Abstract 6591: Antibody-based redirection of meditope-CAR T cells selectively kill antigen bearing tumor cells

BackgroundThe use of CAR T cells for solid tumors has a number of challenges, such as lack of tumor-specific targets, CAR T cell exhaustion, and the immunosuppressive tumor microenvironment. To address these challenges, AffyImmune has developed technologies to affinity tune and track CAR T cells in patients. The targeting moiety is affinity tuned to preferentially bind to tumor cells overexpressing the target while leaving normal cells with low basal levels untouched, thereby increasing the therapeutic window and allowing for more physiological T cell killing. The CAR T cells are designed to express SSTR2 (somatostatin receptor 2), which allows for the tracking of CAR T cells in vivo via PET/CT scan using FDA-approved DOTATATE.MethodsAIC100 was generated by affinity tuning the I-domain of LFA-1, the physiological ligand to ICAM-1. Various mutants with 106-fold difference in affinity were evaluated for affinity. This allowed structure activity relationships to be conducted using CAR T cells expressing the various affinity mutants against targets with varying antigen densities. The variant with micromolar affinity was clearly the most effective in non-clinical animal models. AIC100 is currently being evaluated to assess safety, CAR T expansion, tumor localization, and preliminary activity in patients with advanced thyroid cancer in a phase I study (NCT04420754). Our study uses a modified toxicity probability interval design with three dosage groups of 10 x 106, 100 x 106, and 500 x 106 cells.ResultsPreclinical studies demonstrated greater in vivo anti-tumor activity and safety with lower affinity CAR T cells. A single dose of AIC100 resulted in tumor elimination and significantly improved survival of animals. AIC100 activity was confirmed in other high ICAM-1 tumor models including breast, gastric, and multiple myeloma. In a Phase I patient given 10-million CAR T cells, near synchronous imaging of FDG and DOTATATE revealed preliminary evidence of transient CAR T expansion and tumor reduction at multiple tumor lesions, with the peak of CAR T density coinciding with the spike in CAR T numbers in blood.ConclusionsWe have developed affinity tuned CAR T cells designed to selectively target ICAM-1 overexpressing tumor cells and to spatiotemporally image CAR T cells. Near-synchronous FDG and DOTATATE scans will enhance patient safety by early detection of off-tumor CAR T activity and validation of tumor response. We anticipate that our ‘tune and track’ technology will be widely applicable to developing potent yet safe CAR T cells against hard-to-treat solid cancers.Trial RegistrationNCT04420754Ethics ApprovalIRB number19-12021154IACUC (animal welfare): All animal experiments were performed in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals. Animal handling protocols were approved by the Institutional Laboratory Animal Use and Care Committee of Weill Cornell Medicine (Permit Number: 2012–0063).

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CHIMERIC ANTIGEN RECEPTOR FOR CHRONIC LYMPHOCYTIC LEUKEMIA - A REVIEW

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2019.v12i1.29642 ◽

2019 ◽

Vol 12 (1) ◽

pp. 62

Author(s):

Kiruthiga Raghunathan ◽

Brindha Devi P

Keyword(s):

Bone Marrow ◽

T Cells ◽

Radiation Therapy ◽

Chronic Lymphocytic Leukemia ◽

Tumor Cells ◽

Chimeric Antigen Receptor ◽

Lymphocytic Leukemia ◽

Normal Cells ◽

Car T Cells ◽

Car T

Chronic lymphocytic leukemia cancer is a deadly one which affects the bone marrow from making it to produce more amounts of white blood cells in the humans. This disease can be treated either by radiation therapy, bone marrow transplantation, chemotherapy, or immunotherapy. In radiation therapy, the ionizing radiation is used toward the tumor cells, but the main drawback is the radiation may affect the normal cells as well. To overcome this drawback, immunotherapy chimeric antigen receptor (CAR) is used. These CAR cells will target only the antigen of the tumor cells and not damage the normal cells in the body. In this therapy, the T-cells are taken either from the patients or a healthy donor and are engineered to express the CARs which are called as CAR-T-cells. When these CAR-T-cells come in contact with the antigen present on the surface of the tumor cells, they will get activated and become toxic to the tumor cells. This new class of therapy is having a great prospect in cancer immunotherapy.

