Lentiviral Chimeric Antigen Receptors Research Articles

Efficacy and Safety of Chimeric Antigen Receptor T Cells Therapy Strategy with Dual Targeting of CD19 and CD70 to Treat Relapsed/Refractory Diffuse Large B-Cell Lymphoma

Background: Chimeric antigen receptor (CAR) T cells therapy is highly effective in the treatment of B-cell lymphoma, providing alternative therapeutic options for patients who failed to respond to conventional treatment or relapse. Despite impressive progress, more than 30-50% of patients treated with CD19 CAR-T cells experienced progressive disease. Antigen escape is one of the common causes of relapse, especially CD19-negative relapse. Absent or decreased cell surface CD19 as a mechanism of resistance after CD19 CAR-T cells treatment. CD70 is a novel and promising therapeutic target due to the restricted expression pattern in normal tissues and over expression in some lymphoma tissues. Our center explored the efficacy and safety of CAR-T cells therapy with dual targeting of CD19 and CD70 in the treatment of r/r lymphoma. Patients and Methods: The study included 8 relapsed/refractory diffuse large B-cell lymphoma patients, all patients have exhausted all available treatment options with progressive or stable disease and life expectancy >2 months were enrolled in the study. All patients expressed strong positivity of CD19 and CD70, all of patient details can be found in the table (Fig.1). Autologous T cells were apheresis collected and transduced with an apoptosis-inducible, safety-engineered lentiviral CAR with the following intracellular signaling domains: CD28/CD27/CD3z-iCasp9. All patients received cyclophosphamide/fludarabine chemotherapy conditioning 1-2 days before infusions of CAR-T cells. The quality of apheresis cells, efficiencies of gene transfer and T cell proliferation, CAR-T infusion dose and blood CAR copies were quantitatively documented. Results: In the first months after CAR-T cells infusion, 75.0% (6/8) of patients achieved complete response (CR), while 12.5% (1/8) of patients evaluated as partial response (PR) and 12.5% (1/8) of patients as progressive disease (PD), overall response rate (ORR) is 87.5% (7/8). In the evaluation of safety, 37.5% (3/8) of patients experienced cytokine release syndrome (CRS), and 25% (2/8) of patients experienced grade 1 CRS and 12.5% (1/8) of patients experienced grade 2 CRS, no case of severe CRS defined as≥grade 3 and no immune effector cell-associated neurotoxicity syndrome (ICANS) occurred. After a median follow-up of 19.9 months, 50% (4/8) of patients maintained CR, while 37.5% (3/8) of patients were relapsed, with a duration of response (DOR) of 50% (4/8), median DFS was 10.5 months and median OS was not reached (Fig.2). Conclusions: In summary, our research data confirms that the efficacy and safety of CD19/CD70 dual targeted CAR-T cells infusion, continued follow-up will determine whether the CD19/CD70 CAR-T cells therapy can obtain long term overall survival in our study. How to further improve the efficacy and safety of dual target CAR-T is still worth exploring. It's needed to optimize multi-specific targeting by CAR-T cells to improve the efficacy both in B cell malignancies and other hematological malignancies.

F77 antigen is a promising target for adoptive T cell therapy of prostate cancer

Adoptive immunotherapy using chimeric antigen receptor (CAR) T cells has made significant success in treating hematological malignancies, paving the way for solid tumors like prostate cancer. However, progress is impeded by a paucity of suitable target antigens. A novel carbohydrate antigen, F77, is expressed on both androgen-dependent and androgen-independent prostate cancer cells, making it a potential immunotherapy target. This study entails the generation and evaluation of a second-generation CAR against a carbohydrate antigen on malignant prostate cancer cells. Using a single chain fragment variable (scFv) from an F77-specific mouse monoclonal antibody, we created second-generation CARs with CD28 and CD137 (4-1BB) costimulatory signals. F77 expressing lentiviral CAR T cells produce cytokines and kill tumor cells in a F77 expression-dependent manner. These F77-specific CAR T cells eradicate prostate tumors in a human xenograft model employing PC3 cells. These findings validate F77 as a promising immunotherapeutic target for prostate cancer and other malignancies with this aberrant carbohydrate structure.

Sleeping Beauty kit sets provide rapid and accessible generation of artificial antigen-presenting cells for natural killer cell expansion.

Artificial antigen-presenting cells (aAPCs) offer a cost effective and convenient tool for the expansion of chimeric antigen receptor (CAR)-bearing T cells and NK cells. aAPCs are particularly useful because of their ability to efficiently expand low-frequency antigen-reactive lymphocytes in bulk cultures. Commonly derived from the leukemic cell line K562, these aAPCs lack most major histocompatibility complex expression and are therefore useful for NK cell expansion without triggering allogeneic T-cell proliferation. To combat difficulties in accessing existing aAPC lines, while circumventing the iterative lentiviral gene transfers with antibody-mediated sorting required for the isolation of stable aAPC clones, we developed a single-step technique using Sleeping Beauty (SB)-based vectors with antibiotic selection options. Our SB vectors contain options of two to three genes encoding costimulatory molecules, membrane-bound cytokines as well as the presence of antibiotic-resistance genes that allow for stable transposition-based transfection of feeder cells. Transfection of K562 with SB vectors described in this study allows for the surface expression of CD86, 4-1BBL, membrane-bound (mb) interleukin (IL)-15 and mbIL-21 after simultaneous transposition and antibiotic selection using only two antibiotics. aAPCs successfully expanded NK cells to high purity (80-95%). Expanded NK cells could be further engineered by lentiviral CAR transduction. The multivector kit set is publicly available and will allow convenient and reproducible in-house production of effective aAPCs for the invitro expansion of primary cells.

