Journal: Blood

Article Title: CD38-bispecific antibody pretargeted radioimmunotherapy for multiple myeloma and other B-cell malignancies

doi: 10.1182/blood-2017-09-807610

Figure Lengend Snippet: Structure and characterization of the CD38-bispecific protein. (A) Schematic of the 028-Fc-C825 bispecific (anti-CD38 × anti-Y-DOTA) Fc fusion gene. An anti-human CD38 028 scFv gene and an yttrium-DOTA capturing C825 disulfide-stabilized scFv (ds-scFv) gene were fused to the human immunoglobulin G1 Fc fragment at the amino and carboxyl ends, respectively. An NLG was incorporated between the Fc and C825 ds-scFv domains, as shown. Relevant restriction enzymes for cloning and linearization are indicated (schematic not drawn to scale). (B) SDS-PAGE analysis of the 028-Fc-C825 fusion protein. Bispecific 028-Fc-C825 fusion polypeptides were expressed in CHO-DG44 cells, where they spontaneously formed dimers via the hinge regions and were secreted into the growth medium. The purification fractions and the 028-Fc-C825 fusion protein (5 µg) were analyzed by electrophoresis on a 4% to 20% 2-morpholinoethanesulfonic acid SDS-PAGE gel (Invitrogen). Lane 1, SeeBlue Plus2 marker proteins in kilodaltons (Invitrogen); lane 2, culture supernatant; lane 3, protein A column flow-through; lane 4, wash; lane 5, the nonreduced 028-Fc-C825 fusion protein (samples boiled); lane 7, the monomeric 028-Fc-C825 fusion protein (samples boiled and reduced with 2-mercaptoethanol); lane 6 is empty. The gel was stained with Coomassie blue. (C) Sandwich enzyme-linked immunosorbent assay demonstrating concentration-dependent binding of the CD38 (028-Fc-C825) bispecific protein (red) to the Y-DOTA ligand. A 96-well plate was coated with 70 µL of the bovine serum albumin (BSA)–Y-DOTA conjugate (1 µg/mL in PBS) and then blocked with 200 µL of 2% BSA in PBS buffer. After washing, the wells were treated with 100 µL of bispecific protein at 16 µg/mL followed by serial dilution as indicated. The plate was further treated with horseradish peroxidase (HRP)–anti-human Fc Ab followed by 3,3′,5,5′-tetramethylbenzidine (TMB). Controls demonstrate that binding to Y-DOTA is dependent on the C825 portion of the bispecific protein: the positive control, CD20 2H7-Fc-C825 bispecific (blue), shows binding to Y-DOTA; the negative control, fusion protein-Fc without C825 (black), does not. (D-E) Bifunctional binding assays of the CD38 (028-Fc-C825) bispecific protein demonstrate targeted binding to CD38+ cells and ligand capture of Y-DOTA–biotin. (D) CD38+ target cells (H929) or (E) CD38− control cells (U266) (0.5 × 106) were incubated in 40 µL of HBSS–2% FBS buffer containing 1 µg of biotin–Y-DOTA ligand and 2 µg of either CD38 (red and blue) or CD20 (green) bispecific proteins, or no protein (purple) for 30 minutes at 4°C. For CD38-blocking controls (blue), cells were preincubated for 30 minutes in buffer containing 40 µg of anti-CD38 Ab. Cells were finally washed and resuspended in 40 µL of buffer plus 2 µL of phycoerythrin-SA, incubated 30 minutes at 4°C, washed 3 times, resuspended in 500 µL of PBS buffer containing 1% formaldehyde, and analyzed by flow cytometry. Bsp., bispecific; DHFR, dihydrofolate reductase; FP, fusion protein; Neg., negative; PCMV, (cytomegalovirus) promoter; Pos., positive; SP, signal peptide.

Article Snippet: These lines and the CD38 + , CD20 − human Burkitt lymphoma (BL) cell line Namalwa were authenticated by DNA profiling (ATCC kit 135-XV), tested for mycoplasma, and maintained in log-phase growth at >95% viability (trypan-blue exclusion) in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), 50 U/mL penicillin G, and 50 μg/mL streptomycin sulfate, for no more than 6 weeks after thawing.

