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1.
J Control Release ; 332: 652-659, 2021 04 10.
Article in English | MEDLINE | ID: mdl-33607175

ABSTRACT

Immune checkpoint blockade has revolutionized the treatment of tumors with immunogenic microenvironments. However, low response rate and acquired resistance are still major challenges. Herein we used a more clinically relevant model of transgenic MMTV-PyMT tumor that more closely mimics the development of human breast cancer in an immunocompetent background to investigate a polymer-based chemo-immunotherapy. We have found that tumors acquired an increased degree of immune suppression during progression, rendering them unresponsive to anti-PD-L1 therapy. To treat large tumors at their advanced stage, we applied a combination strategy consisting of two polymer-drug conjugates that could induce immunogenic cell death (ICD) and disrupt the PD-L1/PD-1 interaction, respectively. Although ICD-inducing conjugate remodeled tumor immune microenvironment by facilitating significant CD8+ T cell infiltration, advanced tumor adapted the immune suppressive mechanism of elevating PD-L1 expression on both cancer cells and myeloid cells thereafter to enable continued tumor growth. Concurrent treatment of PD-L1 blocking conjugate not only abrogated the PD-L1 expression from the two disparate cellular sources, but also considerably reduced the number of immunosuppressive myeloid cells, thereby leading to a significant shrinkage of advanced tumors. Our data provide evidence that combinatory strategy of ICD-inducing and PD-L-blocking modalities could reverse immune suppression and establish a basis for the rational design of cancer immunotherapy.


Subject(s)
Breast Neoplasms , Pharmaceutical Preparations , Animals , B7-H1 Antigen , Breast Neoplasms/drug therapy , Female , Humans , Immunotherapy , Mice , Mice, Transgenic , Polymers , Tumor Microenvironment
2.
J Control Release ; 329: 1129-1138, 2021 01 10.
Article in English | MEDLINE | ID: mdl-33098912

ABSTRACT

The architecture of multivalent polymers exerts an amplified interaction between attached ligands and targets. In current research, we reveal that a dendronized polymer augments the efficacy of an oncolytic peptide (OP; KKWWKKWDipK) for immunotherapy by exploiting (i) "flexible" linear polymer backbone to facilitate interactions with biomembrane systems, and (ii) "rigid" dendronized side chains to enhance the membrane lytic property. We show that a dendronized N-(2-hydroxypropyl)methacrylamide (HPMA) polymer-OP conjugate (PDOP) adopts α-helix secondary structure and induces robust immunogenic cell death (ICD) in cancer cells as characterized by multiple damage-associated molecular patterns (DAMPs) which include intracellular formation of reactive oxygen species (ROS) and surface exposure of calreticulin (CRT). These events convert immunosuppressive 4T1 tumor to an immunoresponsive one by recruiting CD8+ cytotoxic T cells into tumor beds. Combination of PDOP with anti-PD-L1 immune checkpoint blockade (ICB) increases the number of effector memory T cells and completely eradicates 4T1 tumors in mice. Our findings suggest that PDOP is a promising platform for oncolytic immunotherapy.


Subject(s)
Immunogenic Cell Death , Neoplasms , Animals , Immunotherapy , Mice , Neoplasms/therapy , Polymers , T-Lymphocytes, Cytotoxic
3.
Adv Funct Mater ; 30(12)2020 Mar 17.
Article in English | MEDLINE | ID: mdl-33071706

ABSTRACT

Checkpoint blockade immunotherapies harness the host's own immune system to fight cancer, but only work against tumors infiltrated by swarms of pre-existing T cells. Unfortunately, most cancers to date are immune-deserted. Here, we report a polymer-assisted combination of immunogenic chemotherapy and PD-L1 degradation for efficacious treatment in originally non-immunogenic cancer. "Priming" tumors with backbone-degradable polymer-epirubicin conjugates elicits immunogenic cell death and fosters tumor-specific CD8+ T cell response. Sequential treatment with a multivalent polymer-peptide antagonist to PD-L1 overcomes adaptive PD-L1 enrichment following chemotherapy, biases the recycling of PD-L1 to lysosome degradation via surface receptor crosslinking, and produces prolonged elimination of PD-L1 rather than the transient blocking afforded by standard anti-PD-L1 antibodies. Together, these findings established the polymer-facilitated tumor targeting of immunogenic drugs and surface crosslinking of PD-L1 as a potential new therapeutic strategy to propagate a long-term antitumor immunity, which might broaden the application of immunotherapy to immunosuppressive cancers.

