Immune checkpoint inhibitors (ICI) are promising in adjuvant settings for solid tumors and hematologic malignancies. They are currently used in the treatment as mAbs in high concentrations, raising concerns of toxicity and adverse side effects. Among various checkpoint molecules, targeting the programmed cell death protein-1 (PD-1)–programmed death-ligand 1 (PD-L1) axis has garnered more clinical utility than others have. To develop a physiologically relevant and systemically stable level of ICIs from a one-time application by genetic antibody engineering, we endeavored using a nonpathogenic, replication-deficient recombinant adeno-associated vector (rAAV) expressing single-chain variable fragments (scFv) of PD-L1 antibody and tested in syngeneic mouse therapy models of MC38 colorectal and EMT6 breast tumors. Results of this study indicated a significant protection against PD-L1–mediated inhibition of CD8+ T-cell function, against the growth of primary and secondary tumors, and durable antitumor CTLs activity by adoptive CD8+ T-cell transfer. Stable maintenance of PD-L1 scFv in vivo resulted in an increase in PD-1 CD8+ T cells and a concomitant decrease in regulatory T cells, M2 macrophages, and myeloid-derived suppressor cells in the tumor microenvironment. Overall, these data demonstrate the potential of rAAV-PD-L1-scFv as an alternative to mAb targeting of PD-L1 for tumor therapy.

This article is featured in Highlights of This Issue, p. 1623

Checkpoint blockade immunotherapy has shown clinical benefit for several tumor types and in a few patients who are refractory to other treatments, demonstrating the power of optimizing the immune system in the fight against cancer. Among immune checkpoint inhibitors (ICI), the engagement of programmed death-ligand 1 (PD-L1) to its cognate receptor, programmed cell death protein 1 (PD-1), remains a major signal that impedes T-cell function in physiologic and pathologic manifestations (1). In many cancers, the checkpoint blockade is accelerated by the production of PD-L1 by tumor cells as a mechanism to evade antitumor T-cell functions (2). Ligation of membrane-bound PD-L1 on tumor cells with PD-1 on tumor-infiltrating T lymphocytes activates Src homology region 2 domain-containing phosphatases (SPH2), resulting in T-cell receptor suppression and activity (3, 4). So far, there are several competing mAbs targeting both PD-1 and PD-L1 that are being used in the clinic, including avelumab, durvalumab, and atezolizumab. Blocking the PD-1/PD-L1 pathway has shown antitumor effects in the last decade in a variety of tumors, especially in adjuvant settings (5, 6). Owing to stability limitations of mAb in vivo, they are mainly included in treatment regimen in high concentrations, raising concerns of toxicity and adverse side effects, including immune-related adverse effects (irAE; ref. 7). This prompts a need to develop better approaches for checkpoint-targeted immunotherapy.

An alternative to using mAbs is single-chain antibody fragments (scFv), consisting of the variable heavy (VH) and variable light (VL) domains of immunoglobulin (Ig), connected by a flexible polypeptide linker. Single-chain antibodies retain specificity and affinity for their antigen and eliminate the complications associated with whole antibodies because of lacking the Fc and other constant regions (8). Moreover, they have demonstrated superior tissue-penetrating capabilities when compared with whole antibodies (9). Unlike mAbs, scFvs are smaller, yet include a large number of accessible epitopes for target antigens (10). Furthermore, to overcome repeated administration, scFvs can be applied by gene delivery methods using viral and nonviral vectors. Recent studies using oncolytic and replication-defective viral vectors for intratumoral delivery of PD-L1 Ab and scFv genes provide promise for adapting gene-based immune checkpoint inhibition for cancer (11–14).

In this context, recombinant adeno-associated virus vectors (rAAV) offer unique advantages as a delivery platform to achieve systemically stable and physiologically relevant concentrations of PD-L1 scFv by a single vector application. Several studies including our own reported the advantages of using rAAV for long-term transgene expression by a single application (15). The ability to generate rAAV particles lacking any viral genes, but containing only transgene sequences of interest minimizes host immune response against viral proteins (16). The use of rAAV for therapeutic gene delivery has attracted a significant amount of attention in preclinical and clinical protocols (15, 16). To date, AAV-based vectors have proven to be one of the safer and effective gene therapy vectors, as approved in over 280 clinical trials for a variety of human diseases (https://clinicaltrials.gov/).

Taking advantage of the long-term transgene expression capabilities of rAAV and exploiting the natural tropism of an AAV serotype vector to the skeletal muscle, the current study developed and tested the potential of a rAAV-expressing murine PD-L1 scFv as a secretory protein against the growth of PD-L1–positive syngeneic colon and breast cancers in immunocompetent mice models. Results of the study indicated that a single application of rAAV-PD-L1-scFv provided significant protection against PD-L1–mediated inhibition of CD8+ T-cell function, and against the growth of MC38 and EMT6 primary and secondary tumors. The durable antitumor CTLs activity from rAAV-PD-L1-scFv was further evident in CD8+ T-cell adoptive transfer, as well as in tumor rechallenge studies. Readouts on immune mechanisms in therapy gains indicated that systemically stable PD-L1 scFv leads to an increase in CD8+ T cells, a decrease in myeloid-derived suppressor cells (MDSC) and M2 macrophages, and a concomitant increase in Th1-associated cytokines and chemokines. Overall, data from this study demonstrate the potential of rAAV-PD-L1-scFv systemic therapy as an alternative to mAb targeting of PD-L1 for tumor therapy.

Mice, cell lines, and reagents

Six to 8 weeks old C57BL/6 and BALB/c mice were purchased from Charles River. Ovalbumin transgenic mice in the C57BL/6 background and the OT-1 peptide were a kind gift of Dr. Frances Lund, Department of Microbiology, University of Alabama at Birmingham. Both male and female mice were used in the experiment. The human embryonic kidney cell line, HEK 293T (CRL-3216) was purchased from ATCC. Murine colon adenocarcinoma cell line, MC38, was a kind gift of Dr. James Primus (Vanderbilt University, Nashville, TN). Murine mammary carcinoma cell line, EMT6, was a gift from Dr. Sophia Ran, Southern Illinois University. In vitro and in vivo studies, authenticating these cell lines have been published by our group (17–19). All cell lines are expanded at low passages and multiple stocks are frozen. Once every 10–12 passages, a new vial is thawed. All cell lines used are routinely tested for Mycoplasma using a PCR-based kit (ATCC, 30-1012K). The cells were maintained in DMEM (Gibco, 11965-092), supplemented with 10% FBS (Omega Scientific, FB-02) and 1% Pen-Strep (Gibco, 15140-122). We routinely use cell lines in their early passage. Recombinant mouse PD-L1 protein was purchased from BioLegend (758206). Anti-His-HRP mAb was purchased from R&D Systems (MAB050H). Streptavidin-HRP was purchased from Cell Signaling Technology (3999). Anti-CD3e (130-092-973), anti-CD28 (130-093-182) antibodies and mouse IL2 (130-120-662) were purchased from Miltenyi Biotec. Antibodies used for flow cytometry analysis and IHC are listed in Supplementary Tables S1 and S2, respectively. The cDNA sequence of the anti-PD-L1 scFv has been published earlier (20), with accession number in GenBank: KF041825.

