Predictive value of pre-infusion tumor growth rate for the occurrence and severity of CRS and ICANS in lymphoma under CAR T-cell therapy.

Chimeric antigen receptor T-cell therapy (CART) can be administered outpatient yet requires management of potential side effects such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). The pre-infusion tumor burden is associated with CRS, yet there is no data on the relevance of pre-infusion tumor growth rate (TGR). Our objective was to investigate TGR for the occurrence and severity of CRS and ICANS. Consecutive patients with available pre-baseline and baseline (BL) imaging before CART were included. TGR was determined as both absolute (abs) and percentage change (%) of Lugano criteria-based tumor burden in relation to days between exams. CRS and ICANS were graded according to ASTCT consensus criteria. Clinical metadata was collected including the international prognostic index (IPI), patient age, ECOG performance status, and LDH. Sixty-two patients were included (median age: 62 years, 40% female). The median pre-BL TGR [abs] and pre-BL TGR [%] was 7.5 mm2/d and 30.9%/d. Pre-BL TGR [abs] and pre-BL TGR [%] displayed a very weak positive correlation with the grade of CRS (r[abs] = 0.14 and r[%] = 0.13) and no correlation with ICANS (r[abs] = - 0.06 and r[%] = - 0.07). There was a weak positive correlation between grade of CRS and grade of ICANS (r = 0.35; p = 0.005) whereas there was no significant correlation of CRS or ICANS to any other of the examined parameters. The pre-infusion TGR before CART was weakly associated with the occurrence of CRS, but not the severity, whereas there were no significant differences in the prediction of ICANS. There was no added information when compared to pre-infusion tumor burden alone. Outpatient planning and toxicity management should not be influenced by the pre-infusion TGR.

[CAR T cells as drugs for novel therapies (advanced therapy medicinal products)]. / CAR-T-Zellen als Arzneimittel für neuartige Therapien (Advanced Therapy Medicinal Products).

BACKGROUND: Two commercial chimeric antigen receptor (CAR) T cell products, axicabtagene-ciloleucel (Yescarta®) and tisagenlecleucel (Kymriah®), are registered for the treatment of B cell neoplasia, for which an increased supply of CAR T cell products is required. PROBLEM: The production of patient-specific CAR T cells as advanced therapy medicinal products (ATMPs) poses considerable challenges with respect to logistics, regulation, and manufacturing. METHOD: Review of the CAR T cell manufacturing process and the regulatory network, the current challenges, and future development capabilities of CAR T cells for adoptive immunotherapy. RESULTS: CAR T cells are manufactured under individualized, laborious, good manufacturing practice-conforming processes in decentralized or in specialized centers. Starting from the patient's leukapheresis product, T cells are genetically engineered ex vivo with a CAR, amplified, and after extensive quality control re-applied to the patient. Most CAR T cell products are manufactured in a manual or semi-automated process; fully automated, supervised, and closed systems are increasingly applied to meet the need for a growing number of CAR T cell products. In this setting, research aims at providing allogeneic CAR T cell products or non-T cells such as natural killer cells for broad applications. CONCLUSION: The significance of CAR T cells in adoptive immunotherapy is continuously growing. As individualized cell products, manufacturing requires highly efficient processes under the control of harmonized protocols and regulations so as to ensure the quality of the ATMP in view of increasing demand and to develop new fields in therapy.

[Multiple myeloma-soon curable?] / Multiples Myelom ­ bald heilbar?

Multiple myeloma (MM) is one of the most frequent cancerous diseases of the hemopoietic system. Over the past 60 years the systemic treatment has undergone multiple changes, from alkylating agents to high-dose therapy followed by autologous peripheral blood stem cell transplantation up to immunomodulating substances and proteasome inhibitors. The treatment of MM is currently undergoing a renewed transition. In recent years monoclonal antibodies have decisively extended the treatment options. Long-term remission is achieved more often. Due to progress in immuno-oncological treatment the prognosis of intensively treated patients with a very short life-expectancy can be improved in the future. It is to be expected that MM will be curable in the medium term. The concentration of free light chains in serum, lesions in magnetic resonance imaging (MRI) and bone marrow infiltration are parameters that are incorporated into the treatment indications. In clinical studies patients with smoldering myeloma are already being treated to delay progression, to increase the remission rates or to achieve long-term remission with negative minimal residual disease. Taking the chromosomal alterations and serological parameters into consideration, the prognosis of patients with MM can nowadays be very well discriminated. In currently running studies high-risk patients are being separately and mostly aggressively treated. Imaging is of great importance in MM. Using MRI focal lesions can be detected even before bone destruction. In this year chimeric antigen receptor (CAR) T cell treatment of MM will be approved for the first time in Germany. Novel antibody constructs, such as belantamab mafodotin, are or will be introduced for a late recurrence.

