Background Ovarian cancer (OC) is the leading cause of death from gynecologic malignancies in the Western world. Contributing factors include a high frequency of late-stage diagnosis, the development of chemoresistance, and the evasion of host immune responses. Currently, debulking surgery and platinum-based chemotherapy are the treatment cornerstones, although recurrence is common. As the clinical efficacy of immune checkpoint blockade is low, new immunotherapeutic strategies are needed. Chimeric antigen receptor (CAR) T cell therapy empowers patients’ own T cells to fight and eradicate cancer, and has been tested against various targets in OC. A promising candidate is the MUC16 ectodomain. This ectodomain remains on the cell surface after cleavage of cancer antigen 125 (CA125), the domain distal from the membrane, which is currently used as a serum biomarker for OC. CA125 itself has not been tested as a possible CAR target. In this study, we examined the suitability of the CA125 as a target for CAR T cell therapy.Methods We tested a series of antibodies raised against the CA125 extracellular repeat domain of MUC16 and adapted them to the CAR format. Comparisons between these candidates, and against an existing CAR targeting the MUC16 ectodomain, identified K101 as having high potency and specificity. The K101CAR was subjected to further biochemical and functional tests, including examination of the effect of soluble CA125 on its activity. Finally, we used cell lines and advanced orthotopic patient-derived xenograft (PDX) models to validate, in vivo, the efficiency of our K101CAR construct.Results We observed a high efficacy of K101CAR T cells against cell lines and patient-derived tumors, in vitro and in vivo. We also demonstrated that K101CAR functionality was not impaired by the soluble antigen. Finally, in direct comparisons, K101CAR, which targets the CA125 extracellular repeat domains, was shown to have similar efficacy to the previously validated 4H11CAR, which targets the MUC16 ectodomain.Conclusions Our in vitro and in vivo results, including PDX studies, demonstrate that the CA125 domain of MUC16 represents an excellent target for treating MUC16-positive malignancies.

A representative case of aggressive ATL for which Mog treatment failed. (a) CD4+CD25+ ATL tumor cells in peripheral blood displayed CCR4. (b) Weekly administration of Mog a total of 3 times failed to suppress ATL disease activity, as represented by an increased LDH value. Thereafter, repeated intensive chemotherapy had been needed to suppress disease activity until allo-HSCT was conducted. At the same time, prolonged lymphocytopenia was noticeable. Clear triangles: lactate dehydrogenase (LDH); solid circles: lymphocyte count; Mog: mogamulizumab; white arrow: infusion of mogamulizumab; gray arrow: intensive chemotherapy. (c) Computed tomograms. Lt: on day -3 before start of Mog, and 26 days after three courses of the mLSG15 regimen24. No lymph node swelling is evident (complete remission). Rt: On day 16 after the start of Mog administration, enlarged lymph nodes were detected systemically (progressive disease), highlighted by yellow circles and yellow arrows.

Chimeric antigen receptor (CAR) T-cell therapy has had considerable success in the treatment of B-cell malignancies. Targeting the B-lineage marker CD19 has brought great advances to the treatment of acute lymphoblastic leukemia and Bcell lymphomas. However, relapse remains an issue in many cases. Such relapse can result from downregulation or loss of CD19 from the malignant cell population or expression of alternate isoforms. Consequently, there remains a need to target alternative B-cell antigens and diversify the spectrum of epitopes targeted within the same antigen. CD22 has been identified as a substitute target in cases of CD19-negative relapse. One anti-CD22 antibody-clone m971-targets a membrane -proximal epitope of CD22 and has been widely validated and used in the clinic. Here, we have compared m971 -CAR with a novel CAR derived from IS7, an antibody that targets a central epitope on CD22. The IS7-CAR has superior avidity and is active and specific against CD22-positive targets, including B-acute lymphoblastic leukemia patientderived xenograft samples. Side -by -side comparisons indicated that while IS7-CAR killed less rapidly than m971 -CAR in vitro, it remains efficient in controlling lymphoma xenograft models in vivo. Thus, IS7-CAR presents a potential alternative candidate for the treatment of refractory B-cell malignancies.

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.

