115 Engineered T cells directed at tumors with defined allelic loss

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A127-A127
Author(s):  
Agi Hamburger ◽  
Breanna DiAndreth ◽  
Jiajia Cui ◽  
Mark Daris ◽  
Melanie Munguia ◽  
...  
Keyword(s):  
T Cells ◽  
Cancer Therapy ◽  
Tumor Cells ◽  
Function Analysis ◽  
Large Body ◽  
Allelic Loss ◽  
Signal Integration ◽  
Engineered T Cells

BackgroundCell therapy, with all its promise as a powerful solid-tumor modality, is still hampered by the fundamental obstacle of cancer therapy: the acute shortage of truly tumor-specific targets. It is well known that an average tumor contains loss of heterozygosity (LOH) at an astonishing frequency: ~20% genome wide. These losses are irreversible and absolutely distinguish the cancer from normal cells.MethodsWe describe a novel approach to cancer immunotherapy that draws on LOH as a large, so far untapped source of cancer targets. To exploit such allelic losses, we focus on polymorphic loci and target the remaining allelic product of a locus that has LOH. We engineer T cells with a modular signal-integration circuit designed to be activated only by tumor cells that have lost expression of one specific allele on their surface.ResultsWe use the HLA locus which undergoes LOH at a frequency of 13%, and the HLA-A*02 allele specifically, as proof of concept. We present a large body of quantitative in vitro data, along with in vivo data, that support the use of a synthetic signal-integration circuit called Tmod as a cancer therapy. We also describe Tmod’s mechanistic properties, including thorough structure/function analysis of its components.ConclusionsLOH is a rich source of new targets, provided a system of sufficient power can be devised to exploit them. Our Tmod signal integration system confers on engineered T cells the capacity to discriminate effectively between normal and tumor cells that contain specific allelic losses.Ethics ApprovalThe animal study was approved by Explora BioLabs’ Ethics Board, protocol number EB17-010-059

scholarly journals Hematopoietic lineage-converted T cells carrying tumor-associated antigen-recognizing TCRs effectively kill tumor cells

2020 ◽  
Vol 8 (2) ◽  
pp. e000498
Author(s):  
Fangxiao Hu ◽  
Dehao Huang ◽  
Yuxuan Luo ◽  
Peiqing Zhou ◽  
Cui Lv ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Tumor Cells ◽  
Cell Receptor ◽  
Histocompatibility Complex ◽  
Tumor Associated Antigen ◽  
Engineered T Cells ◽  
Antitumor Immunotherapy

Tumor-associated antigen (TAA) T-cell receptor (TCR) gene-engineered T cells exhibit great potential in antitumor immunotherapy. Considering the high costs and low availability of patient-derived peripheral blood T cells, substantial efforts have been made to explore alternatives to natural T cells. We previously reported that enforced expression of Hoxb5 converted B cells into induced T (iT) cells in vivo. Here, we successfully regenerated naive OT1 (major histocompatibility complex I restricted ovalbumin antigen) iT cells (OT1-iT) in vivo by expressing Hoxb5 in pro-pre-B cells in the OT1 transgenic mouse. The OT1-iT cells can be activated and expanded in vitro in the presence of tumor cells. Particularly, these regenerated OT1-iT cells effectively eradicated tumor cells expressing the TAA (ovalbumin) both in vitro and in vivo. This study provides insights into the translational applications of blood lineage-transdifferentiated T cells in immunotherapy.

scholarly journals Differential Activation of CD8+Tumor-Specific Tc1 and Tc2 Cells by an IL-10-Producing Murine Plasmacytoma

1998 ◽  
Vol 6 (3-4) ◽  
pp. 331-342 ◽  
Author(s):  
Christoph Specht ◽  
Hans-Gerd Pauels ◽  
Christian Becker ◽  
Eckehart Kölsch
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Tumor Cells ◽  
T Cell Subsets ◽  
Early Stages ◽  
Specific Tolerance

