Chimeric Antigen Receptor Construct Research Articles

Enhancing MHC-Independent T Cell Receptors Using Rational Protein Engineering

The paradigm of synthetic receptors, chimeric antigen receptors (CARs), combine MHC-independence with T cell activation. While T cells engineered to express CARs are successful for some patients, the majority are not cured and clinical failures often result from CAR-driven T cell dysfunction (Lynn R, Nature 2019; Selli ME, Blood 2023). Simultaneously, many patients develop cytokine release syndrome, a hyperinflammatory state resulting from uncontrolled CAR-driven immune activation, indicating that CARs can drive both failure and toxicity. We hypothesized that these limitations result from CAR design which, in its simplicity, bypasses natural TCR regulation. To re-engage endogenous T cell regulatory circuits, we replaced the variable region of the TCR α and β chains with the CD19 binding domain used in several clinical CAR constructs. As shown recently by others (Liu Y, Sci Transl Med 2021; Mansilla-Soto Nature Medicine 2022), we found that this format of synthetic receptor expressed on the cell surface, clustered with endogenous CD3 molecules, and drove T cell cytotoxicity against CD19+ leukemia (Nalm6). Intriguingly, surface expression of these MHC-independent TCRs (miTCR) was markedly reduced in comparison to transgenic full-length TCRs and CARs. Examination of the chimeric interface created by joining the antibody and TCR parent chains revealed significant biochemical conflicts that, based on predictive modeling, disrupted naturally-occurring hydrostatic interactions and associated hydrogen bonds, collectively resulting in steric “strain” on the miTCR. We generated a series of miTCR mutants which resolved these conflicts in a step-wise fashion to more closely approximate the “relaxed” state of endogenous TCRs. We found that most mutations improved miTCR surface expression, and more importantly that some mutations significantly boosted T cell functionality as compared to the wild-type miTCR. Impressively, one mutant that closely approximated endogenous TCR structure (mut035) enabled functionality similar to CD19 CARs in short-term cytotoxicity assays. To further evaluate the functional implications of targeted interface mutation, we subjected miTCRs to chronic in vitro stimulation assays in which engineered T cells are exposed to high quantities of Nalm6 for prolonged periods until T cell lose function. We observed that mut035 outperformed all other miTCRs, but all miTCR T cells lost the ability to kill targets earlier than CAR T cells. To determine if a lack of costimulation was the cause of this reduced efficacy we engineered Nalm6 cells to express ligands for CD28 (CD80) and 41BB (41BBL). In the presence of these ligands, long-term functionality all miTCR T cells was profoundly improved. Notably, mut035 T cells persisted longer and killed Nalm6 better than CD19 CAR T cells under conditions of persistent and high-intensity stimulation. Finally, to determine if CD28 or 41BB engagement was most supportive of miTCR function we designed a “priming” experiment. miTCR T cells were initially cultured with Nalm6 expressing either no, one or both costimulatory ligands and then, after tumor clearance, were re-challenged with WT Nalm6, mirroring endogenous immune responses to infection. mut035 T cells initially exposed to CD80 were unable to clear WT cells on re-challenge, however priming with 41BBL enabled potent and prolonged co-stimulation-independent killing of large Nalm6 burdens, suggesting engagement of endogenous T cell functionality. These data demonstrate that classical approaches to combine protein domains can lead to biochemical conflicts that restrain chimeric protein function. Targeted mutation significantly enhanced miTCR function to exceed CAR under conditions of high-burden, high-antigen tumors.

Rapid In Vitro Cytotoxicity Evaluation of Jurkat Expressing Chimeric Antigen Receptor using Fluorescent Imaging.

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable fragment, scFv), with an accompanying intra-chain linker, followed by a hinge, transmembrane, and costimulatory domain. Modification of the intra-chain linker and hinge domain can have a significant effect on CAR-mediated killing. Considering the many different options for each part of a CAR construct, there are large numbers of permutations. Making CAR-T cells is a time-consuming and expensive process, and making and testing many constructs is a heavy time and material investment. This protocol describes a platform to rapidly evaluate hinge-optimized CAR constructs in Jurkat cells (CAR-J). Jurkat cells are an immortalized T cell line with high lentivirus uptake, allowing for efficient CAR transduction. Here, we present a platform to rapidly evaluate CAR-J using a fluorescent imager, followed by confirmation of cytolysis in PBMC-derived T cells.

