scholarly journals Targeted Fluorogenic Cyanine Carbamates Enable In Vivo Analysis of Antibody-Drug Conjugate Linker Chemistry

2021 ◽  
Author(s):  
Syed Usama ◽  
Sierra Marker ◽  
Donald Caldwell ◽  
Nimit Patel ◽  
Yang Feng ◽  
...  
Keyword(s):  
Near Infrared ◽  
Antibody Drug Conjugate ◽  
Drug Conjugates ◽  
Lysosomal Accumulation ◽  
Cellular Signal ◽  
In Vivo Analysis ◽  
Cellular Permeability ◽  
Therapeutic Platform ◽  
Antibody Drug

Antibody-drug conjugates (ADCs) are a rapidly emerging therapeutic platform. The chemical linker between the antibody and the drug payload plays an essential role in the efficacy and tolerability of these agents. New methods that quantitively assess cleavage efficiency in complex tissue settings could provide valuable insights into the ADC design process. Here we report the development of a near-infrared (NIR) optical imaging approach that measures the site and extent of linker cleavage in mouse models. This approach is enabled by a superior variant of our recently devised cyanine carbamate (CyBam) platform. We identify a novel tertiary amine-containing norcyanine, the product of CyBam cleavage, that exhibits dramatically in-creased cellular signal due to improved cellular permeability and lysosomal accumulation. The resulting cyanine lysosome-targeting carbamates (CyLBams) are ~50X brighter in cells, and we find this strategy is essential for high-contrast in vivo targeted imaging. Finally, we compare a panel of several common ADC linkers across two antibodies and tumor models. These studies indicate that cathepsin-cleavable linkers provide dramatically higher tumor activation relative to hindered or non-hindered disulfides – an observation that is only apparent with in vivo imaging. This strategy enables quantitative com-parisons of cleavable linker chemistries in complex tissue settings with implications across the drug delivery landscape.

scholarly journals Targeted Fluorogenic Cyanine Carbamates Enable In Vivo Analysis of Antibody-Drug Conjugate Linker Chemistry

2021 ◽  
Author(s):  
Syed Usama ◽  
Sierra Marker ◽  
Donald Caldwell ◽  
Nimit Patel ◽  
Yang Feng ◽  
...  
Keyword(s):  
Near Infrared ◽  
Antibody Drug Conjugate ◽  
Drug Conjugates ◽  
Lysosomal Accumulation ◽  
Cellular Signal ◽  
In Vivo Analysis ◽  
Cellular Permeability ◽  
Therapeutic Platform ◽  
Antibody Drug

Antibody-drug conjugates (ADCs) are a rapidly emerging therapeutic platform. The chemical linker between the antibody and the drug payload plays an essential role in the efficacy and tolerability of these agents. New methods that quantitively assess cleavage efficiency in complex tissue settings could provide valuable insights into the ADC design process. Here we report the development of a near-infrared (NIR) optical imaging approach that measures the site and extent of linker cleavage in mouse models. This approach is enabled by a superior variant of our recently devised cyanine carbamate (CyBam) platform. We identify a novel tertiary amine-containing norcyanine, the product of CyBam cleavage, that exhibits dramatically in-creased cellular signal due to improved cellular permeability and lysosomal accumulation. The resulting cyanine lysosome-targeting carbamates (CyLBams) are ~50X brighter in cells, and we find this strategy is essential for high-contrast in vivo targeted imaging. Finally, we compare a panel of several common ADC linkers across two antibodies and tumor models. These studies indicate that cathepsin-cleavable linkers provide dramatically higher tumor activation relative to hindered or non-hindered disulfides – an observation that is only apparent with in vivo imaging. This strategy enables quantitative comparisons of cleavable linker chemistries in complex tissue settings with implications across the drug delivery landscape.