Challenges in HIV-1 Latent Reservoir and Target Cell Quantification in CAR-T Cell and Other Lentiviral Gene Modifying HIV Cure Strategies.

Gene-modification therapies are at the forefront of HIV-1 cure strategies. Chimeric antigen receptor (CAR)-T cells pose a potential approach to target infected cells during antiretroviral therapy or following analytical treatment interruption (ATI). However, there are technical challenges in the quantification of HIV-1-infected and CAR-T cells in the setting of lentiviral CAR gene delivery and also in the identification of cells expressing target antigens. First, there is a lack of validated techniques to identify and characterize cells expressing the hypervariable HIV gp120 in both ART-suppressed and viremic individuals. Second, close sequence homology between lentiviral-based CAR-T gene modification vectors and conserved regions of HIV-1 creates quantification challenges of HIV-1 and lentiviral vector levels. Consideration needs to be taken into standardizing HIV-1 DNA/RNA assays in the setting of CAR-T cell and other lentiviral vector-based therapies to avoid these confounding interactions. Lastly, with the introduction of HIV-1 resistance genes in CAR-T cells, there is a need for assays with single-cell resolution to determine the competence of the gene inserts to prevent CAR-T cells from becoming infected in vivo. As novel therapies continue to arise in the HIV-1 cure field, resolving these challenges in CAR-T-cell therapy will be crucial.

Abstract 5074: Addition of MAPK inhibitors to prime and sensitize poorly differentiated thyroid cancers as a strategy to improve TSHR-CART cell therapy antitumor activity

Abstract Thyroid cancer is the most common endocrine cancer in the US, and its incidence is rising. Most thyroid cancer deaths are attributed to treatment-refractory, metastatic tumors. Thyroid stimulating hormone receptor (TSRH) expression is largely limited to the thyroid gland and is abundantly expressed on thyroid tumor cells, making TSRH a compelling target for advanced thyroid cancer diagnostics and therapeutics. Therefore, we developed a novel TSHR-targeted chimeric antigen receptor (CAR) T cell therapy to treat aggressive thyroid cancers. TSHR-CAR constructs were cloned into a lentiviral CAR construct containing 4-1BB and CD3ζ. First, we demonstrated potent TSHR-CART antigen-specific anti-tumor activity in vitro. Then, NOD-SCID-γ-/- (NSG) mice were inoculated subcutaneously with TSHR+ tumor cells and randomized by tumor volume to treatment with TSHR-CART cells or control Untransduced T cells (UTD). Treatment with TSHR-CART cells resulted in dose-dependent antitumor activity and prolonged survival. De-differentiated anaplastic thyroid cancers (ATC) downregulate TSHR. Our TSHR immunohistochemistry results corroborated these findings and displayed minimal TSHR protein expression, precluding successful TSHR-CART treatment. We therefore sought to sensitize these tumors with MAPK inhibitors, as a strategy to upregulate TSHR expression in patients with metastatic thyroid cancer. TSHR expression was upregulated in patient-derived xenograft (PDX) ATC models after one week of daily administration of the MAPK inhibitors (p=0.0024). After confirming that MAPK inhibition does not dampen TSHR-CART effector functions, we tested sequential and combination therapy of TSHR-CART with MEK and BRAF inhibition in vivo. NSG mice were engrafted with ATC BRAF-mutant PDX tumors and randomized by tumor volume to daily oral treatment with placebo or trametinib (MEK inhibitor) plus dabrafenib (BRAF inhibitor). One week later, mice received either UTD or TSHR-CART. Mice conditioned with trametinib plus dabrafenib (p=0.0018) and subsequently treated with TSHR-CART showed superior antitumor activity. However, the improved antitumor activity in this setting was transient. We therefore tested the durability of TSHR upregulation following MEK/BRAF inhibition and demonstrated that TSHR upregulation lasts less than 48-72 hours after discontinuation. Finally, we tested the combination of TSHR CART cells with MEK/BRAF inhibitors in ATC BRAF-mutant PDX tumors. Here, combining TSHR-CART cells with MEK/BRAF inhibitors result in durable control of the tumors. Collectively, our findings indicate that MEK/BRAF inhibition of de-differentiated thyroid cancers upregulated TSHR expression and enhanced TSHR-CART antitumor activity. This work represents a viable strategy to improve outcomes of patients with aggressive, metastatic thyroid cancers. Citation Format: Claudia Manriquez Roman, Kendall J. Schick, Justyna J. Gleba, Truc N. Huynh, Elizabeth L. Siegler, James L. Miller, Aylin Alasonyalilar Demirer, Matthew L. Pawlush, Ahmet Biligili, Long K. Mai, Erin Tapper, Leo R. Sakemura, Michelle J. Cox, Carli M. Stewart, Ismail Can, Ekene J. Ogbodo, Gaofeng Cui, Georges Mer, Gloria R. Olivier, Yushi Qiu, Robert C. Smallridge, Zubair C. Abba, Han W. Tun, John A. Copland, Saad S. Kenderian. Addition of MAPK inhibitors to prime and sensitize poorly differentiated thyroid cancers as a strategy to improve TSHR-CART cell therapy antitumor activity. [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 5074.

Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies.

The great clinical success of chimeric antigen receptor (CAR) T cells has unlocked new levels of immunotherapy for hematological malignancies. Genetically modifying natural killer (NK) cells as alternative CAR immune effector cells is also highly promising, as NK cells can be transplanted across HLA barriers without causing graft-versus-host disease. Therefore, off-the-shelf usage of CAR NK cell products might allow to widely expand the clinical indications and to limit the costs of treatment per patient. However, in contrast to T cells, manufacturing suitable CAR NK cell products is challenging, as standard techniques for genetically engineering NK cells are still being defined. In this study, we have established optimal lentiviral transduction of primary human NK cells by systematically testing different internal promoters for lentiviral CAR vectors and comparing lentiviral pseudotypes and viral entry enhancers. We have additionally modified CAR constructs recognizing standard target antigens for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) therapy—CD19, CD33, and CD123—to harbor a CD34-derived hinge region that allows efficient detection of transduced NK cells in vitro and in vivo and also facilitates CD34 microbead-assisted selection of CAR NK cell products to >95% purity for potential clinical usage. Importantly, as most leukemic blasts are a priori immunogenic for activated primary human NK cells, we developed an in vitro system that blocks the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46, and NKp80 on these cells and therefore allows systematic testing of the specific killing of CAR NK cells against ALL and AML cell lines and primary AML blasts. Finally, we evaluated in an ALL xenotransplantation model in NOD/SCID-gamma (NSG) mice whether human CD19 CAR NK cells directed against the CD19+ blasts are relying on soluble or membrane-bound IL15 production for NK cell persistence and also in vivo leukemia control. Hence, our study provides important insights into the generation of pure and highly active allogeneic CAR NK cells, thereby advancing adoptive cellular immunotherapy with CAR NK cells for human malignancies further.

A BCL2L1 Armoured BCMA Targeting CAR T Cell to Overcome Exhaustion and Enhance Persistence in Multiple Myeloma

Chimeric antigen receptor (CAR) T cells targeting the B-cell maturation antigen (BCMA) have resulted in deep responses in patients with relapsed MM however most remissions are not sustained. While cellular and molecular mediators of relapse post CAR T therapy in MM are not fully delineated, current data suggest three possible mechanisms including the lack of persistence of the CAR T cell product, acquired exhaustion and less commonly loss of BCMA expression.Using CITE-seq we measured the expansion of variable T cell subsets, T cell specific activation and inhibitor markers and their functional states in serial blood and marrow samples (n=10) collected from patients treated with BCMA targeting CAR T cells. CAR T cells were identified by the expression of the chimeric CAR T cell transcript. With the exception of one patient where biallelic loss of BCMA was identified at relapse, CAR T cells of resistant patients were enriched with terminally exhausted CD45RA+ cells with loss of CD28, low BCL2L1 (gene encoding BCL-XL) expression, high CD57 with co-expression of checkpoint inhibitors (LAG3, TIGIT and PD1). The lack of persistence of the CAR T cells product was notable in all relapsing patients consistent with an activation induced cells death (AICD) specially in the setting of chronic antigenic stimulation. Cognizant of the role BCL-XL plays in T cells survival in response to CD28 co-stimulatory signaling, we postulated that increasing BCL-XL expression is a feasible strategy to enhance CAR T cell resistant to AICD, improve their persistence and anti-BCMA reactivity. To this goal, we designed a 2nd generation lentiviral CAR construct where the anti-BCAM scFV-41BBz CAR and the BCL2L1 cDNA were linked with self-cleaving 2A sequence. The efficiency in eradicating MM cells of this BCL-XL armored CAR (BCMA_BCL2L1_CAR) was compared to that of non-unarmored CAR (BCMA_CAR) in vitro and i n vivo studies. While BCMA_BCL2L1_CAR and BCMA_CAR were equally cytotoxic to OPM2 MM cells, in MM cell lines expressing the FAS death receptor ligand FASLG (MM1S, OCMY5 and H929) BCMA_BCL2L1_CAR viability and cytolytic activity was significantly superior to that of unarmored BCMA_CAR. Of note, the expression of FASLG, a known interferon response gene, was upregulated in H929 cells when co-cultured with CAR T cells. Importantly, under chronic antigenic stimulation conditions (FIG 1A), where CAR T cells were stimulated every 6 days over a 28 days period with irradiated OPM2 cells, we found no phenotypic difference between BCMA_BCL2L1_CAR and BCMA_CAR with respect to the composition of effector memory T cells (Tem: CCR7− CD45RO+ CD45RA−) or central memory T cells (Tcm: CCR7+CD45RO+CD45RA−) or terminal effector / exhausted T cells. However, under these chronic antigenic stimulation conditions, the CAR T cells viability, proliferation (FIG 1B) and anti-MM cytotoxic activities (FIG 1C) of the BCMA_CAR were dramatically reduced compared to that of the BCL2L1 armored CAR. Furthermore, in initial animal studies where NOD-SCID mice were tail vein injected with 2e6 OPM2 MM cells transduced with a luciferin reporter gene, followed 10 days later by control T cells, BCMA_CAR or BCMA_BCL2L1_CAR T cells IV injection, and despite a skewing to a larger initial disease burden in the BCMA-BCL2L1-CAR group, BCL2L1 armored CAR T cells resulted in more prolonged disease control and animal survival compared to the BCMA_CAR treated mice (FIG 1D).Our studies indicate that BCL2L1 blockade of AICD not only enhanced the viability and proliferation of BCMA targeting CAR T cells but surprisingly also reduced their functional exhaustion. Our findings provide an novel approach for CAR T optimization and overcoming disease relapse resulting from lack of persistence and/or T cells exhaustion. [Display omitted] DisclosuresNeri: Amgen: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria. Bahlis: Sanofi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Genentech: Consultancy; Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