Unlocking the potential of Tregs: innovations in CAR technology.

Regulatory T cells (Tregs) adoptive immunotherapy is emerging as a viable treatment option for both autoimmune and alloimmune diseases. However, numerous challenges remain, including limitations related to cell number, availability of target-specific cells, stability, purity, homing ability, and safety concerns. To address these challenges, cell engineering strategies have emerged as promising solutions. Indeed, it has become feasible to increase Treg numbers or enhance their stability through Foxp3 overexpression, post-translational modifications, or demethylation of the Treg-specific demethylated region (TSDR). Specificity can be engineered by the addition of chimeric antigen receptors (CARs), with new techniques designed to fine-tune specificity (tandem chimeric antigen receptors, universal chimeric antigen receptors, synNotch chimeric antigen receptors). The introduction of B-cell targeting antibody receptor (BAR) Tregs has paved the way for effective regulation of B cells and plasma cells. In addition, other constructs have emerged to enhance Tregs activation and function, such as optimized chimeric antigen receptors constructs and the use of armour proteins. Chimeric antigen receptor expression can also be better regulated to limit tonic signaling. Furthermore, various opportunities exist for enhancing the homing capabilities of CAR-Tregs to improve therapy outcomes. Many of these genetic modifications have already been explored for conventional CAR-T therapy but need to be further considered for CAR-Tregs therapies. This review highlights innovative CAR-engineering strategies that have the potential to precisely and efficiently manage immune responses in autoimmune diseases and improve transplant outcomes. As these strategies are further explored and optimized, CAR-Treg therapies may emerge as powerful tools for immune intervention.

Engineering NK-CAR.19 cells with the IL-15/IL-15Rα complex improved proliferation and anti-tumor effect in vivo.

Natural killer 92 (NK-92) cells are an attractive therapeutic approach as alternative chimeric antigen receptor (CAR) carriers, different from T cells, once they can be used in the allogeneic setting. The modest in vivo outcomes observed with NK-92 cells continue to present hurdles in successfully translating NK-92 cell therapies into clinical applications. Adoptive transfer of CAR-NK-92 cells holds out the promise of therapeutic benefit at a lower rate of adverse events due to the absence of GvHD and cytokine release syndrome. However, it has not achieved breakthrough clinical results yet, and further improvement of CAR-NK-92 cells is necessary. In this study, we conducted a comparative analysis between CD19-targeted CAR (CAR.19) co-expressing IL-15 (CAR.19-IL15) with IL-15/IL-15Rα (CAR.19-IL15/IL15Rα) to promote NK cell proliferation, activation, and cytotoxic activity against B-cell leukemia. CAR constructs were cloned into lentiviral vector and transduced into NK-92 cell line. Potency of CAR-NK cells were assessed against CD19-expressing cell lines NALM-6 or Raji in vitro and in vivo in a murine model. Tumor burden was measured by bioluminescence. We demonstrated that a fourth- generation CD19-targeted CAR (CAR.19) co-expressing IL-15 linked to its receptor IL-15/IL-15Rα (CAR.19-IL-15/IL-15Rα) significantly enhanced NK-92 cell proliferation, proinflammatory cytokine secretion, and cytotoxic activity against B-cell cancer cell lines in vitro and in a xenograft mouse model. Together with the results of the systematic analysis of the transcriptome of activated NK-92 CAR variants, this supports the notion that IL-15/IL-15Rα comprising fourth-generation CARs may overcome the limitations of NK-92 cell-based targeted tumor therapies in vivo by providing the necessary growth and activation signals.

Anti-Cancer Potential of Transiently Transfected HER2-Specific Human Mixed CAR-T and NK Cell Populations in Experimental Models: Initial Studies on Fucosylated Chondroitin Sulfate Usage for Safer Treatment.

