Targeted Gene Delivery in CD30+ Hodgkin Lymphoma Cells Using Lentiviral Vectors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5481-5481
Author(s):  
Quang T. Luong ◽  
Kouki Morizono ◽  
Irvin S.Y. Chen ◽  
Sven de Vos
Keyword(s):  
Targeted Therapy ◽  
Gene Delivery ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Lentiviral Vector ◽  
Viral Envelope ◽  
Target Cells ◽  
Lymphoma Cells ◽  
Resistant Disease ◽  
Anti Cd20

Abstract Despite the success of combination chemotherapy for the treatment of Hodgkin lymphoma (HL), chemotherapy-resistant disease still remains an unresolved problem with most patients eventually dying due to progression. HL is an ideal disease for targeted therapy. CD30 is a 120-kDa transmembrane glycoprotein belonging to the tumor necrosis factor (TNF)-receptor superfamily and strongly expressed in HL Reed-Sternberg cells. We hypothesize that specific, antibody-mediated, CD30-directed delivery of lentiviral vector constructs, encoding for a lethal cellular toxin, will be an effective anti-lymphoma strategy. However, gene delivery based therapy is limited by the transduction of non-target cells. The technology developed by Morizono et al. [Cell Cycle2005: 4: p854; Nature Medicine2005: 11: p346] demonstrates that lentiviruses can be specifically and effectively directed to target cells by conjugation of an antibody to a modified ZZ SINDBIS viral envelope (m168). In this study, we have used HL cell lines (CD30+: L591, L428, Hs445, RPMI6666) and Burkitt lymphoma (BL) cell lines (CD20+/CD30−: Raji, Ramos). We show that conjugation of an anti-CD30 antibody to m168 (m168anti-CD30), permits specific targeting and transduction of CD30+ Hodgkin lymphoma cells while avoiding CD30− Raji and Ramos cells [range 11–83% for HL cells versus 1–4% for BL cells]. Similarly, targeting of CD20+ cells can be achieved. Several of our HL cell lines (L591, Hs445 and RPMI6666) are CD30+/CD20+ [range 11–25% by flow cytometry] and we show that we can equally transduce these cells with an anti-CD20 antibody conjugated to our lentivirus [range 11–30% for CD20+ HL cells; 3% for L428 cells; 40–47% for BL cells]. In addition, we show in L591 cells, that the re-targeted viruses can transduce a greater percentage of target cells than an unmodified virus [83% for m168anti-CD30 versus 43% for VSVG]. These results demonstrate that the efficacy and specificity of targeted therapy can be greatly enhanced and lay the foundation for the development of more stable anti-CD30 directed lentiviral constructs expressing cellular toxins.

Src Family Tyrosine Kinases Are Involved in the Transcriptional Regulation of CD20 Levels

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1661-1661
Author(s):  
Magdalena Winiarska ◽  
Jacek Bil ◽  
Kamil Bojarczuk ◽  
Dominika Nowis ◽  
Malgorzata Wanczyk ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Antitumor Activity ◽  
Cell Lines ◽  
Tyrosine Kinases ◽  
Dose Range ◽  
Lymphoma Cells ◽  
Raji Cells ◽  
Src Family Tyrosine Kinases ◽  
Anti Cd20 ◽  
Dasatinib Treatment