Construction of AAV-PD-L1-scFv plasmid, packaging, and purification of rAAV-expressing murine PD-L1 scFv

A scFv against murine PD-L1 was isolated from a phagemid harboring the anti-PD-L1 scFv sequence (kind gift from Dr. Dan Saltzman, University of Minnesota, Minneapolis, MN; GenBank: KF041825; ref. 20). The PD-L1 scFv sequence was amplified by PCR and BamH1 and Not1 overhangs were introduced using 5′-TAAGCAGGATCCCCTGACTCAGCCGTCCT-3′ forward primer and 5′-TAAGCAGCGGCCGCTTAAGAAGCGTAGTCCGG-3′ reverse primers. Following restriction digestion of the PCR product, the PD-L1 scFv sequence was subcloned into pSecTag2/Hygro mammalian expression vector to generate the construct, pSecTag2-PD-L1-scFv. The pSecTag2/Hygro is a cytomegalovirus promoter–driven mammalian expression vector that features a secretory signal from the V-J2-C region of the Igκ chain. For the production of anti-PD-L1-scFv AAV particles, the PD-L1-scFv-6x-His fragment along with the Igκ secretion signal was isolated from pSecTag2-PD-L1-scFv expression vector and subcloned into pAAV-MCS. rAAV was produced using this plasmid by transient transfection in HEK 293T cells with a helper plasmid containing AAV2-rep and AAV1-cap genes and adenovirus helper functions, as described earlier (21). Particle titer of the rAAV was determined by quantitative PCR (22).

Expression and extracellular secretion of PD-L1 scFv

The expression of PD-L1 scFv was confirmed individually from both PD-L1 scFv expression plasmid, and rAAV-PD-L1-scFv in HEK 293T cells. Forty-eight hours after plasmid transfection or rAAV infection, the cells were harvested and media were collected. For protein isolation from the cells, a Pierce RIPA buffer (Thermo Fisher Scientific, 89900) containing protease inhibitors (Thermo Fisher Scientific, 88666) was used and protein concentrations were measured using a Pierce BCA protein assay kit (Thermo Fisher Scientific, 23227). Equal amounts of protein from cell lysates and equal volumes of supernatant media were resolved on a 12% SDS-PAGE gel, and transferred onto nitrocellulose membranes (Bio-Rad, 1620115). The membranes were blocked in 5% nonfat milk in TBS with 0.1% Tween 20 (TBST), and probed with an anti-His-HRP antibody. Following overnight incubation at 4°C and subsequent washes with TBST (3 × 10 minutes), the blots were incubated with a chemiluminescence reagent (Millipore Sigma, WBKLS0500) and images were obtained using a PXi gel imaging system (Syngene).

T-cell proliferation assay

Splenocytes were isolated from OT-1 transgenic mice. Single-cell suspensions of splenocytes were plated at a density of 5 × 105 cells/well in 24-well dishes and cultured overnight. Following overnight culture, the cells were treated with OT-1 peptide (0.5 μg/mL). Concurrently, HEK 293T cells were transduced with rAAV-PD-L1-scFv. The bioactivity of scFv from vector transduced HEK 293T cell conditioned medium (CM) was tested by treating OT-1-pulsed T cells in the presence or absence of recombinant PD-L1 (100 ng/mL) and T-cell proliferation index was quantitated every 24 hours by cell counting. IL2 was maintained in all culture conditions to support T-cell viability.

Analysis of circulating level of PD-L1 scFv in the blood using ELISA

Sera from rAAV-PD-L1-scFv–treated mice were collected at different timepoints (N = 3 per timepoint) and the concentration of PD-L1 scFv was assessed using a His Tag ELISA Detection Kit (GenScript, L00436) following manufacturer's instructions. Sera from untreated mice were used as control.

Plasma levels of PD-L1 scFv and PD-L1 mAb in vivo

Cohorts of C57BL/6 mice were administered with either a one-time intramuscular application of rAAV-PD-L1-scFv [1 × 1012 genomic copies (GC)/mouse] or a four-time intraperitoneal application (once every 3 days) with 200 μg/mouse biotinylated mouse PD-L1 mAb, respectively (N = 3). Blood samples were collected on different timepoints by retro-orbital bleeding and allowed to clot at 4°C. Serum from each sample was separated 4 hours later by centrifugation at 1,500 × g for 10 minutes at 4°C and stored at −80°C until analysis. The stability of rAAV-produced PD-L1 scFv and directly injected PD-L1 mAb in the sera was examined by Western blotting. Equal volumes (20 μL) of serum samples were used for Western blot analysis. For biotinylated PD-L1, membranes were probed with streptavidin-conjugated horseradish peroxidase (HRP). For the detection of PD-L1 scFv, membranes were probed with an HRP-conjugated anti-His antibody. The concentration of PD-L1 scFv in serum was assessed by His Tag ELISA Detection kit. Relative intensity of PD-L1 mAb in serum was calculated on the basis of known concentrations of the injectant. (N = 3 mice/group).

Mouse tumor models and treatment strategy

For the colon cancer model, a syngeneic MC38 colon cancer cells (1 × 105 cells/mouse) were inoculated subcutaneously into 8-week-old female and male C57BL/6 mice. For the breast cancer model, a syngeneic EMT6 cancer cells (2 × 105 cells/mouse) were inoculated into the fourth mammary fat pad of 8-week-old female BALB/c mice. After 2 days, rAAV-PD-L1-scFv or rAAV-GFP was administered by intramuscular injection (1 × 1012 GC/mouse). An untreated group was also used as a negative control. Tumor progression was monitored using a digital caliper every other day and tumor volume was calculated as: volume = (length × width2)/2. All animal studies were conducted in accordance with an approved protocol of the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham (Birmingham, AL).