ADCC activity mediated by cCD16Î-T cells against Mog-opsonized ATL tumor cells was dependent on both effector cell number and Mog dose. However, at a pharmacological dose of Mog, the decline of ADCC was more obviously dependent on the number of effector cells. (a) At a pharmacological dose of Mog (1μg/mL), ADCC activity mediated by cCD16Î-T cells against Mog-opsonized ATL tumor cells declined sharply when the number of effector cells was lower. In order to exclude NK cell activity, K562-A24 was employed in this experiment as a negative control. Each experiment at the indicated dose of Mog was conducted in triplicate using three independent sets of cells from three different healthy donors. LCL, EBV-immortalized B cell line; Mog, mogamulizumab. Each experiment was conducted in triplicate (n=3). Error bars depict SD. (b) In the presence of a sufficient number of effector cells (E/T ratio 5:1), ADCC activity mediated by cCD16Î-T cells against ATL tumor cells was also Mog dose-dependent. However, at pharmacological doses of Mog (>0.1μg/mL), the decline of ADCC activity was limited in comparison to that observed in S2a. LCL, EBV-immortalized B cell line: Mog, mogamulizumab. Each experiment was conducted in triplicate (n=3). Error bars depict SD.

In our limited experience with Mog monotherapy for relapsed/refractory ATL (including the case shown in Fig.S3), excluding patients who received allo-HSCT and combined therapy with Mog and intensified chemotherapy mSLG15(ref.32) had shown that lymphocyte counts during Mog monotherapy in patients for whom the treatment failed to suppress disease progression (failure n=4) tended to be lower than that when Mog monotherapy successfully controlled disease (successful n=2). Total time points when cell counts were measured were shown, respectively. Data indicated as mean {plus minus} SD, respectively.

Background Ovarian cancer (OC) is a heterogeneous disease, often diagnosed at an advanced stage. After an initial encouraging response rate to first-line treatment, comprising cytoreductive surgery combined with platinum-based chemotherapy, most of such cancers recur. The use of advanced, clinically relevant mouse models allows us to study tumor pathogenesis and to evaluate response to new cancer drugs, which may ultimately prevent recurrence and prolong survival. Most preclinical models however do not fully recapitulate the tumor microenvironment as well as heterogeneity, and thus many drugs fail a successful implementation into clinical practice. We developed and characterized humanized patient-derived xenograft (PDX) murine models, comprising of a functional human immune system and an orthotopically implanted primary ovarian cancer tumor. These models were further used to test treatment response of immune checkpoint inhibitor PD-1, combinatorial immunotherapy targeting PD-L1 and CD73, and novel chimeric antigen receptor (CAR) constructs targeting unique biomarker of OC. Methods Humanized PDX mice were generated by co-transplantation of CD34+ hematopoietic cells, isolated from the umbilical cord blood and primary ovarian cancer cell suspensions from treatment naïve OC patients. PDX models were characterized by whole exome sequencing (WES) and the developing human immune system in immunodeficient mice was followed longitudinally by flow cytometry. Bioluminescence and 18F-FDG PET-CT imaging of tumor burden, survival analysis, and characterization of tumor-infiltrating immune cells by a 34-surface mass cytometry were performed to assess the treatment responses. Results Phenotypic and genomic characterization of humanized PDX models was achieved. Mice treated with nivolumab showed a decrease in tumor burden, however no significant survival benefit when compared to untreated controls was identified, nor could a correlation between PD-L1 expression, CD8 T cell infiltration and response parameters been observed. Interestingly, the characterization of immune infiltrating cells identified predominantly myeloid cells as seen in ovarian cancer patients. In a CAR T cell treatment study of OC PDX mice, bioluminescence imaging showed a delayed onset of metastasis and smaller tumor burden, but also severe toxic side effects in CAR T cell treated PDX mice compared to untreated control mice. Results from combinatorial immunotherapy targeting PD-L1 and CD73 are awaited. Conclusions Humanized orthotopic OC PDX models have been established. Together with the promising advances in new immunotherapeutic targets, clinically relevant mouse models facilitate the development of new immunotherapies targeting the ovarian tumor microenvironment. Ethics Approval Patient tumor samples were provided by the Gynecologic Cancer Biobank, Women's Clinic, Haukeland University Hospital, Bergen, Norway (REK ID: 2014/1907, 2015/548, 2018/72). Animal experiments are performed in accordance with the procedures set by the Norwegian State Commission for Laboratory Animals, and the laboratory-animal experiments (FOTS 25412) have been approved by the Norwegian Food Safety Authority. Consent Written informed consent was obtained from all women before collection of fresh tumor tissues. A copy of the written consent and approval is available for review.