4.
J Control Release ; 319: 285-299, 2020 03 10.
Article in English | MEDLINE | ID: mdl-31899273

ABSTRACT

Incorporating targeting moieties that recognize cancer-specific cellular markers can enhance specificity of anticancer nanomedicines. The HER2 receptor is overexpressed on numerous cancers, making it an attractive target. However, unlike many receptors that trigger endocytosis upon ligand binding, HER2 is an internalization-resistant receptor. As most chemotherapeutics act on intracellular targets, this presents a significant challenge for exploiting HER2 overexpression for improved tumor killing. However, hyper-crosslinking of HER2 has been shown to override the receptor's native behavior and trigger internalization. This research co-opts this crosslinking-mediated internalization for efficient intracellular delivery of an anticancer nanomedicine - specifically a HPMA copolymer-based drug delivery system. This polymeric carrier was conjugated with a small (7 kDa) HER2-binding affibody peptide to produce a panel of polymer-affibody conjugates with valences from 2 to 10 peptides per polymer chain. The effect of valence on surface binding and uptake was evaluated separately. All conjugates demonstrated similar (nanomolar) binding affinity towards HER2-positive ovarian carcinoma cells, but higher-valence conjugates induced more rapid endocytosis, with over 90% of the surface-bound conjugate internalized within 4 h. Furthermore, this enhancement was sensitive to crowding - high surface loading reduced conjugates' ability to crosslink receptors. Collectively, this evidence strongly supports a crosslinking-mediated endocytosis mechanism. Lead candidates from this panel achieved high intracellular delivery even at picomolar treatment concentrations; untargeted HPMA copolymers required 1000-fold higher treatment concentrations to achieve similar levels of intracellular accumulation. This increased intracellular delivery also translated to a more potent nanomedicine against HER2-positive cells; incorporation of the chemotherapeutic paclitaxel into this targeted carrier enhanced cytotoxicity over untargeted polymer-drug conjugate.


Subject(s)
Pharmaceutical Preparations , Polymers , Cell Line, Tumor , Doxorubicin , Drug Delivery Systems , Endocytosis
5.
ACS Nano ; 13(10): 11422-11432, 2019 10 22.
Article in English | MEDLINE | ID: mdl-31553883

ABSTRACT

Monoclonal antibody therapy has offered treatment benefits. Nonetheless, a lack of efficacy still exists, partially because monovalent binding of antibodies to specific receptors fails to translate into an active response. Here, we report a pretargeting-postassembly approach that exploits the selective Watson-Crick base pairing properties of oligonucleotides and multivalently tethers receptor-prebound antibodies to albumin at the cell surface. We demonstrate that this two-step self-assembling strategy allows sequential actions of receptor binding and clustering that broadens and strengthens the functions of antibodies. We show that anti-CD20 obinutuzumab (OBN) modified with one morpholino oligonucleotide (OBN-MORF1) maintains the feature of naked OBN antibody upon CD20 binding, and results in actin redistribution, homotypic adhesion, and lysosome-mediated cell death. Consecutive treatment with albumin grafted with multiple copies of a complementary morpholino oligonucleotide (HSA-(MORF2)x) hybridizes with surface-attached OBN-MORF1, manipulates CD20 clustering, and engages additional signals to induce calcium influx and caspase-related apoptosis. With the two types of different mechanisms collaborating in one system, the simple design exerted a notable survival extension of mice bearing disseminated B-cell lymphomas.


Subject(s)
Antibodies, Monoclonal/chemistry , Morpholinos/chemistry , Serum Albumin/chemistry , Actins/chemistry , Antibodies, Monoclonal, Humanized/chemistry , Humans
6.
Nanomedicine ; 16: 217-225, 2019 02.
Article in English | MEDLINE | ID: mdl-30639670

ABSTRACT

Drug-free macromolecular therapeutics (DFMT) is a new paradigm for the treatment of B cell malignancies. Apoptosis is initiated by the biorecognition of complementary oligonucleotide motifs at the cell surface resulting in crosslinking of CD20 receptors. DMFT is composed from two nanoconjugates: 1) bispecific engager, Fab'-MORF1 (anti-CD20 Fab' fragment conjugated with morpholino oligonucleotide), and 2) a crosslinking (effector) component P-(MORF2)X (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer grafted with multiple copies of complementary morpholino oligonucleotide). We evaluated this concept in 44 samples isolated from patients diagnosed with various subtypes of B cell malignancies. Apoptosis was observed in 65.9% of the samples tested. Pretreatment of cells with gemcitabine (GEM) or polymer-gemcitabine conjugate (2P-GEM) enhanced CD20 expression levels thus increasing apoptosis induced by DFMT. These positive results demonstrated that DFMT has remarkable therapeutic potential in various subtypes of B cell malignancies.