Human epidermal growth factor receptor 2 (HER2) is overexpressed in numerous cancer cell types. Therapeutic antibodies and chimeric antigen receptors (CARs) against HER2 were developed to treat human tumors. The major limitation of anti-HER2 CAR-T lymphocyte therapy is attributable to the low HER2 expression in a wide range of normal tissues. Thus, side effects are caused by CAR lymphocyte "on-target off-tumor" reactions. We aimed to develop safer HER2-targeting CAR-based therapy. CAR constructs against HER2 tumor-associated antigen (TAA) for transient expression were delivered into target T and natural killer (NK) cells by an effective and safe non-viral transfection method via nucleofection, excluding the risk of mutations associated with viral transduction. Different in vitro end-point and real-time assays of the CAR lymphocyte antitumor cytotoxicity and in vivo human HER2-positive tumor xenograft mice model proved potent cytotoxic activity of the generated CAR-T-NK cells. Our data suggest transient expression of anti-HER2 CARs in plasmid vectors by human lymphocytes as a safer treatment for HER2-positive human cancers. We also conducted preliminary investigations to elucidate if fucosylated chondroitin sulfate may be used as a possible agent to decrease excessive cytokine production without negative impact on the CAR lymphocyte antitumor effect.

Refining chimeric antigen receptors via barcoded protein domain combination pooled screening

Chimeric antigen receptor (CAR)-T cells represent a promising frontier in cancer immunotherapy. However, the current process for developing new CAR constructs is time consuming and inefficient. To address this challenge and expedite the evaluation and comparison of full-length CAR designs, we have devised a novel cloning strategy. This strategy involves the sequential assembly of individual CAR domains using blunt ligation, with each domain being assigned a unique DNA barcode. Applying this method, we successfully generated 360 CAR constructs that specifically target clinically validated tumor antigens CD19 and GD2. By quantifying changes in barcode frequencies through next-generation sequencing, we characterize CARs that best mediate proliferation and expansion of transduced Tcells. The screening revealed a crucial role for the hinge domain in CAR functionality, with CD8a and IgG4 hinges having opposite effects in the surface expression, cytokine production, and antitumor activity in CD19- versus GD2-based CARs. Importantly, we discovered two novel CD19-CAR architectures containing the IgG4 hinge domain that mediate superior invivo antitumor activity compared with the construct used in Kymriah, a U.S. Food and Drug Administration (FDA)-approved therapy. This novel screening approach represents a major advance in CAR engineering, enabling accelerated development of cell-based cancer immunotherapies.

Optimizing CAR-T Therapy for Glioblastoma.

Chimeric antigen receptor T-cell therapies have transformed the management of hematologic malignancies but have not yet demonstrated consistent efficacy in solid tumors. Glioblastoma is the most common primary malignant brain tumor in adults and remains a major unmet medical need. Attempts at harnessing the potential of chimeric antigen receptor T-cell therapy for glioblastoma have resulted in glimpses of promise but have been met with substantial challenges. In this focused review, we discuss current and future strategies being developed to optimize chimeric antigen receptor T cells for efficacy in patients with glioblastoma, including the identification and characterization of new target antigens, reversal of T-cell dysfunction with novel chimeric antigen receptor constructs, regulatable platforms, and gene knockout strategies, and the use of combination therapies to overcome the immune-hostile microenvironment.

Engineered human pluripotent stem cell-derived natural killer cells with PD-L1 responsive immunological memory for enhanced immunotherapeutic efficacy

Adoptive chimeric antigen receptor (CAR)-engineered natural killer (NK) cells have shown promise in treating various cancers. However, limited immunological memory and access to sufficient numbers of allogenic donor cells have hindered their broader preclinical and clinical applications. Here, we first assess eight different CAR constructs that use an anti-PD-L1 nanobody and/or universal anti-fluorescein (FITC) single-chain variable fragment (scFv) to enhance antigen-specific proliferation and anti-tumor cytotoxicity of NK-92cells against heterogenous solid tumors. We next genetically engineer human pluripotent stem cells (hPSCs) with optimized CARs and differentiate them into functional dual CAR-NK cells. The tumor microenvironment responsive anti-PD-L1 CAR effectively promoted hPSC-NK cell proliferation and cytotoxicity through antigen-dependent activation of phosphorylated STAT3 (pSTAT3) and pSTAT5 signaling pathways via an intracellular truncated IL-2 receptor β-chain (ΔIL-2Rβ) and STAT3-binding tyrosine-X-X-glutamine (YXXQ) motif. Anti-tumor activities of PD-L1-induced memory-like hPSC-NK cells were further boosted by administering a FITC-folate bi-specific adapter that bridges between a programmable anti-FITC CAR and folate receptor alpha-expressing breast tumor cells. Collectively, our hPSC CAR-NK engineering platform is modular and could constitute a realistic strategy to manufacture off-the-shelf CAR-NK cells with immunological memory-like phenotype for targeted immunotherapy.

