Indication-based protocols with different solutions for mechanical stromal-cell transfer

Background Regenerative medicine is the fastest developing branch of plastic surgery in recent times. Adipose tissue is one of the largest and most important sources in the body for stromal cells. Although mechanical isolation methods are both very popular and have many advantages, they still have no accepted protocols. Objective We developed new protocols called indication-based protocols (IPs) for standardization and new techniques called mechanical stromal-cell transfer (MEST) by using ultra-sharp blades and dilution of adipose tissue with different solutions (saline, Ringer and 5% Dextrose) Methods & material: In order to obtain the desired physical structure (liquid, gel, solid) and the desired volume, four different types of IPs have been defined. Adipose tissue was prediluted with different solutions using 10 or 20 cc injectors in IPs 1 and 2, while condensed adipose tissue was used directly in IPs 3 and 4. Results In MEST, stromal cells were obtained from 100 mL of condensed fat using different IPs with 92% mean viability and cell counts of 26.80–91.90 × 106. Stromal cells can be obtained in the desired form and number of cells by using four different IPs. Conclusion Isolation of stromal cells by cutting fat with sharp blades will prevent the death of fat tissue and stromal cells and will allow high viability and cell count with our new technique. Predilution with different solutions: Diluting the condensed adipose tissue with the desired solutions (saline, Ringer or 5% Dextrose) before the adinizing process will provide even more stromal cells. Lay Summary Obtaining regenerative stromal cells from adipose tissue can be done by two methods: Enzymatic and mechanical. Mechanical methods have many advantages. Although mechanical stromal cell extraction from adipose tissue is very popular and many techniques have been described, there are still no accepted protocols, definition for the end product, and no consensus on the status of the stromal cells. In this study, stromal cells were obtained mechanically by using ultra-sharp blade systems, without exposing adipose tissue to blunt trauma. Thus, a higher number of cells and higher viability could be obtained. An “Indication based” protocol has been defined for the first time in order to obtain the desired number and status (solid, semi-solid, liquid) end product. Diluting the condensed adipose tissue with the desired solutions (saline, Ringer or 5% Dextrose) before the adinizing process will provide even more stromal cells. This will provide an opportunity for clinicians to obtain and apply a stromal cell solution for different indications in different anatomical regions.

Cell membrane-anchored and tumor-targeted IL-12 (attIL12)-T cell therapy for eliminating large and heterogeneous solid tumors

BackgroundAdoptive T-cell transfer has become an attractive therapeutic approach for hematological malignancies but shows poor activity against large and heterogeneous solid tumors. Interleukin-12 (IL-12) exhibits potent antitumor efficacy against solid tumors, but its clinical application has been stalled because of toxicity. Here, we aimed to develop a safe approach to IL-12 T-cell therapy for eliminating large solid tumors.MethodsWe generated a cell membrane-anchored IL-12 (aIL12), a tumor-targeted IL-12 (ttIL12), and a cell membrane-anchored and ttIL-12 (attIL12) and a cell membrane-anchored and tumor-targeted ttIL-12 (attIL12) armed T cells, chimeric antigen receptor-T cells, and T cell receptor-T (TCR-T) cells with each. We compared the safety and efficacy of these armed T cells in treating osteosarcoma patient-derived xenograft tumors and mouse melanoma tumors after intravenous infusions of the armed T cells.ResultsattIL12-T cell infusion showed remarkable antitumor efficacy in human and mouse large solid tumor models. Mechanistically, attIL12-T cells targeted tumor cells expressing cell-surface vimentin, enriching effector T cell and interferon γ production in tumors, which in turn stimulates dendritic cell maturation for activating secondary T-cell responses and tumor antigen spreading. Both attIL12- and aIL12-T-cell transfer eliminated peripheral cytokine release and the associated toxic effects.ConclusionsThis novel approach sheds light on the safe application of IL-12-based T-cell therapy for large and heterogeneous solid tumors.

