Weeks Of Expansion Research Articles

Abstract 1338: Novel activation domains coupled to chimeric ILT receptors (CIR) enhance NK cell targeting of HLA-G+leukemic and solid tumor cells

Abstract Human leukocyte antigen-G (HLA-G) suppresses lymphoid and myeloid immune systems by binding ILT2 and ILT4 respectively and activating ITIM signaling. HLA-G is abundantly expressed in placental cytotrophoblasts to block fetal rejection. HLA-G is not expressed in most normal cells but is derepressed in in about 50% of malignancies. We engineered ILT2 and ILT4 to produce activating signals as Chimeric ILT Receptors or CIR. Preliminary results indicated robust killing of Acute Myeloid Leukemia (AML) cells by Natural Killer (NK) cells expressing either ILT2 or ILT4 CIRs. Here, we evaluated the effect of alteration of activating domains in the CIR constructs to produce signals that mimic the effect of IL-18 signaling in CIR-NK cells to enhance cytotoxicity, NK cell growth and persistence. Further, HLA-G expression and CIR-NK cell targeting of primary human cell types were rigorously screened. Activated NK cells derived from peripheral blood (2 to 5 donors) were transduced with γ-retroviruses directing expression of CIR proteins (CIR.X.Y or CIR.Y.X) where X stimulates cytotoxicity through CD3ζ, DAP10 or DAP12, and Y directs coactivation through 4-1BB, fusion of the MyD88 death domain or recruitment of endogenous MyD88 with receptor-derived TIR domains and soluble IL-15. CIR-NK cells were cocultured with GFPffluc-expressing AML and solid tumor target cell lines or normal primary human cells. NK cell proliferation and cytotoxicity of CIR-NK cells was monitored by Incucyte microscopy and supernatants were analyzed for cytokine release by ELISA. At low E:T (1:20 - 1:40) in 7-day cocultures, mock-transduced NK cells displayed innate killing activity against HLA-G+ Molm13 and Kasumi1 AML cells that was augmented by CIR constructs containing direct fusions of MyD88 and DAP10 or DAP12 (mock = 2.06E7 ± 4.53E6 vs. CIR-ILT4.MyD88.DAP10 = 1.03E7 ± 5.46E6) with 7-fold elevation of IFN-γ production. CIR constructs that recruited endogenous MyD88 signaling through the TLR2 TIR domain (4-1BB.DAP10.TLR2) also exhibited enhanced anti-HLA-G activity (9.13E6 ± 5.33E6). Against HLA-G+ HT-1376 bladder cancer cells (E:T = 1:10-1:20), CIR-NK cells also displayed enhanced tumor cell killing with enhanced NK cell growth. Conversely, CIR-enhanced killing was not observed against HLA-G− HCT-116 colorectal cancer cells. The persistence of CIR-NK cell potency after 4 weeks of expansion varied markedly with different activation domains employed. When screened against primary human cells (cardiomyocytes, hepatocytes, hepatic endothelial cells, corneal, colon and lung epithelial cells), no CIR-specific targeting was observed at high E:T ratios (1:1 and 1:5). In conclusion, CIR-NK cells exhibit potent and specific anti-tumor activity against HLA-G+ solid and leukemia cells. Alteration of CIR-NK cell intracellular signaling can optimize the potency and persistence of NK cell anti-tumor activity. Citation Format: MyLinh Duong, Jihyun Park, Raphel Ognar, Simon Wain-Hobson, Henri Bayle. Novel activation domains coupled to chimeric ILT receptors (CIR) enhance NK cell targeting of HLA-G+leukemic and solid tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1338.

CD38 KO/CD38-CAR Human Primary Natural Killer Cells Enhance Cytotoxicity Against CD38-Expressing Primary Lymphoma, Leukemia, and Myeloma

Natural killer (NK) cells have strong cancer immunotherapeutic potential that can be further augmented by engineering with a chimeric antigen receptor (CAR). CD38, a transmembrane glycoprotein involved in signal transduction and cellular adhesion, is highly expressed on the surface of the hematologic malignancies multiple myeloma (MM), acute myeloid leukemia (AML), Burkitt Lymphoma (BL) and T-cell acute lymphoblastic leukemia (T-ALL). FDA approved CD38 monoclonal antibodies (mAb) daratumumab and isatuximab are used to treat MM in patients, and have been tested to treat other CD38 expressing malignancies. NK cells play an imperative role in the efficacy of these antibodies and have natural antitumor activity against these cancers. However, because NK cells also highly express CD38, anti-CD38 mAb can cause NK cell fratricide, limiting their efficacy. Here, we generated multiple CD38 scFv based on isatuximab sequences and generated fratricide-resistant primary CD38-CAR NK cells using CRISPR/Cas9 genome editing and AAV6 gene delivery. Using this approach, we inserted the CD38-CAR gene into the CD38 locus to efficiently generate simultaneous CD38 KO and CD38-CAR KI with a mean expression level of %60. The CAR-expressing NK cells could be expanded on mbIL21-expressing feeder cells to a clinically relevant number of cells with no decline in CAR-expression level during two weeks of expansion confirming the lack of CAR mediated fratricide. We evaluated transduction efficiency and expression of CD38-CAR, and compared the cytotoxic function of two constructs with CD8 transmembrane (TM)/stalk and 41BB signaling domains and reverse heavy and light chains against AML, MM, T-ALL and BL immortalized cell lines and primary patient samples. We enhanced CD38 expression in target cancer cells by pre-treatment with all-trans retinoic acid (ATRA), a metabolite of vitamin A that pushes cells to maturation, consequently upregulating CD38. The sequential treatment of target cells with 10nM ATRA for 48 hours enhanced the cytotoxic activity of the CD38 KO/CD38-CAR-NK cells against primary MM, BL and AML samples. This would be particularly important for patients with low expression of CD38 on their cancers. The enhanced anti-tumor activity of the CAR was also studied and observed by CyTOF. Additionally, to better understand the therapeutic potential of CD38-CAR NK cells compared to CD38-CAR T cells we generated CD38 KO/CD38-CAR T cells using the same engineering approach and comparing their cytotoxic function. We showed that both CD38-CAR-T and CD38-CAR-NK cells have comparable anti-tumor activity compare to their paired non-edited wild type cells. Finally, we investigated the need for a CD38 KO to develop a fratricide-resistant therapy by comparing the cytolytic and metabolic functions of CD38 +/AAVS1 KO and CD38 KO CD38-CAR NK cells. We conclusively report a CD38-CAR NK cell therapy with enhanced metabolism and cytotoxicity toward a variety of hematologic malignancies, which can be further augmented by combination treatment with ATRA to target CD38-low malignancies.