Analysis of the impact of handling and culture on the expansion and functionality of NK cells.

Natural killer (NK) cells are lymphocytes of the innate immune system that play a key role in the elimination of tumor and virus-infected cells. Unlike T cells, NK cell activation is governed by their direct interaction with target cells via the inhibitory and activating receptors present on their cytoplasmic membrane. The simplicity of this activation mechanism has allowed the development of immunotherapies based on the transduction of NK cells with CAR (chimeric antigen receptor) constructs for the treatment of cancer. Despite the advantages of CAR-NK therapy over CAR-T, including their inability to cause graft-versus-host disease in allogenic therapies, a deeper understanding of the impact of their handling is needed in order to increase their functionality and applicability. With that in mind, the present work critically examines the steps required for NK cell isolation, expansion and storage, and analyze the response of the NK cells to these manipulations. The results show that magnetic-assisted cell sorting, traditionally used for NK isolation, increases the CD16+ population of NK cultures only if the protocol includes both, antibody incubation and passage through the isolation column. Furthermore, based on the importance of surface potential on cellular responses, the influence of surfaces with different net surface charge on NK cells has been evaluated, showing that NK cells displayed higher proliferation rates on charged surfaces than on non-charged ones. The present work highlights the relevance of NK cells manipulation for improving the applicability and effectiveness of NK cell-based therapies.

Chimeric antigen receptor T-cell therapy in hematologic malignancies: Successes, challenges, and opportunities.

The success of chimeric antigen receptor T-cell (CAR-T) therapy in hematologic malignancies has realized a longstanding effort toward harnessing the immune system to fight cancer in a truly personalized fashion. Second generation chimeric antigen receptors (CAR) incorporating co-stimulatory molecules like 4-1BB or CD28 were able to overcome some of the hindrances with initial CAR constructs resulting in efficacious products. Many second-generation CAR-T products have been approved in the treatment of relapsed/refractory hematologic malignancies including multiple myeloma (MM), non-Hodgkin lymphoma (NHL), and acute lymphoblastic leukemia. However, challenges remain in optimizing the manufacturing, timely access, limiting the toxicity from CAR-T infusions and improving sustainability of responses derived with CAR-T therapy. Here, we summarize the clinical trial data leading to approval CAR-T therapies in MM and NHL, discuss the limitations with current CAR-T therapy strategies and review emerging strategies for overcoming these limitations.

An In Vitro Model to Assess CRS Potential of CAR T Cells Using a Tumor Cell Line and Autologous Monocytes.

Chimeric antigen receptor (CAR) T cell therapy is an engineered cell therapy where T cells are isolated and genetically modified to contain a synthetic CAR with specificity to a tumor cell antigen. Upon antigen binding, the CAR T cell will initiate signaling cascades that result in lysis of the associated tumor cell. Cytokine release syndrome (CRS) is the primary toxicity associated with CAR T cell therapy and remains a prominent safety issue with currently available commercial products. CRS is driven by interaction of the CAR T cells with endogenous monocytes and macrophages, which can lead to immune cell overactivation and an increase in certain cytokines to supraphysiological levels. Identifying the potential of any given CAR construct to drive toxicities in vivo should be assessed in preclinical models prior to human trials. While there are in vivo mouse models available for this purpose, these are often complex xenograft models available in few centers. Thus, there is a need to develop an in vitro assay for measuring the CRS potential of CAR T cells. The assay described here is a preclinical tool for assessing the propensity of any given CAR construct to produce potentially CRS-driving cytokines following tumor cell and monocyte interactions. This article provides a detailed protocol for target cell preparation and isolation of monocytes from peripheral blood mononuclear cells (PBMCs) autologous to the CAR T cells, as well as protocols for seeding the three cell types in a co-culture assay and collecting/analyzing the cytokines produced via an ELISA or multiplex bead array. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of K562 target cells Basic Protocol 2: Isolation of monocytes from autologous PBMCs Basic Protocol 3: Seeding of CAR T cells, monocytes, and K562 cells in 96-well plates Basic Protocol 4: Analysis of co-culture supernatants by single-cytokine ELISA Alternate Protocol: Analysis of co-culture supernatants by multiplex cytokine bead array.