[Chimeric antigen receptors (CARs): universal tools in adoptive cell therapy]. / Chimäre Antigenrezeptoren (CAR) ­ universelle Werkzeuge in der zellulären Immuntherapie.

BACKGROUND: The observation that tumor-infiltrating lymphocytes (TIL) after ex vivo amplification can control tumors in the long term led to the concept of redirecting patients' cytolytic T­cells by a receptor with defined specificity against the tumor. OBJECTIVES: Development of a recombinant receptor-signal molecule (chimeric antigen receptor, CAR) to increase selectivity and enhance anti-tumor immunity. METHODS: Description of a prototype CAR, overview of the modular composition and further development of CAR technology for use in adoptive immune cell therapy. RESULTS: Intensive research over the last two decades has shown how CAR-mediated T­cell activation is influenced by factors such as binding affinity, the epitope of the target antigen, its expression density and accessibility on the tumor cells, as well as by the signaling domains and their combination to induce T­cell activation. The quality and duration of the T­cell response can be specifically modulated by modifying the modular composition of the CAR; CAR T­cells can act as "biopharmaceutical factories" (T-cells redirected for unrestricted cytokine-mediated killing, TRUCK) in the tissue by CAR-mediated release of transgenic therapeutic proteins. CONCLUSION: Adoptive CAR T­cell therapy has shown clinical efficacy in the treatment of hematological malignancies; the treatment of solid tumors, however, is more challenging. Allogeneic CAR T­cell technology is aimed at generating "off-the-shelf" CAR T­cells that are accessible for a large number of patients. A further promising approach is the use of CAR T­cells for other therapeutic applications such as the treatment of autoimmune diseases.

[CAR T-cell therapy for multiple myeloma]. / CAR-T-Zell-Therapie beim multiplen Myelom.

Chimeric antigen receptor (CAR) T-cell treatment is a novel immunotherapy utilizing the patient's own immune system as a "weapon against tumor cells". In patients with multiple myeloma (MM), CAR T-cell therapy has been investigated in clinical trials. The current data on B­cell maturation antigen (BCMA)-targeted CAR T-cells have shown impressive efficacy, and official approval is expected shortly. However, the majority of patients relapse after CAR T-cell therapy. Moreover, the treatment can cause severe adverse events such as cytokine release syndrome and neurotoxicity with lethal outcome. The cost-benefit ratio of this treatment also needs to be optimized. Despite these limitations, CAR T-cell therapy represents an attractive option for patients with MM and has the potential to be incorporated into the standard of care.

Beware of the CAR-T Hitches.

The two CAR-T therapies approved for certain blood cancers have price tags of $475,000 and $373,000-and those prices don't include other costs for services that are integral to the treatments, such as harvesting a patient's T cells. Moreover, patients typically have a lengthy hospital stay after treatment.

[Killing effect of Robo1 targeted Chimeric Antigen Receptor modified NK92 cells against glioma and neuroblastoma cells].

Objective: To study the cytotoxicity of Robo1-CAR-NK92 cells against U87-MG and SH-SY5Y cells, to explore the effects of IL-15, IL-21 and dexamethasone on the proliferation, survival and cytotoxicity of Robo1-CAR-NK92 cells and to optimize the culture protocol. Methods: Robo1-CAR-NK92 cells were constructed by lentivirus transfection.The Robo1 car positive cells were sorted, expanded and detected by flow cytometry.The levels of Robo1 expression in SH-SY5Y and U87-MG cells were examined by flow cytometry.The cytotoxicity of Robo1-CAR-NK92 or NK92 cells against target cells was tested by CCK-8 and live cell imaging. The levels of cytokines in the supernatant of cultured cells during the cytotoxicity assay were quantified by the multiplex bead-array assay.NK92 and Robo1-CAR-NK92 cells (4×10(4)/ml) were treated with 25 ng/ml of IL-15, 25 ng/ml of IL-21 and/or 50 nmol/L dexamethasone for 3 days and were stained with trypan blue to acquire the viable cell numbers and survival rates. Results: Robo1-CAR-NK92 cells were constructed and tested 98.89% positive after sorting and expansion. While 88.14% of U87-MG cells were Robo1 positive, there were 99.75% of Robo1 positive SH-SY5Y cells.The specific lysis of Robo1-CAR-NK92 cells against target cells was significantly higher than that of NK92 cells (P<0.05). Robo1-CAR-NK92 cells obviously secreted more cytokines including IL-6, IL-10, TNF-α and IFN-γ than parental NK92 cells during cytotoxic activity against U87-MG cells (P<0.05). IL-15 significantly increased the proliferation and survival of Robo1-CAR-NK92 cells, but IL-21 played the opposite role.Remarkably, IL-21 and IL-15+ IL-21 enhanced the cytotoxicity of NK92 and Robo1-CAR-NK92 cells.The combination of dexamethasone and interleukins dramatically promoted the proliferation and survival but obviously impaired the cytotoxicity of NK92 and Robo1-CAR-NK92 cells (except that IL+ 15 and dexamethasone have no effect on the cytotoxicity of Robo1-CAR-NK92 cells). Conclusions: Compared to parental NK92 cells, Robo1-CAR-NK92 cells exhibited more potent targeted killing against glioma and neuroblastoma cells.Collectively, treatment of IL-15 and dexamethasone was the optimized protocol for culture of Robo1 CAR NK cells during our experimental time.