GD2-specific chimeric antigen receptor-modified T cells for the treatment of refractory and/or recurrent neuroblastoma in pediatric patients

PurposeThis study aimed to evaluate the safety and efficacy of chimeric antigen receptor (CAR) disialoganglioside 2 (GD2)-specific (4SCAR-GD2) T cells for treatment of refractory and/or recurrent neuroblastoma (NB) in pediatric patients.Experimental designA phase I clinical study using 4SCAR-GD2 T cells for the treatment of NB in pediatric patients was conducted. This study was registered at www.clinicaltrials.gov (NCT02765243). A lentiviral CAR with the signaling domains of CD28/4-1BB/CD3ζ-iCasp9 was transduced into activated T cells. The response to 4SCAR-GD2 T-cell treatment, and 4SCAR-GD2 T-cell expansion and persistence in patients were evaluated. Toxicities were determined based on the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) v4.03.ResultsTwelve patients were enrolled and finally ten patients were included in this clinical trial which started from January 1, 2016, to August 1, 2017. These patients had progressive disease (PD) before CAR T-cell infusion. After 4SCAR-GD2 T-cell treatment, 6 (6/10) had stable disease (SD) at 6 months, and 4 (4/10) remained SD at 1 year and alive after 3–4 years of follow-up. Six patients died due to disease progression by the end of July 1, 2020. The median overall survival (OS) time was 25 months (95% CI, 0.00–59.43), and the median progression-free survival (PFS) time was 8 months (95% CI, 0.25–15.75). Grade 3 or 4 hematological toxicities were the common adverse events frequently occurred after fludarabine and cyclophosphamide (Flu/cy) chemotherapy. Grade 1–2 toxicities such as cytokine release syndrome (CRS) and neuropathic pain were common, but were transient and mild.ConclusionsThe 4SCAR-GD2 T-cell therapy demonstrated antitumor effect and manageable toxicities, indicating its potential to benefit children with refractory and/or recurrent NB.

OAB-018: A BCL2L1 armoured BCMA-targeting CAR T cells to overcome exhaustion and enhance persistence in multiple myeloma