Abstract Abstract 1661 Introduction: Anti-CD20 monoclonal antibodies (mAbs) have considerably improved the outcomes of patients with B-cell malignancies and reveal promising therapeutic activity in some autoimmune diseases. They eliminate B cells by triggering indirect effector mechanisms of the immune system, namely complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. Unfortunately, the resistance to anti-CD20 mAb-based first line therapies has been a considerable clinical problem. The mechanisms of this resistance are still poorly understood. In an elegant in vitro study by van Meerten et al. a direct positive correlation between rituximab antitumor activity and CD20 levels has been demonstrated. Although for many years CD20 has been described as a stable antigen, accumulating evidence indicates that CD20 can be modulated at several levels, both transcriptional and posttranscriptional. The processes that lead to CD20 downregulation could potentially impair antitumor activity of rituximab-based therapies and lead to rituximab resistance. Src family tyrosine kinases (SFTKs) including Lyn, Fyn and Lck have been already reported by Deans et al. to associate with CD20 localized to lipid rafts. They were shown to be activated during anti-CD20 mAb-mediated apoptosis upon clustering of rafts. However, to the best of our knowledge, the role of SFTKs in the regulation of CD20 expression has not been studied so far. Objectives: The aim of this study was to explore the molecular basis for Src family tyrosine kinases- dependent regulation of CD20 levels in lymphoma cells. Results: In the initial experiments performed using flow cytometry we observed a significantly reduced binding of anti-CD20 mAb to Raji cells incubated for 48h with various inhibitors of Src kinases (dasatinib, PP2, nilotynib, bosutinib, saracatinib) (Fig.1A-E). Dasatinib also impaired the binding of rituximab to Raji cells (Fig.1F). Decreased binding of anti-CD20 mAb upon dasatinib treatment was observed in three additional lymphoma cell lines and primary cells isolated from patients with chronic lymphocytic leukemia. All tested SFTKs inhibitors impaired rituximab-mediated CDC (R-CDC) over a dose range of rituximab concentrations (1–100 ug/ml) in all lymphoma cell lines. Interestingly, in Raji cells incubated for 48h with dasatinib we also observed a dose-dependent reduction of total CD20 protein levels, when assayed by Western blotting (Fig.2A). Moreover, a 48-h incubation with dasatinib significantly reduced the transcription of cd20 gene, as assessed with RT-PCR (Fig.2B). To further elucidate the mechanism of transcriptional regulation of CD20 we performed qRT-PCR studies. A strongly reduced transcription of cd20 gene was observed in Raji cells over a dose range of dasatinib (20–200 nM) after 24- (Fig.2C) and 48h- incubation (Fig.2D). Additionally, the CD20 promoter activity measured with reporter Firefly luciferase assay has been reduced as early as 1 hour after dasatinib treatment (Fig.2E). To elucidate in more detail binding of transcription factors to the promoter of cd20 gene, a chromatin immunoprecipitation assay was performed. Our early results indicate that dasatinib impairs binding of PU.1 transcription factor to its consensus site within cd20 promoter in Raji cells. Conclusions: Our studies indicate for the first time that SFTKs are involved in the transcriptional regulation of CD20 levels in lymphoma cells. Elucidation of the exact mechanism of this phenomena needs further studies. Results of these experiments will help to understand the biology and regulation of CD20 levels in lymphoma cells. The research was supported by Polish Ministry of Science and Higher Education [N N402 352938 (MW), IP2010/046570 (MW), IP2010/028670 (DN)]. Disclosures: No relevant conflicts of interest to declare.

PU.1-Induced Growth Arrest and Apoptosis in Classical Hodgkin Lymphoma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2628-2628
Author(s):  
Hiromichi Yuki ◽  
Shikiko Ueno ◽  
Hiroaki Niiro ◽  
Hiro Tatetsu ◽  
Hiroyuki Hata ◽  
...  
Keyword(s):  
Cell Growth ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Growth Arrest ◽  
Myeloma Cell ◽  
G1 Arrest ◽  
Classical Hodgkin Lymphoma ◽  
Lymphoma Cells ◽  
Down Regulation

