A stable single-chain variable fragment expressing transfectoma demonstrates induction of idiotype-specific cytotoxic T-cells during early growth stages of a murine B-lymphoma

2001 ◽  
Vol 50 (8) ◽  
pp. 437-444 ◽  
Author(s):  
Sreenivas Laxmanan ◽  
Gary W. Stuart ◽  
Swapan K. Ghosh
Keyword(s):  
T Cells ◽  
Cytotoxic T Cells ◽  
Early Growth ◽  
Growth Stages ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
B Lymphoma

Immunotherapy of B-Cell Lymphoma With CD3x19 Bispecific Antibodies: Costimulation via CD28 Prevents “Veto” Apoptosis of Antibody-Targeted Cytotoxic T Cells

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4750-4757 ◽  
Author(s):  
Peter T. Daniel ◽  
Arne Kroidl ◽  
Joachim Kopp ◽  
Isrid Sturm ◽  
Gerhard Moldenhauer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cytotoxic T Cells ◽  
B Cell Lymphoma ◽  
Bispecific Antibodies ◽  
Lymphoma Cells ◽  
B Lymphoma ◽  
B Lymphoma Cells

Bispecific antibodies (CD3x19) against the CD3ɛ-chain of the T-cell–receptor/CD3 complex and the CD19 antigen on B cells can target polyclonal, nontumor-specific T cells to B lymphoma cells. This induces T-cell activation, and generation of cytotoxic T cells (CTLs). These polyclonal CTLs, targeted by the CD3x19 bispecific antibodies, can lyse CD19+ B-lymphoma cells. In a xenotransplant model in severe combined immunodeficiency deficient (SCID) mice, we and others observed that CD28 triggering is required for efficient elimination of B-lymphoma cells and cure from the tumor in addition to CD3x19 administration. We also showed that the activation and targeting of CTLs to the target cell by signal one alone, ie, the CD3x19 mab, induces T-cell death by apoptosis. In blocking experiments we showed that this “veto” apoptosis is mediated by the CD95/Fas ligand. Addition of anti-CD28 (signal 2) renders the T cells resistant for veto apoptosis both in vitro and in vivo. We therefore conclude that the role of costimulation in immunotherapy with bispecific antibodies or other T-cell–based immune strategies is not only to facilitate T-cell activation but also to prevent T-cell deletion by apoptosis.

scholarly journals Targeting the FMS-like Tyrosin Kinase 3 with the Unicar System: Preclinical Comparison of Murine and Humanized Single-Chain Variable Fragment-Based Targeting Modules

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5614-5614
Author(s):  
Frederick Fasslrinner ◽  
Claudia Arndt ◽  
Anja Feldmann ◽  
Stefanie Koristka ◽  
Liliana Raquel Loureiro ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
The Novel ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Cell Therapies ◽  
Myeloid Malignancies ◽  
Car T Cell ◽  
Car T