Loss of metabolic fitness drives tumor resistance after CAR-NK cell therapy and can be overcome by cytokine engineering.

Chimeric antigen receptor (CAR) engineering of natural killer (NK) cells is promising, with early-phase clinical studies showing encouraging responses. However, the transcriptional signatures that control the fate of CAR-NK cells after infusion and factors that influence tumor control remain poorly understood. We performed single-cell RNA sequencing and mass cytometry to study the heterogeneity of CAR-NK cells and their in vivo evolution after adoptive transfer, from the phase of tumor control to relapse. Using a preclinical model of noncurative lymphoma and samples from a responder and a nonresponder patient treated with CAR19/IL-15 NK cells, we observed the emergence of NK cell clusters with distinct patterns of activation, function, and metabolic signature associated with different phases of in vivo evolution and tumor control. Interaction with the highly metabolically active tumor resulted in loss of metabolic fitness in NK cells that could be partly overcome by incorporation of IL-15 in the CAR construct.

T Cells Expressing CAR Equipped With Extracellular Domain of BTLA Are Effective Against HVEM-over-expressing Melanoma Cell Lines.

Several chimeric antigen receptor (CAR) T cells have been used to treat melanoma but have not shown favorable results. This study investigated whether Herpes virus entry mediator (HVEM), which is overexpressed in melanoma, is a potential novel antigen for CAR T cell therapy. A CAR construct, composed of the BTLA extracellular domain for HVEM recognition (BTLA-28z), was developed and tested. Jurkat cells transduced with BTLA-28z exhibited enhanced IL-2 secretion when incubated with HVEM-over-expressing melanoma cells. KHYG-1 cells transduced with BTLA-28z also lysed melanoma cell lines. Using primary T cells, we generated CAR T cells targeting HVEM. BTLA-28z CAR T cells exhibited excellent lytic activities against melanoma cell lines. HVEM-targeting CAR T cells may be useful for the treatment of melanoma.

Development of a bicistronic anti-CD19/CD20 CAR construct including abrogation of unexpected nucleic acid sequence deletions

To address CD19 loss from lymphoma after anti-CD19 chimeric antigen receptor (CAR) Tcell therapy, we designed a bicistronic construct encoding an anti-CD19 CAR and an anti-CD20 CAR. We detected deletions from the expected bicistronic construct sequence in a minority of transcripts by mRNA sequencing. Loss of bicistronic construct transgene DNA was also detected. Deletions of sequence were present at much higher frequencies in transduced Tcell mRNA versus gamma-retroviral vector RNA. We concluded that these deletions were caused by intramolecular template switching of the reverse transcriptase enzyme during reverse transcription of gamma-retroviral vector RNA into transgene DNA of transduced Tcells. Intramolecular template switching was driven by repeated regions of highly similar nucleic acid sequence within CAR sequences. We optimized the sequence of the bicistronic CAR construct to reduce repeated regions of highly similar sequences.This optimization nearly eliminated sequence deletions.This work shows that repeated regions of highly similar nucleic acid sequence must be avoided in complex CAR constructs. We further optimized the bicistronic construct by lengthening the linker of the anti-CD20 single-chain variable fragment. This modification increased CD20-specific interleukin-2 release and reduced CD20-specific activation-induced cell death. We selected an optimized anti-CD19/CD20 bicistronic construct for clinical development.

Mechanisms and strategies for safe chimeric antigen receptor T-cell activity control.

The clinical application of chimeric antigen receptor (CAR) T-cell therapy has rapidly changed the treatment options for terminally ill patients with defined blood-borne cancer types. However, CAR T-cell therapy can lead to severe therapy-associated toxicities including CAR-related hematotoxicity, ON-target OFF-tumor toxicity, cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). Just as CAR T-cell therapy has evolved regarding receptor design, gene transfer systems and production protocols, the management of side effects has also improved. However, because of measures taken to abrogate adverse events, CAR T-cell viability and persistence might be impaired before complete remission can be achieved. This has fueled efforts for the development of extrinsic and intrinsic strategies for better control of CAR T-cell activity. These approaches can mediate a reversible resting state or irreversible T-cell elimination, depending on the route chosen. Control can be passive or active. By combination of CAR T-cells with T-cell inhibiting compounds, pharmacologic control, mostly independent of the CAR construct design used, can be achieved. Other strategies involve the genetic modification of T-cells or further development of the CAR construct by integration of molecular ON/OFF switches such as suicide genes. Alternatively, CAR T-cell activity can be regulated intracellularly through a self-regulation function or extracellularly through titration of a CAR adaptor or of a priming small molecule. In this work, we review the current strategies and mechanisms to control activity of CAR T-cells reversibly or irreversibly for preventing and for managing therapy-associated toxicities.

