NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ Mice Research Articles

Novel Antimitotic Agent SP-1-39 Inhibits Head and Neck Squamous Cell Carcinoma.

Effective management of head and neck cancer (HNC) poses a significant challenge in the field of oncology, due to its intricate pathophysiology and limited treatment options. The most common HNC malignancy is head and neck squamous cell carcinoma (HNSCC). HNSCC treatment includes a combination of surgery, radiation, and chemotherapy. While HNSCC is treatable if diagnosed early, this is often not the case and is considered incurable once in its late stages and metastatic disease has developed. Therapies are also limited once resistant disease has occurred. SP-1-39, a novel colchicine-binding site inhibitor (CBSI), has been recently reported for its potential efficacy in a variety of cancer cell lines including breast, melanoma, pancreatic, and prostate. SP-1-39 also shows abilities to overcome paclitaxel resistance in a paclitaxel-resistant prostate cancer xenograft model. To evaluate the potential of SP-1-39 as a new HNSCC treatment option, herein we systematically performed preclinical studies in HNSCC models using SP-1-39 and demonstrated that, in vitro, SP-1-39 inhibits the proliferation of 2 HNSCC cell lines with low nanomolar IC50 values (1.4 to 2.1 nM), induces HNSCC cell apoptosis in a dose-dependent manner, interferes with migration of HNSCC cells, and leads to HNSCC cell cycle arrest in the G2/M phase. In vivo, SP-1-39 suppresses the primary tumor growth of a Detroit 562 subcutaneous xenograft mouse model in 6- to 8-wk-old, male NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice, with no detectable cytotoxic effects at a low dose of 2.5 mg/kg. This efficacy of SP-1-39 is better when compared with the treatment using a reference chemotherapy drug, paclitaxel at 10 mg/kg. Collectively, these data demonstrate that SP-1-39 is a promising candidate for further development for more efficacious HNSCC treatment.

Abstract 4571: Combination of FOLFIRI (5-FU, Leucovorin, and Irinotecan) and ProAgio on colon cancer growth and metastasis

Abstract Background/Objective: One of the common mechanisms of resistance of solid tumors, including colon cancers, is activation of integrins on endothelial cells (ECs) and cancer-associated fibroblasts (CAFs). ProAgio is a small molecule developed to target integrin alpha V beta 3 (αvβ3), located on surface of abnormal ECs and CAFs. ProAgio enhances apoptosis and inhibits angiogenesis via targeting αvβ3. The aim of the current study is to evaluate the ProAgio combination with FOLFIRI on the immune profile and tumor growth of colon cancer. Material and Methods: Female BALB/CJ and male NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice were acquired from the Jackson Laboratory, and experiments were performed under the approval of The University of Alabama at Birmingham-Institutional Animal Care and Use Committee. For orthotropic tumor efficacy studies, BALB/CJ mice were injected with 2×105 CT-26 wild type and CT26.CL25 cells in 30ul 1:1 Matrigel Matrix and PBS into the Cecum. PDX (10mg) was orthotopically implanted into the NSG mice cecum. At Day five, mice were divided into four treatment groups: including 1) sterile water (vehicle), 2) ProAgio (15mg/kg; IP; once daily for nine days), 3) FOLFIRI (5-FU: 30mg/kg; Leucovorin 50mg/kg; Irinotecan; 20mg/kg - once every three days intraperitoneal) or 4) combination of ProAgio and FOLFIRI. The study completed on day 15, tumor weight was noted, and tumors were processed for histology and flow cytometry. Results: ProAgio plus FOLFIRI significantly (p<0.001) reduced tumor weight in both models compared to individual treatments and vehicles. Combination-treated mice showed a significant (p<0.001) reduction in T-cell exhaustion (CD8+ T cells that are positive for PD-1, TIM-3, and CTLA-4). Analysis of the myeloid compartment showed a significant (p<0.001) increase in the M1 macrophages that express F4/80 as well as CD86, MHC-II and a decrease in the M2 macrophage in the mice treated with ProAgio plus FOLFIRI. The combination therapy exhibited a significant (p<0.001) increase in the dendritic cell infiltration into the tumor microenvironment. Histology analysis showed that ProAgio treatment reduced the incidence of organ site metastasis (disseminated and locoregional spread) such as liver, pancreas, and diaphragm. Conclusion: Our study offers encouraging observations that ProAgio plus FOLFIRI can suppress colon cancer growth and metastasis by inhibiting αvβ3 preclinically and may provide a novel therapeutic combination for the management of colon cancers. Citation Format: Purnachandra Nagaraju Ganji, Jeremy B. Foote, Madhu Sudhana Saddala, Falguni Mishra, Sujith Sarvesh, Midhun Malla, Ashiq Masood, Zhi-Ren Liu, Bassel F. El-Rayes. Combination of FOLFIRI (5-FU, Leucovorin, and Irinotecan) and ProAgio on colon cancer growth and metastasis [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 4571.

Abstract 4002: Investigating CAR-T cell efficacy and activation in the disseminated NALM6-luc human B-cell acute lymphoblastic leukemia model

Abstract Introduction: Chimeric Antigen Receptor T (CAR-T) cell therapy has emerged as a promising approach for treating hematological malignancies, particularly B-cell acute lymphoblastic leukemia (B-ALL). This study aimed to delve into the activation dynamics and therapeutic efficacy of CD19 CAR-T cells in a disseminated NALM6-luc human B-ALL murine model. Experimental design: NALM6-Luc-mCh-Puro cells were implanted intravenously in female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. Animals were treated with CD19 CAR-T cells alone and in combination with ibrutinib. The anti-tumor activity was assessed, and the persistence and activation of CAR-T cells in the blood were evaluated using flow cytometry. Additionally, ddPCR analysis was employed to quantify CAR copy numbers, providing insights into the T-cell persistence over time. Results: Treatment with CD19 CAR-T cells exhibited notable anti-cancer activity and that activity was enhanced in combination with ibrutinib, in line with other reports. The increased time to progression was noted as 69.2% for the CAR-T group and 138.4% for the combination of CAR-T and ibrutinib compared to control. Flow cytometry analysis showed that CAR-T cells persisted in the blood throughout the study. The mean CAR-T absolute cell count for both CAR-T and CAR-T plus ibrutinib treated groups increased from 2 cell/µL blood on Day 0 to 11 and 51 cells/µL of blood, respectively, by Day 39. The progressive increase of PD-1+ and LAG-3+ percentage cells correlates positively with tumor burden, indicating a dynamic link between biomarker upregulation and the presence of tumor cells in mice. ddPCR analysis revealed an initial CAR copy decrease in all groups, followed by gradual increase in tumor-bearing mice treated with CD19 CAR-T cells, with a dramatic increase observed by Day 39. Results from CAR-T treated and CAR-T plus ibrutinib treated groups were comparable, showing mean copy numbers per ng of DNA increasing from less than 1 on day 4 to 17.9 and 23.3, respectively, on day 39. Importantly, no changes were noted in non-tumor implanted mice, emphasizing the tumor dependency for CAR-T cell expansion. Conclusion: This study emphasizes the marked anti-cancer activity of CD19 CAR-T cells in the NALM6-Luc-mCh-Puro B-ALL murine model. Comprehensive analysis of CAR-T cell persistence and activation, including insights from ddPCR, provides a robust understanding of therapeutic response dynamics. The observed in vivo anti-cancer efficacy aligns with the persistence of CAR-T cells revealed by flow cytometry and the dynamic expansion captured through ddPCR analysis. Our results indicate ddPCR and flow cytometry as complementary and independent methods for assessing the systemic CAR-T cell persistence, contributing to optimizing therapy for B-cell acute lymphoblastic leukemia. Citation Format: Prashant Pandey, David Draper, Sheri Barnes, Yewei Xing, Derrik Germain, Lauren Kucharczyk, Roys Stacey. Investigating CAR-T cell efficacy and activation in the disseminated NALM6-luc human B-cell acute lymphoblastic leukemia model [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 4002.

