Chimeric Antigen Receptor Design Research Articles

Novel Adapter Chimeric Antigen Receptor (aCAR) T Cells for Temporally Controllable Targeting of Single and Multiple Tumor Antigens

T cells, engineered to express chimeric antigen receptors (CAR), are capable to generate an extremely potent and highly specific immune response against defined surface-expressed antigens. The very same features however hinder a more general clinical application, first because on-target off-tumor toxicity can cause life-threatening damage to vitally important healthy tissue and second because heterogeneity as well as plasticity of tumor tissue circumvent elimination through modulation or loss of target epitopes.One elegant way to achieve temporal (on/off) as well as qualitative (change and combine target antigens) control of CAR T cell function, is to split antigen recognition and effector cell activation using adapter molecules. Here, we report on the development of a novel adapter CAR (aCAR) system. By harnessing the unique properties of a novel single chain variable fragment (scFv), recognizing a neo-epitope comprising the endogenous vitamin biotin, conjugated to an adapter molecule e.g. a monoclonal antibody, in context of a special linker moiety, the technology can be used to specifically redirect CAR-T cells to a target structure referred to as linker-label epitope (LLE).CAR design was empirically tested using five different extracellular spacer formats combined with a second generation 4-1BB-CD3-ζ signaling backbone. aCAR expression was found to transmit potent T cell activation, cytokine secretion and target cell lysis exclusively in presence of biotinylated antibodies (b-mAbs) or antibody fragments targeting specific antigens at concentrations spanning several log units (10 pg/ml - 100 µg/ml). Importantly, no impairment or inhibition of signaling was detected in human serum or at supra-physiological concentrations of free biotin, providing evidence for specificity against the LLE. Short spacers (IgG4 or CD8 hinge) performed best, independent of the utilized biotinylated antibody format (full-size monoclonal antibodies (mAb), Fab2 or Fab fragments). aCAR T cells were tested in vitro in combination with over 30 different b-mAbs directed against antigens expressed on tumor cells of various cancer entities. With all tested antibodies, aCAR T cells were highly cytotoxic in the presence of a single antibody against an expressed antigen, thusverifying the specificity and applicability of the aCAR system. Further, we were able to demonstrate the simultaneous targeting of multiple antigens can prevent selection of intrinsically resistant antigen-loss variants. Importantly, we found that combining adapters against different antigens expressed on the same target cell mediates aCAR activation at adapter concentrations below the activation threshold for single antigen targeting, allowing target cell recognition and elimination based on multifactorial expression profiles. Finally, in vivo efficacy was demonstrated, targeting CD19 in a NALM-6 NSG mouse xenograft model.In conclusion, we demonstrate a novel aCAR system for maximal flexibility. aCAR T cell activation and cytotoxicity exclusively depends on the presence of biotinylated adapter molecules. Therefore, aCAR-T cell function can be stringently titrated by b-mAb infusion and withdrawal. This could add to an increased safety profile of CAR T cell therapy. Moreover, simultaneous targeting of multiple antigens can reduce selection pressure and the occurrence of resistance to therapy. Finally, since various mAbs have been FDA-approved or are close to enter the clinical setting and since the biotinylation of mAbs can be easily performed in a GMP-compliant manner, future clinical trials in leukemia and solid tumors can be envisioned based on our aCAR system. DisclosuresSeitz:Miltenyi Biotec GmbH: Patents & Royalties, Research Funding. Schlegel:Miltenyi Biotec GmbH: Patents & Royalties, Research Funding. Mittelstaet:Miltenyi Biotec GmbH: Employment. Lock:Miltenyi Biotec GmbH: Employment. Kaiser:Miltenyi Biotec GmbH: Employment, Patents & Royalties. Lang:Miltenyi Biotec GmbH: Patents & Royalties, Research Funding. Handgretinger:Miltenyi Biotec GmbH: Patents & Royalties, Research Funding.

The Combination of 41BB and CD28 Costimulatory Signals Endows Lower Affinity CAR T Cells with the Most Optimal Properties for Selective Targeting of the Tumor Associated Antigen CD38

