Targeted Immuotherapy of Muc1 Positive Carcinomas with Genetically Engineered Human T Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3718-3718
Author(s):  
Nachimuthu Chinnasamy ◽  
James Shaffer ◽  
Dhanalakshmi Chinnasamy

Abstract Mucin1 (Muc1), a tumor-associated antigen, is overexpressed in many adenocarcinomas including breast, pancreas, ovary, prostate, colon, and in multiple myeloma. Therefore, tumor associated Muc1 is an attractive target for immunotherapy. Several types of immunotherapeutic approaches targeting Muc1 are currently being tested in preclinical and clinical settings. A major limitation of vaccination strategies involving tumor antigens is the lack of induction of sufficient number of antigen-specific effector T cells systemically to achieve objective clinical response. In an effort to redirect activated T cells towards tumor cells, we constructed lentiviral vectors expressing chimeric receptors consisting of a novel single chain variable domain (scFv) of a monoclonal antibody against Muc1 combined with various cytoplasmic signaling domains such as CD3 Zeta, CD28 and OX40. Human peripheral blood T cells were lentivirally transduced with chimeric immune receptors in order to redirect them towards Muc1 positive tumor cells. A sufficiently high level of transduction (>80) was achieved without any positive selection. Transduced T cells were stimulated and expanded over a period of 10–15 days in the presence of IL-2. Gene-modified T cells efficiently recognized Muc1 expressing tumor target cells in an MHC-independent manner as demonstrated by antigen-specific tumor cell cytolysis. Furthermore, transduced T cells showed antigen-specific proliferation and secreted interferon gamma upon ligation with Muc1 on tumor cells. These results indicate that generation of large numbers of antigen specific T cells for clinical application is possible through the use of this chimeric immune receptor gene transfer strategy. This approach might be an attractive strategy in a minimal residual disease setting complementing traditional therapies based on surgery, radiation and/or chemotherapy. Therefore, present study suggests a promising new strategy for cancer immunotherapy using gene-modified T cells. Figure Figure

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 151-151
Author(s):  
Christopher C. DeRenzo ◽  
Phuong Nguyen ◽  
Stephen Gottschalk

151 Background: T-cell therapy for cancer faces several challenges, including limited T-cell expansion at tumor sites, and lack of unique tumor antigens that are not expressed in normal tissues. To overcome the first obstacle, we developed Engager (ENG) T cells, which secrete bispecific molecules consisting of single chain variable fragments specific for CD3 and a tumor antigen. ENG T cells have the unique ability to redirect bystander T cells to tumors, amplifying antitumor effects. Costimulatory chimeric antigen receptors (CoCARs) are one potential strategy to restrict full T-cell activation to tumor sites that express a unique "antigen address." The goal of this project was now to generate T cells that express engager molecules and CoCARs (ENG/CoCAR T cells), which recognize distinct tumor antigens, and evaluate their effector function. Methods: We focused on two tumor antigens, EphA2 and HER2, which are expressed in a broad range of solid tumors. RD114-pseudotyped retroviral particles encoding an EphA2-ENG or a HER2-CoCAR were used to transduce CD3/CD28-activated human T cells. Transduced T cells were cocultured with EphA2+/HER2- or EphA2+/HER2+ tumor cells. Results: Both EphA2-ENG and EphA2-ENG/HER2-CoCAR T cells were activated by EphA2+ targets, as judged by IFNγ secretion. EphA2-ENG T cells secreted little IL-2 and died after one stimulation with EphA2+/HER2- or EphA2+/HER2+ tumor cells. In contrast, EphA2-ENG/HER2-CoCAR T cells secreted high levels of IL-2 and proliferated when stimulated with EphA2+/HER2+ cells. Little IL-2 secretion and no proliferation was observed after stimulation of the same T cells with EphA2+/HER2- cells, indicating these T cells are only fully activated in the presence of both target antigens. Upon repeated stimulation with EphA2+/HER2+ tumor cells, EphA2-ENG/HER2-CoCAR T cells continued to secrete IL-2 and proliferate without the addition of external cytokines for at least 10 weeks. Conclusions: EphA2-ENG/HER2-CoCAR T cells demonstrated robust dual antigen dependent IL-2 secretion, and continued proliferation upon repeat stimulation with EphA2+/HER2+ cells. Thus, providing antigen-specific costimulation is a potential strategy to improve the safety and efficacy of T-cell therapy for cancer.