Activated Natural Killer Cell Inoculation Alleviates Fibrotic Liver Pathology in a Carbon Tetrachloride-Induced Liver Cirrhosis Mouse Model

Activated hepatic stellate cells (HSCs) play a detrimental role in liver fibrosis progression. Natural killer (NK) cells are known to selectively recognize abnormal or transformed cells via their receptor activation and induce target cell apoptosis and, therefore, can be used as a potential therapeutic strategy for liver cirrhosis. In this study, we examined the therapeutic effects of NK cells in the carbon tetrachloride (CCl4)-induced liver cirrhosis mouse model. NK cells were isolated from the mouse spleen and expanded in the cytokine-stimulated culture medium. Natural killer group 2, member D (NKG2D)-positive NK cells were significantly increased after a week of expansion in culture. The intravenous injection of NK cells significantly alleviated liver cirrhosis by reducing collagen deposition, HSC marker activation, and macrophage infiltration. For in vivo imaging, NK cells were isolated from codon-optimized luciferase-expressing transgenic mice. Luciferase-expressing NK cells were expanded, activated and administrated to the mouse model to track them. Bioluminescence images showed increased accumulation of the intravenously inoculated NK cells in the cirrhotic liver of the recipient mouse. In addition, we conducted QuantSeq 3' mRNA sequencing-based transcriptomic analysis. From the transcriptomic analysis, 33 downregulated genes in the extracellular matrix (ECM) and 41 downregulated genes involved in the inflammatory response were observed in the NK cell-treated cirrhotic liver tissues from the 1532 differentially expressed genes (DEGs). This result indicated that the repetitive administration of NK cells alleviated the pathology of liver fibrosis in the CCl4-induced liver cirrhosis mouse model via anti-fibrotic and anti-inflammatory mechanisms. Taken together, our research demonstrated that NK cells could have therapeutic effects in a CCl4-induced liver cirrhosis mouse model. In particular, it was elucidated that extracellular matrix genes and inflammatory response genes, which were mainly affected after NK cell treatment, could be potential targets.

Effects of polybrene and retronectin as transduction enhancers on the development and phenotypic characteristics of VHH-based CD19-redirected CAR T cells: a comparative investigation.

Chimeric antigen receptor T cells (CAR T cells) have improved the prognosis of patients with certain hematologic malignancies. However, broader clinical application of this type of therapy is dependent on production protocols. We characterized VHH-based CD19-redirected CAR T cells generated using the transduction enhancers (TEs) polybrene or retronectin. The proliferation rate of activated T cells transduced using polybrene concentrations > 6mg/mL decreased compared with untreated group. There was a direct relationship between polybrene concentration and transduction efficacy. Moreover, we demonstrated the proliferation of retronectin-transduced T cells increased in a dose-dependent manner (4-20μg/mL). Whereas, different retronectin concentrations did not mediate a significant increase in T cell transduction rate. Moreover, lentiviral transduction rate was also dependent on the concentration of lentiviruses. At optimized TE concentrations, multiplicity of infection (MOI) of > 10 decreased livingT cell transduction rate. Additionally, we demonstrated that CAR T cell phenotype is highly affected by TE type. Naïve T cell differentiation to central memory T cell was observed in the beginning of the expansion process and effector memory T cells became the predominant subset in the second week of expansion. Importantly, retronectin increased the proliferation of CAR T cells alongside medicating higher transduction rates, resulting in more naïve and central memory T cells. We demonstrated that a higher percentage of CAR T cells were generated using retronectin (with a less differentiated phenotype) making retronectin a more effective TE than polybrene for long-term CAR T cell processing in preclinical or clinical studies.