T cells equipped with Chimeric Antigen Receptors (CARs) have been emerged as a potent therapy for cancer. The scarcity of tumor-specific surface antigens in several cancer types, however, prompted the research towards improved CAR designs to optimally target tumor associated antigens (TAA), which are also expressed on normal cells at lower densities. To this end, we recently demonstrated the feasibility of targeting CD38, a multiple myeloma (MM) associated TAA, without on-target/off-tumor effects, by designing CARs with optimally lowered affinity for the antigen. It is, however, largely unknown whether lowering the antigen affinity affects the costimulatory requirements of CAR T cells for optimal expansion and in vivo persistence. Therefore we now evaluated the impact of different costimulation designs on CD38-CARs with lower affinity. Previous reports indicate that a CAR design including CD28 costimulation improves the cytotoxic potential of CAR T cells whereas a configuration involving the 4-1BB-costimulation confers more persistent responses. Therefore, we used CARs with 5 different affinities (Kd range 1.8 - >1915 nM), in combination with 3 different described costimulatory designs (28z, 4-1BBz and 28z + 4-1BBL). Lowering the affinity of CAR gradually diminished the cytolytic activity of CAR T cells with a 4-1BB costimulatory domain. Such an effect was however not observed with CARs containing the CD28 costimulatory domain, alone or expressed in combination with 4-1BBL. In vitro as well as in vivo experiments revealed that 28z + 4-1BBL expressing low affinity CD38-CAR T cells possessed a significantly better discriminative capacity between tumor and normal cells, displayed a more clear central memory phenotype, showed better in vitro expansion and improved in vivo persistence compared to the other two configurations tested. More importantly, the 28z + 4-1BBL design potentiated even CARs bearing scFvs with immeasurable affinity (>1915nM) and brought them to the functional level of 4-1BB- and 28z-based CARs of higher affinities. In conclusion, we show that the type of costimulatory domain is of importance when fine-tuning the CAR affinity. A combination of 4-1BB and CD28 signals provides the most optimal costimulation to affinity-tuned CAR T cells and suggests the careful construction of TAA-targeting CARs to enhance their clinical potential. DisclosuresVan De Donk:Janssen, Celgene, Bristol-Myers Squibb, Amgen: Research Funding. Zweegman:Celgene: Other: advisory board participation, Research Funding; Janssen: Other: advisory board participation, Research Funding; Takeda: Other: advisory board participation, Research Funding; Amgen: Other: advisory board participation. Lokhorst:Genmab: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; OncoImmune: Research Funding. Mutis:Novartis: Research Funding; OncoImmune: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Research Funding; Gilead: Research Funding; Celgene: Research Funding.

T Cells Expressing Anti-B-Cell Maturation Antigen (BCMA) Chimeric Antigen Receptors with Antigen Recognition Domains Made up of Only Single Human Heavy Chain Variable Domains Specifically Recognize Bcma and Eradicate Tumors in Mice

Multiple myeloma (MM) is an almost-always incurable malignancy of plasma cells. Chimeric antigen receptors (CARs) are artificial fusion proteins that incorporate antigen-recognition domains and T-cell signaling domains. CAR T cells have been shown to have substantial activity against leukemia and lymphoma, which has generated great interest in developing CAR T-cell therapies for MM. CAR T cells targeting B-cell maturation antigen (BCMA) have been shown to have substantial activity against MM (Ali et al. Blood. 2016. 128(13). 1688-1700). Improvements in CAR T-cell therapies for MM are needed. One possible limitation of CAR T-cell therapies is generation of recipient anti-CAR immune responses. Recipient anti-CAR immune responses have been reported in patients on clinical trials of anti-CD19 CARs (Turtle et al. Journal of Clinical Investigation. 2016. 126 (6). 2123-3138). Recipient anti-CAR immune responses could potentially develop against murine antibody variable region domains that are included in the single-chain variable fragments (scFvs) of most CARs. In addition, recipient anti-CAR immune responses could develop against the artificial linker domain in the scFv or against junctions between different CAR components.We designed CARs with antigen-recognition domains of single fully-human heavy-chain-only variable domains (FHVH). The FHVH CARs do not contain a light chain variable region domain or a linker. The fully-human variable region, lack of a linker, and the minimized number of junctions should all lead to reduced immunogenicity for the FHVH antigen-recognition domain compared with a standard scFv.We constructed CARs with 4 different FHVH domains designated FHVH32, FHVH33, FHVH74, and FHVH93. We designed CARs with each of these FHVH domains along with a CD8-alpha hinge and transmembrane domain, either a CD28 or a 4-1BB costimulatory domain, and a CD3-zeta T-cell activation domain. DNA encoding these CARs was ligated into a gamma-retroviral vector, and primary human T cells were transduced with gamma-retroviruses encoding each of the CARs. We tested the T cells for CAR expression and a variety of T-cell functions. We assessed T-cell surface CAR expression by staining with phycoerythrin-labeled BCMA protein. T cells from 3 MM patients were transduced with genes encoding each of the CD28-containing CARs. The following mean percentages of T cells expressed each CAR after transduction: FHVH32-CD828Z (47.3%), FHVH33-CD828Z (49.6%), FHVH74-CD828Z (51.3%), and FHVH93-CD828Z (46.6%). The mean T-cell surface CAR expression of the 11D5-3-CD828Z CAR containing a standard murine scFv with a heavy chain variable domain and a light chain variable domain connected by a linker was 60.4%. Importantly, we found that each of the FHVH CARs specifically recognized BCMA in functional assays. Specific interferon gamma production by the CARs is shown in the Table. We also demonstrated BCMA-specific tumor-necrosis factor-alpha release, cytotoxicity, and proliferation by T cells expressing each of the 4 FHVH CARs containing CD28. We have previously reported clinical anti-myeloma responses with infusions of T cells expressing the 11D5-3-CD828Z CAR (Ali et al. Blood. 2016. 128(13). 1688-1700). T cells expressing FHVH CARs with CD28 domains, especially FHVH33-CD828Z and FHVH74-CD828Z, had BCMA-specific functions including cytokine release that were equal to or superior to those of T cells expressing 11D5-3-CD828Z (Table). We have demonstrated that human T cells expressing either FHVH33-CD828Z or FHVH74-CD828Z can eliminate established tumors of BCMA-expressing human RPMI8226 cells from immune-deficient mice.We also assessed FHVH CARs with 4-1BB costimulatory domains. Of the 4-1BB CARs, FHVH33-CD8BBZ CAR had the highest percentage of T cells with surface CAR expression (mean 52.1%, n=3). T cells expressing FHVH33-CD8BBZ exhibited BCMA-specific cytokine-release, degranulation, cytotoxicity, and proliferation.In summary, we have designed, constructed, and tested a novel type of CAR with an antigen-recognition domain made up of only single fully-human heavy-chain variable region domains. These CARs specifically recognized BCMA in vitro, and they eradicated tumors from mice. The simple, fully-human antigen-recognition domains in these CARs offer a potential advantage of decreased immunogenicity compared to other CAR designs. [Display omitted] DisclosuresTrinklein:TeneoBio: Employment. Buelow:TeneoBio: Employment. Kochenderfer:Bluebird bio: Research Funding; N/A: Patents & Royalties: I have multiple patents in the CAR field.; Kite Pharma: Research Funding.

