Active targeting of chemotherapy to disseminated tumors using nanoparticle-carrying T cells

Tumor cells disseminate into compartments that are poorly accessible from circulation, which necessitates high doses of systemic chemotherapy. However, the effectiveness of many drugs, such as the potent topoisomerase I poison SN-38, is hampered by poor pharmacokinetics. To deliver SN-38 to lymphoma tumors in vivo, we took advantage of the fact that healthy lymphocytes can be programmed to phenocopy the biodistribution of the tumor cells. In a murine model of disseminated lymphoma, we expanded autologous polyclonal T cells ex vivo under conditions that retained homing receptors mirroring lymphoma cells, and functionalized these T cells to carry SN-38–loaded nanocapsules on their surfaces. Nanocapsule-functionalized T cells were resistant to SN-38 but mediated efficient killing of lymphoma cells in vitro. Upon adoptive transfer into tumor-bearing mice, these T cells served as active vectors to deliver the chemotherapeutic into tumor-bearing lymphoid organs. Cell-mediated delivery concentrated SN-38 in lymph nodes at levels 90-fold greater than free drug systemically administered at 10-fold higher doses. The live T cell delivery approach reduced tumor burden significantly after 2 weeks of treatment and enhanced survival under conditions where free SN-38 and SN-38–loaded nanocapsules alone were ineffective. These results suggest that tissue-homing lymphocytes can serve as specific targeting agents to deliver nanoparticles into sites difficult to access from the circulation, and thus improve the therapeutic index of chemotherapeutic drugs with unfavorable pharmacokinetics.United States. Department of Defense (W81XWH-10-1-0290)National Institutes of Health (U.S.) (CA140476 and CA172164)National Cancer Institute (U.S.) (David H. Koch Institute for Integrative Cancer Research at MIT. Support (Core) Grant P30-CA14051

Nanoparticulate STING agonists are potent lymph node–targeted vaccine adjuvants

Cyclic dinucleotides (CDNs) are agonists of stimulator of IFN genes (STING) and have potential as vaccine adjuvants. However, cyclic di-GMP (cdGMP) injected s.c. shows minimal uptake into lymphatics/draining lymph nodes (dLNs) and instead is rapidly distributed to the bloodstream, leading to systemic inflammation. Here, we encapsulated cdGMP within PEGylated lipid nanoparticles (NP-cdGMP) to redirect this adjuvant to dLNs. Compared with unformulated CDNs, encapsulation blocked systemic dissemination and markedly enhanced dLN accumulation in murine models. Delivery of NP-cdGMP increased CD8[superscript +] T cell responses primed by peptide vaccines and enhanced therapeutic antitumor immunity. A combination of a poorly immunogenic liposomal HIV gp41 peptide antigen and NP-cdGMP robustly induced type I IFN in dLNs, induced a greater expansion of vaccine-specific CD4[superscript +] T cells, and greatly increased germinal center B cell differentiation in dLNs compared with a combination of liposomal HIV gp41 and soluble CDN. Further, NP-cdGMP promoted durable antibody titers that were substantially higher than those promoted by the well-studied TLR agonist monophosphoryl lipid A and comparable to a much larger dose of unformulated cdGMP, without the systemic toxicity of the latter. These results demonstrate that nanoparticulate delivery safely targets CDNs to the dLNs and enhances the efficacy of this adjuvant. Moreover, this approach can be broadly applied to other small-molecule immunomodulators of interest for vaccines and immunotherapy.Bill & Melinda Gates FoundationRagon Institute of MGH, MIT and HarvardNational Institutes of Health (U.S.) (AI091693)National Institutes of Health (U.S.) (AI095109)National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051)National Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award 1F32CA180586)Hertz Foundation (Graduate Fellowship)National Science Foundation (U.S.). Graduate Research Fellowshi

In vivo targeting of adoptively transferred T-cells with antibody- and cytokine-conjugated liposomes

In adoptive cell therapy (ACT), autologous tumor-specific T-cells isolated from cancer patients are activated and expanded ex vivo, then infused back into the individual to eliminate metastatic tumors. A major limitation of this promising approach is the rapid loss of ACT T-cell effector function in vivo due to the highly immunosuppressive environment in tumors. Protection of T-cells from immunosuppressive signals can be achieved by systemic administration of supporting adjuvant drugs such as interleukins, chemotherapy, and other immunomodulators, but these adjuvant treatments are often accompanied by serious toxicities and may still fail to optimally stimulate lymphocytes in all tumor and lymphoid compartments. Here we propose a novel strategy to repeatedly stimulate or track ACT T-cells, using cytokines or ACT-cell-specific antibodies as ligands to target PEGylated liposomes to transferred T-cells in vivo. Using F(ab′)[subscript 2] fragments against a unique cell surface antigen on ACT cells (Thy1.1) or an engineered interleukin-2 (IL-2) molecule on an Fc framework as targeting ligands, we demonstrate that > 95% of ACT cells can be conjugated with liposomes following a single injection in vivo. Further, we show that IL-2-conjugated liposomes both target ACT cells and are capable of inducing repeated waves of ACT T-cell proliferation in tumor-bearing mice. These results demonstrate the feasibility of repeated functional targeting of T-cells in vivo, which will enable delivery of imaging contrast agents, immunomodulators, or chemotherapy agents in adoptive cell therapy regimens.National Institutes of Health (U.S.) (CA140476)National Institutes of Health (U.S.) (CA172164)United States. Dept. of Defense (Contract W81XWH-10-1-0290)National Cancer Institute (U.S.) (Koch Institute Support (core) Grant P30-CA14051

