scholarly journals Oncolytic adenovirus and gene therapy with EphA2-BiTE for the treatment of pediatric high-grade gliomas

2021 ◽  
Vol 9 (5) ◽  
pp. e001930
Author(s):  
Claudia Manuela Arnone ◽  
Vinicia Assunta Polito ◽  
Angela Mastronuzzi ◽  
Andrea Carai ◽  
Francesca Camassei Diomedi ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Tumor Control ◽  
Malignant Neoplasms ◽  
High Grade ◽  
High Grade Gliomas

BackgroundPediatric high-grade gliomas (pHGGs) are among the most common and incurable malignant neoplasms of childhood. Despite aggressive, multimodal treatment, the outcome of children with high-grade gliomas has not significantly improved over the past decades, prompting the development of innovative approaches.MethodsTo develop an effective treatment, we aimed at improving the suboptimal antitumor efficacy of oncolytic adenoviruses (OAs) by testing the combination with a gene-therapy approach using a bispecific T-cell engager (BiTE) directed towards the erythropoietin-producing human hepatocellular carcinoma A2 receptor (EphA2), conveyed by a replication-incompetent adenoviral vector (EphA2 adenovirus (EAd)). The combinatorial approach was tested in vitro, in vivo and thoroughly characterized at a molecular level.ResultsAfter confirming the relevance of EphA2 as target in pHGGs, documenting a significant correlation with worse clinical outcome of the patients, we showed that the proposed strategy provides significant EphA2-BiTE amplification and enhanced tumor cell apoptosis, on coculture with T cells. Moreover, T-cell activation through an agonistic anti-CD28 antibody further increased the activation/proliferation profiles and functional response against infected tumor cells, inducing eradication of highly resistant, primary pHGG cells. The gene-expression analysis of tumor cells and T cells, after coculture, revealed the importance of both EphA2-BiTE and costimulation in the proposed system. These in vitro observations translated into significant tumor control in vivo, in both subcutaneous and a more challenging orthotopic model.ConclusionsThe combination of OA and EphA2-BiTE gene therapy strongly enhances the antitumor activity of OA, inducing the eradication of highly resistant tumor cells, thus supporting the clinical translation of the approach.

Clonal Analyses of Retroviral Vector Integrations in ADA-SCID Patients Treated with Stem Cell Gene Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3249-3249
Author(s):  
Barbara Cassani ◽  
Grazia Andolfi ◽  
Massimiliano Mirolo ◽  
Luca Biasco ◽  
Alessandra Recchia ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Human Genome ◽  
Ex Vivo ◽  
Cd34 Cells

Abstract Gene transfer into hematopoietic stem/progenitor cells (HSC) by gammaretroviral vectors is an effective treatment for patients affected by severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA)-deficiency. Recent studied have indicated that gammaretroviral vectors integrate in a non-random fashion in their host genome, but there is still limited information on the distribution of retroviral insertion sites (RIS) in human long-term reconstituting HSC following therapeutic gene transfer. We performed a genome-wide analysis of RIS in transduced bone marrow-derived CD34+ cells before transplantation (in vitro) and in hematopoietic cell subsets (ex vivo) from five ADA-SCID patients treated with gene therapy combined to low-dose busulfan. Vector-genome junctions were cloned by inverse or linker-mediated PCR, sequenced, mapped onto the human genome, and compared to a library of randomly cloned human genome fragments or to the expected distribution for the NCBI annotation. Both in vitro (n=212) and ex vivo (n=496) RIS showed a non-random distribution, with strong preference for a 5-kb window around transcription start sites (23.6% and 28.8%, respectively) and for gene-dense regions. Integrations occurring inside the transcribed portion of a RefSeq genes were more represented in vitro than ex vivo (50.9 vs 41.3%), while RIS <30kb upstream from the start site were more frequent in the ex vivo sample (25.6% vs 19.4%). Among recurrently hit loci (n=50), LMO2 was the most represented, with one integration cloned from pre-infusion CD34+ cells and five from post-gene therapy samples (2 in granulocytes, 3 in T cells). Clone-specific Q-PCR showed no in vivo expansion of LMO2-carrying clones while LMO2 gene overexpression at the bulk level was excluded by RT-PCR. Gene expression profiling revealed a preference for integration into genes transcriptionally active in CD34+ cells at the time of transduction as well as genes expressed in T cells. Functional clustering analysis of genes hit by retroviral vectors in pre- and post-transplant cells showed no in vivo skewing towards genes controlling self-renewal or survival of HSC (i.e. cell cycle, transcription, signal transduction). Clonal analysis of long-term repopulating cells (>=6 months) revealed a high number of distinct RIS (range 42–121) in the T-cell compartment, in agreement with the complexity of the T-cell repertoire, while fewer RIS were retrieved from granulocytes. The presence of shared integrants among multiple lineages confirmed that the gene transfer protocol was adequate to allow stable engraftment of multipotent HSC. Taken together, our data show that transplantation of ADA-transduced HSC does not result in skewing or expansion of malignant clones in vivo, despite the occurrence of insertions near potentially oncogenic genomic sites. These results, combined to the relatively long-term follow-up of patients, indicate that retroviral-mediated gene transfer for ADA-SCID has a favorable safety profile.

