anti-CAR Antibody Research Articles

CAR-Aptamers Enable Traceless Enrichment and Monitoring of CAR-Positive Cells and Overcome Tumor Immune Escape.

Chimeric antigen receptor (CAR)-positive cell therapy, specifically with anti-CD19 CAR-T (CAR19-T) cells, achieves a high complete response during tumor treatment for hematological malignancies. Large-scale production and application of CAR-T therapy can be achieved by developing efficient and low-cost enrichment methods for CAR-T cells, expansion monitoring in vivo, and overcoming tumor escape. Here, novel CAR-specific binding aptamers (CAR-ap) to traceless sort CAR-positive cells and obtain a high positive rate of CAR19-T cells is identified. Additionally, CAR-ap-enriched CAR19-T cells exhibit similar antitumor capacity as CAR-ab (anti-CAR antibody)-enriched CAR-T cells. Moreover, CAR-ap accurately monitors the expansion of CAR19-T cells in vivo and predicts the prognosis of CAR-T treatment. Essentially, a novel class of stable CAR-ap-based bispecific circular aptamers (CAR-bc-ap) is constructed by linking CAR-ap with a tumor surface antigen (TSA): protein tyrosine kinase 7 (PTK7) binding aptamer Sgc8. These CAR-bc-aps significantly enhance antitumor cytotoxicity with a loss of target antigens by retargeting CAR-T cells to the tumor in vitro and in vivo. Overall, novel CAR-aptamers are screened for traceless enrichment, monitoring of CAR-positive cells, and overcoming tumor cell immune escape. This provides a low-cost and high-throughput approach for CAR-positive cell-based immunotherapy.

Rapid anti-myeloma activity by T cells expressing an anti-BCMA CAR with a human heavy-chain-only antigen-binding domain

Multiple myeloma (MM) is a rarely curable malignancy of plasma cells. MM expresses B cell maturation antigen (BCMA). We developed a fully human anti-BCMA chimeric antigen receptor (CAR) with a heavy-chain-only antigen-recognition domain, a 4-1BB domain, and a CD3ζ domain. The CAR was designated FHVH33-CD8BBZ. We conducted the first-in-humans clinical trial of Tcells expressing FHVH33-CD8BBZ (FHVH-T). Twenty-five patients with relapsed MM were treated. The stringent complete response rate (sCR) was 52%. Median progression-free survival (PFS) was 78weeks. Of 24 evaluable patients, 6 (25%) had a maximum cytokine-release syndrome (CRS) grade of 3; no patients had CRS of greater than grade 3. Most anti-MM activity occurred within 2-4weeks of FHVH-T infusion as shown by decreases in the rapidly changing MM markers serum free light chains, urine light chains, and bone marrow plasma cells. Blood CAR+ cell levels peaked during the time that MM elimination was occurring, between 7 and 15days after FHVH-T infusion. C-C chemokine receptor type 7 (CCR7) expression on infusion CD4+ FHVH-T correlated with peak blood FHVH-T levels. Single-cell RNA sequencing revealed a shift toward more differentiated FHVH-T after infusion. Anti-CAR antibody responses were detected in 4 of 12 patients assessed. FHVH-T has powerful, rapid, and durable anti-MM activity.

Immune Function of Chimeric Antigen Receptor T Cells Quantitatively Assessed Via Molecular Imaging Flow Cytometry

Chimeric antigen receptor (CAR) T cells are an adoptive cell immunotherapy genetically synthesized to reprogram T cells for antigen recognition and intracellular signaling to target tumor cells for destruction. A sensitive and reproducible assay to evaluate CAR T cell function is essential to research and develop novel therapies, optimize manufacturing, and evaluate efficacy in the clinical and non-clinical settings. However, conventional methods, such as cytotoxicity assays and in vitro cytokine production, require a long cell culture processing time with a less quantitative and more time-consuming variability. In the present study, we analyzed the change in localization of CAR molecules immediately following antigen recognition using molecular imaging flow cytometry (MI-FCM) to establish a quantitative method to evaluate the immune functionality of CAR T cells, which is correlated with cytotoxic activity. Anti-CD19 CAR (FMC63-28z) was constructed using anti-CD19 scFv (FMC63) based on the method described by Kochenderfer et al. (J Immunol, 2009). CAR T cells were generated by isolating human peripheral blood mononuclear cells from nine healthy donors (21-69 years old). They were then stimulated on plates pre-coated with CD3 and CD28 antibodies in ALys505N-7 medium containing 700 IU/mL IL-2, transfected with anti-CD19 CAR using a lentivirus on day 3, and subsequently expanded on plates pre-coated with CD3 and CD28 antibodies for maximum to 18 days. Second, each CAR T cell was stimulated with or without CD19 antigen, as well as the K562 cell line stably transfected with CD19 (K562-CD19), fixed with paraformaldehyde, and immunostained with anti-CAR, CD4, and CD8 antibodies. We defined the decrease in the CAR molecule area of expression on the cell surface as concentrated CAR. The existence and number of spots of concentrated CAR on CD4- or CD8-positive CAR T cells were analyzed using MI-FCM. The concentration of superficial CAR antigens on some CAR T cells was observed in every lot from the nine healthy donors when they were stimulated with K562-CD19 cells or CD19 antigen. In contrast, this was not observed in the unstimulated controls. When each lot of CAR T cells was stimulated with CD19 antigen and then quantitatively assessed using MI-FCM, the proportion of cells with concentrated CAR among the CD4-positive and CD8-positive CAR T cells was 83.1 ± 11.7% and 81.3 ± 8.1%, respectively, while the proportion of cells with a single spot fraction of concentrated CAR was 31.2 ± 4.0% and 33.8 ± 6.1%, respectively. Third, the cytotoxic activity of each lot of CAR T cells co-cultured with K562-CD19 cells transfected with the firefly luciferase reporter gene for 15 h was evaluated using a luciferase-mediated bioluminescence imaging assay. Importantly, the proportion of single-spot concentrated CAR among CD8-positive CAR T cells was very strongly positively correlated with killing activity (R = 0.916; 95% CI: 0.644-0.982), while the proportion of single-spot concentrated CAR among CD4-positive CAR T cells was significantly positive correlated (R = 0.693; 95% CI: 0.053-0.930). These results suggest that quantifying the CAR concentration in CAR T cells using MI-FCM could be used to evaluate their immune function.

