human cd19 cdna  (OriGene)

Journal: Cancers

Article Title: CD19-ReTARG TPR : A Novel Fusion Protein for Physiological Engagement of Anti-CMV Cytotoxic T Cells Against CD19-Expressing Malignancies

doi: 10.3390/cancers17142300

Figure Lengend Snippet: CD19-ReTARG TPR selectively binds to CD19 pos cancer cells. ( A ) Schematic illustration of the CD19-ReTARG TPR ( right ) fusion protein compared to a natural peptide-HLA class I complex on human cells ( left ). ( B ) Proposed mechanism of action of CD19-ReTARG TPR . ( C ) SDS-PAGE analysis of CD19-ReTARG TPR stained with Coomassie brilliant blue under non-reducing (NR; lane 1) and reducing (R; lane 2) conditions. The uncropped blots are shown in the . ( D ) Dose-dependent binding of CD19-ReTARG TPR to parental CHO and CHO.CD19 cells. ( E ) Dose-dependent binding of CD19-ReTARG TPR (and Mock-ReTARG TPR ) to CD19 pos B-ALL SEM cells. ( F ) Binding of CD19-ReTARG TPR (1 μg/mL) to CD19 pos SEM cells is abolished in the presence of an excess of the competing antibody, anti-CD19 mAb MOR208. Flow cytometry was used for panels ( D – F ). Graphs show two technical replicates (mean ± SD).

Article Snippet: Stable CD19-expressing CHO and CEM cells were generated via lipofection (Fugene-HD, Promega BNL, Leiden, The Netherlands) with a plasmid encoding human CD19 cDNA (Origene Technologies GmbH, Herford, Germany), followed by clonal selection via limiting dilution.

Techniques: SDS Page, Staining, Binding Assay, Flow Cytometry

Journal: Cancers

Article Title: CD19-ReTARG TPR : A Novel Fusion Protein for Physiological Engagement of Anti-CMV Cytotoxic T Cells Against CD19-Expressing Malignancies

doi: 10.3390/cancers17142300

Figure Lengend Snippet: CD19-ReTARG TPR selectively redirects the cytotoxic activity of anti-CMV pp65 CD8 pos T cells towards CD19 pos cancer cell lines and patient-derived CLL cells. ( A ) Cytotoxic capacity of anti-CMV CD8 pos T cells towards CD19 pos SEM cells (E:T cell ratio = 1:1) treated with increasing concentrations (0–1000 ng/mL) of CD19-ReTARG TPR or Mock-ReTARG TPR . ( B ) Cytotoxic capacity of anti-CMV CD8 pos T cells towards CD19 pos SEM at increasing E:T cell ratios treated with CD19-ReTARG TPR or Mock-ReTARG pp65 (both 100 ng/mL). ( C ) Cytotoxic capacity of anti-CMV CD8 pos T cells towards a range of CD19 pos or CD19 neg cell lines (including AML, CML, B-ALL, B lymphoblast spherocytosis, B-CLL, Mantle cell lymphoma, Burkitt’s lymphoma, and T-ALL; E:T cell ratio = 2:1) treated with CD19-ReTARG TPR (100 ng/mL). Apoptosis (%) is shown as Δ(CD19-ReTARG TPR -anti-CMV pp65 CD8 pos T cells). ( D ) Cytotoxic capacity of anti-CMV CD8 pos T cells towards CD19 pos cancer B cells from a CLL patient (#5) at increasing E:T cell ratios treated with CD19-ReTARG TPR or Mock-ReTARG TPR (both 100 ng/mL). ( E ) Cytotoxic capacity of anti-CMV CD8 pos T cells towards CD19 pos cancer cells from 5 CLL patients (E:T cell ratio = 2:1). Apoptotic cancer cell death was assessed using Annexin V/PI staining after 24 h. Graphs A–E: n = 3 (two technical replicates); mean ± SD are shown. Statistical analysis in A,B,E was performed using unpaired t -test (Mock-ReTARG TPR versus CD19-ReTARG TPR ) (** p < 0.01,*** p < 0.001).

