Payload-delivering engineered γδ T cells display enhanced cytotoxicity, persistence, and efficacy in preclinical models of osteosarcoma.

T cell-based cancer immunotherapy has typically relied on membrane-bound cytotoxicity enhancers such as chimeric antigen receptors expressed in autologous αß T cells. These approaches are limited by tonic signaling of synthetic constructs and costs associated with manufacturing. γδ T cells are an emerging alternative for cellular therapy, having innate antitumor activity, potent antibody-dependent cellular cytotoxicity, and minimal alloreactivity. We present an immunotherapeutic platform technology built around the innate properties of the Vγ9Vδ2 T cell, harnessing specific characteristics of this cell type and offering an allocompatible cellular therapy that recruits bystander immunity. We engineered γδ T cells to secrete synthetic tumor-targeting opsonins in the form of an scFv-Fc fusion protein and a mitogenic IL-15Rα-IL-15 fusion protein (stIL15). Using GD2 as a model antigen, we show that GD2-specific opsonin-secreting Vγ9Vδ2 T cells (stIL15-OPS-γδ T cells) have enhanced cytotoxicity and promote bystander activity of other lymphoid and myeloid cells. Secretion of stIL-15 abrogated the need for exogenous cytokine supplementation and further mediated activation of bystander natural killer cells. Compared with unmodified γδ T cells, stIL15-OPS-γδ T cells exhibited superior in vivo control of subcutaneous tumors and persistence in the blood. Moreover, stIL15-OPS-γδ T cells were efficacious against patient-derived osteosarcomas in animal models and in vitro, where efficacy could be boosted with the addition of zoledronic acid. Together, the data identify stIL15-OPS-γδ T cells as a candidate allogeneic cell therapy platform combining direct cytolysis with bystander activation to promote tumor control.

Protocol for preclinical evaluation of locoregionally delivered CAR T cells in patient-derived xenograft models of brain tumors.

Here, we present a protocol for preclinical evaluation of locoregionally delivered CAR T cells in patient-derived xenograft models of primary, metastatic, and recurrent brain tumors. We provide instructions for isolating peripheral blood mononuclear cells (PBMCs), producing CAR T cells in conjunction with locoregional delivery, and preclinical trial design and analysis involving CAR T cells. Additionally, we describe comprehensive preclinical readouts and guidelines for critical endpoint sample collections. In line with clinical trial procedures, our protocol broadens available treatment modalities for direct clinical translation. For complete details on the use and execution of this protocol, please refer to Donovan et al.1.

A novel class of self-complementary AAV vectors with multiple advantages based on cceAAV lacking mutant ITR.

Self-complementary AAV vectors (scAAV) use a mutant inverted terminal repeat (mITR) for efficient packaging of complementary stranded DNA, enabling rapid transgene expression. However, inefficient resolution at the mITR leads to the packaging of monomeric or subgenomic AAV genomes. These noncanonical particles reduce transgene expression and may affect the safety of gene transfer. To address these issues, we have developed a novel class of scAAV vectors called covalently closed-end double-stranded AAV (cceAAV) that eliminate the mITR resolution step during production. Instead of using a mutant ITR, we used a 56-bp recognition sequence of protelomerase (TelN) to covalently join the top and bottom strands, allowing the vector to be generated with just a single ITR. To produce cceAAV vectors, the vector plasmid is initially digested with TelN, purified, and then subjected to a standard triple-plasmid transfection protocol followed by traditional AAV vector purification procedures. Such cceAAV vectors demonstrate yields comparable to scAAV vectors. Notably, we observed enhanced transgene expression as compared to traditional scAAV vectors. The treatment of mice with hemophilia B with cceAAV-FIX resulted in significantly enhanced long-term FIX expression. The cceAAV vectors hold several advantages over scAAV vectors, potentially leading to the development of improved human gene therapy drugs.

Recombinant AAV genome size effect on viral vector production, purification, and thermostability.

