Chimeric Receptor Research Articles

Continuous Feeding Reduces the Generation of Metabolic Byproducts and Increases Antibodies Expression in Chinese Hamster Ovary-K1 Cells.

Chinese hamster ovary (CHO) cells are the most important host system used for monoclonal antibody (mAb) expression. Moreover, the fed-batch culture mode is the most widely used method to increase mAb expression in CHO cells by increasing the amount of feed. However, a high amount of culture feed results in the production of metabolic byproducts. In this work, we used a continuous feeding strategy to reduce metabolic byproducts and improve mouse–human chimeric anti-epidermal growth factor receptor vIII (EGFRvIII) antibody C12 expression in Chinese hamster ovary-K1 cells. Moreover, the effects of the feeding strategy on the cell culture and monoclonal antibody production were evaluated in chemically defined suspension cultures of recombinant CHO-K1 cells. Compared with bolus feeding methods, the continuous feeding method did not have any advantages when the feeding amount was low, but with a high feeding amount, the continuous feeding method significantly reduced the concentrations of lactate and NH4+ in the later culture stage. At the end of the culture stage, compared with bolus feeding methods, the lactate and NH4+ concentrations under the continuous feeding mode were reduced by approximately 45% and 80%, respectively. In addition, the antibody C12 expression level was also increased by almost 10%. Compared to the bolus feeding method, the antibody C12 produced by the continuous feeding method had a lower content of high-mannose glycoforms. Further analysis found that the osmolality of the continuous feeding method was lower than that of the typical fed-batch bolus feeding method. Conclusively, these results indicate that the continuous feeding method is very useful for reducing metabolic byproducts and achieving higher levels of mAb production.

1007P T-cells transgenic for a highly potent PRAME-specific TCR and a chimeric PD1-41BB co-stimulatory receptor represent a promising approach for the treatment of solid tumors

T-cell receptor (TCR)-based immunotherapies have great potential for the safe and efficacious treatment of patients with unmet needs suffering from various types of cancer. However, novel strategies are needed to overcome the inhibitory tumor microenvironment and to enhance the efficacy of TCR-T-cell (TCR-T) therapy in solid tumors. PRAME is a cancer/testis antigen that is highly expressed in various solid tumor indications while its expression in healthy tissues is mainly restricted to the testis.

Targeting BCMA in Multiple Myeloma.

Despite considerable advances in the treatment of multiple myeloma (MM) in the last decade, a significant number of patients still progress on current available therapies. Here, we review treatment modalities used to target BCMA in the treatment of MM, specifically antibody-drug conjugates (ADC), bispecific antibody constructs, and chimeric antibody receptor (CAR) modified T-cell therapies. We will provide an overview of therapies from these classes that have presented or published clinical data, as well as data on mechanisms of resistance to these novel agents. Clinical trials exploring different BCMA-targeting modalities to treat multiple myeloma are underway and demonstrate promising results. In relapsed/refractory multiple myeloma, anti-BCMA ADCs and bispecific antibody constructs are showing impressive efficacy with manageable side effect profiles. In parallel, adoptive cellular therapy has induced dramatic durable responses in multiply relapsed and refractory myeloma patients. Therapeutic approaches targeting BCMA hold significant potential in the management of multiple myeloma and will soon be incorporated in combination with current standard therapies to improve outcomes for patients with multiple myeloma. In addition, novel approaches are being evaluated to overcome resistance mechanisms to anti-BCMA therapies.

CAR T Cells: Cancer Cell Surface Receptors Are the Target for Cancer Therapy.

Immunotherapy has become a prominent strategy for the treatment of cancer. A method that improves the immune system’s ability to attack a tumor (Enhances antigen binding). Targeted killing of malignant cells by adoptive transfer of chimeric antigen receptor (CAR) T cells is a promising immunotherapy technique in the treatment of cancers. For this purpose, the patient’s immune cells, with genetic engineering aid, are loaded with chimeric receptors that have particular antigen binding and activate cytotoxic T lymphocytes. That increases the effectiveness of immune cells and destroying cancer cells. This review discusses the basic structure and function of CAR-T cells and how antigenic targets are identified to treat different cancers and address the disadvantages of this treatment for cancer.

TGF-β/IL-7 Chimeric Switch Receptor-Expressing CAR-T Cells Inhibit Recurrence of CD19-Positive B Cell Lymphoma.