The involvement of counteractiveCD8+T-cell subsets during tumor-specific immune responses was analyzed in a syngeneic murine plasmacytoma model.CD8+Tc cells against the immunogenic IL-10-producing BALB/c plasmacytoma ADJ-PC-5 can be easily induced by immunization of BALB/c mice with X-irradiated ADJ-PC-5 tumor cellsin vivoandin vitro. However, the failure of recipient mice to mount a protective Tc response against the tumor during early stages of a real or simulated tumor growth is not due to immunological ignorance, but depends on the induction of tumor-specific tolerance, involving a population of tumorinducedCD8+T cells that are able to inhibit the generation of tumor-specific Tc cells in a primary ADJ-PC-5-specific MLTC, using IFN-γas a suppressive factor. Whereas most longterm cultivated CD8+ADJ-PC-5-specific Tc lines produce type-1 cytokines on stimulation, at least two of them, which were derived from a primary MLTC, display a type-2 cytokine spectrum. Furthermore, the primaryin vitroTc response against ADJ-PC-5 cells shows characteristics of a Tc2 response. The Tc response is strictly depending on tumor-derived IL-10.CD8+Tc cells that are induced in a primary MLTC do not produce IFN-γ, and the tumor-specific Tc response is enhanced by IL-4 but suppressed by IFN-γor IL-12. In contrast, ADJ-PC- 5-specificCD8+Tc cells from immunized mice are IFN-γproducing Tc1 cells. Since the primaryin vitroTc response against the tumor is suppressed even by the smallest numbers of irradiated ADJ-PC-5-specific Tc1 cells via IFN-γthese Tc1 cells behave similar to the suppressiveCD8+T cells that are induced during early stages of ADJ-PC-5 tumorigenesis.

scholarly journals 163 Improved anti-tumor activity of next-generation TCR-engineered T cells through CD8 co-expression

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A173-A173
Author(s):  
Gagan Bajwa ◽  
Justin Gunesch ◽  
Inbar Azoulay-Alfaguter ◽  
Melinda Mata ◽  
Ali Mohamed ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Cells ◽  
Solid Tumor ◽  
Cd4 T Cells ◽  
Tumor Response ◽  
Hla Class I ◽  
Next Generation ◽  
Engineered T Cells ◽  
Anti Tumor Activity

BackgroundSuccessful targeting of solid tumors with TCR-engineered T cells (TCR-T) will require eliciting of antigen-specific, multi-dimensional, sustained anti-tumor immune response by infused T cells while overcoming the suppressive tumor microenvironment. First-generation TCR-T approaches have demonstrated clinical efficacy in some solid cancers. However, effective treatment across several solid tumor indications may require engineered T cells with enhanced anti-tumor activity. Here, we show pre-clinical data from one of the engineering approaches currently being developed for next-generation ACTengine® TCR-T product candidates. We evaluated the impact of co-expression of different CD8 co-receptors on functionality of CD4+ and CD8+ T cells genetically modified with an HLA class I-restricted TCR and determined the depth and durability of anti-tumor response in vitro.MethodsHere, we used a PRAME-specific TCR currently being tested in the ACTengine® IMA203 clinical trial. T cells expressing either the TCR alone or co-expressing the TCR and CD8α homodimer (TCR.CD8α) or CD8αβ heterodimer (TCR.CD8αβ) were characterized for transgene expression, antigen-recognition, and functional efficacy in vitro. Comprehensive evaluation of CD4+ T cells expressing TCR.CD8α or TCR.CD8αβ was performed focusing on cytotoxic potential and the breadth of cytokine response against target-positive tumor cell lines.ResultsIntroduction of CD8α or CD8αβ enabled detection of transgenic TCR on the surface of CD4+ T cells via HLA multimer-guided flow cytometry otherwise lacking in the TCR only transduced T cells. Co-expression of either form of CD8 co-receptor endowed CD4+ T cells with the ability to recognize and kill target positive tumor cells; however, genetic modification with TCR.CD8αβ led to more pronounced CD4+ T cell activation as compared to TCR.CD8α. Most distinct differences were observed in the breadth and magnitude of cytokine responses, less in cytotoxic activity against tumor cells. T cells expressing TCR.CD8αβ showed superior induction of Th1 cytokines e.g. IFNγ, TNFα, IL-2, GM-CSF in vitro upon antigen stimulation as compared to TCR.CD8α-T cells. Additionally, TCR.CD8αβ T cells demonstrated more efficient engagement with antigen-presenting cells and consequently, modulation of cytokine response than TCR.CD8α-T cells.ConclusionsOur findings illustrate that engaging CD4+ T cells via CD8 co-expression potentiates anti-tumor activity of HLA class I restricted TCR-T cells in vitro. The pleiotropic effects mediated by activated CD4+ T cells including acquired cytotoxicity may potentially improve outcomes in solid tumor patients when applied clinically. In addition, the differential functional profile of TCR-T cells co-expressing either CD8α or CD8αβ suggests that optimizing the type of co-receptor is relevant to maximize anti-tumor response.