Subject(s)
Apoptosis/drug effects , Deoxycytidine/analogs & derivatives , Lymphoma, B-Cell/drug therapy , Adult , Aged , Aged, 80 and over , Antigens, CD20 , Cell Cycle/drug effects , Deoxycytidine/therapeutic use , Female , Humans , Male , Membrane Potential, Mitochondrial/drug effects , Microscopy, Confocal , Middle Aged , Nanomedicine/methods , Young Adult , Gemcitabine
7.
Macromol Biosci ; 18(11): e1800224, 2018 11.
Article in English | MEDLINE | ID: mdl-30259654

ABSTRACT

A therapeutic platform-drug-free macromolecular therapeutics (DFMT)-that induces apoptosis in B cells by cross-linking of CD20 receptors, without the need for low molecular weight cytotoxic drug, is developed. In this report, a DFMT system is synthesized and evaluated based on human serum albumin (HSA) and two complementary coiled-coil forming peptides, CCE and CCK. Fab' fragment of anti-CD20 monoclonal antibody rituximab is attached to CCE (Fab'-CCE); multiple grafts of CCK are conjugated to HSA (HSA-(CCK)7 ). The colocalization of both nanoconjugates at the surface of non-Hodgkin's lymphoma (NHL) Raji cells is demonstrated by confocal fluorescence microscopy. The colocalization leads to coiled-coil formation, CD20 cross-linking, and apoptosis induction. The apoptotic levels are evaluated by Annexin V, Caspase 3, and terminal deoxynucleotidyl transferase dUTP nick end labeling assays. Selective surface binding of DFMT to CD20+ cells is validated in experiments on a coculture of CD20+ (Raji) and CD20-(DG-75) cells. It is found that DFMT can trigger calcium influx only in Raji cells, but not in DG-75 cells. A highly specific treatment for NHL and other B cell malignancies with considerable translational potential is presented by HSA-based DFMT system.


Subject(s)
Apoptosis/drug effects , Immunoglobulin Fab Fragments , Immunologic Capping/drug effects , Lymphoma, B-Cell/drug therapy , Peptides , Rituximab , Serum Albumin, Human , Cell Line, Tumor , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/pharmacology , Lymphoma, B-Cell/metabolism , Lymphoma, B-Cell/pathology , Peptides/chemistry , Peptides/pharmacology , Rituximab/chemistry , Rituximab/pharmacology , Serum Albumin, Human/chemistry , Serum Albumin, Human/pharmacology
8.
Macromol Biosci ; 17(1)2017 01.
Article in English | MEDLINE | ID: mdl-27198936

ABSTRACT

Fluorescence resonance energy transfer (FRET) is applied to investigate the enzyme-responsive payload release from a macromolecular therapeutic. The donor Cy5 is attached to the N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer backbone and the acceptor Cy7 is bound to the termini of enzyme-sensitive peptide side chains. Upon exposure to an enzyme, the bond between the peptide and Cy7 is cleaved, thereby leading to the loss of FRET signal. This enzyme response is visualized at the cell, tissue and whole-body levels. The in vitro results demonstrate that high expression of cathepsin B in tumor cells induces effective release of the drug model from conjugates resulting in a high concentration of payload inside tumor cells. The in vivo and ex vivo images show that the conjugate releases drug model faster in the ovarian tumor than in the normal tissues. The information will enhance the understanding of enzyme-responsive polymer carriers and help to shape their design.


Subject(s)
Fluorescence Resonance Energy Transfer/methods , Imaging, Three-Dimensional , Methacrylates/chemistry , Administration, Intravenous , Animals , Cell Line, Tumor , Coloring Agents/chemistry , Disease Models, Animal , Female , Methacrylates/chemical synthesis , Mice , Mice, Nude , NIH 3T3 Cells , Papain/metabolism
9.
J Control Release ; 218: 36-44, 2015 Nov 28.
Article in English | MEDLINE | ID: mdl-26410808

