BiTE secretion from in situ-programmed myeloid cells results in tumor-retained pharmacology

Abstract

Bispecific T-Cell Engagers (BiTEs) are effective at inducing remission in hematologic cancers, but their use in solid tumors has been challenging due to their extreme potency and on-target, off-tumor toxicities in healthy tissue. Their deployment against solid tumors is further complicated by insufficient drug penetration, a hostile tumor microenvironment, and immune escape. To address these challenges, we developed targeted nanocarriers that can deliver in vitro-transcribed mRNA encoding BiTEs to host myeloid cells - a cell type that is actively recruited into the tumor microenvironment. We demonstrate in an immunocompetent mouse model of ovarian cancer, that infusion of these nanoparticles directs BiTE expression to tumor sites, which reshapes the microenvironment from suppressive to permissive and triggers disease regression without systemic toxicity. In contrast, conventional injections of recombinant BiTE protein at doses required to achieve anti-tumor activity, induced systemic inflammatory responses and severe tissue damage in all treated animals. Implemented in the clinic, this in situ gene therapy could enable physicians - with a single therapeutic - to safely target tumor antigen that would otherwise not be druggable due to the risks of on-target toxicity and, at the same time, reset the tumor milieu to boost key mediators of antitumor immune responses.

Keywords: Bi-specific T-cell engagers (BiTEs); In situ gene therapy; Nanotechnology.

Fig. 1.

Schematic illustrating how we reprogram circulating myeloid cells to direct BiTE expression to tumor sites, using targeted nanocarriers.

Fig. 2.

Macrophage transfection with BiTE-encoding mRNA nanocarriers results in functional BiTE protein secretion. (a) Design of macrophage-targeted polymeric nanoparticles formulated with mRNAs encoding the CD3 × EpCAM bispecific antibody. The particles consist of a PbAE-mRNA polyplex core coated with a layer of PGA-Di-mannose, which targets the particles to mannose receptors (CD206). Also depicted is the synthetic mRNA encapsulated in the nanoparticles, which is engineered to encode the bispecific antibody. (b) Flow cytometric analysis of gene-transfer efficiencies into RAW 264.7 macrophages. (c) Immunoblot detection with an IRDye 680RD Streptavidin-conjugated antibody to C-tagged CD3 × EpCAM bispecific antibody in supernatants from nanoparticle-transfected RAW 264.7 macrophages. Positive control, 25 ng of the corresponding purified recombinant BiTE protein. (d) ELISA measurements of EpCAM × CD3 BiTE protein production by nanoparticle-transfected (mCherry⁺) RAW264.7 cells. Assays were performed in triplicate. (e-j) Assessing T-cell activation, cytokine secretion and cytotoxicity against tumor cells. (e) Schematic representation of in vitro cell-killing assays conducted by adding escalating doses of rBiTE into co-cultures of tumor cells and naive T cells. ID8_EpCAM cells are lentivirally engineered versions of ID8 ovarian tumor cells that stably express EpCAM. Tumor lysis and T-cell activation were evaluated by bioluminescent imaging and flow cytometry, respectively, after 72 h of incubation. (f) Bioluminescence imaging of ID8 cells or ID8_EpCAM cells cultured in 96-well plates under different conditions. (g) Levels of bioluminescent signals in (f) were summarized as normalized average radiances in a dose-response curve. (h) Representative flow cytometric histograms demonstrating T-cell proliferation after 72 h of co-culturing with tumor cells in the presence of different amounts of rBiTE. (i) Flow cytometric quantitation of activated T cells. (j) Cytokine secretion was determined by Luminex assay. Assays were performed in triplicate. Means ± SD are depicted. NP, nanoparticle; rBiTE, recombinant BiTE protein.

Fig. 3.