Modeling Human T Cell Acute Lymphoblastic Leukemia In Vitro and In Vivo with LMO2.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3648-3648
Author(s):  
James A Kennedy ◽  
Sara Berthiaume ◽  
Frederic Barabe
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Progenitor Cell ◽  
Leukemic Cell ◽  
Human Cells ◽  
Immunodeficient Mice ◽  
Human T Cell

Abstract Abstract 3648 The studies identifying gene translocations and mutations in T-ALL cell lines and/or in patients have contributed significantly to the understanding of the genetic abnormalities involved in T-ALL. However, studies on the biology of these genes, the targeted cells, the sequence and the number of hits required to convert a primary human hematopoietic stem cell (HSC)/progenitor cell into a fully transformed leukemic cell require good experimental models of human T cell development both in vivo and in vitro. The only in vivo model of human T cell leukemogenesis came unexpectedly from the gene therapy trial on patients with X-linked severe combined immunodeficiency (SCID-X1). Three to five years after gene therapy, 4 out of 10 patients in the trial developed clonal T-ALL. In these patients, retroviral integrations were found in proximity to the LMO2 promoter in the malignant clones, leading to aberrant expression of the oncogene. However, little is known on the effect of LMO2 overexpression in human cells and how it facilitates the development of T-ALL. We have developed in vivo and in vitro models to study the role of T cell oncogenes in human cells. Using the OP9-DL1 co-culture system to differentiate human HSC into mature T cells in vitro, we culture human HSC transduced with lentiviruses expressing LMO2. LMO2 overexpressing cells are blocked at the double negative stage (CD4-CD8-) of differentiation when co-cultured on OP9-Delta-Like1 stroma and proliferate 50 to 100 times more than control cells. However, these cells are not immortalized and cultures lasted approximately 80 days. LMO2 overexpression have no effect on myeloid differentiation in vitro. In vivo, LMO2 transduced human HSC/progenitor cells engraft the bone marrow of immunodeficient mice to levels comparable to control cells, while normal myeloid and B cell populations 20–24 weeks post-transplantation. LMO2 transduced cells have an increased capacity to generate T cells in the thymus in comparison to control cells (42% engraftment vs 8%, p<0.0001). Surprisingly, thymic and peripheral LMO2 cells are not blocked in their differentiation. LMO2 cells did not engraft secondary mice, confirming that LMO2 doesn't induce self-renewal of human HSC. However, the increase in thymic repopulation by LMO2 cells and the lack of differentiation block in vivo suggest that LMO2 overexpression generates an abnormal T cell population with an increase repopulation advantage (increase proliferation or decrease apoptosis) in the thymus which becomes the substrate for additional genetic/epigenetic events. To test this hypothesis, we tried to immortalize LMO2 cells in vitro with secondary hits. Our preliminary results show that insertional mutagenesis can immortalized LMO2 cells in vitro. However these cells are not able to engraft immunodeficient mice or generate leukemia in vivo. The addition of intracellular NOTCH to one immortalized LMO2 cell line allows these cells to engraft and generate human T-ALL in vivo. Globally, these results show that T cell oncogenes can be studied in primary human hematopoietic cells both in vitro and in vivo. Also, at least three hits are required to transform a human primary HSC/progenitor cell into a leukemic cell able to engraft and generate leukemia in vivo. It also suggests that a non-engrafting cell can be turned into a leukemic cell generating leukemia in vivo, implying that a cell can regain self-renewing properties. Disclosures: No relevant conflicts of interest to declare.