scholarly journals In vitro-transcribed antigen receptor mRNA nanocarriers for transient expression in circulating T cells in vivo

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
N. N. Parayath ◽  
S. B. Stephan ◽  
A. L. Koehne ◽  
P. S. Nelson ◽  
M. T. Stephan
Keyword(s):  
T Cells ◽  
Ex Vivo ◽  
Human Leukemia ◽  
Antigen Receptors ◽  
Wide Range ◽  
Engineered T Cells ◽  
Circulating T Cells ◽  
The Cost

AbstractEngineering chimeric antigen receptors (CAR) or T cell receptors (TCR) helps create disease-specific T cells for targeted therapy, but the cost and rigor associated with manufacturing engineered T cells ex vivo can be prohibitive, so programing T cells in vivo may be a viable alternative. Here we report an injectable nanocarrier that delivers in vitro-transcribed (IVT) CAR or TCR mRNA for transiently reprograming of circulating T cells to recognize disease-relevant antigens. In mouse models of human leukemia, prostate cancer and hepatitis B-induced hepatocellular carcinoma, repeated infusions of these polymer nanocarriers induce sufficient host T cells expressing tumor-specific CARs or virus-specific TCRs to cause disease regression at levels similar to bolus infusions of ex vivo engineered lymphocytes. Given their ease of manufacturing, distribution and administration, these nanocarriers, and the associated platforms, could become a therapeutic for a wide range of diseases.

Class II-Transfected Tumor Cells Directly Present Endogenous Antigen to CD4+ T Cells In Vitro and Are APCs for Tumor-Encoded Antigens In Vivo

1998 ◽  
Vol 21 (3) ◽  
pp. 218-224 ◽  
Author(s):  
Todd D. Armstrong ◽  
Virginia K. Clements ◽  
Suzanne Ostrand-Rosenberg
Keyword(s):  
T Cells ◽  
Tumor Cells ◽  
Cd4 T Cells ◽  
Class Ii

CD3 limits the efficacy of TCR gene therapy in vivo

Blood ◽  
2011 ◽  
Vol 118 (13) ◽  
pp. 3528-3537 ◽  
Author(s):  
Maryam Ahmadi ◽  
Judith W. King ◽  
Shao-An Xue ◽  
Cécile Voisine ◽  
Angelika Holler ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Cell Receptor ◽  
Surface Expression ◽  
T Cell Function ◽  
Engineered T Cells ◽  
Rate Limiting

Abstract The function of T-cell receptor (TCR) gene modified T cells is dependent on efficient surface expression of the introduced TCR α/β heterodimer. We tested whether endogenous CD3 chains are rate-limiting for TCR expression and antigen-specific T-cell function. We show that co-transfer of CD3 and TCR genes into primary murine T cells enhanced TCR expression and antigen-specific T-cell function in vitro. Peptide titration experiments showed that T cells expressing introduced CD3 and TCR genes recognized lower concentration of antigen than T cells expressing TCR only. In vivo imaging revealed that TCR+CD3 gene modified T cells infiltrated tumors faster and in larger numbers, which resulted in more rapid tumor elimination compared with T cells modified by TCR only. After tumor clearance, TCR+CD3 engineered T cells persisted in larger numbers than TCR-only T cells and mounted a more effective memory response when rechallenged with antigen. The data demonstrate that provision of additional CD3 molecules is an effective strategy to enhance the avidity, anti-tumor activity and functional memory formation of TCR gene modified T cells in vivo.

scholarly journals Ectonucleosidase CD39 Is Highly Expressed on ATLL Cells and Suppresses the Immune Response through the Adenosine Pathway

