Journal: International Journal of Cancer

Article Title: Bispecific CD3 × CD19 diabody for T cell-mediated lysis of malignant human B cells

doi: 10.1002/(sici)1097-0215(19980831)77:5<763::aid-ijc16>3.0.co;2-2

Figure Lengend Snippet: FIGURE 1 – Schematic representation of operons encoding anti-CD19 and anti-CD3 scFvs in plasmid pHOG21 (Kipriyanov et al., 1997a) (a and b, respectively) and dicistronic operon encoding the bispecific anti-human CD3 3 CD19 diabody in plasmid pKID3 3 19 (c). The positions of primers and most important restriction sites used for constructing the plasmids are shown. The locations of ribosome binding sites (rbs), pelB leader sequences (pelB), c-myc epitopes (c-myc), hexahistidinyl tags (His6) and stop codons (Stop) are indicated. Amino acid sequences of linkers connecting V-domains are shown below each drawing. In the linker, amino acids derived from the CH1 domain are indicated by small letters and residues introduced artificially are shown in block letters.

Article Snippet: The CD3 e-chain-specific hybridoma cell line OKT3 secreting an IgG2a MAb (Kung et al., 1979) was obtained from the ATCC (Rockville, MD).

Techniques: Plasmid Preparation, Binding Assay, Derivative Assay, Blocking Assay

Journal: International Journal of Cancer

Article Title: Bispecific CD3 × CD19 diabody for T cell-mediated lysis of malignant human B cells

doi: 10.1002/(sici)1097-0215(19980831)77:5<763::aid-ijc16>3.0.co;2-2

Figure Lengend Snippet: FIGURE 2 – Analyses of purified scFv anti-CD3, scFv anti-CD19 and bispecific diabody. (a) 12% SDS-PAGE under reducing conditions. The gel was stained with Coomassie brilliant blue. (b) Analytical gel filtration on a Superdex 75 column. The elution buffer was PBS, pH 7.0. Sample volume and flow rate were 200 µl and 0.5 ml/min, respectively. Molecular masses were calibrated with BSA (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and ribonuclease A (13.7 kDa).

Article Snippet: The CD3 e-chain-specific hybridoma cell line OKT3 secreting an IgG2a MAb (Kung et al., 1979) was obtained from the ATCC (Rockville, MD).

Techniques: SDS Page, Staining

Journal: International Journal of Cancer

Article Title: Bispecific CD3 × CD19 diabody for T cell-mediated lysis of malignant human B cells

doi: 10.1002/(sici)1097-0215(19980831)77:5<763::aid-ijc16>3.0.co;2-2

Figure Lengend Snippet: FIGURE 4 – Analyses of apparent affinities by flow cytometry. Inhibition of binding of FITC-labeled MAbs HD37 to JOK-1 cells (a) and inhibition of binding of FITC-labeled MAbs OKT3 to Jurkat cells (b) in the presence of MAb OKT3, scFv-aCD19, scFv-aCD3 and bispecific diabody are shown.

Article Snippet: The CD3 e-chain-specific hybridoma cell line OKT3 secreting an IgG2a MAb (Kung et al., 1979) was obtained from the ATCC (Rockville, MD).

Techniques: Cytometry, Inhibition, Binding Assay, Labeling

Journal: International Journal of Cancer

Article Title: Bispecific CD3 × CD19 diabody for T cell-mediated lysis of malignant human B cells

doi: 10.1002/(sici)1097-0215(19980831)77:5<763::aid-ijc16>3.0.co;2-2

Figure Lengend Snippet: FIGURE 5 – Stability of scFv fragments and diabody in human serum at 37°C. The antibody fragments were incubated at 20 µg/ml in human serum at 37°C for the times shown. The CD19 and CD3 binding activity was assessed by flow cytometry using CD191/CD32 JOK-1 and CD31/CD192 Jurkat cells. Activity of the samples at point zero was taken as 100%.

Article Snippet: The CD3 e-chain-specific hybridoma cell line OKT3 secreting an IgG2a MAb (Kung et al., 1979) was obtained from the ATCC (Rockville, MD).

Techniques: Incubation, Binding Assay, Activity Assay, Cytometry

Journal: International Journal of Cancer

Article Title: Bispecific CD3 × CD19 diabody for T cell-mediated lysis of malignant human B cells

doi: 10.1002/(sici)1097-0215(19980831)77:5<763::aid-ijc16>3.0.co;2-2

Figure Lengend Snippet: FIGURE 6 – Diabody-mediated lysis of CD19-positive B cells by activated human PBMC. The human CD19 expressing [51Cr]-labeled target cell lines Raji (a) and Namalwa (b) were co-incubated with effector CTLs at different effector/target ratios for 4 hr. Percent cytotoxicity was calculated based on [51Cr]-release in the presence of 2.5 µg/ml of parental scFvs, 0.25 µg/ml of diabody, 2.5 µg/ml of diabody or 2.5 µg/ml of BsAb OKT3 3 HD37.