Abstract Abstract 2628 PU.1 is an Ets family transcription factor, which is essential for differentiation of both myeloid and lymphoid lineage cells. We have previously shown that PU.1 is down-regulated in various myeloma cell lines and myeloma cells from a subset of myeloma patients. In such cell lines, the promoter and the upstream regulatory element (URE) located in 17 kb 5'-upstream of the PU.1 gene are highly methylated. Furthermore, conditionally expressed PU.1 induces both cell growth arrest and apoptosis in PU.1-low to -negative myeloma cell lines, U266 and KMS12PE. Therefore, we concluded that the down-regulation of PU.1 is necessary for myeloma cell growth. In another B cell malignancy, classical Hodgkin lymphoma, it has been reported that PU.1 is also down-regulated through methylation of its promoter. To evaluate whether down-regulation of PU.1 is essential for growth of classical Hodgkin lymphoma cells, we conditionally expressed PU.1 in two classical Hodgkin lymphoma cell lines, L428 and KMH2, using the tet-off system (designated as L428tetPU.1 and KMH2tetPU.1 cells, respectively). Up-regulation of PU.1 by tetracycline removal induced complete growth arrest in L428tetPU.1 and KMH2tetPU.1 cells. Annexin V staining revealed that up-regulation of PU.1 induced apoptosis in both cell lines. Furthermore, BrdU staining analysis revealed that PU.1 induced G0/G1 arrest in those cells. L428tetPU.1 and KMH2tetPU.1 cells expressing PU.1 showed morphological changes that included the enlargement cytosol and the appearance of various sizes of vacuoles. We next injected L428tetPU.1 and KMH2tetPU.1 cells to immunodeficiency mice (Rag2−/− Jak3−/− bulb/c) subcutaneously. Tumor formation was observed in all those mice with continuous administration of tetracycline (0.5 g/l) in the drinking water. After enlargement of tumor to 1–2 cm diameter, we removed tetracycline in half of the mice. Tetracyclin withdrawal resulted in tumor regression or stable disease, whereas all the mice continuously receiving tetracycline had continuous tumor growth and finally died. These data strongly suggest that PU.1 induced growth arrest and apoptosis of classical Hodgkin lymphoma cells both in vitro and vivo. We next performed DNA microarray analysis to compare gene expression levels of L428tetPU.1 cells before and after PU.1 expression to elucidate the mechanisms of growth arrest and apoptosis induced by PU.1. Among genes related to cell cycle and apoptosis, p21 (CDKN1A) was highly up-regulated in L428tetPU.1 cells after PU.1 induction, and this was also confirmed by mRNA and protein levels. Finally, to clarify the role of p21 up-regulation by PU.1, we stably introduced p21 siRNA in L428tetPU.1 cells. Such stably expressed p21 siRNA rescued L428tetPU.1 cells from growth arrest induced by PU.1, suggesting that the growth arrest in L428tetPU.1 cells by PU.1 should be at least partially dependent on p21 up-regulation. These data suggested that up-regulation of PU.1 by demethylation agents and/or HDAC inhibitors might serve as a possible treatment modality for classical Hodgkin lymphoma. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Sodium stibogluconate and CD47-SIRPα blockade overcome resistance of anti-CD20-opsonized B cells to neutrophil killing

2021 ◽  
Author(s):  
Dieke J. van Rees ◽  
Maximilian Brinkhaus ◽  
Bart Klein ◽  
Paul Verkuijlen ◽  
Anton T.J. Tool ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Tumor Cells ◽  
Target Cells ◽  
Sodium Stibogluconate ◽  
B Cell Malignancies ◽  
Lymphoma Cells ◽  
B Lymphoma ◽  
B Lymphoma Cells ◽  
Anti Cd20

Anti-CD20 antibodies, like rituximab, are broadly used to treat B cell malignancies. These antibodies can induce various effector functions, including immune cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Neutrophils can induce ADCC towards solid cancer cells by trogoptosis, a cytotoxic mechanism known to be dependent on trogocytosis. However, neutrophils appear incapable of killing rituximab-opsonized B lymphoma cells. Nevertheless, neutrophils do trogocytose rituximab-opsonized B lymphoma cells, yet this only reduces CD20 surface expression, and is thought to render tumor cells therapeutically resistant to further rituximab-dependent destruction. Here, we demonstrate that resistance of B lymphoma cells towards neutrophil killing can be overcome by a combination of CD47-SIRPα checkpoint blockade and sodium stibogluconate (SSG), an anti-leishmanial drug and documented inhibitor of the tyrosine phosphatase SHP-1. SSG enhanced neutrophil-mediated ADCC of solid tumor cells, but enabled B lymphoma cell trogoptotic killing, by turning trogocytosis from a resistance-contributing mechanism into a cytotoxic anti-cancer one. The killing in the presence of SSG required both antibody opsonization of the target cells, as well as disruption of CD47-SIRPα interactions. These results provide a more detailed understanding of the role of neutrophil trogocytosis in antibody-mediated destruction of B cells and clues on how to further optimize antibody therapy of B cell malignancies.

scholarly journals A guide in lentiviral vector production for hard-to-transfect cells, using cardiac-derived c-kit expressing cells as a model system

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
V. Kalidasan ◽  
Wai Hoe Ng ◽  
Oluwaseun Ayodeji Ishola ◽  
Nithya Ravichantar ◽  
Jun Jie Tan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Gene Delivery ◽  
Delivery System ◽  
Lentiviral Vector ◽  
Lentiviral Vectors ◽  
Model System ◽  
Selection Marker ◽  
Target Cells ◽  
Primary Cells