Epizootic of enterocolitis and clostridial overgrowth in NSG and NSG-related mouse strains.

While the immunodeficient status of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) and NSG-related mice provides utility for numerous research models, it also results in increased susceptibility to opportunistic pathogens. Over a 9-week period, a high rate of mortality was reported in a housing room of NSG and NSG-related mice. Diagnostics were performed to determine the underlying etiopathogenesis. Mice submitted for evaluation included those found deceased (n = 2), cage mates of deceased mice with or without diarrhea (n = 17), and moribund mice (n = 8). Grossly, mice exhibited small intestinal and cecal dilation with abundant gas and/or digesta (n = 18), serosal hemorrhage and congestion (n = 6), or were grossly normal (n = 3). Histologically, there was erosive to ulcerative enterocolitis (n = 7) of the distal small and large intestine or widespread individual epithelial cell death with luminal sloughing (n = 13) and varying degrees of submucosal edema and mucosal hyperplasia. Cecal dysbiosis, a reduction in typical filamentous bacteria coupled with overgrowth of bacterial rods, was identified in 18 of 24 (75%) mice. Clostridium spp. and Paeniclostridium sordellii were identified in 13 of 23 (57%) and 7 of 23 (30%) mice, respectively. Clostridium perfringens (7 of 23, 30%) was isolated most frequently. Toxinotyping of C. perfringens positive mice (n = 2) identified C. perfringens type A. Luminal immunoreactivity to several clostridial species was identified within lesioned small intestine by immunohistochemistry. Clinicopathologic findings were thus associated with overgrowth of various clostridial species, though direct causality could not be ascribed. A diet shift preceding the mortality event may have contributed to loss of intestinal homeostasis.

Antibiotic Treatment of Corynebacterium bovis-associated Clinical Disease in NSG Mice.

Depending on the strain of immunodeficient mice, Corynebacterium bovis infection can be asymptomatic or cause transient or prolonged skin disease. C. bovis infection of NOD. Cg- Prkdcscid Il2rgtm1Wjl /SzJ (NSG) mice results in clinical skin disease that progresses in severity. Amoxicillin metaphylaxic and prophylaxic therapy prevents transmission and infection of mice after exposure to C. bovis and inhibits the growth of C. bovis isolates at therapeutic doses that are clinically achievable in mice. Amoxicillin is not efficacious for treatment of transient clinical skin disease in athymic nude mice, but the efficacy of amoxicillin treatment has not previously been characterized in C. bovis -infected NSG mice. In the current study, NSG mice were treated with amoxicillin beginning at 5 wk after exposure to C. bovis, at which time they had well-established clinical signs of disease. Clinical signs were scored to assess disease progression, regression, and reappearance. Our results showed that amoxicillin treatment for 3 or 6 wk reduced the clinical scores of NSG mice with C. bovis -associated clinical disease. In addition, withdrawal of treatment led to the recurrence of clinical signs. Collectively, our data suggest that amoxicillin treatment is effective in alleviating the clinical signs associated with C. bovis infection for the duration of treatment in NSG mice. Clinical intervention with antibiotics for C. bovis -infected NSG mice can be an option for management of C. bovis -related clinical disease either before or during facility-wide remediation efforts.

A Novel Fc-Optimized Antibody Drug Conjugate Targeting CD7 As a Therapeutic Strategy in T-Cell Acute Lymphoblastic Leukemia

Despite progress in improving treatment regimens for patients with T-cell acute lymphoblastic leukemia (T-ALL), the therapeutic options are still limited, and especially antibody-based immunotherapy is not established. The CD7 antigen represents a promising target structure in T-ALL since it is strongly expressed in different T-ALL subtypes including early T-cell precursor (ETP)-ALL. Therefore, different approaches are currently pursued for targeting CD7, including CAR-T cell therapy. Due to its high internalization capacity CD7 also represents an ideal target structure for antibody drug conjugates (ADC). Here, a novel antibody engineering approach for CD7-targeting was evaluated in vitro and in xenograft mouse models of T-ALL. A CD7 antibody was optimized for its ability to trigger antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) by introducing two amino acid substitutions (S239D/I332E; DE-variant) in the Fc-domain. In addition, the Fc-engineered antibody was conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) via an enzymatic-cleavable linker (mc-vc-PABC). The Fc-optimized ADC, CD7-DE-vcMMAE, as well as the unconjugated antibody, CD7-DE, showed improved binding to activating Fcγ receptors (FcγRIIa, FcγRIIIa) compared to a CD7 antibody lacking the DE-modification. This resulted in potent ADCC- and ADCP-activity against different T-ALL cell lines mediated by NK-cells or macrophages as effector cells (EC 50 ADCC, 1-7 pM). In contrast to the CD7-DE antibody, CD7-DE-vcMMAE showed strong cytotoxic effects independently of immune effector cell engagement by releasing its cytotoxic compound into T-ALL cells in an antigen-restricted manner, thereby inducing G2/M cell cycle arrest and apoptosis. CD7-DE-vcMMAE was active at subnanomolar concentrations demonstrating dose-dependent cytotoxic effects in six T-ALL cell lines (IC 50 0.2 - 1 nM). The extent of maximum growth inhibition ranged between 54-98 % and correlated with CD7 antigen density. Yet, although CD7-DE-vcMMAE did not kill CD7-negative cells directly, the linker design facilitated bystander killing activity. Thus, in co-culture experiments using CEM cells and CEM-CD7-knockout cells mimicking CD7-antigen escape, the ADC demonstrated significant killing of neighbouring CD7-negative cells, thereby extending its mode of action. The antitumor activity of the CD7-ADC was further investigated in xenograft mouse models of T-ALL. In a first model, CEM cells were injected subcutaneously into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and CD7-DE-vcMMAE treatment was evaluated in comparison to the unconjugated antibody CD7-DE. Treatment with CD7-DE-vcMMAE led to a significantly reduced tumor growth in comparison to CD7-DE or untreated animals (p<0.05; see panel A). A preclinical phase II-like patient-derived xenograft (PDX) study employing eight randomly selected T-ALL-PDX samples from pediatric and adult patients was conducted. PDX-cells were injected intravenously into NSG mice and treatment was started when the leukemia load reached 1 % human blasts in the peripheral blood, reflecting an overt leukemia situation. Animals receiving therapy with CD7-DE-vcMMAE showed significant prolongation of median survival in comparison to animals treated with a similarly designed control ADC targeting an irrelevant antigen (control-DE-vcMMAE) or which were left untreated (median survival time; CD7-DE-vc-MMAE: 82 days; untreated/control-DE-vcMMAE: 60 days; p<0.05; see panel B). Importantly, no leukemic blasts were found in the peripheral blood, spleen or bone marrow in animals treated with CD7-DE-vcMMAE and surviving the experimental period of 150 days. Together, the novel ADC CD7-DE-vcMMAE showed a unique set of Fc effector functions, potent direct growth inhibitory effects, bystander killing activity and efficacy in xenograft models of T-ALL. These results exhibit CD7-DE-vcMMAE as a promising therapeutic strategy and form the basis for new approaches in the treatment of T-ALL.