Development of an anti-CAR antibody response in SIV-infected rhesus macaques treated with CD4-MBL CAR/CXCR5 T cells.

T cells expressing a simian immunodeficiency (SIV)-specific chimeric antigen receptor (CAR) and the follicular homing molecule, CXCR5, were infused into antiretroviral therapy (ART) suppressed, SIV-infected rhesus macaques to assess their ability to localize to the lymphoid follicle and control the virus upon ART interruption. While the cells showed evidence of functionality, they failed to persist in the animals beyond 28 days. Development of anti-CAR antibodies could be responsible for the lack of persistence. Potential antigenic sites on the anti-SIV CAR used in these studies included domains 1 and 2 of CD4, the carbohydrate recognition domain (CRD) of mannose-binding lectin (MBL), and an extracellular domain of the costimulatory molecule, CD28, along with short linker sequences. Using a flow cytometry based assay and target cells expressing the CAR/CXCR5 construct, we examined the serum of the CD4-MBL CAR/CXCR5-T cell treated animals to determine that the animals had developed an anti-CAR antibody response after infusion. Binding sites for the anti-CAR antibodies were identified by using alternative CARs transduced into target cells and by preincubation of the target cells with a CD4 blocking antibody. All of the treated animals developed antibodies in their serum that bound to CD4-MBL CAR/CXCR5 T cells and the majority were capable of inducing an ADCC response. The CD4 antibody-blocking assay suggests that the dominant immunogenic components of this CAR are the CD4 domains with a possible additional site of the CD28 domain with its linker. This study shows that an anti-drug antibody (ADA) response can occur even when using self-proteins, likely due to novel epitopes created by abridged self-proteins and/or the self-domain of the CAR connection to a small non-self linker. While in our study, there was no statistically significant correlation between the ADA response and the persistence of the CD4-MBL CAR/CXCR5-T cells in rhesus macaques, these findings suggest that the development of an ADA response could impact the long-term persistence of self-based CAR immunotherapies.

The Safety and Efficacy of p CAR-19B, a Scfv Humanlized CD19-Directed CAR-T for Patients with Relapsed/Refactory (R/R) CD19+ B-ALL

Introduction: Two murine anti-CD19 scFv based chimeric antigen receptors T cell (CAR-T) products have been approved by FDA for children and adults R/R B-ALL patients, with about 50%-70% complete remission (CR) rates. Humoral and/or cellular immune responses against murine scFv may attenuate the efficacy. To reduce immunogenicity of murine scFv, we developed an optimal humanized anti-CD19 CAR-T product named pCAR-19B, which have a CAR structure including humanized CD19 specific scFv following 4-1BB and CD3ζ cytoplasmic domain. Here we report the preliminary safety and efficacy data for the adults R/R B-ALL patients in a phase I clinical trials. Methods: In the phase Istudy (NCT04888442), Eligible patients were histologically confirmed CD19+ B-ALL, KPS＞60, relapsed or refractory to standard therapies. Patients received single-dose of pCAR-19B at dose level 1 (DL1) (0.6×106 CAR/kg), level 2 (DL2) (2×106 CAR/kg) after fludarabine (25-30 mg/m2 × 3 days) and cyclophosphamide (10-20mg/ kg× 3 days) based pre-condition. Toxicity was graded by CTCAE, CRS and ICANS were graded by ASTCT criteria. Primary endpoint is the incidence of dose-limiting toxicities (DLTs) in the first 28 days. Secondary endpoints are cellular kinetics, overall response rate (CR/CRi), duration of response, and overall survival. Results: As of June 30, 2022, nine patients (3 in DL1, 6 in DL2) were infused with pCAR-19B and all patients have finished at least 6 months follow up visit. Demographics, baseline disease, prior treatment and special concerned adverse events are presented in the table. CAR-T-related adverse events occurred in 9 patients (9/9, 100%), no adverse events leading to patient withdrawal from the study occurred. 8 patients (8/9, 89%) experienced cytokine release syndrome (CRS), including 5 (5/9, 56%) grade 1, 3 (3/9, 33%) grade 2, and no grade 3 or higher CRS occurred. Two patients (2/9, 22%) experienced immune effector cell-associated neurotoxicity syndrome (ICANS), including 1 (1/9, 11%) grade 1, 1 (1/9, 11%) grade 2, and no grade 3 or higher ICANS occurred. No DLTs were reported. 100% (9/9) CR/CRi was achieved in the first month after CAR-T infusion. The relapse-free survival (RFS) is presented in the figure. The median OS and RFS is not reached yet. No anti-CAR antibody was detected in any patients in 1M and 3M after CAR-T infusion. Conclusion: pCAR-19B has demonstrated a good safe profile and 100% CR rate in phase I study for adults R/R CD19+ B-ALL. The muti-center pivotal study is going to recruit patients soon in China. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Long-Term Safety and Efficacy of CD19 Humanized Selective CAR-T Therapy in B-ALL Patients Who Have Previously Received Murine-Based CD19 CAR-T Therapy.