Article Snippet: Stable CD19-expressing CHO and CEM cells were generated via lipofection (Fugene-HD, Promega BNL, Leiden, The Netherlands) with a plasmid encoding human CD19 cDNA (Origene Technologies GmbH, Herford, Germany), followed by clonal selection via limiting dilution.

Techniques: Activity Assay, Derivative Assay, Staining

Journal: Cancers

Article Title: CD19-ReTARG TPR : A Novel Fusion Protein for Physiological Engagement of Anti-CMV Cytotoxic T Cells Against CD19-Expressing Malignancies

doi: 10.3390/cancers17142300

Figure Lengend Snippet: —CD19-ReTARG TPR selectively redirects the cytotoxic activity of anti-CMV pp65 CD8 pos T cells towards CD19 pos cancer cell lines and patient-derived CLL cells. ( A ) CD19 cell surface expression on a panel of CD19-expressing or CD19-negative cell lines (including AML, CML, B-ALL, B lymphoblast spherocytosis, B-CLL, Mantle cell lymphoma, Burkitt’s lymphoma, and T-ALL). ( B ) Cytotoxic capacity of anti-CMV pp65 CD8 pos T cells towards CD19-negative K562 cells at increasing E:T cell ratios treated with CD19-ReTARG TPR (100 ng/mL). Apoptotic cancer cell death was assessed using Annexin V/PI staining after 24 h. ( C ) Capacity of CD19-ReTARG TPR (100 ng/mL) to activate anti-CMV pp65 CD8 pos T cells in the presence of K562. ( D ) Capacity of CD19-ReTARG TPR to activate anti-CMV CD8 pos T cells in the presence of CD19-negative cell line or CD19-expressing cell lines, SEM, JeKo-1, and Wil2S (E:T cell ratio = 1:1). Conditioned culture media were collected after 24 h, and T cell-secreted IFNγ was quantified by ELISA. ( E ) CD19 expression on primary (CLL) patient-derived cancer B cells. Graph –E n = 3 (two technical replicates); mean ± SD are shown.

Article Snippet: Stable CD19-expressing CHO and CEM cells were generated via lipofection (Fugene-HD, Promega BNL, Leiden, The Netherlands) with a plasmid encoding human CD19 cDNA (Origene Technologies GmbH, Herford, Germany), followed by clonal selection via limiting dilution.

Techniques: Activity Assay, Derivative Assay, Expressing, Staining, Enzyme-linked Immunosorbent Assay

Journal: Cancers

Article Title: CD19-ReTARG TPR : A Novel Fusion Protein for Physiological Engagement of Anti-CMV Cytotoxic T Cells Against CD19-Expressing Malignancies

doi: 10.3390/cancers17142300

Figure Lengend Snippet: CD19-ReTARG TPR retains efficacy against cancer cells with low CD19 expression. ( A ) CEM cell surface-expressed CD19 after transfection with the human CD19 plasmid and culturing of single cell clones. Cells were categorized as CD19 neg , low (CD19 + ), intermediate (CD19 ++ ), and high (CD19 +++ ). ( B ) Binding of CD19-ReTARG TPR (1 μg/mL) to CD19 neg or CD19-expressing CEM cells. ( C ) Flow cytometric analysis of CD19 antigen density, semi-quantitatively determined using the BD Quantibrite kit. ( D ) Cytotoxic capacity of anti-CMV CD8 pos T cells towards CD19 neg or CD19-expressing CEM cells at increasing E:T cell ratios treated with CD19-ReTARG TPR (100 ng/mL). ( E ) Cytotoxic capacity of PBMCs towards CD19 neg or CD19-expressing CEM cells at increasing E:T cell ratios treated with blinatumomab (5 ng/mL). ( F ) Cytotoxic capacity of CD19-CAR T cells towards CD19 neg or CD19-expressing CEM cells at increasing E:T cell ratios. For C,D, and E, apoptotic cancer cell death was assessed using Annexin V/PI staining after 24 h. Graphs ( A + B ) show two technical replicates (mean ± SD). Graphs ( D – F ): n = 3 (two technical replicates); mean ± SD are shown. Statistical analysis in C–E was performed using one-way ANOVA (CEM CD19 + vs. CEM CD19 ++ or CEM CD19 +++ ) (ns = non-significant, ** p < 0.01, *** p < 0.001).