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CD19-Targeted Donor T Cells Exert Potent Graft Versus Lymphoma Activity and Attenuated Gvhd

Blood ◽

10.1182/blood.v120.21.451.451 ◽

2012 ◽

Vol 120 (21) ◽

pp. 451-451 ◽

Cited By ~ 2

Author(s):

Arnab Ghosh ◽

Marco L. Davila ◽

Lauren F. Young ◽

Christopher Kloss ◽

Gertrude Gunset ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Tumor Cells ◽

Acute Gvhd ◽

Tcr Signaling ◽

Car T Cells ◽

Donor T Cells ◽

Car T

Abstract Abstract 451 Chimeric antigen receptors (CAR) represent a potent strategy to target T cells against selected tumor antigens. Ongoing clinical trials indicate that autologous T cells expressing CARs targeting CD19, a B cell-associated antigen, can induce complete remission and B cell aplasia in patients with B cell malignancies. Donor CD19-CAR+ T cells could potentially be used to treat recipients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), but the risk of alloreactivity mediated by endogenous T cell receptors (TCR) triggering an acute GVHD is not known. This is partly due to the absence of in vivo models to study the relative effects of CAR and endogenous TCR signaling. For the first time, we have evaluated the relative effects of CD19-targeted donor T cells on the elimination of CD19+ B cells and endogenous TCR-mediated alloreactivity in mouse models of allo-HSCT. We generated a panel of retroviral vectors encoding mouse CD19-specific CARs: as a control, CD19-delta, a tail-less CAR lacking the CD3ζ signaling domain; CD19z1, which signals through its CD3ζ endodomain; and CD19-28z, which signals through CD28 and CD3ζ (Figure 1A). CD19z1+ and CD19-28z+ T cells mediated specific lysis of CD19-expressing tumors in vitro, while CD19-delta+ T cells did not. In order to assess the anti-tumor capacity of CD19-CAR+ T cells in vivo, we transferred the transduced B6 donor T cells into lethally irradiated BALB/c recipients that were administered T cell-depleted allografts and CD19+ lymphoma A20-TGL (B6–> BALB/c+A20-TGL). CD19-CAR+ T cells (CD19z1 and CD19-28z) mediated clearance of A20 tumor cells visualized by in vivo imaging of luciferase-expressing tumor cells (Figure 1B and data not shown) and significantly improved tumor free survival. CD19-CAR+ B6 T cells could sustain prolonged B cell hypoplasia when adoptively transferred into lethally irradiated haploidentical CBF1 recipients of T cell-depleted allografts (B6–> CBF1, Figure 1C). These data indicate that under alloreactive conditions, donor CD19-CAR+ T cell signaled through the CAR leading to specific elimination of CD19+ tumors and B lineage cells. In order to determine the risk of GVHD, we transferred the donor CD19-CAR+ T cells into haploidentical HSCT recipients. Interestingly, CD19-CAR+ T cells mediated significantly less acute GVHD, resulting in improved survival and lower GVHD scores (Figure 1D). Donor CD19-delta+ T cells however mediated lethal GVHD, indicating that the endogenous TCR mediated strong alloreactivity in the absence of CAR signaling. Similar results were obtained from experiments using MHC-mismatched (B6–> BALB/c) models. It is known that signaling through endogenous TCR is accompanied by down-regulation of surface TCR expression. We found significant decreases in surface CD3ϵ, TCRβ and CD90 expressions in donor CD19-delta+ T cells under alloreactive conditions. In contrast, donor CD1928z+ T cells failed to down-regulate surface TCR expression under similar conditions, suggesting that endogenous TCR function was altered in CAR-activated T cells. In the context of allo-HSCT, preferential CAR signaling at the expense of alloreactive endogenous TCR signaling may thus lead to reduced alloreactivity and attenuation of GVHD. These results provide the first pre-clinical evidence suggesting that CAR-modified, unselected donor T cells may be safely applied in an allogeneic context. Disclosures: No relevant conflicts of interest to declare.