Blood ◽  
2005 ◽  
Vol 106 (5) ◽  
pp. 1544-1551 ◽  
Author(s):  
Tong Zhang ◽  
Bethany A. Lemoi ◽  
Charles L. Sentman

Abstract NKG2D is an activating cell-surface receptor expressed on natural killer (NK) cells and some T-cell subsets. Its ligands are primarily expressed on tumor cells. The aim of this study was to determine whether chimeric NK-receptor—bearing T cells would directly kill tumor cells and lead to induction of host immunity against tumors. Chimeric NK receptors were produced by linking NKG2D or DNAX activating protein of 10 kDa (Dap10) to the cytoplasmic portion of the CD3ζ chain. Our results showed that chimeric (ch) NKG2D-bearing T cells responded to NKG2D-ligand–bearing tumor cells (RMA/Rae-1β, EG7) but not to wild-type tumor cells (RMA). This response was dependent upon ligand expression on the target cells but not on expression of major histocompatibility complex (MHC) molecules, and the response could be blocked by anti-NKG2D antibodies. These T cells produced large amounts of T-helper 1 (Th1) cytokines and proinflammatory chemokines and killed ligand–expressing tumor cells. Adoptive transfer of chNKG2D-bearing T cells inhibited RMA/Rae-1β tumor growth in vivo. Moreover, mice that had remained tumor-free were resistant to subsequent challenge with the wild-type RMA tumor cells, suggesting the generation of immunity against other tumor antigens. Taken together, our findings indicate that modification of T cells with chimeric NKG2D receptors represents a promising approach for immunotherapy against cancer.


Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1667-1667
Author(s):  
Joshua B. Bland ◽  
William T. Tse