[Experiment research of natural killer cells amplification in vitro and the killing effect on ovarian cancer cells].

Objective: To amplify natural killer (NK) cells in vitro and explore its killing effect on ovarian cancer cells. Methods: (1) The separation of NK cells and identification. A total of 20 ml peripheral blood of one healthy volunteer was collected in Nov. 2015, Peking University People's Hospital. The peripheral blood mononuclear cells of normal volunteers were isolated, cultured in vitro and amplificated cultivation for 14 days with K562 cells transfected and expressing interleukin 21 (IL-21-K562) as nourish cells. The number and dynamic state of the growth cells were monitored during the cultured process. Cells were harvested and counted after 14 days cultured. The NK cells phenotypes were detected by flow cytometry. (2) The killing effect of NK cells on ovarian cancer cells: the ratio of effector cells (NK cells) and target cells (ovarian cancer cells and its control) was 50∶1, 20∶1, 10∶1, 5∶1 or 1∶1, NK cells killing effect on ovarian cancer cells was detected by the lactate dehydrogenase (LDH) release experiments. Results: (1) The results of NK cells establishment and phenotypic characterization: the cells were induced in vitro for 14 days by amplification culture. With the extension of incubation time, the number of NK cells increased constantly, from 2.0×10(7) on day 0 to 5.1×10(9) on day 14. Obvious amplification of the total number of cells were detected for 255 times. Living cells unstained by trypan blue eventually reached 95% above. Before and after the induction and amplification in vitro, the percentage of NK cells(CD(3)(-)CD(5)(6+)cells) in CD(3)- cells were 2.33% and 85.32%, respectively (P<0.01), which covered the whole lymphocytes 1.06% and 69.42%, respectively (P<0.01), which showed that NK was the main cell type in the amplificated lymphocytes. (2) The killing rate of NK cells on ovarian cancer cells in vitro: the results detected by LDH release experiments showed that NK cells could performed strong nonspecific killing effect on ovarian cancer cell lines SKOV3, HOC1A, 3AO and CAOV3, as well the normal ovarian cell line T29 and NK sensitive cell line K562, and the killing effect increased significantly along with the increase of effector cells and target cells ratio (P<0.01). When the ratio was 1∶1, the killing rate was 37% for K562, while the rate of killing of other cells was around 10% (P<0.05). When the effect-target ratio was 20∶1 and 50∶1, in addition to CAOV3 cells (more than 70%), NK cells had a kill rate of more than 80% for other ovarian cancer cells lines and their control cell K562 and T29 cells (P>0.05). Conclusion: NK cells could be established in vitro and have a good non-specific killing effect on ovarian cancer cells.

Gamma delta T cells in acute myeloid leukemia: biology and emerging therapeutic strategies.

γδ T cells play an important role in disease control in acute myeloid leukemia (AML) and have become an emerging area of therapeutic interest. These cells represent a minor population of T lymphocytes with intrinsic abilities to recognize antigens in a major histocompatibility complex-independent manner and functionally straddle the innate and adaptive immunity interface. AML shows high expression of phosphoantigens and UL-16 binding proteins that activate the Vδ2 and Vδ1 subtypes of γδ T cells, respectively, leading to γδ T cell-mediated cytotoxicity. Insights from murine models and clinical data in humans show improved overall survival, leukemia-free survival, reduced risk of relapse, enhanced graft-versus-leukemia effect, and decreased graft-versus-host disease in patients with AML who have higher reconstitution of γδ T cells following allogeneic hematopoietic stem cell transplantation. Clinical trials leveraging γδ T cell biology have used unmodified and modified allogeneic cells as well as bispecific engagers and monoclonal antibodies. In this review, we discuss γδ T cells' biology, roles in cancer and AML, and mechanisms of immune escape and antileukemia effect; we also discuss recent clinical advances related to γδ T cells in the field of AML therapeutics.