Vaccine delivery with microneedle skin patches in nonhuman primates

Transcutaneous drug delivery from planar skin patches is effective for small-molecule drugs and skin-permeable vaccine adjuvants. However, to achieve efficient delivery of vaccines and other macromolecular therapeutics into the skin, penetration of the stratum corneum is needed. Topically applied skin patches with micron-scale projections ('microneedles') pierce the upper layers of the skin and enable vaccines that are coated on or encapsulated within the microneedles to be dispersed into the skin. Although millimeter-scale syringes have shown promise for vaccine delivery in humans and technologies, such as the Dermaroller (Dermaroller, Wolfenbüttel, Germany), exist for creating microscale punctures in the skin for delivery of solutions of therapeutics, solid microprojection microneedles coated with dry vaccine formulations offer a number of valuable features for vaccination, including reduced risk of blood-borne pathogen transmission or needle-stick injury, the potential for vaccine administration by minimally trained personnel or even self administration and the use of solid-state vaccine formulations that may reduce or eliminate cold-chain requirements in vaccine distribution. Recent studies in mice have demonstrated the ability of microneedles to effectively deliver vaccines to the skin, eliciting protective immunity to influenza, hepatitis C and West Nile virus.Ragon Institute of MGH, MIT and HarvardMassachusetts Institute of TechnologyHarvard UniversityNational Institutes of Health (U.S.) (AI095109)National Institutes of Health (U.S.) (AI096040)National Institutes of Health (U.S.) (AI095985)National Institutes of Health (U.S.) (AI078526)National Institutes of Health (U.S.) (AI060354)United States. Dept. of Defense (Contract W911NF-07-D-0004

Liposomal vaccines incorporating molecular adjuvants and intrastructural T-cell help promote the immunogenicity of HIV membrane-proximal external region peptides

An HIV vaccine capable of inducing high and durable levels of broadly neutralizing antibodies has thus far proven elusive. A promising antigen is the membrane-proximal external region (MPER) from gp41, a segment of the viral envelope recognized by a number of broadly neutralizing antibodies. Though an attractive vaccine target due to the linear nature of the epitope and its highly conserved sequence, MPER peptides are poorly immunogenic and may require display on membranes to achieve a physiological conformation matching the native virus. Here we systematically explored how the structure and composition of liposomes displaying MPER peptides impacts the strength and durability of humoral responses to this antigen as well as helper T-cell responses in mice. Administration of MPER peptides anchored to the surface of liposomes induced MPER-specific antibodies whereas MPER administered in oil-based emulsion adjuvants or alum did not, even when combined with Toll-like receptor agonists. High-titer IgG responses to liposomal MPER required the inclusion of molecular adjuvants such as monophosphoryl lipid A. Anti-MPER humoral responses were further enhanced by incorporating high-Tm lipids in the vesicle bilayer and optimizing the MPER density to a mean distance of ∼10–15 nm between peptides on the liposomes' surfaces. Encapsulation of helper epitopes within the vesicles allowed efficient “intrastructural” T-cell help, which promoted IgG responses to MPER while minimizing competing B-cell responses against the helper sequence. These results define several key properties of liposome formulations that promote durable, high-titer antibody responses against MPER peptides, which will be a prerequisite for a successful MPER-targeting vaccine.Bill & Melinda Gates FoundationNational Institutes of Health (U.S.) (AI091693

Exploiting albumin as a mucosal vaccine chaperone for robust generation of lung-resident memory T cells

Tissue-resident memory T cells (TRMs) can profoundly enhance mucosal immunity, but parameters governing TRM induction by vaccination remain poorly understood. Here, we describe an approach exploiting natural albumin transport across the airway epithelium to enhance mucosal TRM generation by vaccination. Pulmonary immunization with albumin-binding amphiphile conjugates of peptide antigens and CpG adjuvant (amph-vaccines) increased vaccine accumulation in the lung and mediastinal lymph nodes (MLNs). Amph-vaccines prolonged antigen presentation in MLNs over 2 weeks, leading to 25-fold increased lung-resident T cell responses over traditional immunization and enhanced protection from viral or tumor challenge. Mimicking such prolonged exposure through repeated administration of soluble vaccine revealed that persistence of both antigen and adjuvant was critical for optimal TRM induction, mediated through T cell priming in MLNs after prime, and directly in the lung tissue after boost. Thus, vaccine persistence strongly promotes TRM induction, and amph-conjugates may provide a practical approach to achieve such kinetics in mucosal vaccines

The injury response to DNA damage in live tumor cells promotes antitumor immunity