Techniques: Cloning, SDS Page, Purification, Electrophoresis, Marker, Staining, Sandwich ELISA, Concentration Assay, Binding Assay, Serial Dilution, Positive Control, Negative Control, Control, Incubation, Blocking Assay, Flow Cytometry

Journal: Blood

Article Title: CD38-bispecific antibody pretargeted radioimmunotherapy for multiple myeloma and other B-cell malignancies

doi: 10.1182/blood-2017-09-807610

Figure Lengend Snippet: Biodistribution and PK of 90Y-DOTA–biotin using CD38-bispecific PRIT. Athymic nude mice (n = 5 per group) bearing H929 (MM) xenografts (107 cells injected in the right flank) were injected at −24 hours with 2.8 nM pretargeting Ab (either CD38 bispecific = 028-C825, or control bispecific [targeting CD20] = 2H7-C825), then at −1 hour with CA, and at 0 hours with 90Y-DOTA–biotin. (A) Blood, tumor, and normal organ specimens were taken 24 hours after radioactivity injections. (B-C) Comprehensive tissue biodistributions were obtained at sequential time points 6, 24, 48, and 120 hours after 90Y-DOTA–biotin injection, using (B) CD38 or (C) control bispecific PRIT. The panel C description also applies to nontarget tissues in panel B. Error bars = 1 SEM.

Article Snippet: These lines and the CD38 + , CD20 − human Burkitt lymphoma (BL) cell line Namalwa were authenticated by DNA profiling (ATCC kit 135-XV), tested for mycoplasma, and maintained in log-phase growth at >95% viability (trypan-blue exclusion) in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), 50 U/mL penicillin G, and 50 μg/mL streptomycin sulfate, for no more than 6 weeks after thawing.

Techniques: Injection, Control, Radioactivity

Journal: Blood

Article Title: CD38-bispecific antibody pretargeted radioimmunotherapy for multiple myeloma and other B-cell malignancies

doi: 10.1182/blood-2017-09-807610

Figure Lengend Snippet: Effect of CD38-bispecific PRIT on tumor growth rate and survival of mice bearing H929 (MM) xenografts. Athymic nude mice (n = 10 per group) with H929 xenografts were injected at −24 hours with a pretargeting protein (CD38 bispecific or control [anti-CD20] bispecific), then at −1 hour with CA, and at 0 hours with 1200 µCi 90Y-DOTA–biotin. Tumor volume was monitored 3 times weekly and mice were euthanized when tumor size reached IACUC-mandated limits. For tumor volume graphics, data from euthanized mice were retained until all mice in a group died. (A) Treatment with CD38-bispecific PRIT (red) resulted in 100% CRs during days 15 to 30 and 80% long-term complete remissions. This contrasted with control mice, where 90% of untreated (black) and 100% of control bispecific groups (gray) died of tumor progression by day 27 (P ≤ .0001, CD38 bispecific vs either control group, error bars = 1 SEM). One untreated mouse exhibited spontaneous tumor remission. (B) Kaplan-Meier analysis indicates that CD38-bispecific PRIT significantly improved survival over controls (P ≤ .0001), curing 80% of mice. Cure was defined as no sign of tumor recurrence at day 150.

Article Snippet: These lines and the CD38 + , CD20 − human Burkitt lymphoma (BL) cell line Namalwa were authenticated by DNA profiling (ATCC kit 135-XV), tested for mycoplasma, and maintained in log-phase growth at >95% viability (trypan-blue exclusion) in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), 50 U/mL penicillin G, and 50 μg/mL streptomycin sulfate, for no more than 6 weeks after thawing.