Murine-based CD19 CAR-T (CD19m CAR-T) therapy can lead to a relatively high CR rate when administered to B-ALL patients for the first time. However, the DOR is sub-optimal and a subset of patients even show primary resistance to CD19m CAR-T. To address these issues, we employed a humanized selective CD19CAR-T (CD19hs CAR-T) and evaluated the long-term safety and efficacy of treating 8 R/R B-ALL patients who had relapsed or failed to achieve CR following CD19m CAR-T infusion (Clinical trials’ number: ChiCTR1800014761 and ChiCTR1800017439). Of the 8 patients, 7 achieved CR on Day 30 after the 1st infusion of CD19hs CAR-T. The median CRS grade was 1 without significant neurotoxicity seen in any of the 8 patients. The median DOR was 11 months, significantly longer than the DOR following CD19mCAR-T infusions. Anti-CAR antibodies were induced in patients who had received prior CD19m CAR-T infusions but not in those following a single or repeated CD19hsCAR-T treatment, which probably had contributed to the sub-optimal DOR and/or failure of effective response in these patients. CD19hs CAR-T, in contrast, induced low immunogenicity compared with CD19m CAR-T, suggesting that a repeat dosing strategy might be feasible and efficacious for patients who have relapsed and/or show primary resistance to CD19m CAR-T therapy. In this clinical study, CD19hs CAR-T showed a significant clinical efficacy with mild side effect among patients with R/R B-ALL who had previously received CD19m CAR-T.Clinical Trial Registration https://www.chictr.org.cn/showprojen.aspx?proj=25199 (ChiCTR1800014761). https://www.chictr.org.cn/showproj.aspx?proj=29174 (ChiCTR1800017439).

Abstract 561: CAR-targeted IL-2 drives selective CAR-T cell expansion and improves anti-tumor efficacy

Abstract Chimeric antigen receptor T cell (CAR-T) therapies have transformed the treatment of some hematological malignancies and are showing promising preliminary results in solid tumors. Recent studies have shown that in vivo expansion and persistence of CAR-Ts are correlated with improved therapeutic outcomes in patients. Administration of IL-2 enhances CAR-T engraftment, persistence, and functionality in preclinical models. However, the clinical potential of IL-2 stimulation with cell therapies is limited using current molecules due to severe toxicity of high-dose IL-2 and the inadequate selectivity of existing engineered IL-2 variants which expand multiple endogenous cells in addition to CAR-T cells. To address this challenge, we have applied our cis-targeting technology to develop CAR-T selective IL-2 fusion molecules that specifically activate CAR-Ts, while exhibiting minimal activity on CAR-negative cells. Cis-targeted IL-2 fusions are comprised of a fusion between a targeting antibody and an IL-2 mutein having attenuated binding to IL2R⍺ and IL2Rβ. The attenuated cytokine selectively binds and activates IL-2 receptors on CAR-Ts via the avidity provided by the targeting arm. We engineered two cis-targeted CAR-T selective IL-2 fusions, CAR-IL2 or EGFRt-IL2. CAR-IL2 targets the FMC63 CAR directly without blocking CD19 antigen recognition, enabling targeting of approved anti-CD19 CAR-T products, and EGFRt-IL2 targets the EGFRt tag co-expressed with the CAR. Molecules were characterized in vitro using primary human CAR-transduced T cells. The in vivo activity was examined using tumor bearing NSG mice infused with human CAR-Ts. The specificity of the CAR-IL2 or EGFRt-IL2 molecules was demonstrated by their ability to selectively induce pSTAT5 signaling resulting in >1000-fold preferential STAT5 activity in CAR-expressing cells compared to CAR-negative cells. The ability of the fusion proteins to selectively expand CAR-Ts in vivo was shown through a substantial and specific expansion of the infused CAR-Ts. The CAR-T fraction increased from approximately 50% of infused T cells, to over 93% of T cells, demonstrating a nearly 40-fold expansion in vivo. Re-expansion of CAR-Ts was demonstrated after allowing the CAR-Ts to rest in vivo >100 days; re-dosing resulted in significant and selective re-expansion of CAR-T cells. Furthermore, treatment with the CAR-IL2 molecule enhanced tumor regression of CD19+ lymphoma-bearing mice infused with suboptimal doses of CAR-Ts. Together, these data demonstrate that our cis-targeted IL-2 molecules selectively activate CAR-Ts in vitro and enhance CAR-T expansion and anti-tumor efficacy in vivo. Cis-targeted IL-2 fusion molecules directed by anti-CAR or anti-tag antibodies represent a promising approach to confer enhanced CAR-T activity in a specific and temporally controlled manner. Citation Format: Nathan D. Mathewson, Kelly D. Moynihan, Sara Sleiman, Wei Chen, Paul Bessette, Christopher Kimberlin, Eric Wigton, Danielle Pappas, Terrence Park, Ton N. Schumacher, Saar I. Gill, Yik Andy Yeung, Ivana Djuretic. CAR-targeted IL-2 drives selective CAR-T cell expansion and improves anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 561.