Chimeric antigen receptor (CAR)-T cells are effective in the treatment of hematologic malignancies but have shown limited efficacy against solid tumors. Here, we demonstrated an approach to inhibit recurrence of B cell lymphoma by co-expressing both a human anti-CD19-specific single-chain variable fragment (scFv) CAR (CD19 CAR) and a TGF-β/IL-7 chimeric switch receptor (tTRII-I7R) in T cells (CD19 CAR-tTRII-I7R-T cells). The tTRII-I7R was designed to convert immunosuppressive TGF-β signaling into immune-activating IL-7 signaling. The effect of TGF-β on CD19 CAR-tTRII-I7R-T cells was assessed by western blotting. Target-specific killing by CD19 CAR-tTRII-I7R-T cells was evaluated by Eu-TDA assay. Daudi tumor-bearing NSG (NOD/SCID/IL2Rγ-/-) mice were treated with CD19 CAR-tTRII-I7R-T cells to analyze the in vivo anti-tumor effect. In vitro, CD19 CAR-tTRII-I7R-T cells had a lower level of phosphorylated SMAD2 and a higher level of target-specific cytotoxicity than controls in the presence of rhTGF-β1. In the animal model, the overall survival and recurrence-free survival of mice that received CD19 CAR-tTRII-I7R-T cells were significantly longer than in control mice. These findings strongly suggest that CD19 CAR-tTRII-I7R-T cell therapy provides a new strategy for long-lasting, TGF-β-resistant anti-tumor effects against B cell lymphoma, which may lead ultimately to increased clinical efficacy.

Antibody:CD47 ratio regulates macrophage phagocytosis through competitive receptor phosphorylation

SUMMARYCancer immunotherapies often modulate macrophage effector function by introducing either targeting antibodies that activate Fcγ receptors (FcγRs) or blocking antibodies that disrupt inhibitory SIRPα-CD47 engagement. However, how these competing signals are integrated is poorly understood, raising questions about how to effectively titrate immune responses. Here, we find that macrophage phagocytic decisions are regulated by the ratio of activating ligand to inhibitory ligand over a broad range of absolute molecular densities. Using both endogenous and chimeric receptors, we show that activating:inhibitory ligand ratios of at least 10:1 are required to promote phagocytosis of model antibody-opsonized CD47-inhibited targets and that lowering that ratio reduces FcγR phosphorylation because of inhibitory phosphatases recruited to CD47-bound SIRPα. We demonstrate that ratiometric signaling is critical for phagocytosis of tumor cells and can be modified by blocking SIRPα, indicating that balancing targeting and blocking antibodies may be important for controlling macrophage phagocytosis in cancer immunotherapy.

Live visualization of a functional RET-EGFP chimeric receptor in homozygous knock-in mice.

The GDNF Family Ligands (GFLs) regulate neural development and kidney organogenesis by activating the RET receptor tyrosine kinase. Many RET-dependent developmental processes involve long-distance cell-cell communications or cell polarity, which includes cell migration and axon guidance. This suggests that spatiotemporally regulated subcellular localization of RET protein and appropriate propagation of RET signaling in cells are essential for the physiological function of the GFLs. Little is known, however, about the dynamics of RET protein in cells. Addressing this issue requires development of a system that allows visualization of RET in living cells. In this study, we report generation of a novel knock-in mouse line in which the RET-EGFP chimeric receptor is expressed under the Ret promoter. Unlike Ret-deficient mice that die after birth due to the absence of the enteric nervous system (ENS) and kidneys, RetRET-EGFP/RET-EGFP mice were viable and grew to adulthood with no overt abnormality, which indicated that RET-EGFP exerts function comparable to RET. In neurons and ENS progenitors, RET-EGFP signals were detected both on the cell membrane and in the cytoplasm, the latter of which appeared as a punctate pattern. Time-lapse imaging of cultured neural cells and embryos revealed active transport of RET-EGFP puncta in neuronal axons and cell bodies. Immunohistochemical analyses detected RET-EGFP signals in early and recycling endosomes, indicating that RET-EGFP is trafficked via the endocytic pathway. RetRET-EGFP/RET-EGFP mice enable visualization of functional RET protein in vivo for the first time and provide a unique platform to examine the dynamics and physiology of RET trafficking.

Simple monitoring of endocrine-disrupting chemicals using transgenic Arabidopsis plants expressing medaka estrogen receptor

Endocrine-disrupting chemicals (EDCs) are widespread contaminants that severely affect the endocrine systems of living organisms. In addition to the conventional instrument-based approaches for quantifying organic pollutants, a monitoring method using transgenic plants has also been proposed. Plants carrying a recombinant receptor gene combined with a reporter gene represent a system for the easy detection of ligands that specifically bind to the receptor molecule. Here, the EDC detection sensitivity of transgenic Arabidopsis plants expressing the medaka (Oryzias latipes) estrogen receptor (mER) and green fluorescent protein (GFP) genes, was assessed. Four transgenic Arabidopsis lines, obtained by transformation with expression plasmids constructed using combinations of two types of the ligand-binding domains of mER, the DNA-binding domain of LexA and the transactivation domain of VP16 in the chimeric receptors, showed significant induction of GFP when germinated on a medium contaminated with 1 ng/mL 4-t-octylphenol (OP). The most sensitive XmEV19-2 plants detected 0.1 ng/mL OP and 1 pg/mL 17β-estradiol. GFP expression was suppressed by the insecticides imidacloprid and fipronil, whereas perfluorooctanesulfonic acid induced it at 0.1 ng/mL. Experiments with river water-based medium showed that XmEV19-2 can be used for monitoring polluted waters, detecting OP at concentrations as low as 5 ng/mL. Notably, XmEV19-2 showed a significant decrease in root length when grown on 0.1 ng/mL OP. mER transgenic plants can be a promising tool for simple monitoring of EDCs, without the need for extraction and concentration steps in sample preparation.