CD8+ T-cell killing assay

Cytotoxic T-cell assay was performed using a LIVE/DEAD cell-mediated cytotoxicity kit (Molecular Probes, L7010). Briefly, CD8+ T cells from rAAV-PD-L1-scFv–treated mice were flow sorted and plated in anti-CD3 (10 μg/mL)-coated 12-well tissue culture plates and cultured in RPMI1640 medium, containing IL2 (50 U/mL) and anti-CD28 antibody (2 μg/mL) for 3 days with or without the addition of purified cell lysates from MC38 cells (100 μg/mL), and used as effector cells. MC38 cell lysates were prepared by freezing (in liquid nitrogen)/thawing (in 37°C water bath) cells five times. Protein concentration was measured by using the Pierce BCA Protein Assay Kit. After 3 days of culture, CD8+ T cells were cocultured with 3,3′-dioctadecyloxacarbocyanine DiOC18 (3)-labeled MC38 cells as targets at target:effector ratios of 1:1, 1:5, and 1:20 for 24 hours. Next, the cells were harvested, stained with propidium iodide, and subjected to flow cytometry in a BD LSR II instrument (BD Biosciences). The data were analyzed using the FlowJo v.10 software (FlowJo).

Analysis of soluble cytokines and chemokines in the blood

Sera from untreated and rAAV-PD-L1-scFv–treated mice (N = 3) were pooled and 200 μL of samples from each group was used to analyze relative cytokine and chemokine levels using Proteome Profiler Mouse Cytokine Array Kit (R&D Systems, ARY006) following manufacturer's instructions. Briefly, serum samples were mixed with a cocktail of biotinylated detection antibodies for 1 hour and then incubated with Mouse Cytokine Array membranes overnight. After washing, streptavidin-HRP was added and incubated for 30 minutes, followed by chemiluminescent detection in a PXi gel imaging system. Array data were analyzed by densitometry for integral optical density using HLImage++ (Western Vision Software).

Flow cytometry analysis and cell sorting

For flow cytometry analysis, tumor and spleen tissues were harvested and single-cell suspensions were prepared. Tumor tissues were digested with collagenase Type II (Gibco, 17101-015) and spleen tissues were teased with constant aspiration to obtain single-cell suspensions. Next, cells were treated with ACK buffer (Lonza, Basel, 10-548E) to lyse red blood cells, then resuspended in FACS buffer (PBS + 3% FBS). For flow cytometry analysis, MC38 and EMT6 cells were detached from culture plates, then resuspended in FACS buffer. In all experiments, cells were divided into individual tubes with 100 μL FACS buffer and stained with appropriate antibodies (Supplementary Table S1). For intracellular staining, cells were fixed using 2% paraformaldehyde, followed by staining with antibodies in a permeabilization buffer (Thermo Fisher Scientific, 00-8333-56). Flow cytometry data were acquired in a BD LSR II instrument and analyzed using FlowJo v.10 software. Sterile sorting of CD8+ T cells was performed using a BD FACSAria instrument (BD Biosciences).

IHC analysis of CD8+ T cells and macrophages in the tumor microenvironment

IHC was performed on 5 μmol/L sections of paraffin-embedded tumor tissues from untreated or rAAV-PD-L1-scFv–treated mice. After deparaffinization and rehydration, slides were incubated with a citrate buffer for 30 minutes in a steamer for antigen retrieval. Primary antibody incubation was performed overnight at 4°C. Next, slides were stained with fluorophore-conjugated secondary antibodies and incubated with samples for 1 hour at room temperature. After washing the unbound secondary antibodies, cell nuclei were counterstained using DAPI (Thermo Fisher Scientific, D1306). Images were taken using EVOS M7000 Imaging System (Thermo Fisher Scientific). Primary and secondary antibodies used are listed in Supplementary Table S2.

Adoptive CD8+ T-cell transfer and tumor rechallenge studies

For adoptive T-cell transfer, CD8+ T cells were purified by sterile flow cytometry sorting from rAAV-PD-L1-scFv–treated and control mice on day 33. CD8+ T cells from normal C57BL/6 mice without tumor challenge were used as control. Sorted CD8+ T cells were plated in anti-CD3 (10 μg/mL)-coated 12-well tissue culture plates and cultured in RPMI1640 medium plus IL2 (50 U/mL) and anti-CD28 antibody (2 μg/mL) for 3 days. Later, CD8+ T cells were harvested and 1 × 106 cells were injected through the tail vein into recipient mice with preestablished MC-38 tumors. Tumor progression was measured using a digital caliper every other day. For tumor rechallenge studies, 1 × 105 MC38 cells were inoculated at the opposite flank of the C57BL/6 mice that were originally implanted with tumor cells and remained tumor-free following treatment with rAAV-PD-L1-scFv. Measurement and calculation of tumor volumes were performed as described above.

Statistical analysis

Results consisting of three groups or more were analyzed using two-way ANOVA with Tukey multiple comparisons test by GraphPad Prism 8. Analysis of results containing two groups was performed using Student t test. All data are presented as mean ± SD. A P value < 0.05 was considered statistically significant.

Data availability

The data generated in this study are not from any public domain and are available within the article and its Supplementary Data.

Development of a rAAV-PD-L1-scFv expression vector

Because the current study adapted AAV-based gene transfer approach by a single intramuscular vector application to achieve systemically stable levels of PD-L1 scFv, we created a rAAV plasmid containing a murine PD-L1 scFv gene as a secretory protein. To this end, the coding sequence of PD-L1 scFv cDNA (20) was subcloned downstream of the Igκ-chain leader sequence in pSecTag2 vector. Upon verification of high-level secretion of the transgene (Supplementary Fig. S1), the cassette containing Igκ-chain secretory signal and PD-L1 scFv open reading frame (ORF) was subcloned into AAV-MCS vector (Fig. 1A) to generate mature virions. On the basis of the established superiority of AAV1 for intramuscular application, AAV1 serotype capsid was used in this study (23). Packaging and purification of rAAV-PD-L1-scFv was performed by transient transfection in HEK 293T cells by trans-complementing AAV1 serotype capsid, as described earlier (21). The PD-L1-scFv ORF also contained a 6x-His tag that was engineered to enable its detection by immunoblot and ELISA. High-level expression of PD-L1 scFv in cell lysates and its secretion into the medium following transduction of HEK 293T cells were confirmed by immunoblotting with anti-His antibody (Fig. 1A).

Figure 1.