Therapeutic Inducers of Natural Killer cell Killing (ThINKK): preclinical assessment of safety and efficacy in allogeneic hematopoietic stem cell transplant settings.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) remains the standard of care for chemotherapy-refractory leukemia patients, but cure rates are still dismal. To prevent leukemia relapse following HSCT, we aim to improve the early graft-versus-leukemia effect mediated by natural killer (NK) cells. Our approach is based on the adoptive transfer of Therapeutic Inducers of Natural Killer cell Killing (ThINKK). ThINKK are expanded and differentiated from HSC, and exhibit blood plasmacytoid dendritic cell (pDC) features. We previously demonstrated that ThINKK stimulate NK cells and control acute lymphoblastic leukemia (ALL) development in a preclinical mouse model of HSCT for ALL. Here, we assessed the cellular identity of ThINKK and investigated their potential to activate allogeneic T cells. We finally evaluated the effect of immunosuppressive drugs on ThINKK-NK cell interaction. METHODS: ThINKK cellular identity was explored using single-cell RNA sequencing and flow cytometry. Their T-cell activating potential was investigated by coculture of allogeneic T cells and antigen-presenting cells in the presence or the absence of ThINKK. A preclinical human-to-mouse xenograft model was used to evaluate the impact of ThINKK injections on graft-versus-host disease (GvHD). Finally, the effect of immunosuppressive drugs on ThINKK-induced NK cell cytotoxicity against ALL cells was tested. RESULTS: The large majority of ThINKK shared the key characteristics of canonical blood pDC, including potent type-I interferon (IFN) production following Toll-like receptor stimulation. A minor subset expressed some, although not all, markers of other dendritic cell populations. Importantly, while ThINKK were not killed by allogeneic T or NK cells, they did not increase T cell proliferation induced by antigen-presenting cells nor worsened GvHD in vivo. Finally, tacrolimus, sirolimus or mycophenolate did not decrease ThINKK-induced NK cell activation and cytotoxicity. CONCLUSION: Our results indicate that ThINKK are type I IFN producing cells with low T cell activation capacity. Therefore, ThINKK adoptive immunotherapy is not expected to increase the risk of GvHD after allogeneic HSCT. Furthermore, our data predict that the use of tacrolimus, sirolimus or mycophenolate as anti-GvHD prophylaxis regimen will not decrease ThINKK therapeutic efficacy. Collectively, these preclinical data support the testing of ThINKK immunotherapy in a phase I clinical trial.

Cell membrane-anchored and tumor-targeted IL-12 T-cell therapy destroys cancer-associated fibroblasts and disrupts extracellular matrix in heterogenous osteosarcoma xenograft models.

BACKGROUND: The extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs) play major roles in tumor progression, metastasis, and the poor response of many solid tumors to immunotherapy. CAF-targeted chimeric antigen receptor-T cell therapy cannot infiltrate ECM-rich tumors such as osteosarcoma. METHOD: In this study, we used RNA sequencing to assess whether the recently invented membrane-anchored and tumor-targeted IL-12-armed (attIL12) T cells, which bind cell-surface vimentin (CSV) on tumor cells, could destroy CAFs to disrupt the ECM. We established an in vitro model of the interaction between osteosarcoma CAFs and attIL12-T cells to uncover the underlying mechanism by which attIL12-T cells penetrate stroma-enriched osteosarcoma tumors. RESULTS: RNA sequencing demonstrated that attIL12-T cell treatment altered ECM-related gene expression. Immunohistochemistry staining revealed disruption or elimination of high-density CAFs and ECM in osteosarcoma xenograft tumors following attIL12-T cell treatment, and CAF/ECM density was inversely correlated with T-cell infiltration. Other IL12-armed T cells, such as wild-type IL-12-targeted or tumor-targeted IL-12-T cells, did not disrupt the ECM because this effect depended on the engagement between CSV on the tumor cell and its ligand on the attIL12-T cells. Mechanistic studies found that attIL12-T cell treatment elevated IFNγ production on interacting with CSV+ tumor cells, suppressing transforming growth factor beta secretion and in turn upregulating FAS-mediated CAF apoptosis. CAF destruction reshaped the tumor stroma to favor T-cell infiltration and tumor inhibition. CONCLUSIONS: This study unveiled a novel therapy-attIL12-T cells-for targeting CAFs/ECM. These findings are highly relevant to humans because CAFs are abundant in human osteosarcoma.