Progression of melanoma is suppressed by targeting all transforming growth factor‑β isoforms with an Fc chimeric receptor.

Melanoma is an aggressive type of cancer originating from the skin that arises from neoplastic changes in melanocytes. Transforming growth factor-β (TGF-β) is a pleiotropic cytokine and is known to contribute to melanoma progression by inducing the epithelial-mesenchymal transition (EMT) program and creating an environment that favors tumor progression. There are three TGF-β isoforms, TGF-β1, TGF-β2 and TGF-β3, all of which engage in pro-tumorigenic activities by activating SMAD signaling pathways. All TGF-β isoforms activate signaling pathways by binding to their TGF-β type I (TβRI) and type II (TβRII) receptors. Thus, effective targeting of all TGF-β isoforms is of great importance. In the present study, chimeric proteins comprising the extracellular domains of TβRI and/or TβRII fused with the Fc portion of human immunoglobulin (IgG) were validated in the melanoma context. The Fc chimeric receptor comprising both TβRI and TβRII (TβRI-TβRII-Fc) effectively trapped all TGF-β isoforms. Conversely, TβRII-Fc chimeric receptor, that comprises TβRII only, was able to interact with TGF-β1 and TGF-β3 isoforms, but not with TGF-β2, which is a poor prognostic factor for melanoma patients. Accordingly, it was revealed that TβRI-TβRII-Fc chimeric receptor suppressed the EMT program in melanoma cells in vitro induced by any of the three TGF-β isoforms, as revealed by decreased expression of mesenchymal markers. Conversely, TβRII-Fc chimeric receptor inhibited the EMT program induced by TGF-β1 and TGF-β3. In addition, it was established that tumor growth in subcutaneous mouse melanoma was inhibited by TβRI-TβRII-Fc chimeric receptor indicating that Fc chimeric receptor could be applied to modify the tumor microenvironment (TME) of melanoma. Therefore, designing of Fc chimeric receptors targeting TGF-β signals that affect various components of the TME may result in the development of effective anti-melanoma agents.

Real-World Experience of Axicabtagene Ciloleucel and Tisagenlecleucel for Relapsed or Refractory Aggressive B-cell Lymphomas: A Single-Institution Experience

BackgroundCD19-directed chimeric antigen T-cell receptor (CAR-T) therapies have revolutionized the treatment of patients with relapsed/refractory (R/R) aggressive B-cell lymphomas (aBCL). The results of the landmark ZUMA-1 and JULIET trials have been reproducible in real-world settings across multiple institutions, and patients with double (DHL) or triple (THL) hit lymphomas have demonstrated non-inferior outcomes compared to non-DHL/THL counterparts. Materials and MethodsThis retrospective cohort study included 53 patients with R/R aBCL who received CAR-T from October 2017 to June 2020 at the University of California, Los Angeles. Patient characteristics, lymphoma-related variables and outcomes of interest were summarized using descriptive statistics and compared between groups by Fisher's exact test. Kaplan-Meier methods were used for analysis of OS, progression free survival (PFS), and duration of response (DOR). Univariate and multivariate cox regression analysis were performed to evaluate for significant prognostic variables. ResultsWith a median follow-up of 15.2 months, this cohort demonstrated overall response rate and complete response rate of 72% and 64% (n = 34), respectively. The median DOR, PFS and OS were not reached, 7.9 and 17.7 months, respectively. By univariate analysis, DHL/THL status was the only clinical feature significantly associated with relapse post-CAR-T (OR 5.9, P = .015). ConclusionsOur single-institution, real-world cohort of R/R aBCL patients demonstrated similar efficacy outcomes to those of the ZUMA-1 and JULIET trials and published real-world studies. Our findings suggest DHL/THL patients may benefit from novel CAR-T constructs, maintenance strategies with immunomodulatory agents or allogeneic-HCT.

Development and Validation of a Good Manufacturing Process for IL-4-Driven Expansion of Chimeric Cytokine Receptor-Expressing CAR T-Cells.

Adoptive cancer immunotherapy using chimeric antigen receptor (CAR) engineered T-cells holds great promise, although several obstacles hinder the efficient generation of cell products under good manufacturing practice (GMP). Patients are often immune compromised, rendering it challenging to produce sufficient numbers of gene-modified cells. Manufacturing protocols are labour intensive and frequently involve one or more open processing steps, leading to increased risk of contamination. We set out to develop a simplified process to generate autologous gamma retrovirus-transduced T-cells for clinical evaluation in patients with head and neck cancer. T-cells were engineered to co-express a panErbB-specific CAR (T1E28z) and a chimeric cytokine receptor (4αβ) that permits their selective expansion in response to interleukin (IL)-4. Using peripheral blood as starting material, sterile culture procedures were conducted in gas-permeable bags under static conditions. Pre-aliquoted medium and cytokines, bespoke connector devices and sterile welding/sealing were used to maximise the use of closed manufacturing steps. Reproducible IL-4-dependent expansion and enrichment of CAR-engineered T-cells under GMP was achieved, both from patients and healthy donors. We also describe the development and approach taken to validate a panel of monitoring and critical release assays, which provide objective data on cell product quality.