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Targeting An Ancient Retrovirus In Tumor Using Engineered T Cells

Blood ◽

10.1182/blood.v122.21.4206.4206 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4206-4206

Author(s):

Janani Krishnamurthy ◽

Brian Rabinovich ◽

Simon Olivares ◽

Mi Teijuan ◽

Kirsten Switzer ◽

...

Keyword(s):

Clinical Trials ◽

T Cells ◽

Tumor Cells ◽

Treatment Strategy ◽

Conflicts Of Interest ◽

Sleeping Beauty ◽

Normal Organ ◽

Car T Cells ◽

Car T ◽

Env Protein

Abstract Human endogenous retroviruses (HERVs) are ancient viruses forming 8% of human genome. One subset of HERVs, the HERV-K has recently been found to be expressed on tumor cells including melanoma, breast cancer and lymphoma but not on normal body cells. Thus, targeting HERV-K protein as a tumor associated antigen (TAA) may be a potential treatment strategy for tumors that are resistant to conventional therapies. One approach to improve therapeutic outcome is by infusing T cells rendered specific for such TAAs preferentially expressed on tumor cells. Recognition of cell-surface TAAs independent of major histocompatibility complex can be achieved by introducing a chimeric antigen receptor (CAR) on T cells using gene therapy. This approach is currently being used in our clinical trials adoptively transferring CD19-specific CAR+ T cells into patients with B-lineage malignancies. Preliminary analysis of HERV-K env protein expression in 268 melanoma samples and 139 normal organ donor tissues using immunohistochemistry demonstrated antigen expression in tumor cells and absence of expression in normal organ tissues. The scFv region from a mouse monoclonal antibody to target HERV-K env was used to generate a CAR and cloned into Sleeping Beauty (SB) plasmid for stable expression in T cells. HERV-K-specific CAR+T cells were selectively propagated ex vivo on artificial antigen presenting cells (aAPC) using an approach already in our clinical trials. Indeed, after genetic modification of T cells and selection on HERV-K+ aAPC, over 95% of propagated T cells stably expressed the introduced HERV-K-specific CAR and exhibited redirected specificity for HERV-K+ melanoma (Figure 1). Further, the adoptive transfer of HERV-K-specific CAR+T cells killed metastatic melanoma in a mouse xenograph model. While we have chosen melanoma as our tumor model, this study has the potential to be applied to other malignancies, including lymphoma and myeloma due to restricted expression of HERV-K envelope (env) protein on these tumor cells. These data demonstrate that it is feasible to generate T cells expressing a HERV-K-specific CAR using a clinically-appealing approach as a treatment strategy for HERV-K env+ tumors. Disclosures: No relevant conflicts of interest to declare.

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Piggybac Transposon Mediated CD19 CAR-T Cells Derived from CD45RA-Positive PBMCs Possess Potent and Sustained Antileukemic Function

Blood ◽

10.1182/blood-2021-145863 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2807-2807

Author(s):

Masaya Suematsu ◽

Shigeki Yagyu ◽

Nobuyoshi Nagao ◽

Susumu Kubota ◽

Yuto Shimizu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Research Funding ◽