A lentiviral vector expressing the chimeric cCD16 with a 158V/V-CD3Î receptor gene. (a) cDNA encoding the signal peptide and extracellular domain of CD16 gene (accession no.: NM_000569) with a gene alteration at F158V was connected to the CD3Î gene (accession no.: NM_198053) at the second amino acid (P) of the extracellular domain (modified from which we have previously reported.23). SP: signal peptide of CD16 (53 amino acids); aa158: amino acid at position 158; V: replaced to valine; 1ECD: extracellular domain of CD16 (189 amino acids); 2ECD: extracellular domain of CD3Î (2 amino acids); TM: transmembrane portion of CD3Î (21 amino acids); ICP: intracellular portion of CD3Îïº(144 amino acids); aa: amino acid. AscI and SalI are restriction enzymes. (b) Using a lentiviral vector, the cCD16Î receptor gene was successfully introduced into HEK293T being negative for CD3. (c) T cells lentivirally gene-modified using cCD16Î receptor gene transfer (cCD16Î-T cells) successfully displayed cytocidal activity through recognition of the CCR4-Mog complex on target cells, and not via NK cell activity. cCD16Î-T cells successfully killed CCR4-positive MT-4 cells, but not CCR4-negative K562, K562-A24, LCL and Jurkat cells in the presence of 1.0μg/mL Mog. Neither K562 nor K562-A24 were killed by cCD16Î-T cells in the presence or absence of Mog, indicating that NK cell activity was not involved in this ADCC activity mediated by cCD16Î-T cells. K562-A24, HLA-A*24:02-transduced K562; LCL, EBV-immortalized B cell line; NGM-T, non-gene-modified T cells; both NGM-T and cCD16Î-T were generated from the same donor; Mog, mogamulizumab; E/T ratio, effector/target ratio. Each experiment was conducted in triplicate. Error bars depict SD.

Chimeric antigen receptor (CAR) T cells (CAR T) have emerged as a potential therapy for cancer patients. CAR T cells are capable of recognizing membrane proteins on cancer cells which initiates a downstream signaling in T cells that ends in cancer cell death. Continuous antigen exposure over time, activation of inhibitory signaling pathways and/or chronic inflammation can lead to CAR T cell exhaustion. In this context, the design of CARs can have a great impact on the functionality of CAR T cells, on their potency and exhaustion. Here, using CD19CAR as model, we provide a re-challenge protocol where CAR T cells are cultured weekly with malignant lymphoid cell lines BL-41 and Nalm-6 to simulate them with continuous antigen pressure over a four-week period. This protocol can be value for assessing CAR T cell functionality and for the comparison of different CAR constructs.

Therapy employing T cells modified with chimeric antigen receptors (CARs) is effective in hematological malignancies but not yet in solid cancers. CAR T cell activity in solid tumors is limited by immunosuppressive factors, including transforming growth factor β (TGFβ). Here, we describe the development of a switch receptor (SwR), in which the extracellular domains of the TGFβ receptor are fused to the intracellular domains from the IL-2/15 receptor. We evaluated the SwR in tandem with two variants of a CAR that we have developed against STEAP1, a protein highly expressed in prostate cancer. The SwR-CAR T cell activity was assessed against a panel of STEAP1+/− prostate cancer cell lines with or without over-expression of TGFβ, or with added TGFβ, by use of flow cytometry cytokine and killing assays, Luminex cytokine profiling, cell counts, and flow cytometry phenotyping. The results showed that the SwR-CAR constructs improved the functionality of CAR T cells in TGFβ-rich environments, as measured by T cell proliferation and survival, cytokine response, and cytotoxicity. In assays with four repeated target-cell stimulations, the SwR-CAR T cells developed an activated effector memory phenotype with retained STEAP1-specific activity. In conclusion, the SwR confers CAR T cells with potent and durable in vitro functionality in TGFβ-rich environments. The SwR may be used as an add-on construct for CAR T cells or other forms of adoptive cell therapy.

The manufacture of efficacious CAR T cells represents a major challenge in cellular therapy. An important aspect of their quality concerns energy production and consumption, known as metabolism. T cells tend to adopt diverse metabolic profiles depending on their differentiation state and their stimulation level. It is therefore expected that the introduction of a synthetic molecule such as CAR, activating endogenous signaling pathways, will affect metabolism. In addition, upon patient treatment, the tumor microenvironment might influence the CAR T cell metabolism by compromising the energy resources. The access to novel technology with higher throughput and reduced cost has led to an increased interest in studying metabolism. Indeed, methods to quantify glycolysis and mitochondrial respiration have been available for decades but were rarely applied in the context of CAR T cell therapy before the release of the Seahorse XF apparatus. The present review will focus on the use of this instrument in the context of studies describing the impact of CAR on T cell metabolism and the strategies to render of CAR T cells more metabolically fit.