Abstract Expression of immune checkpoint ligands is a mechanism that many tumors use to escape attack by host immune cells. PD-L1, the ligand for checkpoint receptor PD-1 on T cells, is often expressed on tumor cells. Engagement of PD-1 on T cells by PD-L1 on tumor cells attenuates T-cell receptor signaling and suppresses anti-tumor response. PD-1 and PD-L1 blocking antibodies have been implemented clinically as treatment for many cancers, but the pattern of PD-L1 expression on AML is not well characterized. To answer this question, we studied how PD-L1 expression on AML is regulated under in vitro conditions that simulate the leukemia-host microenvironment. We examined surface expression of PD-L1 by flow cytometry on 4 AML lines, THP-1, KG1, KG1a, HL60, and a CML line, K562. Under basal conditions, these lines expressed no or low levels of PDL1. The AML cells were then subjected to conditions that mimic the leukemia-host microenvironment. AML cells were stained with the green fluorescent dye CFSE and co-cultured with Ficoll-separated PMNCs from healthy donors. After a day of co-culture, expression of PD-L1 was analyzed on AML cells and PD-1, CD25 and CD69 activation markers on PMNCs. Only a small increase of PD-L1, up to 2-4 fold, was seen on AML cells under this condition. To simulate the pro-inflammatory milieu in the tumor microenvironment, anti-CD3/CD28 microbeads were then added in culture to activate T cells. We observed a marked up-regulation of PD-L1 on AML cells, up to 5-60 fold, plus prominent expression of PD-1, CD25 and CD69 on T cells. These findings were confirmed by an alternative method of T cell activation in which AML cells were first coated with an anti-CD123 antibody, linked to anti-CD3/CD28 antibodies via a biotin-streptavidin bridge, and then cultured with PMNCs. To test whether pro-inflammatory cytokines were the sole inducers of PD-L1 expression, AML cells were treated with IFN-γ or TNF-α alone. IFN-γ treatment enhanced PD-L1 expression by 2-10 fold, while TNF-α showed a <2-fold increase. These results show that expression of PD-L1 on AML is dynamically regulated through interaction with activated T cells, by multiple mechanisms including cytokine production and cell-cell interaction. MYC has been shown to regulate PD-L1 expression on T-ALL and solid tumors (Science 2016; 352:227). We asked whether MYC inhibition would suppress PD-L1 on AML. AML and PMNCs were co-cultured in the presence of anti-CD3/CD28 beads, with JQ1, a BET bromodomain inhibitor that blocks MYC expression. JQ1 inhibited PD-L1 expression by >90%. Dose-effect titration showed sigmoidal curves with ED50 of 0.03 to 0.1 μM for the 5 AML lines. Treatment with another MYC inhibitor, CPI-203, yielded similar results. These observations indicate that MYC inhibition can suppress PD-L1 expression on AML induced by activated T cells. TP53 has been shown to regulate PD-L1 expression on non-small cell lung cancer (JNCI 2016; 108:djv303). We asked whether TP53 activation in AML would also affect PD-L1 expression. Since the AML lines we used did not express wild-type TP53, we overexpressed TP53 in these cells by transfecting with a TP53-GFP plasmid. Expression of TP53 in the cells decreased PD-L1 levels by >80%. Treatment of the cells with pifithrin, an inhibitor that blocks trans-activating function of TP53, did not rescue PD-L1 expression, suggesting that the effect of TP53 on PD-L1 expression is independent of its canonical trans-activating pathway. We asked if MYC and TP53 would synergistically affect PD-L1 expression on AML. We transfected AML cells with the TP53-GFP plasmid and co-cultured the cells with PMNCs and anti-CD3-/CD28 beads, in the presence or absence of JQ1. We found that JQ1 treatment of TP53-transfected cells further decreased PD-L1 expression by another 15%, indicating that MYC and TP53 independently and synergistically affect PD-L1 expression on AML. In summary, PD-L1 expression on AML cells is dynamically up-regulated upon interaction with activated T cells and suppressed by perturbation of the MYC and TP53 pathways. These findings have implications in the use of immune effector cell therapy against AML, since the activated effector cells could up-regulate PD-L1 expression on target cells and attenuate anti-leukemia effects. MYC inhibitors and TP53 activators could potentially be used in combination to suppress PD-L1 up-regulation and abrogate the ability of AML cells to escape host immune elimination. Disclosures No relevant conflicts of interest to declare.


Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1747-1747 ◽  
Author(s):  
Martin A. Pule ◽  
Karin C. Straathof ◽  
Gianpietro Dotti ◽  
Helen E. Heslop ◽  
Cliona M. Rooney ◽  
...  