AbstractGene therapy revolves around modifying genetic makeup by inserting foreign nucleic acids into targeted cells via gene delivery methods to treat a particular disease. While the genes targeted play a key role in gene therapy, the gene delivery system used is also of utmost importance as it determines the success of gene therapy. As primary cells and stem cells are often the target cells for gene therapy in clinical trials, the delivery system would need to be robust, and viral-based entries such as lentiviral vectors work best at transporting the transgene into the cells. However, even within lentiviral vectors, several parameters can affect the functionality of the delivery system. Using cardiac-derived c-kit expressing cells (CCs) as a model system, this study aims to optimize lentiviral production by investigating various experimental factors such as the generation of the lentiviral system, concentration method, and type of selection marker. Our findings showed that the 2nd generation system with pCMV-dR8.2 dvpr as the packaging plasmid produced a 7.3-fold higher yield of lentiviral production compared to psPAX2. Concentrating the virus with ultracentrifuge produced a higher viral titer at greater than 5 × 105 infectious unit values/ml (IFU/ml). And lastly, the minimum inhibitory concentration (MIC) of puromycin selection marker was 10 μg/mL and 7 μg/mL for HEK293T and CCs, demonstrating the suitability of antibiotic selection for all cell types. This encouraging data can be extrapolated and applied to other difficult-to-transfect cells, such as different types of stem cells or primary cells.

Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3900-3908 ◽  
Author(s):  
Josée Golay ◽  
Luisella Zaffaroni ◽  
Thomas Vaccari ◽  
Manuela Lazzari ◽  
Gian-Maria Borleri ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Lines ◽  
Low Grade ◽  
Lymphoma Cells ◽  
B Lymphoma Cells ◽  
Biologic Response ◽  
Anti Cd20 ◽  
Hodgkin's Lymphomas

Abstract The chimeric anti-CD20 MAb rituximab has recently become a treatment of choice for low-grade or follicular non-Hodgkin's lymphomas (FL) with a response rate of about 50%. In this report, we have investigated the mechanism of action of rituximab on 4 FL and 1 Burkitt's lymphoma (BL) cell lines, 3 fresh FL samples and normal B cells in vitro. Rituximab efficiently blocks the proliferation of normal B cells, but not that of the lymphoma lines. We did not detect significant apoptosis of the cell lines in response to rituximab alone. All cell lines were targets of antibody-dependent cellular cytotoxicity (ADCC). On the other hand, human complement-mediated lysis was highly variable between cell lines, ranging from 100% lysis to complete resistance. Investigation of the role of the complement inhibitors CD35, CD46, CD55, and CD59 showed that CD55, and to a lesser extent CD59, are important regulators of complement-mediated cytotoxicity (CDC) in FL cell lines as well as in fresh cases of FL: Blocking CD55 and/or CD59 function with specific antibodies significantly increased CDC in FL cells. We conclude that CDC and ADCC are major mechanisms of action of rituximab on B-cell lymphomas and that a heterogeneous susceptibility of different lymphoma cells to complement may be at least in part responsible for the heterogeneity of the response of different patients to rituximab in vivo. Furthermore, we suggest that the relative levels of CD55 and CD59 may become useful markers to predict the clinical response.

scholarly journals A Divalent Hapten-Peptide Induces Apoptosis in Human Non–Hodgkin Lymphoma Cell Lines Targeted by Anti-CD20 × Anti-Hapten Bispecific Antibodies

2007 ◽  
Vol 13 (18) ◽  
pp. 5564s-5571s ◽  
Author(s):  
Pierre-Yves Brard ◽  
Habibe Karacay ◽  
Rhona Stein ◽  
Robert M. Sharkey ◽  
M. Jules Mattes ◽  
...  
Keyword(s):  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Lymphoma Cell ◽  
Bispecific Antibodies ◽  
Non Hodgkin Lymphoma ◽  
Hodgkin Lymphoma Cell ◽  
Anti Cd20

scholarly journals CD19 Target Activated Natural Killer (CD19.TaNK) Cellular Therapy: A Novel immunotherapeutic Approach to the Treatment of Non-Hodgkin Lymphoma (NHL)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4174-4174
Author(s):  
Ravi Dashnamoorthy ◽  
Afshin Beheshti ◽  
Saheli Sarkar ◽  
Pooja Sabhachandani ◽  
Frank C. Passero ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Resistant Cell ◽  
Cytolytic Activity ◽  
Lymphoma Cells ◽  
Cd20 Antibody ◽  
Anti Cd20 ◽  
Targeted Immunotherapy ◽  
Cd20 Antibodies