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3720-3720
Author(s):  
Yasuhiro Nagate ◽  
Sachiko Ezoe ◽  
Jiro Fujita ◽  
Takafumi Yokota ◽  
Michiko Ichii ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Tumor Cells ◽  
Research Funding ◽  
Poly I:C ◽  
Acute Type ◽  
Atll Cell

Abstract Background: Adult T-cell leukemia/lymphoma (ATLL) is a mature T-cell neoplasm, linked to the human T-cell lymphotropic virus, HTLV-1. Patients with ATLL are often at the risk of opportunistic infections. Some studies suggested that ATLL cells originate from HTLV-1-infected regulatory T cells (Tregs). It could be possible that this immunocompromised state is caused by the function of ATLL cells having similar phenotypes with Tregs. In this study, we examined the expression of immunosuppressive molecules associated with Tregs in ATLL cells, and analyzed their roles in the function of ATLL cells. Methods: The protocol of this study was approved by the Investigational Review Board of Osaka University Hospital. Peripheral blood mononuclear cells (PBMCs) were collected from 10 asymptomatic HTLV-1 carriers and 22 ATLL patients (1 with smoldering type, 5 with chronic type, 2 with lymphoma type, and 14 with acute type) after getting informed consent. PBMCs from 3 ATLL patients were separated into CD4+ CD7- CADM1+ATLL cells and adjacent CD4+CD7+ CADM1-normal T cells using Fluorescence-activated Cell Sorter (FACS), and cells in each fraction were subjected to total RNA sequencing experiments. Based on the results, we examined the expression patterns of CD39 and CD73 in HTLV-1 carriers or each type of ATLL patients, and also analyzed the immune functions of these molecules in ATLL tumor cells. Results: We compared whole transcriptome of ATLL cells and normal CD4+cells. Bioinformatic analyses showed that many genes associated with immunosuppressive functions were elevated or downregulated in ATLL cells. Among these genes we focused on CD39, CD73 and CD26, because they have recently been reported to be strongly associated with the functions of Tregs. CD39, expressed on normal Tregs, and extrinsic CD73 have immunosuppressive potential by catalyzing adenosine from extracellular ATP, and CD26 has opposite potential by resolving adenosine, which have a strong anti-inflammatory function and plays major role in Treg-mediated immunosuppression. We found that all of 4 ATLL cell lines (MJ, MT1, MT2, MT4) expressed CD39, but not CD73 just as human effector Tregs. Tumor cells from 12 acute ATLL patients (86%) and 2 chronic ATLL patients (40%) expressed CD39, but the expressions of CD73 were various. Also in asymptomatic carriers, we could detect CD39 and/or CD73 positive in CD7- CADM1+ abnormal fraction of CD4+cells. On the other hand, CD26, normally expressed on human CD4+Th cells other than effector Tregs, was negative in ATLL cell lines and primary ATLL cells except for cells in abnormal fraction of one asymptomatic carrier. CD39 negative cases in chronic/smoldering type tended to show slower disease progression after the blood collection. Next, the role of CD39 and/or CD73 in ATLL cells was assessed in vitro and in vivo. As expected, CD39+ ATLL cells converted significantly more extracellular ATP than CD39- ATLL cells, and mass spectrometry analysis of AMP/adenosine concentration identified the AMPase activity of CD73+ ATLL cells. Furthermore, we established CD39 knockout (KO) cells from ATL cell-line MJ using CRISPR/Cas9 system, and performed in vitro suppression assays for assessment of immunosuppressive function. Although wild type MJ suppressed the growth of normal CD4+ and CD8+ T cells, KO MJ did little. Next, we analyzed the role of CD39 in the progression of tumor cells in vivo. We transplanted mouse T-cell lymphoma cell-line EG7-OVA artificially expressing CD39 or mock into mice subcutaneously. The coinjection of immunoadjuvant poly(I:C) significantly suppressed the tumor growth of mock cells, but the tumor sizes of CD39 expressing cells were almost the same as those of mock cells without poly(I:C) injection (Figure). Conclusion: In this study, we reported that most of ATLL cells in acute type patients express CD39+ CD26- just as Tregs, and that CD39- KO of ATLL cell line cancelled its immunosuppressive effects, and forcibly expressed CD39 on tumor cells rejected the anti-tumor immunity in vivo. From these data, we clarified the pathological mechanism of immunosuppressive function in ATLL cells, and also showed that CD39 expression could be used as a prognostic clue and be a new therapeutic target of ATLL. Disclosures Ezoe: TAIHO Phamaceutical Co., Ltd.: Research Funding. Yokota:Celgene: Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer Inc.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; MSD K.K.: Research Funding. Ichii:Novartis Pharma K.K.: Speakers Bureau; Kowa Pharmaceutical Co.,LTD.: Speakers Bureau; Celgene K.K.: Speakers Bureau. Shibayama:Novartis Pharma K.K.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Fujimoto Pharmaceutical: Honoraria, Research Funding; Jansen Pharmaceutical K.K: Honoraria; Ono Pharmaceutical Co.,LTD: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Bristol-Meyer Squibb K.K: Honoraria, Research Funding. Oritani:Novartis Pharma: Speakers Bureau. Kanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.