A Novel Switchable CAR-T Platform for Acute Lymphoblastic Leukemia Treatment

Treatment of Acute Lymphoblastic Leukemia (ALL) with Chimeric Antigen Receptor (CAR) -T cells has shown tremendous results in clinical trials achieving complete responses in 90% of heavily pretreated patients. However serious side effects, most commonly Cytokine Release Syndrome (CRS), have also been encountered. Mechanisms to control CAR-T cell activity and adverse events by steroidal immune-suppression or specific CAR-T cell depletion may mitigate safety concerns, however antigen loss relapse remains a major source of resistance. Current CAR-T constructs in the clinic are fixed in their ability to target a single antigen, limiting the anti-tumor efficacy once loss or mutation of the targeted antigen occurs.We have previously shown that T cells can be redirected towards specific antigens via transduction with a “switchable” CAR (sCAR) construct that recognizes a short peptide engrafted into a Fab antibody fragment which recognizes the tumor (the switch) when dosed separately. In preclinical models this platform has demonstrated potent in vivo activity and tumor regression targeting multiple antigens and exhibits exquisite control of the CAR-T cell activity by switch titration. This may control the severity of side effects in the clinic.Herein, we have taken advantage of synthetic E/K coiled-coil peptidic structures to create a new species of switchable CAR that do not rely on the conventional scFv structure. The coiled-coil system is based on the specific interaction of two peptidic alpha helices, the sequence which is characterized by a heptad repeat and a continuous hydrophobic core that stabilizes the helix and generates a left handed supercoil. By linking one peptide-coil to the end of the extracellular part of the CAR and the other one to a Fab antibody fragment we have created a unique and versatile platform for switchable CAR design. In silico analysis of each coil-peptide linked to the Fab antibody fragment or to the N-terminus of the CAR showed a low probability of causing an immunogenic response in humans. This novel coiled-coil switchable CAR-T cell afforded highly specific killing of CD19 positive leukemia cells in vitro in a dose titratable manner with no non-specific killing of CD19 negative cell lines. Optimization of the overall distance and orientation of the immunological synapse and the switch valency resulted in pico Molar EC50 range with robust multi-cytokine release in in vitro cytotoxicityassays. Moreover, in an aggressive xenograft model of human B-cell leukemia the switchable CAR-T efficiently controlled tumor growth in the presence of switch.The coiled-coil switchable CAR (ccsCAR) platform shows great potential for tuning switch-CAR affinity and specificity. The design of coiled-coil pairs with variable iterations of helices and/or peptide sequences determine their specificity and affinity for each other. This may increase the effector functions of the ccsCAR while generating new specific coiled-coil pairs. A shorter coiled-coil would decrease the genetic content of the CAR construct and increase CAR expression on the T cell surface while decreasing the potential for immunogenicity.In summary, we show here that the coiled-coil platform can be used to design fully controllable CAR-T cells without the use of a scFv. This is expected to allow the targeting of multiple antigens (ie. CD19, CD20, and CD22 can be simultaneously or sequentially targeted) with the same CAR-T cell construct to potentially decrease tumor immune escape and prevent relapse prevalence while increasing the safety of CAR-T cell based therapies. Finally, this platform could be extended to other malignancies and applied under a personalized medicine criteria by characterizing the tumor phenotype prior to treatment initiation. DisclosuresNo relevant conflicts of interest to declare.