An antibody-drug conjugate directed to the ALK receptor demonstrates efficacy in preclinical models of neuroblastoma

2019 ◽  
Vol 11 (483) ◽  
pp. eaau9732 ◽  
Author(s):  
Renata Sano ◽  
Kateryna Krytska ◽  
Colleen E. Larmour ◽  
Pichai Raman ◽  
Daniel Martinez ◽  
...  
Keyword(s):  
Cell Surface ◽  
Neuroblastoma Cells ◽  
In Vivo Studies ◽  
Wild Type ◽  
Antibody Drug Conjugate ◽  
Cell Surface Antigens ◽  
Drug Conjugates ◽  
Bona Fide ◽  
Antibody Drug

Enthusiasm for the use of antibody-drug conjugates (ADCs) in cancer therapy has risen over the past few years. The success of this therapeutic approach relies on the identification of cell surface antigens that are widely and selectively expressed on tumor cells. Studies have shown that native ALK protein is expressed on the surface of most neuroblastoma cells, providing an opportunity for development of immune-targeting strategies. Clinically relevant antibodies for this target have not yet been developed. Here, we describe the development of an ALK-ADC, CDX-0125-TEI, which selectively targets both wild-type and mutated ALK-expressing neuroblastomas. CDX-0125-TEI exhibited efficient antigen binding and internalization, and cytotoxicity at picomolar concentrations in cells with different expression of ALK on the cell surface. In vivo studies showed that CDX-0125-TEI is effective against ALK wild-type and mutant patient-derived xenograft models. These data demonstrate that ALK is a bona fide immunotherapeutic target and provide a rationale for clinical development of an ALK-ADC approach for neuroblastomas and other ALK-expressing childhood cancers such as rhabdomyosarcomas.

scholarly journals Monodisperse polysarcosine-based highly-loaded antibody-drug conjugates

2019 ◽  
Vol 10 (14) ◽  
pp. 4048-4053 ◽  
Author(s):  
Warren Viricel ◽  
Guy Fournet ◽  
Sabine Beaumel ◽  
Emeline Perrial ◽  
Sébastien Papot ◽  
...  
Keyword(s):  
Drug Loading ◽  
Antibody Drug Conjugate ◽  
Antibody Drug Conjugates ◽  
Drug Conjugate ◽  
Drug Conjugates ◽  
Highly Loaded ◽  
Antibody Drug

A new antibody-drug conjugate (ADC) chemical drug-linker platform based on polysarcosine enables increased drug-loading, improved pharmacokinetics and exquisite in vivo potency.

scholarly journals ATRT-15. LY6D – A CANDIDATE FOR NANOPARTICLE-BASED TARGETED THERAPIES OF ATRT

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii278-iii278
Author(s):  
Lea Hagemeier ◽  
Marthe Sönksen ◽  
Natalia Moreno ◽  
Romy Ettlinger ◽  
Hana Bunzen ◽  
...  
Keyword(s):  
Targeted Therapies ◽  
Surface Proteins ◽  
Antibody Drug Conjugate ◽  
Drug Conjugates ◽  
Interesting Candidate ◽  
Atypical Teratoid Rhabdoid Tumors ◽  
Rhabdoid Tumors ◽  
Antibody Drug

Abstract Atypical Teratoid Rhabdoid Tumors (ATRT) are aggressive brain malignancies of the infant. Despite intensive multimodal therapy, the overall prognosis remains poor, making investigations on targeted therapies crucial. Arsenic trioxide (ATO) is known to inhibit cell growth of ATRT in vitro and in vivo but its efficacy in solid tumors is limited by its adverse effects. We aimed to characterize whether a nanoparticle-based drug delivery could overcome these limitations. Therefore metal-organic frameworks containing ATO (MOF-ATO) were constructed. To improve drug specificity further, we searched for unique proteins on the surface of ATRT, in order to create antibody-drug-conjugates out of MOF-ATO and an ATRT-specific ligand. ATRT are marked by a biallelic loss of SMARCB1, which results in an activation of the repressive histone methyltransferase EZH2. After chemical inhibition of EZH2 with GSK126, a mass spectrometric based screening for differentially expressed surface proteins was performed. Treatment with ATO, as well as MOF-ATO and GSK126 each reduces the cell viability of ATRT cell lines. It results in a cell cycle arrest and an induction in apoptosis, being analysed via MTT test and flow cytometry. GSK126 treatment causes a significant upregulation of several cell surface proteins, upon them the Lymphocyte antigen 6 family member D (LY6D). Being rarely expressed on other human cells, this protein is an interesting candidate. An antibody-drug-conjugate consisting of MOF-ATO and LY6D-ligands could be a promising approach for future targeted therapies of ATRT.

scholarly journals Targeted Fluorogenic Cyanine Carbamates Enable In Vivo Analysis of Antibody–Drug Conjugate Linker Chemistry