scholarly journals NKG2D-CAR Transduced Primary Natural Killer Cells Efficiently Target Multiple Myeloma Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 590-590 ◽  
Author(s):  
Alejandra Leivas ◽  
Paula Rio ◽  
Rebeca Mateos ◽  
Mari Liz Paciello ◽  
Almudena Garcia-Ortiz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Cell Therapy ◽  
Nk Cells ◽  
Tumor Cells ◽  
Research Funding ◽  
Car T ◽  
Equity Ownership

Abstract Introduction Immunotherapy represents a new weapon in the fight against multiple myeloma. Current clinical outcomes using CAR-T cell therapy against multiple myeloma show promise in the eradication of the disease. However, these CARs observe relapse as a common phenomenon after treatment due to the reemergence of neoantigens or negative cells. CARs can also be targeted using non-antibody approaches, including the use of receptors, as NKG2D with a wider range of ligands, and ligands to provide target specificity. Different cell types have been used to improve CAR cell therapy. CAR-T cells are the most commonly used. However, despite its effectiveness, there are still problems to face. The toxicity of the cytokine release syndrome is well known, that is why memory CD45RA- T cells are used to avoid collateral effects, although having lower efficacy. However, CAR-NK cells may have less toxicity and provide a method to redirect these cells specifically to refractory cancer. The objective of this work was to compare the anti-tumor activity of CAR-T, NKAEs and CAR-NK cells from multiple myeloma patients. Methods The activated and expanded NK cells (NKAE) were generated by coculture of peripheral blood mononuclear cells with the previously irradiated CSTX002 cell line. The CD45RA- T cells were obtained by depletion with CD45RA magnetic beads and subsequent culture. The NKAE and T were transduced with an NKG2D-CAR with signaling domains of 4-1BB and CD3z. The expansion of NKAE and the expression of NKG2D-CAR were evaluated by flow cytometry based on the percentage of NK cell population and transduction efficiency by the expression of NKG2D. Europium-TDA release assays (2-4 hours) were performed to evaluate in vitro cytotoxic activity. The antitumor activity of the NKAE (n=4) and CD45RA- (n=4) cells against MM U-266 cells was studied. Methylcellulose cultures were performed to assess the activity against the clonogenic tumor cell. In vivo studies were carried out in NSG mice receiving 5.106 of U266-luc MM cells i.v. injected at day 1. At day 4, mice received 15.106 i.v. injected of either CAR-NKAE or untransduced NKAE cells. Results In vitro. The killing activity of primary NKAE cells (n=4) was 86.6% (± 13.9%), considerably higher than that of CD45RA- lymphocytes (16.7% ± 13.6%) from the same patient (n=4). Even CD45RA- T cells from healthy donors (n=4) exhibit lower anti tumoral capacity (28.2% ± 9.7%) than NKAE cells. The transduction with an NKG2D CAR (MOI=5) improved the activity of autologous NKAE cells by 10% (96.4% ± 19%) leading to a nearly complete destruction of U-266 MM cells, and that of CD45RA- allogenic healthy cells in 19% (47.4% ± 12.6%). Nevertheless, CD45RA- autologous T cells transduced with NKG2D-CAR minimally improved their activity by 5.8% (22.5% ± 10.6%). Additionally, the CAR-NKAE cells were able to destroy the clonogenic tumor cell responsible for the progression of the MM from RPMI-8226 cell line. At an 8:1 ratio the CAR-NKAE cells were able to destroy 71.2% ± 2.5% of the clonogenic tumor cells, while the NKAE reached 56.5% ± 2.6% at a maximum ratio of 32: 1. The toxicity of the CAR-NKAE cells on healthy tissue from the same patient was assessed, and no activity against autologous PBMCs was observed, 1,8% at a maximun ratio of 32:1 (effector:target). In vivo. NKAE cells and CAR-NKAE cells were efficient in abrogating MM growth. However, CAR-NKAE cells treatment showed higher efficiency 14 days after tumor cells injection. Forty-two days after tumor cells injection, only animals receiving CAR-NKAE cells treatment remain free of disease (Figure 1). Conclusions It is feasible to modify primary NKAE cells and CD45RA- T cells from primary MM cells to safely express an NKG2D-CAR. Our data show that CD45RA- T cells from patients are not effective in vitro against MM even once transduced with our CAR. The resulting CAR-NKG2D NKAE cells are the most appropriate strategy for the destruction of MM in vitro and in vivo in our model. These results form the basis for the development of an NKG2D-CAR NK cell therapy in MM. Disclosures Rio: Rocket Pharmaceuticals Inc: Equity Ownership, Patents & Royalties, Research Funding. Lee:Merck, Sharp, and Dohme: Consultancy; Courier Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CytoSen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Martinez-Lopez:Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Vivia: Honoraria; Pfizer: Research Funding; BMS: Research Funding; Novartis: Research Funding.