Inducing a DNA damage response in tumor cells ex vivo creates an immunogenic live-cell adjuvant.</jats:p

Optimization of an alum-anchored clinical HIV vaccine candidate

Abstract In the ongoing effort to develop a vaccine against HIV, vaccine approaches that promote strong germinal center (GC) responses may be critical to enable the selection and affinity maturation of rare B cell clones capable of evolving to produce broadly neutralizing antibodies. We previously demonstrated an approach for enhancing GC responses and overall humoral immunity elicited by alum-adjuvanted protein immunization via the use of phosphoserine (pSer) peptide-tagged immunogens that stably anchor to alum particles via ligand exchange with the alum particle surface. Here, using a clinically relevant stabilized HIV Env trimer termed MD39, we systematically evaluated the impact of several parameters relevant to pSer tag composition and trimer immunogen design to optimize this approach, including phosphate valency, amino acid sequence of the trimer C-terminus used for pSer tag conjugation, and structure of the pSer tag. We also tested the impact of co-administering a potent saponin/monophosphoryl lipid A (MPLA) nanoparticle co-adjuvant with alum-bound trimers. We identified MD39 trimer sequences bearing an optimized positively-charged C-terminal amino acid sequence, which, when conjugated to a pSer tag with four phosphates and a polypeptide spacer, bound very tightly to alum particles while retaining a native Env-like antigenicity profile. This optimized pSer-trimer design elicited robust antigen-specific GC B cell and serum IgG responses in mice. Through this optimization, we present a favorable MD39-pSer immunogen construct for clinical translation

Nanoparticulate STING agonists are potent lymph node–targeted vaccine adjuvants

Cyclic dinucleotides (CDNs) are agonists of stimulator of IFN genes (STING) and have potential as vaccine adjuvants. However, cyclic di-GMP (cdGMP) injected s.c. shows minimal uptake into lymphatics/draining lymph nodes (dLNs) and instead is rapidly distributed to the bloodstream, leading to systemic inflammation. Here, we encapsulated cdGMP within PEGylated lipid nanoparticles (NP-cdGMP) to redirect this adjuvant to dLNs. Compared with unformulated CDNs, encapsulation blocked systemic dissemination and markedly enhanced dLN accumulation in murine models. Delivery of NP-cdGMP increased CD8[superscript +] T cell responses primed by peptide vaccines and enhanced therapeutic antitumor immunity. A combination of a poorly immunogenic liposomal HIV gp41 peptide antigen and NP-cdGMP robustly induced type I IFN in dLNs, induced a greater expansion of vaccine-specific CD4[superscript +] T cells, and greatly increased germinal center B cell differentiation in dLNs compared with a combination of liposomal HIV gp41 and soluble CDN. Further, NP-cdGMP promoted durable antibody titers that were substantially higher than those promoted by the well-studied TLR agonist monophosphoryl lipid A and comparable to a much larger dose of unformulated cdGMP, without the systemic toxicity of the latter. These results demonstrate that nanoparticulate delivery safely targets CDNs to the dLNs and enhances the efficacy of this adjuvant. Moreover, this approach can be broadly applied to other small-molecule immunomodulators of interest for vaccines and immunotherapy.Bill & Melinda Gates FoundationRagon Institute of MGH, MIT and HarvardNational Institutes of Health (U.S.) (AI091693)National Institutes of Health (U.S.) (AI095109)National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051)National Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award 1F32CA180586)Hertz Foundation (Graduate Fellowship)National Science Foundation (U.S.). Graduate Research Fellowshi

Enhancement of Peptide Vaccine Immunogenicity by Increasing Lymphatic Drainage and Boosting Serum Stability

Antitumor T-cell responses have the potential to be curative in cancer patients, but the induction of potent T-cell immunity through vaccination remains a largely unmet goal of immunotherapy. We previously reported that the immunogenicity of peptide vaccines could be increased by maximizing delivery to lymph nodes (LNs), where T-cell responses are generated. This was achieved by conjugating the peptide to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG (DSPE-PEG) to promote albumin binding, which resulted in enhanced lymphatic drainage and improved T-cell responses. Here, we expanded upon these findings and mechanistically dissected the properties that contribute to the potency of this amphiphile-vaccine (amph-vaccine). We found that multiple linkage chemistries could be used to link peptides with DSPE-PEG, and further, that multiple albumin-binding moieties conjugated to peptide antigens enhanced LN accumulation and subsequent T-cell priming. In addition to enhancing lymphatic trafficking, DSPE-PEG conjugation increased the stability of peptides in serum. DSPE-PEG peptides trafficked beyond immediate draining LNs to reach distal nodes, with antigen presented for at least a week in vivo, whereas soluble peptide presentation quickly decayed. Responses to amph-vaccines were not altered in mice deficient in the albumin-binding neonatal Fc receptor (FcRn), but required Batf3-dependent dendritic cells (DCs). Amph-peptides were processed by human DCs equivalently to unmodified peptides. These data define design criteria for enhancing the immunogenicity of molecular vaccines to guide the design of next-generation peptide vaccines.National Cancer Institute (U.S.) (Grant P30-CA14051