Chimeric Receptor Research Articles

Abstract 5513: Chimeric CD3 fusion receptors expressed on iPSC-derived universal TCR-less CAR-T and -NK cells synergize with bispecific engagers to enhance antitumor activity and limit antigen escape

Abstract Despite success in hematologic malignancies, chimeric antigen receptor (CAR) T cells have been less effective in solid tumors, in part because of the heterogeneous expression of the CAR-specific target antigen (1° Ag) found within the tumor mass. In combination with a CAR, bispecific T cell engagers (BiTEs) can target additional tumor antigens (2° Ag) while engaging CD3 signaling molecules on T cells, providing a unique way to address tumor heterogeneity, mitigate antigen escape and further potentiate durable effector function. However, in generating off-the-shelf universal CAR-T and NK cells, a combination strategy is not feasible as neither modified cell product is compatible to specifically engage with a BiTE. In developing allogeneic CAR-T cells, the T cell receptor (TCR) surface expression must be eliminated to prevent graft versus host disease, but the absence of surface TCR expression leads to loss of surface CD3 expression and BiTE compatibility. Similarly, NK cells naturally lack TCR expression and have no surface CD3 molecules for BiTE engagement. Here we discuss the development of a novel chimeric CD3ε fusion receptor (CD3-CFR) with a modified transmembrane and endodomain enabling surface expression in TCR-less T or NK cells, allowing for a novel combinatorial solution between universal CAR-T or NK cells with BiTEs in an allogeneic setting. CD3-CFR signal transduction was initially investigated in TRAC knockout NFAT reporter (T-KO) Jurkat cells engineered with CD3-CFR and co-cultured with EpCAM+ target cells. When soluble EpCAM-BiTE was added to the co-culture, the observed 6.9-fold increase in NFAT activity indicated successful engagement associated with the interaction of CD3-CFR, target cells and the BiTE. CD3-CFR T-KO Jurkat cells were further modified to secrete the EpCAM-BiTE and showed increased NFAT activity, demonstrating the feasibility of self-secreting BiTE-mediated targeting through CD3-CFR signaling. We next engineered CD3-CFR expressing iPSC-derived CAR-T (iT) cells to show preserved T cell differentiation kinetics and maintained CAR activity against 1° Ag positive targets (>90% cytolysis). Importantly, while CAR-iT cells showed only background lysis of 1° Ag negative tumor cells, CD3-CFR+ CAR-iT cells were able to lyse ~99% of 1° Ag negative tumor cells with the addition of soluble EpCAM-BiTE targeting the 2° Ag. When CD3-CFR+ CAR-iT cells were further engineered to secrete the EpCAM-BiTE, CD3-CFR+ BiTE+ CAR-iT cells demonstrated superior killing of heterogenous tumors compared to CD3-CFR+ BiTE- CAR-iT cells (70% vs. 5% cytolysis). Taken together these data demonstrate for the first time that CD3 expression and BiTE engagement can be combined in a universal iPSC-derived CAR-iT cell product to overcome antigen heterogeneity and enhance efficacy in solid tumor settings. Citation Format: Eigen Peralta, Dan Lu, Mark Landon, Hui-Yi Chu, Amit Mehta, Philip Chu, Alec Witty, Tom Lee, Bahram Valamehr. Chimeric CD3 fusion receptors expressed on iPSC-derived universal TCR-less CAR-T and -NK cells synergize with bispecific engagers to enhance antitumor activity and limit antigen escape [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 5513.

Abstract CT247: A phase I/II trial investigating safety and efficacy of autologous TAC T cells targeting HER2 in relapsed or refractory solid tumors (TACTIC-2)

Abstract Background: Despite recent therapeutic advances for patients (pts) with breast, colorectal and gastroesophageal cancers with HER2 overexpression, there is still a significant unmet medical need for better treatment options for HER2-positive solid tumors, especially those with low or intermediate HER2 expression (1+ and 2+ by immunohistochemistry (IHC)). T Cell Antigen-Coupler (TAC) technology is a novel way to genetically modify T cells and to redirect these T cells to target cancer antigens and to activate T cells naturally by co-opting the natural T cell Receptor (TCR). TAC01-HER2 is an autologous T-cell product comprising T cells expressing the HER2 TAC, a chimeric receptor that is genetically engineered into T cells via lentiviral transduction to furnish T cells with two main functions: redirection to and specific recognition of HER2-positive cells, and T cell activation via the endogenous TCR. In vivo models, TAC T-cells can infiltrate tumors and persist for extended period of time, leading to robust and persistent anti-tumor efficacy, with a favorable safety profile. The safety and efficacy of TAC01-HER2 is investigated in the ongoing (TACTIC-2) first-in-human phase I-II study (NCT04727151). Methods: Eligible pts are ≥ 18 years with HER2-positive solid tumors (1+, 2+ or 3+ by IHC, regardless of amplification status) who progressed after at least two lines of systemic therapy. Upon enrollment, pts undergo leukapheresis to obtain T cells for TAC01-HER2 manufacturing. Bridging therapy can be administered prior to lymphodepleting chemotherapy (LDC). TAC01-HER2 is a single infusion after LDC with a starting dose of 1-3 x 105 cells/kg and escalating based on the keyboard statistical dose escalation method. Data safety monitoring committee meetings will be held after each dose level. Once the recommended phase 2 dose (RP2D) is identified, an additional 3-6 pts will be treated at that dose level. Current planned phase II expansion cohorts include HER2 3+ breast cancer (N=20), HER2 3+ other solid tumors (N=20), and HER2 2+ breast and other solid tumors (N=10). The primary objective for the trial is to determine the safety and tolerability of TAC01-HER2 with primary endpoints of incidence of dose limiting toxicities (DLTs) and type, frequency, and severity of adverse events (AEs). Secondary endpoints include the RP2D, overall response rate, duration of response, overall survival and pharmacokinetics. Exploratory endpoints include assessment of potential biomarkers of response or resistance and characterization of soluble immune factors and relationship to cytokine release syndrome (CRS), neurotoxicity and TAC T cell engraftment. Tumor response assessments are performed at 4 weeks, then at months 3, 6, 9, 12, 18 and 24. After study completion, subjects are followed for survival and long-term safety for up to 15 years. The trial opened in January 2021 and it is actively enrolling pts. Citation Format: Ecaterina E. Dumbrava, Daniel Olson, Benjamin L. Schlechter, Sam Saibil, Donna Rill, Andreas Bader, Deyaa Adib, Michael Bishop. A phase I/II trial investigating safety and efficacy of autologous TAC T cells targeting HER2 in relapsed or refractory solid tumors (TACTIC-2) [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 CT247.