Clinical translation of chimeric antigen receptor (CAR) T cell therapy in myeloid malignancies is progressing slowly compared to its success in treatment of B cell malignancies. Clinical experiences with CAR T cell therapies against the currently investigated tumor-associated antigens (TAA) (e.g. CD33, CD123 and FMS-like tyrosine kinase 3 (FLT3)) were discouraging and severe side effects occurred (cytokine release syndrome, neurotoxicity and myeloid aplasia) (Hoffmann et al. Journal of Clinical Medicine 2019). Probably targeting a single TAA is insufficient to treat high risk myeloid malignancies with CAR T cell therapies. Therefore, combined targeting of two or even more TAAs seems to be a promising approach. In order to implement such a multiple tumor targeting strategy, we developed a modular CAR T cell system termed UniCAR. The system consists of a universal CAR (UniCAR) directed against the La peptide epitope E5B9 combined with single-chain variable fragment (scFv) -based target modules (TM). In contrast to conventional CARs, anti-tumor activity of UniCAR T cells is only turned on in the presence of the TMs. Thus, this approach will allow UniCAR T cell control due to the short half-life of the TM and therefore has a favorable safety profile. Furthermore, different TMs against several TAAs can be administered both sequentially or in parallel to increase the anti-tumor efficacy or face disease relapse due to antigen escape mechanisms. In the field of myeloid malignancies our group developed retargeting strategies against the TAAs CD33 and CD123 (Cartellieri et al. Blood Cancer Journal 2016). In addition, we have developed a new TM for the UniCAR system that is directed against the TAA FLT3. FLT3 is highly expressed on acute myeloid leukemia (AML) cells and also present on CD123low AML samples (Riccioni et al. British Journal of Haematology 2011). The novel FLT3 TM was constructed by fusion of the variable domain of the heavy and the light chain of the murine anti-FLT3 monoclonal antibody (4G8) to the E5B9 UniCAR epitope. In light of a potential clinical application, we in parallel generated a humanized FLT3 TM to further decrease its immunogenicity. Both FLT3 TMs were tested in vitro against different AML cell lines, by using flow cytometry based killing assays as described elsewhere (Fasslrinner et al. British Journal of Haematology 2019). The functionality of the FLT3 TMs in vitro was highly effective. Both FLT3 TMs were able to redirect UniCAR T cells for AML cell lysis already in the picomolar range and were moreover comparable effective than the previously developed CD123 TM. Thus, humanization of the FLT3 TM did not lead to a decrease in anti-tumor efficacy. In summary, we could show that both the novel murine FLT3 TM and the humanized counterpart redirected UniCAR T cells and induced highly effective elimination of AML cells in vitro. Thus, the flexible application of the FLT3-based UniCAR system seems to be a promising tool for cell-based AML therapy alone or even in combination with other AML-specific TMs (e.g. CD33, CD123). Disclosures Koristka: Intellia Therapeutics: Employment. Jung:Synimmune: Other: shareholder interest. Bachmann:GEMoaB Monoclonals: Equity Ownership, Patents & Royalties.

Redirection of CMV Specific CTL towards B-CLL Via CD20 Targeted HLA/CMV Complexes.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 449-449 ◽  
Author(s):  
Rogier Mous ◽  
Philip Savage Savage ◽  
Ester BM Remmerswaal ◽  
Rene A.W. van Lier ◽  
Eric Eldering ◽  
...  
Keyword(s):  
T Cells ◽  
B Cell ◽  
Immunological Synapse ◽  
Ex Vivo ◽  
Cytotoxic T Cells ◽  
Interferon Γ ◽  
Lymphocytic Leukemia ◽  
Viral Immunity ◽  
Single Chain ◽  
Effector Cells