Adeno-associated virus (AAV) has shown great promise as a viral vector for gene therapy in clinical applications. The present work studied the effect of genome size on AAV production, purification, and thermostability by producing AAV2-GFP using suspension-adapted HEK293 cells via triple transfection using AAV plasmids containing the same GFP transgene with DNA stuffers for variable-size AAV genomes consisting of 1.9, 3.4, and 4.9 kb (ITR to ITR). Production was performed at the small and large shake flask scales and the results showed that the 4.9 kb GFP genome had significantly reduced encapsidation compared to other genomes. The large shake flask productions were purified by AEX chromatography, and the results suggest that the triple transfection condition significantly affects the AEX retention time and resolution between the full and empty capsid peaks. Charge detection-mass spectrometry was performed on all AEX full-capsid peak samples showing a wide distribution of empty, partial, full length, and copackaged DNA in the capsids. The AEX-purified samples were then analyzed by differential scanning fluorimetry, and the results suggest that sample formulation may improve the thermostability of AAV genome ejection melting temperature regardless of the packaged genome content.

Harnessing Nanomedicine to Potentiate the Chemo-Immunotherapeutic Effects of Doxorubicin and Alendronate Co-Encapsulated in Pegylated Liposomes.

Encapsulation of Doxorubicin (Dox), a potent cytotoxic agent and immunogenic cell death inducer, in pegylated (Stealth) liposomes, is well known to have major pharmacologic advantages over treatment with free Dox. Reformulation of alendronate (Ald), a potent amino-bisphosphonate, by encapsulation in pegylated liposomes, results in significant immune modulatory effects through interaction with tumor-associated macrophages and activation of a subset of gamma-delta T lymphocytes. We present here recent findings of our research work with a formulation of Dox and Ald co-encapsulated in pegylated liposomes (PLAD) and discuss its pharmacological properties vis-à-vis free Dox and the current clinical formulation of pegylated liposomal Dox. PLAD is a robust formulation with high and reproducible remote loading of Dox and high stability in plasma. Results of biodistribution studies, imaging with radionuclide-labeled liposomes, and therapeutic studies as a single agent and in combination with immune checkpoint inhibitors or gamma-delta T lymphocytes suggest that PLAD is a unique product with distinct tumor microenvironmental interactions and distinct pharmacologic properties when compared with free Dox and the clinical formulation of pegylated liposomal Dox. These results underscore the potential added value of PLAD for chemo-immunotherapy of cancer and the relevance of the co-encapsulation approach in nanomedicine.

Partial genome content within rAAVs impacts performance in a cell assay-dependent manner.

Recombinant adeno-associated viruses (rAAVs) deliver DNA to numerous cell types. However, packaging of partial genomes into the rAAV capsid is of concern. Although empty rAAV capsids are studied, there is little information regarding the impact of partial DNA content on rAAV performance in controlled studies. To address this, we tested vectors containing varying levels of partial, self-complementary EGFP genomes. Density gradient cesium chloride ultracentrifugation was used to isolate three distinct rAAV populations: (1) a lighter fraction, (2) a moderate fraction, and (3) a heavy fraction. Alkaline gels, Illumina Mi-Seq, size exclusion chromatography with multi-angle light scattering (SEC-MALS), and charge detection mass spectrometry (CD-MS) were used to characterize the genome of each population and ddPCR to quantify residual DNA molecules. Live-cell imaging and EGFP ELISA assays demonstrated reduced expression following transduction with the light fraction compared with the moderate and heavy fractions. However, PCR-based assays showed that the light density delivered EGFP DNA to cells as efficiently as the moderate and heavy fractions. Mi-Seq data revealed an underrepresentation of the promoter region for EGFP, suggesting that expression of EGFP was reduced because of lack of regulatory control. This work demonstrates that rAAVs containing partial genomes contribute to the DNA signal but have reduced vector performance.

Antibody Binding to Recombinant Adeno Associated Virus Monitored by Charge Detection Mass Spectrometry.