Article Snippet: The CD3 e-chain-specific hybridoma cell line OKT3 secreting an IgG2a MAb (Kung et al., 1979) was obtained from the ATCC (Rockville, MD).

Techniques: Lysis, Expressing, Labeling, Incubation

Journal: International journal of cancer

Article Title: Immunotherapy of B-cell non-Hodgkin lymphoma by targeting the chemokine receptor CXCR5 in a preclinical mouse model.

doi: 10.1002/ijc.28893

Figure Lengend Snippet: Figure 1. Purification of CXCR5::CD3 by cationic exchange chromatography. (a) Elution profile for the separation of the parental mouse anti- bodies (peak no. 1–5) from bsAbs CXCR5::CD3 (peak no. 6–8) by cationic exchange chromatography. The left y-axis represents the absorb- ance of light (280 nm). The right y-axis represents the conductivity (mS/cm). (b) Analysis of the eluted fractions [corresponding to the peaks in (a)] for their identity by reducing SDS-PAGE and western blotting with anti-mouse- and anti-rat-detection antibodies (left and right panels, respectively). Only fractions 6–8 are comprised of rat as well as mouse-IgG. (c) Evaluation of the purity of the bsAb and parental antibodies by SDS-PAGE and Coomassie staining under reducing condition. The arrows mark the two different light chains of the bsAb. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: The quadroma cell line CXCR5::CD3 was generated by fusion of the CD3-specific hybridoma OKT3 (mouse-IgG2a; ATCC, Rockville, MD) and the CXCR5-specific hybridoma 8B2 (ratIgG2b)11 using standard protocols.21 Quadroma cells were grown in CELLineTM CL-1000 Flasks (Becton Dickinson) and the antibody purified from cell culture supernatant on a Biologic DuoflowTM System (Bio-Rad) first by affinitychromatography on Protein-A Sepharose Cartridges (Bio-Rad Laboratories) to remove the parental rat-IgG2b antibodies and subsequently by FPLC on SP-high performance sepharose XK16/20 columns (GE Healthcare, Sweden), for a separation of the bsAbs from parental mouse-IgG2a antibodies.18 The purity and identity of the bsAb was determined by SDSPAGE under reducing conditions and by Western blotting.

Techniques: Chromatography, SDS Page, Western Blot, Staining

Journal: International journal of cancer

Article Title: Immunotherapy of B-cell non-Hodgkin lymphoma by targeting the chemokine receptor CXCR5 in a preclinical mouse model.

doi: 10.1002/ijc.28893

Figure Lengend Snippet: Figure 2. Proof of identity and functional activity of the purified bsAb. (a) Schematic picture of the proof-of-identity test. Binding of the bsAb CXCR5::CD3 to CD3 expressed on Jurkat T cells, mediated by the mouse-derived heavy and light chain from the parental OKT3 anti- body, is detected by an anti-rat secondary antibody binding the rat-derived heavy chain of the bsAb (left). Similarly, binding of the bsAb CXCR5::CD3 to CXCR5 on Raji cells, mediated by the rat-derived heavy and light chain from the parental 8B2 antibody, is detected by a sec- ondary anti-mouse antibody (right). (b and c) Binding of the bsAb (green) to CD31 Jurkat T cells (left) or CXCR51 Raji cells (right) is compa- rable to the parental antibodies CD3 and CXCR5. (d–e) The bsAb promotes in vitro T-cell recruitment to B cells. Fluorescence imaging shows that in the presence of bsAb Jurkat T cells (green) tend to cluster around Raji B cells (red) when compared to the corresponding parental anti-CXCR5 or anti-CD3 antibody, isotype control antibodies or the medium control (Supporting Information Fig. S1).

Article Snippet: The quadroma cell line CXCR5::CD3 was generated by fusion of the CD3-specific hybridoma OKT3 (mouse-IgG2a; ATCC, Rockville, MD) and the CXCR5-specific hybridoma 8B2 (ratIgG2b)11 using standard protocols.21 Quadroma cells were grown in CELLineTM CL-1000 Flasks (Becton Dickinson) and the antibody purified from cell culture supernatant on a Biologic DuoflowTM System (Bio-Rad) first by affinitychromatography on Protein-A Sepharose Cartridges (Bio-Rad Laboratories) to remove the parental rat-IgG2b antibodies and subsequently by FPLC on SP-high performance sepharose XK16/20 columns (GE Healthcare, Sweden), for a separation of the bsAbs from parental mouse-IgG2a antibodies.18 The purity and identity of the bsAb was determined by SDSPAGE under reducing conditions and by Western blotting.