2021 ◽  
Author(s):  
Syed Muhammad Usama ◽  
Sierra C. Marker ◽  
Donald R. Caldwell ◽  
Nimit L. Patel ◽  
Yang Feng ◽  
...  
Keyword(s):  
Antibody Drug Conjugate ◽  
Drug Conjugate ◽  
In Vivo Analysis ◽  
Antibody Drug

scholarly journals A Novel Antibody-Drug Conjugate (ADC) Delivering a DNA Mono-Alkylating Payload to Chondroitin Sulfate Proteoglycan (CSPG4)-Expressing Melanoma

Cancers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 1029
Author(s):  
Ricarda M. Hoffmann ◽  
Silvia Crescioli ◽  
Silvia Mele ◽  
Eirini Sachouli ◽  
Anthony Cheung ◽  
...  
Keyword(s):  
Tumor Regression ◽  
Chondroitin Sulphate ◽  
Alkylating Agents ◽  
Residual Tumor ◽  
Similar Efficacy ◽  
Antibody Drug Conjugate ◽  
Drug Conjugates ◽  
Antibody Drug

Despite emerging targeted and immunotherapy treatments, no monoclonal antibodies or antibody-drug conjugates (ADCs) directly targeting tumor cells are currently approved for melanoma therapy. The tumor-associated antigen chondroitin sulphate proteoglycan 4 (CSPG4), a neural crest glycoprotein over-expressed on 70% of melanomas, contributes to proliferative signaling pathways, but despite highly tumor-selective expression it has not yet been targeted using ADCs. We developed a novel ADC comprising an anti-CSPG4 antibody linked to a DNA minor groove-binding agent belonging to the novel pyrridinobenzodiazepine (PDD) class. Unlike conventional DNA-interactive pyrrolobenzodiazepine (PBD) dimer payloads that cross-link DNA, PDD-based payloads are mono-alkylating agents but have similar efficacy and substantially enhanced tolerability profiles compared to PBD-based cross-linkers. We investigated the anti-tumor activity and safety of the anti-CSPG4-(PDD) ADC in vitro and in human melanoma xenografts. Anti-CSPG4-(PDD) inhibited CSPG4-expressing melanoma cell growth and colony formation and triggered apoptosis in vitro at low nanomolar to picomolar concentrations without off-target Fab-mediated or Fc-mediated toxicity. Anti-CSPG4-(PDD) restricted xenograft growth in vivo at 2 mg/kg doses. One 5 mg/kg injection triggered tumor regression in the absence of overt toxic effects or of acquired residual tumor cell resistance. This anti-CSPG4-(PDD) can deliver a highly cytotoxic DNA mono-alkylating payload to CSPG4-expressing tumors at doses tolerated in vivo.

A homogeneous high-DAR antibody-drug conjugate platform combining THIOMABTM antibodies and XTEN polypeptides

2021 ◽  
Author(s):  
Neelie Zacharias ◽  
Vladimir Podust ◽  
Kimberly Kajihara ◽  
Douglas leipold ◽  
Geoffrey Del Rosario ◽  
...  
Keyword(s):  
Cancer Therapeutics ◽  
Therapeutic Index ◽  
Tumor Xenograft ◽  
Antibody Drug Conjugate ◽  
Drug Conjugates ◽  
Anti Cancer ◽  
Rapid Clearance ◽  
Antibody Ratio ◽  
Antibody Drug

Abstract Antibody-drug conjugates (ADCs) enable cell-specific delivery of small molecules and are validated anti-cancer therapeutics. One factor limiting ADC advancement and broader application is the drug-to-antibody ratio (DAR), which dictates the number of payloads that can be delivered per antibody. With few exceptions, efficacious ADCs with DAR > 4 are inaccessible due to aggregation or rapid clearance in vivo. Here, we report a versatile platform for the generation of homogeneous ADCs with DAR up to 18, combining Cys-engineered THIOMAB antibodies and XTEN polypeptides to give “TXCs”. We show that high-DAR TXCs are stable biochemically and in vivo. We demonstrate that two different cytotoxic TXCs directed toward a tumor xenograft and one TXC targeting Staphylococcus aureus have comparable pharmacokinetics, but significantly enhanced efficacy in vivo versus conventional low-DAR ADCs. Our data suggest that high-DAR TXCs may ultimately offer several advantages versus conventional ADCs, including increased therapeutic index and efficacious delivery of less potent payloads.