Abstract CT215: LB1901: A phase 1, open-label, multicenter, multicohort study of CD4-targeted chimeric antigen receptor T cells (CD4-CAR-T) in relapsed or refractory T-cell lymphoma (TCL)

Abstract Background: Disease relapse is common with peripheral T-cell lymphomas (PTCL) or cutaneous T-cell lymphomas (CTCL). Adoptive immunotherapy with CD19 directed CAR-T cells is a standard option for B cell lymphoid malignancies in the relapsed setting. This study assesses the safety and preliminary efficacy of CD4 targeting autologous CAR-T, LB1901, in patients (pts) with CD4+ relapsed or refractory (R/R) TCL. A pre-clinical study showed LB1901 exhibited potent anti-tumor activity without off-target effects (Zeng 2021). Methods: This is a phase 1, open-label, multicenter, multicohort study of LB1901 (NCT04712864; currently enrolling). Eligible pts are ≥18 years with histologically confirmed CD4+ PTCL-NOS (not otherwise specified); or CD4+ AITL (angioimmunoblastic T-cell lymphoma); or CD4+ CTCL (either MF [mycosis fungoides] or SS [Sézary syndrome]). In Part A (dose escalation), pts with SS must have ≤2,000 circulating Sézary cells/µL to ensure safety of pts with higher disease burden. In Part B, pts with >2,000 Sézary cells/µL is allowed. CD4 expressed on tumor cells must be confirmed within 3 months prior to apheresis. Other inclusion criteria include: R/R disease with ≥2 prior lines of systemic antineoplastic therapy; for pts with PTCL-NOS or AITL, ≥1 measurable lesion according to IWG Response Criteria (Cheson 2014) and for pts with CTCL, ≥stage IIB on TNMB-ISLC/EORTC staging system; identified HSCT donor available prior to enrollment (in the event of severe recurrent infections and prolonged lymphopenia for which pt may need an allogeneic HSCT as a safety rescue); and ECOG status of 0 or 1. Response will be evaluated based on IWG response criteria for PTCL and Global Composite Response for CTCL (Olsen 2007). The study will be conducted in 2 parts (Part A: dose escalation and Part B: dose expansion). Three dose levels (0.3 X 106, 1.0 X 106, and 3.0 X 106 CAR+ viable T cells/kg) will be evaluated (N=3-6 for each dose level) with an optional dose level minus 1 (0.1 X 106 CAR+ viable T cells/kg). Part B will include 2 cohorts, PTCL and CTCL (N=12-20 for each), after the recommended dose for expansion (RDE) has been identified in Part A. Enrolled pts will undergo apheresis for PBMC collection. LB1901 will be manufactured from autologous CD8+ T cells. Pts may receive optional bridging therapy and receive lymphodepleting chemotherapy with fludarabine 30mg/m2/day and cyclophosphamide 300mg/m2/day (Flu-Cy), for 3 days followed by LB1901. Primary endpoints are incidence, duration, and severity of AEs and laboratory abnormalities (Parts A and B) and DLT at each dose level (Part A). Secondary endpoints include overall response rate; duration of response; CAR-positive T cell counts and CAR transgene level in blood; and presence of anti-CAR antibody response. Exploratory endpoints include preliminary efficacy, pharmacokinetics, and CD4+ T cell counts. Citation Format: Swaminathan P. lyer, David G. Maloney, Nora Bennani, Auris Huen, Muhammad Akram, Soo Kim, Chuan Wang, Zhiyin Liang, Henry Castro, Lida Pacaud, Mehdi Hamadani. LB1901: A phase 1, open-label, multicenter, multicohort study of CD4-targeted chimeric antigen receptor T cells (CD4-CAR-T) in relapsed or refractory T-cell lymphoma (TCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT215.

Immunogenicity of CAR-T Cell Therapeutics: Evidence, Mechanism and Mitigation.

