scholarly journals Spatio-temporal biodistribution of 89Zr-oxine labeled huLym-1-A-BB3z-CAR T-cells by PET imaging in a preclinical tumor model

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Naomi S. Sta Maria ◽  
Leslie A. Khawli ◽  
Vyshnavi Pachipulusu ◽  
Sharon W. Lin ◽  
Long Zheng ◽  
...  
Keyword(s):  
T Cells ◽  
Real Time ◽  
Pet Imaging ◽  
Dose Group ◽  
Low Dose ◽  
High Dose ◽  
Car T Cells ◽  
Nsg Mice ◽  
Car T

AbstractQuantitative in vivo monitoring of cell biodistribution offers assessment of treatment efficacy in real-time and can provide guidance for further optimization of chimeric antigen receptor (CAR) modified cell therapy. We evaluated the utility of a non-invasive, serial 89Zr-oxine PET imaging to assess optimal dosing for huLym-1-A-BB3z-CAR T-cell directed to Lym-1-positive Raji lymphoma xenograft in NOD Scid-IL2Rgammanull (NSG) mice. In vitro experiments showed no detrimental effects in cell health and function following 89Zr-oxine labeling. In vivo experiments employed simultaneous PET/MRI of Raji-bearing NSG mice on day 0 (3 h), 1, 2, and 5 after intravenous administration of low (1.87 ± 0.04 × 106 cells), middle (7.14 ± 0.45 × 106 cells), or high (16.83 ± 0.41 × 106 cells) cell dose. Biodistribution (%ID/g) in regions of interests defined over T1-weighted MRI, such as blood, bone, brain, liver, lungs, spleen, and tumor, were analyzed from PET images. Escalating doses of CAR T-cells resulted in dose-dependent %ID/g biodistributions in all regions. Middle and High dose groups showed significantly higher tumor %ID/g compared to Low dose group on day 2. Tumor-to-blood ratios showed the enhanced extravascular tumor uptake by day 2 in the Low dose group, while the Middle dose showed significant tumor accumulation starting on day 1 up to day 5. From these data obtained over time, it is apparent that intravenously administered CAR T-cells become trapped in the lung for 3–5 h and then migrate to the liver and spleen for up to 2–3 days. This surprising biodistribution data may be responsible for the inactivation of these cells before targeting solid tumors. Ex vivo biodistributions confirmed in vivo PET-derived biodistributions. According to these studies, we conclude that in vivo serial PET imaging with 89Zr-oxine labeled CAR T-cells provides real-time monitoring of biodistributions crucial for interpreting efficacy and guiding treatment in patient care.

scholarly journals Feasibility of real-time in vivo89Zr-DFO-labeled CAR T-cell trafficking using PET imaging

2019 ◽  
Author(s):  
Suk Hyun Lee ◽  
Hyunsu Soh ◽  
Jin Hwa Chung ◽  
Eun Hyae Cho ◽  
Sang Joo Lee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Real Time ◽  
Pet Imaging ◽  
Cell Trafficking ◽  
T Cell Trafficking ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

AbstractIntroductionChimeric antigen receptor (CAR) T-cells have been developed recently, producing impressive outcomes in patients with hematologic malignancies. However, there is no standardized method for cell trafficking and in vivo CAR T-cell monitoring. We assessed the feasibility of real-time in vivo89Zr-p-Isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS, DFO) labeled CAR T-cell trafficking using positron emission tomography (PET).ResultsThe 89Zr-DFO radiolabeling efficiency of Jurkat/CAR and human peripheral blood mononuclear cells (hPBMC)/CAR T-cells was 70–79%, and cell radiolabeling activity was 98.1–103.6 kBq/106 cells. Cell viability after radiolabeling was >95%. Compared with unlabeled cells, cell proliferation was not significantly different during the early period after injection; however, the proliferative capacity decreased over time (p = 0.02, day 7 after labeling). IL-2 or IFN-γ secretion was not significantly different between unlabeled and labeled CAR T-cells. PET/magnetic resonance images in the xenograft model showed that most of the 89Zr-DFO-labeled Jurkat/CAR T-cells were distributed in the lung (24.4% ± 3.4%ID) and liver (22.9% ± 5.6%ID) by 1 hour after injection. The cells gradually migrated from lung to the liver and spleen by day 1, and remained stably until day 7 (on day 7: lung 3.9% ± 0.3%ID, liver 36.4% ± 2.7%ID, spleen 1.4% ± 0.3%ID). No significant accumulation of labeled cells was identified in tumors. A similar pattern was observed in ex vivo biodistributions on day 7 (lung 3.0% ± 1.0%ID, liver 19.8% ± 2.2%ID, spleen 2.3% ± 1.7%ID). 89Zr-DFO-labeled hPBMC/CAR T-cells showed the similar distribution on serial PET images as Jurkat/CAR T-cells. The distribution of CAR T-cells was cross-confirmed by flow cytometry, Alu polymerase chain reaction, and immunohistochemistry.ConclusionUsing PET imaging of 89Zr-DFO-labeled CAR T-cells, real time in vivo cell trafficking is feasible. It can be used to investigate cellular kinetics, initial in vivo biodistribution, and the safety profile in future CAR T-cell development.

