Efficient and Highly Specific Gene Transfer Using Mutated Lentiviral Vectors Redirected with Bispecific Antibodies.

Despite their exceptional potencies, the broad tropism of most commonly used lentivirus (LV) vectors limits their use for targeted gene delivery in vivo We hypothesized that we could improve the specificity of LV targeting by coupling (i) reduction of their binding to off-target cells with (ii) redirection of the vectors with a bispecific antibody (bsAb) that binds both LV and receptors on target cells. As a proof of concept, we pseudotyped nonreplicating LV using a mutated Sindbis envelope (mSindbis) with ablated binding to native receptors, while retaining the capacity to facilitate efficient fusion and endosomal escape. We then evaluated the transduction potencies of the mSindbis LV for HER2-positive (HER2+) (SKBR3) breast and HER2-negative (HER2-) (A2780) cells when redirected with different bsAbs. mSindbis LV alone failed to induce appreciable green fluorescent protein (GFP) expression in either cell. When mixed with HER2-targeting bsAb, mSindbis LV was exceptionally potent, transducing 12% to 16% of the SKBR3 cells at a multiplicity of infection (MOI [ratio of viral genome copies to target cells]) of 3. Transduction was highly specific, resulting in ∼50-fold-greater selectivity toward SKBR3 cells versus A2780 cells. Redirecting mSindbis LV led to a 10-fold improvement in cell-specific targeting compared to redirecting wild-type Sindbis LV with the same bsAb, underscoring the importance of ablating native virus tropism in order to maximize targeting specificity. The redirection of mutated LV using bsAb represents a potent and highly versatile platform for targeted gene therapy.IMPORTANCE The goal of gene therapy is specific delivery and expression of therapeutic genes to target cells and tissues. Common lentivirus (LV) vectors are efficient gene delivery vehicles but offer little specificity. Here, we report an effective and versatile strategy to redirect LV to target cells using bispecific antibodies (bsAbs) that bind both cell receptors and LV envelope domains. Importantly, we ablated the native receptor binding of LV to minimize off-target transduction. Coupling bsAb specificity and ablated native LV tropism synergistically enhanced the selectivity of our targeted gene delivery system. The modular nature of our bsAb-based redirection enables facile targeting of the same LV to diverse tissues/cells. By abrogating the native broad tropism of LV, our bsAb-LV redirection strategy may enable lentivirus-based gene delivery in vivo, expanding the current use of LV beyond ex vivo applications.

Pretargeted delivery of PEG-coated drug carriers to breast tumors using multivalent, bispecific antibody against polyethylene glycol and HER2.

Pretargeting is an increasingly explored strategy to improve nanoparticle targeting, in which pretargeting molecules that bind both selected epitopes on target cells and nanocarriers are first administered, followed by the drug-loaded nanocarriers. Bispecific antibodies (bsAb) represent a promising class of pretargeting molecules, but how different bsAb formats may impact the efficiency of pretargeting remains poorly understood, in particular Fab valency and Fc receptor (FcR)-binding of bsAb. We found the tetravalent bsAb markedly enhanced PEGylated nanoparticle binding to target HER2+ cells relative to the bivalent bsAb in vitro. Pretargeting with tetravalent bsAb with abrogated FcR binding increased tumor accumulation of PEGylated liposomal doxorubicin (PLD) 3-fold compared to passively targeted PLD alone, and 5-fold vs pretargeting with tetravalent bsAb with normal FcR binding in vivo. Our work demonstrates that multivalency and elimination of FcRn recycling are both important features of pretargeting molecules, and further supports pretargeting as a promising nanoparticle delivery strategy.

Engineering Polymer-Binding Bispecific Antibodies for Enhanced Pretargeted Delivery of Nanoparticles to Mucus-Covered Epithelium.