Characterization of rAAV-expressing PD-L1 scFv as a secretory protein, functional validation in overcoming T-cell exhaustion, and concentration of PD-L1 scFv in the blood in vivo. A, scFvs against murine PD-L1 was subcloned into a rAAV vector. The expression of PD-L1 scFv was confirmed by transducing rAAV-PD-L1-scFv in HEK 293T cells and detecting high-level expression and extracellular secretion in CM using an anti-His antibody by Western blot analysis. B, Splenocytes from OT-1 transgenic mice were plated at a density of 5 × 105 cells/well in 24-well tissue culture plates. Following overnight culture, cells in replicates were stimulated with the OT-1 peptide (0.5 μg/mL). The bioactivity of PD-L1 scFv from rAAV-PD-L1-scFv–transduced 293T CM was examined by treating OT-1 peptide–pulsed T cells in the presence or absence of purified recombinant PD-L1 protein (100 ng/mL). Live splenocytes were counted at 24-hour intervals to monitor kinetics of splenocytes in response to the peptide stimulation (*, P < 0.05; **, P < 0.01; ***, P < 0.001). C, Cohorts of C57BL/6 mice were administered with either a one-time intramuscular application of rAAV-PD-L1-scFv (left) or a four-time application (once every 3 days) with 200 μg/mouse biotinylated mouse PD-L1 mAb (right). Sera were collected from mice on indicated days after completion of a one-time application of rAAV-PD-L1-scFv or PD-L1 mAb applications. The systemic level of PD-L1 scFv was detected by Western blotting using an anti-His-HRP antibody (left) and the level of PD-L1 mAb was detected using streptavidin-HRP (right). Quantitation of PD-L1 scFv and PD-L1 mAb was performed by ELISA.

Figure 1.

Characterization of rAAV-expressing PD-L1 scFv as a secretory protein, functional validation in overcoming T-cell exhaustion, and concentration of PD-L1 scFv in the blood in vivo. A, scFvs against murine PD-L1 was subcloned into a rAAV vector. The expression of PD-L1 scFv was confirmed by transducing rAAV-PD-L1-scFv in HEK 293T cells and detecting high-level expression and extracellular secretion in CM using an anti-His antibody by Western blot analysis. B, Splenocytes from OT-1 transgenic mice were plated at a density of 5 × 105 cells/well in 24-well tissue culture plates. Following overnight culture, cells in replicates were stimulated with the OT-1 peptide (0.5 μg/mL). The bioactivity of PD-L1 scFv from rAAV-PD-L1-scFv–transduced 293T CM was examined by treating OT-1 peptide–pulsed T cells in the presence or absence of purified recombinant PD-L1 protein (100 ng/mL). Live splenocytes were counted at 24-hour intervals to monitor kinetics of splenocytes in response to the peptide stimulation (*, P < 0.05; **, P < 0.01; ***, P < 0.001). C, Cohorts of C57BL/6 mice were administered with either a one-time intramuscular application of rAAV-PD-L1-scFv (left) or a four-time application (once every 3 days) with 200 μg/mouse biotinylated mouse PD-L1 mAb (right). Sera were collected from mice on indicated days after completion of a one-time application of rAAV-PD-L1-scFv or PD-L1 mAb applications. The systemic level of PD-L1 scFv was detected by Western blotting using an anti-His-HRP antibody (left) and the level of PD-L1 mAb was detected using streptavidin-HRP (right). Quantitation of PD-L1 scFv and PD-L1 mAb was performed by ELISA.

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PD-L1 scFv protects CD8+ T cells from PD-1/PD-L1 checkpoint blockade

We next tested the bioactivity of rAAV-produced PD-L1 scFv using spleen cells from C57BL/6-Tg (TcraTcrb)1100Mjb model (referred to as OT1 mouse; ref. 24). This transgenic mouse model contains sequences of mouse Tcra-V2 and Tcrb-V5 genes, designed to recognize ovalbumin peptide residues 257–264 (OVA257–264) in the context of coreceptor interaction with MHC class I (24). This results in MHC class I–restricted, ovalbumin-specific, CD8+ T cells (OT-1 cells). The CD8+ T cells thus generated primarily recognize OVA257–264 when presented by the MHC I molecule. To examine bioactivity of rAAV-produced PD-L1 scFv; peptide-pulsed splenocytes from OT1 mice were treated with purified recombinant PD-L1 protein in the presence or absence of CM from rAAV-PD-L1-scFv–tranduced HEK 293T cells. Equal volume of CM from HEK 293T cells was added to OT-1 peptide stimulated and untreated splenocytes and cell proliferation was monitored every 24 hours, for up to 72 hours. As expected, stimulation with OT-1 peptide resulted in a robust and time-dependent proliferation of OT-1 splenocytes for 72 hours. The proliferation of OT-1 splenocytes in response to OT-1 peptide was significantly blunted by recombinant PD-L1 protein after 48 hours and further, by 72 hours after peptide pulsing (P < 0.01 and P < 0.05, for 24 and 72 hours, respectively). CM from rAAV-PD-L1-scFv–transduced cells sequestered recombinant PD-L1 protein, resulting in a significant increase in proliferation of OT-1 splenocytes, allowing the proliferation of OT-1 splenocytes to progress at a significantly higher rate than was observed with OT-1 alone (P < 0.01 and P < 0.002 for recombinant PD-L1 vs. recombinant PD-L1 + PD-L1 scFv at 48 and 72 hours, respectively). Treatment of OT-1 splenocytes with the scFv-CM in the absence of recombinant PD-L1 did not alter the kinetics of peptide-pulsed OT-1 splenocytes (Fig. 1B).

Concentration of PD-L1 scFv in the blood of mice by a single application of rAAV

We next examined the relative changes in plasma stability of PD-L1 scFv from a single application of rAAV-PD-L1-scFv, as compared with multiple infusions of an anti-PD-L1 mAb. Cohorts of C57BL/6 mice were administered with either a one-time intramuscular application of 1 × 1012 GC of rAAV-PD-L1-scFv or four intraperitoneal applications of, biotinylated murine anti-PD-L1-mAb (Fig. 1C), once every 3 days and serum samples were collected at different timepoints. The time-dependent stability of respective antibodies was assessed by Western blotting and quantitation of relative concentrations was determined by ELISA (Fig. 1C). Results indicated detectable amounts of mAb (∼0.8 μg/mL serum) one day after the final mAb application, which rapidly declined to < 0.1 μg/mL serum on day 4. In contrast, the group of mice that received a one-time application of rAAV-PD-L1-scFv showed detectable levels of the scFv in serum by 1 week approximately, reaching a maximum level (∼0.3 μg/mL serum) around day 16, which persisted more than 1 month. These data suggest that a single vector application maintains a systemically stable amount of PD-L1 scFv, at a much lesser concentration compared with multiple applications of the mAb.