Abstract 5187: Development of multiomics approaches to evaluate NKG2D ligand dynamics and anti-tumor immune responses during CAR-NK treatment

Abstract NK cells are highly potent, fast-acting cytolytic cells capable of eradicating cancer with limited risk of adverse effects such as cytokine release syndrome or GVHD. NKX101 is a NK cell therapy product engineered to overexpress a chimeric receptor consisting of NKG2D ectodomain, costimulatory signaling motifs, and a membrane-bound form of IL-15. NKX101 is currently under clinical evaluation for treatment of relapsed/refractory AML and MDS. To better understand patterns of response to NKX101, we describe the development of (i) a single-cell (sc) RNAseq approach to assess gene expression pattern changes in NKX101 and patient cells, (ii) a multiplex IHC panel to monitor NKG2D-ligand expression by cancer cells, and (iii) an ELISA method to detect NKG2D-ligand shedding in serum. To verify that our scRNAseq approach can distinguish the NKX101 product cells from patient cells, healthy human PBMCs were pre-mixed with NKX101 cells at set ratios. 10x Genomics hybrid capture baits were prepared to enrich for exogene reads, first via single-cell 5’ gene expression assays and then further processed for target enrichment using a human immunology gene set panel. Multiplex IHC was carried out using the Vectra Polaris Phenoptics platform. FFPE biopsy samples were stained with an 8-marker multiplex panel. Digital image analysis was conducted using the inForm analysis software. For the detection of shed NKG2D-ligands, a sandwich ELISA was employed using recombinant human NKG2D to capture the ligands, followed by detection using ligand-specific antibodies and HRP-conjugated secondary antibodies. We can successfully separate NKX101 product cells from the host PBMC background using our scRNAseq data. NKX101 cell proportions in the transcriptome datasets correlated with the expected titration ratios against host PBMC, with samples titrated at 25% NKX101+ cells yielding approximately 25% NKX101 exogene expressing cells by scRNAseq. Our internally developed multiplex IHC methods can quantify NKG2D-ligand expression levels in AML and solid tumors. We show examples of NKG2D-ligand expression on blast cells from AML solid bone marrow biopsies. In solid tumors, we demonstrate that NKG2D-ligands are upregulated on PanCK+ cancer cells compared to PanCK- cells or healthy normal tissues. Finally, we found certain commercial kits for the detection of soluble NKG2D ligands to potentially under-report the NKG2D-ligand concentrations due to their propensity for aggregation. Using recombinant NKG2D as a capture reagent, we have developed sandwich ELISA method to detect NKG2D-ligands shed into the culture supernatant or serum. In summary, we describe here the development of IHC and ELISA methods to determine NKG2D-ligand expression and shedding as well as a scRNAseq assay to identify NKX101+ product cells and evaluate the anti-tumor immune response during CAR-NK treatment. Citation Format: Kyle Hansen, Sombeet Sahu, Bao Duong, Cynthia Cho, Qi Zhang, James Trager, Joanne Tan. Development of multiomics approaches to evaluate NKG2D ligand dynamics and anti-tumor immune responses during CAR-NK treatment [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 5187.