Effects of Botulinum Toxin A on the Blood Flow in Expanded Rat Skin

Background Poor blood supply can easily lead to expander extrusion and necrosis at the distal expanded flap. Botulinum toxin A (BTX-A) has been previously found to improve pedicled flap blood flow perfusion, but its effects on the blood supply of expanded skin remain unclear. Therefore, this study aimed to evaluate the effects of BTX-A on blood flow perfusion during and after expansion. Methods Eighteen Sprague-Dawley rats were randomly divided into a BTX-A group and a control group. BTX-A or normal saline was injected intradermally into the marked skin on the back immediately. Then expanders were implanted in the rats. One week later, inflation of the expander with normal saline was started and performed twice a week to reach an intracapsular pressure of 8 kPa. The skin blood flow was measured before each injection. After 4 weeks of expansion, the sample was harvested for histological staining to measure the diameter and density of blood vessels; meanwhile, a 2 cm× 8 cm expanded random flap was elevated and sutured in situ. Blood flow perfusion and flap survival were observed. Results Compared with the control group, the BTX-A group had more blood flow, a larger blood vessel diameter, and higher blood vessel density in the expanded skin. Additionally, the flap of the BTX-A group had good blood flow perfusion and a high proportion of flap survival area within 7 days after expanded flap transfer. Data were analyzed using an independent t-test. Conclusion Pre-surgical BTX-A treatment may increase angiogenesis and vasodilatation, with subsequent blood perfusion elevation during and after expansion, and obtain a greater proportion of survival area of the transferred expanded flap.

QS10: Morphological Changes Of Skin Related To Acellular Dermal Matrix Incorporation In Tissue Expansion

Purpose:Acellular dermal matrix (ADM) is used to create an inferolateral sling nearly three-quarters of the time in breast reconstruction and has proven a valuable alternative to total submuscular coverage of the implant. Previous work has demonstrated decreased inflammation and fibrosis of the pocket lining the ADM sling; however there has been minimal investigation into the role of ADM in skin growth and regeneration. The present study evaluates morphologic and molecular changes mediated by use of ADM in tissue expansion.Methods:Two tissue expanders, one wrapped in ADM, were placed subcutaneously on the back of Yucatan minipigs. All expanders were inflated with two weekly fills of 60cc of normal saline and skin biopsies were harvested after two weeks of expansion from each condition: control, tissue expansion (TE), and tissue expansion with ADM (TE+ADM). Three biopsies per condition were embedded in paraffin or OCT medium and stained with Russell Movat Pentachrome and Immunofluorescence of CD31, respectively. Collagen in the papillary dermis of pentachrome-stained images were analyzed using an ImageJ plug-in, Fibril Tool, that applies circular statistics to estimate average fibril orientation as the direction angle from -90 to 90 with respect to the x-axis. One-way ANOVA evaluated seventy-two measurements per condition and post-hoc analysis with Tukey’s HSD test identified significant comparisons between the groups. Number of fluorescent cells expressing CD31 (a marker of endothelial cells) were counted on 12 photographs per condition. P-values ≤ .05 were considered significant. Total deformation was calculated using a computational model and isogeometric analysis.Results:The mean fibril orientation of TE and TE+ADM underwent -85% change (P < .001) and -15% change (P = .65), respectively, compared to control. Three times more CD31+ cells were observed in TE+ADM compared to control (P < .001), but no significant changes were detected in TE alone. Histogram of total deformation revealed more even distribution of forces in TE+ADM compared to TE and control.Conclusions:The use of ADM in a porcine tissue expansion model appears to mitigate disarray of the collagen network in adjacent tissue, thereby creating a more extensive, yet even, distribution of stretched skin. This observation, combined with the finding of increased angiogenesis, suggests it is the incorporation of ADM that confers these protective benefits. Future studies will evaluate whether the protective effects of ADM can serve to improve TE in compromised tissue beds, as seen in patients undergoing TE concurrent with perioperative radiation therapy.

IL-2 Plus IL-15 Leads to Enhanced Ex Vivo Expansion of Human Invariant Natural Killer T Cells

Invariant natural killer T (iNKT) cells have been shown to play an important role in immune regulation, defense against pathogens, and cancer immunity, despite representing a minority population of lymphocytes. In particular, emerging murine and human data demonstrate that within this immunoregulatory capacity, iNKT cells function to suppress graft-versus-host disease without loss of the graft-versus-tumor effect, making them ideal to exploit in hematopoietic cell transplantation. Given the key functions of this relatively understudied population, the ability to ex vivo expand human iNKT cells has vital scientific and clinical applications. Therefore, we sought to determine the optimal conditions for iNKT cell expansion and the effects of expansion method on the iNKT cell cytokine production profile. Several expansion culture conditions were explored following initial magnetic bead-based iNKT enrichment and stimulation with α-galactosylceramide (α-GalCer)-loaded irradiated autologous peripheral blood mononuclear cells (PBMCs) on day 0: inclusion of either IL-2 or IL-2 plus IL-15 and day 10 restimulation with either α-GalCer-loaded irradiated autologous PBMCs or α-GalCer-loaded irradiated K562 cells engineered to express CD1d, CD86, 4-1BBL, and OX40L (“B82” cells; Tian G et al. J Clin Invest. 2016 Jun 1;126(6):2341-55). We found significant heterogeneity in the numbers of circulating iNKT cells in healthy donors, ranging from 0.05-0.3% of CD3+ cells, as well as in the fold expansion obtained following two weeks of in vitro culture. Ex vivo expansion using B82 cells for day 10 stimulation and IL-2 alone routinely yielded viability >90% and purity >95% (Figure 1A), and we obtained a median of 3.5x107 iNKT cells starting from a median of 7.2x108 PBMCs following two weeks of expansion. However, we found that when iNKT cells were expanded with autologous PBMCs used on day 10 for restimulation, fold expansion was higher than when B82 cells were used on day 10 and the inclusion of IL-15 along with IL-2 in the culture media led to substantially enhanced expansion (mean fold expansion for PBMC with IL-2: 458; B82 with IL-2: 314; PBMC with IL-2 plus IL-15: 634; B82 with IL-2 plus IL-15: 375; Figure 1B). Following PMA/ionomycin stimulation, cytokine production profiles were similar regardless of expansion conditions, with nearly all iNKT cells producing IFNγ (ranging 94.4-99.9%), and a smaller fraction of iNKT cells producing IL-4 (ranging 21.0-36.1%). These data describe the ex vivo expansion capacity of iNKT cells and culture conditions which lead to optimal expansion. These results contribute to our understanding of human iNKT cells and could inform the design of future clinical trials utilizing iNKT cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