Abstract Background: Continued improvement in the treatment of NHL is desired, especially via the incorporation of 'targeted' immunotherapy agents. This is especially important in B-cell NHL (bNHL) as resistance to rituximab anti-CD20 antibody, and now second-generation antibodies (e.g., obinutuzumab), may occur. Activated NK-92 (aNK-92) is a continuously growing cell line consisting of "pure" (100%) activated NK cells. These cells were subsequently bioengineered to express human anti-CD19 chimeric antigen receptor (CAR) recognizing CD19+ B cells. The goal of this project was to investigate the specificity and the efficacy of a novel 'off the shelf' targeted immunotherapy, CD19.TaNK, in a multitude of B-cell NHL cell lines, including anti-CD20 antibody resistant cell lines. Methods: Using gene expression profiling, Gene Set Enrichment Analysis and Ingenuity Pathway Analysis, we first investigated the expression of NK activation and inhibitory ligands in varied lymphoma cells. The bNHL cell lines, SUDHL10 (DLBCL), L540, L428 (Hodgkin lymphoma), HF1 (follicular), Raji (Burkitt's), and Mino (mantle cell) were purchased from ATCC and maintained in RPMI1640 medium. aNK and CD19.TaNK were supplied by NantKwest, Inc and were maintained in Myelocult supplemented with recombinant human IL-2 (500IU/ml). NK cell mediated cytotoxicity was determined using lactate dehydrogenase (LDH) release glucose-6-phosphate dehydrogenase (G6PD) release (aCella-tox assay). Briefly, 10,000 target bNHL cells were co-cultured with effector NK cells, at clinically relevant effector to target ratios (E:T 1:1-10:1) for 4 hours, and the supernatant was assayed for LDH or G6PD release. Percent cytotoxicity was determined based on the experimental levels of LDH or G6PD release from NK mediated bNHL cell lysis compared to maximum LDH or G6PD release from target cells. To determine if resistance to anti-CD20 antibodies would interfere with sensitivity to CD19.TaNK therapy, rituximab and obinutuzumab resistant bNHL cell lines (SUDHL4, SHUDHL10, and Raji) were established; cells were exposed to incremental increasing concentrations of antibody drugs (5-20μg/ml) over a period of 8 weeks. CD19, CD20 and CD30 expression in bNHL cells was determined by flow cytometry. Additionally, the efficacy of primary NK cells were determined against CD20 monoclonal antibody sensitive and resistant cell lines utilizing droplet microfluidics based assessment. Results: We observed that bNHL cell lines expressed a multitude of ligands associated with stimulating NK cell activity, while expression of inhibitory ligands was minimal. This indicates that NK cell interaction with bNHL cells is predicted to lead to overall robust antitumor immune response (Figure). Using LDH and G6PD release assays in bNHL cell lines, we observed increased cytolytic activity in an E:T ratio dependent manner, with Raji and L428 cells being the most sensitive to CD19.TaNK at 1:1 E:T ratio. Development of resistance to anti-CD20 antibodies (rituximab and obinutuzumab) resulted in significantly decreased down regulation of CD20, but not CD19 or CD30, as detected by flow cytometry. After direct contact with primary NK cells, we observed that rituximab resistant SUDHL10 cells were poorly sensitive (7%), while in rituximab sensitive cells, there was 22% cell loss. Moreover, at 4 hours using CD19.TaNK therapy (1:5 ratios), there was marked cytolytic activity with consistent high LDH release seen across all bNHL cell lines without differences noted regardless of rituximab or obinutuzumab resistance (ie, SUDHL4, SHUDHL10, and Raji). These results were further confirmed using live cell video microscopy measuring the cytolytic activity of CD19.TaNK versus bNHL cells. Conclusion: We identified that bNHL cells contain high expression levels of NK activation ligands and low amounts of inhibitory ligands and that CD19.TaNK immunotherapy had potent single-agent anti-tumor activity against a spectrum of bNHL cells. Furthermore, CD19.TaNK maintained high cytolytic activity in bNHL cells resistant to standard CD20 antibody therapy, which were poorly sensitive to innate NK cells. Ongoing analyses include systems biology studies to determine potential biologic mechanisms of activity of CD19.TaNK therapy as well as well as to help guide optimum combinatorial therapy. Figure Expression of NK activation and inhibitory ligands in lymphoma cells. Figure. Expression of NK activation and inhibitory ligands in lymphoma cells. Disclosures Boissel: NantKwest, Inc.: Employment. Evens:Takeda: Other: Advisory board.

scholarly journals Targeted alpha-therapy using [Bi-213]anti-CD20 as novel treatment option for radio- and chemoresistant non-Hodgkin lymphoma cells

