Dina Schneider

A tandem CD19/CD20 CAR lentiviral vector drives on-target and off-target antigen modulation in leukemia cell lines

BackgroundClinical success with chimeric antigen receptor (CAR)- based immunotherapy for leukemia has been accompanied by the associated finding that antigen-escape variants of the disease are responsible for relapse. To target hematologic malignancies with a chimeric antigen receptor (CAR) that targets two antigens with a single vector, and thus potentially lessen the chance of leukemic escape mutations, a tandem-CAR approach was investigated.MethodsAntigen binding domains from the FMC63 (anti-CD19) and Leu16 (anti-CD20) antibodies were linked in differing configurations to transmembrane and T cell signaling domains to create tandem-CARs. Expression on the surface of primary human T cells was induced by transduction with a single lentiviral vector (LV) encoding the tandem-CAR. Tandem-CARs were compared to single antigen targeting CARs in vitro and in vivo, and to an admixture of transduced cells expressing each CAR in vivo in immunodeficient (NSG) disease-bearing mice.ResultsTandem constructs efficient killed the Raji leukemia cell line both in vitro and in vivo. Tandem CARs generated less cytokine than the CD20 CAR, but similar to CD19 CARs, on their own. In co-culture experiments at low effector to target ratios with both single- and tandem- CAR-T cells, a rapid down-modulation of full-length CD19 expression was seen on leukemia targets. There also was a partial down-modulation of CD22, and to a lesser degree, of CD20. Our data also highlight the extreme sensitivity of the NALM-6 cell line to general lymphocyte-mediated cytotoxicity. While single and tandem constructs were effective in vivo in a standard setting, in a high-disease burden setting, the tandem CAR proved both effective and less toxic than an admixture of transduced T cell populations expressing single CARs.ConclusionTandem CARs are equally effective in standard disease models to single antigen specificity CARs, and may be both more effective and less toxic in a higher disease burden setting. This may be due to optimized cell killing with more moderate cytokine production. The rapid co-modulation of CD19, CD20, and CD22 may account for the ability to rapidly evolve escape mutants by selecting for leukemic clones that not require these target antigens for continued expansion.

Minimizing leukemia escape: implementing a dual anti-CD20- and CD19-scFv-based chimeric antigen receptor (CAR)

Adoptive immunotherapy with chimeric antigen receptor (CAR) transduced T lymphocytes has shown promising results in both pediatric and adult B cell malignancies. Nevertheless, both CAR-based and antibody-based anti-CD19 therapies, e.g. blinatumomab, have seen treatment failures attributed to the loss of CD19 or an epitope of CD19 on the surface of the malignant B cell. It may be possible to overcome antigen escape by targeting two tumor antigens simultaneously, i.e. CD19 and CD20 using a tandem construct with two scFv-based CAR binding domains. Lentiviral vectors encoding chimeric antigen receptors comprised of anti-CD19 and anti-CD20 targeting domains expressed alone or in tandem were transduced into T cells from healthy donors to generate the corresponding CAR19, CAR20, CAR19_20 (CD19 scFv more distal to the T cell plasma membrane) and CAR20_19 T cells (CD20 scFv distal). The transduced T cells were 50-70% CAR positive as determined by protein L flow cytometric analysis. Expression of CAR proteins of the expected molecular weight was confirmed by Western blot analysis of transduced T cells. When CAR-transduced T cells were combined with CD19+CD20+ Raji target cells, but not CD19-CD20- K562 cells, all four CAR T cell types demonstrated comparable efficient killing of leukemia targets (E:T ratio >2), and target-dependent induction of IFN-γ, as measured in co-culture supernatants by ELISA. We then began a series of in vitro co-culture experiments where we used very low E:T ratios to examine the potential for CAR-induced antigen loss on surviving leukemia cells. All CAR-T cells expressing an anti-CD19 scFv induced rapid loss of CD19 surface expression. In contrast, the CD20 surface marker was less prone to down-regulation by CAR-T cells expressing anti-CD20 scFv. Upon flow cytometric analysis of surviving leukemia cells on day 5, CD19 expression was reduced to 3%, 48%, 73%, 90% and 93% of control when co-cultured with CAR T cells expressing CAR19_20, CAR20_19, CAR19, CAR20, and control T cells, respectively. Similar results were seen when experiments were of longer, 7 days, or shorter, 1 day, duration. In conclusion, tandem CAR T cells are as effective as single CAR19 or CAR20 T cells in leukemia cell killing. Importantly, dual scFv-expressing CARs are more potent in preventing tumor antigen escape via target antigen down-regulation.