Abstract 41: Subcutaneous growth and immune cell profiling of BT-474 human breast carcinoma in four strains of triple immunodeficient mice

Abstract Triple immunodeficient mouse models, that lack functional T cells, B cells, and natural killer cells, are necessary when evaluating human cancer cell growth and cell-based therapy activity, allowing for assessment without innate mouse immune system involvement. Due to limited colony sizes at any given vendor, study timelines can sometimes become compromised unless a sufficient alternative is validated. Site location can also be a factor, as licensing restrictions may be an issue for international corporations. In an effort to prove overall similarities across four strains of triple immunodeficient mice, the growth characteristics of BT-474 were compared in female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, NOD.CB17-Prkdcscid IL2rgtm1/BcgenHsd (B-NDG) mice, NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl (NCG) mice, and NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac (CIEA NOG) mice. An immune cell profile was also performed across these four mouse strains and evaluated via flow cytometry to observe any immune cell subset differences in blood, spleen, and tumor samples. Samples analyzed post implant were collected when subcutaneous tumors were ~500 mm3. Model development studies were completed previously to optimize tumor growth kinetics. Historic growth conditions were BT-474 cells implanted at 1.0E+07 cells/implant in the presence of an extracellular matrix into the high right axilla of female NSG mice. Using the same optimized growth conditions, NSG, B-NDG, NCG, and CIEA NOG mice were implanted with BT-474 cells. BT-474 grew well in all the tested mouse strains, producing 100% take rate. Compared to NSG mice, median tumor volume doubling times differed by 1-5 days, median times to an evaluation size of 150 mm3 (typical study enrollment tumor volume) differed by +/- ~4 days and median time to an evaluation size of 1250 mm3 (tumor volume at the end of life) differed by +/- ~10 days. BT-474 did not induce body weight loss in any of the tested mouse strains and all clinical observations were similar. Flow cytometry was performed to quantify lymphocyte and myeloid immune subsets in blood, spleen, and tumor. Overall, compared to NSG mice, the absolute counts were similar for all immune subsets measured except for regulatory T cells (Tregs). B-NDG, NCG, and CIEA NOG mice had reduced Treg numbers in the spleen of tumor-bearing mice. In the tumor, Treg numbers were lower in the B-NDG and CIEA NOG mice. No differences were observed for any immune subset in the blood. Overall, the tumor growth kinetics of BT-474 in B-NDG mice was most similar to historical data using NSG mice. The flow data also confirmed that each strain’s immune cell population generally looked very similar, with limited differences between the four strains of triple immunodeficient mice. Citation Format: Justin Snider, Derrik Germain, Lauren Kucharczyk, Anita Zaitouna, Erin Trachet, Scott Wise. Subcutaneous growth and immune cell profiling of BT-474 human breast carcinoma in four strains of triple immunodeficient mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 41.

Human Hematopoietic Stem Cell Engrafted IL-15 Transgenic NSG Mice Support Robust NK Cell Responses and Sustained HIV-1 Infection.

Mice reconstituted with human immune systems are instrumental in the investigation of HIV-1 pathogenesis and therapeutics. Natural killer (NK) cells have long been recognized as a key mediator of innate anti-HIV responses. However, established humanized mouse models do not support robust human NK cell development from engrafted human hematopoietic stem cells (HSCs). A major obstacle to human NK cell reconstitution is the lack of human interleukin-15 (IL-15) signaling, as murine IL-15 is a poor stimulator of the human IL-15 receptor. Here, we demonstrate that immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice expressing a transgene encoding human IL-15 (NSG-Tg(IL-15)) have physiological levels of human IL-15 and support long-term engraftment of human NK cells when transplanted with human umbilical-cord-blood-derived HSCs. These Hu-NSG-Tg(IL-15) mice demonstrate robust and long-term reconstitution with human immune cells, but do not develop graft-versus-host disease (GVHD), allowing for long-term studies of human NK cells. Finally, we show that these HSC engrafted mice can sustain HIV-1 infection, resulting in human NK cell responses in HIV-infected mice. We conclude that Hu-NSG-Tg(IL-15) mice are a robust novel model to study NK cell responses to HIV-1.

Degenerative Myelopathy and Neuropathy in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) Mice Caused by Lactate Dehydrogenase-Elevating Virus (LDV).

Following implantation of patient-derived xenograft (PDX) breast carcinomas from three separate individuals, 33/51 female NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice presented with progressive, unilateral to bilateral, ascending hindlimb paresis to paralysis. Mice were mildly dehydrated, in thin to poor body condition, with reduced to absent hindlimb withdrawal reflex and deep pain sensation. Microscopically, there was variable axonal swelling, vacuolation, and dilation of myelin sheaths within the ventral spinal cord and spinal nerve roots of the thoracolumbar and sacral spinal cord, as well as within corresponding sciatic nerves. Results of PCR screening of PDX samples obtained at necropsy and pooled environmental swabs from the racks housing affected animals were positive for lactate dehydrogenase-elevating virus (LDV). LDV is transmitted through animal-animal contact or commonly as a contaminant of biologic materials of mouse origin. Infection is associated with progressive degenerative myelopathy and neuropathy in strains of mice harboring endogenous retrovirus (AKR, C58), or in immunosuppressed strains (NOD-SCID, Foxn1nu), and can interfere with normal immune responses and alter engraftment and growth of xenograft tumors in immunosuppressed mice. This is the first reported series of LDV-induced poliomyelitis in NSG mice and should be recognized as a potentially significant confounder to biomedical studies utilizing immunodeficient xenograft models.