Stress Test ◽

Transduction Efficiency ◽

Antigen Stimulation ◽

Car T Cells ◽

Car T

Abstract Background: The quality of chimeric antigen receptor (CAR)-T cell products, including the expression of memory and exhaustion markers, has been shown to influence their long-term functionality. We previously demonstrated that piggyBac (PB) transposon-mediated CD19 CAR-T cells exhibit memory-rich phenotype that is characterized by a high proportion of CD45RA+/CCR7+ T cell fraction. To further investigate the favorable phenotype of PB-CD19 CAR-T cells, we generated PB-CD19 CAR-T cells from CD45RA+ and CD45RA− peripheral blood mononuclear cells (PBMCs) (RA+ CAR and RA− CAR, respectively), and compared their phenotype and antitumor function. Methods: CD45RA+ and CD45RA− PBMCs were isolated by magnetic selection from whole PBMCs, then the CD19-CAR transgene was transduced into these cells using the PB transposon system, as described previously. Transduction efficiency of CD19 CAR transgene was determined 24 hours by flow cytometry after transduction. The phenotype of CD19 CAR-T was evaluated by flow cytometry on day 14. High throughput RNA sequencing was performed to see the T cell activation/exhaustion profile upon antigen stimulation. Sequential killing assays were performed by adding fresh tumor cells into CAR-T cells co-cultured with tumor cells every three days by restoring an effector target ratio of 1:1. To see the durable antitumor efficacy in vivo, we performed in vivo stress test, in which CAR T-cells dosage was lowered to the functional limits, so that these CAR-T cells should be maintained and expanded in vivo, to achieve the antitumor efficacy. We injected 5 x 10 5 of firefly luciferase-labeled CD19+ tumor cells (REH) into NSG mice via tail vein, then these mice were treated with 1 x 10 5 of CD19 RA+ CAR-T, RA− CAR-T, or control CAR-T cells, respectively, at day 6 after the tumor injection. Results: RA+ CAR T cells demonstrated better transient transduction efficiency 24 h after transduction (RA+ CAR-T: 77.5 ± 9.8% vs RA− CAR-T: 39.7 ± 3.8%), and superior expansion capacity after 14 days of culture than RA− CAR-T cells (RA+ CAR-T: 32.5 ± 9.3-fold vs RA− CAR-T: 11.1 ± 5.4-fold). RA+ CAR-T cells exhibited dominant CD8 expression (RA+ CAR-T: 84.0 ± 3.4% vs RA− CAR-T: 34.1 ± 10.6%), less expression of exhaustion marker PD-1 (RA+ CAR-T: 3.1 ± 2.5% vs RA− CAR-T: 19.2 ± 6.4%) and T cell senescence marker CD57 (RA+ CAR-T: 6.8 ± 3.6% vs RA− CAR-T: 20.2 ± 6.9%), and enrichment of naïve/stem cell memory fraction (CAR+/CD45RA+CCR7+ fraction; RA+ CAR-T: 71.9 ± 9.7% vs RA− CAR-T: 8.0 ± 5.3%), which were associated with longevity of CAR-T cells. Transcriptome analysis revealed that RA+ CAR-T cells exhibited the enrichment of naïve/memory phenotype and less expression of canonical exhaustion markers, and these exhaustion profiles even maintained after the antigen stimulation. RA+ CAR-T cells demonstrated sustained killing activity even after multiple tumor rechallenges in vitro, without inducing exhaustion marker expression of PD-1. Although antigen stimulation could increase CAR expression, leading to tonic CAR signaling and exhaustion, in our study, the expression of CAR molecule on the cell surface following antigen stimulation in RA+ CAR was controlled at a relatively lower level that in RA− CAR-T cells. RA+ CAR-T cells achieved prolonged tumor control with expansion of CAR-T cells than RA− CAR-T cells in in vivo stress test (Fig.1A-C). On day15, bone marrow studies in RA+ CAR group exhibited abundant human CD3 positive T cells with less expression of PD-1, and relatively smaller amount of REH cells than RA− CAR group (Fig.1D). Furthermore, in two of long-lived mice in RA+ CAR group, human CD3 positive T cells were expanded even day 50 after treatment as confirmed by sequential bone marrow studies (Fig.1E), which indicated the antigen-induced proliferation and long-term functionality of RA+ CAR-T cells in vivo. Conclusion: Our results suggest that PB-mediated RA+ CAR-T cells exhibit memory-rich phenotype and superior antitumor function, thereby indicating the usefulness of CD45RA+ PBMC as a starting material of PB-CAR-T cells. Figure 1 Figure 1. Disclosures Yagyu: AGC Inc.: Research Funding. Nagao: AGC Inc.: Current Employment. Kubota: AGC Inc.: Current Employment. Shimizu: AGC Inc.: Current Employment. Nakazawa: AGC Inc.: Research Funding; Toshiba Corporation: Research Funding.