Pro-Resolving Factors Released by Macrophages After Efferocytosis Promote Mucosal Wound Healing in Inflammatory Bowel Disease

Nonresolving inflammation is a critical driver of several chronic inflammatory diseases, including inflammatory bowel diseases (IBD). This unresolved inflammation may result from the persistence of an initiating stimulus or from the alteration of the resolution phase of inflammation. Elimination of apoptotic cells by macrophages (a process called efferocytosis) is a critical step in the resolution phase of inflammation. Efferocytosis participates in macrophage reprogramming and favors the release of numerous pro-resolving factors. These pro-resolving factors exert therapeutic effects in experimental autoimmune arthritis. Here, we propose to evaluate the efficacy of pro-resolving factors produced by macrophages after efferocytosis, a secretome called SuperMApo, in two IBD models, namely dextran sodium sulfate (DSS)-induced and T cell transfer-induced colitis. Reintroducing these pro-resolving factors was sufficient to decrease clinical, endoscopic and histological colitis scores in ongoing naive T cell-transfer-induced colitis and in DSS-induced colitis. Mouse primary fibroblasts isolated from the colon demonstrated enhanced healing properties in the presence of SuperMApo, as attested by their increased migratory, proliferative and contractive properties. This was confirmed by the use of human fibroblasts isolated from patients with IBD. Exposure of an intestinal epithelial cell (IEC) line to these pro-resolving factors increased their proliferative properties and IEC acquired the capacity to capture apoptotic cells. The improvement of wound healing properties induced by SuperMApo was confirmed in vivo in a biopsy forceps-wound colonic mucosa model. Further in vivo analysis in naive T cell transfer-induced colitis model demonstrated an improvement of intestinal barrier permeability after administration of SuperMApo, an intestinal cell proliferation and an increase of α-SMA expression by fibroblasts, as well as a reduction of the transcript coding for fibronectin (Fn1). Finally, we identified TGF-β, IGF-I and VEGF among SuperMApo as necessary to favor mucosal healing and confirmed their role both in vitro (using neutralizing antibodies) and in vivo by depleting these factors from efferocytic macrophage secretome using antibody-coated microbeads. These growth factors only explained some of the beneficial effects induced by factors released by efferocytic macrophages. Overall, the administration of pro-resolving factors released by efferocytic macrophages limits intestinal inflammation and enhance tissue repair, which represents an innovative treatment of IBD.

Enhancing Adoptive Cell Transfer with Combination BRAF-MEK and CDK4/6 Inhibitors in Melanoma

Despite the success of immune checkpoint inhibitors that target cytotoxic lymphocyte antigen-4 (CTLA-4) and programmed-cell-death-1 (PD-1) in the treatment of metastatic melanoma, there is still great need to develop robust options for patients who are refractory to first line immunotherapy. As such there has been a resurgence in interest of adoptive cell transfer (ACT) particularly derived from tumor infiltrating lymphocytes. Moreover, the addition of cyclin dependent kinase 4/6 inhibitors (CDK4/6i) have been shown to greatly extend duration of response in combination with BRAF-MEK inhibitors (BRAF-MEKi) in pre-clinical models of melanoma. We therefore investigated whether combinations of BRAF-MEK-CDK4/6i and ACT were efficacious in murine models of melanoma. Triplet targeted therapy of BRAF-MEK-CDK4/6i with OT-1 ACT led to sustained and robust anti-tumor responses in BRAFi sensitive YOVAL1.1. We also show that BRAF-MEKi but not CDK4/6i enhanced MHC Class I expression in melanoma cell lines in vitro. Paradoxically CDK4/6i in low concentrations of IFN-γ reduced expression of MHC Class I and PD-L1 in YOVAL1.1. Overall, this work provides additional pre-clinical evidence to pursue combination of BRAF-MEK-CDK4/6i and to combine this combination with ACT in the clinic.