Next Generation Sequencing of Immunized Mouse Splenocytes to Develop an Anti-TIM3 Chimeric Antigen Receptor for Acute Myeloid Leukemia

The field of anti-cancer T cell therapy has had major advances in recent years with the development of the chimeric antigen receptor (CAR). Our group has developed and demonstrated that CARs against CD19 have complete remission rates of greater than 90% against B cell acute lymphoblastic leukemia (B-ALL). However, the progress against B-ALL has not been matched in other types of cancers. Acute Myeloid Leukemia (AML) is the most common acute leukemia in adults, presenting greater than 20,000 new cases per annum and representing 80% of acute leukemia. Additionally, prognosis is poor with as much as 70% of patients, 65 years or older, dying within 1 year of diagnosis. A recent study examining AML has shown that a TIM3 monoclonal antibody (mAb) blocks AML engraftment and eliminates leukemic stem cells (LSC). Additionally, TIM3 mAbs have anti-tumor effects such as slowing tumor progression. Gene-expression analysis demonstrates an 8-fold increase of TIM3 in LSC compared to hematopoietic stem cells. Our own evaluation of TIM3 expression on AML blasts and LSC demonstrated that TIM3 is commonly co-expressed on CD33+ blasts. These results led us to hypothesize anti-TIM3 CAR T cells can target and kill AML, which we will evaluate by the development of novel anti-TIM3 CARs. Generation of new CARs rely on a time consuming and/or costly process of phage display screening or hybridoma production, hybridoma screening, and immunoglobulin (Ig) sequencing. Here, we describe a novel CAR design strategy by next generation sequencing (NGS) followed by CAR synthesis and functionalvalidation against TIM3+ targets. NGS allows for direct identification of rearranged immunoglobulin genes, mutation and repertoire analysis, and comparison of multiple immunized mice. Furthermore, by eliminating the need for hybridoma production and screening we have developed a rapid, economical system for the development of novel CARs.After immunization of four separate mice with CHO-TIM3, NGS demonstrated that nearly 70% of Ig heavy (IgH) and 61% of Ig light (IgL) sequences were accounted for by 2 and 3 V-J rearrangements, respectively. This suggested that these rearrangements were enriched for TIM3-reactivity and we designed pairs of these IgH and IgL rearrangements. By this process we generated 8 anti-TIM3 CARs (4 VHS1-based CARs and 4 VH8-based CARs). After their gene-synthesis and cloning into a genetic construct we produced retrovirus and transduced Jurkat, as well as primary T cells. Robust gene-transfer was observed with most constructs (14-64%). Furthemore, we detected antigen-specific cytokine production by Jurkat stimulated with TIM3+ targets (Fig. 1). We also examined in vitro killing by primary anti-TIM3 human T cells using a Real-Time cell Analysis cytotoxicity assay. We determined that anti-TIM3 CAR VH8-461 had the highest transduction efficiency, IFNγ production, and in vitro killing of target cells. We are now evaluating the in vivo efficacy of anti-TIM3 CAR T cells in immune deficient animal models implanted with TIM3+ targets. This work demonstrates NGS can be used to identify new sequences that can be developed rapidly and inexpensively into CARs. Using this process we created 8 anti-TIM3 CARs and by in vitro screening selected 1 of them (VH8-461) for in vivo validation. We are using this same NGS CAR generation system to develop additional anti-AML CARs which can be used in tandem with anti-TIM3 CARs to treat AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CAR T-Cells for CNS Lymphoma: Driving into New Terrain?

Simple SummaryPrimary or secondary central nervous system (CNS) lymphoma is frequently associated with a poor prognosis. CAR T-cells are being established as a relevant treatment approach in hematological B-cell malignancies. Unfortunately, most clinical studies on chimeric antigen-receptor (CAR) T-cells have excluded patients with CNS involvement but several clinical trials on CAR T-cell therapy in CNS lymphoma patients are currently ongoing. Preclinical and preliminary clinical data suggest an overall acceptable safety profile and considerable anti-tumor effects might be extrapolated for CAR T-cell therapy in CNS lymphoma.Primary CNS lymphomas (PCNSL) represent a group of extranodal non-Hodgkin lymphomas and secondary CNS lymphomas refer to secondary involvement of the neuroaxis by systemic disease. CNS lymphomas are associated with limited prognosis even after aggressive multimodal therapy. Chimeric antigen receptor (CAR) T-cells have proven as a promising therapeutic avenue in hematological B-cell malignancies including diffuse large B-cell lymphoma, B-cell acute lymphoblastic leukemia, and mantle-cell lymphoma. CARs endow an autologous T-cell population with MHC-unrestricted effectivity against tumor target antigens such as the pan B-cell marker CD19. In PCNSL, compelling and long-lasting anti-tumor effects of such therapy have been shown in murine immunocompromised models. In clinical studies on CAR T-cells for CNS lymphoma, only limited data are available and often include both patients with PCNSL but also patients with secondary CNS lymphoma. Several clinical trials on CAR T-cell therapy for primary and secondary CNS lymphoma are currently ongoing. Extrapolated from the available preliminary data, an overall acceptable safety profile with considerable anti-tumor effects might be expected. Whether these beneficial anti-tumor effects are as long-lasting as in animal models is currently in doubt; and the immunosuppressive tumor microenvironment of the brain may be among the most pivotal factors limiting efficacy of CAR T-cell therapy in CNS lymphoma. Based on an increasing understanding of CAR T-cell interactions with the tumor cells as well as the cerebral tissue, modifications of CAR design or the combination of CAR T-cell therapy with other therapeutic approaches may aid to release the full therapeutic efficiency of CAR T-cells. CAR T-cells may therefore emerge as a novel treatment strategy in primary and secondary CNS lymphoma.