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Low CD19 Antigen Density Diminishes Efficacy of CD19 CAR T Cells and Can be Overcome By Rational Redesign of CAR Signaling Domains

Blood ◽

10.1182/blood-2018-99-115558 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 963-963 ◽

Cited By ~ 6

Author(s):

Robbie G. Majzner ◽

Skyler P. Rietberg ◽

Louai Labanieh ◽

Elena Sotillo ◽

Evan W. Weber ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Cytokine Production ◽

Xenograft Model ◽

Target Antigen ◽

Tumor Escape ◽

Car T Cells ◽

Car T Cell ◽

Car T

Abstract Target antigen density has emerged as a major factor influencing the potency of CAR T cells. Our laboratory has demonstrated that the activity of numerous CARs is highly dependent on target antigen density (Walker et al., Mol Ther, 2017), and high complete response rates in a recent trial of CD22 CAR T cells for B-ALL were tempered by frequent relapses due to decreased CD22 antigen density on lymphoblasts (Fry et al., Nat Med, 2018). To assess if antigen density is also a determinant of CD19 CAR T cell therapeutic success, we analyzed CD19 antigen density from fifty pediatric B-ALL patients treated on a clinical trial of CD19-CD28ζ CAR T cells. We found that patients whose CD19 expression was below a threshold density (2000 molecules/lymphoblast) were significantly less likely to achieve a clinical response than those whose leukemia expressed higher levels of CD19. In order to further understand this limitation and how it may be overcome, we developed a model of variable CD19 antigen density B-ALL. After establishing a CD19 knockout of the B-ALL cell line NALM6, we used a lentivirus to reintroduce CD19 and then FACS sorted and single cell cloned to achieve a library of NALM6 clones with varying CD19 surface densities. CD19-CD28ζ CAR T cell activity was highly dependent on CD19 antigen density. We observed decreases in cytotoxicity, proliferation, and cytokine production by CD19 CAR T cells when encountering CD19-low cells, with an approximate threshold of 2,000 molecules of CD19 per lymphoblast, below which, cytokine production in response to tumor cells was nearly ablated. Given that a CD19-4-1BBζ CAR is FDA approved for children with B-ALL and adults with DLBCL, we wondered whether CARs incorporating this alternative costimulatory domain would have similar antigen density thresholds for activation. Surprisingly, CD19-4-1BBζ CAR T cells made even less cytokine, proliferated less, and had further diminished cytolytic capacity against CD19-low cells compared to CD19-CD28ζ CAR T cells. Analysis by western blot of protein lysates from CAR T cells stimulated with varying amounts of antigen demonstrated that CD19-CD28ζ CAR T cells had higher levels of downstream signals such as pERK than CD19-4-1BBζ CAR T cells at lower antigen densities. Accordingly, calcium flux after stimulation was also significantly higher in CD19-CD28ζ than CD19-4-1BBζ CAR T cells. In a xenograft model of CD19-low B-ALL, CD19-4-1BBζ CAR T cells demonstrated no anti-tumor activity, while CD19-CD28ζ CAR T cells eradicated CD19-low leukemia cells. Therefore, the choice of costimulatory domain in CAR T cells plays a major role in modulating activity against low antigen density tumors. CD28 costimulation endows high reactivity towards low antigen density tumors. We confirmed the generalizability of this finding using Her2 CAR T cells; Her2-CD28ζ CAR T cells cleared tumors in an orthotopic xenograft model of Her2-low osteosarcoma, while Her2-4-1BBζ CAR T cells had no effect. This finding has implications for CAR design for lymphoma and solid tumors, where antigen expression is more heterogeneous than B-ALL. To enhance the activity of CD19-4-1BBζ CAR T cells against CD19-low leukemia, we designed a CAR with two copies of intracellular zeta in the signaling domain (CD19-4-1BBζζ). T cells expressing this double-zeta CAR demonstrated enhanced cytotoxicity, proliferation, cytokine production, and pERK signaling in response to CD19-low cells compared to single-zeta CARs. Additionally, in a xenograft model, CD19-4-1BBζζ CAR T cells demonstrated enhanced activity against CD19-low leukemia compared to CD19-4-1BBζ CAR T cells, significantly extending survival. The addition of a third zeta domain (CD19-4-1BBζζζ) further enhanced the activity of CAR T cells. However, inclusion of multiple copies of the costimulatory domains did not improve function. In conclusion, CD19 antigen density is an important determinant of CAR T cell function and therapeutic response. CD19-CD28ζ CARs are more efficient at targeting CD19-low tumor cells than CD19-4-1BBζ CARs. The addition of multiple zeta domains to the CAR enhances its ability to target low antigen density tumors. This serves as proof of concept that rational redesign of CAR signaling endodomains can result in enhanced function against low antigen density tumors, an important step for extending the reach of these powerful therapeutics and overcoming a significant mechanism of tumor escape. Disclosures Lee: Juno: Consultancy.