Functional SCD1 Is Critical for Acute Lymphoblastic Leukaemia Maintenance in the Central Nervous System

The largest advance in survival rates for childhood acute lymphoblastic leukaemia (ALL) came with the recognition that eradication of disease in the central nervous system (CNS) is needed to achieve long-term cure. All modern ALL protocols include large amounts of CNS-directed therapy, predominantly with methotrexate, which can result in significant acute and chronic neurotoxicity. In addition, CNS relapse remains a clinical challenge with a lack of effective drugs for recurrent disease. Despite this, little is known about the mechanisms by which leukaemic blasts enter the CNS and survive in this different microenvironment. Identifying specific mechanisms that support maintenance of ALL in the CNS should facilitate more effective targeted therapies.CNS ALL blasts reside in the leptomeninges, bathed in cerebrospinal fluid (CSF) which is low in glucose, protein, lipids and oxygen. Since blasts need building blocks and energy to survive and proliferate, and CSF nutrient supplies are scarce, we hypothesized that changes in their metabolism would occur. Such metabolic remodelling (flexible adaptation of metabolism according to tissue location and environmental conditions) is acknowledged as a hallmark of cancers. In a nutrient-deprived environment such as the CSF, metabolic adaptations are likely to be key to ALL survival.Thus, we investigated specific CNS-related metabolic signatures of ALL blasts. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were xenotransplanted with two human ALL cell lines: SEM (t(4;11), MLL-AF4) and REH (t(12;21), ETV6-RUNX1). After engraftment, cells were isolated from the CNS and spleen and RNA sequencing performed (Next Seq500). Transcript quantification was done against both human and mouse transcriptomes, in order to remove any possible mouse RNA contamination. Stearoyl-CoA Desaturase (SCD1) and Fatty Acid Synthase (FASN), key enzymes involved in fatty acid synthesis, were among the most upregulated genes in the CNS. Single sample gene set enrichment analysis confirmed that fatty acid and lipid synthesis showed a clear and significant upregulation in blasts from the CNS compared to the spleen for both cell lines, whereas the opposite result was observed for fatty acid and lipid oxidation. These results were replicated using a publicly available human dataset comparing bone marrow (BM) vs. CNS relapse samples from ALL patients (GEO dataset GSE60926).We went on to characterise the fatty acid profile of normal CSF and paired plasma from both non-leukaemic NSG mice and healthy human volunteers. Both murine and human CSF are about 1000-fold less rich in fatty acids than plasma. Immunostaining of SCD1 and FASN confirmed that both enzymes were strongly expressed on ALL blasts in the leptomeninges of post-mortem samples from patients with CNS-relapse and xenograft models. Murine BM showed equivalent levels of FASN expression but lower SCD1, and western blots confirmed that SCD1 was overexpressed at the protein level by about 2-fold in CNS compared to spleen or BM. We therefore chose to pursue SCD1 as a potential therapeutic target for CNS leukaemia.Two groups of NSG mice were xenografted with SEM cells and treated from day 15 post-transplant with either vehicle or 5mg/kg of the oral small-molecule SCD1 inhibitor MF-438 daily for 11 days. This dose strongly impaired SCD1 activity, since the ratios of oleate to stearate in plasma from vehicle- treated mice were 6 times higher than in plasma from MF-438 treated mice. We confirmed that the drug also reduced intra-cellular ratios of oleate:stearate by approximately 2-fold. Measurement of ALL burden in the leptomeninges showed an approximately 50% reduction in the area of leukaemic infiltrates in brain-skull sections. No difference in the burden of leukaemia in BM could be identified, supporting a specific vulnerability to SCD1 inhibition in the CNS microenvironment.In conclusion, we report a strong fatty acid and lipid signature in ALL blasts retrieved from the CNS environment, and identify the enzyme SCD1 as a critical target. We show a significant upregulation of the enzyme, from RNA levels to enzymatic activity, both in murine and human samples of CNS ALL. Using a small-molecule inhibitor in vivo, we show that inhibiting SCD1 activity reduces the amount of CNS leukaemia. Altogether, our data identify fatty acid metabolism as a novel therapeutic target for CNS leukaemia. DisclosuresHalsey:Jazz Pharmaceuticals: Consultancy, Other: Support to attend educational meetings.

Live cell imaging of highly activated natural killer cells against human hepatocellular carcinoma in vivo

Background aimsTracking administered natural killer (NK) cells in vivo is critical for developing an effective NK cell-based immunotherapy against human hepatocellular carcinoma (HCC). Here the authors established a new molecular imaging using ex vivo-activated NK cells and investigated real-time biodistribution of administered NK cells during HCC progression. MethodsEx vivo-expanded NK cells from healthy donors were labeled with a near-infrared lipophilic cytoplasmic dye, and their proliferation, surface receptor expression and cytotoxicity activity were evaluated. Human HCC HepG2 cells were implanted into the livers of NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) mice. The authors administered 1,1’-dioctadecyltetramethyl indotricarbocyanine iodide (DiR)-labeled NK cells intravenously to non-tumor-bearing and intrahepatic HCC tumor-bearing NSG mice. Fluorescent imaging was performed using a fluorescence-labeled organism bioimaging instrument. Single cell suspensions from the resected organs were analyzed using flow cytometry. ResultsThe fluorescent DiR dye was nontoxic and did not affect the proliferation or surface receptor expression levels of the NK cells, even at high doses. The administered DiR-labeled NK cells immediately migrated to the lungs of the non-tumor-bearing NSG mice, with increased NK cell signals evident in the liver and spleen after 4 h. NK cells migrated to the intrahepatic tumor-bearing livers of both early- and late-stage HCC mice within 1 h of injection. In early-stage intrahepatic tumor-bearing mice, the fluorescence signal increased in the liver until 48 h post-injection and decreased 7 days after NK injection. In late-stage HCC, the NK cell fluorescence signal was the highest in the liver for 7 days after NK injection and persisted for 14 days. The purity of long-term persistent CD45+CD56+CD3− NK cells was highest in early- and late-stage HepG2-bearing liver compared with normal liver 2 weeks after NK injection, whereas highest purity was still observed in the lungs of non-tumor-bearing mice. In addition, Ki-67 expression was detected in migrated human NK cells in the liver and lung up to 72 h after administration. With HepG2 tumor progression, NK cells reduced the expression of NKp30 and NKG2D. ConclusionsAdministered NK cells were successfully tracked in vivo by labeling the NK cells with near-infrared DiR dye. Highly expanded, activated NK cells migrated rapidly to the tumor-bearing liver, where they persisted for 14 days after administration, with high purity of CD45+CD56+CD3− NK cells. Liver biodistribution and persistence of administered NK cells showed significantly different accumulation patterns during HCC progression.