BATF3/MYC axis drives hyper-proliferation of TET2 deficient CAR T cells

Abstract A recent report described a patient presenting a dominant chimeric antigen receptor (CAR) T cell clone at the height of anti-tumor response wherein a TET2 allele was disrupted by the integration of the lentiviral 4-1BB CAR vector (Fraietta et. al. Nature 2018). The enhanced proliferative ability conferred by this chance TET2 disruption opened up the possibility of generating TET2 edited CAR T cells for an enhanced anti-tumor efficacy. We modeled and evaluated loss of TET2 in human T cells engineered to express different CAR receptors in a murine model of human acute lymphoblastic leukemia (ALL). Our results indicate that CAR design plays a critical role in determining anti-tumor efficacy and early stage T cell phenotype upon TET2 disruption. We further observe that TET2 edited CAR T cells, over time, enter a hyper-proliferative state with a near total loss of effector function. The frequency with which TET2 edited T cells achieve hyper-proliferation is dependent on the signaling properties of the CAR receptor. This state is in contrast to the canonical exhaustion state wherein loss of proliferative ability precedes loss of effector function. This hyper-proliferative state shares some transcriptional features with T cell leukemia/lymphoma with sustained upregulation of cell cycle associated factors. Exome analysis identified point mutations and chromosomal aberrations in hyper-proliferative cells, but they were not conserved across different populations. Chromatin accessibility analysis revealed that loss of TET2 sets an epigenetic state that allows for sustained elevated levels of BATF3 which in turn drives a MYC-dependent proliferative program in TET2 deficient CAR T cells.

In Like a Lamb; Out Like a Lion: Marching CAR T Cells Toward Enhanced Efficacy in B-ALL.

Combining synthetic biology with adoptive T-cell transfer has led to promising advances in the treatment of relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL), diffuse large B-cell lymphoma (DLBCL), and mantle cell lymphoma (MCL). Chimeric antigen receptors (CARs) are synthetic receptors that redirect T-cell specificity against cancer. CARs include "built-in" signaling domains that reprogram T-cell metabolism, enhance effector function, and support long-term persistence. Despite their success in blood-based malignancies, relapse can occur in CD19-redirected CAR T-cell therapies for several reasons, including poor engraftment, impaired in vivo proliferation, and T-cell senescence. Herein, we explain how subtle alterations in CAR design may overcome barriers to effective adoptive immunotherapy. We also discuss how the physiochemical properties of the single-chain variable fragment (scFv) affect differentiation and persistence. Moreover, we describe innovative advances in CAR engineering and provide insight into the development of humanized scFvs whose proposed benefits include increased persistence and improved clinical outcomes. Tumor cells can evade CAR T-cell-mediated detection and elimination due to the emergence or presence of CD19-negative leukemic cell subpopulations. We also discuss the opportunities and challenges in targeting other B-ALL-associated antigens. Identifying alternate targets is fundamentally necessary to restore the success of CAR T-cell therapies in CD19-negative patients with B-ALL.

Chimeric Antigen Receptor Design and Efficacy in Ovarian Cancer Treatment.

Our increased understanding of tumour biology gained over the last few years has led to the development of targeted molecular therapies, e.g., vascular endothelial growth factor A (VEGF-A) antagonists, poly[ADP-ribose] polymerase 1 (PARP1) inhibitors in hereditary breast and ovarian cancer syndrome (BRCA1 and BRCA2 mutants), increasing survival and improving the quality of life. However, the majority of ovarian cancer (OC) patients still do not have access to targeted molecular therapies that would be capable of controlling their disease, especially resistant or relapsed. Chimeric antigen receptors (CARs) are recombinant receptor constructs located on T lymphocytes or other immune cells that change its specificity and functions. Therefore, in a search for a successful solid tumour therapy using CARs the specific cell surface antigens identification is crucial. Numerous in vitro and in vivo studies, as well as studies on humans, prove that targeting overexpressed molecules, such as mucin 16 (MUC16), annexin 2 (ANXA2), receptor tyrosine-protein kinase erbB-2 (HER2/neu) causes high tumour cells toxicity and decreased tumour burden. CARs are well tolerated, side effects are minimal and they inhibit disease progression. However, as OC is heterogenic in its nature with high mutation diversity and overexpression of different receptors, there is a need to consider an individual approach to treat this type of cancer. In this publication, we would like to present the history and status of therapies involving the CAR T cells in treatment of OC tumours, suggest potential T cell-intrinsic determinants of response and resistance as well as present extrinsic factors impacting the success of this approach.

CD28 Co-Stimulus Achieves Superior CAR T Cell Effector Function against Solid Tumors Than 4-1BB Co-Stimulus.