Chimeric antigen receptor T cell (CAR-T) therapy demonstrated remarkable success in long-term remission of cancers and other autoimmune diseases. Currently, six products (Kymriah, Yescarta, Tecartus, Breyanzi, Abecma, and Carvykti) are approved by the US-FDA for treatment of a few hematological malignancies. All the six products are autologous CAR-T cell therapies, where delivery of CAR, which comprises of scFv (single-chain variable fragment) derived from monoclonal antibodies for tumor target antigen recognition is through a lentiviral vector. Although available CAR-T therapies yielded impressive response rates in a large number of patients in comparison to conventional treatment strategies, there are potential challenges in the field which limit their efficacy. One of the major challenges is the induction of humoral and/or cellular immune response in patients elicited due to scFv domain of CAR construct, which is of non-human origin in majority of the commercially available products. Generation of anti-CAR antibodies may lead to the clearance of the therapeutic CAR-T cells, increasing the likelihood of tumor relapse and lower the CAR-T cells efficacy upon reinfusion. These immune responses influence CAR-T cell expansion and persistence, that might affect the overall clinical response. In this review, we will discuss the impact of immunogenicity of the CAR transgene on treatment outcomes. Finally, this review will highlight the mitigation strategies to limit the immunogenic potential of CARs and improve the therapeutic outcome.

USING A NONVIRAL AND NONINTEGRATING DNA VECTOR TO GENERATE SAFE CAR-T CELLS

CAR-T cell therapy has proven its importance in cancer treatment and currently there are five approved products on the market. However, these products are based on viral vectors, which presents a risk of insertional mutagenesis, it can also be immunogenic and its manufacture is extremely expensive. The development of a nonviral vector, suitable for an efficient and stable transfection of NK and T cells is the goal of this study. In this study we use a special episomal vector (extrachromosomal vectors) with i) scaffold/matrix attachment region (S/MAR) motifs that can actively mediate the episomal maintenance, ii) a plasmid replication enhancer, the replication-Initiation Region (IR) from the β-globin locus (pEP-IR) and iii) a CAR anti CD19 (pEP-IR-CAR). We electroporated activated and non-activated T cells and NK cells with different plasmid concentrations. We evaluated transfection efficiency and viability. Our findings showed that unstimulated T cells are transfected more efficiently than activated T cells. Transfection of unstimulated T cells with 20 ug of supercoiled pEP-IR-GFP plasmid reached 22 to 63% of GFP and viability around 54% depending on the culture condition used after electroporation (FBS% and IL2). We then accessed the efficacy of pEP-IR-CAR and CAR expression, confirmed in 3,2% of the cells with 89% of viability. Cells expressing CAR on their surface were marked with anti-CAR antibody Biotin-SP-conjugated and selected by anti-Biotin microbeads, achieving an enrichment of 54,9% CAR+ cells. Our results showed that we established an optimized protocol resulting in an acceptable transfection efficiency and the transfected cells can be selected. So, the next step is to perform in vitro cytotoxicity and in vivo assays with the episomal CAR+ cells. Financially supported by FAPESP 2020/09206-4, FAPESP 2019/25309-0, CTC Center for Cell-based Therapy (FAPESP 2013/08135-2), and National Institute of Science and Technology in Stem Cell and Cell Therapy (CNPq 573754-2008-0 and FAPESP 2008/578773).

Anti B-cell maturation antigen CAR T-cell and antibody drug conjugate therapy for heavily pretreated relapsed and refractory multiple myeloma.

DISCLOSURES: Funding for this summary was contributed by Arnold Ventures, California Health Care Foundation, The Donaghue Foundation, Harvard Pilgrim Health Care, and Kaiser Foundation Health Plan to the Institute for Clinical and Economic Review (ICER), an independent organization that evaluates the evidence on the value of health care interventions. ICER's annual policy summit is supported by dues from AbbVie, Aetna, America's Health Insurance Plans, Anthem, Alnylam, AstraZeneca, Biogen, Blue Shield of CA, Boehringer-Ingelheim, Cambia Health Services, CVS, Editas, Evolve Pharmacy, Express Scripts, Genentech/Roche, GlaxoSmithKline, Harvard Pilgrim, Health Care Service Corporation, HealthFirst, Health Partners, Humana, Johnson & Johnson (Janssen), Kaiser Permanente, LEO Pharma, Mallinckrodt, Merck, Novartis, National Pharmaceutical Council, Pfizer, Premera, Prime Therapeutics, Regeneron, Sanofi, Spark Therapeutics, uniQure, and United Healthcare. Beinfeld, Fluetsch, and Pearson are employed by ICER. Ollendorf received funding from ICER for work on this summary and reports consulting and other personal fees from EMD Serono, Amgen, Analysis Group, Aspen Institute/University of Southern California, GalbraithWight, Cytokinetics, Executive Insight, Sunovion, University of Colorado, World Health Organization, and Eli Lilly, unrelated to this work. Lee and McQueen received funding from ICER for work on this summary.