Abstract 5186: Human stem cell humanized mouse model for in vivo evaluation of bispecific antibody efficacy and drug-induced cytokine release

Abstract Bispecific antibodies have shown several promising advantages over monoclonal antibodies and are emerging as one of the growing cancer immunotherapies. Preclinical in vivo models to assess efficacy and cytokine release associated with immune cell engagement are needed. We have developed a human stem cell (HSC) humanized mouse model for testing efficacy and for quantifying cytokine release in response to bispecific antibody treatment. Here we evaluated an EGFR bispecific T cell engager (BiTE), using EGFR-positive MDA-MB-231 orthotopic breast cancer model in CD34+ HSC humanized NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG™) mice. BiTE treatment for 2 weeks resulted in complete tumor regression when dosing was initiated at an average tumor volume of around 100 mm3. In addition, BiTE treatment for 4 weeks effectively inhibited the tumor growth when dosing mice bearing larger tumors of around 400mm3 in volume. Sixteen hours after the first two doses as compared to PBS control, human cytokines IFN-γ, IL-6, and IL-10 were elevated. Cytokine levels then returned to the baselines following continuous dosing. We observed a similar time course and levels of cytokine release between these two BiTE-treated groups with different tumor sizes. T cell activation in the peripheral blood after the last dose was assessed by flow cytometry analysis. In the BiTE-treated group, T cell percentage decreased, as compared to the PBS control. Both CD4+ and CD8+ T cell subsets showed an increased frequency of CD69+ cells after BiTE treatment. Evaluation of drug efficacy and cytokine release in response to BiTE treatment using PBMC-humanized NSG™ mice with and without MDA-MB-231 tumors are underway and results will be discussed. Citation Format: Li-Chin Yao, Danying Cai, Roberta Aliperta, Allison Colthart, Werner Meier, Mingshan Cheng, James G. Keck. Human stem cell humanized mouse model for in vivo evaluation of bispecific antibody efficacy and drug-induced cytokine release [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5186.

Immunodeficient mice are better for modeling the transfusion of human blood components than wild-type mice.

Animal models are vital to the study of transfusion and development of new blood products. Post-transfusion recovery of human blood components can be studied in mice, however, there is a need to identify strains that can best tolerate xenogeneic transfusions, as well as to optimize such protocols. Specifically, the importance of using immunodeficient mice, such as NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, to study human transfusion has been questioned. In this study, strains of wild-type and NSG mice were compared as hosts for human transfusions with outcomes quantified by flow cytometric analyses of CD235a+ erythrocytes, CD45+ leukocytes, and CD41+CD42b+ platelets. Complete blood counts were evaluated as well as serum cytokines by multiplexing methods. Circulating human blood cells were maintained better in NSG than in wild-type mice. Lethargy and hemoglobinuria were observed in the first hours in wild-type mice along with increased pro-inflammatory cytokines/chemokines such as monocyte chemoattractant protein-1, tumor necrosis factor α, keratinocyte-derived chemokine (KC or CXCL1), and interleukin-6, whereas NSG mice were less severely affected. Whole blood transfusion resulted in rapid sequestration and then release of human cells back into the circulation within several hours. This rebound effect diminished when only erythrocytes were transfused. Nonetheless, human erythrocytes were found in excess of mouse erythrocytes in the liver and lungs and had a shorter half-life in circulation. Variables affecting the outcomes of transfused erythrocytes were cell dose and mouse weight; recipient sex did not affect outcomes. The sensitivity and utility of this xenogeneic model were shown by measuring the effects of erythrocyte damage due to exposure to the oxidizer diamide on post-transfusion recovery. Overall, immunodeficient mice are superior models for xenotransfusion as they maintain improved post-transfusion recovery with negligible immune-associated side effects.

Abstract D110: Same patient, different tumor. Does heterogeneity of pancreatic cancer matter? Differences in growth rates of patient-derived xenografts from individual patients

Abstract Abstract Background Pancreatic cancer (PC) is one of the leading causes of cancer mortality and soon to be the second-leading cause of cancer death in the US by 2030. Patient derived xenografts (PDX) represent an opportunity to better understand the biology driving higher incidence and mortality. Racial disparities exist and Blacks have worse clinical outcomes than any other race. We hypothesize that the PDX tumors from the same patient will grow at differing rates including PDX development from patients of different races/ethnicities. Methods Under an IRB approved protocol, patients with the diagnosis of pancreatic adenocarcinoma (PDAC) were recruited for tissue donation. After surgical harvest of PC specimen, patient-derived xenograft (PDX) models were derived in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. After initial passage from operation room, tumors from each individual patient were allowed to grow and subsequently divided and passed into 5 NSG mice and tumor growth rates were assessed by digital caliper. Patient race/ethnicity were de-identified as G and number of passages (p) from initial implantation were considered. Growth was assessed from 41 to 56 days. Coefficient of variation was attained and demonstrated the variability in growth values within each graft. Results PDX grafts (GXpX) were measured from 41 to 56 days. G1p5 and G2p1 were from non-Hispanic White patients and G3p3 was from a Black patient with PDAC. G1p5 grew an average of 0.25 mm per day and tumor size at 55 days after implantation were 1.0 cm, 1.4 cm, and 1.7 cm with a coefficient of variation of 20.98%. G2p1 grew an average of 0.17 mm per day and tumor sizes at 56 days were 0.7 cm and 1.3 cm with a coefficient of variation of 42.42%. G3p3 grew an average of 0.16 mm per day and tumor size at 41 days were 1.3 cm, 1.3 cm, and 0.7 cm with a coefficient of variation of 31.49%. The growth rate of mice implanted with patient tumor G2p1 demonstrated more growth variation than G3p3 and G1p5. Conclusion PDX from the same patient and different races exhibit differing growth rates over time. Each PDX should be considered as a unique tumor given its gross heterogeneity and possible genetic drift within the same tumor. Recommendations for PDX model use should include tumor measurements of each individual xenograft throughout time intervals throughout experiments especially when considering therapeutics. These important observations will leverage our understanding of much needed preclinical tumor models in PDAC and help improve our understanding of the biology of racial disparities in pancreatic cancer. Citation Format: Michael U Maduka, Patrick W Underwood, Andrea N Riner, Miles E Cameron, Jose G Trevino. Same patient, different tumor. Does heterogeneity of pancreatic cancer matter? Differences in growth rates of patient-derived xenografts from individual patients [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr D110.