Techniques: Functional Assay, Activity Assay, Binding Assay, Derivative Assay, In Vitro, Fluorescence, Imaging, Control

Journal: International journal of cancer

Article Title: Immunotherapy of B-cell non-Hodgkin lymphoma by targeting the chemokine receptor CXCR5 in a preclinical mouse model.

doi: 10.1002/ijc.28893

Figure Lengend Snippet: Figure 3. CXCR5::CD3 efficiently activates T cells. (a) Activation of CD31 T cells was determined by CD25 expression following incuba- tion of PBMCs with the bsAb in the presence or absence of anti- CD28 antibody, IL-2 or PMA. CD31 T-cell activation was determined by CD25 expression by flow cytometry. (b) The bsAb CXCR5::CD3 is highly efficient in activating CD81 T cells compared to its parental anti-CD3 antibody OKT3. PBMCs were incubated with IL-2 and the bsAb CXCR5::CD3, its parental CD3 antibody OKT3 or isotype con- trol antibodies. CD81 T-cell activation was determined by co- expression of CD25 and CD69 on CD81 T cells by flow cytometry. (c) The bsAb leads to a stronger activation of CD81 T cells than the parental anti-CD3 antibody. The figure shows data from two representative donors on the third day (n 5 4, *p < 0.05, **p < 0.02 paired Student’s t-test).

Article Snippet: The quadroma cell line CXCR5::CD3 was generated by fusion of the CD3-specific hybridoma OKT3 (mouse-IgG2a; ATCC, Rockville, MD) and the CXCR5-specific hybridoma 8B2 (ratIgG2b)11 using standard protocols.21 Quadroma cells were grown in CELLineTM CL-1000 Flasks (Becton Dickinson) and the antibody purified from cell culture supernatant on a Biologic DuoflowTM System (Bio-Rad) first by affinitychromatography on Protein-A Sepharose Cartridges (Bio-Rad Laboratories) to remove the parental rat-IgG2b antibodies and subsequently by FPLC on SP-high performance sepharose XK16/20 columns (GE Healthcare, Sweden), for a separation of the bsAbs from parental mouse-IgG2a antibodies.18 The purity and identity of the bsAb was determined by SDSPAGE under reducing conditions and by Western blotting.

Techniques: Activation Assay, Expressing, Cytometry, Incubation

Journal: International journal of cancer

Article Title: Immunotherapy of B-cell non-Hodgkin lymphoma by targeting the chemokine receptor CXCR5 in a preclinical mouse model.

doi: 10.1002/ijc.28893

Figure Lengend Snippet: Figure 4. Efficient B-cell depletion by CXCR5::CD3 at low antibody con- centrations. (a1) PBMCs were cultured with CXCR5::CD3, its parental antibodies, isotype control antibodies or rituximab for 3 days at the con- centrations indicated. The number of remaining B cells was determined by flow cytometry. (a2) Depletion of B cells by autologous NK cells. B cells and NK cells were mixed at a ratio of (1:5) and incubated for 3 days with CXCR5::CD3 or its parental antibodies at the concentration indicated. (b) Lysis of Raji cells by the bsAb CXCR5::CD3, its parental CD3 antibody and rituximab compared to the isotype antibodies and the parental CXCR5 antibody. Raji cells were incubated with CD81 T cells isolated from PBMCs at an effector to target ratio of (5:1) and an antibody concentration of 4 ng/ml for up to 4 days. (n5 4, *p< 0.05, **p< 0.02 paired Student’s t-test).

Article Snippet: The quadroma cell line CXCR5::CD3 was generated by fusion of the CD3-specific hybridoma OKT3 (mouse-IgG2a; ATCC, Rockville, MD) and the CXCR5-specific hybridoma 8B2 (ratIgG2b)11 using standard protocols.21 Quadroma cells were grown in CELLineTM CL-1000 Flasks (Becton Dickinson) and the antibody purified from cell culture supernatant on a Biologic DuoflowTM System (Bio-Rad) first by affinitychromatography on Protein-A Sepharose Cartridges (Bio-Rad Laboratories) to remove the parental rat-IgG2b antibodies and subsequently by FPLC on SP-high performance sepharose XK16/20 columns (GE Healthcare, Sweden), for a separation of the bsAbs from parental mouse-IgG2a antibodies.18 The purity and identity of the bsAb was determined by SDSPAGE under reducing conditions and by Western blotting.