T Cells Retain Pivotal Antitumoral Functions under Tumor-Treating Electric Fields.

Tumor-treating fields (TTFields) are a localized, antitumoral therapy using alternating electric fields, which impair cell proliferation. Combining TTFields with tumor immunotherapy constitutes a rational approach; however, it is currently unknown whether TTFields' locoregional effects are compatible with T cell functionality. Healthy donor PBMCs and viably dissociated human glioblastoma samples were cultured under either standard or TTFields conditions. Select pivotal T cell functions were measured by multiparametric flow cytometry. Cytotoxicity was evaluated using a chimeric Ag receptor (CAR)-T-based assay. Glioblastoma patient samples were acquired before and after standard chemoradiation or standard chemoradiation + TTFields treatment and examined by immunohistochemistry and by RNA sequencing. TTFields reduced the viability of proliferating T cells, but had little or no effect on the viability of nonproliferating T cells. The functionality of T cells cultured under TTFields was retained: they exhibited similar IFN-γ secretion, cytotoxic degranulation, and PD1 upregulation as controls with similar polyfunctional patterns. Glioblastoma Ag-specific T cells exhibited unaltered viability and functionality under TTFields. CAR-T cells cultured under TTFields exhibited similar cytotoxicity as controls toward their CAR target. Transcriptomic analysis of patients' glioblastoma samples revealed a significant shift in the TTFields-treated versus the standard-treated samples, from a protumoral to an antitumoral immune signature. Immunohistochemistry of samples before and after TTFields treatment showed no reduction in T cell infiltration. T cells were found to retain key antitumoral functions under TTFields settings. Our data provide a mechanistic insight and a rationale for ongoing and future clinical trials that combine TTFields with immunotherapy.

Rationally Designed Transgene-Encoded Cell-Surface Polypeptide Tag for Multiplexed Programming of CAR T-cell Synthetic Outputs.

Synthetic immunology, as exemplified by chimeric antigen receptor (CAR) T-cell immunotherapy, has transformed the treatment of relapsed/refractory B cell-lineage malignancies. However, there are substantial barriers-including limited tumor homing, lack of retention of function within a suppressive tumor microenvironment, and antigen heterogeneity/escape-to using this technology to effectively treat solid tumors. A multiplexed engineering approach is needed to equip effector T cells with synthetic countermeasures to overcome these barriers. This, in turn, necessitates combinatorial use of lentiviruses because of the limited payload size of current lentiviral vectors. Accordingly, there is a need for cell-surface human molecular constructs that mark multi-vector cotransduced T cells, to enable their purification ex vivo and their tracking in vivo. To this end, we engineered a cell surface-localizing polypeptide tag based on human HER2, designated HER2t, that was truncated in its extracellular and intracellular domains to eliminate ligand binding and signaling, respectively, and retained the membrane-proximal binding epitope of the HER2-specific mAb trastuzumab. We linked HER2t to CAR coexpression in lentivirally transduced T cells and showed that co-transduction with a second lentivirus expressing our previously described EGFRt tag linked to a second CAR efficiently generated bispecific dual-CAR T cells. Using the same approach, we generated T cells expressing a CAR and a second module, a chimeric cytokine receptor. The HER2txEGFRt multiplexing strategy is now being deployed for the manufacture of CD19xCD22 bispecific CAR T-cell products for the treatment of acute lymphoblastic leukemia (NCT03330691).

Acute Myeloid Leukemia Mutations and Future Mechanistic Target to Overcome Resistance.

Cytogenetics and mutation identification in acute myeloid leukemia have allowed for more targeted therapy. Many therapies have been approved by the FDA in the last 3 years including gilteritinib and azacitidine but the overall survival has remained stagnant at 25%. The inability to achieve complete remission was related to the residual leukemic stem cells (LSCs). Thus, the relationship between bone marrow niche and LSCs must be further explored to prevent treatment relapse/resistance. The development of immunotherapy and nanotechnology may play a role in future therapy to achieve the complete remission. Nano-encapsulation of drugs can improve drugs' bioavailability, help drugs evade resistance, and provide combination therapy directly to the cancer cells. Studies indicate targeting surface antigens such as CLL1 and CD123 using chimeric antibody receptor T cells can improve survival outcomes. Finally, new discoveries indicate that inhibiting integrin αvβ3 and acid ceramidase may prove to be efficacious.