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.

Development of human NKG2D-CD3ε chimeric antigen receptor (CAR) for T-cell-mediated cancer immunotherapy.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 150-150
Author(s):  
Sergei Kusmartsev ◽  
Johaness Vieweg ◽  
Victor Prima
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Adaptor Protein ◽  
T Cell Subsets ◽  
Human T Cells

150 Background: NKG2D is a lectin-like type 2 transmembrane receptor that expressed by natural killer cells and some T cell subsets. Stimulation of NKG2D receptor with specific agonistic ligands produces activating signals through signaling adaptor protein DAP10 leading to the enhanced cytokine production, proliferation, and cytotoxicity against tumor cells. There is strong evidence that NKG2D ligands are expressed in many human tumors, including melanoma, leukemia, myeloma, glioma, and carcinomas of the prostate, breast, lung, and colon. Recent studies also demonstrated that T cells bearing chimeric antigen receptor (CAR) NKG2D linked to CD3ζ (zeta) chain produce marked in vitro and in vivo anti-tumor effects. The aim of current study was to determine whether human T cells bearing chimeric antigen receptor (CAR) NKGD2 linked to CD3ε (epsilon) chain could be activated by the NKG2D-specific stimulation and able to kill human cancer cells. Given the important role of CD3ε in activation and survival of T cells, we hypothesized that NKG2D-CDε-bearing T cells could exert strong in vitro and in vivo anti-tumor effects. Methods: NKG2D CAR was produced by linking human NKG2D to DAP10 and the cytoplasmic portion of the CD3ε chain. Original full-length human cDNA clones were obtained from NIH Mammalian Gene Collection (MGC). Functional domain analysis and oligonucleotide design in the in-Fusion system of DNA cloning (Clontech) was used to generate the retroviral expression constructs. Results: Human PBMC-derived T cells were retrovirally transduced with newly generated NKG2D-CD3ε CAR DNA construct. These NKG2D CAR-expressing human T cells responded to NKG2D-specific activation by producing IFN-γ and exhibited significant cellular cytotoxicity against human tumor cells in vitro. In vivo studies demonstrated that NKG2D-CD3ε-bearing cells are capable of inhibiting growth of DU-145 human prostate cancer in the immunodeficient mice. Conclusions: Collectively, our data indicate the feasibility of developing chimeric antigen receptor NKG2D-CD3ε for T cells and suggest that adoptive transfer of T cells bearing NKG2D-CD3ε CAR could be potentially effective for immunotherapy of cancer patients.

TNB-486, a Novel Fully Human Bispecific CD19 x CD3 Antibody That Kills CD19-Positive Tumor Cells with Minimal Cytokine Secretion

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4070-4070
Author(s):  
Harbani Malik ◽  
Ben Buelow ◽  
Udaya Rangaswamy ◽  
Aarti Balasubramani ◽  
Andrew Boudreau ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Cytokine Secretion ◽  
Employment Equity ◽  
Equity Ownership