Techniques: Injection, Control

Journal: Blood

Article Title: CD38-bispecific antibody pretargeted radioimmunotherapy for multiple myeloma and other B-cell malignancies

doi: 10.1182/blood-2017-09-807610

Figure Lengend Snippet: Comparative effects of CD38-bispecific PRIT and CD38-SA PRIT on tumor growth and survival of mice bearing Namalwa (CD38+BL) xenografts. Athymic nude mice (n = 8 per group) with Namalwa xenografts were injected at −24 hours with a pretargeting protein (CD38 bispecific, CD38-SA, or control [anti-CD20] bispecific), then at −1 hour with CA, and at 0 hours with 1200 µCi 90Y-DOTA–biotin. Tumor volumes were monitored 3 times weekly and mice were euthanized when tumor size reached IACUC-mandated limits. For tumor volume graphics, data for euthanized mice were retained until all mice in a group died. (A) CD38-bispecific PRIT (red) and CD38-SA PRIT (blue) each reduced tumor volumes to undetectable levels by day 21, followed by a single tumor recurrence in the CD38-SA group and no recurrences in the CD38-bispecific group (P < .0001, either CD38 PRIT group vs either control group, error bars = 1 SEM). (B) Kaplan-Meier survival analysis. CD38-bispecific PRIT cured 75% of mice (all mortality due to early weight loss), whereas CD38-SA PRIT cured 88% of mice (all mortality due to tumor progression), demonstrating that at this 1200-µCi dose, the 2 CD38 treatments each benefitted survival with high and equivalent efficacy (P < .0001 for either CD38 treatment vs either control, P = .48 for CD38 bispecific vs CD38-SA). Cure was defined as the absence of tumor recurrence through day 150.

Article Snippet: These lines and the CD38 + , CD20 − human Burkitt lymphoma (BL) cell line Namalwa were authenticated by DNA profiling (ATCC kit 135-XV), tested for mycoplasma, and maintained in log-phase growth at >95% viability (trypan-blue exclusion) in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), 50 U/mL penicillin G, and 50 μg/mL streptomycin sulfate, for no more than 6 weeks after thawing.

Techniques: Injection, Control

Journal: Blood

Article Title: CD38-bispecific antibody pretargeted radioimmunotherapy for multiple myeloma and other B-cell malignancies

doi: 10.1182/blood-2017-09-807610

Figure Lengend Snippet: Comparative dose-response effects of CD38-bispecific PRIT vs CD38-SA PRIT on tumor growth and survival of mice bearing CD38+BL xenografts. This figure presents data from 2 replicate experiments, each using 8 to 10 athymic nude mice per group. In total, n = 18 mice per treatment were injected at −24 hours with a pretargeting protein (CD38 bispecific, CD38-SA, or control [anti-CD20] bispecific), then at −1 hour with CA, and at 0 hours with 600 or 1000 µCi 90Y-DOTA–biotin. An additional n = 10 mice per group received only pretargeting protein (CD38 bispecific or CD38-SA) and CA, with no 90Y-DOTA–biotin. (A) All control mice including untreated (black), pretargeting protein without 90Y (maroon and navy), and control bispecific 1000-µCi groups (gray) experienced rapid tumor progression and died by day 14. All CD38 PRIT mice showed CR by day 11, and subsequent strongly reduced tumor progression relative to controls (P < .0001, any CD38 PRIT group vs any control group). However, CD38-bispecific PRIT (reds) outperformed CD38-SA PRIT (blues), the former showing later and fewer tumor progressions in the 600- and 1000-µCi treatment groups (P < .003, CD38 bispecific vs CD38-SA). (B) Kaplan-Meier survival analyses reflect the tumor volume results, showing greatly improved survival in each CD38 PRIT group relative to each control (P < .0001), and improved survival of CD38-bispecific PRIT mice relative to CD38-SA PRIT mice, across 600- and 1000-µCi levels (P < .004, CD38 bispecific vs CD38-SA).

Article Snippet: These lines and the CD38 + , CD20 − human Burkitt lymphoma (BL) cell line Namalwa were authenticated by DNA profiling (ATCC kit 135-XV), tested for mycoplasma, and maintained in log-phase growth at >95% viability (trypan-blue exclusion) in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), 50 U/mL penicillin G, and 50 μg/mL streptomycin sulfate, for no more than 6 weeks after thawing.

Techniques: Injection, Control