ABSTRACT

To develop a biodegradable polymeric drug delivery system for the treatment of ovarian cancer with the capacity for non-invasive fate monitoring, we designed and synthesized N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-epirubicin (EPI) conjugates. The polymer backbone was labeled with acceptor fluorophore Cy5, while donor fluorophores (Cy3 or EPI) were attached to HPMA copolymer side chains via an enzyme-cleavable GFLG linker. This design allows elucidating separately the fate of the drug and of the polymer backbone using fluorescence resonance energy transfer (FRET). The degradable diblock conjugate (2P-EPI) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization using a bifunctional chain transfer agent (Peptide2CTA). The pharmacokinetics (PK) and therapeutic effect of 2P-EPI (Mw ~100 kDa) were determined in mice bearing human ovarian carcinoma A2780 xenografts. Compared to 1st generation conjugate (P-EPI, Mw <50 kDa), 2P-EPI demonstrated remarkably improved PK such as fourfold terminal half-life (33.22 ± 3.18 h for 2P-EPI vs. 7.55 ± 3.18 h for P-EPI), which is primarily attributed to the increased molecular weight of the polymer carrier. Notably, complete tumor remission and long-term inhibition of tumorigenesis (100 days) were achieved in mice (n=5) treated with 2P-EPI. Moreover, in vitro cell uptake and intracellular drug release were determined via FRET intensity changes. The results establish a solid foundation for future in vivo tracking of drug delivery and chain scission of polymeric conjugates by FRET imaging.


Subject(s)
Antibiotics, Antineoplastic/administration & dosage , Epirubicin/administration & dosage , Methacrylates/administration & dosage , Ovarian Neoplasms/drug therapy , Animals , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/pharmacology , Antibiotics, Antineoplastic/therapeutic use , Carbocyanines/administration & dosage , Carbocyanines/chemistry , Carbocyanines/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Epirubicin/chemistry , Epirubicin/pharmacology , Epirubicin/therapeutic use , Female , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/administration & dosage , Fluorescent Dyes/chemistry , Fluorescent Dyes/pharmacology , Humans , Methacrylates/chemistry , Methacrylates/pharmacology , Methacrylates/therapeutic use , Mice , Mice, Nude , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Tumor Burden/drug effects
10.
Nano Lett ; 14(5): 2890-5, 2014 May 14.
Article in English | MEDLINE | ID: mdl-24738626

ABSTRACT

This paper demonstrates the first example of targeting a solid tumor that is externally heated to 42 °C by "heat seeking" drug-loaded polypeptide nanoparticles. These nanoparticles consist of a thermally responsive elastin-like polypeptide (ELP) conjugated to multiple copies of a hydrophobic cancer drug. To rationally design drug-loaded nanoparticles that exhibit thermal responsiveness in the narrow temperature range between 37 and 42 °C, an analytical model was developed that relates ELP composition and chain length to the nanoparticle phase transition temperature. Suitable candidates were designed based on the predictions of the model and tested in vivo by intravital confocal fluorescence microscopy of solid tumors, which revealed that the nanoparticles aggregate in the vasculature of tumors heated to 42 °C and that the aggregation is reversible as the temperature reverts to 37 °C. Biodistribution studies showed that the most effective strategy to target the nanoparticles to tumors is to thermally cycle the tumors between 37 and 42 °C. These nanoparticles set the stage for the targeted delivery of a range of cancer chemotherapeutics by externally applied mild hyperthermia of solid tumors.


Subject(s)
Antineoplastic Agents/chemistry , Colonic Neoplasms/drug therapy , Elastin/chemistry , Nanoparticles/chemistry , Animals , Antineoplastic Agents/administration & dosage , Cell Line, Tumor , Drug Delivery Systems , Elastin/administration & dosage , Humans , Hyperthermia, Induced , Mice , Nanoparticles/administration & dosage , Peptides/administration & dosage , Peptides/chemistry , Temperature
11.
Biomacromolecules ; 14(8): 2866-72, 2013 Aug 12.
Article in English | MEDLINE | ID: mdl-23808597

ABSTRACT

Elastin-like polypeptides (ELPs) are stimulus-responsive peptide polymers that exhibit inverse temperature phase transition behavior, causing an ELP to aggregate above its inverse transition temperature (T(t)). Although this property has been exploited in a variety of biotechnological applications, de novo design of ELPs that display a specific T(t) is not trivial because the T(t) of an ELP is a complex function of several variables, including its sequence, chain length, polypeptide concentration, and the type and concentration of cosolutes in solution. This paper provides a quantitative model that predicts the T(t) of a family of ELPs (Val-Pro-Gly-Xaa-Gly, where Xaa = Ala and/or Val) from their composition, chain length, and concentration in phosphate buffered saline. This model will enable de novo prediction of the amino acid sequence and chain length of ELPs that will display a predetermined T(t) in physiological buffer within a specified concentration regime, thereby greatly facilitating the design of new ELPs for applications in medicine and biotechnology.


Subject(s)
Elastin/chemistry , Models, Molecular , Algorithms , Amino Acid Sequence , Molecular Weight , Protein Engineering , Solutions , Transition Temperature
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