Validation of a High-Sensitivity Assay for Detection of Chimeric Antigen Receptor T-Cell Vectors Using Low-Partition Digital PCR Technology

Although invivo engraftment, expansion, and persistence of chimeric antigen receptor (CAR) T cells are pivotal components of treatment efficacy, quantitative monitoring has not been implemented in routine clinical practice. We describe the development and analytical validation of a digital PCR assay for ultrasensitive detection of CAR constructs after treatment, circumventing known technical limitations of low-partitioning platforms. Primers and probes, designed for detection of axicabtagene, brexucabtagene, and Memorial Sloan Kettering CAR constructs, were employed to validate testing on the Bio-Rad digital PCR low-partitioning platform; results were compared with Raindrop, a high-partitioning system, as reference method. Bio-Rad protocols were modified to enable testing of DNA inputs as high as 500 ng. Using dual-input reactions (20 and 500 ng) and a combined analysis approach, the assay demonstrated consistent target detection around 1 × 10-5 (0.001%) with excellent specificity and reproducibility and 100% accuracy compared with the reference method. Dedicated analysis of 53 clinical samples received during validation/implementation phases showed the assay effectively enabled monitoring across multiple time points of early expansion (day 6 to 28) and long-term persistence (up to 479 days). CAR vectors were detected at levels ranging from 0.005% to 74% (vector versus reference gene copies). The highest levels observed in our cohort correlated strongly with the temporal diagnosis of grade 2 and 3 cytokine release syndrome diagnosis (P<0.005). Only three patients with undetectable constructs had disease progression at the time of sampling.

ALPL-1 is a target for chimeric antigen receptor therapy in osteosarcoma

Osteosarcoma (OS) remains a dismal malignancy in children and young adults, with poor outcome for metastatic and recurrent disease. Immunotherapies in OS are not as promising as in some other cancer types due to intra-tumor heterogeneity and considerable off-target expression of the potentially targetable proteins. Here we show that chimeric antigen receptor (CAR) T cells could successfully target an isoform of alkaline phosphatase, ALPL-1, which is highly and specifically expressed in primary and metastatic OS. The target recognition element of the second-generation CAR construct is based on two antibodies, previously shown to react against OS. T cells transduced with these CAR constructs mediate efficient and effective cytotoxicity against ALPL-positive cells in in vitro settings and in state-of-the-art in vivo orthotopic models of primary and metastatic OS, without unexpected toxicities against hematopoietic stem cells or healthy tissues. In summary, CAR-T cells targeting ALPL-1 show efficiency and specificity in treating OS in preclinical models, paving the path for clinical translation.

VHH-based chimeric antigen receptor T cells against CD70 to eliminate primary and re-challenge renal cell carcinoma tumors in preclinical study.