Abstract Artificial T-cell receptors (TCR) are generated by connecting an antigen recognizing ectodomain to a signal transducing endodomain. Most frequently the variable chains of Immunoglobulin molecules expressed as a single chain (ScFv) are utilized as ectodomains and the intracellular portion of CD3-ζ is used as endodomain. When expressed by primary T-cells these molecules can redirect the cellular immune response to almost any surface target molecule for which a monoclonal antibody can be made. However, clinical studies with these chimeric T-cells have been disappointing, with no clear clinical benefit, and only minimal in vivo persistence of infused T-cells. Transmitted CD3-ζ signal is only sufficient to activate cell-killing and Inteferon-γ release but fails to induce IL-2 release or proliferation. Full T cell activation requires co-stimulatory signals that are rarely provided by the tumor cells and therefore may need to be incorporated in the endodomain of the artificial TCR. Indeed, inclusion of a CD28 signaling component resulted in IL-2 release and limited proliferation, but T cell activation appears still incomplete. OX40 is a TNFR family molecule expressed by activated T-cells. It transmits a potent and prolonged activation signal and has been found to be an important molecule for maintaining a prolonged immunological response e.g. in chronic inflammation. We held the hypothesis that an artificial TCR providing 3 signals - CD3-ζ, CD28 and OX40 in cis would result in more potent activation and more prolonged proliferation. We generated and compared a number of constructs based on GD2 recognizing scFv 14g2a: 14g2a-ζ, 14g2a-CD28-ζ, 14g2a-OX40-ζ, 14g2a-CD28-OX40-ζ. We first co-immunoprecipitated TRAF-2 with OX40 containing constructs. This demonstrated that the OX40 binding site was unaffected by fusion with other proteins. Incorporating 3 signals - CD3ζ, CD28 and OX40 in cis from a single endodomain of an artificial TCR recruited a 10 fold higher level of NFkB quantified by Luciferase-reporter than two signals (14g2a.CD28-ζ) and over 50 fold higher than a single signal (14g2a.ζ). T-cells transduced with all of these constructs were capable of lysing GD2+ neuroblastoma cells. Only limited expansion (1.6 fold, range 0.9–3) was induced upon stimulation with tumor cells in T cells transduced with 14g2a.OX40.ζ. Adding a CD28 domain resulted in a 5.2 fold (range: 1.6–7.2) expansion within 7 days but this proliferation could not be maintained. In contrast, 14g2a.CD28.OX40.ζ transduced T cells expanded 10.7 fold (range: 4–17) within 7 days and continued to proliferate with weekly stimulations with tumor cells, even in the absence of exogenous IL-2. This increased proliferation of 14g2a.CD28.OX40.ζ transduced T cells was accompanied by a >10-fold increase in IL-2 and 5-fold increase in TNF-a secretion as compared to the 14g2a.CD28-ζ construct. Sustained proliferation was accompanied by persisting function - T-cells transduced with 14g2a.CD28-OX40-ζ were still capable of killing GD2+ targets after 35 days of culture. These improved functional characteristics should favor the overall utility of chimeric T-cells.


Antibodies ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 25
Author(s):  
Violet Y. Tu ◽  
Asma Ayari ◽  
Roddy S. O’Connor

T cell therapies, including CAR T cells, have proven more effective in hematologic malignancies than solid tumors, where the local metabolic environment is distinctly immunosuppressive. In particular, the acidic and hypoxic features of the tumor microenvironment (TME) present a unique challenge for T cells. Local metabolism is an important consideration for activated T cells as they undergo bursts of migration, proliferation and differentiation in hostile soil. Tumor cells and activated T cells both produce lactic acid at high rates. The role of lactic acid in T cell biology is complex, as lactate is an often-neglected carbon source that can fuel TCA anaplerosis. Circulating lactate is also an important means to regulate redox balance. In hypoxic tumors, lactate is immune-suppressive. Here, we discuss how intrinsic- (T cells) as well as extrinsic (tumor cells and micro-environmental)-derived metabolic factors, including lactate, suppress the ability of antigen-specific T cells to eradicate tumors. Finally, we introduce recent discoveries that target the TME in order to potentiate T cell-based therapies against cancer.


Leukemia ◽  
2021 ◽  
Author(s):  
Mohamed-Reda Benmebarek ◽  
Bruno L. Cadilha ◽  
Monika Herrmann ◽  
Stefanie Lesch ◽  
Saskia Schmitt ◽  
...  

AbstractTargeted T cell therapy is highly effective in disease settings where tumor antigens are uniformly expressed on malignant cells and where off-tumor on-target-associated toxicity is manageable. Although acute myeloid leukemia (AML) has in principle been shown to be a T cell-sensitive disease by the graft-versus-leukemia activity of allogeneic stem cell transplantation, T cell therapy has so far failed in this setting. This is largely due to the lack of target structures both sufficiently selective and uniformly expressed on AML, causing unacceptable myeloid cell toxicity. To address this, we developed a modular and controllable MHC-unrestricted adoptive T cell therapy platform tailored to AML. This platform combines synthetic agonistic receptor (SAR) -transduced T cells with AML-targeting tandem single chain variable fragment (scFv) constructs. Construct exchange allows SAR T cells to be redirected toward alternative targets, a process enabled by the short half-life and controllability of these antibody fragments. Combining SAR-transduced T cells with the scFv constructs resulted in selective killing of CD33+ and CD123+ AML cell lines, as well as of patient-derived AML blasts. Durable responses and persistence of SAR-transduced T cells could also be demonstrated in AML xenograft models. Together these results warrant further translation of this novel platform for AML treatment.