Aged Plasma and Plasma‐Derived Factors Drive Detrimental Effects in the CNS of Young Mice

Aging results in cognitive decline and molecular changes that contribute to the risk of developing aging‐associated disease. As the aging population is rapidly increasing, with those over 60‐years‐old set to double by 2050, there is urgent need for a better understanding of the aging process and development of therapeutics for diseases of aging. Recent publications have shown that some aging‐associated phenotypes can be driven by plasma and plasma‐derived factors. In these studies, we sought to identify and validate detrimental effects of aged plasma and plasma‐derived factors on cognitive, histological, and molecular readouts in order to discover novel therapeutic targets for age‐related diseases. Our data show that administration of aged human plasma into young immunodeficient NSG (NOD.Cg‐Prkdcscid Il2rgtm1Wjl/SzJ) mice results in a decrease in neurogenesis and mild impairment in cognition. These results indicate that factors in aged human plasma can drive detrimental effects in the brains of young NSG mice. Plasma proteomics have identified aging signatures and specific proteins that are potential drivers of these negative effects in aging, and beta‐2 microglobulin (B2M) has been shown to be a pro‐aging factor with deleterious effects on cognition and neurogenesis. To confirm B2M as a negative factor, we treated young mice (WT; C57BL/6J) with recombinant human B2M and assessed changes in behavioral performance and histological markers. We found that systemic administration of B2M resulted in cognitive impairment during contextual fear conditioning and decreased neurogenesis, suggesting that it is a driver of negative effects during aging. Our data elucidate molecular pathways that contribute to the aging process and provide promising targets for developing translational therapeutics.Support or Funding InformationStudies were funded by Alkahest.

Spectrum of Posttransplant Lymphoproliferations in NSG Mice and Their Association With EBV Infection After Engraftment of Pediatric Solid Tumors.

Pediatric patients receiving solid organ transplants may develop lymphoproliferative diseases, including graft-versus-host disease (GvHD) and posttransplant lymphoproliferative diseases (PTLDs). We characterized lesions in 11 clinically ill NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice that received pediatric-patient-derived solid tumors (PDXs) and developed immunodeficiency-associated lymphoproliferations comparable to GvHD and PTLDs over a period of 46 to 283 days after implantation. Lymphoproliferations were diffusely positive for human-specific biomarkers, including NUMA1, CD45, and CD43, but lacked immunoreactivity for murine CD45. Human immune cells were CD3-positive, with subsets having immunoreactivity for CD4 and CD8 as well as PAX5, CD79a, and IRF4, resulting from populations of human T and B cells present within the xenotransplants. Tissues and organs infiltrated included mucocutaneous zones (oral cavity and perigenital and perianal regions), haired skin, tongue, esophagus, forestomach, thyroid, salivary glands, lungs, liver, kidneys, spleen, lymph nodes, bone marrow, and brain. In 4 of 5 mice with PTLD, Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) were detected by in situ hybridization in PAX5+ human B cells associated with the PDX (n = 1/4) or with engrafted human immune cells at other anatomic locations (n = 4/11). One of the 4 mice had an EBV-associated human large B-cell lymphoma. NSG mice receiving xenotransplants can develop combinations of GvHD, EBV-driven PTLD, and B-cell lymphoma similar to those occurring in human pediatric patients. Therefore, pediatric xenotransplants should undergo histopathologic and immunohistochemical assessment upon collection to ensure that the specimen is not a lymphoma and does not contain lymphoma cells because these neoplasms can morphologically mimic small round blue cell pediatric solid tumors.

Targeted Inhibition of the NUP98-NSD1 Fusion Oncogene in AML

Background: NUP98-NSD1 positive AML is a poor prognostic subgroup within pediatric and adult AML (Thol et al., 2013). However, targeted therapeutics for these AML patients are not available to date. As a result of the NUP98-NSD1 fusion, NSD1 causes H3K36 hypermethylation of HOXA genes, which contributes to myeloid progenitor cell immortalization and results in AML (Wang et al., 2007). Therefore, we hypothesized that inhibition of the methyltransferase activity of NSD1 could be an effective treatment strategy for NUP98-NSD1 AML patients. Here, we assessed the efficacy of NSD1 inhibitor suramin and NUP98-NSD1-directed siRNA-containing lipid nanoparticles (LNP) in a preclinical patient-derived xenograft (PDX) model of NUP98-NSD1 leukemia. Methods: A NUP98-NSD1 positive AML patient was screened through nested PCR and Sanger sequencing. Bone marrow cells from this patient were serially transplanted into NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice to establish a NUP98-NSD1 PDX model. During serial transplantations, an alternative NUP98-NSD1 fusion gene appeared. Flow-cytometry was used to check the engraftment and immunophenotype of engrafted cells. Effective siRNAs against each of the two fusion genes were developed. The microfluidic mixing technology, Nanoassemblr was used to package siRNA into LNPs and the Zetasizer was used to characterize them. Fifteen days after transplantation, suramin and solvent treatment were initiated in 9 mice per group with 10 mg/kg suramin (2 days/week for 10 weeks). For LNP treatment, treatment was initiated 3 weeks after transplantation with a once daily dose of 2.5 mg/kg on days 1-3 and then every third day thereafter for a total of 17 injections. Results: Besides the NUP98-NSD1 translocation, our patient had a FLT3-ITD mutation and a normal karyotype. In our PDX model, the engraftment of human leukemia cells reached up to 90% after ten weeks of transplantation. In successive transplantations up to the 6th generation, the NUP98-NSD1 fusion was confirmed in leukemic cells, supporting the importance of the fusion for leukemia development and stability of the model. In later transplantations, a minor clone of NUP98-NSD1 was identified. A high blast count, high WBC count, increased spleen weight, and a low hemoglobin and platelet count at death demonstrated the development of acute leukemia. High expression of myeloid markers (e.g. CD33, 99%, N=9) and negligible expression of lymphoid markers (CD3, 2%; CD19, 2%; N=9) confirmed acute myeloid leukemia. In the suramin treatment study, the mean human leukemic cell engraftment was similar between the control and treatment groups at the start of treatment (0.51%, N=9 and 0.71%, N=9, respectively), but was lower in suramin treated mice after 4 and 8 weeks of treatment (4 weeks: CTRL, 4,8%; suramin, 2,66%, P=0.1; 8 weeks: CTRL, 87,3%; suramin: 66,5%, P=0.016). No significant effect was seen on the immunophenotype of suramin and control treated leukemia cells. Suramin treatment significantly prolonged the median survival of mice compared to control mice (126 vs 114 days after transplantation, P=0.008). To establish the siRNA-LNP treatment, we identified one siRNA against each NUP98-NSD1 clone that reduced expression levels by 78% and 89.5% in the major and minor clones, respectively. The effective siRNAs were modified to increase their in vivo stability and were packaged into LNPs and used in vivo. We started the treatment when the engraftment was similar in both control LNP and NUP98-NSD1 LNP groups (0.93%, N=7 and 1.25%, N=6, respectively). After 3 weeks of treatment, LNP uptake was 99.3% and 99.2% in the CTRL LNP and NUP98-NSD1 LNP groups, respectively. The mean engraftment was lower in NUP98-NSD1 LNP mice after 5 and 8 weeks of treatment (5 weeks: CTRL LNP, 15%; NUP98-NSD1 LNP, 4.6%, P= 0.08; 8 weeks: CTRL LNP, 94.8%; NUP98-NSD1 LNP, 55.83%, P=0.007). Importantly, the NUP98-NSD1 siRNA-LNP treated mice showed a significant survival benefit compared to CTRL siRNA-LNP treated mice (106 vs 82 days after transplantation, P=0.02). Conclusions: In summary, our findings demonstrate that targeted inhibition of NUP98-NSD1 either through siRNA-LNP or suramin delays leukemia development in vivo and prolongs the survival of mice carrying a NUP98-NSD1 positive AML. Our results provide the rationale for the evaluation of NSD1 methyltransferase inhibitors and siRNA-LNP formulations in NUP98-NSD1 positive AML. Disclosures Heuser: Bayer Pharma AG, Berlin: Research Funding; Synimmune: Research Funding.