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New Generation Chimeric Antigen Receptor T-Cell Therapy (CoupledCAR) Induces High Rate Remissions in Solid Tumor

Blood ◽

10.1182/blood-2019-130703 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4631-4631

Author(s):

Lei Xiao

Keyword(s):

Clinical Trials ◽

T Cells ◽

Thyroid Cancer ◽

T Cell ◽

Cell Therapy ◽

Solid Tumors ◽

Tumor Cells ◽

T Cell Therapy ◽

Car T Cells ◽

Car T

New Generation Chimeric Antigen Receptor T-Cell Therapy ( CoupledCAR ) Induces High Rate Remissions in Solid Tumor Yu Liu1,Song Li2,Youli Luo3,Haixia Song4,Chengfei Pu5, Zhiyuan Cao 5, Cheng Lu5,Yang Hang5,Xi Huang5,Xiaogang Shen5 ,Xiaojun Hu3 , Renbin Liu1,Xiuwen Wang2,Junjie Mao3,Shihong Wei4 ,Zhao Wu5and Lei Xiao5* 1.The Third Affiliated Hospital, SUN YAT-SEN University 2.Qilu Hospital of Shandong University 3.The Fifth Affiliated Hospital, SUN YAT-SEN University 4.Gansu Procincial Cancer Hospital 5.Innovative Cellular Therapeutics *Corresponding to: Lei Xiao, [email protected] Chimeric antigen receptor (CAR) T cell therapy made significant progress for treating blood cancer such as leukemia, lymphoma, and myeloma. However, the therapy faces many challenges, such as physical barrier, tumor microenvironment immunosuppression, tumor heterogeneity, target specificity, and cell expansion in vivo for treatment of solid tumors Conventional CAR T cell therapy showed weak CAR T expansion in patients and thus achieved no or little response for treating solid tumors. Here, we generated "CoupledCAR" T cells including an anti-TSHR CAR molecule. Compared with conventional CART cells,these "CoupledCAR" T cells successfully improved the expansion of CART cells more than 100 times and enhanced CAR T cells' migration ability, allowing the CAR T cells to resist and infiltrate the tumor microenvironment and killed tumor cells. To verify the effect of "CoupledCAR" T cells on solid tumors, we have completed several clinical trials for different solid tumors, including two patients with thyroid cancer. Immunohistochemistry (IHC) results showed that thyroid stimulating hormone receptors (TSHR) were highly expressed in thyroid cancer cells. In vitro co-culture experiments showed that TSHR CAR T cells specifically recognized and killed TSHR-positive tumor cells. Animal experiments showed that TSHR CAR T cells inhibited the proliferation of TSHR-positive tumor cells. Therefore, we designed "CoupledCAR" T cells expressing a binding domain against TSHR. Further,we did clinical trials of two group patients that were successfully treated using conventional TSHR CAR T cells and the "CoupledCAR" T cells, respectively. In the first group using conventional TSHR CAR T cells, patients showed weak cell expansion and less migration ability. In the group using TSHR "CoupledCAR" T cells, patients showed rapid expansion of CAR T cells and killing of tumor cells. One month after infusion (M1), the patient was evaluated as PR(Partial Response): the lymph node metastasis disappeared, and thoracic paratracheal tumors decreased significantly. Three months after infusion (M3), the patient was evaluated as a durable response, and the tumor tissue was substantially smaller than M1. Further, two patients with colonrectal cancer were enrolled in this trial and infused "CoupledCAR" T cells. One patient achieved PR and the other one achieved SD (Stable Disease). Therefore, "CoupledCAR" T cells can effectively promote expansion, migration and killing ability of CAR T cells in patients with thyroid cancer. "CoupledCAR" T cell technology is a technological platform, which may be used to treat other cancer types. Next, we are recruiting more patients with solid tumors in clinical trials using "CoupledCAR" T cells. Disclosures No relevant conflicts of interest to declare.