Characterisation and optimisation of nitroreductase-prodrug combinations for bacterial-directed enzyme-prodrug therapy

<p>Gene-directed enzyme-prodrug therapy (GDEPT) employs tumour-tropic vectors including viruses (VDEPT) and bacteria (BDEPT) to deliver a genetically-encoded prodrug-converting enzyme to the tumour environment, thereby sensitising the tumour to a prodrug. Bacterial nitroreductases, which are able to activate a range of anti-cancer nitroaromatic prodrugs to genotoxic metabolites, are of particular interest for GDEPT. The bystander effect is crucial to the success of GDEPT. The bystander effect is a measure of how efficiently activated prodrug metabolites are transferred from gene-expressing cells to neighbouring tissues. This promotes more extensive tumour cell killing. The bystander effect has been quantified for multiple nitroaromatic prodrugs in mixed multilayer human cell cultures. Although this is a good model for VDEPT it cannot simulate the ability of these prodrug metabolites to exit the bacterial vectors relevant to BDEPT. Prior to this work there was an unmet need for an in vitro method of quantifying the bystander effect as it occurs in BDEPT, i.e. a bacterial model of cell-to-cell transfer of activated prodrug metabolites. This thesis presents a method for measuring the bacterial bystander effect in vitro in a microplate based assay that was validated by flow cytometry. In this assay two Escherichia coli strains are grown in co-culture; an activator strain expressing the nitroreductase E. coli nfsA and a recipient strain containing an SOS-GFP DNA damage responsive gene construct. In this system, induction of GFP by reduced prodrug metabolites can only occur following their transfer from the activators to the recipients. Using this method, the bacterial bystander effect of the clinically relevant prodrugs, metronidazole, CB1954, nitro-CBI-DEI, PR-104A and SN27686, was assessed. Consistent with the bystander efficiencies in human cell multilayers, reduced metronidazole exhibited little bacterial cell-to-cell transfer, whereas nitro-CBI-DEI was passed very efficiently from activator to recipient cells post-reduction. In contrast with observations in human cell multilayers, the PR-104A and SN27686 metabolites were not effectively passed between the two bacterial strains, whereas reduced CB1954 was transferred efficiently. Using nitroreductase enzymes that exhibit different biases for the 2- versus 4-nitro substituents of CB1954, I further showed that the 2-nitro reduction products exhibit substantially higher levels of bacterial cell-to-cell transfer than the 4-nitro reduction products. The outcomes of this investigation highlighted the importance of evaluating enzyme-prodrug combinations in models relevant to the intended GDEPT vector, as there can evidently be profound differences in efficacy in different settings. These findings motivated an investigation into the influence of the bystander effect on certain screening strategies used for directed evolution of nitroreductases. It was observed that the bacterial bystander effect can occur during fluorescence activated cell sorting (FACS) of a nitroreductase variant library and negatively impact the recovery of more active variants. Significantly fewer nfsA-expressing cells were recovered from FACS when using CB1954 and nitro-CBI-DEI, when the bystander effect was given time to occur, as compared to controls in which the bystander effect was given no time to occur. In contrast, at the preferred challenge concentrations the mustard prodrugs PR-104A and SN27686 did not yield significantly lower proportions of nfsA-expressing cells under bystander condition. A subsequent investigation compared the evolutionary outcomes arising from screening a nitroreductase variant library using FACS, in which the bystander effect can occur, in parallel to a manual pre-selection method of individual clones for detoxification of structurally divergent nitroaromatic antibiotics. Overall the results of this investigation were inconclusive after just a single round of selection, but there is some evidence that the FACS strategy was more effective than niclosamide/chloramphenicol pre-selection in enriching for superior CB1954-reducing variants. Finally, a panel of nitroreductase candidates was evaluated with the next generation prodrugs PR-104A and SN36506 for possible Clostridia-DEPT development. It was found that the Vibrio vulnificus NfsB F70A/F108Y variant displayed the highest catalytic efficiency with PR-104A reported thus far compared to any other nitroreductase, and was the only NfsB family nitroreductase to exhibit any activity with SN36506 at the purified protein level. At the time this research was performed only NfsB family nitroreductases had been successfully expressed in C. sporogenes by our collaborators, hence the V. vulnificus NfsB F70A/F108Y variant was selected as a promising lead enzyme for further development.</p>

Characterisation and optimisation of nitroreductase-prodrug combinations for bacterial-directed enzyme-prodrug therapy