1995 ◽  
Vol 181 (1) ◽  
pp. 71-77 ◽  
Author(s):  
M R Alderson ◽  
T W Tough ◽  
T Davis-Smith ◽  
S Braddy ◽  
B Falk ◽  
...  

A significant proportion of previously activated human T cells undergo apoptosis when triggered through the CD3/T cell receptor complex, a process termed activation-induced cell death (AICD). Ligation of Fas on activated T cells by either Fas antibodies or recombinant human Fas-ligand (Fas-L) also results in cytolysis. We demonstrate that these two pathways of apoptosis are causally related. Stimulation of previously activated T cells resulted in the expression of Fas-L mRNA and lysis of Fas-positive target cells. Fas-L antagonists inhibited AICD of T cell clones and staphylococcus enterotoxin B (SEB)-specific T cell lines. The data indicate AICD in previously stimulated T cells is mediated by Fas/Fas-L interactions.


1976 ◽  
Vol 143 (3) ◽  
pp. 601-614 ◽  
Author(s):  
J W Schrader ◽  
G M Edelman

Cytotoxic T lymphocytes were generated in vitro against H-2 compatible or syngeneic tumor cells. In vitro cytotoxic activity was inhibited by specific anti-H2 sera, suggesting that H-2 antigens are involved in cell lysis. Two observations directly demonstrated the participation of the H-2 antigens on the tumor cells in their lysis by H-2-compatible T cells. First, coating of the H-2 antigens on the target tumor cell reduced the number of cells lysed on subsequent exposure to cytotoxic T cells. Second, when cytotoxic T cells were activated against an H-2 compatible tumor and assayed against an H-2-incompatible tumor, anti-H-2 serum that could bind to the target cell, but not to the cytotoxic lymphocyte, inhibited lysis. H-2 antigens were also shown to be present on the cytotoxic lymphocytes. Specific antisera reacting with these H-2 antigens, but not those of the target cell, failed to inhibit lysis when small numbers of effector cells were assayed against H-2-incompatible target cells or when effector cells of F1-hybrid origin and bearing two H-2 haplotypes were assayed against a tumor cell of one of the parental strains. These findings suggest that it is the H-2 antigens on the tumor cell and not those on the cytotoxic lymphocytes that are important in cell-mediated lysis of H-2-compatible tumor cells.


2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A743-A743
Author(s):  
Tomoyoshi Yamano ◽  
Xiabing Lyu ◽  
Rikinari Hanayama