<h3>Background</h3> Chimeric antigen receptor (CAR) T cells targeting BCMA have resulted in deep responses in patients with relapsed MM however most remissions are not sustained. While cellular and molecular mediators of relapse post CAR T therapy are not fully delineated, current data suggest three possible mechanisms including the lack of persistence of the CAR T cell product, acquired exhaustion and less commonly loss of BCMA expression. <h3>Methods</h3> Using CITE-seq we measured the expansion of variable T cell subsets, T cell specific activation and inhibitor markers and their functional states in serial blood and marrow samples (n=10) collected from patients treated with anti BCMA CAR T cells. <h3>Results</h3> CAR T cells were identified by the expression of the chimeric CAR T cell transcript. With the exception of one patient where biallelic loss of BCMA was identified at relapse, CAR T cells of resistant patients were enriched with terminally exhausted CD45RA+ cells with loss of CD28, low BCL2L1 (gene encoding BclxL) expression, high CD57 with co-expression of checkpoint inhibitors (LAG3, TIGIT and PD1). The lack of persistence of the CAR T cells product was notable in all relapsing patients consistent with an activation induced cells death (AICD). <h3>Conclusions</h3> Cognizant of the role BclxL plays in T cells survival in response to CD28 co-stimulatory signaling, we postulated that increasing BclxL expression is a feasible strategy to enhance CAR T cell resistant to AICD, improve their persistence and anti-BCMA reactivity. To this goal, we designed a 2nd generation lentiviral CAR construct where the anti-BCAM scFV-41BBz CAR and the BCL2L1 cDNA were linked with self-cleaving 2A sequence. The efficiency in eradicating MM cells of this BclxL armored CAR (BCL2L1_CAR) was compared to that of non-unarmored CAR (BCMA_CAR) in vitro and in vivo studies. While BCL2L1_CAR and BCMA_CAR were equally cytotoxic to OPM2 MM cells, in MM cell lines expressing the FAS death receptor ligand FASLG (MM1S, OCMY5 and H929) BCL2L1_CAR viability and cytotoxic activity was significantly superior to that of unarmored BCMA_CAR. Of note, the expression of FASLG was upregulated in H929 cells when co-cultured with CAR T cells. Importantly, under chronic antigenic stimulation conditions, where CAR T cells were stimulated every 2 days over a 28 days period with irradiated OPM2 cells, we found no phenotypic difference between BCL2L1_CAR and BCMA_CAR with respect to the composition of Tem cells (CCR7–CD45RO+CD45RA–) or Tcm cells (CCR7+CD45RO+CD45RA–). However, under these chronic antigenic stimulation conditions, the CAR T cells viability, proliferation and anti-MM cytotoxic activities of the BCMA_CAR were dramatically reduced compared to that of the BCL2L1 armored CAR. Therefore BCL2L1 blockade of AICD not only enhanced the viability and cytotoxicity of CAR-T cells but surprisingly also reduced their functional exhaustion. Our findings provide a novel approach for CAR-T optimization and overcoming relapse resulting from lack of persistence.

Updated Results from an Ongoing Phase 1 Clinical Study of bb21217 Anti-Bcma CAR T Cell Therapy

Updated Results from an Ongoing Phase 1 Clinical Study of bb21217 Anti-Bcma CAR T Cell Therapy

Cellular Immunotherapies for Leukemia Patients

Cellular Immunotherapies for Leukemia Patients

649. Developing FGFR4 Chimeric Antigen Receptor CAR T Cell Therapy Against Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in pediatrics with an annual incidence of 4.5 cases per 1 million. Patients with high-risk metastatic disease have dismal prognosis and newer treatments are needed. We identified, fibroblast growth factor receptor 4 (FGFR4) as an overexpressed cell surface protein in RMS by mRNA expression analysis. Furthermore, activating mutations in FGFR4 are associated with metastatic disease. FGFR4 protein overexpression in RMS provides a specific target for immune-based therapy of RMS. We are developing T cells genetically modified to express chimeric antigen receptors (CARs) that target FGFR4.To verify FGFR4 RNA expression at the protein level we performed both immunohistochemistry (IHC) and electrochemillumescence (ECL) ELISA assays. Using IHC analysis we measured FGFR4 protein levels on tissue microarrays (TMA) of normal tissue and primary tumor from RMS patients, increased staining for FGFR4 protein on RMS primary tumors, compared to normal tissues was demonstrated. FGFR4 expression measured using ECL ELISA assay, shows a range of 300 - 800pg FGFR4 per 1mg of total lysate in RMS cell lines. The range for normal tissues was 30 - 40 pg/mg for all tissues with the exception of liver, which expressed 70 pg/mg. A single-chain variable fragment (scFv) cDNA library derived from a human B cells was screened, and clones that showed binding to recombinant FGFR4 extracellular domain (ECD) selected. We identified ten specific human anti-FGFR4 scFv binders. The scFvs were cloned into prokaryotic expression vector containing the human IgG1 Fc region. Anti-FGFR4 scFv-Fc were expressed in 293FT cells by transient transfection and purified using Protein A affinity chromatography. The binding of scFv-Fcs to recombinant FGFR4 ECD was verified by ELISA. scFv-Fc binders were then assayed for binding to cell surface FGFR4 on RMS cell lines using flow cytometry. Anti-FGFR4 scFv-Fc bound to 293T cells transfected to express FGFR4 but not 293T control cells. Anti-FGFR4 scFv-Fc also bound FGFR4 on three RMS cell lines. The first two anti-FGFR4 scFv binder sequences evaluated, M410 and M412, were used to make short (S, extracellular scFv only) and long format (L, scFv with a CH2CH3 domain of IgG1) CAR constructs. Activated T cells were transduced with lentiviral CAR expression vector (LV) encoding M410-L, M412-S and M412-L CAR constructs and cell surface expression of FGFR4 CAR on transduced T cells was measured using flow cytometry. The M410 and M412 FGFR4 CARs, both short and long constructs, were tested for cell-mediated cytotoxicity against RMS cell lines. M410-L showed higher cytotoxic activity compared to M412-L. M412-S showed greater cytotoxic activity compared to M412-L CAR. Thus, overall CAR structure format may be important for its functional activity. The remaining scFv will be further analyzed in various CAR formats for its functional activity including cytotoxicity and interferon-gamma production. In summary, the overexpression of FGFR4 protein in RMS versus normal cell lines demonstrates that FGFR4 may be a suitable target for immune-based therapy. FGFR4 CAR-T cell therapy offers the potential of a novel therapeutic intervention for high-risk, refractory and relapsed RMS.