Recombinant adeno-associated virus (rAAV) is a leading gene therapy vector. However, neutralizing antibodies reduce its efficacy. Traditional methods used to investigate antibody binding provide limited information. Here, charge detection mass spectrometry (CD-MS) was used to investigate the binding of monoclonal antibody ADK8 to AAV serotype 8 (AAV8). CD-MS provides a label-free approach to antibody binding. Individual binding events can be monitored as each event is indicated by a shift of the antibody-antigen complex to a higher mass. Unlike other methods, the CD-MS approach reveals the distribution of antibodies bound on capsids, allowing AAV8 subpopulations with different affinities to be identified. The charge state generated by the electrospray of large ions is normally correlated with the structure, and the charge is expected to increase when an antibody binds to the capsid exterior. Surprisingly, binding of the first ADK8 to AAV8 causes a substantial decrease in the charge, suggesting that the first antibody binding event causes a significant structural change. The charge increases for subsequent binding events. Finally, high ADK8 concentrations cause agglutination, where ADK8 links AAV capsids to form dimers and higher order multimers.

Analysis of AAV-Extracted DNA by Charge Detection Mass Spectrometry Reveals Genome Truncations.

Adeno-associated virus (AAV) is a widely used gene therapy vector. The intact packaged genome is a critical quality attribute and necessary for an effective therapeutic. In this work, charge detection mass spectrometry (CDMS) was used to measure the molecular weight (MW) distribution for the genome of interest (GOI) extracted from recombinant AAV (rAAV) vectors. The measured MWs were compared to sequence masses for a range of rAAV vectors with different GOIs, serotypes, and production methods (Sf9 and HEK293 cell lines). In most cases, the measured MWs were slightly larger than the sequence masses, a result attributed to counterions. However, in a few cases, the measured MWs were significantly smaller than the sequence masses. In these cases, genome truncation is the only reasonable explanation for the discrepancy. These results suggest that direct analysis of the extracted GOI by CDMS provides a rapid and powerful tool to evaluate genome integrity in gene therapy products.

Quantitative analysis of preferential utilization of AAV ITR as the packaging terminal signal.

Genetic engineering advances have led to recombinant adeno-associated virus (rAAV) becoming an invaluable tool for the development of effective gene therapies. The production of rAAV is susceptible to off-target heterogeneous packaging, the effects of which are still being understood. Here, rAAV vectors with four-genome lengths were produced using both adherent and suspension HEK293 cells to understand the 5'ITR termination. AAV8 vectors were produced from the human FVIII plasmid for a full-length cargo of 4,707 nucleotides with specific truncations, creating smaller genomes. Conventionally, rAAV is characterized by differentiating empty capsids from full capsids, but for this work, that description is incomplete. The small genomes in this study were characterized by charge detection-mass spectrometry (CD-MS). Using CD-MS, packaged genomes in the range conventionally attributed to partials were resolved and quantified. In addition, alkaline gels and qPCR were used to assess the identity of the packaged genomes. Together, these results showed a propensity for unit-length genomes to be encapsidated. Packaged genomes occurred as replication intermediates emanating from the 5'ITR, indicating that HEK293 cells prefer unit-length genomes as opposed to the 5'ITR termination and heterogeneous DNA packaging observed previously from Sf9 cell systems. As both manufacturing processes are used and continually assessed to produce clinical material, such an understanding will benefit rAAV design for basic research and gene therapy.

Analysis of thermally driven structural changes, genome release, disassembly, and aggregation of recombinant AAV by CDMS.

Charge detection mass spectrometry (CDMS) was used to analyze recombinant adeno-associated virus serotype 8 (rAAV8) vectors after incubation at elevated temperatures. rAAV8 vectors with a range of genomes of interest (GOIs) from 2.22 to 4.84 kb were investigated. For the shorter GOIs, GOI release occurred at surprisingly low temperatures (15 min at 45°C for cytomegalovirus [CMV]-GFP). The released DNA and intermediates with the GOI extruded from the capsid were detected. The temperature required to release the short GOIs is well below the 65°C incubation temperature required to disassemble the empty rAAV8 capsid. The temperature for GOI release increased with its GOI length. With the longer GOIs, the GOI stabilized the capsid so that it remained intact under conditions that would disassemble the empty particle. After incubation at 65°C, the main species in the CDMS mass distributions for the longer GOIs was the vector with the GOI. However, for GOIs longer than the wild-type genome (∼4.7 kb), the stability diminished, and genome release occurred at a lower temperature. Heterogeneous DNA fragments from the host cells or plasmids is released at a lower temperature than the longer GOIs, suggesting that the GOIs have a feature that resists early release.