scholarly journals Anti-CD19/CD22 Dual CAR-T Therapy for Refractory and Relapsed B-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 284-284 ◽  
Author(s):  
Junfang Yang ◽  
Pengfei Jiang ◽  
Xian Zhang ◽  
Xiaobin Zhu ◽  
Qi Dong ◽  
...  
Keyword(s):  
T Cells ◽  
Dose Group ◽  
Low Dose ◽  
Lymphoblastic Leukemia ◽  
Observation Time ◽  
High Dose ◽  
Car T Cells ◽  
Car T ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Medium Dose

Introduction Chimeric antigen receptor (CAR) T cell therapy targeting CD19 has demonstrated high success for B-cell acute lymphoblastic leukemia (B-ALL). Despite the initial high complete remission (CR) rate, about half of patients (pts) relapse at 1 year. CD19 antigen loss was observed in a significant number of relapsed patients. CD22 is another leukemic marker that often expressed on the surface of CD19- relapsed B-ALL blasts. We have developed a bispecific CAR construct targeting CD19 and CD22. Here we report results from a phase Ⅰ clinical trial of CD19/CD22 (GC022) dual CAR-T to evaluate the safety and feasibility of treating patients with relapsed/refractory B-ALL. Methods The CD19/CD22 dual CAR-T cells were manufactured in a cGMP facility. Patients' peripheral blood (PB) mononuclear cells were first collected, and CD3+ T cells were separated. The cells were then transfected by lentivirus encoded with CD19 CD22 bispecific scFv sequences. The CAR-T cells produced in this way contained a 4-1BB co-stimulatory signal domain. The CAR-T cells were then cultured for 8-14 days until sufficient cells were harvested for infusion. All pts received conditioning regimen of fludarabine and cyclophosphamide intravenously for 3 consecutive days with doses of 30 mg/m2/day and 250 mg/m2/day, respectively before a single infusion of CAR-T cells. The level of infused CAR-T cell proliferation in PB was analyzed by qPCR and flow cytometry. The primary end points were to evaluate feasibility and toxicity, and the secondary end points included disease response and engraftment/persistence of infused CD19/CD22 dual CAR-T cells. Results From Feb. 2019 to 23 July. 2019, 17 patients (pts) with relapsed/refractory B-ALL including 4 pts who previously treated with CD19 CAR-T cells were enrolled and pts were treated with CD19/CD22 dual CAR-T GC022 (US NIH Clinical#: NCT03825731). Four were adults, 13 pediatrics (age 1-45, Table 1). The median bone marrow (BM) blasts was 19.09 (0.36-87.82) %. Four patients received a low-dose (2.5-5×105/kg) dual CAR-T, 7 received a medium-dose (1-2.5×106/kg) and 5, a high-dose (3-5×106/kg). One patient withdrew immediately before CAR-T infusion due to his personal issue. Anti-leukemic efficacy was evaluated in 11/16 pts (5 pts have not yet reached D15). The 3/4 pts received low dose of GC022 had no response to treatment and 1 had MRD-positive CR. Seven patients who received medium dose achieved 100% CR on D15, highlighting the dose-dependent anti-leukemic activity. Six out of seven pts had MRD negative CR in this medium dose group. Five pts in high dose group have not reached the time for evaluation. No one relapsed with a median observation time of 60 (7-139) days. Cellular kinetic data was analyzed. Median peak of CAR-T copies was 1.09 (0.0022-4.98) x105 copy number/µg PB genomic DNA (Fig.1). The proliferation of medium or high dose groups was significantly better than the low dose group 3.47(0.43-4.98) x105 vs. 0.023(0.0022-0.81) x105(P=0.02) and 2.02(1.89-2.16) x105 vs. 0.023(0.0022-0.81) x105(P=0.004), (Fig.2). The peaks of IL-6, IFN-γ, IL-10, and CD25 were observed around day 7-10. Sixteen out of seventeen pts had grade 0-1 cytokine release syndrome (CRS) and only 1 patient experienced grade 2 CRS. None developed neurotoxicity. Conclusion Our study demonstrates safety and technical feasibility of CD19 and CD22 dual CAR-T in treating patients with CD19+CD22+ relapsed/refractory B-ALL. A low toxicity with dose-dependent high CR rate including pts who previously treated with CD19 CAR-T cells were observed. Longer observation time and more patients are needed to evaluate a beneficial advantage of the CD19/CD22 dual CAR-T over CD19 CAR-T product. Disclosures No relevant conflicts of interest to declare.