T-cell receptor (TCR) redirected T cells are considered as the next generation of care for the treatment of numerous solid tumors. KRAS mutations are driver neoantigens that are expressed in over 25% of all cancers and are thus regarded as ideal targets for Adoptive Cell Therapy (ACT). We have isolated four KRAS-specific TCRs from a long-term surviving pancreatic cancer patient vaccinated with a mix of mutated KRAS peptides. The sequence of these TCRs could be identified and expressed in primary cells. We demonstrated stable expression of all TCRs as well as target-specific functionality when expressing T cells were co-incubated with target cells presenting KRAS peptides. In addition, these TCRs were all partially co-receptor independent since they were functional in both CD4 and CD8 T cells, thus indicating high affinity. Interestingly, we observed that certain TCRs were able to recognize several KRAS mutations in complex with their cognate Human leukocyte antigen (HLA), suggesting that, here, the point mutations were less important for the HLA binding and TCR recognition, whereas others were single-mutation restricted. Finally, we demonstrated that these peptides were indeed processed and presented, since HLA-matched antigen presenting cells exogenously loaded with KRAS proteins were recognized by TCR-transduced T cells. Taken together, our data demonstrate that KRAS mutations are immunogenic for CD4 T cells and are interesting targets for TCR-based cancer immunotherapy.

Ovarian Cancer (OC) is currently difficult to cure, mainly due to its late detection and the advanced state of the disease at the time of diagnosis. Therefore, conventional treatments such as debulking surgery and combination chemotherapy are rarely able to control progression of the tumour, and relapses are frequent. Alternative therapies are currently being evaluated, including immunotherapy and advanced T-cell based therapy. In the present review, we will focus on a description of those Chimeric Antigen Receptors (CARs) that have been validated in the lab, or are being tested in the clinic. Numerous target antigens have been defined due to the identification of OC biomarkers, and many are being used as CAR targets. We provide an exhaustive list of these constructs and their current status. Despite being innovative and efficient, the OC-specific CARs face a barrier to their clinical efficacy: the tumour microenvironment (TME). Indeed, effector cells expressing CARs have been shown to be severely inhibited, rendering the CAR T cells useless once at the tumour site. Herein we give a thorough description of the highly immunosuppressive OC-TME, and present recent studies and innovations that have enabled CAR T cells to counteract this negative environment and to destroy tumours.

Ovarian Cancer (OC) is currently difficult to cure, mainly due to its late detection and the advanced state of the disease at the time of diagnosis. Therefore, conventional treatments such as debulking surgery and combination chemotherapy are rarely able to control progression of the tumour, and relapses are frequent. Alternative therapies are currently being evaluated, including immunotherapy and advanced T cell-based therapy. In the present review, we will focus on a description of those Chimeric Antigen Receptors (CARs) that have been validated in the laboratory or are being tested in the clinic. Numerous target antigens have been defined due to the identification of OC biomarkers, and many are being used as CAR targets. We provide an exhaustive list of these constructs and their current status. Despite being innovative and efficient, the OC-specific CARs face a barrier to their clinical efficacy: the tumour microenvironment (TME). Indeed, effector cells expressing CARs have been shown to be severely inhibited, rendering the CAR T cells useless once at the tumour site. Herein, we give a thorough description of the highly immunosuppressive OC TME and present recent studies and innovations that have enabled CAR T cells to counteract this negative environment and to destroy tumours.

The gene transfer of T-cell receptors (TCRs) is an attractive strategy for adoptive cell therapy, allowing the transfer of reactivity against antigens that may not otherwise engender an immune response. The TCRs recognize intracellular or extracellular antigens presented in the context of MHC class I or II, respectively. This broadens the range of targets considerably, compared to antibodies and chimeric antigen receptors, that are generally confined to surface antigens. However, TCR transfer must overcome some technical hurdles, relating to interference with endogenous alpha- and beta-TCR chains and competition with other existing TCR infrastructure of T cells. In this review, we will outline the challenges facing TCR gene transfer and compare several approaches to address them. We will then focus upon one of the most promising amongst these-RNA interference-and detail the methods involved in designing and using this technology.

