand, or prostate cancer cell-targeting ligand. In some embodiments, the hydrophilic targeting ligand is a HER2 ligand, a prodrug for a HER2 ligand, an integrin-targeting ligand, epidermal growth factor receptor-targeting ligand, ovarian cancer cell targeting ligand, or prostate cancer cell targeting ligand.

In some embodiments, the hydrophilic targeting ligand is a HER2 ligand. In some embodiments, the HER2 ligand is an anti-HER2 antibody peptide. In some embodiments, the hydrophilic targeting ligand is the HER2 ligand, wherein the HER2 ligand is an anti-HER2 antibody peptide mimic derived from the primary sequence of the CDR-H3 loop of the anti-HER2 rhumAb 4D5. In some embodiments, the HER2 ligand is as described in “Rationally designed anti-HER2/neu peptide mimetic disables P185HER2/neu tyrosine kinases in vitro and in vivo” (Park et al. Nat Biotechnol. 2000 February; 18(2):194-8.)

In some embodiments, the HER2 ligand has at least 40% sequence identity to SEQ ID NO:4. In some embodiments, the HER2 ligand has at least 50% sequence identity to SEQ ID NO:4. In some embodiments, the HER2 ligand has at least 60% sequence identity to SEQ ID NO:4. In some embodiments, the HER2 ligand has at least 80% sequence identity to SEQ ID NO:4. In some embodiments, the HER2 ligand is SEQ ID NO:4.

In some embodiments, the hydrophilic targeting ligand is an integrin-targeting ligand, epidermal growth factor receptor-targeting ligand, ovarian cancer cell targeting ligand, or prostate cancer cell targeting ligand. In some embodiments, the hydrophilic targeting ligand is a prodrug for an integrin-targeting ligand, epidermal growth factor receptor-targeting ligand, ovarian cancer cell targeting ligand, or prostate cancer cell targeting ligand.

In some embodiments, the hydrophilic targeting ligand is a LLP2A prodrug, LLP2A, LXY30, DUPA, folate, a LHRH peptide, or an EGFR ligand. Any one of the carboxylic acid groups in the DUPA structure can be used to link to the beta-sheet peptide. LHRH analog peptide comprises the following peptide sequence: H-Glp-His-Trp-Ser-Thr-Lys-Leu-Arg-Pro-Gly-NH₂or H-Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH₂. The Lys side chain NH2 group of the LHRH peptides can be used to link to the beta-peptide sheet. In some embodiments, the NH₂group is used to covalently link to the beta-peptide sheet.

EGFR ligands useful in the present invention includes any EGFR ligand known by one of skill in the art. In some embodiments, the EGFR ligand can be EGF, TGF-alpha, HB-EGF, amphiregulin, betacellulin, epigen, epiregulin, neuregulin 1, neuregulin 2, neuregulin 3, and neuregulin 4.

In some embodiments, the hydrophilic targeting ligand is a LLP2A prodrug, LLP2A, or LXY30. The LLP2A prodrug can include any cleavable functional group to be metabolized in situ known by one of skill in the art. In some embodiments, the LLP2A prodrug comprises an ester, amide, carbamate, oxime, imine, ether, phosphate, or beta-amino-ketone functional group. In some embodiments, the LLP2A prodrug comprises an ester, amide, carbamate, ether, or phosphate functional group. In some embodiments, the LLP2A prodrug comprises an ester, amide, carbamate or phosphate functional group. In some embodiments, the LLP2A prodrug comprises an ester group.

In some embodiments, the hydrophilic targeting ligand is a LLP2A prodrug, with the following structure:

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In some embodiments, the hydrophilic targeting ligand is LLP2A, with the following structure:

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In some embodiments, the hydrophilic targeting ligand is LXY30, with the following structure:

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