Abstract LB148: Gamma delta T cells engineered with a chimeric PD-1 receptor effectively control PD-L1 positive tumors in vitro and in vivo with minimal toxicities

Abstract Introduction. Despite the overwhelming success of anti-CD19 CAR T cell therapies in recent years, it is still impossible to date to replicate comparable results in patients with solid tumors. Such obstacle is due to many factors, but the three most relevant ones are: a) the complex and immune-suppressive tumor microenvironment, b) the lack of optimal tumor targets that are not also expressed on normal cells and c) the use of autologous, patient-derived cells, which are frequently sub-optimal due to chemotherapy. Here we investigated a novel strategy to overcome these three barriers by using unconventional, MHC-independent gamma delta T cells and a chimeric PD-1 protein that was previously validated in murine conventional alpha-beta T cells. We tested the hypothesis that our strategy could specifically target cancer cells without a CAR for a specific tumor-associated antigen by turning PD-1 immune suppression into T-cell activation and using donor-derived, “off-the-shelf” effector cells. Experimental procedures. We built a chimeric PD-1 receptor consisting of the extracellular portion of PD-1 fused to the intracellular domains of DAP10 and CD3z. Gamma delta T cells from healthy donors were expanded ex vivo and stably transduced with a gamma-retrovirus-derived viral vector carrying the chimeric construct. Cells were tested for killing efficiency, cytokines secretion, and memory differentiation in vitro. We then evaluated the in vivo efficacy and tolerability in murine xenografts. Results. chPD1-gdT cells selectively killed PDL-1+ tumor cells, with minimal on-target/off-tumor toxicities and without off-target toxicities. Cells were well tolerated in mice, without damage to normal PD-L1+ cells. Upon contact with PD-L1+ cancers, chPD1-gdT cells, but not untransduced gdT cells, expressed markers of memory phenotype and secreted inflammatory cytokines. In vivo, chPD1-gdT cells, but not untransduced gdT cells, cleared PD-L1+ tumors without significant elevation of CRS-related cytokines nor off-tumor toxicities. Conclusions. chPD1-gdT cells are potent and safe in vitro and in vivo and will be assessed in a Phase I/II clinical trial. Citation Format: Amorette Barber, Xiaohong Wang, Anupama Gopisetty, Leonardo Mirandola, Maurizio Chiriva-Internati. Gamma delta T cells engineered with a chimeric PD-1 receptor effectively control PD-L1 positive tumors in vitro and in vivo with minimal toxicities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB148.

Abstract 332: Chimeric fusion receptors provide a CD3-mediated activation signal to off-the-shelf iPSC-derived TCR-less CAR-T cells for enhanced efficacy

Abstract T cells require a combination of signals to properly activate and exhibit their full potential; signal 1 from the T cell receptor (TCR), signal 2 from costimulatory receptors and signal 3 from cytokine receptors can shape the function of T cells. Second generation chimeric antigen receptor (CAR) T-cells targeting CD19 have shown success in hematologic malignancies, in part because signals 1 and 2 are provided by the CAR endodomain. As previously reported, we have developed an off-the-shelf iPSC-derived CAR19 T (CAR19-iT) cell product where the CAR is inserted into the T cell receptor alpha constant (TRAC) locus for enhanced efficacy and disruption of surface TCR expression. While the TCR-less CAR19-iT cells are ideal for allogeneic CAR-T cell therapies, as a consequence, they lack CD3 surface expression and the means by which to mediate CD3 signaling. To further improve CAR-iT cell performance and facilitate combinatorial strategies with T cell engagers such as bispecific T cell engagers (BiTEs), we designed chimeric fusion receptors (CFRs) comprising an ectodomain of CD3 or CD28, a transmembrane domain of CD28, and various endodomains that provide combinations of signals 1, 2, and 3. In addition to providing co-stimulatory signaling, the CFR designs can be controlled via agonistic antibodies or BiTEs to initiate signal transduction for precision control of the cell product. Furthermore, a series of CFRs with various CD3 endodomains were also developed to augment costimulatory signaling and the ER retention and endocytosis motifs found in CD3 endodomains were mutated to prevent downregulation of the CFRs from the cell surface of TCR-less cells. Selected CFRs were engineered into TRAC knockout Jurkat cells expressing a luciferase based NFAT reporter gene. After 48 hours, surface expression of the CFRs was confirmed by flow cytometry. Signal transduction via the CFRs was evaluated following exposure to agonistic antibodies or BiTEs. Measurement of the luciferase activity showed comparable levels to activated wildtype reporter cells. After construct validation, the CFRs were introduced into TCR-less CAR19-iT cells and co-cultured overnight with Nalm6 target cells in the presence of agonistic antibodies. Compared to control CAR19-iT cells, the armed CFR-CAR19 iT cells demonstrated superior killing of target cells. These results support the notion that enhancing signal 1 can increase CAR-T cell efficacy with current studies underway to investigate CFRs with endodomains that provide additional signal 2 or 3. We expect these constructs to further enhance CAR-iT cell persistence and engagement with BiTEs for activation and multi-antigen targeting. Collectively, the data suggest that TRC-less CAR-iT cells can be further armed with CFRs containing appropriate endodomains to provide superior antitumor function in a regulated manner. Citation Format: Eigen Peralta, Dan Lu, Mark Landon, Helen Chu, Shohreh Sikaroodi, Emily Carron, Natalie Navarrete, Alec Witty, Tom Lee, Anhco Nguyen, Bahram Valamehr. Chimeric fusion receptors provide a CD3-mediated activation signal to off-the-shelf iPSC-derived TCR-less CAR-T cells for enhanced efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 332.