Mucus represents a major barrier to sustained and targeted drug delivery to mucosal epithelium. Ideal drug carriers should not only rapidly diffuse across mucus, but also bind the epithelium. Unfortunately, ligand-conjugated particles often exhibit poor penetration across mucus. In this work, we explored a two-step "pretargeting" approach through engineering a bispecific antibody that binds both cell-surface ICAM-1 and polyethylene glycol (PEG) on the surface of nanoparticles, thereby effectively decoupling cell targeting from particle design and formulation. When tested in a mucus-coated Caco-2 culture model that mimics the physiological process of mucus clearance, pretargeting increased the amount of PEGylated particles binding to cells by around 2-fold or more compared to either non-targeted or actively targeted PEGylated particles. Pretargeting also markedly enhanced particle retention in mouse intestinal tissues. Our work underscores pretargeting as a promising strategy to improve the delivery of therapeutics to mucosal surfaces.

Multivalent interactions between streptavidin-based pretargeting fusion proteins and cell receptors impede efficient internalization of biotinylated nanoparticles.

Pretargeting represents a promising strategy to enhance delivery of nanoparticles. The strategy involves first introducing bispecific antibodies or fusion proteins (BFP) that can bind specific epitopes on target cells with one arm, and use the other arm to capture subsequently administered effector molecules, such as radionuclides or drug-loaded nanoparticles. Nevertheless, it remains unclear whether BFP that bind slowly- or non-internalizing epitopes on target cells can facilitate efficient intracellular delivery. Here, we investigated the cellular uptake of biotin-functionalized nanoparticles with streptavidin-scFv against TAG-72, a membrane protein on Jurkat T-cell leukemia cells. Unlike conventional active-targeted nanoparticles, we found that pretargeting resulted in preferential retention of ∼100nm nanoparticles at the plasma membrane rather than internalization into cells. We found no improvement in nanoparticle internalization by simply reducing nanoparticle concentration or surface biotin density. Interestingly, by adding both the BFP and a monoclonal antibody against TAG-72, we observed a twofold improvement in internalization of pretargeted nanoparticles. Our work illustrates that the cellular fate of pretargeted nanoparticles can be controlled by carefully tuning the interactions between pretargeting molecules and nanoparticles on the cell surface. STATEMENT OF SIGNIFICANCE: Pretargeting is a multi-step strategy that utilizes bispecific proteins that recognize both cellular epitopes and subsequently administered therapeutic molecules. This approach has been extensively studied for radiotherapy of blood cancers; however, pretargeting remains largely underexplored for nanoparticle targeting, including whether pretargeting can facilitate efficient intracellular delivery. Here, we found that high density of targeting proteins on the cell surface can effectively limit internalization of pretargeted nanoparticles. Our work underscores the need to carefully assess specific cell-pretargeting molecule pairs for applications requiring intracellular delivery, and the key design requirements for such bispecific pretargeting molecules.

Pretargeting with bispecific fusion proteins facilitates delivery of nanoparticles to tumor cells with distinct surface antigens.

Tumor heterogeneity, which describes the genetically and phenotypically distinct subpopulations of tumor cells present within the same tumor or patient, presents a major challenge to targeted delivery of diagnostic and/or therapeutic agents. An ideal targeting strategy should deliver a given nanocarrier to the full diversity of cancer cells, which is difficult to achieve with conventional ligand-conjugated nanoparticles. We evaluated pretargeting (i.e., multistep targeting) as a strategy to facilitate nanoparticle delivery to multiple target cells by measuring the uptake of biotinylated nanoparticles by lymphoma cells with distinct surface antigens pretreated with different bispecific streptavidin-scFv fusion proteins. Fusion proteins targeting CD20 or tumor-associated glycoprotein 72 (TAG-72) mediated the specific in vitro uptake of 100nm biotin-functionalized nanoparticles by Raji and Jurkat lymphoma cells (CD20-positive and TAG-72-positive cells, respectively). Greater uptake was observed for pretargeted nanoparticles with increasing amounts of surface biotin, with 6- to 18-fold higher uptake vs. non-biotinylated nanoparticle and fusion protein controls. Fully biotin-modified particles remained resistant to cultured macrophage cell uptake, although they were still quickly cleared from systemic circulation in vivo (t1/2<1h). For single Raji tumor-bearing mice, pretargeting with CD20-specific FP significantly increased nanoparticle tumor targeting. In mice bearing both Raji and Jurkat tumors, pretargeting with both fusion proteins markedly increased nanoparticle targeting to both tumor types, compared to animals dosed with nanoparticles alone. These in vitro and in vivo observations support further evaluations of pretargeting fusion protein cocktails as a strategy to enhance nanoparticle delivery to a diverse array of molecularly distinct target cells.