Article Snippet: Stable CD19-expressing CHO and CEM cells were generated via lipofection (Fugene-HD, Promega BNL, Leiden, The Netherlands) with a plasmid encoding human CD19 cDNA (Origene Technologies GmbH, Herford, Germany), followed by clonal selection via limiting dilution.

Techniques: Expressing, Transfection, Plasmid Preparation, Clone Assay, Binding Assay, Staining

Journal: Cancers

Article Title: CD19-ReTARG TPR : A Novel Fusion Protein for Physiological Engagement of Anti-CMV Cytotoxic T Cells Against CD19-Expressing Malignancies

doi: 10.3390/cancers17142300

Figure Lengend Snippet: CD19-ReTARG TPR induces effective lysis of CD19-expressing cancer B cells with reduced proinflammatory cytokine release compared to blinatumomab and CD19 CAR T cells. A – C : Comparison of T cell-secreted IFNγ levels after treatment of CEM CD19 + ( A ), CEM CD19 ++ ( B ), and CEM CD19 +++ ( C ) by CD19-ReTARG TPR (+ anti-CMV CD8 pos T cells, E:T = 2:1), blinatumomab (+ PBMCs, E:T = 5:1), and CD19-CAR T cells (E:T = 1:1). Conditioned culture media were collected after 24 h, and T cell-secreted IFNγ was quantified by ELISA. ( D ) Cytotoxic capacity of CD19-ReTARG TPR (+ anti-CMV pp65 CD8 pos T cells, E:T = 2:1), blinatumomab (+ PBMCs, E:T = 5:1), and CD19-CAR T cells (E:T = 1:1) to eliminate SEM B-ALL cell line. Apoptotic cancer cell death was assessed using Annexin V/PI staining after 24 h. ( E ) Conditioned culture media were collected after 24 h, and T cell-secreted proinflammatory cytokines were quantified using a cytokine array. Graphs ( A – C ) n = 3 with two technical replicates (mean ± SD). Statistical analysis in ( A – C ) was performed using one-way ANOVA (CD19-ReTARG TPR vs. blinatumomab or CD19-CAR T cells) (** p < 0.01, *** p < 0.001).

Article Snippet: Stable CD19-expressing CHO and CEM cells were generated via lipofection (Fugene-HD, Promega BNL, Leiden, The Netherlands) with a plasmid encoding human CD19 cDNA (Origene Technologies GmbH, Herford, Germany), followed by clonal selection via limiting dilution.

Techniques: Lysis, Expressing, Comparison, Enzyme-linked Immunosorbent Assay, Staining

Journal: Cancers

Article Title: CD19-ReTARG TPR : A Novel Fusion Protein for Physiological Engagement of Anti-CMV Cytotoxic T Cells Against CD19-Expressing Malignancies

doi: 10.3390/cancers17142300

Figure Lengend Snippet: CD19-ReTARG TPR induces effective lysis of CD19-expressing cancer B cells with reduced proinflammatory cytokine release compared to blinatumomab and CD19 CAR T cells. Heatmap of cytokine array comparing T cell-secreted IFNγ levels after treatment of SEM by CD19-ReTARG TPR (+ anti-CMV CD8 pos T cells, E:T = 2:1), blinatumomab (+ PBMCs, E:T = 5:1), and CD19-CAR T cells (E:T = 1:1). Conditioned culture media were collected after 24 h.

Article Snippet: Stable CD19-expressing CHO and CEM cells were generated via lipofection (Fugene-HD, Promega BNL, Leiden, The Netherlands) with a plasmid encoding human CD19 cDNA (Origene Technologies GmbH, Herford, Germany), followed by clonal selection via limiting dilution.