<p>Gene-directed enzyme-prodrug therapy (GDEPT) employs tumour-tropic vectors including viruses (VDEPT) and bacteria (BDEPT) to deliver a genetically-encoded prodrug-converting enzyme to the tumour environment, thereby sensitising the tumour to a prodrug. Bacterial nitroreductases, which are able to activate a range of anti-cancer nitroaromatic prodrugs to genotoxic metabolites, are of particular interest for GDEPT. The bystander effect is crucial to the success of GDEPT. The bystander effect is a measure of how efficiently activated prodrug metabolites are transferred from gene-expressing cells to neighbouring tissues. This promotes more extensive tumour cell killing. The bystander effect has been quantified for multiple nitroaromatic prodrugs in mixed multilayer human cell cultures. Although this is a good model for VDEPT it cannot simulate the ability of these prodrug metabolites to exit the bacterial vectors relevant to BDEPT. Prior to this work there was an unmet need for an in vitro method of quantifying the bystander effect as it occurs in BDEPT, i.e. a bacterial model of cell-to-cell transfer of activated prodrug metabolites. This thesis presents a method for measuring the bacterial bystander effect in vitro in a microplate based assay that was validated by flow cytometry. In this assay two Escherichia coli strains are grown in co-culture; an activator strain expressing the nitroreductase E. coli nfsA and a recipient strain containing an SOS-GFP DNA damage responsive gene construct. In this system, induction of GFP by reduced prodrug metabolites can only occur following their transfer from the activators to the recipients. Using this method, the bacterial bystander effect of the clinically relevant prodrugs, metronidazole, CB1954, nitro-CBI-DEI, PR-104A and SN27686, was assessed. Consistent with the bystander efficiencies in human cell multilayers, reduced metronidazole exhibited little bacterial cell-to-cell transfer, whereas nitro-CBI-DEI was passed very efficiently from activator to recipient cells post-reduction. In contrast with observations in human cell multilayers, the PR-104A and SN27686 metabolites were not effectively passed between the two bacterial strains, whereas reduced CB1954 was transferred efficiently. Using nitroreductase enzymes that exhibit different biases for the 2- versus 4-nitro substituents of CB1954, I further showed that the 2-nitro reduction products exhibit substantially higher levels of bacterial cell-to-cell transfer than the 4-nitro reduction products. The outcomes of this investigation highlighted the importance of evaluating enzyme-prodrug combinations in models relevant to the intended GDEPT vector, as there can evidently be profound differences in efficacy in different settings. These findings motivated an investigation into the influence of the bystander effect on certain screening strategies used for directed evolution of nitroreductases. It was observed that the bacterial bystander effect can occur during fluorescence activated cell sorting (FACS) of a nitroreductase variant library and negatively impact the recovery of more active variants. Significantly fewer nfsA-expressing cells were recovered from FACS when using CB1954 and nitro-CBI-DEI, when the bystander effect was given time to occur, as compared to controls in which the bystander effect was given no time to occur. In contrast, at the preferred challenge concentrations the mustard prodrugs PR-104A and SN27686 did not yield significantly lower proportions of nfsA-expressing cells under bystander condition. A subsequent investigation compared the evolutionary outcomes arising from screening a nitroreductase variant library using FACS, in which the bystander effect can occur, in parallel to a manual pre-selection method of individual clones for detoxification of structurally divergent nitroaromatic antibiotics. Overall the results of this investigation were inconclusive after just a single round of selection, but there is some evidence that the FACS strategy was more effective than niclosamide/chloramphenicol pre-selection in enriching for superior CB1954-reducing variants. Finally, a panel of nitroreductase candidates was evaluated with the next generation prodrugs PR-104A and SN36506 for possible Clostridia-DEPT development. It was found that the Vibrio vulnificus NfsB F70A/F108Y variant displayed the highest catalytic efficiency with PR-104A reported thus far compared to any other nitroreductase, and was the only NfsB family nitroreductase to exhibit any activity with SN36506 at the purified protein level. At the time this research was performed only NfsB family nitroreductases had been successfully expressed in C. sporogenes by our collaborators, hence the V. vulnificus NfsB F70A/F108Y variant was selected as a promising lead enzyme for further development.</p>

Adoptive Cell Transfer of Allogeneic Epstein–Barr Virus-Specific T Lymphocytes for Treatment of Refractory EBV-Associated Posttransplant Smooth Muscle Tumors: A Case Report