Abstract 572: Pre-clinical evaluation of Claudin 18.2 TAC T cells for the treatment of gastric cancer

Abstract Background: The T cell antigen coupler (TAC) is a novel, proprietary chimeric receptor that facilitates the re-direction of T cells to tumor cells and activates T cells by co-opting the endogenous T cell receptor complex with the goal to elicit a safe and durable anti-tumor response. Based on this preclinical pharmacology and toxicology data, TAC01-HER2, a first-in-class TAC T product targeting HER2 (ERBB2), has entered a phase I/II clinical trial in patients with HER2-positive solid tumors. Here, we present the development of a new TAC T product targeting Claudin 18.2 (CLDN18.2) to treat gastric cancer. CLDN18.2 belongs to a family of Claudin tight junction proteins whose expression is naturally exclusive to normal stomach. In gastric cancer cells, however, CLDN18.2 surface expression is upregulated and perturbed, leading to tumor-selective surface expression of CLDN18.2. Thus, CLDN18.2 is a preferred antigen for the specific targeting of tumor cells via TAC T cells. Materials and Methods: CLDN18.2-TAC receptor functionality was characterized using a variety of in vitro and in vivo assays. In vitro assays were based on flow cytometric analysis of TAC surface staining and cytokine release. Cytotoxicity was assessed via luminescence-based co-culture assays and real-time microscopy. In vivo studies examined the anti-tumor effect of TAC-engineered T-cells against established solid CLDN18.2 expressing tumor xenografts. Results: The CLDN18.2-TAC receptor showed strong surface expression and specific activation when co-cultured with a variety of cancer cells expressing CLDN18.2 in vitro. Secretion of IL2, IFNg and TNFa were comparable with cytokine levels produced by activated control TAC T cells. In vitro cytotoxicity assays demonstrated a strong anti-CLDN18.2 response and killing of CLDN18.2 expressing target cell lines. No increases in cytokine levels and no cytotoxicity were observed in non-transduced T cells and CLDN18.2-TAC T cells co-cultured with CLDN18.2-negative target cells, indicating that the T cell response is specific to the CLDN18.2 antigen. Intravenous administration of CLDN18.2-TAC T cells in mice carrying CLDN18.2-positive solid tumor xenografts led to a sustained anti-tumor response. Conclusion: The in vitro and in vivo data confirm strong and specific activity of CLDN18.2-targeted TAC T cells against CLDN18.2-expressing solid tumor models and highlight the versatility of the TAC platform for therapeutic applications in solid tumors. Citation Format: Tania Benatar, Ling Wang, Thanyashanthi Nitya-Nootan, Heather MacGregor, Suzanna Prosser, Philbert Ip, Prabha Lal, Laura Shaver, Sadhak Sengupta, Christopher W. Helsen, Andreas G. Bader. Pre-clinical evaluation of Claudin 18.2 TAC T cells for the treatment of gastric cancer [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 572.

Potentiating adoptive cell therapy using synthetic IL-9 receptors

Synthetic receptor signalling has the potential to endow adoptively transferred T cells with new functions that overcome major barriers in the treatment of solid tumours, including the need for conditioning chemotherapy1,2. Here we designed chimeric receptors that have an orthogonal IL-2 receptor extracellular domain (ECD) fused with the intracellular domain (ICD) of receptors for common γ-chain (γc) cytokines IL-4, IL-7, IL-9 and IL-21 such that the orthogonal IL-2 cytokine elicits the corresponding γc cytokine signal. Of these, T cells that signal through the chimeric orthogonal IL-2Rβ-ECD–IL-9R-ICD (o9R) are distinguished by the concomitant activation of STAT1, STAT3 and STAT5 and assume characteristics of stem cell memory and effector T cells. Compared to o2R T cells, o9R T cells have superior anti-tumour efficacy in two recalcitrant syngeneic mouse solid tumour models of melanoma and pancreatic cancer and are effective even in the absence of conditioning lymphodepletion. Therefore, by repurposing IL-9R signalling using a chimeric orthogonal cytokine receptor, T cells gain new functions, and this results in improved anti-tumour activity for hard-to-treat solid tumours.

DAP10 integration in CAR-T cells enhances the killing of heterogeneous tumors by harnessing endogenous NKG2D

Although chimeric antigen receptor T (CAR-T) cells have achieved remarkable successes in hematological malignancies, the efficacies of CAR-T cells against solid tumors remains unsatisfactory. Heterogeneous antigen expression is one of the obstacles on its effective elimination of solid cancer cells. DNAX-activating protein 10 (DAP10) interacts with natural killer group 2D (NKG2D), acting as an adaptor that targets various malignant cells for surveillance. Here, we designed a DAP10 chimeric receptor that utilized native NKG2D on T cells to target NKG2D ligand-expressing cancer cells. We then tandemly incorporated it with anti-glypican 3 (GPC3) single-chain variable fragment (scFv) to construct a dual-antigen-targeting system. T cells expressing DAP10 chimeric receptor (DAP10-T cells) displayed with an enhancement on both cytotoxicity and cytokine secretion against solid cancer cell lines, and its tandem connection with anti-GPC3 scFv (CAR GPC3-DAP10-T cells) exhibited a dual-antigen-targeting capacity on eliminating heterogeneous cancer cells in vitro and suppressing the growth of heterogeneous cancer in vivo. Thus, this novel dual-targeting system enabled a high efficacy on killing cancer cells and extended the recognition profile of CAR-T cells toward tumors, which providing a potential strategy on treatment of solid cancer clinically.

NKG2A-checkpoint inhibition and its blockade critically depends on peptides presented by its ligand HLA-E.