Abstract 1537: Polatuzumab Vedotin alone or in-combination with anti-CD20 antibody significantly enhanced overall survival in xenografted NSG mice against rituximab sensitive and resistant Burkitt Lymphoma (BL) and Primary Mediastinal B-cell Lymphoma (PMBL)

Abstract Background: Patients with relapsed or refractory B-cell Non-Hodgkin Lymphoma (B-NHL) have an unfavorable prognosis with few treatment options (Goldman/Cairo et al. Leukemia, 2013). It is therefore critical to investigate and to develop targeted translational strategies in BL/PMBL in order to reduce acute morbidities, decrease late effects, and provide new options for those with recurrent disease. Polatuzumab Vedotin (PV) has been demonstrated to possess significant preclinical activity against indolent CD79b+NHL (Polson et. al. Can. Res. 2009). We previously observed that obinutuzumab vs. rituximab (anti-CD20 mAb) significantly increased overall survival against xenografted NSG mice in BL (Awasthi/Cairo et al., BJH 2015). However, synergistic effects of PV with obinutuzumab against mature PMBL/BL are unknown. Objective: To determine the efficacy of the PV alone or in-combination with rituximab/obinutuzumab against rituximab-sensitive/resistant BL (Raji & Raji4RH) and PMBL (Karpas1106P) cell lines. Methods: Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were xenografted with intravenous injections of Luc+ BL and PMBL cells. Mice were divided into following groups: PBS, isotype control, PV, anti-CD79B and MMAE (5mg/kg) and or/with obinutuzumab/RTX (20mg/kg) for 8 weeks with 106 ex.PBNK cells. Animals were monitored for tumor burden progression/regression and survival for up to 12-30 weeks via BLI using the IVIS spectrum system as previously described (Awasthi/Cairo et al BJH, 2015). Results: The probability of OS in B-NHL xenografts NSG mice receiving PV alone was significantly increased compared to anti-CD79b or isotype control in Raji (35.5 vs.17 vs.19.5 days, p=0.0001, 0.0003), Raji4RH (50 vs.18 vs.18.5 days, p=0.0001, 0.0001) and Karpas1106P (150 vs 89 vs 64 days, p=0.03, 0.003), respectively. Furthermore, The probability of OS of B-NHL xenogrfats NSG mice receiving PV+obinutuzumab+NK significantly increased compared to PV+rituximab+NK in Raji (95.5 vs. 50.4 days, p=0.03 ) and Raji4RH (185 vs.47 days, p=0.05). Moreover, obinutuzumab+NK vs. rituximab+NK also significantly increased over all survival both in Raji (92.5 vs. 52 days, p=0.05) and Raji4RH (80.5 vs. 40.0 days, p=0.04) respectively. Conclusion: Our preliminary data indicates that PV significantly increased overall survival in BL and PMBL NSG xenografts compared to anti-CD79b Ab alone. Furthermore, PV in combination with obinutuzumab+NK-cells significantly enhance improved OS in BL and PMBL NSG xenografts compared to obinutuzumab or PV alone. Citation Format: Aradhana Awasthi Tiwari, Dina Edani, Janet Ayello, Christian Klein, Mitchell S. Cairo. Polatuzumab Vedotin alone or in-combination with anti-CD20 antibody significantly enhanced overall survival in xenografted NSG mice against rituximab sensitive and resistant Burkitt Lymphoma (BL) and Primary Mediastinal B-cell Lymphoma (PMBL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1537.

Abstract A023: Highly enriched memory stem T-cell subsets (TSCM) expressing a novel CAR30 have enhanced antitumor effect in Hodgkin lymphoma