A single application of rAAV-PD-L1-scFv confers protection against the growth of syngeneic tumors in therapy models in vivo

To evaluate the antitumor potential of PD-L1 scFv, two syngeneic murine cancer models were adopted using the MC-38 colon and EMT6 breast cancer cell lines, which were characterized to be positive for PD-L1 expression (Fig. 2A). Cohorts of C57BL/6 mice were inoculated subcutaneously with 1 × 105 MC38 cells. In the breast cancer model, 2 × 105 EMT6 cells were inoculated into the fourth mammary fat pad of BALB/c mice. Two days later, mice were given a one-time intramuscular application of 1 × 1012 GC of rAAV-PD-L1-scFv, based on the kinetics of PD-L1 scFv expression, showing PD-L1 scFv in serum on day 8 (Fig. 1C, left). rAAV-GFP was used as a vector control. Tumor-challenged mice without any treatment were included as an additional control. Results of the study indicated a significant decrease in tumor growth and a significant increase in tumor-free survival in the rAAV-PD-L1-scFv group, compared with mice without treatment or treatment with rAAV-GFP (Fig. 2A; Supplementary Fig. S2). We also confirmed that a one-time genetic application of PD-L1 scFv was as efficient as multiple PD-L1 mAb treatments (Supplementary Fig. S3). We further assessed the concentration of rAAV-produced PD-L1 scFv in the EMT6 tumor model by immunoblot and quantitative ELISA. Results of this analysis showed a similar kinetics of PD-L1 scFv expression to that observed in the MC38 model (Supplementary Fig. S4), with detectable levels of scFv in the serum, beginning day 8, and reaching a steady state around day 16.

Figure 2.

Single application of rAAV-PD-L1-scFv confers protection against the growth of syngeneic tumors and decreases tumor-derived PD-L1 in vivo. A, MC38 or EMT6 cells were tested for membrane-bound PD-L1 expression by flow cytometry by staining single-cell suspensions with a PE-conjugated PD-L1 antibody or a PE-conjugated isotype control antibody. Unstained MC38 cells were also used as control (left). A total of 1 × 105 of MC38 cells were subcutaneously implanted into C57BL/6 mice, and 2 × 105 of EMT6 cells were inoculated into fourth mammary fat pad of female BALB/c mice. After 2 days, rAAV-PD-L1-scFv or rAAV-GFP (1 × 1012 GC/mouse) was administered by intramuscular injection in the hind limb. An untreated group was used as a negative control. Growth kinetics and tumor volumes at the endpoint are shown from each group. N = 5–8; ***, P < 0.001. B, Mice were sacrificed on day 33 following initiation of therapy. MC38 tumors were isolated and an equal number of tumor cells were analyzed for PD-L1 expression by flow cytometry. Tumor cells were gated as CD45 population. PD-L1 expression was analyzed using a PE-conjugated antibody. CD45 cell percentage within live cell population and percentage of PD-L1+ cells within the CD45 population are shown. ***, P < 0.001

Figure 2.

Single application of rAAV-PD-L1-scFv confers protection against the growth of syngeneic tumors and decreases tumor-derived PD-L1 in vivo. A, MC38 or EMT6 cells were tested for membrane-bound PD-L1 expression by flow cytometry by staining single-cell suspensions with a PE-conjugated PD-L1 antibody or a PE-conjugated isotype control antibody. Unstained MC38 cells were also used as control (left). A total of 1 × 105 of MC38 cells were subcutaneously implanted into C57BL/6 mice, and 2 × 105 of EMT6 cells were inoculated into fourth mammary fat pad of female BALB/c mice. After 2 days, rAAV-PD-L1-scFv or rAAV-GFP (1 × 1012 GC/mouse) was administered by intramuscular injection in the hind limb. An untreated group was used as a negative control. Growth kinetics and tumor volumes at the endpoint are shown from each group. N = 5–8; ***, P < 0.001. B, Mice were sacrificed on day 33 following initiation of therapy. MC38 tumors were isolated and an equal number of tumor cells were analyzed for PD-L1 expression by flow cytometry. Tumor cells were gated as CD45 population. PD-L1 expression was analyzed using a PE-conjugated antibody. CD45 cell percentage within live cell population and percentage of PD-L1+ cells within the CD45 population are shown. ***, P < 0.001

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Systemically stable PD-L1 scFv promotes antitumor T-cell response and dampens immune suppressor cells in vivo

To gain more insights into possible mechanisms of observed antitumor response induced by rAAV-PD-L1-scFv, we first characterized the immune cells both in the tumor microenvironment (TME) and in the spleen from treated and untreated mice by flow cytometry. Gating strategies used in flow cytometry are shown in Supplementary Figs. S5 and S6, respectively. Results of the study indicated that rAAV-PD-L1-scFv treatment significantly decreased membrane-bound PD-L1 on CD45 tumor cells (Fig. 2B). To rule out that PD-L1 scFv does not block the binding of the particular clone of anti-PD-L1 antibody used in flow cytometry, MC38 cells were preincubated with CM containing PD-L1 scFv for 2 hours, prior to detecting membrane-bound PD-L1 by flow cytometry. The results confirmed PD-L1 scFv did not change PD-L1 levels (Supplementary Fig. S7). Furthermore, rAAV-PD-L1-scFv treatment significantly enhanced CD8+ and CD4+ T cells in the TME, compared with untreated mice and in mice that were treated with rAAV-GFP (Fig. 3A). The increase in CD8+ and CD4+ T cells was accompanied by a significant decrease in regulatory T cells (Treg) in the rAAV-PD-L1-scFv–treated mice (Fig. 3A). Among cells of the innate immune system, MDSCs possess strong immunosuppressive activity in the TME, along with polarization of macrophages, favoring an M2 phenotype over M1 macrophages (25–28). Hence, we analyzed both MDSCs and macrophages in the TME following treatment with rAAV-PD-L1-scFv and compared it with untreated and rAAV-GFP–treated groups. Results of the analysis indicated a significant decrease in MDSCs at the TME of rAAV-PD-L1-scFv–treated mice (Fig. 3A). Furthermore, total macrophages were significantly decreased following rAAV-PD-L1-scFv treatment at the TME (Fig. 3A).

Figure 3.