Abstract A164: Genetically modified cytokine-induced killer (CIK) cells for targeted cancer therapy

Abstract Pre-emptive immunotherapy after HSC transplantation based on minimal residual disease (MRD) status with donor lymphocyte infusions (DLI) using cytokine-induced killer (CIK) cells may be beneficial to prevent relapse with a reduced risk of causing graft-versus-host-disease (GvHD) compared to conventional T cell infusion. CIK cells are a heterogeneous effector cell population including T cells (CD3+CD56-), natural killer (NK) cells (CD3-CD56+) and natural killer T (T-NK) cells (CD3+CD56+), that exhibit non-MHC-restricted cytotoxic activity and are generated by ex vivo expansion of peripheral blood mononuclear cells (PBMC) through the addition of interferon (IFN)-γ, anti-CD3 antibody, IL-2 and IL-15. While CIK cells have shown potent in vivo activity against various cancer types such as lymphomas or colorectal cancer, their cytotoxicity against B-ALL, characterized by the expression of CD19, has been limited. Hence, retargeting of CIK cells using chimeric antigen receptors (CARs) to facilitate selective target cell recognition and enhance specific cytotoxicity represents a promising approach. CAR comprise an extracellular scFv antibody fragment as an antigen-binding domain, linked via a flexible hinge region and a transmembrane domain to an intracellular signaling moiety such as CD3 zeta chain (first generation CAR), or zeta chain fused to a co-stimulatory protein domain such as CD28 (second generation CAR). We established an optimized protocol for transduction of CIK cells with CD19-specific lentiviral CAR constructs, and characterized cells for expression of an EGFP marker gene and CAR surface expression. Effects of exposure to lentiviral vector particles on the development of CIK cell subpopulations were monitored. In in vitro cytotoxicity assays we could demonstrate potent and selective cytotoxicity of retargeted CIK cells towards established cancer cell lines expressing CD19 and primary pre-B-ALL blasts. In addition, we observed significantly enhanced degranulation of CAR-CIK cells upon target cell contact and showed increased secretion of pro-inflammatory cytokines, while no secretion of immunosuppressive IL-10 could be detected. Cytotoxity towards non-malignant allo-PBMCs remained low. In addition, we investigated anti-leukemic activity of retargeted CIK cells in vivo in NOD/SCID common gamma chain knockout (NSG) mouse models using bioluminescence and fluorescence imaging. We observed potent reduction of tumor load after only two CIK cell applications in mice with an engrafted primary pre-B-ALL yielding long-term survivors with a negative MRD status (4/6). In a separate model, we demonstrated migration of CD19-specific CIK cells into local, subcutaneous tumor sites. In conclusion, CAR-CIK cells represent a promising alternative to CAR-T cell therapy, as relatively high numbers of conventional CIK cells have already been infused in clinical trials, which demonstrated a low risk of causing severe side effects. Citation Format: Sarah Oelsner, Juliane Wagner, Miriam E. Friede, Verena Pfirrmann, Eva Rettinger, Ralf Schubert, Heike Pfeifer, Evelyn Ullrich, Peter Bader, Winfried S. Wels. Genetically modified cytokine-induced killer (CIK) cells for targeted cancer therapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A164.

First-in-Man CD123-Specific Chimeric Antigen Receptor-Modified T Cells for the Treatment of Refractory Acute Myeloid Leukemia

Introduction: Adoptive immunotherapy using T-cells endowed with chimeric antigen receptors (CARs) has emerged as a promising new approach to treating CD19+ acute lymphoblastic leukemia (ALL). However,treatments for relapse/refractory acute myeloid leukemia (AML) have remained largely unchanged for nearly 50 years, and some AML patients have very poor prognosis. The interleukin-3 receptor alpha chain (CD123) has been identified as a potential immunotherapeutic target due to its overexpression in AML cells compared with normal hematopoietic stem cells. Antibodies targeting CD123 for the treatment of AML have demonstrated promising anti-leukemic activity in murine models but showed limited efficacy in clinical trials, suggesting that alternative and more potent therapies targeting CD123 are required.Methods: We have generated a 4th generation, apoptosis-inducible lentiviral CAR targeting CD123: CD123-scFv/CD28/CD137/CD27/CD3ζ-iCasp9 (4SCAR123), and demonstrated its high AML killing and AP1903-inducible apoptosis functions in ex vivo analyses. In a pilot trial of 4SCAR123, we enrolled a 47-year-old male patient with AML-M2 (FLT3/ITD+). The patient underwent allogeneic hematopoietic stem cell transplantation and relapsed. After 3 chemotherapies combined with sorafenib, his AML cells kept at 59% in bone marrow. He received CTX 250mg/kg/day for 3 days as conditioning regimen followed by 1.8x106/kg 4SCAR123 T cell infusion. Serum cytokine levels were measured by flow cytometric bead assay.Results: At day 1 after 4SCAR123 T infusion, the patient experienced rigorous chills and fevers, low blood pressure and hypoxemia. We detected elevated serum cytokine levels including interleukin-6 (2,500pg/ml) and tumor necrosis factor-α (33.8 pg/ml) at day 8, and the patient suffered from severe cytokine release syndrome (CRS) on day 4, which was controlled by one dose of Tocilizumab. BM examination detected a decrease of blasts from 59% to 45% 20 days after CAR-T therapy.Conclusion: Here we report a first-in-man pilot safety study of CD123 CAR-T therapy for AML patients. The 4SCAR123 exhibited potent cytotoxicity against AML in vitro, and in this pilot trial, the patient developed a rapid response consistent with CRS and achieved partial remission within 20 days. Importantly, although CD123 is universally expressed in myeloid and endothelial cells, we did not observe overt off-target cytotoxicity from the 4SCAR123 T cells, except for a controllable CRS. Our pilot study warrants further exploration of CD123 CAR for the management of refractory AML. DisclosuresDong:America Yuva Biomed: Consultancy.