Development of a compact bidirectional promoter-driven dual chimeric antigen receptor (CAR) construct targeting CD19 and CD20 in the Sleeping Beauty (SB) transposon system.

BACKGROUND: A bidirectional promoter-driven chimeric antigen receptor (CAR) cassette provides the simultaneous expression of two CARs, which significantly enhances dual antigen-targeted CAR T-cell therapy. METHODS: We developed a second-generation CAR directing CD19 and CD20 antigens, incorporating them in a head-to-head orientation from a bidirectional promoter using a single Sleeping Beauty transposon system. The efficacy of bidirectional promoter-driven dual CD19 and CD20 CAR T cells was determined in vitro against cell lines expressing either, or both, CD19 and CD20 antigens. In vivo antitumor activity was tested in Raji lymphoma-bearing immunodeficient NOD-scid IL2Rgammanull (NSG) mice. RESULTS: Of all tested promoters, the bidirectional EF-1α promoter optimally expressed transcripts from both sense (CD19-CAR) and antisense (GFP.CD20-CAR) directions. Superior cytotoxicity, cytokine production and antigen-specific activation were observed in vitro in the bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells. In contrast, a unidirectional construct driven by the EF-1α promoter, but using self-cleaving peptide-linked CD19 and CD20 CARs, showed inferior expression and in vitro function. Treatment of mice bearing advanced Raji lymphomas with bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells effectively controlled tumor growth and extended the survival of mice compared with group treated with single antigen targeted CAR T cells. CONCLUSION: The use of bidirectional promoters in a single vector offers advantages of size and robust CAR expression with the potential to expand use in other forms of gene therapies like CAR T cells.

CHMP2A regulates broad immune cell-mediated antitumor activity in an immunocompetent in vivo head and neck squamous cell carcinoma model.

BACKGROUND: Natural killer (NK) cells are key effector cells of antitumor immunity. However, tumors can acquire resistance programs to escape NK cell-mediated immunosurveillance. Identifying mechanisms that mediate this resistance enables us to define approaches to improve immune-mediate antitumor activity. In previous studies from our group, a genome-wide CRISPR-Cas9 screen identified Charged Multivesicular Body Protein 2A (CHMP2A) as a novel mechanism that mediates tumor intrinsic resistance to NK cell activity. METHODS: Here, we use an immunocompetent mouse model to demonstrate that CHMP2A serves as a targetable regulator of not only NK cell-mediated immunity but also other immune cell populations. Using the recently characterized murine 4MOSC model system, a syngeneic, tobacco-signature murine head and neck squamous cell carcinoma model, we deleted mCHMP2A using CRISPR/Cas9-mediated knock-out (KO), following orthotopic transplantation into immunocompetent hosts. RESULTS: We found that mCHMP2A KO in 4MOSC1 cells leads to more potent NK-mediated tumor cell killing in vitro in these tumor cells. Moreover, following orthotopic transplantation, KO of mCHMP2A in 4MOSC1 cells, but not the more immune-resistant 4MOSC2 cells enables both T cells and NK cells to better mediate antitumor activity compared with wild type (WT) tumors. However, there was no difference in tumor development between WT and mCHMP2A KO 4MOSC1 or 4MOSC2 tumors when implanted in immunodeficient mice. Mechanistically, we find that mCHMP2A KO 4MOSC1 tumors transplanted into the immunocompetent mice had significantly increased CD4+T cells, CD8+T cells. NK cell, as well as fewer myeloid-derived suppressor cells (MDSC). CONCLUSIONS: Together, these studies demonstrate that CHMP2A is a targetable inhibitor of cellular antitumor immunity.

Armored TGFßRIIDN ROR1-CAR T cells reject solid tumors and resist suppression by constitutively-expressed and treatment-induced TGFß1.