Incorporation of bacterial immunoevasins to protect cell therapies from host antibody-mediated immune rejection

Cellular therapies are engineered using foreign and synthetic protein sequences, such as chimeric antigen receptors (CARs). The frequently observed humoral responses to CAR Tcells result in rapid clearance, especially after re-infusions. There is an unmet need to protect engineered cells from host-versus-graft rejection, particularly for the advancement of allogeneic cell therapies. Here, utilizing the immunoglobulin G (IgG) protease "IdeS," we programmed CAR Tcells to defeat humoral immune attacks. IdeS cleavage of host IgG averted Fc-dependent phagocytosis and lysis, and the residual F(ab')2 fragments remained on the surface, providing cells with an inert shield from additional IgG deposition. "Shield" CAR Tcells efficiently cleaved cytotoxic IgG, including anti-CAR antibodies, detected in patient samples and provided effective anti-tumor activity in the presence of anti-cell IgG invivo. This technology may be useful for repeated human infusions of engineered cells, more complex engineered cells, and expanding widespread use of "off-the-shelf" allogeneic cellular therapies.

Coxsackievirus B3 Infection of Human iPSC Lines and Derived Primary Germ-Layer Cells Regarding Receptor Expression.

The association of members of the enterovirus family with pregnancy complications up to miscarriages is under discussion. Here, infection of two different human induced pluripotent stem cell (iPSC) lines and iPSC-derived primary germ-layer cells with coxsackievirus B3 (CVB3) was characterized as an in vitro cell culture model for very early human development. Transcriptomic analysis of iPSC lines infected with recombinant CVB3 expressing enhanced green fluorescent protein (EGFP) revealed a reduction in the expression of pluripotency genes besides an enhancement of genes involved in RNA metabolism. The initial distribution of CVB3-EGFP-positive cells within iPSC colonies correlated with the distribution of its receptor coxsackie- and adenovirus receptor (CAR). Application of anti-CAR blocking antibodies supported the requirement of CAR, but not of the co-receptor decay-accelerating factor (DAF) for infection of iPSC lines. Among iPSC-derived germ-layer cells, mesodermal cells were especially vulnerable to CVB3-EGFP infection. Our data implicate further consideration of members of the enterovirus family in the screening program of human pregnancies. Furthermore, iPSCs with their differentiation capacity into cell populations of relevant viral target organs could offer a reliable screening approach for therapeutic intervention and for assessment of organ-specific enterovirus virulence.

Phase 1/2 Study of the Safety and Response of P-BCMA-101 CAR-T Cells in Patients with Relapsed/Refractory (r/r) Multiple Myeloma (MM) (PRIME) with Novel Therapeutic Strategies

Phase 1/2 Study of the Safety and Response of P-BCMA-101 CAR-T Cells in Patients with Relapsed/Refractory (r/r) Multiple Myeloma (MM) (PRIME) with Novel Therapeutic Strategies

Abstract 3714: Stealth liposomes encapsulating plasmid DNA of telomerase specific oncolytic adenovirus

Abstract Background: Oncolytic virotherapy has the potential to become the next breakthrough in cancer therapy. We previously established a telomerase-specific oncolytic adenovirus (Telomelysin), which is controlled to replicate and induce oncolytic cell death selectively in tumor cells, and its safety for humans was proven in the phase I clinical trial performed in the USA. While oncolytic adenoviruses have promising antitumor effects, the application is basically limited to local administration because of non-specific trapping in the liver and immune elimination after systemic delivery. Liposomes are among the most common nanoparticles used as drug carriers, and liposome-encapsulated therapeutic agents enhance the efficacy by increasing their stability in the bloodstream. We investigated the potential of liposome-encapsulated plasmid DNA of telomerase-specific oncolytic adenovirus as an oncolytic adenoviral agent suitable for systemic delivery. Methods: TelomeScan, a modified Telomelysin expressing the green fluorescence protein (GFP), was used in this study. Cytotoxic effects of TelomeScan plasmid DNA-lipid complexes (Lipo-pTS) and control GFP plasmid DNA-lipid complexes (Lipo-pGFP) against HCT116 colon carcinoma cells were evaluated in vitro and in vivo. Coxsackie and adenovirus receptor (CAR) blocking assay using anti-CAR antibodies and neutralizing assay using adenovirus type 5-specific antibodies (AdNAbs) were performed to assess the CAR-independency of Lipo-pTS in infectivity and the influence of AdNAbs on Lipo-pTS cytotoxicity, respectively. Immunogenecity of Lipo-pTS was evaluated by assessing the productivity of AdNAbs after systemic administration in immune-competent BALB/c mice. Results: Lipo-pTS, which has a diameter of 40-50 nm, showed potent antitumor effects on HCT116 cells compared to Lipo-pGFP in vitro and in vivo, by producing actual viable adenoviruses after Lipo-pTS treatment, leading to tumor-specific oncolytic cell death. Infectivity of Lipo-pTS was not affected by pretreatment of anti-CAR antibodies, while infectivity of TelomeScan was reduced to less than half by anti-CAR antibodies, which suggested that tumor selectivity of Lipo-pTS was independent of CAR expression. Importantly, Lipo-pTS reduced production of AdNAbs significantly after intravenous administration into BALB/c mice compared to TelomeScan, and even in the presence of AdNAbs, Lipo-pTS maintained strong cytotoxicity in contrast to TelomeScan whose cytotoxicity was almost nullified by AdNAbs. These findings proved that Lipo-pTS possesses a stealth effect on the immune system in vivo environment, meaning that systemic delivery of Lipo-pTS may be possible. Conclusion: Lipo-pTS has the potential to become an oncolytic adenoviral agent suitable for systemic delivery with the characteristics of CAR-independent antitumor activity and a stealth effect on the immune system. Citation Format: Yoshihiko Kakiuchi. Stealth liposomes encapsulating plasmid DNA of telomerase specific oncolytic adenovirus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3714.