Abstract 3157: Novel GD2-specific chimeric antigen receptor-modified T cells targeting osteosarcoma

Abstract Despite intensive multi-modality therapies employed in the treatment of osteosarcoma (OS) the prognosis for children with high-risk disease remains suboptimal. Since standard systemic therapy for OS has remained unchanged in the past thirty years, with no new agents identified to complement conventional regimens, there has been a significant plateau in the survival of OS patients thus necessitating the development of more targeted and efficacious therapeutics. Chimeric antigen receptor (CAR) modified T cells can meet this need by utilizing the immune system's surveillance capacity and potent cytotoxic mechanisms against tumor specific antigen targets with exquisite specificity. Since OS highly expresses the GD2 antigen, a viable immunotherapeutic target, we sought to assess if a novel 4th generation CAR modified T cell could target GD2 and induce cytotoxicity against OS. We have engineered a newer generation of CAR T cells that harness the vital co-stimulatory domains imperative for T cell activation while embedding the safety features of suicide induction. The latter, an advanced safety design iCasp9, is based on AP1903-induced caspase 9 dimer formation to trigger apoptosis, which has been incorporated in our CAR design. The 4th generation lentiviral CAR (4S-CAR) encodes a combination of CD28/CD137/CD27 T cell co-stimulatory domains and CD3zeta domain fused with a mutated FKBP12 (F36V) and caspase 9. To illustrate the self-destructive function, 4S-CAR T cells were treated with AP1903 to induce iCasp9 dimerization. High efficiency of apoptosis was observed within 30 min, and in 2 hours, more than 80% CAR T cells died. After demonstrated the ability to screen patient OS samples for GD2 expression, we found that OS cells expressing high levels of GD2 were effectively targeted and killed by 4S-GD2-CAR-T cells. 4S-GD2 CAR modified T cells were shown to overexpress the exhaustion marker PD-1 when in contact with target tumor cells, and multiple OS expressing cell lines were shown to overexpress PD-L1, and the PD-L1 level increased after cocultured with 4S-GD2 CAR modified T cells. The combination effects of immune checkpoint inhibitors such as anti-PD-L1 and anti-PD-1 antibodies with CAR T cells will be further investigated. Anti-GD2 expressing CAR T cells provide a novel targeted approach to eliminate residual disease with consummate specificity. As cellular therapeutics using CAR T cells become further entrenched in the forefront against refractory malignancies, OS provides a new and compulsory niche to further test the feasibility, safety and potential efficacy of this promising new technology. Citation Format: Monrat Chulanetra, Elias Sayour, Lamis Eldjerou, Joanne Lagmay, Rowan Milner, William Slayton, Lung-Ji Chang. Novel GD2-specific chimeric antigen receptor-modified T cells targeting osteosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3157. doi:10.1158/1538-7445.AM2015-3157

Abstract 3972: Carpet bombing with CARs: Influence of dose, timing, lymphodepletion and costimulatory domain on efficacy of mRNA engineered anti-CD19 Chimeric Antigen Receptor T cells in preclinical xenograft models.

