scholarly journals Biotin as a Reactive Handle to Selectively Label Proteins and DNA with Small Molecules

2021 ◽  
Author(s):  
Adam Cotton ◽  
James Wells ◽  
Ian Seiple
Keyword(s):  
Flow Cytometry ◽  
Small Molecules ◽  
Room Temperature ◽  
Efficient Synthesis ◽  
Antibody Drug Conjugate ◽  
Drug Conjugate ◽  
Powerful Approach ◽  
Chemical Tagging ◽  
Aqueous Conditions ◽  
Antibody Drug

<p>Here we report the reaction between biotin and azide-labelled oxaziridine reagents in aqueous conditions at room temperature. This method, which we call biotin redox-activated chemical tagging (BioReACT), achieves efficient and stable labelling of proteins with oxaziridine reagents. We functionally validate the method by generating an antibody-drug conjugate and numerous flow-cytometry reagents. Finally, we conjugate a functional click handle to a biotinylated oligonucleotide. These studies show that the biotin–oxaziridine reaction is a powerful approach for the efficient synthesis of stable protein and DNA bioconjugates.</p>

scholarly journals Biotin as a Reactive Handle to Selectively Label Proteins and DNA with Small Molecules

2021 ◽  
Author(s):  
Adam Cotton ◽  
James Wells ◽  
Ian Seiple
Keyword(s):  
Flow Cytometry ◽  
Small Molecules ◽  
Room Temperature ◽  
Efficient Synthesis ◽  
Antibody Drug Conjugate ◽  
Drug Conjugate ◽  
Powerful Approach ◽  
Chemical Tagging ◽  
Aqueous Conditions ◽  
Antibody Drug

<p>Here we report the reaction between biotin and azide-labelled oxaziridine reagents in aqueous conditions at room temperature. This method, which we call biotin redox-activated chemical tagging (BioReACT), achieves efficient and stable labelling of proteins with oxaziridine reagents. We functionally validate the method by generating an antibody-drug conjugate and numerous flow-cytometry reagents. Finally, we conjugate a functional click handle to a biotinylated oligonucleotide. These studies show that the biotin–oxaziridine reaction is a powerful approach for the efficient synthesis of stable protein and DNA bioconjugates.</p>

scholarly journals Discovery and Development of MEDI7247, a Novel Pyrrolobenzodiazepine (PBD)-Based Antibody Drug Conjugate Targeting ASCT2, for Treating Hematological Cancers

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4071-4071 ◽  
Author(s):  
Nabendu Pore ◽  
Kevin P Schifferli ◽  
Noel R Monks ◽  
Ravinder Tammali ◽  
Martin Borrok ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Standard Of Care ◽  
High Expression ◽  
Antibody Drug Conjugate ◽  
Drug Conjugate ◽  
Normal Tissues ◽  
Significant Survival ◽  
Hematological Cancers ◽  
Antibody Drug