BiTE mRNA nanoparticles improve the narrow therapeutic window of EpCAM × CD3 BiTE recombinant protein and extend the survival of mice with ovarian cancer. (a) Timelines and dosing regimens for intraperitoneal (i.p.)-delivered rBiTE and BiTE nanoparticles. Escalating doses of rBiTE (0.5 μg, 2 μg, 10 μg, and 50 μg) and BiTE nanoparticles containing 50 μg mRNA were injected i.p. into mice with ovarian cancer at 2 doses per week for a total of 9 doses. Treatment started at 7 days after tumor inoculation. (b) Representative sequential bioluminescence imaging of tumor growth and the quantified tumor burden over time (as radiance from luciferase activity from each mouse). (c) Kaplan-Meier survival curves. Statistical analysis was performed using the log-rank test. N = 8 biologically independent animals. (d) Timelines and dosing regimens for intravenously (i.v.)-delivered rBiTE and BiTE nanoparticles. (e) Representative sequential bioluminescence imaging of tumor growth and the quantified tumor burden over time (as radiance from luciferase activity from each mouse). (f) Kaplan-Meier survival curves. Statistical analysis was performed using the log-rank test.

Fig. 4.

EpCAM is expressed in non-pathological tissue as well as malignant lesions. Confocal microscopy of various healthy tissues isolated from C57BL/6 mice and ID8-VEGF-EpCAM ovarian tumor lesions. The expression of EpCAM was assessed by immunofluorescence, and representative staining results for each tissue are shown. Scale bars, liver and kidney: 500 μm; all other organs: 50 μm.

Fig. 5.

Secretion of T-cell engagers from in situ-programmed myeloid cells overcomes autoimmune toxicities of conventional injections of recombinant BiTE protein. (a) Schematic representation of the experimental timeline. (b) Serum chemistry and blood counts. (c) Representative H&E-stained sections of various organs isolated from PBS controls, recombinant BiTE- or nanoparticle-treated animals. Scale bars, 100 μm. Lesions found in the animals are shown and they are described beneath each image. (d) Serial Luminex assay measurements of serum IL-2, IFN-ɣ, TNF-α and IL-6 cytokines.

Fig. 6.

Limited systemic drug exposure following secretion of BiTE antibodies by nanoparticle-transfected macrophages. Quantitative analysis of EpCAM × CD3 BiTE protein in the serum or peritoneal fluid of mice with established ID8-VEGF-EpCAM ovarian tumor lesions. Mice received a single-bolus injection of recombinant BiTE protein (rBiTE) or BiTE-encoding mRNA nanoparticles (BiTE NPs) administered intravenously or intraperitoneally. At the indicated time points, EpCAM × CD3 BiTE protein amounts were quantified by c-tag ELISA. N = 9 biologically independent samples. Shown are mean values ± SD.

Fig. 7.

BiTE nanoparticle-transfected macrophages infiltrate solid tumors and recruit cytotoxic T cells. (a) Immunohistochemical (IHC) staining (top panel) and immunofluorescence staining (bottom panel) of ID8-VEGF-EpCAM ovarian tumors isolated from Ai14 reporter mice treated with PBS or Cre mRNA nanoparticles (3 weekly 50 μg mRNA doses for 2 weeks). Tissues were stained for the indicated markers. Tu = Tumor, Me = Mesentery. Scale bars: 100 μm. (b, d) Representative confocal images of peritoneal metastases of ID8-VEGF-EpCAM ovarian cancer cells in the mesentery. Tissues were collected after 6 twice-weekly i.p. injections of PBS or EpCAM × CD3 BiTE mRNA nanoparticles (50 μg mRNA/dose), and were stained for the indicated lymphocyte- and myeloid-markers (Tu = Tumor, Me = Mesentery. Scale bars: 100 μm). (c, e) Bar graphs showing fluorescent signals for each phenotypic marker using Halo™ image analysis software. N = 5.

Fig. 8.

BiTE-encoding nanoparticles can imprint a pro-inflammatory M1-like phenotype. (a) Experimental timeline of the gene expression study. (b) Heat map of signature gene expression in nanoparticle-transfected (mCherry+) myeloid cells sorted from ovarian tumor-bearing mice following EpCAM × CD3 BiTE mRNA nanoparticle therapy or PBS control treatment. Abundances of mRNAs were normalized by z-scores created with the equation $\text{z}=(\text{x-}\overline{\text{x}})/\mathrm{SD}(\text{x})$. (c-e) Violin plots showing counts for the indicated genes. N = 6 biologically independent samples.