Generation of human parallel chimeric antigen receptor (pCAR) T cells to achieve synergistic T cell co-stimulation.

Dual co-stimulation may be harnessed using parallel chimeric antigen receptors (pCARs) in which two distinct co-stimulatory units are adjacently localized on the plasma membrane. This protocol summarizes construct design, human T cell isolation, retroviral transduction, tissue culture expansion, and preclinical testing of pCAR T cells, exemplified by receptors that co-target avb6 integrin and ErbB dimers. For complete details on the use and execution of this protocol, please refer to Muliaditan et al. (2021).

Analysis of Recombinant Adenovirus Vectors by Ion Trap Charge Detection Mass Spectrometry: Accurate Molecular Weight Measurements beyond 150 MDa.

Adenovirus is one of the largest nonenveloped, double-stranded DNA viruses. It is widely used as a gene therapy vector and has recently received a lot of attention as a novel vaccine platform for SARS-CoV-2. Human adenovirus 5 (HAdV5) contains over 2500 protein molecules and has a 36 kbp genome. Adenovirus is well beyond the range of conventional mass spectrometry, and it was unclear how well such a large complex could be desolvated. Here, we report molecular weight (MW) distributions measured for HAdV5 and for 11 recombinant AdV vectors with genomes of varying lengths. The MW distributions were recorded using ion trap charge detection mass spectrometry (CDMS), a single-particle technique where m/z and charge are measured for individual ions. The results show that ions as large as 150 MDa can be effectively desolvated and accurate MW distributions obtained. The MW distribution for HAdV5 contains a narrow peak at 156.1 MDa, assigned to the infectious virus. A smaller peak at 129.6 MDa is attributed to incomplete particles that have not packaged a genome. The ions in the 129.6 MDa peak have a much lower average charge than those in the peak at 156.1 MDa. This is attributed to the empty particles missing some or all of the fibers that decorate the surface of the virion. The MW measured for the mature virus (156.1 MDa) is much larger than that predicted from sequence masses and copy numbers of the constituents (142.5 MDa). Measurements performed for recombinant AdV as a function of genome length show that for every 1 MDa increase in the genome MW, the MW of the mature virus increases by around 2.3 MDa. The additional 1.3 MDa is attributed to core proteins that are copackaged with the DNA. This observation suggests that the discrepancy between the measured and expected MWs for mature HAdV5 is due to an underestimate in the copy numbers of the core proteins.

Characterization of Recombinant Chimpanzee Adenovirus C68 Low and High-Density Particles: Impact on Determination of Viral Particle Titer.

We observed differential infectivity and product yield between two recombinant chimpanzee adenovirus C68 constructs whose primary difference was genome length. To determine a possible reason for this outcome, we characterized the proportion and composition of the empty and packaged capsids. Both analytical ultracentrifugation (AUC) and differential centrifugation sedimentation (DCS, a rapid and quantitative method for measuring adenoviral packaging variants) were employed for an initial assessment of genome packaging and showed multiple species whose abundance deviated between the virus builds but not manufacturing campaigns. Identity of the packaging variants was confirmed by charge detection mass spectrometry (CDMS), the first known application of this technique to analyze adenovirus. The empty and packaged capsid populations were separated via preparative ultracentrifugation and then combined into a series of mixtures. These mixtures showed the oft-utilized denaturing A260 adenoviral particle titer method will underestimate the actual particle titer by as much as three-fold depending on the empty/full ratio. In contrast, liquid chromatography with fluorescence detection proves to be a superior viral particle titer methodology.

Quantitative analysis of genome packaging in recombinant AAV vectors by charge detection mass spectrometry.