Abstract Background: Up to 20% of patients with advanced Hodgkin lymphoma (HL) are not cured with current chemo-immunotherapy treatments. Adoptive immunotherapy (AIT) with mature T-cells modified with a chimeric antigen receptor (CAR) holds promise for the treatment of various types of B cell lymphoma. The CD30 antigen, expressed in virtually all HL tumor cells, is absent in most healthy tissues, thus representing an ideal target for AIT of HL. In contrast to CAR19 for B cell malignancies, efficacy of CAR30 T-cells for Hodgkin and other CD30 lymphomas remains modest. Thus, novel approaches are needed to significantly improve the clinical efficacy of this approach. To this end, we used a novel antigen binding domain of a CD30 mAb, unaffected by soluble CD30 protein, as part of a chimeric antigen receptor (CAR) coupled to the 4.1BB and ζ chain endodomains, and transduced memory stem T-cells (TSCM) in an effort to ensure efficient engraftment, prolonged persistence, and enhancement of in vivo antitumor activity. To evaluate the feasibility of ex vivo expansion and antitumor efficacy of genetically-modified TSCM with a novel CD30-specific CAR that recognizes a membrane-proximal epitope of the CD30 molecule for the treatment of HL. Methods: A second-generation CD30-41BBz-EGFRt CAR was developed using a scFv that recognizes a membrane-proximal epitope of the CD30 protein. TSCM cells were generated with IL-7, IL-15 and IL-21, and transduced on day 2 of culture with a third-generation lentiviral vector encoding the CD30-CAR. The HL derived cell line L540 was used as tumor model. Tumor-specific cytotoxicity and cytokines were analysed at 24 hours. The in vivo antitumor efficacy was tested in a NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mouse model of HL xenografted with 2,5x10^6 L450 cells. Results: After 10 days, TSCM represented the majority of T-cells in culture (50.38±5.47 % for CD4+ and 70.84 ± 3.36 % for CD8+ TSCM, respectively) with a very high expression of CD30-CAR (77 ± 3.64 % in CD4+ and 83.12 ± 4.25 % in CD8+ TSCM, respectively). Expression of CD30 protein in TSCM increased over the culture period, peaking at day 8 in both CD4+ (17.5 ± 4.17 %) and CD8+ TSCM (42.37 ± 5.75 %), respectively. Despite these high levels of CD30 expression, TSCM expanded ex vivo very efficiently (CD4+ CD30-CAR: 97.24 ± 26.2 fold expansion, and CD8+ CD30-CAR: 96.79 ± 22.84 fold expansion, respectively), although lower than TSCM transduced with a CD19-CAR (CD4+ CD19-CAR: 161.96±35.9 fold expansion, and CD8+ CD19-CAR: 163.25 ± 26.51, fold expansion, respectively). Bulky CD30-CAR TSCM mediated very efficient in vitro tumor cytotoxicity (tumor cell death at 5:1 ratio: 85.25% vs. 0% with untransduced TSCM), while control CD30- targeT-cells were not killed. In addition, we evaluated the antitumor efficacy of CD30-CAR TSCM in vivo. Upon adoptive transfer of 10x10^6 bulk (CD4+ and CD8+) TSCM into NSG mice engrafted with L540 (2,5x10^6 subcutaneously), complete eradication of HL was observed. In addition, in a stress L540 model where equal numbers of CAR30 T-cells were infused at a low, suboptimal dose (i.e, 50% of effective T-cell dose to induce complete remission), highly enriched CAR30 TSCM (>70% of TSCM) had significantly antitumor effect compared to the infusion of CAR30 T-cells predominantly represented by effector cells (<30% TSCM) (mean survival 54 days vs 40 days, respectively; P< 0.01). Conclusions: We demonstrate that TSCM can be efficiently transduced and ex vivo expanded with a novel CD30-CAR that surpasses fratricide killing of CD30 expressing T-cells and confers potent in vivo antitumor efficacy against HL. Importantly, highly enriched CAR30 TSCM products have enhanced antitumor properties compared to those comprising predominantly effector cell subsets. These proof-of-concept studies support the use of a refined CAR30 with highly enriched TSCM cell products for improving clinical efficacy of adoptive immunotherapy of patients with HL. Citation Format: Carmen Alvarez-Fernández, Laura Escribà-Garcia, Ana Carolina Caballero, Jorge Sierra, Javier Briones. Highly enriched memory stem T-cell subsets (TSCM) expressing a novel CAR30 have enhanced antitumor effect in Hodgkin lymphoma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A023.

CD79a Is Associated with Central Nervous System Infiltration of Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

▪Despite the advances in the treatment of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), infiltration of the central nervous system (CNS) remains a clinical challenge. Certain cytogenetic subtypes such as E2A-PBX1-and BCR-ABL-positive BCP-ALL confer a higher risk for CNS involvement initially and for CNS relapse. Novel strategies to predict CNS and to eradicate leukemic cells from the CNS are subjects of ongoing research.In order to identify targets with diagnostic and therapeutic relevance, comparative RNA-sequencing was performed with patient derived xenograft (PDX) blasts from 5 E2A-PBX1-positive patients, recovered from the bone marrow (BM) and from the CNS of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Differential gene expression analysis revealed the upregulation of various genes of the pre-B cell receptor complex, particularly the signaling component CD79a (Igα) in blasts recovered from the CNS as compared to blasts from the BM. We then investigated the impact of CD79a on CNS infiltration in vivo and in patients. CD79a was downregulated by short-hairpin RNA (shRNA) mediated knockdown in the E2A-PBX1 positive cell line 697. Proliferation rates of 697-shCD79a cells and control-transfected 697 cells (697-shCtrl) in vitro were similar. Furthermore, NSG mice injected with 697-shCD79a cells showed comparable survival times, as well as similar blast infiltration in spleen and BM as animals injected with 697-shCtrl cells. However, downregulation of CD79a led to a significantly lower number of CNS-positive mice (4/15, 26%) as compared to control animals (7/10, 70%) (p=0.0486, Figure A). This indicates that CD79a is not critically involved in proliferation and peripheral engraftment, but in CNS infiltration of E2A-PBX1 positive 697 cells in vivo.To test if CD79a also affects CNS involvement in BCR-ABL-positive leukemia, a murine/murine transplantation model was used. B-cells isolated from CD79a-knockout (CD79a-KO) or wildtype mice (CD79a-Ctrl) were stably transfected with a BCR-ABL fusion gene and cultured independent of cytokines, thereby inducing malignant transformation. Both cell lines were subsequently injected into recipient NSG mice (n=8/group) and leukemic development was followed. The experiment was terminated when all control mice had developed leukemic symptoms and mice were analyzed for leukemic engraftment. A further CD79a-KO group was included for survival analysis. Median spleen volume as a surrogate of leukemic infiltration was significantly lower in mice injected with CD79a-KO as compared to CD79a-Ctrl cells (0.35 cm³ vs. 0.06 cm³; p=0.0001). Median blast percentages in spleens and BM were also markedly reduced (75.3% vs. 5.8%; p=0.0001 and 61.0% vs. 4.5%; p=0.0001, respectively). Importantly, none of the animals in the CD79a-KO group showed blasts in the CNS as assessed by histology whereas blasts were present in all of the animals in the CD79a-Ctrl group. Finally and most importantly, NSG-mice injected with CD79a-KO cells showed a highly significant prolongation in median survival as compared to mice with CD79a-Ctrl cells (29 days vs. 95 days; p=0.0001, Figure B). Altogether, these data suggest that in a model of BCR-ABL-positive leukemia, absence of CD79a impacts the engraftment of blasts in vivo, in the CNS and other leukemic niches.To further validate our findings in patient material, we measured CD79a protein expression in PDX cells from an E2A-PBX1- and a BCR-ABL-positive patient serially transplanted into NSG mice for three passages. For both entities and in all passages, CNS blasts showed a higher CD79a expression than blasts isolated from the bone marrow. In order to assess if CD79a can be used as a marker to predict CNS involvement in patients, CD79a mRNA levels were measured in a selected cohort of 98 pediatric BCP-ALL patients, which contained 26 CNS-positive patients matched to 72 CNS-negative patients. CNS-positive patients showed significantly higher mRNA levels of CD79a than CNS-negative patients (p=0.0225, unpaired t-test, Figure C) suggesting that CD79a may be of value as a potential diagnostic marker for initial CNS involvement in BCP-ALL.Our results indicate a role of CD79a in proliferation and CNS infiltration of BCP-ALL blasts in experimental settings and patients. We intend to prospectively evaluate CD79a as a prognostic marker, which may also be a therapeutic target in CNS-positive BCP-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.