BackgroundExosomes are vesicular granules of about 100 nm and are secreted by many types of cells. Exosomes contain various proteins, lipids, and RNAs that are transported to target cells which induce functional and physiological changes. Exosomes are promising nano-vesicles for clinical application, owing to their high biocompatibility, low immunogenicity, and high drug delivery efficacy. Recent studies have demonstrated that exosomes from tumor cells or antigen presenting cells (APCs) regulate immune responses. Tumor derived exosomes express PD-L1 on their surface and suppress tumor immunity systemically. On the other hand, mature dendritic cells derived exosomes exert immune activation, and tumor immunotherapy using DCs exosome has been developed. However, few studies have been found to exert a significant effect on cancer treatment, may be because of low expression of costimulatory molecules and lack of cytokines on DCs derived exosomes.MethodsIt has been demonstrated that GFP can be conveyed into exosomes by conjugating GFP with tetraspanins, exosome-specific surface proteins. First, we generated a tetraspanin fusion protein with a single-chain MHCI trimer (scMHCI). IL-2 is inserted on the second extracellular loop of CD81, allowing robust and functional expression of IL-2 on the exosome. We collected exosomes from HEK293 cells culture, which stably express scMHCI-CD81-IL2 and CD80-MFGE8, and used as Antigen-presenting exosome(AP-Exo).ResultsAP-Exo expresses high expression of MHCI-peptide complex, costimulatory molecule, and cytokine, activating cognate CD8 T cells as dendritic cells do. AP-Exo selectively delivered co-stimulation and IL-2 to antigen-specific CD8 T cells, resulting in a massive expansion of antigen-specific CD8 T cells without severe adverse effects in mice. AP-Exo can expand endogenous tumor-specific CD8 T cells and induce the potent anti-tumor effect.ConclusionsOur strategy for building engineered exosomes that work like APCs might develop novel methods for cancer immunotherapy.Ethics ApprovalAll mice were housed in a specific pathogen-free facility, and all animal experiments were performed according to a protocol approved by Kanazawa University, Kanazawa, Japan.


2021 ◽  
Vol 11 ◽  
Author(s):  
Hirva Mamdani ◽  
Bryan Schneider ◽  
Susan M. Perkins ◽  
Heather N. Burney ◽  
Pashtoon Murtaza Kasi ◽  
...  

BackgroundMost patients with resectable locally advanced esophageal and gastroesophageal junction (GEJ) adenocarcinoma (AC) receive concurrent chemoradiation (CRT) followed by esophagectomy. The majority of patients do not achieve pathologic complete response (pCR) with neoadjuvant CRT, and the relapse rate is high among these patients.MethodsWe conducted a phase II study (ClinicalTrials.gov Identifier: NCT02639065) evaluating the efficacy and safety of PD-L1 inhibitor durvalumab in patients with locally advanced esophageal and GEJ AC who have undergone neoadjuvant CRT followed by R0 resection with evidence of persistent residual disease in the surgical specimen. Patients received durvalumab 1500 mg IV every 4 weeks for up to 1 year. The primary endpoint was 1-year relapse free survival (RFS). Secondary endpoint was safety and tolerability of durvalumab following trimodality therapy. Exploratory endpoints included correlation of RFS with PD-L1 expression, HER-2 expression, and tumor immune cell population.ResultsThirty-seven patients were enrolled. The majority (64.9%) had pathologically positive lymph nodes. The most common treatment related adverse events were fatigue (27%), diarrhea (18.9%), arthralgia (16.2%), nausea (16.2%), pruritus (16.2%), cough (10.8%), and increase in AST/ALT/bilirubin (10.8%). Three (8.1%) patients developed grade 3 immune mediated adverse events. One-year RFS was 73% (95% CI, 56–84%) with median RFS of 21 months (95% CI, 14–40.4 months). Patients with GEJ AC had a trend toward superior 1-year RFS compared to those with esophageal AC (83% vs. 63%, p = 0.1534). There was a numerical trend toward superior 1-year RFS among patients with PD-L1 positive disease compared to those with PD-L1 negative disease, using CPS of ≥10 (100% vs. 66.7%, p = 0.1551) and ≥1 (84.2% vs. 61.1%, p = 0.1510) cutoffs. A higher relative proportion of M2 macrophages and CD4 memory activated T cells was associated with improved RFS (HR = 0.16; 95% CI, 0.05–0.59; p = 0.0053; and HR = 0.37; 95% CI, 0.15–0.93, p = 0.0351, respectively).ConclusionsAdjuvant durvalumab in patients with residual disease in the surgical specimen following trimodality therapy for locally advanced esophageal and GEJ AC led to clinically meaningful improvement in 1-year RFS compared to historical control rate. Higher PD-L1 expression may have a correlation with the efficacy of durvalumab in this setting. Higher proportion of M2 macrophages and CD4 memory activated T cells was associated with superior RFS.


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