Techniques: Cell Culture, Control, Cytometry, Incubation, Concentration Assay, Lysis, Isolation

Journal: International journal of cancer

Article Title: Immunotherapy of B-cell non-Hodgkin lymphoma by targeting the chemokine receptor CXCR5 in a preclinical mouse model.

doi: 10.1002/ijc.28893

Figure Lengend Snippet: Figure 5. Cytokine analyses of CXCR5::CD3-treated PBMC culture supernatants. (a) PBMC were treated with different concentrations of CXCR5::CD3 or the corresponding parental anti-CD3 or anti-CXCR5 antibodies. On Day 3, cytokine concentrations (IL-4, IL-6, IL-10, IFN-g and TNF-a) were measured using flow cytometry-based cytokine bead arrays (n 5 3). (b) To evaluate cytokine release in vivo, blood samples of mice were collected 24 hr after the first antibody treatment (n 5 3). The concentrations of IL-10 and TNF-a were below the detection limit (data not shown).

Article Snippet: The quadroma cell line CXCR5::CD3 was generated by fusion of the CD3-specific hybridoma OKT3 (mouse-IgG2a; ATCC, Rockville, MD) and the CXCR5-specific hybridoma 8B2 (ratIgG2b)11 using standard protocols.21 Quadroma cells were grown in CELLineTM CL-1000 Flasks (Becton Dickinson) and the antibody purified from cell culture supernatant on a Biologic DuoflowTM System (Bio-Rad) first by affinitychromatography on Protein-A Sepharose Cartridges (Bio-Rad Laboratories) to remove the parental rat-IgG2b antibodies and subsequently by FPLC on SP-high performance sepharose XK16/20 columns (GE Healthcare, Sweden), for a separation of the bsAbs from parental mouse-IgG2a antibodies.18 The purity and identity of the bsAb was determined by SDSPAGE under reducing conditions and by Western blotting.

Techniques: Cytometry, In Vivo

Journal: International journal of cancer

Article Title: Immunotherapy of B-cell non-Hodgkin lymphoma by targeting the chemokine receptor CXCR5 in a preclinical mouse model.

doi: 10.1002/ijc.28893

Figure Lengend Snippet: Figure 6. CXCR5 targeted immunotherapy inhibits tumor growth in vivo. (a) Experimental scheme of the B-cell lymphoma model. On Day 0, NOD/SCID CB17 mice received luciferase transduced Raji (2 3 106) cells together with preactivated PBMCs (7.5 3 106). Subsequently, mice received either 50 mg of the bsAb CXCR5::CD3, 50 mg of the parental antibodies anti-CXCR5 or anti-CD3 antibodies, 100 mg CD19::CD3, 100 mg of a 1:1 mixture of isotype control antibodies or 100 ml PBS as negative control. Antibodies were applied daily up to Day 5 and tumor development measured once per week over a period of 6 weeks. (b) Three-dimensional reconstruction of tumor develop- ment in a PBS-treated mouse 5 weeks after tumor cell inoculation. (c–e). Immunotherapy with either CXCR5::CD3 or the parental CXCR5 antibody compared to treatment with parental CD3 antibody, isotype control antibodies or PBS. (f and g) In an additional study, the thera- peutic effect of CXCR5::CD3 was compared to the effect of bsAb CD19::CD3 (positive control). Mice (n 5 7–10 per group) were then moni- tored for tumor development (***p < 0.001, two-way ANOVA test) and overall survival [***p < 0.0002 for CXCR5 antibody 1 CD3 antibody, p < 0.0006 for CXCR5::CD3 and **p < 0.0015 for bsAbCD19::CD3, log-rank (Mantel–Cox) test].

Article Snippet: The quadroma cell line CXCR5::CD3 was generated by fusion of the CD3-specific hybridoma OKT3 (mouse-IgG2a; ATCC, Rockville, MD) and the CXCR5-specific hybridoma 8B2 (ratIgG2b)11 using standard protocols.21 Quadroma cells were grown in CELLineTM CL-1000 Flasks (Becton Dickinson) and the antibody purified from cell culture supernatant on a Biologic DuoflowTM System (Bio-Rad) first by affinitychromatography on Protein-A Sepharose Cartridges (Bio-Rad Laboratories) to remove the parental rat-IgG2b antibodies and subsequently by FPLC on SP-high performance sepharose XK16/20 columns (GE Healthcare, Sweden), for a separation of the bsAbs from parental mouse-IgG2a antibodies.18 The purity and identity of the bsAb was determined by SDSPAGE under reducing conditions and by Western blotting.

Techniques: In Vivo, Luciferase, Control, Negative Control, Positive Control