ADAM17-Deficient Pluripotent Stem Cell-Derived Natural Killer Cells Possess Improved Antibody-Dependent Cellular Cytotoxicity and Antitumor Activity

Antibody-dependent cellular cytotoxicity (ADCC) is a key pathway that mediates natural killer (NK) cell cytotoxicity against antibody-opsonized target cells. This process helps mediate the therapeutic efficacy of anti-tumor antibodies. On NK cells, ADCC occurs via engagement of antibody-coated target cells with activating receptor FcγRIIIa, or CD16a, leading to proinflammatory cytokine upregulation, degranulation, and target cell death. Upon cellular activation, the CD16a ectodomain is cleaved from the NK cell surface by A Disintegrin and Metalloprotease-17 (ADAM17). Cleavage of the ectodomain prevents further antibody binding and signaling through CD16a, which dampens NK cell activity. Blocking activation-induced ADAM17-mediated CD16a cleavage has been previously demonstrated to augment ADCC activity and provides a novel strategy to improve efficacy of therapeutic antibodies in combination with adoptive transfer of engineered NK cells. To further define the ability of ADAM17 to regulate NK cell activity, we have generated and characterized ADAM17-deficient (ADAM17-KO) NK cells derived from CRISPR/Cas9-modified human induced pluripotent stem cells (iPSCs). ADAM17-KO iPSCs successfully differentiate into hematopoietic progenitor cells, then to NK cells that uniformly express typical NK cell surface markers including CD56, CD94, NKG2D, NKp44, and NKp46. ADAM17-KO iPSC-NKs are functional and kill K562 erythroleukemia cells comparable to wildtype iPSC-derived NK cells (WT iPSC-NK cells) and healthy donor-derived peripheral blood NK cells (PB-NK cells) in vitro. Surprisingly, upon differentiation, ADAM17-KO iPSC-NK cells express ~20% lower CD16a surface expression compared to WT iPSC-NK cells, but stably retain CD16a expression after enrichment for CD16a+ cells and over 6 weeks of expansion in culture. WT iPSC-NKs and PB-NKs rapidly lose CD16a surface expression upon stimulation with phorbol esters, while ADAM17 KO iPSC-NK cells maintain over 90% CD16a expression after this stimulation. Additionally, a significantly higher proportion of ADAM17-KO iPSCs express TNF-α (71%) and CD62L (L-Selectin) (36%) - two other known ADAM17 substrates, on the cell surface after stimulation with phorbol esters for 4 hours compared to WT iPSC-NK (7% TNF-α+, 2% L-Selectin+) and PB-NK (2% TNF-α+, 1% L-Selectin+). CD16a+ ADAM17-KO iPSC-NK cells mediate increased CD107a (45%) and IFNγ (39%) expression when co-incubated with RAJI B-lymphoma cells in the presence of the anti-CD20 antibody rituximab, compared to CD16a+ WT iPSC-NK (32% CD107a+, 11% IFNγ) and PB-NK (37% CD107a+, 7% IFNγ) cells. Similarly, CD16a+ ADAM17-KO iPSC-NK cells upregulate increased CD107a (29%) and IFNγ (42%) expression when co-incubated with CAL27 squamous cell carcinoma cells in the presence of the anti-EGFR antibody cetuximab, compared to CD16a+ WT iPSC-NK (12% CD107a+, 8% IFNγ) and PB-NK (14% CD107a+, 6% IFNγ). Long-term (24 hour) cytotoxicity assay against RAJI cells in the presence of rituximab demonstrates higher cytotoxicity in CD16a+ ADAM17-KO iPSC-NK cells compared to CD16a+ WT iPSC-NK and CD16a+ PB-NK cells over time (see associated figure). In vivo studies to determine the therapeutic efficacy of ADAM17-KO iPSC-NK cells compared to WT iPSC-NK and PB-NK cells are ongoing. Together, these studies demonstrate ADAM17-KO iPSC-NK cells derived from a renewable source of gene-edited iPSCs possess enhanced ADCC potential, and provide a promising candidate to be used for standardized, off-the-shelf NK cell-based therapies in conjunction with therapeutic antibodies. Figure Disclosures Blum: Fate Therapeutics: Current Employment. Kaufman:Fate Therapeutics: Consultancy.

Large-Scale Generation of Human Allospecific Induced Tregs With Functional Stability for Use in Immunotherapy in Transplantation.