Impact of treatment with rAAV-PD-L1-scFv on immune cell phenotype at the TME. A, Thirty-three days after the MC38 tumor challenge, mice were sacrificed and tumor tissues were harvested. Explanted tumors were divided into two parts. From one portion, single-cell suspensions were analyzed by flow cytometry for CD8+ (CD45+CD3e+CD8+) and CD4+ (CD45+CD3e+CD4+) T cells, Tregs (CD45+CD3e+CD4+CD25+FoxP3+), MDSCs (CD45+CD11b+Gr1+), and macrophages (CD45+CD11b+F4/80+). Relative percentages of immune cells are shown (*, P < 0.05; **, P < 0.01). Gating strategy used in the flow cytometry is shown in Supplementary Fig. S5. B, The other portion of the tumor tissues were formalin fixed and 5-μm sections were analyzed by IHC for CD8+ T cells, and M1 and M2 macrophages (Supplementary Fig. S8). CD8+ T cells were stained with anti-CD3e (red), CD8 (green), and DAPI (blue). Images were taken using EVOS M7000 Imaging System.

Figure 3.

Impact of treatment with rAAV-PD-L1-scFv on immune cell phenotype at the TME. A, Thirty-three days after the MC38 tumor challenge, mice were sacrificed and tumor tissues were harvested. Explanted tumors were divided into two parts. From one portion, single-cell suspensions were analyzed by flow cytometry for CD8+ (CD45+CD3e+CD8+) and CD4+ (CD45+CD3e+CD4+) T cells, Tregs (CD45+CD3e+CD4+CD25+FoxP3+), MDSCs (CD45+CD11b+Gr1+), and macrophages (CD45+CD11b+F4/80+). Relative percentages of immune cells are shown (*, P < 0.05; **, P < 0.01). Gating strategy used in the flow cytometry is shown in Supplementary Fig. S5. B, The other portion of the tumor tissues were formalin fixed and 5-μm sections were analyzed by IHC for CD8+ T cells, and M1 and M2 macrophages (Supplementary Fig. S8). CD8+ T cells were stained with anti-CD3e (red), CD8 (green), and DAPI (blue). Images were taken using EVOS M7000 Imaging System.

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The findings of flow cytometry analysis were further confirmed by IHC of tumor tissues using fluorescent antibody combinations for CD8+ T cells and M1 and M2 macrophages. The immune landscape within the tumor tissue demonstrated an increase in CD8+ cells that appeared to have infiltrated into the tumor tissue following treatment with rAAV-PD-L1-scFv (Fig. 3B). The increase in CD8+ T cells also corresponded to a decrease in M2 macrophages, but interestingly, there was no change in M1 macrophages in mice treated with rAAV-PD-L1-scFv, compared with untreated tumors (Supplementary Fig. S8). Similar results of a significant increase in CD4+ and CD8+ T cells and a concomitant decrease in Tregs, MDSCs, and macrophages were observed in the spleen of mice that were treated with rAAV-PD-L1-scFv, compared with untreated and rAAV-GFP–treated mice (Fig. 4A; Supplementary Fig. S9). PD-1 plays a pivotal role in regulating T-cell function, as its upregulation on T cells is associated with T-cell exhaustion upon binding to PD-L1 (29, 30). Because treatment with rAAV-PD-L1-scFv resulted in a significant increase in CD8+ T cells, we analyzed their functional status, based on PD-1 expression, by flow cytometry. Results of the analysis indicated a significant decrease in PD-1+ cells in both CD4+ and CD8+ T-cell populations (Fig. 4B). The changes affecting PD-1/PD-L1 axis were also noted in splenic MDSCs and macrophages, which showed a significant decrease in PD-L1 expression in the rAAV-PD-L1-scFv treatment group (Fig. 4C). Together with a decrease in PD-L1 expression on tumor cells in the rAAV-PD-L1-scFv–treated mice (Fig. 2B), the above results confirm a significant immune checkpoint inhibitory effect involving the PD-1/PD-L1 axis.

Figure 4.

The impact of rAAV-PD-L1-scFv treatment on immune cells in the spleen. A, Thirty-three days after MC38 tumor inoculation, mice were sacrificed and spleens were isolated. Single-cell suspensions of explanted spleens were analyzed by flow cytometry for CD8+ and CD4+ T cells, Tregs, MDSCs, and macrophages, using markers listed in Fig. 3 legend. Relative percentages of each immune cell type are shown. B, PD-1 expression on CD8+ and CD4+ T cells were analyzed by staining with an anti–PD-1 antibody. Relative percentages of PD-1+ cells within the CD8+ and CD4+ T-cell populations are shown. C, PD-L1 expression on MDSCs and macrophages were analyzed by staining with an anti–PD-L1 antibody. Gating strategy used in flow cytometry is shown in Supplementary Fig. S6. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4.

The impact of rAAV-PD-L1-scFv treatment on immune cells in the spleen. A, Thirty-three days after MC38 tumor inoculation, mice were sacrificed and spleens were isolated. Single-cell suspensions of explanted spleens were analyzed by flow cytometry for CD8+ and CD4+ T cells, Tregs, MDSCs, and macrophages, using markers listed in Fig. 3 legend. Relative percentages of each immune cell type are shown. B, PD-1 expression on CD8+ and CD4+ T cells were analyzed by staining with an anti–PD-1 antibody. Relative percentages of PD-1+ cells within the CD8+ and CD4+ T-cell populations are shown. C, PD-L1 expression on MDSCs and macrophages were analyzed by staining with an anti–PD-L1 antibody. Gating strategy used in flow cytometry is shown in Supplementary Fig. S6. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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Because a majority of tumor-suppressive cytokines and chemokines are soluble proteins, we reasoned that the observed antitumor effects following systemically stable PD-L1 scFv level should reflect in the homeostasis of tumor-produced and immune cell–produced soluble cytokines and chemokines. To corroborate this, cytokine and chemokine signals in rAAV-PD-L1-scFv–treated mice sera were analyzed using Proteome Profiler Mouse Cytokine Array Kit. Sera from rAAV-GFP–treated or untreated mice were used as controls. Serum samples from each group of mice were pooled and equal volumes of sera was used in the assay. Results showed that treatment with rAAV-PD-L1-scFv induced remarkable changes in cytokine and chemokine profiles. Following rAAV-PD-L1-scFv treatment, there was a notable upregulation of Th1 cytokines, including IL12, IL23, IL16, and IL1α. In addition, rAAV-PD-L1-scFv treatment resulted in an increase in IL1ra, and chemokines, macrophage inflammatory protein-1α [MIP-1α (CCL3)] and thymus and activation-regulated chemokine [TARC (CCL17)] (Fig. 5), indicating the potential of rAAV-PD-L1-scFv in dampening protumorigenic functions. The increase in antitumor cytokines and chemokines was also associated with a decrease in protumorigenic cytokines and chemokines, including IL13, GCSF, and MCSF (Fig. 5).

Figure 5.