Closed-system manufacturing of CD19 and dual-targeted CD20/19 chimeric antigen receptor T cells using the CliniMACS Prodigy device at an academic medical center

BACKGROUND AIMS Multiple steps are required to produce chimeric antigen receptor (CAR)-T cells, involving subset enrichment or depletion, activation, gene transduction and expansion. Open processing steps that increase risk of contamination and production failure are required. This complex process requires skilled personnel and costly clean-room facilities and infrastructure. Simplified, reproducible CAR-T-cell manufacturing with reduced labor intensity within a closed-system is highly desirable for increased availability for patients. METHODS The CliniMACS Prodigy with TCT process software and the TS520 tubing set that allows closed-system processing for cell enrichment, transduction, washing and expansion was used. We used MACS-CD4 and CD8-MicroBeads for enrichment, TransAct CD3/CD28 reagent for activation, lentiviral CD8 TM-41BB-CD3 ζ-cfrag vectors expressing scFv for CD19 or CD20/CD19 antigens for transduction, TexMACS medium-3%-HS-IL2 for culture and phosphate-buffered saline/ethylenediaminetetraacetic acid buffer for washing. Processing time was 13 days. RESULTS Enrichment (N = 7) resulted in CD4/CD8 purity of 98 ± 4.0%, 55 ± 6% recovery and CD3+ T-cell purity of 89 ± 10%. Vectors at multiplicity of infection 5-10 resulted in transduction averaging 37%. An average 30-fold expansion of 108 CD4/CD8-enriched cells resulted in sufficient transduced T cells for clinical use. CAR-T cells were 82-100% CD3+ with a mix of CD4+ and CD8+ cells that primarily expressed an effector-memory or central-memory phenotype. Functional testing demonstrated recognition of B-cells and for the CAR-20/19 T cells, CD19 and CD20 single transfectants were recognized in cytotoxic T lymphocyte and interferon-γ production assays. DISCUSSION The CliniMACS Prodigy device, tubing set TS520 and TCT software allow CAR-T cells to be manufactured in a closed system at the treatment site without need for clean-room facilities and related infrastructure.

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.

648. Mitigating Tumor Escape: Tandem Anti-CD20- and CD19 SCFV-Based Chimeric Antigen Receptors (CARs) in Leukemia/Lymphoma

In recent years, adoptive cell therapy using T cells engineered with chimeric antigen receptors (CAR T) specific for CD19 has shown marked clinical efficacy. Nevertheless, cases of tumor escape due to tumor escape in B cell malignancies could be mitigated by CAR T the loss of CD19 antigen have been reported. We hypothesized that approaches that target the CD19 and CD20 tumor antigens simultaneously. Second generation CARs containing scFv specific for CD19 and CD20 were generated by lentiviral transduction of human primary T cells. The configurations were: a) single targeting domain, b) tandem targeting domains, or c) two full-length CAR receptors co-expressed bicistronically in the same T cell. CAR T expression for all constructs confirmed by Western blotting and protein L flow cytometry. CAR T cytolytic activity and IFN gamma secretion were evaluated by killing assays and ELISA, respectively. Single, tandem, and bicistronic CAR T cells exhibited CAR surface expression of 50-70%. All anti- CD19, anti-CD20 CAR T cells demonstrated target-specific cells lysis and induction of IFN-gamma. Interestingly, bicistronic CAR19, CAR20 construct yielded high CAR T surface expression and IFN-gamma production, but inferior in vitro cytolytic activity. To confirm CAR T specificity, K562 CD19+ and K562 CD20+ cell lines were generated. In flow-based 1h killing assays, CAR19 and CAR20 lysed their respective target lines only, whereas tandem CAR constructs lysed both K562 CD19+ and K562 CD20+ cells, but not K562 control. In a subsequent series of co-culture experiments, Raji target cells were combined with very low ratios of CAR T cells, to allow for the evolution of escape variants. On day 5 of co-incubation in the presence of CAR19 T cells, the surviving Raji cells numbers were comparable to control (85,000 cells/well vs 77, 000 cells/well in sham transduction). In comparison, viable Raji numbers decreased drastically post co-incubation with CAR20-, CAR19_20- and CAR20_19-T cells (546, 1,355 and 1,543 cells/well, respectively). Among the surviving Raji population, CD19 surface expression was reduced to 1.6%, vs 93.29% for sham control, whereas the expression of CD20 and CD22 remained high (97% and 78%, respectively). By contrast, the CD20 surface marker was less prone to down-regulation by CAR-T cells expressing anti-CD20 scFv. Similar results were seen when experiments were of longer, 7 days, or shorter, 1 day, duration. Thus, targeting two tumor antigens simultaneously using tandem CAR T cells is a promising new strategy for mitigation of tumor escape via antigen down-regulation. Moreover, expression of two scFv in a single CAR format is superior to expression of two independent CARs from a bicitsronic vector. It also appears that the order of the targeting domains within the tandem CAR impacts anti-tumor activity. These findings are currently being explored further in animal model systems.