Regulatory T cells play an important role in the control of autoimmune diseases and maintenance of tolerance. In the context of transplantation, regulatory T cells (Tregs) have been proposed as new therapeutic tools that may induce allospecific tolerance toward the graft, avoiding the side effects induced by generalized immunosuppressors. Although most clinical trials are based on the use of thymic Tregs in adoptive therapy, some reports suggest the potential use of in vitro induced Tregs (iTregs), based on their functional stability under inflammatory conditions, indicating an advantage in a setting of allograft rejection. The aim of this work was to generate and expand large numbers of allospecific Tregs that maintain stable suppressive function in the presence of pro-inflammatory cytokines. Dendritic cells were derived from monocytes isolated from healthy donors and were co-cultured with CTV-labeled naïve T cells from unrelated individuals, in the presence of TGF-β1, IL-2, and retinoic acid. After 7 days of co-culture, proliferating CD4+CD25++CTV− cells (allospecific iTregs) were sorted and polyclonally expanded for 6 weeks in the presence of TGF-β1, IL-2, and rapamycin. After 6 weeks of polyclonal activation, iTregs were expanded 230,000 times, giving rise to 4,600 million allospecific iTregs. Allospecific iTregs were able to specifically suppress the proliferation of autologous CD4+ and CD8+ T cells in response to the allo-MoDCs used for iTreg generation, but not to third-party allo-MoDCs. Importantly, 88.5% of the expanded cells were CD4+CD25+FOXP3+, expressed high levels of CCR4 and CXCR3, and maintained their phenotype and suppressive function in the presence of TNF-α and IL-6. Finally, analysis of the methylation status of the FOXP3 TSDR locus demonstrated a 40% demethylation in the purified allospecific iTreg, prior to the polyclonal expansion. Interestingly, the phenotype and suppressive activity of expanded allospecific iTregs were maintained after 6 weeks of expansion, despite an increase in the methylation status of the FOXP3 TSDR. In conclusion, this is the first report that demonstrates a large-scale generation of allospecific iTregs that preserve a stable phenotype and suppressor function in the presence of pro-inflammatory cytokines and pave the way for adoptive cell therapy with iTregs in transplanted patients.

Abstract A23: Identification of prevalent targets for the development of tailored sarcoma immunotherapies using a rapid clinic-to-bench immunoprofiling pipeline

Abstract Sarcomas are rare malignancies of mesenchymal origin for which current treatment options are very limited. Due to the vast heterogeneity of the various subtypes, the development of new targeted therapies requires a customized approach. Checkpoint blockade therapies are a promising alternative that still have not been systematically investigated for sarcoma patients. Thus, utilizing the infrastructure of the Cell Therapy Core Facility (CTC) at Nova Southeastern University (NSU), we developed a clinic-to-bench pipeline that enables the processing of fresh tumor material after surgical excision to generate primary sarcoma cell lines for biobanking and research purposes. Using this pipeline, we have extensively characterized mRNA, miRNA and cell surface expression profiles from more than 40 sarcoma cell lines to date, as well as validated their tumorigenicity by assessing expression of sarcoma/cancer associated genes such as Birc5, Birc2, Bcl2, Ewsr1-fli1, Syt/Ssx and Pcna. Moreover, we assessed the expression of immunomodulatory molecules that may suppress antitumor responses, such as PD-L1, PD-L2, OX40L and CD40L, using multicolor flow cytometry. Detailed characterization of the immune profile of 7 bone-related and 7 soft tissue sarcoma cell lines, assessed after 12 weeks of in vitro serial passaging, revealed a common signature in the cell surface expression of proliferative marker PCNA, and lymphocyte ligands CD112 and CD115. Moreover, immunoprofiling of the PCNA/CD112/CD155+ sarcoma cell lines (n=14) demonstrated persistent expression of PD-L2 in all cell lines and the potential applications of currently clinically approved anti-PD-1 checkpoint inhibitors for sarcoma treatment. Importantly, the conserved expression of these proteins enabled the distinction of sarcoma tumor cells from other cells in the fresh primary tumor mix directly after isolation. Thus, we next performed a comparative analysis of the immune profile of 6 freshly isolated sarcoma tumors and their respective cell lines. Comparison of the surface expression profiles of the generated cell lines and the corresponding primary tumor cells showed that the initial phenotype was preserved after more than 12 weeks of expansion and after cryopreservation. This proves that the generated cell lines are an accurate tool that can be used for the development of novel personalized immunotherapies. Most importantly, our results enabled the design of a translational research pipeline that allows the systematic characterization of the immune profile of understudied sarcomas for the first time in order to gain insight into which patients may directly benefit from available immunotherapies and which may be eligible to enroll in clinical trials assessing the efficacy of checkpoint inhibitors. In summary, the generated sarcoma biobank serves as a unique and highly representative primary resource that can be utilized to identify potential candidates for novel targeted and personalized immunotherapies for the treatment of sarcomas. Citation Format: Anna-Maria Georgoudaki, Robin Krueger, Michelle Hartman, Tamara Chinn, Dustin Tran, Reneé Potens, Wendy Weston, H. Thomas Temple, Adil D. Duru. Identification of prevalent targets for the development of tailored sarcoma immunotherapies using a rapid clinic-to-bench immunoprofiling pipeline [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A23.