Simple SummaryEfficient trafficking and survival of CAR T cells within the hostile tumor microenvironment are important prerequisites for potent solid tumor attack that have not yet been achieved. We deployed monospecific murine instead of polyclonal human T cells for CAR T cell generation to evaluate second generation L1CAM- and HER2-specific CARs with different spacer length and either the CD28 or 4-1BB co-stimulatory domain in mouse models of neuroblastoma and ovarian carcinoma. This mouse-in-mouse approach ensured CAR T cell trafficking unhindered by species-specific discrepancies and demonstrated superior solid tumor attack by CAR T cells harboring the CD28 compared to 4-1BB co-stimulatory domain. Our approach has the potential to improve prediction and selection of promising clinical CAR candidates against solid tumors in the future.Spacer or co-stimulatory components in chimeric antigen receptor (CAR) design influence CAR T cell effector function. Few preclinical mouse models optimally support CAR candidate pre-selection for clinical development. Here we use a model in which murine CAR T cells can be exploited with human tumor xenografts. This mouse-in-mouse approach avoids limitations caused by species-specific factors crucial for CAR T cell survival, trafficking and function. We compared trafficking, expansion and tumor control for T cells expressing different CAR construct designs targeting two antigens (L1CAM or HER2), structurally identical except for spacer (long or short) or co-stimulatory (4-1BB or CD28) domains to be evaluated. Using monoclonal, murine-derived L1CAM-specific CAR T cells in Rag-/- mice harboring established xenografted tumors from a human neuroblastoma cell line revealed a clear superiority in CAR T cell trafficking using CD28 co-stimulation. L1CAM-targeting short spacer-CD28/ζ CAR T cells expanded the most at the tumor site and induced initial tumor regression. Treating patient-derived neuroblastoma xenografts with human L1CAM-targeting CAR T cells confirmed the superiority of CD28 co-stimulus. CD28 superiority was also demonstrated with HER2-specific CAR T cells (targeting ovarian carcinoma xenografts). Our findings encourage incorporating CD28 signaling into CAR design for adoptive T cell treatment of solid tumors.

Chimeric Antigen Receptor Design Today and Tomorrow.

The US Food and Drug Administration has approved 3 chimeric antigen receptor (CAR) T-cell therapies. For continued breakthroughs, novel CAR designs are needed. This includes different antigen-binding domains such as antigen-ligand binding partners and variable lymphocyte receptors. Another recent advancement in CAR design is Boolean logic gates that can minimize on-target, off-tumor toxicities. Recent studies on the optimization of costimulatory signaling have also shown how CAR design can impact function. By using specific signaling pathways and transcription factors, CARs can impact T-cell gene expression to enhance function. By using these techniques, the promise of CAR T-cell therapies for solid tumors can be fulfilled.

Phenotypic Models of CAR T-Cell Activation Elucidate the Pivotal Regulatory Role of CAR Downmodulation.

Adoptive cell immunotherapy with chimeric antigen receptor (CAR) showed limited potency in solid tumors, despite durable remissions for hematopoietic malignancies. Therefore, an investigation of ways to enhance the efficacy of CARs' antitumor response has been engaged upon. We previously examined the interplay between the biophysical parameters of CAR binding (i.e., affinity, avidity, and antigen density), as regulators of CAR T-cell activity and detected nonmonotonic behaviors of affinity and antigen density and an interrelation between avidity and antigen density. Here, we built an evolving phenotypic model of CAR T-cell regulation, which suggested that receptor downmodulation is a key determinant of CAR T-cell function. We verified this assumption by measuring and manipulating receptor downmodulation and intracellular signaling processes. CAR downmodulation inhibition, via actin polymerization inhibition, but not inhibition of regulatory inhibitory phosphatases, was able to increase CAR T-cell responses. In addition, we documented trogocytosis in CAR T cells that depends on actin polymerization. In summary, our study modeled the parameters that govern CAR T-cell engagement and revealed an underappreciated mechanism of T-cell regulation. These results have a potential to predict and therefore advance the rational design of CAR T cells for adoptive cell treatments.See related article on p. 872.

Robust Antitumor Activity and Low Cytokine Production by Novel Humanized Anti-CD19 CAR T Cells.

Recent studies have described the remarkable clinical outcome of anti-CD19 chimeric antigen receptor (CAR) T cells in treating B-cell malignancies. However, over 50% of patients develop life-threatening toxicities associated with cytokine release syndrome which may limit its utilization in low-resource settings. To mitigate the toxicity, we designed a novel humanized anti-CD19 CAR T cells by humanizing the framework region of single-chain variable fragment (scFv) derived from a murine FMC63 mAb and combining it with CD8α transmembrane domain, 4-1BB costimulatory domain, and CD3ζ signaling domain (h1CAR19-8BBζ). Docking studies followed by molecular dynamics simulation revealed that the humanized anti-CD19 scFv (h1CAR19) establishes higher binding affinity and has a flexible molecular structure with CD19 antigen compared with murine scFv (mCAR19). Ex vivo studies with CAR T cells generated from healthy donors and patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) expressing either h1CAR19 or mCAR19 showed comparable antitumor activity and proliferation. More importantly, h1CAR19-8BBζ T cells produced lower levels of cytokines (IFNγ, TNFα) upon antigen encounter and reduced the induction of IL6 cytokine from monocytes than mCAR19-8BBζ T cells. There was a comparable proliferation of h1CAR19-8BBζ T cells and mCAR19-8BBζ T cells upon repeated antigen encounter. Finally, h1CAR19-8BBζ T cells efficiently eliminated NALM6 tumor cells in a preclinical model. In conclusion, the distinct structural modification in CAR design confers the novel humanized anti-CD19 CAR with a favorable balance of efficacy to toxicity providing a rationale to test this construct in a phase I trial.