Techniques: Lysis, Expressing

Journal: Cancers

Article Title: CD19-ReTARG TPR : A Novel Fusion Protein for Physiological Engagement of Anti-CMV Cytotoxic T Cells Against CD19-Expressing Malignancies

doi: 10.3390/cancers17142300

Figure Lengend Snippet: CD19-ReTARG TPR induces minimal activation-induced cell death in redirected anti-CMV CD8 pos T cells. ( A ) Apoptosis of effector anti-CMV pp65 CD8 pos T cells after co-culture with CEM, CEM CD19 + , CEM CD19 ++ , or CEM CD19 +++ cells at increasing E:T cell ratios treated with CD19-ReTARG TPR (100 ng/mL). ( B ) Apoptosis of PBMCs after co-culture with CEM, CEM CD19 + , CEM CD19 ++ , or CEM CD19 +++ cells at increasing E:T cell ratios treated with blinatumomab (5 ng/mL). ( C ) Apoptosis of CD19-CAR T cells after co-culture with CEM, CEM CD19 + , CEM CD19 ++ , or CEM CD19 +++ cells at increasing E:T cell ratios. Effector cell death was assessed using PI staining after 24 h. Graphs ( A – C ): n = 3 (two technical replicates); mean ± SD are shown. Statistical analysis in ( A – C ) was performed using one-way ANOVA (CEM vs. CEM CD19 + , CEM CD19 ++ , or CEM CD19 +++ ) (ns = non-significant, * p < 0.05,** p < 0.01, *** p < 0.001).

Article Snippet: Stable CD19-expressing CHO and CEM cells were generated via lipofection (Fugene-HD, Promega BNL, Leiden, The Netherlands) with a plasmid encoding human CD19 cDNA (Origene Technologies GmbH, Herford, Germany), followed by clonal selection via limiting dilution.

Techniques: Activation Assay, Co-Culture Assay, Staining

Journal: Cellular and Molecular Immunology

Article Title: Streamlined human antibody generation and optimization by exploiting designed immunoglobulin loci in a B cell line

doi: 10.1038/s41423-020-0440-9

Figure Lengend Snippet: Construction of PCLs. a Knock-in of hV H plasmid libraries into humanized DT40 cells. The expression of surface hIgG on the cells in each step was analyzed by flow cytometry. Parental cells (L30H45(fff), leftmost) were transfected with hV H plasmid libraries (second from the left), which led to the loss of cell surface IgG due to insertion of the blasticidin resistance marker between V H and C H . The IgG-cells were sorted (middle) to concentrate the knocked-in cells. The knocked-in cells were transfected with the Cre recombinase expression vector (second from the right), and those cells that regained their surface IgG cells were collected by further magnetic sorting (rightmost). b Distribution of the amino acid length in CDR-H3 determined by NGS data from plasmid libraries (cDNA: closed bars) and the poly-clone-derived library (PCL: open bars). The distribution pattern of the CDR3-H3 length of the knocked-in library is similar to that of the plasmid library, except that the frequency of the 11-amino-acid sequence is noticeably higher. c The distribution of amino acid usage at each position in CDR-H3 was analyzed with respect to 14-residue and 11-residue CDR-H3. The results for the hVH plasmid library (left), PCL (right), 14 amino acids (upper) and 11 amino acids (lower), are shown. The sequence with the most abundant amino acid at each position of the 11-amino-acid-long CDR-H3 of the knocked-in cells was GYSGYDYYFDY. This sequence was identical to the CDR-H3 of the parental cells (L30H45(fff)), possibly derived from nontargeted parental cells during negative IgG selection

Article Snippet: The germline-derived divergent hV H harboring CDR3 was prepared from normal human peripheral blood CD19 + B-cell cDNA (ALLCELLS) using degenerate primers that annealed to FR3 (F13) and FR4 (R13).

Techniques: Knock-In, Plasmid Preparation, Expressing, Flow Cytometry, Transfection, Marker, Derivative Assay, Sequencing, Residue, Selection