Introduction The restricted expression of CD19 in the B-cell lineage makes it an attractive target for the therapeutic treatment of B-cell malignancies. Many monoclonal antibodies and antibody drug conjugates targeting CD19 have been developed, including bispecific T-cell redirecting antibodies (T-BsAbs). In addition, anti-CD19 chimeric antigen receptor T-cells (CAR-T) have been approved to treat leukemia and lymphoma. However, despite the impressive depth of responses achieved by T-cell redirecting approaches such as T-BsAbs and CAR-T cells, toxicity from over-activation of T-cells remains a substantial limitation for this type of therapy, in particular neurotoxicity. In designing TNB-486, a novel CD19 x CD3 T-BsAb, we endeavored to retain activity against CD19-positive tumor cells while limiting the cytokine secretion thought to underlie toxicity from T-cell redirecting therapies. Utilizing TeneoSeek, a next generation sequencing (NGS)-based discovery pipeline that leverages in silico analysis of heavy chain only/fixed light chain antibody (HCA/Flic, respectively) sequences to enrich for antigen specific antibodies, we made a high affinity αCD19 HCA and a library of αCD3 Flic antibodies that showed a >2 log range of EC50s for T cell activation in vitro. Of note, the library contained a low-activating αCD3 that induced minimal cytokine secretion even at concentrations that mediated saturating T-cell dependent lysis of lymphoma cells (when paired with an αCD19 HCA). We characterized the relative efficacy and potential therapeutic window of this unique molecule, TNB-486, in vitro and in vivo and compared it to two strongly activating bispecific CD19 x CD3 antibodies similar to those currently available and in clinical development. Methods Affinity measurements of the αCD19 moiety were made via Biacore (protein) and flow cytometry (cell surface). Stability measurements were made by subjecting the molecule to thermal stress and the %aggregation was measured by Size Exclusion Chromatography. T-cell activation was measured via flow cytometry (CD69 and CD25 expression) and cytokine was measured by ELISA (IL-2, IL-6, IL-10, INF-ɣ, and TNFα) in vitro. Lysis of B-cell tumor cell lines (Raji, RI-1, and Nalm6) was measured via flow cytometry in vitro. In vivo, NOG mice were engrafted subcutaneously with NALM-6 or SUDHL-10 cells and intravenously with human peripheral blood mononuclear cells (huPBMC), and the mice treated with multiple doses of TNB-486 or negative or positive control antibody. Tumor burden was evaluated via caliper measurement. Pharmacodynamic/Pharmacokinetic (PK/PD) studies were performed in NOG mice. A pharmacokinetic (PK) study was performed in BALB/c mice, and a tolerability and PK study are ongoing in cynomolgus monkeys. Results TNB-486 bound to cell surface CD19 with single digit nanomolar affinity (~3nM). EC50s for cytotoxicity were in the single-digit nanomolar range for TNB-486, and sub-nanomolar for the strongly activating controls; TNB-486 maximum achievable lysis was identical to the positive controls. TNB-486 induced significantly less cytokine release for all cytokines tested compared to the positive controls even at doses saturating for tumor lysis. No off-target activation was observed in the absence of CD19 expressing target cells. In vivo, TNB-486 eradicated all CD19-positive tumors tested (NALM-6 and SUDHL10) at doses as little as 1µg administered every four days after tumors had reached ~200mm3. TNB-486 showed a PK profile consistent with other IgG molecules in mice (T1/2 ~6 days in mice). Conclusions TNB-486 induced comparable lysis of CD19-positive tumor cells as the strongly activating control bispecific antibodies while inducing significantly reduced cytokine secretion, even at doses saturating for tumor lysis in vitro. In vivo TNB-486 eradicated all tested CD19 positive tumor cell lines in established tumor models. No off-target binding was observed. In summary, TNB-486 shows promise as a lymphoma therapeutic differentiated from T-cell targeted therapies currently in the clinic and in clinical trials. Disclosures Malik: Teneobio, Inc.: Employment, Equity Ownership. Buelow:Teneobio, Inc.: Employment, Equity Ownership. Rangaswamy:Teneobio, Inc.: Employment, Equity Ownership. Balasubramani:Teneobio, Inc.: Employment, Equity Ownership. Boudreau:Teneobio, Inc.: Employment, Equity Ownership. Dang:Teneobio, Inc.: Employment, Equity Ownership. Davison:Teneobio, Inc.: Employment, Equity Ownership. Force Aldred:Teneobio, Inc.: Equity Ownership. Iyer:Teneobio, Inc.: Employment, Equity Ownership. Jorgensen:Teneobio, Inc.: Employment, Equity Ownership. Pham:Teneobio, Inc.: Employment, Equity Ownership. Prabhakar:Teneobio, Inc.: Employment, Equity Ownership. Schellenberger:Teneobio, Inc.: Employment, Equity Ownership. Ugamraj:Teneobio, Inc.: Employment, Equity Ownership. Trinklein:Teneobio, Inc.: Employment, Equity Ownership. Van Schooten:Teneobio, Inc.: Employment, Equity Ownership.