Oncotarget ◽  
2013 ◽  
Vol 4 (2) ◽  
pp. 218-230 ◽  
Author(s):  
Mareike Roscher ◽  
Inis Hormann ◽  
Oliver Leib ◽  
Sebastian Marx ◽  
Josue Moreno ◽  
...  
Keyword(s):  
Hodgkin Lymphoma ◽  
Treatment Option ◽  
Lymphoma Cells ◽  
Targeted Alpha Therapy ◽  
Non Hodgkin Lymphoma ◽  
Novel Treatment ◽  
Anti Cd20 ◽  
Alpha Therapy

scholarly journals Nanobody-armed T cells endow CAR-T cells with cytotoxicity against lymphoma cells

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Hongxia Wang ◽  
Liyan Wang ◽  
Yanning Li ◽  
Guangqi Li ◽  
Xiaochun Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
Phage Display ◽  
Cell Lines ◽  
Tumour Cell ◽  
Phage Display Library ◽  
Target Cells ◽  
Lymphoma Cells ◽  
Car T Cells ◽  
Car T ◽  
Tumour Cell Lines

Abstract Background Taking advantage of nanobodies (Nbs) in immunotherapy, we investigated the cytotoxicity of Nb-based chimeric antigen receptor T cells (Nb CAR-T) against lymphoma cells. Methods CD19 Nb CAR-T, CD20 Nb CAR-T, and Bispecific Nb CAR-T cells were generated by panning anti-human CD19- and CD20-specific nanobody sequences from a natural Nb-expressing phage display library, integrating Nb genes with a lentiviral cassette that included other CAR elements, and finally transducing T cells that were expanded under an optimization system with the above generated CAR lentivirus. Prepared Nb CAR-T cells were cocultured with tumour cell lines or primary tumour cells for 24 h or 5 days to evaluate their biological function. Results The nanobodies that we selected from the natural Nb-expressing phage display library had a high affinity and specificity for CD19 and CD20. CD19 Nb CAR-T, CD20 Nb CAR-T and Bispecific Nb CAR-T cells were successfully constructed, and these Nb CAR-T cells could strongly recognize Burkitt lymphoma cell lines (Raji and Daudi), thereby leading to activation, enhanced proliferation, and specific killing of target cells. Furthermore, similar results were obtained when using patient samples as target cells, with a cytotoxicity of approximately 60%. Conclusions Nanobody-based CAR-T cells can kill both tumour cell lines and patient-derived tumour cells in vitro, and Nb-based CAR-T cells may be a promising therapeutic strategy in future immunotherapy.

Homodimers but not monomers of Rituxan (chimeric anti-CD20) induce apoptosis in human B-lymphoma cells and synergize with a chemotherapeutic agent and an immunotoxin

Blood ◽  
2001 ◽  
Vol 97 (5) ◽  
pp. 1392-1398 ◽  
Author(s):  
Maria-Ana Ghetie ◽  
Helen Bright ◽  
Ellen S. Vitetta
Keyword(s):  
Cell Growth ◽  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cells ◽  
B Lymphoma ◽  
B Lymphoma Cells ◽  
Anti Cd20

In 1997, a chimeric anti-CD20 monoclonal antibody (mAb) (Rituxan) was approved for the treatment of low-grade/follicular B-cell lymphoma. Rituxan has a long half-life and low immunogenicity, and it mediates effector function. Rituxan induces apoptosis in some tumor cell lines in vitro. Previous studies with mAbs that react with neoplastic B cells have demonstrated that homodimers of immunoglobulin G ([IgG]2) often inhibit cell growth more effectively than their monomeric (IgG)1counterparts. In this study, the ability of IgG or F(ab′)2 homodimers vs monomers of Rituxan were compared for their ability to inhibit the growth of several different B-lymphoma cell lines in vitro. It was found that homodimers of Rituxan had superior antigrowth activity in vitro and that F(ab′)2 homodimers were the most active. Homodimers, but not monomers, of Rituxan induced both apoptosis and necrosis of several B-cell lymphoma lines in vitro; the inhibition of cell growth was not dependent upon the presence of Fc receptors or upon 10-fold or greater differences in the density of CD20 on the target cells. Rituxan homodimers, compared with monomers, also rendered drug-resistant CD20+ B-lymphoma cells more sensitive to chemotherapeutic agents and synergized with an anti-CD22 immunotoxin in vitro.

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