Abstract T cells engineered to express chimeric antigen receptors (CARs), consisting of an anti-tumor targeting domain fused to Signal 1 and 2 activation domains, are a promising approach to cell therapy. We compared T cells engineered to stably express CARs using integrating viral vectors with T cells transiently expressing CARs via direct transduction with non-integrating mRNA. In early clinical testing, this CAR design using lentiviral transduction has been tested in patients with advanced CLL and relapsed ALL. Dramatic clinical responses have been observed in these refractory patients, speaking to the power of this approach. Transient expression has several advantages: i) short-term expression may allow initial clinical testing while limiting toxicity, and ii) RNA is easier to manufacture and modify, expediting manufacturing and scaling to GMP of changing CAR designs, allowing rapid iterative testing of and improvements to CAR design. To evaluate the in vivo efficacy of mRNA CAR+T cells compared to permanently modified (lentiviral transduction) T cells, we used xenograft models of human ALL (Nalm-6 and a primary patient leukemia, both CD19+) and high-level human T cell engraftment. NSG mice were injected with ALL at a dose producing 100% BM engraftment by 5d with 100% mortality by 25d (Nalm-6) or 36d (Primary). T cells were expanded ex vivo using CD3/CD28 beads and underwent lentiviral gene transfer or mRNA electroporation. Short-term efficacy of a single dose of mRNA vs. lentiviral CAR+ T cells was similar, and unlike permanently modified CAR T cells the costimulatory domain (CD28, 41BB or both) in mRNA engineered cells had no effect on efficacy in this model. Interval lymphodepletion with cyclophosphamide (CTX, 60mg/kg1) significantly improved efficacy with subsequent infusions of RNA CAR T cells. Neither CTX nor untargeted repeat CAR T cells infusions alone had an effect on leukemia burden or survival. While lentiviral CAR+ T cells persisted and were able to demonstrate a memory response on leukemia rechallenge, median overall survival was not different between repeat mRNA CARs (80d) and a single dose of lentiviral transduced CARs (77d). Importantly, both models produced long term (&amp;gt;120 days) survivors, suggesting that repeat infusion of RNA CAR T cells with interval lymphodepletion may approximate the efficacy of permanently modified T cells in this liquid tumor model. In our clinical experience with permanently modified cells, disease clearance happens over 14-21 days, which correlates with the window of efficacy of mRNA CARs. This suggests that though they do not persist, mRNA CARs may have significant potential for clinical efficacy. These pre-clinical results demonstrate an important advance in T cell engineering, and are leading directly to clinical trials testing mRNA CAR CTLs. Citation Format: David M. Barrett, Yangbing Zhao, Xiaojun Liu, Shuguang Jiang, Gregor S D Reid, Alix E. Seif, Carl H. June, Stephan A. Grupp. Carpet bombing with CARs: Influence of dose, timing, lymphodepletion and costimulatory domain on efficacy of mRNA engineered anti-CD19 Chimeric Antigen Receptor T cells in preclinical xenograft models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3972. doi:10.1158/1538-7445.AM2013-3972

Abstract 3505: Preclinical testing of mRNA engineered chimeric antigen receptor T cells: Comparison of a multi-infusion protocol to a single injection of stably expressed CARs in ALL xenograft models

Abstract T cells engineered to express chimeric antigen receptors (CARs), consisting of an anti-tumor targeting domain fused to Signal 1 and 2 activation domains, are a promising approach to cell therapy. We compared T cells engineered to stably express CARs using integrating viral vectors with T cells transiently expressing CARS via direct transduction with non-integrating mRNA. In early clinical testing, this CAR design using lentiviral transduction has been tested in 3 patients with advanced CLL. In these refractory patients with very high disease burdens, dramatic clinical responses have been observed, with two patients achieving CRs and one a PR, all sustained at &amp;gt;12 months. Transient expression has several advantages: short-term expression may allow initial clinical testing while limiting toxicity, and expediting manufacturing and scaling to GMP of CAR modifications, allowing rapid iterative improvements of CAR design. To evaluate the in vivo efficacy of mRNA CAR+T cells, we used xenograft models of human ALL (Nalm-6 and a primary patient leukemia, both CD19+) and high-level human T cell engraftment. NSG mice were injected with ALL at a dose producing 100% BM engraftment by 5d with 100% mortality by 25d (Nalm-6) or 36d (Primary). T cells were expanded ex vivo using CD3/CD28 beads and underwent lentiviral gene transfer or mRNA electroporation. mRNA transfection produced high but transient CAR surface expression, peaking at 4 hours and then declining over 8d. Using a novel method of simultaneous bioluminescent tracking of CARs and leukemia, we observed rapid migration of CAR T cells to all sites of disease and 100-fold reduction in ALL burden after only 24h. Using low dose cyclophosphamide (CTX; 60 mg/kg IP) 24h before the next infusion, we were able to deplete non-CAR expressing T cells and observe continued effects on leukemia from subsequent mRNA CAR infusions resulting in significantly prolonged survival in mice treated with mRNA CARs. Short-term efficacy of a single dose of mRNA vs. lentiviral CAR+ T cells was similar. Neither CTX nor untargeted repeat CAR T cells infusions alone had an effect on leukemia burden or survival. While lentiviral CAR+ T cells persisted and were active longer, median overall survival was not different between repeat mRNA CARs (52d Nalm-6, 76d primary) and a single dose of lentiviral transduced CARs (54d Nalm-6, 87d primary). Only stably expressed CARs resulted in any long term cures in this model. Relapse patterns suggest that the efficacy of mRNA and lentivirus CAR T cells are similar, as peripheral disease can be repeatedly cleared with subsequent mRNA CAR infusions but immune privileged xenograft artifact sites such as the tooth root are more resistant. These pre-clinical results demonstrate an important advance in T cell engineering, and are leading directly to clinical trials testing mRNA CAR CTLs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3505. doi:1538-7445.AM2012-3505