A new approach to CAR T-cell gene engineering and cultivation using piggyBac transposon in the presence of IL-4, IL-7 and IL-21

Background aimsClinical-grade chimeric antigenic receptor (CAR)19 T cells are routinely manufactured by lentiviral/retroviral (LV/RV) transduction of an anti-CD3/CD28 activated T cells, which are then propagated in a culture medium supplemented with interleukin (IL)-2. The use of LV/RVs for T-cell modification represents a manufacturing challenge due to the complexity of the transduction approach and the necessity of thorough quality control. MethodsWe present here a significantly improved protocol for CAR19 T-cell manufacture that is based on the electroporation of peripheral blood mononuclear cells with plasmid DNA encoding the piggyBac transposon/transposase vectors and their cultivation in the presence of cytokines IL-4, IL-7 and IL-21. ResultsWe found that activation of the CAR receptor by either its cognate ligand (i.e., CD19 expressed on the surface of B cells) or anti-CAR antibody, followed by cultivation in the presence of cytokines IL-4 and IL-7, enables strong and highly selective expansion of functional CAR19 T cells, resulting in >90% CAR+ T cells. Addition of cytokine IL-21 to the mixture of IL-4 and IL-7 supported development of immature CAR19 T cells with central memory and stem cell memory phenotypes and expressing very low amounts of inhibitory receptors PD-1, LAG-3 and TIM-3. ConclusionsOur protocol provides a simple and cost-effective method for engineering high-quality T cells for adoptive therapies.

Study of Coxsackie B viruses interactions with Coxsackie Adenovirus receptor and Decay-Accelerating Factor using Human CaCo-2 cell line.

BackgroundDecay Accelerating Factor (DAF) and Coxsackievirus-Adenovirus Receptor (CAR) have been identified as cellular receptors for Coxsackie B viruses (CV-B). The aim of this study is to elucidate the different binding properties of CV-B serotypes and to find out if there are any amino acid changes that could be associated to the different phenotypes.Twenty clinical CV-B isolates were tested on CaCo-2 cell line using anti-DAF (BRIC216) and anti-CAR (RmcB) antibodies. CV-B3 Nancy prototype strain and a recombinant strain (Rec, CV-B3/B4) were tested in parallel. The P1 genomic region of 12 CV-B isolates from different serotypes was sequenced and the Trans-Epithelial Electrical Resistance (TEER) along with the virus growth cycle was measured.ResultsInfectivity assays revealed clear differences between CV-B isolates with regard to their interactions with DAF and CAR. All tested CV-B isolates showed an absolute requirement for CAR but varied in their binding to DAF. We also reported that for some isolates of CV-B, DAF attachment was not adapted. Genetic analysis of the P1 region detected multiple differences in the deduced amino acid sequences.ConclusionWithin a given serotype, variations exist in the capacity of virus isolates to bind to specific receptors, and variants with different additional ligands may arise during infection in humans as well as in tissue culture.

Abnormal Expression Of The Major Coxsackie-Adenovirus Receptor CAR On Immature Dysplastic CD105+ Erythroid Progenitor Cells In Patients With MDS and Related Bone Marrow Neoplasms