Abstract Because B cell chronic lymphocytic leukemia (B-CLL) can not be cured with current therapies, but in general has a slow progression and rather long median survival, it is considered an attractive candidate for active T cell mediated immunotherapy. However B-CLL cells have poor antigen presenting capacity because they express low levels of co-stimulatory molecules. Moreover, most immunotherapeutic strategies require knowledge of the eliciting tumor antigen and/or ex vivo manipulation of patient cells. To circumvent these drawbacks we aim to redirect existing viral immunity towards B-CLL. Previously, we have shown that in patients with B-CLL considerably expanded numbers of cytomegalovirus (CMV)-specific CD45RA+CD27 CD8+ cytotoxic T cells are present (W. Mackus et al. Blood2003; 102:1057). These cells are potent cytotoxic effector cells when directed against B-CLL cells loaded with CMV peptide (A.Kater et al. Br.J. Haematol.2004; 126:512). In the current study, we apply a novel bridging reagent to redirect CMV-specific CTL to specifically target B-CLL. The targeting complex is composed of a streptavidin fused anti-CD20 single chain variable fragment (scFv) in combination with biotinylated MHC class I molecules containing CMV pp65 peptide (HLA/CMV). We demonstrate that this complex is stable on the cell surface for ≥24 hours, and that B-CLL cells coated with this CD20-HLA/CMV complex can be lysed by autologous CMV-specific CD8+ CTL with similar efficiency as B-CLL cells directly loaded with CMV-peptide. Killing occurs at scFv CD20 concentrations of ≥100 ng ml−1 and HLA/CMV concentrations of ≥20 ng ml−1. Lysis of CD20-HLA/CMV complex coated CLL cells could not be blocked by anti-LFA1 antibodies, in contrast to B-CLL cells directly loaded with CMV-peptide, indicating a different immunological synapse. HLA-A2 positive B-CLL cells coated with HLA-B7 /CMV complexes were only lysed by HLA-B7 positive CMV-specific CTL, whereas HLA-A2/CMV complex targeted HLA-A2 positive B-CLL cells were unaffected by HLA-B7 positive CMV specific CTL, proving HLA restriction of the killing.. Furthermore, CD20-HLA/CMV complex coated B-CLL cells induce both proliferation and cytokine production (interferon γ, tumor necrosis factor α, and macrophage inflammatory protein-1 β) in CMV-specific CD8+ T cells. Thus, CD20-HLA/CMV complexes elicit both immune activation and direct cytotoxicity towards B-CLL cells. The findings of our study constitute a necessary step towards possible application of CD20-HLA/CMV complexes for immunotherapy of B cell malignancies. It is obvious that this recently recognized capacity to redirect existing antiviral immunity towards tumor cells has a utility in cancer immunotherapy far beyond CMV and B-CLL.

scholarly journals Titan Cells and Yeast Forms of Cryptococcus neoformans and Cryptococcus gattii Are Recognized by GXMR-CAR

2021 ◽  
Vol 9 (9) ◽  
pp. 1886
Author(s):  
Matheus Henrique dos Santos ◽  
Michele Procópio Machado ◽  
Pappanaicken R. Kumaresan ◽  
Thiago Aparecido da Silva
Keyword(s):  
T Cells ◽  
Cryptococcus Neoformans ◽  
Cytotoxic T Cells ◽  
Fungal Spores ◽  
Cell Types ◽  
Cryptococcus Gattii ◽  
Jurkat Cells ◽  
Single Chain ◽  
Cryptococcus Spp ◽  
Polysaccharide Capsules

Cryptococcosis, a systemic mycosis that affects both the immunocompromised and immunocompetent, is caused by the inhalation of dehydrated yeasts or fungal spores of Cryptococcus gattii or Cryptococcus neoformans. The Cryptococcus spp. polysaccharide capsule is composed mainly of glucuronoxylomannan—GXM, its major virulence factor. The capsule thickness increases to more than 15 μm during titanization, favoring the pathogenesis of cryptococcosis. Previous studies demonstrated that cytotoxic T cells that had been bioengineered with GXM-targeting chimeric antigen receptor (GXMR-CAR) were able to recognize C. neoformans by promoting the control of titanization. GXMR-CAR, a second-generation CAR, contains a single-chain variable fragment that originates from a 18B7 clone: a human IgG4 hinge, followed by a human CD28 (transmembrane/cytoplasmic domains) and a CD3ς chain. In the current study, we redirected T cells to target distinct C. neoformans and C. gattii cell types by GXMR-CAR. Lentiviral particles carrying the GXMR-CAR sequence were used to transduce Jurkat cells, and these modified cells interacted with the GXM of the C. gattii R265 strain. Moreover, GXMR-CAR mediated the recognition of C. gattii and C. neoformans yeasts with both thin and thick polysaccharide capsules, and GXMR-CAR Jurkat cells interacted with titan cells sourced from both Cryptococcus spp. Thus, bioengineered cells using CAR can improve the treatment of cryptococcosis.