Serum cytokine analysis following rAAV-PD-L1-scFv treatment. Thirty-three days after the MC38 tumor challenge, mice were sacrificed and sera were collected. To determine the effects of soluble PD-L1 scFv in altering the immune milieu, equal volumes of sera from rAAV-PD-L1-scFv– or rAAV-GFP–treated mice and untreated mice (N = 3) were analyzed using a Proteome Profiler Mouse Cytokine Assay Kit and relative changes in individual cytokine and/or chemokine were determined by quantitation of acquired signal intensity using an HLImage++ software. Representative images (top) and the fold change in notable cytokine and/or chemokine levels in rAAV-PD-L1-scFv–treated mice compared with tumor-challenged untreated mice and rAAV-GFP–treated mice (bottom) are shown.

Figure 5.

Serum cytokine analysis following rAAV-PD-L1-scFv treatment. Thirty-three days after the MC38 tumor challenge, mice were sacrificed and sera were collected. To determine the effects of soluble PD-L1 scFv in altering the immune milieu, equal volumes of sera from rAAV-PD-L1-scFv– or rAAV-GFP–treated mice and untreated mice (N = 3) were analyzed using a Proteome Profiler Mouse Cytokine Assay Kit and relative changes in individual cytokine and/or chemokine were determined by quantitation of acquired signal intensity using an HLImage++ software. Representative images (top) and the fold change in notable cytokine and/or chemokine levels in rAAV-PD-L1-scFv–treated mice compared with tumor-challenged untreated mice and rAAV-GFP–treated mice (bottom) are shown.

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Antitumor effects of CD8+ T cells following rAAV-PD-L1-scFv treatment in adoptive transfer and tumor rechallenge studies

To determine the effects of anti-PD-L1 therapy using rAAV-PD-L1-scFv on long-term T-cell function, we first performed an adoptive transfer study wherein splenetic CD8+ T cells from control mice (without tumor challenge), tumor-bearing mice that were untreated, and tumor-bearing mice that were treated with rAAV-PD-L1-scFv, were individually sorted by flow cytometry and adoptively transferred into C57BL/6 mice with preestablished MC38 tumors and the tumor kinetics was monitored by digital caliper measurements. Results of this study indicated a significant decrease in tumor growth following administration of CD8+ T cells from rAAV-PD-L1-scFv–treated mice (Fig. 6A). However, there was no protection against the growth of tumors following adoptive transfer of T cells from control, or tumor-bearing untreated mice. We further tested the antigen specificity of CD8+ T cells from rAAV-PD-L1-scFv–treated mice by T-cell killing assay. CD8+ T cells (effector) were cocultured with labeled MC38 tumor cells (target) at different target: effector ratios with or without addition of MC38 cell lysates. MC38 cell lysates were added to increase proliferation of antigen-specific T cells. Proliferation index of labeled tumor cells was determined by flow cytometry and used as a measure of CD8+ T-cell cytolytic function. Results of this study indicated that MC38 cell lysates enhanced CD8+ T-cell activity (Supplementary Fig. S10).

Figure 6.

Effects of rAAV-PD-L1-scFv treatment in long-term antitumor immunity and memory T-cell function. A, Splenic CD8+ T cells from control mice without tumors, or mice bearing MC38 tumors that were either untreated or treated with rAAV-PD-L1-scFv were purified by flow sorting. Approximately 1 × 106 CD8+ T cells were adoptively transferred to female C57BL/6 mice that were previously challenged with MC38 tumors (N = 5). B, To determine long-term antitumor effects against the regrowth of secondary tumors, groups of mice that received rAAV-PD-L1-scFv and remained tumor free were rechallenged 60 days later with 1 × 105 MC38 cells in the opposite flank. To compare growth kinetics of rechallenged tumors, one group of control mice was freshly challenged with the same number of MC38 cells on the day of tumor rechallenge in the rAAV-PD-L1-scFv group. Growth kinetics and tumor volumes at the endpoint are shown from each group. *, P < 0.05; **, P < 0.01.

Figure 6.

Effects of rAAV-PD-L1-scFv treatment in long-term antitumor immunity and memory T-cell function. A, Splenic CD8+ T cells from control mice without tumors, or mice bearing MC38 tumors that were either untreated or treated with rAAV-PD-L1-scFv were purified by flow sorting. Approximately 1 × 106 CD8+ T cells were adoptively transferred to female C57BL/6 mice that were previously challenged with MC38 tumors (N = 5). B, To determine long-term antitumor effects against the regrowth of secondary tumors, groups of mice that received rAAV-PD-L1-scFv and remained tumor free were rechallenged 60 days later with 1 × 105 MC38 cells in the opposite flank. To compare growth kinetics of rechallenged tumors, one group of control mice was freshly challenged with the same number of MC38 cells on the day of tumor rechallenge in the rAAV-PD-L1-scFv group. Growth kinetics and tumor volumes at the endpoint are shown from each group. *, P < 0.05; **, P < 0.01.

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Finally, to test the potential of antitumor effector and memory T cells against tumor relapse, cohorts of tumor-free mice following rAAV-PD-L1-scFv treatment were rechallenged 60 days after primary tumor challenge with MC38 cells on opposite flank from the site of primary tumor challenge. Results of this study demonstrated a significant protection against recurrence of secondary tumors in mice that were tumor-free following rAAV-PD-L1-scFv treatment (Fig. 6B). These data indicate that rAAV-PD-L1-scFv therapy induces tumor-specific T-cell effector and memory functions and may be effective against tumor relapse.

Expression of checkpoint molecules by tumors allows them to escape T cell–mediated killing (31, 32). As a consequence of immune escape mechanisms, tumors additionally recruit tumor-associated macrophages (TAM) and MDSCs, which also express PD-L1, and promote Treg development and enrichment at the TME (33, 34), further dampening the effector T-cell function. The presence of TAMs with high PD-L1 is associated with poor survival in several malignancies, including glioblastoma, non–small cell lung cancer, ovarian cancer, and gastrointestinal cancers (35–38). Among ICIs, those targeting the PD-1/PD-L1 axis have been more commonly used with varying degrees of positive outcome in several solid cancers and hematologic malignancies (1, 2). In recent immunotherapy studies, PD-L1 antibody treatments have been shown to decrease tumor burden, MDSCs, and TAMs (39–41). To date, many FDA-approved anti-PD-1 and anti-PD-L1 mAbs are being incorporated in clinical regimen (6). Despite the potential of ICIs, currently used as mAbs, concerns relating toxicity and adverse effects, including irAEs, are posing clinical challenges (7). To prevent or limit such adverse reactions from using high concentrations of ICIs, gene-based delivery systems offer a useful alternative. Furthermore, the use of scFvs of antibodies, over full-length antibody cDNA, may offer potential advantages for gene-based therapeutics as ICIs, including higher systemic stability and superior tissue-penetrating capabilities, especially in the context of solid tumor therapy (10).