Posttransplant smooth muscle tumors (PTSMTs) are rare Epstein–Barr virus (EBV)-associated neoplasms, mostly occurring after solid organ transplantation. Current therapeutic strategies include surgery and reduction of immunosuppressive medication. We describe for the first time a novel treatment approach for PTSMT by adoptive cell transfer (ACT) of EBV-specific T cells to a 20-year-old patient with a medical history of cardiac transplantation, posttransplant lymphoproliferative disease, and multilocular PTSMT. During ACT, mild cytokine release syndrome occurred, while no unexpected safety signals were recorded. We observed in vivo expansion of EBV-specific T cells and reduction of EBV viremia. Best response was stable disease after 4 months with reduction of EBV viremia and normalization of lactate dehydrogenase levels. ACT with EBV-specific T cells may be a safe and efficacious therapeutic option for PTSMT that warrants further exploration.

385 Identification and enrichment of neoantigen-reactive T cells to optimize adoptive cell transfer with tumor-infiltrating lymphocytes (TIL)

BackgroundAdoptive cell transfer (ACT) using tumor-infiltrating lymphocytes (TIL) has achieved an overall response rate of 39% in metastatic melanoma patients at Moffitt Cancer Center. In these trials, a substantial fraction of patients were non-responders by RECIST, but demonstrated a mixed response to therapy. These results suggest that the infused TIL product contained tumor-reactive T cells with therapeutic potential, which could be further optimized to improve ACT with TIL. We hypothesized that outcomes might be improved by identifying and enriching neoantigen-reactive TIL within bulk products. The purpose of this study is to define approaches to optimize ACT with TIL, by identifying, enriching, and analyzing neoantigen reactive TIL from the ACT infusion product of previously treated metastatic melanoma patients.MethodsPatient-derived cryopreserved tumor tissue, PBMC, and TIL from completed metastatic melanoma TIL trials were used for this study. Whole exome and RNA sequencing were performed on DNA and RNA extracted from tumor tissue and compared to DNA from autologous PBMC. Genetic sequencing and gene expression data were utilized to determine protein-modifying somatic mutations. Peptides were then predicted for their ability to be presented on MHC molecules, prioritized, and up to 192 custom 25-mers were synthesized per patient sample. Neoantigen peptides were loaded onto patient-derived dendritic cells (DC) and co-cultured with autologous TIL. These TIL were then sorted by FACS on their ability to upregulate 41BB and OX40 and expanded through the rapid expansion protocol (REP). Enriched TIL were subsequently screened for neoantigen reactivity by 41BB/OX40 upregulation, cytokine release, and degranulation.ResultsProtein-altering somatic mutations from metastatic melanoma tissues ranged from 49 to 1631 mutations (median = 389). On average, 16.2% of TIL were sorted for upregulation of 41BB/OX40 upon co-culture with DC pulsed with the neoantigen peptide pool (range: 2.7–31.1%). CD4+ TIL displayed a 3.75-fold upregulation of 41BB/OX40, while CD8+ TIL saw a 1.88-fold increase (n=6). This coincided with substantial production of IFNγ, TNFα, and granzyme B (n=6). Neoantigen-reactive (41BB+/OX40+) and non-reactive (41BB-/OX40-) TIL expanded to similar degrees in REP (average of 639-fold vs. 611-fold; n=6). Restimulation of enriched neoantigen-specific TIL resulted in superior pro-inflammatory functionality (granzyme B, IFNγ, and TNFα) when compared to non-reactive TIL.ConclusionsTIL from metastatic melanoma patient samples were successfully enriched for neoantigen-reactive TIL, which maintained increased reactivity against these predicted peptides upon restimulation when compared non-reactive TIL. These data support further investigation into the use of neoantigen-enriched TIL products to enhance efficacy of ACT.Trial RegistrationNCT01005745, NCT01659151, NCT01701674Ethics ApprovalNCT01005745 was approved by USF IRB approval number Ame5_107905.NCT01659151 was approved by Advarra IRB approval number 14.03.0083.NCT01701674 was approved by USF IRB approval number Ame13_Pro00009061.All participants gave informed consent before taking part.