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapy target receptor tyrosine kinase-like orphan receptor 1 (ROR1) is broadly expressed in hematologic and solid tumors, however clinically-characterized ROR1-CAR T cells with single chain variable fragment (scFv)-R12 targeting domain failed to induce durable remissions, in part due to the immunosuppressive tumor microenvironment (TME). Herein, we describe the development of an improved ROR1-CAR with a novel, fully human scFv9 targeting domain, and augmented with TGFßRIIDN armor protective against a major TME factor, transforming growth factor beta (TGFß). METHODS: CAR T cells were generated by lentiviral transduction of enriched CD4+ and CD8+ T cells, and the novel scFv9-based ROR1-CAR-1 was compared with the clinically-characterized ROR1-R12-scFv-based CAR-2 in vitro and in vivo. RESULTS: CAR-1 T cells exhibited greater CAR surface density than CAR-2 when normalized for %CAR+, and produced more interferon (IFN)-γ tumor necrosis factor (TNF)-α and interleukin (IL)-2 in response to hematologic (Jeko-1, RPMI-8226) and solid (OVCAR-3, Capan-2, NCI-H226) tumor cell lines in vitro. In vivo, CAR-1 and CAR-2 both cleared hematologic Jeko-1 lymphoma xenografts, however only CAR-1 fully rejected ovarian solid OVCAR-3 tumors, concordantly with greater expansion of CD8+ and CD4+CAR T cells, and enrichment for central and effector memory phenotype. When equipped with TGFß-protective armor TGFßRIIDN, CAR-1 T cells resisted TGFß-mediated pSmad2/3 phosphorylation, as compared with CAR-1 alone. When co-cultured with ROR-1+ AsPC-1 pancreatic cancer line in the presence of TGFß1, armored CAR-1 demonstrated improved recovery of killing function, IFN-γ, TNF-α and IL-2 secretion. In mouse AsPC-1 pancreatic tumor xenografts overexpressing TGFß1, armored CAR-1, in contrast to CAR-1 alone, achieved complete tumor remissions, and yielded accelerated expansion of CAR+ T cells, diminished circulating active TGFß1, and no apparent toxicity or weight loss. Unexpectedly, in AsPC-1 xenografts without TGFß overexpression, TGFß1 production was specifically induced by ROR-1-CAR T cells interaction with ROR-1 positive tumor cells, and the TGFßRIIDN armor conferred accelerated tumor clearance. CONCLUSIONS: The novel fully human TGFßRIIDN-armored ROR1-CAR-1 T cells are highly potent against ROR1-positive tumors, and withstand the inhibitory effects of TGFß in solid TME. Moreover, TGFß1 induction represents a novel, CAR-induced checkpoint in the solid TME, which can be circumvented by co-expressing the TGßRIIDN armor on T cells.

Adoptive transfer of CMV-specific TCR-T cells for the treatment of CMV infection after haploidentical hematopoietic stem cell transplantation.

BACKGROUND: Cytomegalovirus (CMV) reactivation after unmanipulated haploidentical stem cell transplantation (SCT) frequently occurs, causing life-threatening morbidities and transplantation failure. Pre-emptive therapy upon the detection of CMV viremia using antiviral agents is currently the standard of care but it was associated with significant toxicity. The CMV antigen-specific cytotoxic T lymphocyte therapy was limited by the time-consuming manufacture process and relatively low success rate. More effective and safer approaches for the treatment of CMV reactivation after haploidentical SCT are in urgent need. METHODS: A single-arm, open-label, phase I clinical trial evaluating the safety and efficacy of CMV-targeting T cell receptor-engineered T (CMV-TCR-T) cell therapy as the first-line pre-emptive therapy for patients with CMV reactivation after haploidentical peripheral blood SCT (PBSCT) was conducted in the Chinese PLA General Hospital. Six patients with CMV reactivation after haploidentical SCT were adoptively transferred by one to three doses of SCT donors-derived CMV-TCR-T cells. This trial was a dose-escalation study with doses ranging from 1×103 CMV-TCR-T cells/kg body weight per dose to 5×105 CMV-TCR-T cells/kg per dose. RESULTS: Except for the grade 1 cytokine release syndrome observed in one patient and mild fever in two patients, no other adverse events were observed. Four patients had response within a month after CMV-TCR-T cell infusion without the administration of any antiviral agents. The other two patients who initially did not respond to CMV-TCR-T cell therapy had salvage ganciclovir and foscarnet administration and then had rapid CMV clearance. The CMV-TCR-T cells displayed overall robust expansion and persistence in the peripheral blood after infusion. The CMV-TCR-T cells were first detected in the peripheral blood of these patients 3-7 days after the first dose of CMV-TCR-T infusion, rapidly expanded and persisted for at least 1-4 months, providing long-term protection against CMV reactivation. In one patient, the CMV-TCR-T cells started to expand even when the anti-graft-versus-host disease reagents were still being used, further indicating the proliferation potential of CMV-TCR-T cells. CONCLUSIONS: Our study first showed CMV-TCR-T cell as a highly feasible, safe and effective first-line pre-emptive treatment for CMV reactivation after haploidentical PBSCT. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Registry (NCT05140187).

Preclinical evaluation and first-in-dog clinical trials of PBMC-expanded natural killer cells for adoptive immunotherapy in dogs with cancer.