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BAY 60-6583 Enhances the Antitumor Function of Chimeric Antigen Receptor-Modified T Cells Independent of the Adenosine A2b Receptor

Frontiers in Pharmacology ◽

10.3389/fphar.2021.619800 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jiaxing Tang ◽

Yan Zou ◽

Long Li ◽

Fengping Lu ◽

Hongtao Xu ◽

...

Keyword(s):

T Cells ◽

Tumor Cells ◽

Chimeric Antigen Receptor ◽

Antitumor Activities ◽

Adenosine A2b Receptor ◽

Car T Cells ◽

Car T ◽

A2b Receptor ◽

Adenosine A2b

Chimeric antigen receptor (CAR) T cells are powerful in eradicating hematological malignancies, but their efficacy is limited in treating solid tumors. One of the barriers is the immunosuppressive response induced by immunomodulatory signaling pathways. Pharmacological targeting of these immunosuppressive pathways may be a simple way to improve the efficacy of CAR T cells. In this study, anti-CD133 and anti-HER2 CAR T cells were generated from healthy donors, and combination therapy using CAR T cells and small molecules targeting adenosine receptors was performed in vitro and in vivo with the goal of probing for potential synergistic antitumor activities. The adenosine A2b receptor agonist, BAY 60-6583, was found to significantly increase cytokine secretion of CD133-or HER2-specific CAR T cells when co-cultured with the respective target tumor cells. The in vitro cytotoxicity and proliferation of CAR T cells were also enhanced when supplied with BAY 60-6583. Furthermore, the combination with this small molecule facilitated the anti-HER2 CAR T cell-mediated elimination of tumor cells in a xenograft mouse model. However, the enhanced antitumor activities could not be suppressed by knockout of the adenosine A2b receptor in CAR T cells. Furthermore, mass spectrometry and computational methods were used to predict several potential alternative targets. Four potential targets (pyruvate kinase M (PKM), Talin-1, Plastin-2, and lamina-associated polypeptide 2) were captured by a photo-affinity probe, of which PKM and Talin-1 were predicted to interact with BAY 60-6583. Overall, our data suggest that BAY 60-6583 upregulates T cell functions through a mechanism independent of the adenosine A2b receptor.

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IL-18 Secreting CAR T Cells Enhance Cell Persistence, Induce Prolonged B Cell Aplasia and Eradicate CD19+ Tumor Cells without Need for Prior Conditioning

Blood ◽

10.1182/blood.v128.22.816.816 ◽

2016 ◽

Vol 128 (22) ◽

pp. 816-816 ◽

Cited By ~ 10

Author(s):