A CD19/CD3 Bispecific Tandab, AFM11, Recruits T Cells To Potently and Safely Kill CD19+ Tumor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4405-4405
Author(s):  
Eugene Zhukovsky ◽  
Uwe Reusch ◽  
Carmen Burkhardt ◽  
Stefan Knackmuss ◽  
Ivica Fucek ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cancer Cells ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Cross Reactivity ◽  
Equity Ownership

Abstract To harness the potent tumor-killing capacity of T cells for the treatment of CD19+ malignancies, we developed a humanized bispecific tetravalent antibody, with two binding sites for CD3 and CD19, the CD19/CD3 RECRUIT-TandAb AFM11. CD19 is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies such as Non Hodgkin Lymphoma. Since native antibodies cannot recruit T cells, we engineered a bispecific anti-CD19/anti-CD3 TandAb. The tumor-specific CD19 antigen module targets the TandAb to cancer cells, while simultaneously, the CD3 effector module recruits and activates T cells, leading to cancer cell lysis. The advantages of the TandAb technology, relative to other bi-functional fragment antibody scaffolds, include: improved pharmacokinetics (PK) enabling intravenous dosing, more drug-like properties, and avidity-enhanced efficacy for the targeting and killing of tumor cells. We evaluated in vitro efficacy and safety using CD19+ cell lines, and in vivo efficacy in a murine NOD/scid xenograft model reconstituted with human PBMC. Further, we used standard preclinical IND enabling assays to evaluate tissue cross reactivity, PK, and toxicological profile (local tolerance, hematocompatibility, effects on hematopoesis, etc). In vitro assays demonstrated the higher potency and efficacy of target cell lysis by AFM11 relative to a bispecific tandem scFv (that is currently in clinical evaluation). CD8+ T cells dominate early AFM11-mediated cytotoxicity (4 hrs) while after 24 hrs both CD4+ and CD8+ T cells equally contribute to tumor lysis with EC50 between 0.5 – 5 pM; cytotoxicity was independent of CD19 cell-surface density. AFM11 exhibited similar cytotoxicity over effector:target ratios ranging from 5:1 to 1:5, and facilitated serial T cell-killing of its targets. The advantage of AFM11 over the bispecific tandem scFv was most pronounced at lower effector:target ratios. AFM11 activated T cells only in the presence of CD19+ cells. In PBMC cultures, AFM11 induced CD69 and CD25 expression, T cell proliferation, and production of IFN-γ, TNF-α, IL-2, IL-6, and IL-10. Depletion of CD19+ cells from PBMC abrogated these effects, demonstrating that the T cell activation is strictly CD19+ target-dependent. Thus, AFM11 should not elicit the devastating cytokine release observed when full-length antibodies bind CD3. Up to one week co-incubation with AFM11 did not inhibit T cell cytotoxicity, suggesting that the TandAb does not induce anergy. In vivo, AFM11 induced dose-dependent growth inhibition of Raji tumors; a single 0.5 mg/kg dose exhibited efficacy similar to 5 daily injections. In the tissue cross reactivity study, only tissues containing CD19+ and CD3+ cells were stained by AFM11; all other tissues, including vital organs, displayed no cross reactivity. Similarly, no local intolerance was observed in rabbits, and no effect on myeloid and erythroid progenitors was observed in a colony-forming assay. Strong accumulation of 125I-labeled AFM11 was observed in the tumors of mice engrafted with CD19+ cancer cells, and no unspecific organ accumulation was observed. Finally, evaluated on the basis of Cmax and the area under the curve (AUC), AFM11 exhibited dose linearity (20 – 500 mg AFM11 dose range) upon single i.v. bolus administration in mice; half-life (T1/2) ranged from 18.4 to 22.9 hr. In summary, AFM11 is a highly efficacious novel drug candidate for the treatment of CD19+ malignancies with an advantageous safety profile and anticipated dosing regimen. Disclosures: Zhukovsky: Affimed Therapeutics AG: Employment, Equity Ownership. Reusch:Affimed Therapeutics AG: Employment. Burkhardt:Affimed Therapeutics AG: Employment. Knackmuss:Affimed Therapeutics AG: Employment. Fucek:Affimed Therapeutics AG: Employment. Eser:Affimed Therapeutics AG: Employment. McAleese:Affimed Therapeutics AG: Employment. Ellwanger:Affimed Therapeutics AG: Employment. Little:Affimed Therapeutics AG: Consultancy, Equity Ownership.