Ex vivo expansion of regulatory T cells from long-term Belatacept-treated kidney transplant patients restores their phenotype and suppressive function but not their FOXP3 TSDR demethylation status

We recently reported that Tregs from long-term Belatacept-treated kidney transplant patients displayed an altered phenotype and impaired suppressive function compared to Tregs from healthy controls. However, it remains unknown whether ex vivo expansion of Tregs from patients who underwent long-term immunosuppression may be feasible to be used in their treatment. In this work, Tregs from Belatacept-treated patients were polyclonally expanded in vitro in the presence of rapamycin and IL-2. After four weeks of expansion, Tregs from patients expressed high levels of FOXP3, CD25, CTLA-4, Helios and CCR7, and showed strong suppressive activity, even in the presence of pro-inflammatory cytokines. However, FOXP3 TSDR demethylation remained lower in expanded Tregs from Belatacept-treated patients compared to healthy control Tregs. These data suggest that ex vivo expansion of Tregs from patients undergoing long-term immunosuppression may require the use of epigenetic modifying agents to stabilize FOXP3 expression to be considered as treatment in kidney transplant patients.

Perforator artery response to tissue expansion: An experimental study in rabbits.

The reconstruction of defects is a major area of interest in plastic surgery. Tissues are expanded to extend the tissue surface area and to prepare more reliable perforator flaps. Because expanded perforator flaps have become more popular, the aim of this study is to determine the response of the perforator artery to tissue expansion. We used a rabbit S1 perforator (first perforator branch of the thoracodorsal arteries) flap model. In 12 New Zealand White rabbits, left flaps were used as the experimental group (n = 12) and right flaps were used as the control group (n = 12). Both flaps were constructed in the dorsal skin. The experimental group was further divided into 3 subgroups according to expansion volume: 150 mL (n = 4), 200 mL (n = 4) and 250 mL (n = 4). We evaluated the responses of the perforator arteries to tissue expansion using the resistivity index (RI), the pulsatility index (PI), vessel diameter (D), histopathological examinations, and angiography. After 3 weeks of expansion, the perforator artery diameter had increased (p = 0.002) and the RI had decreased (p = 0.031) in the experimental group. The perforator artery diameter (p = 0.006) and RI had increased (p = 0.003) in the control group. No significant changes were observed in the PI in either group (p > 0.05) and no significant differences in post-expansion measurements were observed between experimental subgroups (p > 0.05). Suprafascial expansion of a perforator flap leads to an increase in diameter and a decrease in the RI of the perforator artery. The decrease in RI may indicate increased flap perfusion.

S910 A NEW MEMBRANE‐BOUND IL‐21/4–1BBL‐EXPANDED NK PRODUCT EXHIBITS ACTIVITY AGAINST ACUTE MYELOID LEUKEMIA ACTIVITY IN VIVO

Background:Adoptive NK cells infusion is a promising immunotherapy for acute myeloid leukemia (AML) patients.Aims:The aim of this study was to explore that two weeks of clinical grade expansion could provide sufficient numbers and purity of NK cells for clinical application with an enhanced ant‐leukemia response in vitro.Methods:NK cells were expanded in the large‐scale expansion platform based on membrane‐bound interleukin‐21/4–1BBL K562 feeder cells (mbIL21/4‐1BBL). The phenotype as well as responsitveness of NKcells were evaluated before and post NK cells expansion. NCG mouse model were used to assess the in vivo persistence and in vivo anti‐leukemia effect of NK products. Finally, 20 minimal residual disease persistent or relapse (MRD+) AML were enrolled to evaluate the safety of adoptive expanded NK products infusion.Results:In this study, using the large‐scale expansion platform, we first demonstrated that 2 weeks of expansion could satisfy clinical use in number (∼4.1 × 109 NK cell) and purity (median 81%) from 2 × 107 peripheral blood mononuclear cells based on membrane‐bound interleukin‐21/4‐1BBL K562 feeder cells (mbIL21/4‐1BBL), with expansion folds of 1318 and 2876 at 2 and 3 weeks, respectively. The CD107α production as well as TNF‐α secretion capacity of NK products against AML cell lines, or primary AML blasts, were comparable between 2 weeks and 3 weeks expanded NK products, both of which were higher than those resting NK cells before expansion. The MbIL21/4‐1BBL expanded NK product xenografted into immunodeficient leukemia mice could be found in bone marrow, spleen, liver, lung and peripheral blood, persisting for at least 2 weeks and reducing the AML burden in vivo. NK cells infusions once, or three times every other days exhibited similar tumor control. Compared with the case‐paired 20 minimal residual disease persistent or relapse (MRD+) AML patients with regular consolidation therapy, 20 AML MRD+ patients who accepted the expanded NK treatment showed better leukemia‐free survival and overall survival.Summary/Conclusion:In conclusion, this study demonstrated that mbIL‐21/4‐1BBL expanded NK cells exhibited anti‐leukemia activity against AML in vitro and in a mouse model, and the infusion of expanded NK product was potentially efficacious for AML patient without adverse effects.

Reshapable hydrogel tissue expander for ridge augmentation: Results of a series of successive insertions at the same intraoral site.