Among viral delivery systems, rAAV-based vectors are gaining popularity for clinical use owing to their nonpathogenic nature, relatively less immunogenicity, and long-term transgene expression capabilities (16). These advantages have led to the approval and testing of AAV-based vectors in various clinical trials of genetic and nongenetic diseases, including cancer (15). The potential advantage of rAAV-mediated systemically stable expression of PD-L1 scFv as a secretory protein from skeletal muscle, as adopted in this work, over intratumoral vector application attempted so far using AAV, lentivirus, and retrovirus-based vectors for genetic antibody therapy (11–14) is overcoming limitations of tissue penetrance through elevated angiogenesis in the TME and more importantly, reaching multiple tumor foci in the metastatic setting. In addition, maintaining PD-L1 scFv in systemically stable and physiologically relevant concentrations could help prevent relapse. In addition to tumor cells, PD-L1 expression is also found on protumorigenic myeloid cells (35, 42), hence, stable systemic levels of PD-L1 scFv from intramuscular rAAV application would also sequester membrane-bound PD-L1 expressed on protumorigenic myeloid cells.

In the current study, we observed that following rAAV-PD-L1-scFv treatment, there was not only a significant decrease in tumor growth, but also a decrease in TAMs, MDSCs, and Tregs. A significant increase in CD8+ T cells following PD-L1-scFv therapy was also reflected in higher infiltration of the cells within the tumor tissue. Although many antitumor immunotherapy approaches, including those with viral vectors have resulted in transient antitumor responses as indicated by an increase in elevated CTL activity, the durability of the response has only been transient (43). While some of that can be attributed to insufficient antigen dosing, others include anergy and exhaustion of antigen-specific T cells (44). To test the durability of antitumor T cells following rAAV-PD-L1-scFv therapy, we conducted two experiments wherein in one, CD8+ T cells from mice that demonstrated significant antitumor activity were adoptively transferred into naïve mice bearing MC38 tumors, and in other experiment, mice that showed a significant antitumor activity following a single application of rAAV-PD-L1-scFv were rechallenged with MC38 cells. In both cases, there was a significant protection against tumor growth, suggesting that rAAV-PD-L1-scFv may benefit against tumor relapse and when appropriately combined with other frontline therapies, may delay or prevent metastasis.

Cytokines and chemokines play an important role in either suppressing or supporting tumor growth and metastasis. Antitumor Th1 cytokines, including IFNγ, TNFα, IL12, IL2, and IL23 are produced by both innate and adaptive immune cells in response to tumor growth (45). Because stable expression of PD-L1 scFv in our study not only resulted in a significant decrease in tumor growth, but also protected against the growth of tumor rechallenge, we reasoned that checkpoint inhibition of the PD-1/PD-L1 axis should extend to activation of Th1 responses. Data from soluble cytokine and chemokine analysis confirmed this, with an increase in IL12 and IL16, IL1ra in mice treated with rAAV-PD-L1-scFv. IL12 is known to be involved in the differentiation and proliferation of helper and cytotoxic T cells (46), and IL12 is known to play a role in the reversal of immunosuppressive signatures in the TME by Th1 mechanisms and causing Treg apoptosis (47). IL16 is known to recruit and activate cells expressing CD4, including naïve T cells, monocytes, and dendritic cell (DC; refs. 48, 49); and IL1α plays a role in Th-1 and Th-17–specific T-cell responses (50). IL1ra is known to modulate IL1-related immune and inflammatory responses (51). In addition to these antitumor cytokines, we also observed an increase in the level of chemokine, MIP-1α (CCL3) and CCL17, which functions as a potent activator of both innate and adaptive responses. Specifically, MIP-1α plays a critical role in recruiting distinct immune phenotypes to intratumoral sites and functions as an important chemokine in regulating lymph node homing of DC subsets, and induces antigen-specific T-cell responses (52). Along with an increase in antitumor cytokines and chemokine, rAAV-PD-L1-scFv treatment also resulted in a notable decrease in antitumor cytokines including IL13, IL23, TREM, GCSF, and MCSF. Although stable expression of rAAV transgene occurs for a longer duration, following a single application of the vector, preexisting immunity could dampen the levels of PD-L1 scFv over time. Unlike application of rAAV in the correction of monogenic diseases that requires expression of transgenes for the life of patients, efficacious tumor therapy requires a relatively shorter time frame. Nonetheless, a balance between PD-L1 scFv expression and receptor occupancy needs to be considered during clinical translation of this approach. An additional concern from stable expression of rAAV-PD-L1-scFv, beyond tumor eradication, could be imbalance in T-cell homeostasis, including autoimmunity. Although this point is beyond the scope of the current study, if such effects are observed in future studies, rAAV with regulatory switch to inactivate transgene expression should be considered (53). Together, our study demonstrates that a single application of rAAV-PD-L1-scFv can produce a systemically stable level of anti-PD-L1 scFv for durable antitumor effects.

A.G. Sorace reports grants from the NIH NCI during the conduct of the study, grants from the American Cancer Society, personal fees from InfoSoft, and nonfinancial support from Imaginab outside the submitted work. No disclosures were reported by the other authors.

H. Wang: Data curation, formal analysis, investigation, methodology, writing–review and editing. V. Khattar: Investigation, methodology. J.A. Hensel: Investigation. R. Ashton: Investigation. Y. Lu: Methodology. A.G. Sorace: Resources, methodology. Y. Wang: Investigation. J.S. Deshane: Resources. J.L. Mieher: Data curation. C. Deivanayagam: Resources, investigation. S. Ponnazhagan: Conceptualization, formal analysis, supervision, funding acquisition, validation, methodology, writing–review and editing.

This work was supported by NIH grants: R01CA184770 to S. Ponnazhagan and P42ES027723 to J.S. Deshane, and grants from the Breast Cancer Research Foundation of Alabama and the METAvivor Research and Support Inc. to S. Ponnazhagan. We thank Dr. France Lund, Department of Microbiology, The University of Alabama, Birmingham, for kindly sharing the OVA transgenic mice and the OT-1 peptide. We thank the Pathology Research Core and the Comprehensive Flow Cytometry Core at UAB for support with histology and flow cytometry analyses, respectively.

The publication costs of this article were defrayed in part by the payment of publication fees. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

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Supplementary data