Mauro P. Avanzi ◽

Dayenne G. van Leeuwen ◽

Xinghuo Li ◽

Kenneth Cheung ◽

Hyebin Park ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Tumor Cells ◽

Car T Cells ◽

Car T Cell ◽

Cytokine Analysis ◽

Car T ◽

Ifn Γ

Abstract Chimeric antigen receptor (CAR) T cell therapy has consistently shown significant results against acute lymphoblastic leukemia (ALL) in clinical trials1. However, results with other hematological or solid malignancies have been far more modest2. These disparate outcomes could be partially due to an inhibitory tumor microenvironment that suppresses CAR T cell function3. Thus, in order to expand the anti-tumor CAR T cell applications, a novel strategy in which these cells are capable of overcoming the hostile tumor microenvironment is needed. The cytokine interleukin-18 (IL-18) induces IFN-γ secretion, enhances the Th1 immune response and activates natural killer and cytotoxic T cells4. Early phase clinical trials that utilized systemic administration of recombinant IL-18 for the treatment of both solid and hematological malignancies have demonstrated the safety of this therapy5. We hypothesize that CAR T cells that constitutively secrete IL-18 could enhance CAR T cell survival and anti-tumor activity, and also activate cells from the endogenous immune system. To generate CAR T cells that constitutively secrete IL-18, we modified SFG-1928z and SFG-19m28mz CAR T cell constructs and engineered bicistronic human and murine vectors with a P2A element to actively secrete the IL-18 protein (1928z-P2A-hIL18 and 19m28mz-P2A-mIL18, respectively). Human and mouse T cells were transduced with these constructs and in vitro CAR T cell function was validated by coculturing the CAR T cells with CD19+ tumor cells and collecting supernatant for cytokine analysis. Both human and mouse CAR T cells secreted increased levels of IL-18, IFN-γ and IL-2. Proliferation and anti-tumor cytotoxic experiments were conducted with human T cells by coculturing CAR T cells with hCD19+ expressing tumor cells. 1928z-P2A-hIL18 CAR T cells had enhanced proliferation over 7 days and enhanced anti-tumor cytotoxicity over 72 hours when compared to 1928z CAR T cells (p=0.03 and 0.01, respectively) Next, the in vivo anti-tumor efficacy of the IL-18 secreting CAR T cell was tested in xenograft and syngeneic mouse models. Experiments were conducted without any prior lympho-depleting regimen. In the human CAR T cell experiments, Scid-Beige mice were injected with 1x106 NALM-6 tumor cells on day 0 and 5x106 CAR T cells on day 1. Survival curves showed a significant improvement in mouse survival with the 1928z-P2A-hIL18 CAR T cell treatment when compared to 1928z CAR T cell (p=0.006). Subsequently, to determine if IL-18 secreting CAR T cells could also improve anti-tumor efficacy in immunocompetent mice, we tested the murine 19m28mz-P2A-mIL18 CAR T cells in a syngeneic mouse model. The C57BL/6 hCD19+/- mCD19+/- mouse model was utilized and injected with 1x106 EL4 hCD19+ tumor cells on day 0 and 2.5 x106 CAR T cells on day 1. Mice treated with 19m28mz-P2A-mIL18 CAR T cells had 100% long-term survival, when compared to 19m28mz (p<0.0001). 19m28mz-P2A-mIL18 CAR T cells were detected in peripheral blood for up to 30 days after injection, whereas the 19m28mz CAR T cells were not detectable at any time point. In addition, 19m28mz-P2A-mIL18 CAR T cells were capable of inducing B cell aplasia for greater than 70 days, whereas 19m28mz treatment was not capable of inducing B cell aplasia. In vivo serum cytokine analysis demonstrated that 19m28mz-P2A-mIL18 CAR T cells, as compared to 19m28mz, significantly increased the levels of IFN-γ and TNF-α in the peripheral blood for up to 14 days after injection (p<0.0001 and 0.01, respectively). Despite the increase in IFN-γ and TNF-α cytokines, there was no increase in IL-6 levels. Our findings demonstrate that anti-CD19 CAR T cells that constitutively secrete IL-18 significantly increase serum cytokine secretion, enhance CAR T cell persistence, induce long-term B cell aplasia and improve mouse survival, even without any prior preconditioning. To our knowledge, this is the first description of an anti-CD19 CAR T cell that constitutively secretes IL-18 and that induces such high levels of T cell proliferation, persistence and anti-tumor cytotoxicity. We are currently investigating other mechanisms by which this novel CAR T cell functions, its interactions with the endogenous immune system, as well as testing its applicability in other tumor types. We anticipate that the advances presented by this new technology will expand the applicability of CAR T cells to a wider array of malignancies. Disclosures Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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