scholarly journals Engineered red blood cells as an off-the-shelf allogeneic anti-tumor therapeutic

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xuqing Zhang ◽  
Mengyao Luo ◽  
Shamael R. Dastagir ◽  
Mellissa Nixon ◽  
Annie Khamhoung ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Checkpoint Inhibitors ◽  
Critical Role ◽  
Cellular Immunotherapy ◽  
Tumor Control

AbstractCheckpoint inhibitors and T-cell therapies have highlighted the critical role of T cells in anti-cancer immunity. However, limitations associated with these treatments drive the need for alternative approaches. Here, we engineer red blood cells into artificial antigen-presenting cells (aAPCs) presenting a peptide bound to the major histocompatibility complex I, the costimulatory ligand 4-1BBL, and interleukin (IL)-12. This leads to robust, antigen-specific T-cell expansion, memory formation, additional immune activation, tumor control, and antigen spreading in tumor models in vivo. The presence of 4-1BBL and IL-12 induces minimal toxicities due to restriction to the vasculature and spleen. The allogeneic aAPC, RTX-321, comprised of human leukocyte antigen-A*02:01 presenting the human papilloma virus (HPV) peptide HPV16 E711-19, 4-1BBL, and IL-12 on the surface, activates HPV-specific T cells and promotes effector function in vitro. Thus, RTX-321 is a potential ‘off-the-shelf’ in vivo cellular immunotherapy for treating HPV + cancers, including cervical and head/neck cancers.

scholarly journals GD2 or HER2 targeting T cell engaging bispecific antibodies to treat osteosarcoma

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Jeong A. Park ◽  
Nai-Kong V. Cheung
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Immune Checkpoint ◽  
Immune Checkpoint Inhibitors ◽  
Checkpoint Inhibitors ◽  
Tumor Control ◽  
Osteosarcoma Cell

Abstract Background The cure rate for metastatic osteosarcoma has not substantially improved over the past decades. Clinical trials of anti-HER2 trastuzumab or anti-GD2 dinutuximab for metastatic or refractory osteosarcoma were not successful, and neither was immune checkpoint inhibitors (ICIs). Methods We tested various target antigen expressions on osteosarcoma cell lines using flow cytometry and analyzed in vitro T cell engaging BsAb (T-BsAb)-dependent T cell-mediated cytotoxicity using 4-h 51Cr release assay. We tested in vivo anti-tumor activities of T-BsAb targeting GD2 or HER2 in established osteosarcoma cell line or patient-derived xenograft (PDX) mouse models carried out in BALB-Rag2−/−IL-2R-γc-KO (BRG) mice. We also generated ex vivo BsAb-armed T cells (EATs) and studied their tumor-suppressive effect against osteosarcoma xenografts. In order to improve the anti-tumor response, ICIs, anti-human PD-1 (pembrolizumab) or anti-human PD-L1 (atezolizumab) antibodies were tested their synergy with GD2- or HER2-BsAb against osteosarcoma. Results GD2 and HER2 were chosen from a panel of surface markers on osteosarcoma cell lines and PDXs. Anti-GD2 BsAb or anti-HER2 BsAb exerted potent anti-tumor effect against osteosarcoma tumors in vitro and in vivo. T cells armed with anti-GD2-BsAb (GD2-EATs) or anti-HER2-BsAb (HER2-EATs) showed significant anti-tumor activities as well. Anti-PD-L1 combination treatment enhanced BsAb-armed T cell function in vivo and improved tumor control and survival of the mice, when given sequentially and continuously. Conclusion Anti-GD2 and anti-HER2 BsAbs were effective in controlling osteosarcoma. These data support the clinical investigation of GD2 and HER2 targeted T-BsAb treatment in combination with immune checkpoint inhibitors, particularly anti-PD-L1, in patients with osteosarcoma to improve their treatment outcome.