BACKGROUND: Natural killer (NK) cells are cytotoxic cells capable of recognizing heterogeneous cancer targets without prior sensitization, making them promising prospects for use in cellular immunotherapy. Companion dogs develop spontaneous cancers in the context of an intact immune system, representing a valid cancer immunotherapy model. Previously, CD5 depletion of peripheral blood mononuclear cells (PBMCs) was used in dogs to isolate a CD5dim-expressing NK subset prior to co-culture with an irradiated feeder line, but this can limit the yield of the final NK product. This study aimed to assess NK activation, expansion, and preliminary clinical activity in first-in-dog clinical trials using a novel system with unmanipulated PBMCs to generate our NK cell product. METHODS: Starting populations of CD5-depleted cells and PBMCs from healthy beagle donors were co-cultured for 14 days, phenotype, cytotoxicity, and cytokine secretion were measured, and samples were sequenced using the 3'-Tag-RNA-Seq protocol. Co-cultured human PBMCs and NK-isolated cells were also sequenced for comparative analysis. In addition, two first-in-dog clinical trials were performed in dogs with melanoma and osteosarcoma using autologous and allogeneic NK cells, respectively, to establish safety and proof-of-concept of this manufacturing approach. RESULTS: Calculated cell counts, viability, killing, and cytokine secretion were equivalent or higher in expanded NK cells from canine PBMCs versus CD5-depleted cells, and immune phenotyping confirmed a CD3-NKp46+ product from PBMC-expanded cells at day 14. Transcriptomic analysis of expanded cell populations confirmed upregulation of NK activation genes and related pathways, and human NK cells using well-characterized NK markers closely mirrored canine gene expression patterns. Autologous and allogeneic PBMC-derived NK cells were successfully expanded for use in first-in-dog clinical trials, resulting in no serious adverse events and preliminary efficacy data. RNA sequencing of PBMCs from dogs receiving allogeneic NK transfer showed patient-unique gene signatures with NK gene expression trends in response to treatment. CONCLUSIONS: Overall, the use of unmanipulated PBMCs appears safe and potentially effective for canine NK immunotherapy with equivalent to superior results to CD5 depletion in NK expansion, activation, and cytotoxicity. Our preclinical and clinical data support further evaluation of this technique as a novel platform for optimizing NK immunotherapy in dogs.

Bispecific antibodies redirect synthetic agonistic receptor modified T cells against melanoma.

BACKGROUND: Melanoma is an immune sensitive disease, as demonstrated by the activity of immune check point blockade (ICB), but many patients will either not respond or relapse. More recently, tumor infiltrating lymphocyte (TIL) therapy has shown promising efficacy in melanoma treatment after ICB failure, indicating the potential of cellular therapies. However, TIL treatment comes with manufacturing limitations, product heterogeneity, as well as toxicity problems, due to the transfer of a large number of phenotypically diverse T cells. To overcome said limitations, we propose a controlled adoptive cell therapy approach, where T cells are armed with synthetic agonistic receptors (SAR) that are selectively activated by bispecific antibodies (BiAb) targeting SAR and melanoma-associated antigens. METHODS: Human as well as murine SAR constructs were generated and transduced into primary T cells. The approach was validated in murine, human and patient-derived cancer models expressing the melanoma-associated target antigens tyrosinase-related protein 1 (TYRP1) and melanoma-associated chondroitin sulfate proteoglycan (MCSP) (CSPG4). SAR T cells were functionally characterized by assessing their specific stimulation and proliferation, as well as their tumor-directed cytotoxicity, in vitro and in vivo. RESULTS: MCSP and TYRP1 expression was conserved in samples of patients with treated as well as untreated melanoma, supporting their use as melanoma-target antigens. The presence of target cells and anti-TYRP1 × anti-SAR or anti-MCSP × anti-SAR BiAb induced conditional antigen-dependent activation, proliferation of SAR T cells and targeted tumor cell lysis in all tested models. In vivo, antitumoral activity and long-term survival was mediated by the co-administration of SAR T cells and BiAb in a syngeneic tumor model and was further validated in several xenograft models, including a patient-derived xenograft model. CONCLUSION: The SAR T cell-BiAb approach delivers specific and conditional T cell activation as well as targeted tumor cell lysis in melanoma models. Modularity is a key feature for targeting melanoma and is fundamental towards personalized immunotherapies encompassing cancer heterogeneity. Because antigen expression may vary in primary melanoma tissues, we propose that a dual approach targeting two tumor-associated antigens, either simultaneously or sequentially, could avoid issues of antigen heterogeneity and deliver therapeutic benefit to patients.

Apoptosis: a Janus bifrons in T-cell immunotherapy.