Minimally Ex Vivo Manipulated Gene-Modified T Cells Display Enhanced Tumor Control

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4549-4549 ◽  
Author(s):  
Saba Ghassemi ◽  
Patel Prachi ◽  
John Scholler ◽  
Selene Nunez-Cruz ◽  
David M. Barrett ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Low Dose ◽  
Ex Vivo ◽  
University Of Pennsylvania ◽  
Car T Cells ◽  
Car T ◽  
Equity Ownership

Abstract Adoptive cell therapy employing T cells equipped with a chimeric antigen receptor (CAR) containing a single chain antibody fragment fused to T cell signaling domains 4-1BB and CD3zeta (CTL019) has shown great potency against various hematopoietic malignancies, e.g. B cell acute lymphoblastic leukemia (ALL). However, it has not shown the same response rate in other malignancies such as chronic lymphocytic leukemia (CLL). We recently demonstrated that the in vivo expansion and persistence of CAR T cells is an important predictor of response to CTL019 in CLL (PMID: 26333935) and ALL (Thudium et al., ASH 2016; Fraietta et al., ASH 2016). Furthermore, it is well known that prolonged culture of T cells negatively impacts the in vivo expansion of the adoptively transferred cells. We therefore hypothesized that minimizing the ex vivo manipulation of T cells would improve the efficacy of CAR T cells. We tested this hypothesis by generating CART19 cells using our standard 9-day manufacturing process plus two abbreviated versions. Cells from normal donors (n=9) and from patients with adult ALL (n=6) were stimulated on day 0 followed by transduction with the CAR19-encoding lentiviral vector on day 1. Cells were harvested on days 3, 5, and 9. Cryopreserved aliquots were evaluated for T cell differentiation using polychromatic flow cytometry, cytokine secretion profile using Luminex, cytolytic ability against a leukemia cell line (NALM6), proliferative ability upon restimulation with CD19-expressing target cells, and in vivo control of our well-established xenogeneic ALL model employing NALM6 as the target. Our data show that all cultures contain a substantial proportion (40%-80%) of na•ve-like CD45RO-CCR7+ T cells that progressively differentiate leading to the accumulation of predominantly (60%-90%) central memory T cells by the end of expansion. Comparative assessment of the CART19 cells at all three time points demonstrated that the cells from the shorter cultures displayed a superior in vitrocytolytic activity, and proliferative response compared to the standard process. In addition,the cells from our standard and shortened cultures all secreted comparable levels of type I cytokines (i.e. IFN-g, IL-2, and TNF-α). Importantly, we investigated the therapeutic potential of cells harvested at day 3 versus later time points. We treated NALM6 xenograftmice with a low dose (0.5 x106 CAR+ T cell I.V.) or standard dose (3 x106 CAR+ T cell I.V.).We demonstrate that day 3 CART19 cells show superior anti-leukemic activity compared to day 5 or day 9 cells. Additionally, we show that mice treated at a low dose with day 3 cells exhibit the greatest anti-leukemic efficacy compared with day 9 cells where the latter fail to control leukemia (Figure 1). Our preclinical findings provide evidence that extended ex vivo manipulation of T cells negatively affects their in vivo potency.In summary, we show that limiting T cell culture ex vivo to the minimum required for lentiviral transduction provides the most efficacious T cells for adoptive T cell immunotherapy. Figure 1 Figure 1. Disclosures Ghassemi: Novartis: Research Funding. Scholler:Novartis: Patents & Royalties; University of Pennsylvania: Patents & Royalties: FAP-CAR US Patent 9,365,641 for targeting tumor microenvironment. Nunez-Cruz:Novartis: Research Funding. Barrett:Novartis: Research Funding. Bedoya:Novartis: Patents & Royalties. Fraietta:Novartis: Patents & Royalties: Novartis, Research Funding. Lacey:Novartis: Research Funding. Levine:GE Healthcare Bio-Sciences: Consultancy; Novartis: Patents & Royalties, Research Funding. Grupp:Novartis: Research Funding. June:Johnson & Johnson: Research Funding; Tmunity: Equity Ownership, Other: Founder, stockholder ; University of Pennsylvania: Patents & Royalties; Pfizer: Honoraria; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; Immune Design: Consultancy, Equity Ownership; Celldex: Consultancy, Equity Ownership. Milone:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding.

scholarly journals 105 4–1BB and optimized CD28 co-stimulation enhances function of human mono- and bi-specific third-generation CAR T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A115-A116
Author(s):  
Emiliano Roselli ◽  
Justin Boucher ◽  
Gongbo Li ◽  
Hiroshi Kotani ◽  
Kristen Spitler ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
The Other ◽  
Target Cells ◽  
Third Generation ◽  
Car T Cells ◽  
Nsg Mice ◽  
Car T