scholarly journals T Cells Expressing Engager and Costimulatory Molecules for the Immunotherapy of CD19+ Malignancies

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2433-2433
Author(s):  
Mireya Paulina Velasquez ◽  
Kota Iwahori ◽  
David L Torres ◽  
Sunitha Kakarla ◽  
Caroline Arber ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Xenograft Model ◽  
Retroviral Vector ◽  
Effector Function ◽  
Target Cells ◽  
Antitumor Effects

Abstract Background: Immunotherapy with anti-CD19/anti-CD3 bispecific engager molecules has shown promise in clinical studies for CD19+ malignancies. However engager molecules have short half-lives and do not accumulate at tumor sites. In addition, co-delivery of other immunostimulatory molecules to enhance antitumor effects is difficult to achieve. We have recently shown that T cells can be genetically modified to secrete bispecific engager molecules (ENG-T cells). ENG-T cells are activated by tumor cells in an antigen-dependent manner, redirect bystander T cells to tumor cells, and have antitumor activity in preclinical models. We now wanted to explore if additional genetic modifications of ENG-T cells can enhance their effector function in vitro and in vivo. Since bispecific engager molecules do not provide co-stimulation, we focused on the provision of co-stimulatory signals by coexpressing CD80 and CD137L on the cell surface of ENG-T cells. Thus, the aim of the study was to compare the effector function of CD19-specific T-cell engagers (CD19-ENG T cells) and CD19-ENG T cells co-expressing CD80 and 41BBL (CD19-ENG/Costim T cells). Methods: CD19-ENG T cells were generated by transducing T cells with a retroviral vector encoding a CD19-specific T-cell engager and mOrange separated by an IRES (SFG.CD19-ENG-I-mO), and CD19-ENG/Costim T cells were generated by double transducing T cells with SFG.CD19-ENG-I-mO and a 2nd retroviral vector encoding 41BBL and CD80 separated by an IRES. The effector function of ENG T-cells was evaluated in vitro and in a leukemia xenograft model. Results: After single or double transduction 60-80% of T cells were positive for mOrange, and ~80% of CD19-ENG/Costim T cells were positive for CD80 and 30-40% positive for 41BBL. In coculture assays CD19-ENG and CD19-ENG/Costim T cells recognized CD19+ lymphoma (Daudi, Raji) and acute leukemia (BV173) cells as judged by IFN-g secretion in contrast to negative controls. While CD19+ target cells that express CD80 and CD86 (Daudi and Raji) induced robust IL2 production of CD19-ENG and CD19-ENG/Costim T cells, CD19-ENG/Costim T cells produced significantly higher levels of IL2 in comparison to CD19-ENG T cells after stimulation with CD19+/CD80-/CD86- negative target cells (BV173). Cytokine production was antigen dependent since ENG and ENG/Costim T cells specific for an irrelevant antigen (EphA2) did not produce cytokines. Specificity was confirmed in cytotoxicity assays. In transwell assays containing inserts preventing T-cell migration, only ENG T cells redirected bystander T cells in the bottom well to CD19+ tumor cells. To assess in vivo anti-tumor activity of CD19-ENG T cells and CD19-ENG/Costim T cells we used the BV173/NSG mouse xenograft model in which BV173 cells are genetically modified with firefly luciferase (ffLuc-BV173) to allow for serial bioluminescence imaging. While therapy with CD19-ENG T cells on day 7 post ffLuc-BV173 injection resulted in the cure of all mice, when therapy was delayed to day 14, only 1/10 mice was alive on day 80. In contrast therapy of mice on day 14 with CD19-ENG/Costim T cells resulted in long-term survival of 7/10 mice. Control T cells (EphA2-ENG T cells or EphA2-ENG/Costim T cells) had no antitumor effects. Conclusions: We have generated CD19-ENG T cells and CD19-ENG/Costim T cells with the ability to direct bystander T cells to CD19+ malignancies. Both ENG T-cell populations had potent antitumor activity in a preclinical ALL model, and provision of costimulation further enhanced antitumor effects. Genetically modifying T cells to express engager molecules and additional molecules to enhance their effector function may present a promising alternative to current CD19-targeted immunotherapies. Disclosures Velasquez: Celgene, Bluebird bio: Other. Iwahori:Celgene, Bluebird bio: Other. Kakarla:Celgene, Bluebird bio: Other. Song:Celgene, Bluebird bio: Other. Gottschalk:Celgene, Bluebird bio: Other.