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of myeloid neoplasms characterized by peripheral cytopenia, bone marrow (BM) dysplasia in one or more hematopoietic lineages, and clonal instability with an increased risk to transform to secondary acute myeloid leukemia (AML). In most patients, the erythroid lineage is affected. During the past few years our knowledge about the pathogenesis of MDS, has increased substantially. However, most of these studies focused on CD34+ stem and progenitor cells or granulomonocytic cells, whereas only little is known about genes aberrantly expressed in dysplastic erythroid cells in MDS.In an attempt to define abnormal gene expression patterns and molecular pathways contributing to erythroid dysplasia in MDS and to identify disease-specific phenotypic and molecular abnormalities in dysplastic erythoid cells, we compared gene expression profiles of erythroid progenitor cells (EryPC) in 10 patients with low-risk MDS (IPSS-R score≤7) and 15 control BM samples. EryPC were defined as CD45low/CD105+ cells and purified from BM samples by multicolor flow cytometry and cell sorting (purity>95%). Gene expression levels were analyzed by Affymetrix array technology (GeneChip U133 Plus 2.0 arrays) and confirmed for a panel of select genes by qPCR. A stringent cut-off (p<0.01; fold-change >3.0) was used to define differences in mRNA expression levels. A total number of 1,180 mRNA species were found to be differently expressed in EryPC in MDS (72 up-regulated, 1,108 down regulated) compared to normal BM EryPC. Among these, a number of genes regulating proliferation and differentiation in lymphohematopoietic cells, including TCEB1 transcription elongation factor B (SIII), the transcriptional repressor YY1, E2F3, and the ATP-dependent RNA helicase DDX5, were found to be downregulated in EryPC in MDS compared to normal BM.In the validation phase of our project, we identified the major Coxsackie-Adenovirus Receptor (CAR) as a rather specifically regulated protein-product in dysplastic EryPC. In fact, as assessed by flow cytometry, a significantly lower or even absent expression of CAR was found on EryPC in 20 of 30 patients with MDS (67%), including 3/4 patients with RCUD, 1/4 with RARS, 6/10 with RCMD, 8/9 with RAEB, and 2/3 with 5q- syndrome, compared to normal BM or non-neoplastic controls (p<0.001). In addition, reduced expression of CAR on EryPC was seen in 11/25 patients with myeloproliferative neoplasms (MPN) with dysplastic BM cells, in 4/8 patients with MDS/MPN overlap syndromes, and in 14/19 patients with AML. By contrast, expression levels of CAR on EryPC were normal in almost all non-neoplastic conditions, including renal anemia (n=6), hepatopathy (n=1), drug-related anemia (n=10), folic acid or vitamin B12 deficiency (n=3), intestinal bleeding (n=1), autoimmune hemolytic anemia (n=2), aplastic anemia (n=2), spherocytosis (n=1), chronic inflammation (n=4), and viral infections (n=2). Moreover, CAR expression on EryPC remained normal in most anemic patients suffering from lymphoid malignancies (n=36). Within the group of patients suffering from infection-related anemia, the lowest expression of CAR was observed in a case of adenovirus infection mimicking MDS. An overview of staining results obtained with anti-CAR antibody in various groups of patients and in healthy controls (normal BM, n=20) is shown in Figure 1. [Display omitted] In summary, our data show that the major Coxsackie-Adenovirus Receptor CAR is expressed at abnormally low levels on dysplastic EryPC in a substantial subset of patients with MDS and in most patients with related BM neoplasms. Since CAR expression remains normal in almost all reactive conditions, deficiency states and lymphoid malignancies, abnormal CAR expression may contribute as a potential indicator of (clonal) dysplastic erythropoiesis in myeloid malignancies, including MDS. The biochemical basis of abnormal expression and the possible role CAR may play in the pathogenesis of MDS and in related BM neoplasms, is currently under investigation. Disclosures:Valent:Novartis: Consultancy, Honoraria, Research Funding.

Hepatic damage caused by coxsackievirus B3 is dependent on age-related tissue tropisms associated with the coxsackievirus-adenovirus receptor

Coxsackievirus B (CVB) and enterovirus 71 (EV71) are important causes of severe enteroviral diseases in neonates or young children in Taiwan. CVB can cause fulminant hepatitis, myocarditis or meningoencephalitis. This study was designed to explore the role of coxsackievirus-adenovirus receptor (CAR) in the pathogenesis of CVB3-infected hepatocytes via in vitro and mice studies. CVB3 (CVB3/2630) was isolated from liver tissue of a neonate with fulminant hepatitis. Cell lines A549, HeLa, HEp2 and Huh-7 were maintained in Dulbecco's modified Eagle's medium. Mice progeny 1 or 7 days old were used in the experiments. Viremia was noted in 7-day-old ICR mice 2 h after intraperitoneal injection. The highest viral titers were detected in blood, liver and spleen. Histopathological studies of the liver demonstrated polymorphonuclear cell infiltration, massive hepatic cell necrosis and apoptosis. CAR was expressed more in liver than in other tissues. Expression of CAR decreased with mouse age. Anti-CAR monoclonal antibody prevented infection of Huh-7 cells from CVB3. Furthermore, anti-CAR monoclonal antibody pretreatment can reduce mortality and decrease the level of liver enzymes in CVB3-infected mice. These findings indicate that CAR plays an important role in the initiation of CVB infections and is closely associated with hepatotropism and age-specific susceptibility.

Adenovirus Serotype 26 Utilizes CD46 as a Primary Cellular Receptor and Only Transiently Activates T Lymphocytes following Vaccination of Rhesus Monkeys

The cellular receptor utilized by adenovirus serotype 26 (Ad26) has remained unclear. Here we show that Ad26 transduction is CD46-dependent and is efficiently blocked by anti-CD46 but not anti-CAR antibodies, demonstrating that Ad26 utilizes CD46 as a primary cellular receptor. Moreover, following Ad26 vaccination of rhesus monkeys, we did not observe sustained activation of peripheral or mucosal vector-specific CD4(+) T lymphocytes. These data contribute to our understanding of Ad26 as a candidate vaccine vector.