Recombinant adeno-associated virus (rAAV) has emerged as an important gene therapy vector with many clinical trials currently in progress. Analytical characterization and quantitation of particle content remain challenges in both the development and production of rAAV vectors. In this study, charge detection mass spectrometry (CDMS) and gel electrophoresis are used to characterize the DNA content of recombinant AAV8 (rAAV8) vectors with a wide range of target genome sizes. We show that the differences between the masses of empty particles and particles with the genome of interest (GOI) are correlated with the expected genome mass. A small systematic deviation (around 2%) is attributed to the packaging of counterions along with the DNA. In addition to the GOI, a broad distribution of heterogeneous DNA is packaged. The distribution peaks are close to the packaging capacity of the rAAV8 vectors. There is also evidence for the co-packaging of small DNA fragments along with the GOI. Finally, we present evidence that incubation at an elevated temperature can reduce the heterogeneity of the packaged DNA. Taken together, these results show that CDMS is a viable tool for characterization of the packaged genome.

Synergistic T cell signaling by 41BB and CD28 is optimally achieved by membrane proximal positioning within parallel chimeric antigen receptors.

Second generation (2G) chimeric antigen receptors (CARs) contain a CD28 or 41BB co-stimulatory endodomain and elicit remarkable efficacy in hematological malignancies. Third generation (3G) CARs extend this linear blueprint by fusing both co-stimulatory units in series. However, clinical impact has been muted despite compelling evidence that co-signaling by CD28 and 41BB can powerfully amplify natural immune responses. We postulate that effective dual co-stimulation requires juxta-membrane positioning of endodomain components within separate synthetic receptors. Consequently, we designed parallel (p)CARs in which a 2G (CD28+CD3ζ) CAR is co-expressed with a 41BB-containing chimeric co-stimulatory receptor. We demonstrate that the pCAR platform optimally harnesses synergistic and tumor-dependent co-stimulation to resist T cell exhaustion and senescence, sustaining proliferation, cytokine release, cytokine signaling, and metabolic fitness upon repeated stimulation. When engineered using targeting moieties of diverse composition, affinity, and specificity, pCAR T cells consistently elicit superior anti-tumor activity compared with T cells that express traditional linear CARs.

TGF-ß1 potentiates Vγ9Vδ2 T cell adoptive immunotherapy of cancer.

Despite its role in cancer surveillance, adoptive immunotherapy using γδ T cells has achieved limited efficacy. To enhance trafficking to bone marrow, circulating Vγ9Vδ2 T cells are expanded in serum-free medium containing TGF-ß1 and IL-2 (γδ[T2] cells) or medium containing IL-2 alone (γδ[2] cells, as the control). Unexpectedly, the yield and viability of γδ[T2] cells are also increased by TGF-ß1, when compared to γδ[2] controls. γδ[T2] cells are less differentiated and yet display increased cytolytic activity, cytokine release, and antitumor activity in several leukemic and solid tumor models. Efficacy is further enhanced by cancer cell sensitization using aminobisphosphonates or Ara-C. A number of contributory effects of TGF-ß are described, including prostaglandin E2 receptor downmodulation, TGF-ß insensitivity, and upregulated integrin activity. Biological relevance is supported by the identification of a favorable γδ[T2] signature in acute myeloid leukemia (AML). Given their enhanced therapeutic activity and compatibility with allogeneic use, γδ[T2] cells warrant evaluation in cancer immunotherapy.

CAR T-Cells Targeting the Integrin αvß6 and Co-Expressing the Chemokine Receptor CXCR2 Demonstrate Enhanced Homing and Efficacy against Several Solid Malignancies.