NKG2A has emerged as a new immunotherapy target and its blockade with the novel immune checkpoint inhibitor (ICI) monalizumab can boost both NK cell and CD8+ T cell responses. NKG2A forms heterodimers with CD94 and binds to the human non‐classical MHC class I molecule HLA‐E. HLA‐E forms complexes with a limited set of peptides mainly derived from the leader sequences of the classical MHC class I molecules (HLA‐A, HLA‐B and HLA‐C) and the non‐classical class I paralogue HLA‐G, and it is well established that the interaction between CD94/NKG2x receptors and its ligand HLA‐E is peptide‐sensitive. Here, we have evaluated peptide dependence of NKG2A‐mediated inhibition and the efficiency of interference by monalizumab in a transcriptional T cell reporter system. NKG2A inhibition was mediated by cell‐expressed HLA‐E molecules stably presenting disulfate‐trapped peptide ligands. We show that different HLA‐class I leader peptides mediate varying levels of inhibition. We have used NKG2A/NKG2C chimeric receptors to map the binding site of NKG2A and NKG2C blocking antibodies. Furthermore, we determined the functional EC50 values of blocking NKG2A antibodies and show that they greatly depend on the HLA‐leader peptide presented by HLA‐E. Monalizumab was less effective in augmenting NK cell‐mediated killing of target cells displaying HLA‐G peptide on HLA‐E, than cells expressing HLA‐E complexed with HLA‐A, HLA‐B and HLA‐C peptides. Our results indicate that peptides displayed by HLA‐E molecules on tumour cells might influence the effectivity of NKG2A‐ICI therapy and potentially suggest novel approaches for patient stratification, for example, based on tumoral HLA‐G levels.

IMMU-20. Effective CAR-T cell medulloblastoma therapy in an immunocompetent mouse model

Immunotherapy with chimeric antibody receptor (CAR) T cells is effective for previously incurable hematologic cancers and may transform treatment for refractory brain tumors. However, CAR-T cell therapy for solid tumors has not yet been as successful as for leukemias, and animal models are needed to improve implementation. At present, preclinical studies of CAR-T cell therapy for brain tumors have typically used exogenous tumors, xenografted into immunocompromised mice, because primary mouse brain tumors do not express antigens that match brain tumor-specific antigens in humans. To advance preclinical development of CAR-T brain tumor therapy, we engineered mice to develop medulloblastomas that express B7-H3, an antigen specifically expressed on human medulloblastomas and other pediatric brain tumors. We show that treating these tumors with B7-H3-directed CAR-T cells provokes anti-tumor responses both in vitro and in vivo. Administering B7-H3 CAR-T cells by intracranial injection increased the event-free survival time of mice with medulloblastoma, in a dose-dependent manner. CAR-T cell treatment was not curative as an isolated intervention, suggesting that cure will require pairing with surgical resection and additional adjuvant therapy. Our model presents new opportunities to study the mechanisms of CAR-T cell efficacy and recurrence in an immunocompetent host with intact vasculature and blood-brain barrier. Our ongoing studies using scRNA-seq will allow us to define therapy-induced changes in tumor cells, CAR-T cells and cells of the tumor microenvironment and to test new T-cell modifications and combinations of therapeutic modalities, toward a goal of optimizing CAR-T cell therapy for pediatric brain tumors.

DEVELOPMENT OF AN ADRENOCORTICAL CELL MODEL OF CALCIUM SIGNALING MODULATION TO DECIPHER THE MOLECULAR MECHANISMS RESPONSIBLE FOR PRIMARY ALDOSTERONISM

Objective: Primary aldosteronism (PA) is the most frequent form of secondary hypertension. The identification of germline or somatic mutations in different genes coding for ion channels (KCNJ5, CACNA1D, CACNA1H and CLCN2) and ATPases (ATP1A1 and ATP2B3) defines PA as a channelopathy. These mutations promote activation of calcium signaling, the main trigger for aldosterone biosynthesis. The objective of our work was to elucidate, using chemogenetic tools, the molecular mechanisms underlying the development of PA by modulating sodium entry into the cells, mimicking some of known mutations identified in PA. Design and method: We have developed an adrenocortical H295R_S2 cell line stably expressing a chimeric ion channel receptor formed by the extracellular ligand-binding domain of the α7 nicotinic acetylcholine receptor fused to the ion pore domain of the serotonin receptor 5HT3α and named α7–5HT3. Mutations have been introduced in the ligand binding domain to allow only synthetic drugs to activate this channel receptor. Activation of α7–5HT3 by a specific drug, PSEM-817 leads to sodium entry into the cells. This cell line was characterized in terms of intracellular calcium concentrations, cell proliferation, aldosterone production, steroidogenic expression and electrophysiological properties. Results: Treatment of α7–5HT3 expressing cells with increasing concentration of PSEM-817 (from 10–9 to 10–5 M) induced a significant increase in intracellular calcium concentrations, similarly to potassium (12 mM) or angiotensin II (10–8 M). This stimulation of calcium signaling did not affect cell proliferation, but was responsible for an increase in CYP11B2 expression and aldosterone production after 24 h of treatment. However, while increased intracellular calcium concentrations were observed starting from 10–8 M of PSEM-817, CYP11B2 expression and aldosterone production were only affected starting from 10–7 M, suggesting a dose dependent effect. Finally, whereas cells were hyperpolarized in absence of stimulation (around -60 mV), PSEM-817 induced a strong depolarization, cells rising to a membrane potential around -10 mV. Conclusions: This cell line, in which we can modulate the intracellular calcium concentration “on demand”, is a useful tool for a better understanding of the alterations of intracellular ion balance and calcium signaling in the pathophysiology of PA.