BackgroundCo-stimulatory signals regulate the expansion, persistence, and function of chimeric antigen receptor (CAR) T cells. Most studies have focused on the co-stimulatory domains CD28 or 4-1BB. CAR T cell persistence is enhanced by 4-1BB co-stimulation leading to NF-κB signaling, while resistance to exhaustion is enhanced by mutations of the CD28 co-stimulatory domain.MethodsWe hypothesized that a third-generation CAR containing 4-1BB and CD28 with only PYAP signaling motif (mut06) would provide beneficial aspects of both. We designed CD19-specific CAR T cells with 4-1BB or mut06 together with the combination of both (BB06). We evaluated their immune-phenotype, cytokine secretion, real-time cytotoxic ability and polyfunctionality against CD19-expressing cells. We analyzed LCK recruitment by the different constructs by immunoblotting. We further determined their ability to control growth of Raji cells in NSG mice. Additionally, we engineered bi-specific CARs against CD20/CD19 combining 4-1BB and mut06 and performed repeated in vitro antigenic stimulation experiments to evaluate their expansion, memory phenotype and phenotypic (PD1+CD39+) and functional exhaustion. Bi-specific CAR T cells were transferred into Raji or Nalm6-bearing mice to study their ability to eradicate CD20/CD19-expressing tumors.ResultsCo-stimulatory domains combining 4-1BB and mut06 confers CAR T cells with an increased polyfunctionality and LCK recruitment to the CAR (figure 1A), after repeated-antigen stimulation these cells expanded significantly better than second-generation CAR T cells (figure 1B). A bi-specific CAR targeting CD20/CD19, incorporating 4-1BB and mut06 co-stimulation, showed enhanced antigen-dependent in vitro expansion with lower exhaustion-associated markers (figure 1C). Bi-specific CAR T cells exhibited improved in vivo anti-tumor activity with increased persistence and decreased exhaustion (figure 1D).Abstract 105 Figure 1A. pLCK is increased in h19BB06z CAR T cells and associated with the CAR. CAR T cells were stimulated with irradiated 3T3-hCD19 cells at a 10:1 E:T ratio for 24hr. Cells were lysed and CAR bound and unbound fractions were western blotted. A single-cell measure of polyfunctional strength index (PSI) of CAR T cells. B. h19BB06z CAR T cells have the highest proliferation after repeated antigen stimulations. 5x105 CAR T cells were stimulated with 1x105 irradiated 3T3-hCD19 cells. After 1 week, half of the cells were enumerated by flow cytometry and the other half was re-stimulated with 1x105 fresh irradiated 3T3-hCD19 cells. This was repeated for a total of 4 weeks. C. 5x105 CAR T cells were co-cultured with 5x105 target cells (Raji-CD19High). After 1 week half the cells were harvested enumerated and stained by flow cytometry while the other half was re-stimulated with 5x105 fresh target cells (indicated by arrows). This was repeated for a total of 4 weeks. Frequency of PD1+CD39+ cells within CD8+ CAR T cells. D. Raji-FFLuc-bearing NSG mice were treated with 1x106 CAR T cells 5 days after initial tumor cell injection. Tumor burden (average luminescence) of mice treated with bi-specific or monospecific CAR T cells, UT and tumor control. Each line represents an individual mouse. (n = 7 mice per group).ConclusionsThese results demonstrate that co-stimulation combining 4-1BB with an optimized form of CD28 is a valid approach to optimize CAR T cell function. Cells with both mono- and bi-specific versions of this design showed enhanced in vitro and in vivo features such as expansion, persistence and resistance to exhaustion. Our observations validate the approach and justify clinical studies to test the efficacy and safety of this CAR in patients.

scholarly journals Safety and Response of Incorporating CD19 Chimeric Antigen Receptor T Cell Therapy in Typical Salvage Regimens for Children and Young Adults with Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 684-684 ◽  
Author(s):  
Daniel W. Lee ◽  
Maryalice Stetler-Stevenson ◽  
Constance M. Yuan ◽  
Terry J. Fry ◽  
Nirali N Shah ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Disease Burden ◽  
Low Dose ◽  
High Dose ◽  
T Cell Therapy ◽  
High Burden ◽  
Car T Cells ◽  
Car T ◽  
Preparative Regimen