e14540 Background: CD70 is not expressed in normal tissue, but highly expressed on various hematopoietic and solid tumors, including renal cell carcinoma (RCC). It is an ideal target for cancer therapy and recently was proved by good results in clinical trials of CD70 monoclonal antibody. Methods: We obtained 4 VHH antibody（2A5, 8B4, 11C9 and 9D8）against CD70 by screen nanobody library derived from an antigen-immunized alpaca. Generated a panel of CAR-T cells by transduce retrovirus expressing CAR (chimeric antigen receptor) to human T cells isolated from health donors. These CAR constructs include 4 VHH sequences and a human scFv containing VH and VL derived from cusatuzumab. We evaluated CAR-T cell function in vitro included cell phenotype, proliferation, cytotoxicity, and cytokine release. Furthermore, RCC tumor cell-xenograft NOG mice models were used to test the anti-tumor activity of CAR-T cells in vivo. Results: All anti-CD70 CAR-T cells eliminate CD70-positive tumor cells but not CD70-negative cells in vitro. Although there were no significant differences between the killing efficiency of the CAR-T cells, more abundant IL-2 production in two VHH-based CAR-T cells (11C9 and 2A5) were observed in multi-round co-culture assay. CAR-T cells eradicated RCC tumor cell in our Xenograft model. After infusion, human T cells expanded over 250 folds in 11C9 group and over 30 folds in 2A5 group, but only nearly 6 folds in human scFv CAR-T treated group. In our second in vivo experiment, all mice treated with higher dose CAR-T, the VHH groups maintained tumor free statue much longer time than human scFv group. Cured mice were re-challenged by subcutaneously infuse RCC tumor cells, observed and measured tumor in the next 53 days. For 11C9 CAR-T treated group, 3of 7 mice maintained tumor-free, 1 of 7 mice were tumor-palpable but too small to measurement; For 2A5 treated group only 1 of 7 mice maintained tumor-free. A significantly greater expansion of human T cells in 11C9 group but not 2A5 group after re-challenge. Conclusions: Our results indicate a promising VHH-based CD70 CAR-T cells for RCC therapy, supporting further development for future clinical trial and application.

Targeting Stage-Specific Embryonic Antigen 4 (SSEA-4) in Triple Negative Breast Cancer by CAR T Cells Results in Unexpected on Target/off Tumor Toxicities in Mice.

Due to the paucity of targetable antigens, triple-negative breast cancer (TNBC) remains a challenging subtype of breast cancer to treat. In this study, we developed and evaluated a chimeric antigen receptor (CAR) T cell-based treatment modality for TNBC by targeting stage-specific embryonic antigen 4 (SSEA-4), a glycolipid whose overexpression in TNBC has been correlated with metastasis and chemoresistance. To delineate the optimal CAR configuration, a panel of SSEA-4-specific CARs containing alternative extracellular spacer domains was constructed. The different CAR constructs mediated antigen-specific T cell activation characterized by degranulation of T cells, secretion of inflammatory cytokines, and killing of SSEA-4-expressing target cells, but the extent of this activation differed depending on the length of the spacer region. Adoptive transfer of the CAR-engineered T cells into mice with subcutaneous TNBC xenografts mediated a limited antitumor effect but induced severe toxicity symptoms in the cohort receiving the most bioactive CAR variant. We found that progenitor cells in the lung and bone marrow express SSEA-4 and are likely co-targeted by the CAR T cells. Thus, this study has revealed serious adverse effects that raise safety concerns for SSEA-4-directed CAR therapies because of the risk of eliminating vital cells with stem cell properties.

Targeting CD70 using CAR NK cells to enhance NK cells cytolytic effect against osteosarcoma

Abstract Background: Cure rates for osteosarcoma (OS) have not improved in &amp;gt;30 years and no new effectivetherapies have been identified for metastatic disease. Natural killer (NK) cells have become an attractive tool for cancer immunotherapy. Efficay is improved by direct tumor targeting introducing a specific chimeric antigen receptor (CAR). Emerging knowledge suggests CD70 to be a viable candidate as expression is higher in lung metastases and it has been correlated with tumor escape from immune surveillance. Objective: To assess whether using CD70 CAR-NK cells we can enhance trafficking, homing and proliferation of NK cells and therefore therapeutic efficacy against OS. Methods: We analyzed the cBioportal database to verify CD70 expression in OS. Surface expression of CD70 on human OS cell lines and normal osteoblasts was determined by flow cytometry. CD70 CAR constructs were sequenced, verified and transduced into NK cells. CD70 CAR NK and control NK cells cytolytic activity against OS cells was evaluated by IncuCyte. In vitrocytokine release was measured upon CD70 CAR NK/NK cells exposure to OS cells (IsoPlexis). Results: Database analysis confirmed OS CD70 gene amplification. Flow cytometry showed variable expression of CD70 on OS cells and no expression on human osteoblasts. There was an increase in CD70 CAR NK percent lysis against OS cells at the highest effector:target ratios compared to control NK cells. We found a predominant release in the chemoattractive/stimulatory cytokines upon OS cells exposure to CD70 CAR NK cells. Conclusion: CD70 has immunotherapy potential against OS. CD70 CAR NK cells have increased cytolytic activity against OS cells in vitro. Cytolytic activity may be influenced by the release of specific cytokines.