Adult T-cell leukemia/lymphoma (ATLL) is a distinct type of peripheral T-cell neoplasm characterized by transformed T-lymphocytes with a pleomorphic (flower-like) nucleus and is resulted due to an ...

Aurora Kinase A is a cancer-associated protein normally involved in the regulation of mitosis. Being over-expressed in a range of cancers, it is a suitable target for cell-based immunotherapy. Gene transfer of T-cell receptor sequences cognisant of HLA-A*0201-restricted Aurora Kinase A antigen has previously been shown to transfer specific immunoreactivity against the target peptide in a Human Lymphocyte Antigen-restricted manner. While T cell receptor gene-transfer has great potential in overcoming the difficulties of isolating and expanding tumour-reactive lymphocytes from a patient's own cells, one hurdle is potential mispairing and competition between exogenous and endogenous T cell receptor chains. We have used a retroviral vector design bearing a short-interfering RNA that downregulates endogenous T cell receptor chains, without affecting expression of the transgenic T cell receptor sequences. The T cell receptor expression cassette also includes a 2A self-cleaving peptide, resulting in equimolar expression of the T cell receptor alpha and beta chains, further enhancing formation of the desired T cell receptor. Via a simple, modular cloning method, we have cloned the alpha and beta chains of the anti-Aurora Kinase A-reactive T cell receptor into this 'siTCR' vector. We then compared the activity of this vector against the original, 'conventional' vector across a panel of assays. T cell receptors expressed from the siTCR-vector retained the cytotoxic functionality of the original vector, with evidence of reduced off-target reactivity. The rate of expression of correctly-formed T cell receptors was superior using the siTCR design, and this was achieved at lower vector copy numbers. Maintaining T cell receptor efficacy with a reduced vector copy number reduces the risk of genotoxicity. The siTCR design also reduces the risk of mispairing and cross-reactivity, while increasing the functional titre. Such improvements in the safety of T cell receptor gene-transfer will be crucial for clinical applications of this technology.

Abstract Purpose: Mogamulizumab (Mog), a humanized anti-CC chemokine receptor 4 (CCR4) mAb that mediates antibody-dependent cellular cytotoxicity (ADCC) using FcγR IIIa (CD16)-expressing effector cells, has recently been approved for treatment of CCR4-positive adult T-cell leukemia (ATL) in Japan. However, Mog failure has sometimes been observed in patients who have accompanying chemotherapy-associated lymphocytopenia. In this study, we examined whether adoptive transfer of artificial ADCC effector cells combined with Mog would overcome this drawback. Experimental Design: We lentivirally gene-modified peripheral blood T cells from healthy volunteers and ATL patients expressing the affinity-increased chimeric CD16-CD3ζ receptor (cCD16ζ-T cells). Subsequently, we examined the ADCC effect mediated by those cCD16ζ-T cells in the presence of Mog against ATL tumor cells both in vitro and in vivo. Results: cCD16ζ-T cells derived from healthy donors killed in vitro Mog-opsonized ATL cell line cells (n = 7) and primary ATL cells (n = 4) depending on both the number of effector cells and the dose of the antibody. cCD16ζ-T cells generated from ATL patients (n = 3) also exerted cytocidal activity in vitro against Mog-opsonized autologous ATL cells. Using both intravenously disseminated model (n = 5) and subcutaneously inoculated model (n = 4), coadministration of Mog and human cCD16ζ-T cells successfully suppressed tumor growth in xenografted immunodeficient mice, and significantly prolonged their survival (P < 0.01 and P = 0.02, respectively). Conclusions: These data strongly suggest clinical feasibility of the novel combined adoptive immunotherapy using cCD16ζ-T cells and Mog for treatment of aggressive ATL, particularly in patients who are ineligible for allogeneic hematopoietic stem cell transplantation. Clin Cancer Res; 22(17); 4405–16. ©2016 AACR.

[Background] Aurora Kinase A (AURKA) is a cancer-associated protein normally involved in the regulation of mitosis. It is over-expressed in a range of cancers, and has a 'cancer-testis' expression profile, making it a suitable target for cell-based immunotherapy. Gene transfer of T-cell receptor (TCR) sequences cognisant of HLA-A*0201-restricted AURKA antigen (aa207-215: YLILEYAPL) has previously been shown to transfer specific immunoreactivity against the target peptide in a HLA-restricted manner (Nagai K et al. Blood, 2012).