Design and Assessment of Novel Anti-CD30 Chimeric Antigen Receptors with Human Antigen-Recognition Domains.

Chimeric antigen receptors (CARs) are artificial fusion proteins that incorporate antigen-recognition domains and T cell signaling domains. CD30 is a cell surface protein expressed on Hodgkin's lymphoma, some T cell lymphomas, and some B cell lymphomas. CD30 has a restricted expression pattern in normal cells, so CD30 has good potential as a clinical target for CAR T cells. We compared three different anti-CD30 CAR designs incorporating a single-chain variable fragment derived from the 5F11 fully human monoclonal antibody. 5F11-28Z has hinge, transmembrane, and costimulatory domains from CD28 and a CD3ζ T cell activation domain. 5F11-CD828Z has hinge and transmembrane domains from CD8α, a CD28 costimulatory domain, and a CD3ζ T cell activation domain. 5F11-CD8BBZ is identical to 5F11-CD828Z, except for the replacement of the CD28 moiety with a 4-1BB moiety. We found that T cells expressing 5F11-CD8BBZ had lower levels of CD30-specific degranulation and cytokine release compared with CD28-containing CARs. When compared to the CD28-containing CARs, T cells expressing 5F11-CD8BBZ had higher levels of nonspecific functional activity, including degranulation, cytokine release, and proliferation, when stimulated with CD30-negative target cells. We established tumors in nod-scid common gamma-chain deficient (NSG) mice and treated the tumors with T cells expressing different CARs. T cells expressing 5F11-28Z were most effective at eradicating tumors. T cells expressing 5F11-CD828Z had intermediate effectiveness, and T cells expressing 5F11-CD8BBZ were least effective. CD30+ T cells are lost from cultures of T cells containing 5F11-28Z-expressing T cells. This indicated the killing of CD30+ T cells by the 5F11-28Z-expressing T cells. Despite this, the number of T cells in the cultures consistently accumulated to numbers needed for use in a clinical trial. Based on all in vitro and murine experiments comparing the different CARs, we selected 5F11-28Z for further development, and we have initiated a clinical trial testing 5F11-28Z T cells.

Challenges and Clinical Strategies of CAR T-Cell Therapy for Acute Lymphoblastic Leukemia: Overview and Developments.

Chimeric antigen receptor (CAR) T-cell therapy exhibits desirable and robust efficacy in patients with acute lymphoblastic leukemia (ALL). Stimulated by the revolutionized progress in the use of FDA-approved CD19 CAR T cells, novel agents with CAR designs and targets are being produced in pursuit of superior performance. However, on the path from bench to bedside, new challenges emerge. Accessibility is considered the initial barrier to the transformation of this patient-specific product into a commercially available product. To ensure infusion safety, profound comprehension of adverse events and proactive intervention are required. Additionally, resistance and relapse are the most critical and intractable issues in CAR T-cell therapy for ALL, thus precluding its further development. Understanding the limitations through up-to-date insights and characterizing multiple strategies will be critical to leverage CAR T-cell therapy flexibly for use in clinical situations. Herein, we provide an overview of the application of CAR T-cell therapy in ALL, emphasizing the main challenges and potential clinical strategies in an effort to promote a standardized set of treatment paradigms for ALL.

Characterization and Functional Analysis of CD44v6.CAR T Cells Endowed with a New Low-Affinity Nerve Growth Factor Receptor-Based Spacer.

Effectiveness of adoptively transferred chimeric antigen receptor (CAR) T cells strongly depends on the quality of CAR-mediated interaction of the effector cells with the target antigen on tumor cells. A major role in this interaction is played by the affinity of the single-chain variable fragment (scFv) for the antigen, and by the CAR design. In particular, the spacer domain may impact on the CAR T cell function by affecting the length and flexibility of the resulting CAR. This study addresses the need to improve the manufacturing process and the antitumor activity of CD44v6-specific CAR T cells by defining the optimal structure of a spacer region derived from the extracellular domain of the human low-affinity nerve growth factor receptor (LNGFR). We tailored the LNGFR spacer to modulate CAR length to efficiently recognize distal or proximal epitopes and to allow selection of transduced CAR T cells by the use of clinical-grade validated manufacturing systems. The different LNGFR spacers investigated in this study are responsible for the generation of CAR T cells with a different memory phenotype, which is mainly related to the level of CAR expression and the extent of the associated tonic signaling. In particular, the CD44v6-NWN2.CAR T cells are enriched in central memory cells and show improved in vitro functions in terms of killing capability, and in vivo antitumor activity against hematological and solid tumors. Clinical Trial Registration numbers: clinicaltrial.gov NCT04097301; ClinicalTrials.gov, NCT00423124.