CD19 chimeric antigen receptor (CAR) T cells have shown significant promise in multiple early phase trials including our own (Lancet 385:517-28). We manufacture CAR T cells containing CD28 and CD3z domains in 7 days using a retroviral platform. Several challenges remain to its widespread use: 1) reduction in the incidence of grade 4 cytokine release syndrome (CRS) and 2) incorporation with standard salvage regimens. Here, we update our experience with 39 patients. In the first 21 patients we defined the maximally tolerated dose as 1x106 CAR T cells/kg, grade 4 CRS occurred in 16%, and noted that severity of CRS correlated with disease burden. We stratified the current cohort (n=18) by disease burden. Subjects 1-21 and subsequent patients with low burden disease (Arm 1: isolated CNS disease or <25% marrow blasts) received a low dose preparative regimen of fludarabine (25 mg/m2/day Days-4 to -2) and cyclophosphamide (900 mg/m2 Day-2). Those with high burden disease (Arm 2: ³25% marrow blasts, circulating blasts or lymphomatous disease) received a high dose regimen to reduce tumor burden prior to cell infusion in an attempt to decrease severity of CRS. Arm 2 regimens were individualized based on prior therapies and risk from comorbidities. FLAG (n=6), ifosfamide/etoposide per AALL0031 (IE; n=2) and high dose fludarabine (30 mg/m2/day Days -6 to -3) with cyclophosphamide (1200 mg/m2/day Days -4 and -3) (HD flu/cy; n=3) were used. All products in the second cohort met cell dose though contaminating monocytes tended to inhibit maximal growth and transduction (see companion abstract by Stroncek). All patients received 1x106 CAR T cells/kg. Using grading criteria and an algorithm for early intervention to prevent grade 4 CRS (Blood 124:188-95) no grade 3 and only 1 grade 4 (5.6%) CRS occurred. Having significant comorbidities, Pt 34 was electively intubated for airway protection, did not require vasopressors, and rapidly recovered after tocilizumab and steroids. A brief seizure occurred, though he had a history of seizures. None others in the current cohort had neurotoxicity. Using intent to treat analysis, the complete response (CR) rate was 59% overall and 61% in ALL. 13/16 (81%) low burden and 10/22 (46%) high burden ALL patients had a CR across both cohorts. Low burden patients treated on either cohort had similar CR rate of 8/10 (80%) and 5/6 (83%). Although not statistically significant and underpowered, 7/11 (64%) high burden patients treated with low dose flu/cy had a CR while 3/11 (27%) had a CR with high dose regimens. Specifically, 3/6 (50%) receiving FLAG achieved MRD-CR while none receiving IE or HD flu/cy responded. 8/8 with primary refractory ALL had MRD-CR regardless of disease burden or preparative regimen raising the prospect that T cell fitness in these patients was superior to others. Of the 20 patients achieving an MRD-CR, the median leukemia free survival (LFS) is 17.7 months with 45.5% probability of LFS beginning at 18 months. Only 3 did not have a subsequent hematopoietic stem cell transplant as their referring oncologist determined the risk of such was unacceptable. Two relapsed with CD19-leukemia at 3 and 5 months, while 1 remains in CR with detectable CAR T cells at 5 months. Reliance on multiple infusions of cells is problematic as 0/5 CD19+ patients receiving a second dose responded. Preclinical models have demonstrated that T cell exhaustion has a role in limiting the efficacy of CAR T cells. We evaluated CAR products and the T cells used to generate them for phenotypic markers of exhaustion and will present data evaluating the relationship between these and response. Our results demonstrate that CD19 CAR T cell therapy is safe and effective with aggressive supportive care and use of an early intervention algorithm to prevent severe CRS and provides a potential for cure in primary refractory ALL. Table. Patient Characteristics, Response, and Toxicity Pt Age/ Sex/Risk # Relapses Arm/Prep Regimen(if Arm 2) Marrow Blasts Response CRS Grade Pre-Therapy Post CAR 22 17M 3 1 20 0 MRD- 2 23 13M 2 2 IE 99 98 SD 0 24 12M MLL 2 1 8.5 3 CR 1 25 25F 1 2 FLAG 95 0 MRD- 2 26 4M DS 2 2 IE (60%) 89 NA PD 0 27 8F 2 2 FLAG 77 69 SD 0 28 4M 2 2 FLAG (60%) 99 99 PD 0 29 12M PR 1 0.15 0 MRD- 1 30 15M Ph+ CNS2 3 1 0.08 0 MRD- 1 31 22M 3 2 FLAG 97 99 SD 0 32 15M CNS2 3 2 FLAG 0.04 + Lymphoma 0 MRD- 2 33 6M PR 1 0.15 0 MRD- 0 34 14M DS 3 2 Arm 1 Flu/Cy 90 0 MRD- 4 35 25M 2 2 HD Flu/Cy 30 87 PD 2 36 6M 2 1 1.5 91 PD 0 37 4F MLL 1 2 HD Flu/Cy 90 99 SD 0 38 7M 1 2 HD Flu/Cy 99 99 SD 1 Disclosures Off Label Use: Off-label use of tocilizumab will be discussed in managing cytokine release syndrome.. Rosenberg:Kite Pharma: Other: CRADA between Surgery Branch-NCI and Kite Pharma. Mackall:Juno: Patents & Royalties: CD22-CAR.

First in-human study of in vivo imaging of ex vivo labeled CAR T cells with dual PET-MR.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 3557-3557
Author(s):  
Ritu Singla ◽  
Dominic M Wall ◽  
Samuel Anderson ◽  
Nicholas Zia ◽  
James C Korte ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Real Time ◽  
Ex Vivo ◽  
Time Points ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Insight Into