Abstract The neutral amino acid transporter, ASCT2, is frequently overexpressed in several cancers to sustain "glutamine addiction" of cancer cells. High expression of ASCT2 is often associated with poor disease prognosis. Immuno-histochemistry (IHC) on formalin-fixed paraffin embedded (FFPE) tissue samples revealed high prevalence of membranous ASCT2 expression in several hematological cancers, including MM, AML, and DLBCL summarized in Table1. ASCT2 expression was predominantly on the plasma membrane of the carcinoma cells. ASCT2 staining was observed in almost all the samples with high positivity (>2+ and in >50% of tumor cells) in 98, 74 and 55% of MM, AML and DLBCL samples respectively. Additionally, flow cytometry analyses suggest significantly high expression of ASCT2 in bone marrow (BM) samples from AML and MM patients in comparison to BM from healthy donors. Also, our data suggest relatively less expression of ASCT2 in LT-HSC (long term hematopoietic stem cells) in comparison to prominent myeloid-associated marker CD33. In contrast, we found similar expression profile of ASCT2 and CD33 in downstream lineage-committed progenitor cells, such as MPP (multi potent progenitors) and CP (common progenitors). Furthermore, IHC evaluated across normal tissues suggest restricted ASCT2 expression in the normal tissues of vital organs. Broad expression across various unmet cancers and restricted expression in normal tissues warrant ASCT2 as an attractive candidate for an antibody drug conjugate. MEDI7247 is a novel investigational antibody-drug conjugate (ADC) comprising an anti-ASCT2 human monoclonal antibody site-specifically conjugated to pyrrolobenzodiazepine (PBD) dimer via a protease-cleavable linker, with a drug to antibody ratio (DAR) of 2. MEDI7247 specifically binds to the cell surface ASCT2 while exhibiting no affinity to the other members of the family, including ASCT1. Following binding to the extracellular region of ASCT2 antigen, MEDI7247 is internalized and trafficked to the lysosomes to subsequently release the PBD warhead. The release of PBD induces DNA damage and results in tumor cell death. MEDI7247 shows potent in vitro cytotoxic killing in several heme cancer cell lines (IC50 range of 0.05 ng/ml to ~65 ng/ml) with variable levels of ASCT2 expression (e.g. H929 (high) ~ 1.1 X106 copies/cell; Nomo (low) ~ 1.3 X105 copies/cell). In vivo anti-tumor activity of MEDI7247 was evaluated in a panel of hematological tumor models (CDx) representing AML, MM, DLBCL, cALL, and Burkitt's lymphoma. Briefly, MEDI7247 (dosed: single dose or Q1Wx4) demonstrated a significant survival advantage in disseminated or subcutaneous tumor models, when compared to the untreated control or standard of care (SOC) at the various dose levels examined: 0.05, 0.1 and 0.4 mg/kg, respectively. Likewise, MEDI7247 was also tested in disseminated AML PDX (ASCT2-low) models. Significant improvement in survival was observed at both 0.1 and 0.4 mg/kg with the higher dose level extending survival by >80 days and PDX models showed robust antitumor effect and significant survival benefit compared to standard of care (SOC). We used flow cytometry sorting of CD34+ and CD38+ populations from AML BM and ran colony forming assays in methocult culture conditions to confirm that CD34+CD38+ cells are leukemic stem cells (LSC) of AML BM samples. Furthermore, we confirmed high expression of ASCT2 in LSC and in the bulk population of AML BM. Similar analyses suggest relatively high expression of ASCT2 in MM plasma cells (CD138+CD19-) and modest to low expression in MM stem cells (CD19+ CD138-). This may yield opportunity for more durable clinical response in AML, genetically heterogeneous diseases. Furthermore, MEDI7247 demonstrated acceptable safety profile in toxicity studies with non-human primates to support first in human trials. In conclusion, based on its combined efficacy and safety, MEDI7247, a first-in class ADC is currently being evaluated in clinic for the treatment of ASCT2 positive hematological malignancies (NCT03106428). Disclosures Pore: Medimmune: Employment. Schifferli:Medimmune: Employment. Monks:Medimmune: Employment. Tammali:Medimmune: Employment. Borrok:Medimmune: Employment. Hurt:Medimmune: Employment. Flynn:Medimmune: Employment. Rebelatto:Medimmune: Employment. Townsley:Medimmue: Employment. Hinrichs:Medimmune: Employment. Dixit:Medimmune: Employment. Coats:Medimmune: Employment. Herbst:Medimmune: Employment. Tice:Medimmune: Employment.

Evaluation Of Bcma As a Therapeutic Target In Multiple Myeloma Using An Antibody-Drug Conjugate

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4447-4447 ◽  
Author(s):  
Kwee L Yong ◽  
Fiona M Germaschewski ◽  
Manuel Rodriguez-Justo ◽  
Danton Bounds ◽  
Lydia Lee ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Confocal Microscopy ◽  
Surface Expression ◽  
Tumour Cells ◽  
Time Dependent ◽  
Antibody Drug Conjugate ◽  
Drug Conjugate ◽  
Antibody Drug