Immunotherapy has revolutionized the treatment of cancer. In particular, immune checkpoint blockade, bispecific antibodies, and adoptive T-cell transfer have yielded unprecedented clinical results in hematological malignancies and solid cancers. While T cell-based immunotherapies have multiple mechanisms of action, their ultimate goal is achieving apoptosis of cancer cells. Unsurprisingly, apoptosis evasion is a key feature of cancer biology. Therefore, enhancing cancer cells' sensitivity to apoptosis represents a key strategy to improve clinical outcomes in cancer immunotherapy. Indeed, cancer cells are characterized by several intrinsic mechanisms to resist apoptosis, in addition to features to promote apoptosis in T cells and evade therapy. However, apoptosis is double-faced: when it occurs in T cells, it represents a critical mechanism of failure for immunotherapies. This review will summarize the recent efforts to enhance T cell-based immunotherapies by increasing apoptosis susceptibility in cancer cells and discuss the role of apoptosis in modulating the survival of cytotoxic T lymphocytes in the tumor microenvironment and potential strategies to overcome this issue.

TIPE2 deletion improves the therapeutic potential of adoptively transferred NK cells.

BACKGROUND: To enhance the efficacy of adoptive NK cell therapy against solid tumors, NK cells must be modified to resist exhaustion in the tumor microenvironment (TME). However, the molecular checkpoint underlying NK cell exhaustion in the TME remains elusive. METHODS: We analyzed the correlation between TIPE2 expression and NK cell functional exhaustion in the TME both in humans and mice by single-cell transcriptomic analysis and by using gene reporter mice. We investigated the effects of TIPE2 deletion on adoptively transferred NK cell therapy against cancers by using NK cells from NK-specific Tipe2-deficient mice or peripheral blood-derived or induced pluripotent stem cell (iPSC)-derived human NK cells with TIPE2 deletion by CRISPR/Cas9. We also investigated the potential synergy of double deletion of TIPE2 and another checkpoint molecule, CISH. RESULTS: By single-cell transcriptomic analysis and by using gene reporter mice, we found that TIPE2 expression correlated with NK cell exhaustion in the TME both in humans and mice and that the TIPE2 high NK cell subset correlated with poorer survival of tumor patients. TIPE2 deletion promoted the antitumor activity of adoptively transferred mouse NK cells and adoptively transferred human NK cells, either derived from peripheral blood or differentiated from iPSCs. TIPE2 deletion rendered NK cells with elevated capacities for tumor infiltration and effector functions. TIPE2 deletion also synergized with CISH deletion to further improve antitumor activity in vivo. CONCLUSIONS: This study highlighted TIPE2 targeting as a promising approach for enhancing adoptive NK cell therapy against solid tumors.

Use of cytokine-induced killer cell therapy in patients with colorectal cancer: a systematic review and meta-analysis.

BACKGROUND: The number of clinical studies evaluating the benefit of cytokine-induced killer cell (CIK) therapy, an adoptive immunotherapy, for colorectal cancer (CRC) is increasing. In many of these trials, CIK therapy was coadministered with conventional cancer therapy. The aim of this review is to systematically assess the available literature, in which the majority were only in Chinese, on CIK therapy for the management of CRC using meta-analysis and to identify parameters associated with successful CIK therapy implementation. METHODS: Prospective and retrospective clinical studies which compared CIK therapy to non-CIK therapy in patients with CRC were searched for electronically on MEDLINE, Embase, China National Knowledge Infrastructure, and Wanfang Data databases. The clinical endpoints of overall survival (OS), progression-free survival (PFS), OS and PFS rates, overall response rate (ORR), and toxicity were meta-analyzed using HR and relative ratio (RR), and subgroup analyses were performed using chi-square (χ2) test and I-squared (I2) statistics for study design, disease stage, cotherapy type, and timing of administration. RESULTS: In total, 70 studies involving 6743 patients were analyzed. CIK therapy was favored over non-CIK therapy for OS (HR=0.59, 95% CI: 0.53 to 0.65), PFS (HR=0.55, 95% CI: 0.47 to 0.63), and ORR (RR=0.65, 95% CI: 0.57 to 0.74) without increasing toxicity (HR=0.59, 95% CI: 0.16 to 2.25). Subgroup analyses on OS and PFS by study design (randomized vs non-randomized study design), disease stage (Stage I-III vs Stage IV), cotreatment with dendritic cells (DCs) (CIK vs DC-CIK therapy), or timing of therapy administration (concurrent vs sequential with coadministered anticancer therapy) also showed that the clinical benefit of CIK therapy was robust in any subgroup analysis. Furthermore, cotreatment with DCs did not improve clinical outcomes over CIK therapy alone. CONCLUSION: Compared with standard therapy, patients who received additional CIK cell therapy had favorable outcomes without increased toxicity, warranting further investigation into CIK therapy for the treatment of CRC.