Oral mucosa expansion before ridge augmentation is a procedure to reduce soft tissue exposure and to improve bone graft density and volume after augmentation. This study explored a novel, shapeable hydrogel tissue expander (HTE) in intraoral sites that had undergone previous expansion and surgery. Nine beagle dogs had all premolar teeth extracted and adjacent alveolar bone reduced. After at least 3 months healing hydrogels were placed at 4 sites in each dog: maxilla and mandible, right and left. After 6 weeks of expansion, the hydrogels were removed and measured for volume expansion and physical condition. Punch biopsies were taken of the expanded oral mucosa. After 3 months, a second hydrogel insertion was performed at each of the same sites. After this second expansion cycle, volume and hydrogel condition were recorded. Three dogs received ultrasound imaging of the hydrogels during the second expansion. Necropsy specimens were taken of both expanded and non-expanded oral mucosa. Within 2 weeks after HTE insertion in both first and second insertions, blood flow returned to the pre-insertion level. The first and second insertions resulted in linear oral mucosa gain of 8.13 mm, and 6.44 mm, respectively. First and second insertion hydrogels erupted from 4% of the first expansion sites, and 3% of the second expansion sites. There was no directional migration of the expanding hydrogel at any site. Histology found little inflammatory reaction to any hydrogel implant. Oral mucosa can be consistently and successfully expanded before bone graft for ridge augmentation even at sites with a history of prior surgeries.

Relaxin 2 carried by magnetically directed liposomes accelerates rat midpalatal suture expansion and subsequent new bone formation

Relaxin (RLN) is an insulin-like peptide hormone that enables softening and lengthening of the pubic symphysis and uterine cervix. Here, we analyzed the effects of RLN2 on the expansion of rat midpalatal suture (MPS) using a magnetically directed liposome-based drug delivery system. Thirty-six male rats were divided into three groups: control (MPS was not expanded), lipo (expanded for 1 week with vehicle liposomes encapsulating ferric oxide and Cy5.5), and RLN-lipo (expanded for 1 week with the liposomes coated with RLN2). Rats were sacrificed after 1 week of expansion or after 2 weeks of retention. To accumulate RLN2-liposomes, a magnetic sheet was fixed to the palatal mucosa of the MPS. In vivo imaging showed magnetically controlled accumulation of liposomes in the MPS for 72 h. Immunohistochemistry revealed RLN2 expression in the MPS after expansion and relaxin receptor (RXFP) 2 expression at the osteogenic front (OF) in the RLN-lipo group; all groups expressed RXFP1 in the MPS. MPS expansion and bone formation were significantly accelerated at the OF in RLN-lipo group compared with the other groups. In the RLN-lipo group, significantly accelerated serrate bone deposition and elevated periostin (POSTN), iNOS, and MMP-1 levels were observed in the MPS. Sclerostin (SOST) expression was significantly reduced in newly formed bone in the RLN-lipo group. Our data revealed that RLN2 enhanced suture expansion via MMP-1 and iNOS secretion in the sutural fibroblasts and new bone formation via POSTN expression in osteoblasts at the OF. These properties may be useful for developing a new less-invasive orthopedic treatment aiming at sutural modification of cranio- and maxillofacial deformity patients.

Development of Potency Assays for Quality Assessment of an Advanced Therapy Medicinal Product: Mitomycin C-Induced Peripheral Blood Mononuclear Cell (MIC) Product

▪Introduction:Quality assessment in terms of product specificity and product potency is obligatory and strictly required by EMA and FDA to provide a platform for analysis of product comparability, stability and compatibility, and for clinical development to help predict product clinical efficacy. Mitomycin-treated donor peripheral blood mononuclear cells (MICs) could induce donor-specific tolerance via generation of recipient-derived tolerogenic dendritic cells (tDCs). To evaluate the immunomodulating capacity of the MIC product, we assessed the phenotype of tDCs, the immunosuppressive capacity of tDCs on allo-reactive CD4+ and CD8+ T cells as well as on CMV-specific CD8+ T cells.Methods:For standardization of the potency assay, third-party PBMCs positive for HLA-A2 and anti-CMV IgG were used. Immature DCs (iDCs) were generated 3 days before MIC production. MICs were generated under Good Manufacturing Practice (GMP) conditions. Each batch of MICs was introduced to iDCs at different ratios (1:0, 1:1, 1:10, 1:20) for a two-hour interaction followed by adding a DC maturation cocktail for overnight co-culture. Thereafter tDCs were purified by magnetic negative separation. The morphology of tDCs was observed by microscopy. The expression of HLA-DR, CD80, CD83, CD86 and CD103 was analyzed by flow cytometry. The inhibitory capacity on allo-reactive T cells and virus specific T cells was determined by mixed lymphocyte reaction (MLR) assay, by ELISpot assay and by Tetramer staining assay after one week of expansion in mixed lymphocyte peptide culture (MLPC).Results:In light microscopy (magnification: x 40), tDCs showed a relative smooth membrane surface. While the conventional mDCs were significantly larger in size with rough surface, richer ruffles on the cell membrane, and bigger, longer protrusions or pseudopodia. MIC products could inhibit the expression of costimulatory molecules on tDCs with an inhibition of 41% of CD80, 27% of CD83 and 23% of CD86. In parallel, the inhibitory marker CD103 was up-regulated about 65% on tDCs. Functionally, both tDCs and MICs could inhibit the IFN-γ secretion by CMV-specific CD8+ T cells, respectively. Moreover, the proliferation of allo-reactive T cells and CMV-specific T cells could be inhibited by tDCs and MICs.Conclusions:In summary, the potency assays, including the measurement of physicochemical parameters, the morphology and marker expression of tDCs, as well as the biologic characterization, the functionally immunosuppressive capacity of tDCs, comprise valuable parameters for the evaluation of clinically used advanced therapeutic cellular products. DisclosuresNo relevant conflicts of interest to declare.