Abstract 1516: Inclusion of a Dap10 costimulatory domain enhances anti-tumor efficacy of chimeric PD1-expressing T cells in multiple types of solid tumors

Abstract Adoptive transfer of T cells is a promising anti-tumor therapy for many cancers. To enhance tumor recognition by T cells, chimeric antigen receptors (CAR) consisting of signaling domains fused to receptors that recognize tumor antigens can be expressed in T cells. One receptor that is a prospective target for a new chimeric antigen receptor is PD1 because the ligands for the PD1 receptor are expressed on many cancer types. Therefore, we developed a murine chimeric PD1 receptor (chPD1) consisting of the PD1 receptor extracellular domain and the activation domain of CD3 zeta. In addition, current chimeric antigen receptor therapies utilize various costimulatory domains to enhance anti-tumor efficacy. Therefore, we also compared the inclusion of CD28, Dap10, 4-1BB, GITR, ICOS, or OX40 costimulatory domains in the chPD1 receptor to determine which costimulatory domain induced optimal anti-tumor immunity. To determine if this novel CAR could potentially target a wide variety of tumors, the anti-tumor efficacy of chPD1 T cells against murine lymphoma, melanoma, kidney, pancreatic, liver, colon, breast, ovarian, prostate, and bladder cancer cell lines was measured. Of the eighteen cell lines tested, all expressed PD1 ligands on their cell surface, making them potential targets for chPD1 T cells. Regardless of the costimulatory domain in the CAR, all of the chPD1 T cells induced similar levels of T cell proliferation and tumor cell lysis. However, differences were observed in the cytokine secretion profiles depending on which costimulatory receptor was included in the CAR. While most of the chPD1 T cell receptor combinations secreted both pro-inflammatory (IFNγ, TNFα, IL-2, GM-CSF, IL-17, and IL-21) and anti-inflammatory cytokines (IL-10), chPD1 T cells containing a Dap10 costimulatory domain secreted high levels of proinflammatory cytokines but did not secrete a significant amount of anti-inflammatory cytokines. Furthermore, T cells expressing chPD1 receptors with a Dap10 domain also had the strongest anti-tumor efficacy in vivo. ChPD1 T cells did not survive for longer than 14 days in vivo, however treatment with chPD1 T cells induced long-lived protective host-anti-tumor immune responses in tumor-bearing mice. Therefore, adoptive transfer of chPD1 T cells could be a novel therapeutic strategy to treat multiple types of cancer and inclusion of the Dap10 costimulatory domain in chimeric antigen receptors may induce a preferential cytokine profile for anti-tumor therapies. Citation Format: Amorette E. Barber. Inclusion of a Dap10 costimulatory domain enhances anti-tumor efficacy of chimeric PD1-expressing T cells in multiple types of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1516.

Abstract 1810: Anti-human TNFR2 antagonist induces T cell proliferation and restores T cell balance to promote tumor killing in TNFR2 HuGEMM™ engrafted with MC38/OVA tumor cells