Functional genomic screening reveals asparagine dependence as a metabolic vulnerability in sarcoma.

Current therapies for sarcomas are often inadequate. This study sought to identify actionable gene targets by selective targeting of the molecular networks that support sarcoma cell proliferation. Silencing of asparagine synthetase (ASNS), an amidotransferase that converts aspartate into asparagine, produced the strongest inhibitory effect on sarcoma growth in a functional genomic screen of mouse sarcomas generated by oncogenic Kras and disruption of Cdkn2a. ASNS silencing in mouse and human sarcoma cell lines reduced the percentage of S phase cells and impeded new polypeptide synthesis. These effects of ASNS silencing were reversed by exogenous supplementation with asparagine. Also, asparagine depletion via the ASNS inhibitor amino sulfoximine 5 (AS5) or asparaginase inhibited mouse and human sarcoma growth in vitro, and genetic silencing of ASNS in mouse sarcoma cells combined with depletion of plasma asparagine inhibited tumor growth in vivo. Asparagine reliance of sarcoma cells may represent a metabolic vulnerability with potential anti-sarcoma therapeutic value.

Addressing challenges of heterogeneous tumor treatment through bispecific protein-mediated pretargeted drug delivery.

Tumors are frequently characterized by genomically and phenotypically distinct cancer cell subpopulations within the same tumor or between tumor lesions, a phenomenon termed tumor heterogeneity. These diverse cancer cell populations pose a major challenge to targeted delivery of diagnostic and/or therapeutic agents, as the conventional approach of conjugating individual ligands to nanoparticles is often unable to facilitate intracellular delivery to the full spectrum of cancer cells present in a given tumor lesion or patient. As a result, many cancers are only partially suppressed, leading to eventual tumor regrowth and/or the development of drug-resistant tumors. Pretargeting (multistep targeting) approaches involving the administration of 1) a cocktail of bispecific proteins that can collectively bind to the entirety of a mixed tumor population followed by 2) nanoparticles containing therapeutic and/or diagnostic agents that can bind to the bispecific proteins accumulated on the surface of target cells offer the potential to overcome many of the challenges associated with drug delivery to heterogeneous tumors. Despite its considerable success in improving the efficacy of radioimmunotherapy, the pretargeting strategy remains underexplored for a majority of nanoparticle therapeutic applications, especially for targeted delivery to heterogeneous tumors. In this review, we will present concepts in tumor heterogeneity, the shortcomings of conventional targeted systems, lessons learned from pretargeted radioimmunotherapy, and important considerations for harnessing the pretargeting strategy to improve nanoparticle delivery to heterogeneous tumors.

Evading immune cell uptake and clearance requires PEG grafting at densities substantially exceeding the minimum for brush conformation.

Coating nanoparticles with polyethylene glycol (PEG), which reduces particle uptake and clearance by immune cells, is routinely used to extend the circulation times of nanoparticle therapeutics. Nevertheless, due to technical hurdles in quantifying the extent of PEG grafting, as well as in generating very dense PEG coatings, few studies have rigorously explored the precise PEG grafting density necessary to achieve desirable "stealth" properties. Here, using polymeric nanoparticles with precisely tunable PEG grafting, we found that, for a wide range of PEG lengths (0.6-20 kDa), PEG coatings at densities substantially exceeding those required for PEG to adopt a "brush" conformation are exceptionally resistant to uptake by cultured human macrophages, as well as primary peripheral blood leukocytes. Less than 20% of these nanoparticles were cleared from the blood after 2 h (t1/2 ∼ 14 h) in BALB/c mice, whereas slightly less densely PEGylated and uncoated control particles were both virtually eliminated within 2 h. Our results suggest that the stealth properties of PEG-coated nanoparticles are critically dependent on achieving PEG grafting at densities exceeding those required for brush conformation.