scholarly journals PD-L1 chimeric costimulatory receptor improves the efficacy of CAR-T cells for PD-L1-positive solid tumors and reduces toxicity in vivo

2020 ◽  
Author(s):  
Qibin Liao ◽  
Yunyu Mao ◽  
Huan He ◽  
Xiangqing Ding ◽  
Xiaoyan Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
Solid Tumors ◽  
Jurkat T Cells ◽  
Costimulatory Receptor ◽  
Car T Cells ◽  
Car T ◽  
Engineered T Cells ◽  
Therapeutic Safety

Abstract Background: On-target off-tumor toxicity impedes the clinical application of chimeric antigen receptor-modified T cells (CAR-T cells) in the treatment of solid tumors. The combinatorial antigen recognition strategy can improve the therapeutic safety of CAR-T cells by targeting two different tumor-associated antigens (TAAs) using a CAR and a chimeric costimulatory receptor (CCR). Although programmed death-ligand 1 (PD-L1, also known as B7-H1) is expressed on multiple tumors, the potential of PD-L1 as a universal target for designing CCR remains unknown.Methods: A first-generation CD19 or HER2 CAR and a PD-L1 CCR containing the CD28 signaling domain were constructed and delivered into Jurkat T cells or primary T cells by a pseudotyped lentivirus. The release of cytokines, including IL-2, IFN-γ and TNF-α, was quantified using enzyme-linked immunosorbent assay (ELISA) kits or a cytometric bead array (CBA). The in vitro cytotoxicity of CAR-T cells was detected with a luciferase-based killing assay. The in vitro proliferation of CAR-T cells was assessed by flow cytometry. The therapeutic safety and efficacy of CAR-T cells was evaluated using a subcutaneous dual-tumor model in vivo.Results: Jurkat T cells or primary T cells expressing both the CD19/HER2 CAR and PD-L1 CCR produced higher levels of cytokines in the presence of CD19/HER2 and PD-L1 than in the presence of HER2/CD19. Compared to HER2-z-engineered T cells, HER2-z-PD-L1-28-engineered T cells had higher in vitro cytotoxicity potential against PD-L1-positive tumor cells. CD19/HER2-z-PD-L1-28-engineered T cells showed higher proliferation potential in the presence of CD19/HER2 and PD-L1 than in the absence of PD-L1. CD19/HER2-z-PD-L1-28-engineered T cells preferably destroyed xenograft tumors expressing CD19/HER2 and PD-L1 in vivo and did not significantly affect CD19/HER2-expressing tumors. The PD-L1 CCR improved the antitumor efficacy of low-affinity HER2 CAR-T cells against PD-L1-positive tumors expressing high levels of HER2.Conclusion: Our findings confirmed that PD-L1 can be used as a universal target antigen for designing CCR, improving the efficacy of CAR-T cells in the treatment of PD-L1-positive solid tumors but reducing toxicity within PD-L1-negative normal tissues expressing low levels of TAA in vivo.