Serial killing of tumor cells by cytotoxic T cells redirected with a CD19-/CD3-bispecific single-chain antibody construct

2005 ◽  
Vol 115 (1) ◽  
pp. 98-104 ◽  
Author(s):  
Patrick Hoffmann ◽  
Robert Hofmeister ◽  
Klaus Brischwein ◽  
Christian Brandl ◽  
Sandrine Crommer ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Cells ◽  
Cytotoxic T Cells ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Serial Killing

Immunotherapy of B-Cell Lymphoma With CD3x19 Bispecific Antibodies: Costimulation via CD28 Prevents “Veto” Apoptosis of Antibody-Targeted Cytotoxic T Cells

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4750-4757 ◽  
Author(s):  
Peter T. Daniel ◽  
Arne Kroidl ◽  
Joachim Kopp ◽  
Isrid Sturm ◽  
Gerhard Moldenhauer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cytotoxic T Cells ◽  
B Cell Lymphoma ◽  
Bispecific Antibodies ◽  
Lymphoma Cells ◽  
B Lymphoma ◽  
B Lymphoma Cells

Abstract Bispecific antibodies (CD3x19) against the CD3ɛ-chain of the T-cell–receptor/CD3 complex and the CD19 antigen on B cells can target polyclonal, nontumor-specific T cells to B lymphoma cells. This induces T-cell activation, and generation of cytotoxic T cells (CTLs). These polyclonal CTLs, targeted by the CD3x19 bispecific antibodies, can lyse CD19+ B-lymphoma cells. In a xenotransplant model in severe combined immunodeficiency deficient (SCID) mice, we and others observed that CD28 triggering is required for efficient elimination of B-lymphoma cells and cure from the tumor in addition to CD3x19 administration. We also showed that the activation and targeting of CTLs to the target cell by signal one alone, ie, the CD3x19 mab, induces T-cell death by apoptosis. In blocking experiments we showed that this “veto” apoptosis is mediated by the CD95/Fas ligand. Addition of anti-CD28 (signal 2) renders the T cells resistant for veto apoptosis both in vitro and in vivo. We therefore conclude that the role of costimulation in immunotherapy with bispecific antibodies or other T-cell–based immune strategies is not only to facilitate T-cell activation but also to prevent T-cell deletion by apoptosis.

scholarly journals Prolactin gene expression in mouse spleen helper T cells

2004 ◽  
Vol 183 (3) ◽  
pp. 639-646 ◽  
Author(s):  
K Horiguchi ◽  
S Yagi ◽  
K Ono ◽  
Y Nishiura ◽  
M Tanaka ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Helper T Cells ◽  
Mouse Spleen ◽  
Growth Stages ◽  
Single Chain ◽  
Messenger Ribonucleic Acid ◽  
Prolactin Gene ◽  
Old Mice

Prolactin (PRL) is a single-chain polypeptide hormone that is generally secreted from prolactin cells of the anterior pituitary gland into the blood circulation. However, recent studies indicate that the gene expression of prolactin is ectopic in several tissues across several species. These studies found that lymphocytes also produce PRL, which is involved in the immunoregulatory system. Here, we searched for PRL messenger ribonucleic acid (mRNA), using the reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting in the spleens of mice at various growth stages. We also localized mouse prolactin (mPRL) and its mRNA in the spleens of 30- and 60-day-old mice by immunohistochemistry and in situ hybridization respectively. The mPRL gene was expressed in all spleen samples at 0–60 days postpartum. We localized mPRL mRNA in the sheathed artery, periarterial lymphatic sheath and the marginal zone of the spleen. Moreover, we detected mPRL in essentially the same area as its mRNA. Furthermore, we performed double-fluorescence immunohistochemical staining for mPRL and mouse CD4 that is specifically produced in helper T cells, or for mPRL and mouse CD19 or CD40 specified B cells. We colocalized mPRL immunoreactivity only in some CD4-immunopositive cells. These results clearly suggest that T cells synthesize mPRL in the mouse spleen.