Development of an adrenocortical cell model of calcium signaling modulation to decipher the molecular mechanisms responsible for primary aldosteronism

Primary aldosteronism (PA) is the most frequent form of secondary hypertension. The identification of germline or somatic mutations in different genes coding for ion channels and defines PA as a channelopathy. These mutations promote activation of calcium signaling, the main trigger for aldosterone biosynthesis. The objective of our work was to elucidate, using chemogenetic tools, the molecular mechanisms underlying the development of PA by modulating sodium entry into the cells, mimicking some of known mutations identified in PA. We have developed an adrenocortical H295R-S2 cell line stably expressing a chimeric ion channel receptor formed by the extracellular ligand-binding domain of the α7 nicotinic acetylcholine receptor fused to the ion pore domain of the serotonin receptor 5HT3α and named α7-5HT3. Mutations have been introduced in the ligand-binding domain to allow only synthetic drugs to activate this channel receptor. Activation of α7-5HT3 by a specific drug, PSEM-817 leads to sodium entry into the cells. This cell line was characterized in terms of intracellular calcium concentrations, cell proliferation, aldosterone production, steroidogenic expression and electrophysiological properties. Treatment of α7-5HT3 expressing cells with increasing concentration of PSEM-817 (from 10-9 to 10-5 M) induced a significant increase in intracellular calcium concentrations, similarly to potassium (12 mM) or angiotensin II (10-8 M). This stimulation of calcium signaling did not affect cell proliferation, but was responsible for an increase in CYP11B2 expression and aldosterone production after 24 h of treatment. However, while increased intracellular calcium concentrations were observed starting from 10-8 M of PSEM-817, CYP11B2 expression and aldosterone production were only affected starting from 10-7 M, suggesting a dose-dependent effect. Finally, whereas cells were hyperpolarized in absence of stimulation (around −60 mV), PSEM-817 induced a strong depolarization, cells rising to a membrane potential around −10 mV. This cell line, in which we can modulate the intracellular calcium concentration “on demand”, is a useful tool for a better understanding of the alterations of intracellular ion balance and calcium signaling in the pathophysiology of PA.

SO-32 Entrectinib in NTRK fusion-positive gastrointestinal cancers: Updated integrated analysis

Neurotrophic tyrosine receptor kinase ( NTRK ) gene fusions, coding for constitutively active chimeric tyrosine receptor kinase (TRK) proteins, are oncogenic drivers found in a large range of tumour types, including gastrointestinal cancers. Entrectinib is a potent TRK inhibitor with activity in the CNS, that has demonstrated deep and durable responses in patients with a range of NTRK fusion-positive solid tumours from the phase 1/2 ALKA-372-001 (EudraCT 2012-000148-88), STARTRK-1 (NCT02097810) and STARTRK-2 (NCT02568267) clinical trials.

Pharmacotherapy of Generalized Myasthenia Gravis with Special Emphasis on Newer Biologicals.

Myasthenia gravis (MG) is a chronic, fluctuating, antibody-mediated autoimmune disorder directed against the post-synaptic neuromuscular junctions of skeletal muscles, resulting in a wide spectrum of manifestations ranging from mild to potentially fatal. Given its unique natural course, designing an ideal trial design for MG has been wrought with difficulties and evidence in favour of several of the conventional agents is weak as per current standards. Despite this, acetylcholinesterases and corticosteroids have remained the cornerstones of treatment for several decades with intravenous immunoglobulins (IVIG) and therapeutic plasma exchange (PLEX) offering rapid treatment response, especially in crises. However, the treatment of MG entails long-term immunosuppression and conventional agents are viable options but take longer to act and have a number of class-specific adverse effects. Advances in immunology, translational medicine and drug development have seen the emergence of several newer biological agents which offer selective, target-specific immunotherapy with fewer side effects and rapid onset of action. Eculizumab is one of the newer agents that belong to the class of complement inhibitors and has been approved for the treatment of refractory general MG. Zilucoplan and ravulizumab are other agents in this group in clinical trials. Neisseria meningitis is a concern with all complement inhibitors, mandating vaccination. Neonatal Fc receptor (FcRn) inhibitors prevent immunoglobulin recycling and cause rapid reduction in antibody levels. Efgartigimod is an FcRn inhibitor recently approved for MG treatment, and rozanolixizumab, nipocalimab and batoclimab are other agents in clinical trial development. Although lacking high quality evidence from randomized clinical trials, clinical experience with the use of anti-CD20 rituximab has led to its use in refractory MG. Among novel targets, interleukin 6 (IL6) inhibitors such as satralizumab are promising and currently undergoing evaluation. Cutting-edge therapies include genetically modifying T cells to recognise chimeric antigen receptors (CAR) and chimeric autoantibody receptors (CAAR). These may offer sustained and long-term remissions, but are still in very early stages of evaluation. Hematopoietic stem cell transplantation (HSCT) allows immune resetting and offers sustained remission, but the induction regimens often involve serious systemic toxicity. While MG treatment is moving beyond conventional agents towards target-specific biologicals, lack of knowledge as to the initiation, maintenance, switching, tapering and long-term safety profile necessitates further research. These concerns and the high financial burden of novel agents may hamper widespread clinical use in the near future.