Despite the unprecedented clinical success of chimeric antigen receptors (CAR) T-cells against haematological malignancy, solid tumors impose a far greater challenge to success. Largely, this stems from an inadequate capacity of CAR T-cells that can traffic and maintain function within a hostile microenvironment. To enhance tumor-directed T-cell trafficking, we have engineered CAR T-cells to acquire heightened responsiveness to interleukin (IL)-8. Circulating IL-8 levels correlate with disease burden and prognosis in multiple solid tumors in which it exerts diverse pathological functions including angiogenesis, support of cancer stem cell survival, and recruitment of immunosuppressive myeloid cells. To harness tumor-derived IL-8 for therapeutic benefit, we have co-expressed either of its cognate receptors (CXCR1 or CXCR2) in CAR T-cells that target the tumor-associated αvß6 integrin. We demonstrate here that CXCR2-expressing CAR T-cells migrate more efficiently towards IL-8 and towards tumor conditioned media that contains this cytokine. As a result, these CAR T-cells elicit superior anti-tumor activity against established αvß6-expressing ovarian or pancreatic tumor xenografts, with a more favorable toxicity profile. These data support the further engineering of CAR T-cells to acquire responsiveness to cancer-derived chemokines in order to improve their therapeutic activity against solid tumors.

In Vivo PET Tracking of 89Zr-Labeled Vγ9Vδ2 T Cells to Mouse Xenograft Breast Tumors Activated with Liposomal Alendronate.

Gammadelta T (γδ-T) cells are strong candidates for adoptive immunotherapy in oncology due to their cytotoxicity, ease of expansion, and favorable safety profile. The development of γδ-T cell therapies would benefit from non-invasive cell-tracking methods and increased targeting to tumor sites. Here we report the use of [89Zr]Zr(oxinate)4 to track Vγ9Vδ2 T cells in vivo by positron emission tomography (PET). In vitro, we showed that 89Zr-labeled Vγ9Vδ2 T cells retained their viability, proliferative capacity, and anti-cancer cytotoxicity with minimal DNA damage for amounts of 89Zr ≤20 mBq/cell. Using a mouse xenograft model of human breast cancer, 89Zr-labeled γδ-T cells were tracked by PET imaging over 1 week. To increase tumor antigen expression, the mice were pre-treated with PEGylated liposomal alendronate. Liposomal alendronate, but not placebo liposomes or non-liposomal alendronate, significantly increased the 89Zr signal in the tumors, suggesting increased homing of γδ-T cells to the tumors. γδ-T cell trafficking to tumors occurred within 48 hr of administration. The presence of γδ-T cells in tumors, liver, and spleen was confirmed by histology. Our results demonstrate the suitability of [89Zr]Zr(oxinate)4 as a cell-labeling agent for therapeutic T cells and the potential benefits of liposomal bisphosphonate treatment before γδ-T cell administration.

An APRIL-based chimeric antigen receptor for dual targeting of BCMA and TACI in multiple myeloma.

B-cell maturation antigen (BCMA) is a promising therapeutic target for multiple myeloma (MM), but expression is variable, and early reports of BCMA targeting chimeric antigen receptors (CARs) suggest antigen downregulation at relapse. Dual-antigen targeting increases targetable tumor antigens and reduces the risk of antigen-negative disease escape. "A proliferation-inducing ligand" (APRIL) is a natural high-affinity ligand for BCMA and transmembrane activator and calcium-modulator and cyclophilin ligand (TACI). We quantified surface tumor expression of BCMA and TACI on primary MM cells (n = 50). All cases tested expressed BCMA, and 39 (78%) of them also expressed TACI. We engineered a third-generation APRIL-based CAR (ACAR), which killed targets expressing either BCMA or TACI (P < .01 and P < .05, respectively, cf. control, effector-to-target [E:T] ratio 16:1). We confirmed cytolysis at antigen levels similar to those on primary MM, at low E:T ratios (56.2% ± 3.9% killing of MM.1s at 48 h, E:T ratio 1:32; P < .01) and of primary MM cells (72.9% ± 12.2% killing at 3 days, E:T ratio 1:1; P < .05, n = 5). Demonstrating tumor control in the absence of BCMA, we maintained cytolysis of primary tumor expressing both BCMA and TACI in the presence of a BCMA-targeting antibody. Furthermore, using an intramedullary myeloma model, ACAR T cells caused regression of an established tumor within 2 days. Finally, in an in vivo model of tumor escape, there was complete ACAR-mediated tumor clearance of BCMA+TACI- and BCMA-TACI+ cells, and a single-chain variable fragment CAR targeting BCMA alone resulted in outgrowth of a BCMA-negative tumor. These results support the clinical potential of this approach.