3557 Background: This is a first in human in-vivo biodistribution of ex-vivo labelled CAR T cells assessed in a cohort of patients. Cells were labelled with novel Cu-64 labelled superparamagnetic iron oxide nanoparticles (SPION) and infused IV into patients with solid tumors & tracked using clinical dual PET-MR. The study validates the clinical translation of CAR T cell in-vivo tracking in real time. Methods: Cu-64 radioisotope was bound to silica coated SPION using electrolysis plating with tin & palladium seeding. Cellular uptake of Cu-64 SPION was facilitated with a transfecting agent. Functional assays including 51Chromium release, cytometric bead array demonstrated that labelling process did not affect cytotoxicity & cytokine secretion (TNFα & IFN-g). T cells were transduced with retroviral vector constructs encoding for second-generation chimeric T-cell receptor specific for carbohydrate Lewis Y antigen. Modified T-cells were expanded ex-vivo & were labelled with Cu-64 (~300 MBq) prior to re-infusion (3 x108 labelled cells). Scanning is performed with Siemens 3T dual PET-MR scanner. Results: In this first in human in-vivo study (HREC/16/PMCC/30) a cohort of patients received ex-vivo labelled CAR T cells to determine how many labelled cells distribute to solid tumor sites within 3-5 days. Our results demonstrate that cells can be efficiently labelled (≤60%) with high cell viability (≥85%) at a sensitivity sufficient to detect labelled cells at tumor site for up to 5 days. An observed trend in SUVmean & SUVmax provided insight into efficacy & individual response to therapy. Early time points showed moderate uptake of labelled cells in lungs posterior basal segments without increased activity over next few days, suggesting a transient process. Mild, diffuse bone marrow & relatively intense uptake of labelled cells in liver & spleen suggests margination of cells to reticulo-endothelial system. Distinct PET signal at some of the tumor sites at 24 h suggests antigen specific localization & time taken to reach these sites. Excretion via hepatobiliary indicated reabsorption from GI tract & re-circulation of labelled cells. Minimal uptake in brain & heart supported safety profile of labeling agent. Conclusions: This is first in human in-vivo study to provide highly valuable visual and dynamic data in real time and provides insight into individual responses to therapy. CAR T cell functionality largely remain unchanged due to labeling process. The findings indicate that labelled cells traffic to tumor sites at later time points & remain persistent for extended period of time.

769 CAR T cells undergoing epigenetic reprogramming by low-dose decitabine enhances persistent anti-tumor efficacy in vivo

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A817-A817
Author(s):  
Yao Wang ◽  
Weidong Han ◽  
Chuan Tong ◽  
Zhiqianag Wu ◽  
Hanren Dai
Keyword(s):  
T Cells ◽  
T Cell ◽  
Low Dose ◽  
Cytokine Production ◽  
De Novo ◽  
T Cell Exhaustion ◽  
Cell Exhaustion ◽  
Car T Cells ◽  
Car T

BackgroundAnti-CD19-directed chimeric antigen receptor (CAR) T-cell therapy has had a resounding effect on the treatment of B-ALL. However, CAR T cells have been less effective against B-cell non-Hodgkin lymphoma (B-NHL), in part because they become a exhausted state triggered by chronic antigen stimulation and characterized by upregulation of inhibitory receptors and loss of effector function.1-4 It has recently been demonstrated that de novo DNA methylation promoted T-cell exhaustion, whereas methylation inhibition enhanced ICB-mediated T-cell rejuvenation in vivo.5 6 FDA-approved DNA demethylating agents, such as decitabine (DAC), may provide a means to modify exhaustion-associated DNA methylation programs that restrict ICB-responsiveness.MethodsWe treated CAR (CAR-CD19-expressing) T cells with low-dose DAC (dCAR T cells), to determine its effects on antitumor activities, exhaustion- and memory-associate cell phenotype change, cell cytokine production, and cell proliferation. Its impact on antitumor activities was evaluated in vitro functional assays and mouse in vivo studies. We also conducted western blot, flow cytometry, methylation analysis, RNA in situ hybridization and high throughput RNA sequencing to determine the underlying mechanisms of dCAR T cell function.ResultsThe low-dose, short-term DAC treatment in vitro enhanced the central memory (Tcm) population and the ration of CD4/CD8, and induced degradation of DNMT3a.CAR T cell treated by DAC developing into less-differention status by enhancing memory. dCAR T cells exhibit enhanced antitumour reactivity and the maintenance of a memory-like phenotype at low effector:target ratios. Especially shown by the ‘stress test’, the dCAR T cells at very low doses could efficiently control tumours with a very large burden, and have effective recall responses upon tumour re-challenge in vivo. Importantly, the dCAR T cells maintained a higher proportion of cells with a memory phenotype than did the CAR T cells under long-term tumour stimulation. Transcription of gene sets involved in memory maintenance, proliferation, cytokine production and anti-inhibitor processes was triggered by antigen-expressing target cells upon DAC exposure before antigen stimulation. dCAR T cells avoided the exhaustion programme induced during tumour cell stimulation; they did not upregulate the expression of genes encoding inhibitory receptors and retained relatively high expression of memory related transcription factors and genes.ConclusionsCAR T cells underwent DNA reprogramming after DAC treatment, which induced significant sustained cell expansion, cytotoxicity, and cytokine production and reduced exhaustion after antigen exposure.AcknowledgementsWe thank Professor Lin Xin of Tsinghua University and Professor Mingzhou Guo of Chinese PLA General Hospital for support of data analysis.ReferencesWherry EJ, Kurachi M. Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol 2015;15:486–499. doi:10.1038/nri3862Wherry EJ, et al. Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity 2007;27:670–684. doi:10.1016/j.immuni.2007.09.006Schietinger A, et al. Tumor-specific T cell dysfunction is a dynamic antigen-driven differentiation program initiated early during tumorigenesis. Immunity 2016;45:389–401. doi:10.1016/j.immuni.2016.07.011Schietinger A, Greenberg PD. Tolerance and exhaustion: defining mechanisms of T cell dysfunction. Trends Immunol 2014;35:51–60. doi:10.1016/j.it.2013.10.001Ghoneim HE, et al. De novo epigenetic programs inhibit PD-1 blockade-mediated T cell rejuvenation. Cell 2017;170:142–157.e119. doi:10.1016/j.cell.2017.06.007Pauken KE, et al. Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science 2016;354:1160–1165. doi:10.1126/science.aaf2807