Hemodynamic Assessment with SPY-Indocyanine Green Angiography in Expansion Period: A Study for Expansion Capsule Pressure Optimization.

Tissue expansion-related severe complication exists, and the poor blood supply is one of the causes. After decades of negative correlation between capsule pressure and blood flow was verified, the widely recognized expansion capsule pressure is still absent. A prospective randomized controlled trial was performed to explore the optimal expanding capsule pressure. Thirty subjects were randomly divided into 5 groups, received a weekly expanding on 60-, 70-, 80-, 90-, and 100-mm Hg capsule pressure, respectively. All patients achieved 8-week follow-up; hemodynamic assessment by SPY-indocyanine green (ICG) angiography was taken every 2 weeks. Standardized indexes from SPY-ICG angiography were used to assess the blood supply, in which the ratio of ingress rate and the ingress rate of normal skin (IR/NIR) reflects the arterial perfusion level, and the ratio of Engress Rate and IR (ER/IR) indicates the venous reflux level. The expansion-related adverse events during the trial were recorded. The IR/NIR and the ER/IR are both obviously negatively correlated to the capsule pressure of tissue expander (P ≤ 0.05, the ER/IR's correlation coefficient = -0.453; the IR/NIR's correlation coefficient = -0.482). The post-expansion IR/NIR increased significantly after 8 weeks of expansion (P ≤ 0.05) in 90-mm Hg group. And the post-expansion ER/IR was significantly elevated (P ≤ 0.05) in 80 and 90-mmHg groups. There were 2 expansion-related complications reported in 100-mm Hg group, whereas no complication occurred in 4 other lower groups. SPY-ICG angiography is an objective measurement for tissue expansion hemodynamic monitoring. The expanding capsule pressure of 80 to 90 mm Hg is a reasonable upper limit.

Th17 cells are refractory to senescence and retain robust antitumor activity after long-term ex vivo expansion.

Adoptive immunotherapy for solid tumors relies on infusing large numbers of T cells to mediate successful antitumor responses in patients. While long-term rapid-expansion protocols (REPs) produce sufficient numbers of CD8+ T cells for treatment, they also cause decline in the cell's therapeutic fitness. In contrast, we discovered that IL-17-producing CD4+ T cells (Th17 cells) do not require REPs to expand 5,000-fold over 3 weeks. Also, unlike Th1 cells, Th17 cells do not exhibit hallmarks of senescence or apoptosis, retaining robust antitumor efficacy in vivo. Three-week-expanded Th17 cells eliminated melanoma as effectively as Th17 cells expanded for 1 week when infused in equal numbers into mice. However, treating mice with large recalcitrant tumors required the infusion of all cells generated after 2 or 3 weeks of expansion, while the cell yield obtained after 1-week expansion was insufficient. Long-term-expanded Th17 cells also protected mice from tumor rechallenge including lung metastasis. Importantly, 2-week-expanded human chimeric antigen receptor-positive (CAR+) Th17 cells also retained their ability to regress human mesothelioma, while CAR+ Th1 cells did not. Our results indicate that tumor-reactive Th17 cells are an effective cell therapy for cancer, remaining uncompromised when expanded for a long duration owing to their resistance to senescence.

Leaf Demography of Some Evergreen and Deciduous Tree and Shrub Species of Kumaun Himalaya, India

Leaf demography was studied in 9 trees (6 Evergreen (ES), 1 Semideciduous (SD) and 2 Deciduous (DS)) and 10 shrubs (7 Evergreen (ES) and 3 Deciduous (DS)) occurring between 350 to 2500 m elevation in the Kumaun Himalaya, India. Although each species had its own pattern about seasonality of leaf recruitment, it was possible to group the species based on leaf expansion behavior. The result shows the leaf expansion rate was greater for deciduous species compared to Evergreen species. The percent leaf area after four weeks of expansion was also greater for deciduous species.

High herbivore pressure favors constitutive over induced defense.

Theoretical and empirical studies show that, when past or current herbivory is a reliable cue of future attack and defenses are costly, defenses can be induced only when needed and thereby permit investment in other functions such as growth or reproduction. Theory also states that, in environments where herbivory is constantly high, constitutive defenses should be favored. Here, we present data to support the second aspect of the induced resistance hypothesis. We examined herbivore‐induced responses for four species of Inga (Fabaceae), a common canopy tree in Neotropical forests. We quantified chemical defenses of expanding leaves, including phenolic, saponin and toxic amino acids, in experimental field treatments with and without caterpillars. Because young leaves lack fiber and are higher in protein than mature leaves, they typically lose >25% of their leaf area during the few weeks of expansion. We predicted that the high rates of attack would select for investment in constitutive defenses over induction. Our data show that chemical defenses were quite unresponsive to herbivory. We demonstrated that expanding leaves showed no or only small increases in investment in secondary metabolites, and no qualitative changes in the phenolic compound profile in response to herbivory. The proteinogenic amino acid tyrosine, which can be toxic at high concentrations, showed the greatest levels of induction. Synthesis: These results provide some of the first support for theoretical predictions that the evolution of induced vs. constitutive defenses depends on the risk of herbivory. In habitats with constant and high potential losses to herbivores, such as tropical rainforests, high investments in constitutive defenses are favored over induction.