Expression of CD20-specific chimeric antigen receptor (CD20-CAR) on Jurkat T cellsand evaluation its biological activity

CD20 is a potential target for immunotherapy treatments of haematological malignancies due to its specific expression and unique biological property. Over the years, many anti-CD20 monoclonal antibodies (mAbs) have been developed, such as Rituximab, Ibritumomab, Tositumomab, etc., and applied in B cells related to cancer treatments. Recently, a new immunological therapy, namely Chimeric antigen receptor (CAR) T-cell therapy, in which the patient’s T cells are reprogrammed ex vivo with a transgene encoding a CAR in order to active them against the tumour, has emerged as a promising treatment for B-cell malignancies. In this study, the authors generated and expressed two variants of chimeric receptor antigen-specific to CD20 on Jurkat T cells, in which one variant referenced the sequence from the patent (SEQ01) and the other was our own designed sequence (SEQ02). The transient expression after electroporation 24 hours confirmed the presence of mRNA and protein of CD20-CAR in both sequences. Moreover, these chimeric receptors were also detected on the membrane of Jurkat cells using flow cytometer analysis, indicating that the author could successfully transduce and express CD20-CAR in Jurkat cells. Co-culture CD20-Jurkat-CAR-T cells with CD20-expressing Raji B cells show that the generated Jurkat could be activated via their chimeric receptors through increasing the secretion of Interleukin-2 in both sequences. Taken together, our preliminary data suggest that the authors have been able to express the CD20-activating functional transgenic receptor on Jurkat cells, which will be applied for generating CD20-CAR-expressing CD4+/CD8+ T cells.

Structure-guided optimization of light-activated chimeric G-protein-coupled receptors

G-protein-coupled receptors (GPCRs) are the largest human receptor family and involved in virtually every physiological process. One hallmark of their function is specific coupling to selected signaling pathways. The ability to tune this coupling would make development of receptors with new capabilities possible. Complexes of GPCRs and G-proteins have recently been resolved at high resolution, but this information was in only few cases harnessed for rational receptor engineering. Here, we demonstrate structure-guided optimization of light-activated OptoXRs. Our hypothesis was that incorporation of GPCR-Gα contacts would lead to improved coupling. We first evaluated structure-based alignments for chimeric receptor fusion. We then show in a light-activated β2AR that including Gα contacts increased signaling 7- to 20-fold compared with other designs. In turn, contact elimination diminished function. Finally, this platform allowed optimization of a further OptoXR and spectral tuning. Our work exemplifies structure-based OptoXR development for targeted cell and network manipulation.

Characterization of Novel A3 Adenosine Receptor Allosteric Modulators

IntroductionA3 adenosine receptor (A3AR) agonists are currently being evaluated in clinical trials for treatment of inflammation, cancer, and neuropathic pain. To circumvent complications associated with the use of direct agonists of GPCRs (selectivity, dose‐limiting side‐effects), we have pursued development of A3 adenosine receptor positive allosteric modulators (PAMs). These compounds potentiate signaling while allowing for spatiotemporal specificity and decreased off target‐effects.MethodsPrior structure‐activity‐relationship (SAR) studies identified the 1H‐imidazo[4,5]quinoline‐4‐amine, LUF6000, and the 2,4‐disubstituted quinoline, LUF6096, as exhibiting human A3AR PAM activity. We observed a ~2‐3‐fold increased maximal effect in G protein‐dependent signaling assays, although they had the tendency to reduce agonist potency. In addition, these lead compounds were substantially less potent at rodent A3ARs. The purpose of the present investigation was to re‐evaluate two expanded compound series based on LUF6000 and LUF6096 in greater detail, with emphasis on examining effects on orthosteric ligand potency, G protein‐dependent versus G protein‐independent signaling, and efficacy versus mouse A3 adenosine receptors. Based on known species differences, human/mouse chimeric receptors were prepared to determine the region of the receptor comprising the allosteric ligand binding pocket.ResultsOur evaluation centered on a series of 2‐substituted imidazoquinolinamine and comparable 4‐substituted quinoline derivatives consisting of 3–12‐membered cycloalkyl rings and their carbon‐bridged analogues. This was based on our observation during an initial screen that the 2‐cycloheptyl and 2‐cyclooctyl imidazoquinolinamine analogues increased affinity of the orthosteric radioligand ([125I]I‐AB‐MECA) in equilibrium binding assays in a manner that did not slow its (apparent) association binding rate. Among this series, compounds with larger 2‐substituents (c4‐9) were found to increase the maximal efficacy of the orthosteric agonist Cl‐IB‐MECA in [35S]GTPγS binding assays ~2‐fold. Most prominently, the 2‐cyclononanyl derivative increased the efficacy of Cl‐IB‐MECA (~2.5‐fold) while also increasing its potency. 4‐substituted quinoline derivatives displayed a similar SAR with larger 4‐substituents (c4‐9) increasing maximal efficacy – however, none of the tested derivatives increase orthosteric radioligand binding affinity at equilibrium. While human A3 G‐protein coupling profiles were similar for all agonists examined, with robust coupling being detected to all Gαi proteins, probe‐dependent effects were observed for β‐arrestin‐2 recruitment. Moreover, while LUF6000 and LUF6096 potentiated agonist‐induced Gαi protein activation, enhancement of β‐arrestin‐2 recruitment was agonist‐dependent. Studies with human/mouse chimeric receptors indicated that modulators based on the imidazoquinolinamine template do not function as ‘vestibule’ modulators but instead bind intracellularly.ConclusionsWe have identified improved PAMs for the human A3 receptor that: 1) not only increase agonist efficacy, but also increase agonist potency (and binding affinity), and 2) may bias receptor signaling towards G protein‐dependent signaling. Furthermore, we have evidence supporting a novel intracellular allosteric binding pocket on the A3AR.