117 Rapid point-of-care subcutaneous CAR-T from blood draw to injection in 4 hours with modified LV encoding CARs and synthetic driver elements enables efficient CAR-T expansion and tumor regression

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A129-A129
Author(s):  
Frederic Vigant ◽  
Qun He ◽  
Wei Zhang ◽  
Hongliang Zong ◽  
Anirban Kundu ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Tumor Regression ◽  
Subcutaneous Administration ◽  
Viral Envelope ◽  
Blood Draw ◽  
Car T Cells ◽  
Nsg Mice ◽  
Car T

BackgroundAdoptive cellular therapy with chimeric antigen receptor (CAR)-T cells has demonstrated remarkable clinical activity in a number of hematologic malignancies, but product chain of custody, individualized manufacturing, preparative chemotherapy, and patient management present technical and logistical hurdles to broader implementation.MethodsLentiviral constructs for CARs (either CD19- or CD22-directed) co-expressed with a synthetic driver domain were identified from a >6 × 10 diversity combinatorial library of proliferative elements, transmembrane domains, leucine zippers, and an EGFR epitope screened for cellular expansion in a lymphoreplete model. Modified serum-free-lentiviral manufacturing process was developed to reduce complexity of CAR-T and to introduce CD3-activating elements into the viral envelope allowing activation and transduction of resting lymphocytes from peripheral blood.ResultsFour-hour exposure of as little as 1 ml of blood to the CD3-directed CD19-targeted CAR encoding lentivirus followed by subcutaneous injection in NSG mice bearing CD19+/CD22+ Raji cells resulted in tumor regression (figure 1) and robust CAR-T cell expansion as determined by flow cytometry (figure 2) and qPCR (table 1), with peak levels >10,000 CAR-T cells/µl and less than three CAR copies per genome. In contrast, administration of the same products intravenously failed to support significant CAR-T expansion or control tumor growth (figure 3). Regression of established Raji tumors was also observed in NSG-(KbDb) (IA) animals following SC administration of CD19 or CD22 CARs with driver domains. CAR-T cells contracted in peripheral blood following tumor regression.Abstract 117 Figure 1Tumor RegressionRegression of Raji tumor from the initial median volume of 151 mm3 throughout 40 days post subcutaneous administration of the LV transduced (at MOI 1 or 5) CD19-directed CAR T product (1M or 5M cells) in the NSG miceAbstract 117 Figure 2CAR-T Cells expansion in Vivo Post SC injectionExpansion of CAR-T cells throughout 35 days post subcutaneous administration of the LV transduced (at MOI 1 or 5) CD19-directed CAR T product (1M or 5M cells) in the NSG mice bearing Raji tumor (groups G1-G4) or w/o tumor (G5-7G)Abstract 117 Figure 3CAR-T Cells expansion in vivo post IV injectionExpansion of CAR-T cells throughout 35 days post intravenous administration of the LV transduced (at MOI 1 or 5) CD19-directed CAR T product (1M or 5M cells) in the NSG mice bearing Raji tumor (groups G1-G7).Abstract 117 Table 1qPCR Analysis of LV IntegrationNumber of LV integrations (LV copies) in the PBMC genome (RNase P copies) post transduction at MOI 1 or 5.ConclusionsWe conclude that through a synthetic subcutaneous lymph node approach with modified lentiviruses and driver domains, rPOC SC may enable CAR-T generation with reduced complexity, while maintaining the ability of CAR-T cells to expand, persist and exert anti-tumor activity.Ethics ApprovalAll animal studies were IACUC approved.

scholarly journals Low-dose decitabine priming endows CAR T cells with enhanced and persistent antitumour potential via epigenetic reprogramming

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yao Wang ◽  
Chuan Tong ◽  
Hanren Dai ◽  
Zhiqiang Wu ◽  
Xiao Han ◽  
...  
Keyword(s):  
Dna Methylation ◽  
T Cells ◽  
T Cell ◽  
Dna Methyltransferase ◽  
Low Dose ◽  
Cytokine Production ◽  
Car T Cells ◽  
Car T