Abstract 2297: Tandem anti-CD20 and -CD19 scFV-based chimeric antigen receptors (CARs) mitigate tumor escape in hematologic malignancies

In recent years, adoptive cell therapy using T cells engineered with chimeric antigen receptors (CAR T) specific for CD19 has shown marked clinical efficacy. Nevertheless, cases of tumor escape due to the loss of CD19 antigen have been reported. We hypothesized that tumor escape in B cell malignancies could be mitigated by CAR T approaches that target the CD19 and CD20 tumor antigens simultaneously. Second generation CARs containing scFv specific for CD19 and CD20 were generated by lentiviral transduction of human primary T cells. The configurations were: a) single targeting domain, b) tandem targeting domains, or c) two full-length CAR receptors co-expressed bicistronically in the same T cell. CAR T expression for all constructs was confirmed by Western blotting and protein L flow cytometry. CAR T cytolytic activity and IFN gamma secretion were evaluated by killing assays and ELISA, respectively. Single, tandem, and bicistronic CAR T cells exhibited CAR surface expression of 50-70%. All anti-CD19 and anti-CD20 CAR T cells demonstrated target-specific cells lysis and induction of IFN gamma. Interestingly, the bicistronic CAR19 CAR20 construct yielded high CAR T surface expression and IFN gamma production, but inferior in vitro cytolytic activity. To confirm CAR T specificity, K562 CD19+ and K562 CD20+ cell lines were generated. In flow-based 1h killing assays, CAR19 and CAR20 lysed their respective target lines only, whereas tandem CAR constructs lysed both K562 CD19+ and K562 CD20+ cells, but not K562 control. In a subsequent series of co-culture experiments, Raji target cells were combined with very low ratios of CAR T cells, to allow for the evolution of escape variants. On day 5 of co-incubation in the presence of CAR19 T cells, the surviving Raji cells numbers were comparable to control (85,000 cells/well vs 77, 000 cells/well in sham control). In comparison, viable Raji numbers decreased drastically post co-incubation with CAR20-, CAR19_20- and CAR20_19-T cells (546, 1,355 and 1,543 cells/well, respectively). Among the surviving Raji population, CD19 surface expression was reduced to 1.6%, vs 93.29% for sham control, whereas the expression of CD20 and CD22 remained high (97% and 78%, respectively). By contrast, the CD20 surface marker was less prone to down-regulation by CAR-T cells expressing anti-CD20 scFv. Similar results were seen when experiments were of longer, 7 days, or shorter, 1 day, duration. Thus, targeting two tumor antigens simultaneously using tandem CAR T cells is a promising new strategy for mitigation of tumor escape via antigen down-regulation. Moreover, expression of two scFv in a single CAR format is superior to expression of two independent CARs from a bicitsronic vector. It also appears that the order of the targeting domains within the tandem CAR impacts anti-tumor activity. These findings are currently being explored further in animal model systems. Citation Format: Dina Schneider, Ying Xiong, Darong Wu, Boro Dropulic, Rimas Orentas. Tandem anti-CD20 and -CD19 scFV-based chimeric antigen receptors (CARs) mitigate tumor escape in hematologic malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2297.