Abstract Introduction: Tumor necrosis factor receptor 2 (TNFR2) is a member of the TNF-receptor superfamily, which is abundantly expressed in regulatory T cells (Treg) and effector T cells (Teff) in the tumor microenvironment (TME) upon T cell activation. TNFR2 is also highly expressed on the surface of many human tumors. For hot tumors, the TME is infiltrated by immunosuppressive Treg cells that suppress Teff cells to allow tumor growth. However, antagonistic anti-TNFR2 inhibitors are believed to break this imbalance by promoting T cell activation and eliminating Tregs to improve cytotoxic T cell function. Taken together, TNFR2 is becoming a promising potential immunotherapy target in the TNF-receptor superfamily along with GITR, OX40, 4-1BB, etc. whether by stimulating T cells to restore Teff/Treg balance in the TME or by directly inducing tumor cell killing though NF-kappaB signaling. We have developed a humanized TNFR2 mouse model (TNFR2 HuGEMM) to evaluate antagonistic anti-TNFR2 inhibitors to induce tumor killing. Methods: We established TNFR2 HuGEMM in both C57BL/6 background and BALB/c background by inserting chimeric h/m TNFR2 CDS in the mouse Tnfrsf1b locus via CRISPR/Cas9-based gene engineering, in which the mouse TNFR2 extracellular domain was replaced by human counterparts, leading to expression of chimeric TNFR2 receptors with human extracellular domain and mouse trans-membrane and intracellular domains. Results: We profiled and characterized the surface expression of human/mouse chimeric TNFR2 on FoxP3+ Treg cells as well as on granulocytes (CD11b+Gr1hi/F4/80- and CD11b+Gr1-/F4/80+) and monocytes (CD11blowGr1-/F4/80low) by FACS analysis of peripheral blood and spleen in the heterozygous and homozygous TNFR2 HuGEMM mice. The expression of chimeric m/hTNFR2 on Tregs in HuGEMM mice can proportionally recapitulate the expression of mouse TNFR2 in wild-type mice as well as human TNFR2 expression in human PBMC. We then inoculated MC38-OVA syngeneic tumors into the female homozygous HuGEMM mice and treated the mice with anti-human TNFR2 antagonists (Ab1 and Ab2). Anti-hTNFR2 Ab1 led to ~80% tumor growth inhibition (TGI) and 14% of the mice (1/7) were eventually cured. By contrast, anti-hTNFR2 Ab2 only led to ~50% TGI and 28% of the mice (2/7) were cured, but 3 out of 7 mice were found dead upon treatment with Ab2. At the efficacy endpoint, FACS analysis of tumor infiltrating lymphocytes indicated that both anti-hTNFR2 antibodies promoted T cell proliferation and elevated Teff/Treg ratio in tumors. Conclusions: Here, we report the in vivo tumor killing by anti-hTNFR2 antibodies in a humanized TNFR2 mouse model engrafted with MC38-OVA. The TNFR2 HuGEMM is a useful preclinical animal model to evaluate the in vivo efficacy and safety of human TNFR2 agonists or antagonists potentially in combination with other immune modulators. Citation Format: Daniel X. He, Lei Zheng, Ruilin Sun, Annie X. An, Henry Q.x. Li, Davy Xuesong Ouyang. Anti-human TNFR2 antagonist induces T cell proliferation and restores T cell balance to promote tumor killing in TNFR2 HuGEMM™ engrafted with MC38/OVA tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1810.

CAR-T Cells Targeting BAFF-Receptor for B-Cell Malignancies: A Potential Alternative to CD19

Background: Chimeric antigen receptor (CAR) T-cell adoptive therapies have the potential to revolutionize the treatment of cancers. Particularly in hematological malignancies, there are reports of promising clinical outcomes in advanced non-Hodgkin lymphomas (NHLs) with CD19-CAR T-cell therapy. However, disease relapse is problematic, and is thought to be caused by poor long-term persistence of the CAR T-cells, and loss of the CD19 target on tumors. Thus, there is an urgent need for improved novel CAR T-cell therapies directed at alternative targets.The CAR T-cell platform relies on antibody-derived single chain fragments (scFv), which are genetically engineered into chimeric T-cell receptors, and that recognize target cell surface proteins on tumors. One potential target is B-cell activating factor receptor (BAFF-R), a tumor necrosis factor receptor superfamily protein (TNFRSF13C) specifically involved in B lymphocyte development and mature B-cell survival, that is primarily expressed on B cells and various subtypes of B-cell NHLs and ALL. We have recently developed a humanized therapeutic BAFF-R antibody with strong anti B-cell tumor activity.Method and Results: We adapted a scFv based on our humanized anti-BAFF-R antibody onto a second generation CAR platform containing CD3ζ and 4-1BB intracellular signaling domains. In response to BAFF-R-expressing malignant human B cells (NHLs, acute lymphoblastic leukemias, and chronic lymphocytic leukemias), our CAR T cells readily proliferated and secreted cytotoxic cytokines. We demonstrated both significant levels of BAFF-R CAR T-cell activation and malignant B-cell killing in vitro .Established human NHL tumors in xenogeneic models were eliminated following BAFF-R CAR T-cell treatments in vivo . Remarkable tumor-free survival was repeatedly observed in human lymphoma xenograft models including JeKo-1 (mantle cell lymphoma) and Raji (Burkitt lymphoma) in NSG mice. Moreover, upon tumor re-challenge and without any further treatment tumors failed to develop, due to persisting CAR T cells conferring continued anti-lymphoma activity. We pursued optimization of CAR T-cell persistence by comparing three subsets of early stage T cells (central memory, TCM; memory stem, TSCM; and naïve, TN) as potential starting material for CAR T cell generation. Our in vivo studies show CAR T cells from the TN population retained highest potency eradicating established tumors at a minimal therapeutic dose compared to other subtypes; 80% of TN CAR treated mice achieved long-term survival compared to 20% of TCM and 40% of TSCM CAR treated mice. We also observed the long term anti-tumor effects conferred by CD8+ CAR T cells required the addition of CD4+ CAR T cells. Finally, we performed a head-to-head comparison of BAFF-R CAR T cells with CD19 CAR T cells in the Raji model (Figure). Comparable expression levels of BAFF-R and CD19 were observed on the tumor cells (A), and both treatments elicited significant antitumor effects compared to T-cell and saline controls (B-C). However, the BAFF-R CAR T-cell treatment demonstrated long-term tumor free survival in all treated mice compared to the CD19 CAR T-cell treated cohort, which only showed delayed tumor growth (P<0.01, B-C).Conclusion: BAFF-R CAR T cells demonstrate remarkable efficacy against B-cell malignancies. Unexpectedly, we found that naïve T cells were superior to TCM or TSCM subpopulations as starting material in vivo . Naïve T-cell starting products, after transduction and expansion, generated sufficient numbers of TCM and TSCM CAR T cells for activity. Targeting BAFF-R combined with our strategy to improve CAR T-cell persistence potentially addresses unmet clinical needs in B-cell NHLs and ALL, particularly in the setting of CD19 CAR-T-resistance or CD19-negative relapse. [Display omitted] DisclosuresNeelapu:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Karus: Research Funding; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis Inc.: Research Funding; Poseida Therapeutics, Inc: Research Funding. Kwak:InnoLifes: Consultancy, Equity Ownership; Pepromene Bio: Consultancy, Equity Ownership; Celltrion, Inc: Consultancy.