AbstractInsufficient eradication capacity and dysfunction are common occurrences in T cells that characterize cancer immunotherapy failure. De novo DNA methylation promotes T cell exhaustion, whereas methylation inhibition enhances T cell rejuvenation in vivo. Decitabine, a DNA methyltransferase inhibitor approved for clinical use, may provide a means of modifying exhaustion-associated DNA methylation programmes. Herein, anti-tumour activities, cytokine production, and proliferation are enhanced in decitabine-treated chimeric antigen receptor T (dCAR T) cells both in vitro and in vivo. Additionally, dCAR T cells can eradicate bulky tumours at a low-dose and establish effective recall responses upon tumour rechallenge. Antigen-expressing tumour cells trigger higher expression levels of memory-, proliferation- and cytokine production-associated genes in dCAR T cells. Tumour-infiltrating dCAR T cells retain a relatively high expression of memory-related genes and low expression of exhaustion-related genes in vivo. In vitro administration of decitabine may represent an option for the generation of CAR T cells with improved anti-tumour properties.

scholarly journals Use of CD70 Targeted Chimeric Antigen Receptor (CAR) T Cells for the Treatment of Acute Myeloid Leukemia (AML)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4443-4443 ◽  
Author(s):  
Mark Leick ◽  
Irene Scarfò ◽  
Bryan D. Choi ◽  
Rebecca Larson ◽  
Amanda A Bouffard ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Synergistic Activity ◽  
Leukemic Stem Cells ◽  
Cytotoxicity Assays ◽  
Car T Cells ◽  
Nsg Mice ◽  
Car T

Background: CAR-T cells have led to a revolution in the treatment of advanced hematologic malignancies. Since these cells target antigens that are expressed on the cellular surface, it is imperative that there is near ubiquitous tumor expression with minimal expression vital human tissues. Finding targets with these characteristics in myeloid malignancies has been challenging. Typical markers expressed on the surface of AML are also expressed on essential innate immune effector cells (e.g. neutrophils) which, if targeted, could lead to prolonged absence of this immune arm, which is not survivable or replaceable. Current approaches rely on the use of CAR-T cells against common myeloid targets (e.g. CD123, CD33) as an ablative strategy with a planned allogeneic stem cell transplant rescue to eradicate the CAR-T cells afterwards. These solutions have resulted in significant toxicity with several deaths resulting from CD123-targeted CAR-T cells. Another approach has involved gene editing donor progenitor cells to delete CD33, repopulation of the marrow with these CD33 negative cells, and then treatment with CD33-targeted CAR-T cells. (Kim, Cell 2018). However, this approach is challenging, costly, and genomic editing of stem cells remains a concern. CD70 is an immune checkpoint found on antigen presenting cells and activated T cells. Multiple studies have shown a strong degree of expression on AML blasts and leukemic stem cells, with minimal normal tissue expression (Perna, Cell 2017, Riether J Exp Med 2017). A Phase 1 study of a CD70 targeted antibody drug conjugate in combination with azacitidine (which has been shown to increase CD70 expression on leukemic stem cells) for untreated AML patients has shown impressive results (Blood 2018 132:2680, Blood 2017 130:2652). Based on these findings, we explored CD70-targeting CARs for the treatment of AML. Methods: Based on our success with a trimeric ligand-based CAR of another TNFα family member, APRIL, for multiple myeloma (Schmidt Blood 2018 132:2059), we generated monomeric and trimeric second-generation ligand-based CAR constructs to target CD70 on AML. In vitro effector function was compared by cytotoxic potency and cytokine production. In vivo anti-tumor efficiency was assessed in a xenograft mouse model of AML. Effect of surface CD70 expression on AML cell lines after co-culture with azacitidine was assessed. Results: CAR T cell manufacturing of both constructs was accomplished successfully (transduction efficiency 70-93%) from three different healthy donors with no apparent fratricide. CD70 CARs were efficacious in in vitro cytotoxicity assays targeting an AML cell line Molm13. Unexpectedly, monomeric CD70 targeted CAR-T cells were superior to trimeric in cytotoxicity assays and, thus, were carried forward for in vivo assays. Next, we treated NSG mice that had been engrafted with Molm13 and demonstrated a substantial dose-dependent therapeutic effect with prolonged survival of CAR treated mice compared to those treated with untransduced T-cells (UTD). Treated mice demonstrated a CAR-T robust expansion in the peripheral blood assessed by flow cytometry that was commensurate with individual animal treatment responses. Bone marrow from these mice revealed substantially reduced CD70 in all groups. Preliminary in vitro co-culture of AML cells with azacitidine showed increased CD70 expression. Conclusion: CD70 based CAR-T targeting of AML is effective in vitro and in vivo. Combination treatment with azacitidine may increase target antigen expression and lead to synergistic activity and represents a viable therapeutic strategy that warrants further investigation. Treatment of AML engrafted NSG mice with CD70 CAR-T cells in conjunction with azacitidine is ongoing. Disclosures Frigault: Xenetic: Consultancy; Novartis: Consultancy; Juno/Celgene: Consultancy; Foundation Medicine: Consultancy; Incyte: Consultancy; Nkarta: Consultancy; Kite/Gilead: Honoraria. Maus:INFO PENDING: Other: INFO PENDING.

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