Recombinant Endoglin-Single-Chain Variable Fragment/ Induced Protein 10 Fusion Protein Potently Boosts the Anti-Tumor Efficacy of Adoptively Transferred TRP2-Specific CD8+ CD28+ Cytotoxic T Lymphocytes in Mice

2020 ◽  
Vol 16 (7) ◽  
pp. 1119-1134
Author(s):  
Yangzi Li ◽  
Xiaomei Yang ◽  
Xiaoling Lu ◽  
Zhengui Peng ◽  
Chunhui Lai ◽  
...  
Keyword(s):  
T Cells ◽  
Fusion Protein ◽  
Tumor Growth ◽  
Therapeutic Efficacy ◽  
Therapeutic Potential ◽  
Suppressor Cells ◽  
Single Chain Variable Fragment ◽  
Single Chain

In this research, we studied the therapeutic efficacy of a newly designed fusion protein containing Endoglin single-chain variable fragment and IP10 (Endoglin-scFv/IP10), together with our recently generated TRP2-specific CD8+ CD28+ CTLs (CD8+ CD28+ CTLs) in controlling melanoma growth in mice. The recombinant Endoglin-scFv/IP10 was expressed in E. coli, purified by affinity chromatography, and characterized in vitro for its chemotactic movement and immunoreactivity with endoglin-expressing cells. In vivo, melanoma xenografts were established in mice (C57BL/6) using B16F10 cells. After that, mice were treated with intravenous injections of vehicle (PBS), Endoglin-scFv/IP10 alone, CD8+ CD28+ CTLs alone, or Endoglin-scFv/IP10+ CD8+ CD28+ CTLs. The therapeutic efficacy was assessed by monitoring tumor growth, mouse survival and cellular biomarkers. Endoglin-scFv/IP10 fusion protein combined with CD8+ CD28+ CTLs observed a reduction in tumor growth, resulting in improved survival. On the cellular level, the combination treatment dramatically reduced the number of systemic and tumor associated myeloid-derived suppressor cells or regulatory T cells, increased tumor-responsive interferon-γ-producing lymphocytes and tumor-associated CD8+ CXCR3+ T cells, and inhibited proliferation and angiogenesis but stimulated apoptosis within melanoma tissue. This study demonstrates the therapeutic potential of Endoglin-scFv/IP10 fusion protein in combination with CD8+ CD28+ CTLs in melanoma treatment.

TREM1/Dap12-based CAR-T cells show potent antitumor activity

Immunotherapy ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1043-1055 ◽  
Author(s):  
Bing Chen ◽  
Min Zhou ◽  
Hai Zhang ◽  
Chen Wang ◽  
Xiaocui Hu ◽  
...  
Keyword(s):  
T Cells ◽  
Antitumor Activity ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Car T Cells ◽  
Car T ◽  
Clinical Benefits ◽  
Chimeric Immunoreceptor

Aim: Chimeric antigen receptor-engineered T (CAR-T) cells have gained huge success in treating hematological malignancies, yet the CD3ζ-based CAR-T therapies have not shown comparable clinical benefits in solid tumors. We designed an alternative chimeric immunoreceptor in which a single-chain variable fragment was fused to the transmembrane-cytoplasmic domains of triggering receptor expressed on myeloid (TREM1), which may show potent antitumor activity. Methods: To generate TREM1/DNAX activation protein of 12 kDa (Dap12)-based CAR-T cells, TREM1 along with DAP12 was transduced into T cells. Results: TREM1/Dap12-based CAR-T cells showed more lysis in vitro and a similar antitumor effect in mouse models compared with CD19BBζ CAR-T cells. Conclusion: In this study, we designed a TREM1/Dap12-based CAR, which was not reported previously and demonstrated that TREM1/Dap12-based CAR-T cells had potent antitumor activity in vitro and in vivo.

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