ISB 1342: A first-in-class CD38 T cell engager for the treatment of relapsed refractory multiple myeloma.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 8044-8044
Author(s):  
Marie-Agnès Doucey ◽  
Blandine Pouleau ◽  
Carole Estoppey ◽  
Cian Stutz ◽  
Amelie Croset ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Killing ◽  
Fragment Antigen Binding ◽  
Tumor Cell Killing

8044 Background: ISB 1342 is a bispecific antibody heterodimer based on the Ichnos proprietary Bispecific Engagement by Antibodies based on T cell receptor (BEAT) platform. ISB 1342 is a first-in-class CD38 T cell engager under investigation in subjects with relapsed multiple myeloma refractory to proteasome inhibitors (PIs), immunomodulators (IMiDs) and daratumumab (study ISB 1342-101). Methods: ISB 1342 was engineered with a single chain variable fragment (scFv) arm that specifically recognizes a cluster of differentiation (CD)3-epsilon (CD3ε) and a fragment antigen binding (Fab) arm which specifically recognizes CD38 and does not compete with daratumumab. By co-engaging CD3ε on T cells and CD38 on tumor cells, ISB 1342 redirects T cells to kill CD38-expressing tumor cells. This mechanism of action is differentiated from existing monospecific CD38 targeting therapies and was designed to overcome resistance to daratumumab in multiple myeloma. Results: In vitro, ISB 1342 killed a large range of CD38-expressing tumor cell lines (EC50:12 to 90 pM) with 8 to 239-fold superior efficacy than daratumumab. ISB 1342 was also able to efficiently kill CD38 low-intermediate-expressing tumor cells that were poorly killed by daratumumab. ISB 1342 retained the potency to kill CD38 low-intermediate-expressing tumor cells when used in sequential or concomitant combination with daratumumab. In addition, the presence of soluble CD38 or glucocorticoid did not impact ISB 1342 killing potency. ISB 1342 was constructed with a double LALA mutation that dampens the binding to Fcγ receptors and C1q. Consistently, ISB 1342 showed only residual Fc-mediated effector functions and its mechanism of tumor cell killing critically relies on the engagement and the activation of T lymphocytes. ISB 1342 showed a favorable on target specificity profile in vitro and was unable to activate T cells in the absence of CD38 positive target cells. Further, ISB 1342-induced tumor cell killing was not associated with a detectable T cell fratricide in vitro. Finally, the potency of ISB 1342 was assessed in vivo in a therapeutic model of a subcutaneously established Daudi tumor co-xenografted with human PBMCs. In marked contrast to daratumumab, which induced only a partial tumor control, ISB 1342 induced complete tumor eradication when injected intravenously weekly at 0.5 mg/kg. As anticipated, the ISB 1342 control molecule (ISB 1342_13DU) made of an irrelevant CD38 binder failed to control tumor growth. The release of the Granzyme A and B, TNF-alpha and CXCL-10 in the tumor micro-environment one week post-treatment was strongly and significantly increased by ISB 1342 but not by daratumumab and ISB 1342_13DU; this represents a correlate of anti-tumor immunity associated with ISB 1342 efficacy in vivo. Conclusions: Hence the higher potency of ISB 1342 relative to daratumumab supports the ongoing clinical development in multiple myeloma patients.

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