Engineering advanced logic and distributed computing in human CAR immune cells

The immune system is a sophisticated network of different cell types performing complex biocomputation at single-cell and consortium levels. The ability to reprogram such an interconnected multicellular system holds enormous promise in treating various diseases, as exemplified by the use of chimeric antigen receptor (CAR) T cells as cancer therapy. However, most CAR designs lack computation features and cannot reprogram multiple immune cell types in a coordinated manner. Here, leveraging our split, universal, and programmable (SUPRA) CAR system, we develop an inhibitory feature, achieving a three-input logic, and demonstrate that this programmable system is functional in diverse adaptive and innate immune cells. We also create an inducible multi-cellular NIMPLY circuit, kill switch, and a synthetic intercellular communication channel. Our work highlights that a simple split CAR design can generate diverse and complex phenotypes and provide a foundation for engineering an immune cell consortium with user-defined functionalities.

New CAR Designs: Highlights From SOHO 2020.

Kelly Valla, PharmD, BCOP, of Winship Cancer Institute of Emory University, considers exciting research on novel chimeric antigen receptor designs presented by Renier J. Brentjens, MD, PhD, of Memorial Sloan Kettering Cancer Center, at the 2020 SOHO Annual Meeting.

Chimeric Antigen Receptor beyond CAR-T Cells.

Simple SummaryChimeric antigen receptors (CAR) are engineered molecules expressed on the cell surface that can recognise specific proteins and deliver an activation signal to the cells. Human T lymphocytes equipped with CAR, also called CAR-T cells, can target and kill tumour cells. This technology has been successfully used in treating some of the blood cancers in the last decade. Although the majority of research interest in CAR technology has been focused on CAR-T cells to date, the CAR design has also been used in other types of immune cells to fight against cancers. In this review, we discuss recent advances in CAR design beyond that used in conventional CAR-T cells and their novel indications to develop more potent CAR-based therapy for cancers.Chimeric antigen receptors (CAR) are genetically engineered receptors that can recognise specific antigens and subsequently activate downstream signalling. Human T cells engineered to express a CAR, also known as CAR-T cells, can target a specific tumour antigen on the cell surface to mediate a cytotoxic response against the tumour. CAR-T cell therapy has achieved remarkable success in treating hematologic malignancies, but not in solid tumours. Currently, extensive research is being carried out to make CAR-T cells a therapy for solid tumours. To date, most of the research interest in the field has focused on cytotoxic T lymphocytes as the carrier of CAR products. However, in addition to T cells, the CAR design can be introduced in other immune cells, such as natural killer (NK)/NKT cells, γδ T cells, mucosal-associated invariant T (MAIT) cells, dendritic cells (DC), macrophages, regulatory T cells (Treg), B cells, etc. Some of the CAR-engineered immune cells, such as CAR- γδ T and CAR-NK/NK-T cells, are directly involved in the anti-tumour response, demonstrated in preclinical studies and/or clinical trials. CAR-Tregs showed promising therapeutic potential in treating autoimmune diseases. In particular, B cells engineered with chimeric receptors can be used as a platform for long-term delivery of therapeutic proteins, such as recombinant antibodies or protein replacement, in an antigen-specific manner. CAR technology is one of the most powerful engineering platforms in immunotherapy, especially for the treatment of cancers. In this review, we will discuss the recent application of the CAR design in non-CAR-T cells and future opportunities in immunotherapy.

The Influence of Chimeric Antigen Receptor Structural Domains on Clinical Outcomes and Associated Toxicities.

Simple SummaryThe development and refinement chimeric antigen receptor (CAR)-T cell immunotherapy has significantly improved the prognosis of patients with B cell malignancies. Severe treatment related toxicities however remain a significant challenge for the field. This perspective reviews 17 clinical trials of the most widely used anti-CD19 (FMC63) CAR-T cell therapies, with the aim of dissecting the contribution of the structural and costimulatory domains of the CAR to clinical outcomes and toxicities. The CD28 structural hinge and transmembrane CAR domains are highlighted as strongly associated with both clinical efficacy and severe toxicity. This perspective supports further investigation into the structural CAR domains for improved CAR design and safer CAR-T cell therapies.Chimeric antigen receptor (CAR)-T cell therapy has transformed the treatment of B cell malignancies, improving patient survival and long-term remission. Nonetheless, over 50% of patients experience severe treatment-related toxicities including cytokine release syndrome (CRS) and neurotoxicity. Differences in severity of toxic side-effects among anti-CD19 CARs suggest that the choice of costimulatory domain makes a significant contribution to toxicity, but comparisons are complicated by additional differences in the hinge and transmembrane (TM) domains of the most commonly used CARs in the clinic, segments that have long been considered to perform purely structural roles. In this perspective, we examine clinical and preclinical data for anti-CD19 CARs with identical antigen-binding (FMC63) and signalling (CD3ζ) domains to unravel the contributions of different hinge-TM and costimulatory domains. Analysis of clinical trials highlights an association of the CD28 hinge-TM with higher incidence of CRS and neurotoxicity than the corresponding sequences from CD8, regardless of whether the CD28 or the 4-1BB costimulatory domain is used. The few preclinical studies that have systematically varied these domains similarly support a strong and independent role for the CD28 hinge-TM sequence in high cytokine production. These observations highlight the value that a comprehensive and systematic interrogation of each of these structural domains could provide toward developing fundamental principles for rational design of safer CAR-T cell therapies.