RXFP2 Small Molecule Agonists: Potential Therapeutics for Osteoporosis

Osteoporosis is a chronic bone disease characterized by decreased bone mass and increased risk of developing fractures, predominantly observed in the elderly. Osteoporosis affects approximately 10 million people in the US, and the number is expected to increase exponentially as the elderly population continues to grow. The pathophysiological cause of the disease is a decrease in the activity of the bone‐forming cells (osteoblasts) that alters bone remodeling in favor of bone resorption, leading to a decrease in bone mass. Recent studies identified the G protein‐coupled receptor (GPCR) for insulin‐like 3 peptide (INSL3), relaxin family peptide receptor 2 (RXFP2), as an attractive target for the treatment of osteoporosis. The goal of this study is to develop small molecule agonists of RXFP2, expressed in osteoblast cells, to promote bone growth and counteract the deleterious effects of osteoporosis. Currently, the most effective available treatment for osteoporosis is an expensive hormone therapy that requires daily injections. We aim to create drugs that are stable and can be delivered orally. Several low molecular weight compounds were identified as agonists of the RXFP2 receptor using a high‐throughput screen of the NCATS small molecule library. The screening assay measured cAMP response in RXFP2‐transfected HEK293T cells. An extensive structure‐activity relationship campaign resulted in highly potent and efficient full RXFP2 agonists. The selectivity and specificity of the compounds for human and mouse RXFP2 was shown using orthogonal cAMP assays, counter‐screening against the related relaxin receptor RXFP1, and a GPCRome screen using the PRESTO‐Tango assay. The drug candidates were further tested in primary human osteoblasts to demonstrate that they promote mineralization by measuring hydroxyapatite deposition in the cell matrix. We showed that the compounds had low cytotoxicity in various cell types. Using a series of RXFP2/RXFP1 chimeric receptors, we established that the compounds are allosteric agonists of the RXFP2 receptor and identified the GPCR transmembrane domains as the specific region for compound interaction. The RXFP2 agonist with the highest activity in vitro was selected for pharmacokinetics (PK) profiling in mice, showing optimal oral bioavailability and bone exposure. An efficacy study using 2‐month‐old WT female mice treated orally with 10 mg/kg of compound 3 times a week for 8 weeks showed a significant increase of the vertebral trabecular number and thickness by micro‐CT analysis compared to vehicle treated controls. We expect that the further characterization of these compounds may lead to the development of a new class of cost‐effective drugs for the treatment of osteoporosis and other diseases associated with bone loss.

Chimeric Cytokine Targeting NKG2D and IL-2 Receptor Augments CD8+ T Cell-Metabolic Fitness and Improves Immunotherapy

Abstract T cell mediated immunotherapy has gained credible traction for cancer treatment. To date experimental and clinical strategies have relied on antibody-mediated co-stimulatory blockade, or the administration of high doses of naturally occurring or engineered cytokines to increase T cell activation. Cytokine therapy is by limited undesirable off-target side effects as well as terminal differentiation and exhaustion of chronically stimulated T cells. Here we describe functionality of a unique chimeric cytokine by design, called OMCPmutIL-2, binds with high affinity to the cytotoxic lymphocyte-defining immunoreceptor NKG2D but signals through the common γ-chain cytokine receptor, thus eliminating IL-2Ra mediated off-target side effects. In addition to precise activation of cytotoxic T cells due to redirected binding we describe that OMCPmutIL-2 results in superior activation of both human and murine CD8+ T cells by improving their survival, decreasing exhaustion, and improving memory cell generation. This functional improvement is the result altered signal transduction based on the reorganization of surface membrane lipid rafts resulting in JAK-mediated phosphorylation of the T cell receptor (TCR) rather than canonical STAT/AKT signaling intermediates activated by other common γ-chain cytokines. This altered signaling pathway increases CD8+ T cell response to low affinity antigens, activates Nuclear Factor of Activated T Cells (NFAT) transcription factors, and enhances mitochondrial biogenesis with improved T cell survival, cytotoxicity, and memory cell generation. OMCPmutIL-2 thus outperforms other common γ-chain cytokines as both a reagent for T cell expansion and in vivo CD8+ T cell-based immunotherapy. Supported by grant I01 BX002299

Evolving Strategies to Eliminate the CD4 T Cells HIV Viral Reservoir via CAR T Cell Immunotherapy.