B-cell maturation antigen (BCMA) is upregulated at the terminal stages of plasma cell (PC) differentiation, and is expressed on normal and malignant PC. Apart from low levels of mRNA detected on dendritic cells, expression appears absent on other tissues, indicating the potential as a target for novel antibody therapeutics for multiple myeloma (MM). We generated a humanised anti-BCMA antibody, modified for Fc-enhanced function, and conjugated to mcMMAF (anti-BCMA antibody drug conjugate, ADC). Flow cytometric studies on human myeloma cell lines (HMCL) showed rapid internalisation of anti-BCMA antibody by flow cytometry and confocal microscopy. The internalised antibody was transported to the lysosome and was nearly undetectable by confocal microscopy after 6 hours, indicating the suitability of BCMA as a target for an antibody drug conjugate (ADC). BCMA expression reached original surface levels by 6 hours post antibody treatment, thus maintaining the cell as a target for effector mediated killing. Evaluation of BCMA expression on HMCL revealed variable surface expression (1/11 high, 5/11 moderate, 5/11 low). Tumour cell killing by the ADC was expression level, dose and time dependent. The highest expressing HMCL, H929, showed significant killing (60% at 100ng/mL) at 24 hours, and up to 90% after 2 days. Cells expressing moderate levels of BCMA required incubation for up to 4 days to show maximal levels of cell death, suggesting the importance of continued internalisation of the antibody/antigen complexes over this period. ARH77 cells were transduced to express varying levels of BCMA, and killing at 3 days (200ng/ml) was directly proportional to level of surface expression (NT 0% killing, Low BCMA 75% killing, High BCMA 90%). We studied surface antigen levels in a cohort of patients to ascertain the need for patient selection. Like the HMCL, patient CD138+ plasma cells (PC) displayed a range of expression. Of 67 patients tested, CD138+ PC from 12 expressed high levels, 52 expressed intermediate, and 3 had low/negative surface BCMA as determined by MFI ratio of specific antibody to isotype control. Non-CD138+ cells from the bone marrow (BM) were negative for BCMA. Immuno-histochemistry (IHC) on paraffin-embedded BM sections, using a murine antibody and dual staining with anti-Blimp1 to identify tumour cells, revealed both membrane and diffuse, or punctate, cytoplasmic staining. Expression levels varied, from high uniform, to heterogeneous and patchy, to uniform low level. In no patient were the tumour cells entirely negative for BCMA. There was broad correlation between FACS analysis and IHC, thus patients were divided into high, moderate and low expressing groups. Examination of patient and disease characteristics revealed no correlation between BCMA expression and disease stage, response to last treatment, time from diagnosis, isotype, CD56 expression, or cyclin D-type, but there was a trend towards higher BCMA levels in tumours with adverse genetics (90% of patients with adverse genetics had high/moderate levels cf 64% of patients with standard CGN (p=0.06, Fisher’s exact test, 2-tailed). CD138+ cells in LPL (n=3) were positive for BCMA, but CD20+ lymphocytes were negative. Serum BCMA levels in MM patients (175.6±242.6ng/mL; mean±SD, n=34) were higher than in normal subjects (9.28±1.9ng/mL; n=38) but no correlation with bone marrow plasmacytosis or surface BCMA was noted. Levels appeared similar between new diagnosis (147.6±190.8ng/mL; mean±SD, n=8) and relapsed disease (184.3±259.1ng/mL; n=26). We tested ADC activity on primary tumour cells in whole BM cultures, enumerating viable CD138+ cells by flow cytometry. As with the HMCL, ADC mediated cytotoxicity was expression level, dose and time dependent, with a slower time course than with HMCL, perhaps reflecting the slower turn-over of these cells. In samples expressing moderate levels of BCMA, ADC-mediated cytotoxicity increased from 23.1±2.9% (mean±SEM, n=6) at 1-2 days to 48.3±5.1% at 4 days, and 61.2±6.2% by 6-7 days. Optimal doses of ADC ranged from 500ng-1ug/ml. In summary, these preclinical data in MM support the potential utility of an anti-BCMA ADC across the whole MM population, perhaps with particular efficacy in patients with adverse genetics, for whom an unmet need remains. Disclosures: Yong: GSK: Research Funding. Germaschewski:GSK: Employment. Mayes:GlaxoSmithKline: Employment. Sully:GlaxoSmithKline: Employment. Seestaller-Wehr:GlaxoSmithKline: Employment. Fieles:GlaxoSmithKline: Employment. Tunstead:GlaxoSmithKline: Employment. McCahon:GlaxoSmithKline: Employment. Craigen:GlaxoSmithKline: Employment.

scholarly journals Anti-CD47 Monoclonal Antibody–Drug Conjugate: A Targeted Therapy to Treat Triple-Negative Breast Cancers

Vaccines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 882
Author(s):  
Yingnan Si ◽  
Ya Zhang ◽  
Jia-Shiung Guan ◽  
Hanh Giai Ngo ◽  
Angela Totoro ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Targeted Therapy ◽  
Triple Negative ◽  
Imaging System ◽  
Blood Analysis ◽  
Breast Cancers ◽  
Antibody Drug Conjugate ◽  
Drug Conjugate ◽  
Antibody Drug

Triple-negative breast cancers (TNBCs) are frequently recurrent due to the development of drug resistance post chemotherapy. Both the existing literature and our study found that surface receptor CD47 (cluster of differentiation 47) was upregulated in chemotherapy-treated TNBC cells. The goal of this study was to develop a monoclonal antibody (mAb)-based targeting strategy to treat TNBC after standard treatment. Specifically, a new mAb that targets the extracellular domain of receptor CD47 was developed using hybridoma technology and produced in fed-batch culture. Flow cytometry, confocal microscopy, and in vivo imaging system (IVIS) showed that the anti-CD47 mAb effectively targeted human and mouse TNBC cells and xenograft models with high specificity. The antibody–drug conjugate (ADC) carrying mertansine was constructed and demonstrated higher potency with reduced IC50 in TNBC cells than did the free drug and significantly inhibited tumor growth post gemcitabine treatment in MDA-MB-231 xenograft NSG model. Finally, whole blood analysis indicated that the anti-CD47 mAb had no general immune toxicity, flow cytometry analysis of lymph nodes revealed an increase of CD69+ NK, CD11c+ DC, and CD4+ T cells, and IHC staining showed tumoral infiltration of macrophage in the 4T1 xenograft BALB/cJ model. This study demonstrated that targeting CD47 with ADC has great potential to treat TNBCs as a targeted therapy.

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