A Novel TGF-β2/Interleukin Receptor Signal Conversion Platform That Protects CAR/TCR T Cells from TGF-β2-Mediated Immune Suppression and Induces T Cell Supportive Signaling Networks

Numerous immune-suppressive mechanisms exist within the tumor microenvironment that may hinder the efficacy of adoptively transferred T cells. One such mechanism is mediated by TGF-β, a cytokine secreted by tumor cells and infiltrating suppressive immune cells that directly inhibits effector T cell activity. Effector T cells express the TGF-β receptors TGFBR1 and TGFBR2, and exposure of T cells to TGF-β induces hetero-dimerization of these receptors and phosphorylation of the major TGF-β signal mediators SMAD2 and SMAD3. Phosphorylated SMAD proteins (pSMADs) induce a suppressive transcriptional program that ultimately leads to reduced cytokine production, reduced cytotoxicity, and a failure to proliferate in response to antigen stimulation. In several previous reports, a dominant negative receptor (DNR) version of TGFBR2, that does not contain signaling domains, was shown to protect T cells from the impacts of TGF-β by blocking the ability of TGF-β to induce pSMADs.Herein, we report the development of a novel TGF-β signal conversion platform that provides a T cell supportive signal upon exposure to TGF-β. This platform utilizes co-expression of chimeric variants of TGFBR2 and TGFBR1 where the TGF-β-binding domain of each receptor is fused to the transmembrane and intracellular signaling domains of a T cell stimulating interleukin receptor. The signaling domains of interleukin receptors that were chosen for this platform are naturally activated via cytokine-induced hetero-dimerization and induce STAT transcription factor signaling pathways beneficial to effector T cell function and persistence. Based on the ability of lentiviral vectors to express large and complex gene expression cassettes, we constructed single vectors encoding both chimeric TGF-β receptors (CTBR) in the context of either a CAR or transgenic TCR. This required expression of four unique proteins from a single lentiviral vector when CTBR was combined with a transgenic TCR consisting of unique alpha and beta chains.To demonstrate successful signal conversion, we evaluated the impact of TGF-β exposure on multiple functional attributes of CTBR expressing T cells, including gene expression, cytokine production, cytotoxicity, and metabolic function. As anticipated, the STAT signaling signature induced by a particular CTBR was predictive of the functional consequence of exposure to TGF-β. In the case of a CTBR based on IL-12 signaling domains (CTBR12) that strongly induced STAT4 phosphorylation, CTBR12 expressing T cells secreted significantly greater amounts of IFNγ than control T cells following activation in the presence of TGF-β. This result was consistent with IL-12-mediated STAT4 signaling, which induces IFNγ production in effector T cells. We next evaluated CTBR expressing T cells in a stringent serial re-stimulation assay in the presence of TGF-β but absence of exogenous IL-2 cytokine support. Antigen driven T cell expansion was severely limited by exposure to TGF-β in this assay, and either CAR or transgenic TCR expressing cells ultimately lost cytotoxic activity over time. T cells overexpressing the dominant negative TGF-β receptor failed to expand and clear tumor cells following multiple rounds of antigen-driven stimulation in the presence of TGF-β, recapitulating the impact of tumor-derived TGF-β on infiltrating lymphocyte function. On the other hand, CTBR expressing cells maintained their ability to expand and kill tumor targets in the presence of TGF-β.Together, these data demonstrate the successful development of a TGF-β signal conversion platform that transforms the inhibitory effects of TGF-β exposure into a STAT signal that supports T cell effector function. The specific impact on T cell function is dependent on the nature of the signaling domains utilized. This platform is relevant in the context of both CAR T cells and transgenic TCR T cells, and has the potential to produce superior T cell responses in the immunosuppressive tumor microenvironment. DisclosuresBoyerinas:bluebird bio: Employment, Equity Ownership. Miller:bluebird bio: Employment, Equity Ownership. Murray:bluebird bio: Employment, Equity Ownership. Evans:bluebird bio: Employment, Equity Ownership. Parsons:bluebird bio: Employment, Equity Ownership. Seidl:bluebird bio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership, Patents & Royalties.