Although the advent of ART has significantly reduced the morbidity and mortality associated with HIV infection, the stable pool of HIV in latently infected cells requires lifelong treatment adherence, with the cessation of ART resulting in rapid reactivation of the virus and productive HIV infection. Therefore, these few cells containing replication-competent HIV, known as the latent HIV reservoir, act as the main barrier to immune clearance and HIV cure. While several strategies involving HIV silencing or its reactivation in latently infected cells for elimination by immune responses have been explored, exciting cell based immune therapies involving genetically engineered T cells expressing synthetic chimeric receptors (CAR T cells) are highly appealing and promising. CAR T cells, in contrast to endogenous cytotoxic T cells, can function independently of MHC to target HIV-infected cells, are efficacious and have demonstrated acceptable safety profiles and long-term persistence in peripheral blood. In this review, we present a comprehensive picture of the current efforts to target the HIV latent reservoir, with a focus on CAR T cell therapies. We highlight the current challenges and advances in this field, while discussing the importance of novel CAR designs in the efforts to find a HIV cure.

Chimeric anti-GPC3 sFv-CD3ε receptor-modified T cells with IL7 co-expression for the treatment of solid tumors

Chimeric antigen receptor (CAR) T cells targeting glypican-3 (GPC3) demonstrated early signs of therapeutic efficacy to hepatocellular carcinoma patients with a risk of cytokine release syndrome (CRS). Several adoptive cell therapies (ACTs) with T cells using the natural T cell receptor (TCR) signaling induced more efficient antitumor function and reduced cytokine production relative to CARs in solid tumors. To improve the efficacy and safety of GPC3-targeted ACTs, T cells were modified with anti-GPC3 single-chain fragment variable(sFv) linked to CD3ε, which could be incorporated into the entire TCR/CD3 complex to form chimeric sFv-CD3ε receptor (sFv-ε). sFv-ε T cells showed competitive antitumor activity and lower cytokine release compared to 28ζ or BBζ CAR T cells, which may be ascribed to moderately less activated Ca2+-calcineurin-NFAT signaling pathway. We further generated murine sFv-ε T cells with interleukin-7 co-expression (7sFv-ε) to promote T cell survival and to mobilize the endogenous immune system. In immunocompetent mouse models, 7sFv-ε T cells showed superior persistence, antitumor efficacy, and immunological memory while preserving the low production of cytokines associated with CRS compared to conventional sFv-ε T cells. These results indicate that GPC3-specific 7sFv-ε T cells could serve as a promising therapeutic strategy for solid tumors.

T-Cells Expressing a Highly Potent PRAME-Specific T-Cell Receptor in Combination with a Chimeric PD1-41BB Co-Stimulatory Receptor Show a Favorable Preclinical Safety Profile and Strong Anti-Tumor Reactivity.

Simple SummaryThe development of effective adoptive T-cell therapies (ATCs) to treat solid tumors has several challenges: the choice of a suitable target antigen, the generation of a specific T-cell receptor (TCR) directed against this target, and the hostile tumor microenvironment (TME). The cancer/testis antigen Preferentially Expressed Antigen in Melanoma (PRAME) is a promising target for ATCs since it is highly expressed in several solid tumor indications, while its expression in normal tissues is mainly restricted to the testis. Using our well-established high throughput TCR generation and characterization process, we identified a highly potent PRAME-specific TCR. To convert the inhibitory PD-1 signal in T-cells to an activating signal, we designed a chimeric receptor consisting of the extracellular domain of PD-1 and the signaling domain of 4-1BB. Combining this PD1-41BB receptor with our lead PRAME-TCR generated a very promising T-cell product with a favorable preclinical in vitro safety profile and enhanced in vitro and in vivo anti-tumor efficacy.The hostile tumor microenvironment (TME) is a major challenge for the treatment of solid tumors with T-cell receptor (TCR)-modified T-cells (TCR-Ts), as it negatively influences T-cell efficacy, fitness, and persistence. These negative influences are caused, among others, by the inhibitory checkpoint PD-1/PD-L1 axis. The Preferentially Expressed Antigen in Melanoma (PRAME) is a highly relevant cancer/testis antigen for TCR-T immunotherapy due to broad expression in multiple solid cancer indications. A TCR with high specificity and sensitivity for PRAME was isolated from non-tolerized T-cell repertoires and introduced into T-cells alongside a chimeric PD1-41BB receptor, consisting of the natural extracellular domain of PD-1 and the intracellular signaling domain of 4-1BB, turning an inhibitory pathway into a T-cell co-stimulatory pathway. The addition of PD1-41BB to CD8+ T-cells expressing the transgenic PRAME-TCR enhanced IFN-γ secretion, improved cytotoxic capacity, and prevented exhaustion upon repetitive re-challenge with tumor cells in vitro without altering the in vitro safety profile. Furthermore, a single dose of TCR-Ts co-expressing PD1-41BB was sufficient to clear a hard-to-treat melanoma xenograft in a mouse model, whereas TCR-Ts without PD1-41BB could not eradicate the PD-L1-positive tumors. This cutting-edge strategy supports development efforts to provide more effective TCR-T immunotherapies for the treatment of solid tumors.