scholarly journals T Cells Engineered with a Novel Chimeric Receptor Demonstrate Durable In Vivo Efficacy Against Disseminated Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 962-962 ◽  
Author(s):  
Ksenia Bezverbnaya ◽  
Vivian Lau ◽  
Craig Aarts ◽  
Galina Denisova ◽  
Arya Afsahi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Inhibitory Effect ◽  
In Vivo Efficacy ◽  
Engineered T Cells

Abstract Despite recent therapeutic developments, multiple myeloma remains an incurable plasma cell malignancy. Poor prognosis for myeloma patients relapsing post-transplant calls for the need for novel treatment options. Immunotherapy with engineered T cells has proven highly efficacious against B-cell cancers, and early-phase clinical trials suggest that multiple myeloma is susceptible to this form of therapy. We designed a new chimeric T cell receptor, T cell antigen coupler (TAC), which relies upon activation through endogenous T cell receptor complex, thus allowing engineered T cells to auto-regulate their activity (Helsen et al, Nat. Comm., 2018). Using published single-chain antibody fragments (scFvs) C11D5.3 and J22.9-xi, we generated B cell maturation antigen (BCMA)-specific TAC receptors for targeting multiple myeloma. Primary human T cells were transduced with lentiviral vectors carrying different BCMA TAC constructs and assessed for in vitro functionality via cytokine production, cytotoxicity, and proliferation assays. In vivo efficacy and T cell tracking were performed in an established orthotopic xenograft mouse model based on a BCMA-positive KMS-11 cell line. C11D5.3 and J22.9-xi TAC T cells demonstrated comparable in vitro performance with both types of cultures efficiently killing BCMA-expressing targets, producing IFN-γ, TNF-α, and IL-2 cytokines, and undergoing multiple rounds of proliferation. In vivo, TAC T cells carrying either scFv were capable of curing mice bearing disseminated myeloma; however, the TAC T cells carrying J22.9-xi scFv were more potent on a per-cell basis (Figure 1A, top panel). Mice in remission 3 months post-treatment with a single dose of 106 TAC-positive T cells showed evidence of sustained anti-tumor protection upon rechallenge with a fresh dose of 106 KMS-11 tumor cells (Figure 1B). Mice treated with low-dose J22.9-xi T cells were more resistant to rechallenge than mice treated with a comparable dose of C11D5.3 TAC T cells. Tracking of the TAC T cells in vivo revealed that the J22.9-xi TAC T cells expanded to a much larger extent than the C11D5.3 TAC T cells (Figure 1A, bottom panel), indicating that there were likely more J22.9-xi TAC T cells present at the time of tumor rechallenge. To understand whether biological aspects of BCMA may influence the proliferative response of the TAC T cells, we explored the influence of APRIL, the soluble ligand for BCMA, on TAC T cell proliferation in vitro. Strikingly, despite comparable proliferation of both TAC T cell populations following stimulation with KMS-11 tumor cells in the absence of APRIL in vitro, the presence of APRIL had a strong inhibitory effect on proliferation of C11D5.3 TAC T cells and only a modest inhibitory effect on J22.9-xi TAC T cells. Our preclinical findings support further development of TAC T cells for the treatment of multiple myeloma and underscore the importance of T cell expansion in determining the therapeutic activity of engineered T cells. This work further reveals a novel observation that the natural ligand of BCMA can impair the therapeutic impact of T cells engineered with chimeric receptors directed against BCMA and provide a basis for advancing BCMA-specific TAC T cells into the clinic. Disclosures Denisova: Triumvira Immunologics: Patents & Royalties. Afsahi:Triumvira Immunologics: Patents & Royalties. Helsen:Triumvira Immunologics: Employment, Patents & Royalties. Bramson:Triumvira Immunologics: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

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