scholarly journals 620 Tumor cell-intrinsic STING pathway is activated in the presence of cues from immune cells and contributes to the anti-tumor activity of tumor cell-targeted STING agonist antibody-drug conjugates

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A656-A656
Author(s):  
Naniye Malli Cetinbas ◽  
Travis Monnell ◽  
Winnie Lee ◽  
Kalli Catcott ◽  
Chen-Ni Chin ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Cancer Cell ◽  
Immune Cells ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
Primary Monocyte ◽  
Sting Pathway ◽  
Anti Tumor Activity ◽  
Antibody Drug

BackgroundSTING pathway agonism has emerged as a potential therapeutic mechanism to stimulate an innate anti-tumor immune response. While in principle systemic administration of a STING agonist would have many therapeutic benefits, including the delivery of STING to all tumor lesions, such an approach may be limited by toxicity. Antibody-drug conjugates (ADCs) constitute a proven therapeutic modality that is ideally suited to allow systemic administration while stimulating the innate immunity in a targeted manner. We have previously demonstrated that targeted delivery of a STING agonist with an ADC induces robust anti-tumor immune responses.MethodsHerein we investigated the mechanism of action of tumor cell-targeted STING agonist ADCs. We evaluated STING pathway activation and anti-tumor activity elicited by ADCs harboring either wild type (wt) or mutant Fc deficient in Fcγ receptor (FcγR) binding in wt or STING knockout (ko) cancer cell mono-cultures, immune cell co-cultures, and in in vivo tumor models.ResultsConsistent with previous reports, the majority of cancer cell lines tested failed to induce STING pathway following STING agonist payload treatment in mono-cultures. In cancer cell:THP1 monocytic cell co-cultures, tumor-targeted STING agonist ADCs with wt Fc exhibited robust STING activation, whereas Fc-mutant ADCs or non-targeted control ADCs had minimal activity. Similar results were obtained when THP1 cells were treated in plates coated with target antigen without cancer cells, demonstrating STING activation in THP1 cells following FcγR-mediated uptake of antigen-bound ADCs. Tumor-targeted Fc-wt ADCs led to marked induction of STING pathway and cancer cell-killing in cancer cell:PBMC or primary monocyte co-cultures, and complete tumor regressions in in vivo tumors. Surprisingly, while at reduced levels relative to the Fc-wt ADCs, Fc-mutant ADCs exhibited significant activity in these in vitro and in vivo models, suggesting that tumor cell-intrinsic STING pathway may be activated in the presence of cues from immune cells. Consistently, STING agonist payload treatment in the presence of conditioned media from PBMC and primary monocyte but not from THP1 cultures, led to STING activation in cancer cell mono-cultures. Moreover, Fc-mutant ADCs had diminished activity in STING ko cancer cell:PBMC or primary monocyte co-cultures, demonstrating the contribution of tumor cell-intrinsic STING activation to the anti-tumor activity elicited by tumor cell-targeted STING agonist ADCs.ConclusionsIn conclusion, we demonstrated that tumor cell-targeted STING agonist ADCs induce robust anti-tumor activity through mechanisms involving both FcγR and tumor antigen-mediated ADC internalization and subsequent induction of STING pathway in immune cells and tumor cells.

Axially-substituted silicon phthalocyanine payloads for antibody-drug conjugates

Synlett ◽  
2021 ◽  
Author(s):  
Kazuki Takahashi ◽  
Akira Sugiyama ◽  
Kei Ohkubo ◽  
Toshifumi Tatsumi ◽  
Tatsuhiko Kodama ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Antibody Drug Conjugate ◽  
Antibody Drug Conjugates ◽  
Drug Conjugate ◽  
Drug Conjugates ◽  
Antibody Drug ◽  
Silicon Phthalocyanine

IR700, a silicon phthalocyanine (SiPc) photosensitizer, is an antibody-drug conjugate payload used clinically. It is, however, the sole SiPc payload to date, possibly due to the difficulty of its synthesis, resulting from its asymmetric phathalocyanine skeleton. Here we report a new axially-substituted SiPc payload with easier synthesis. Trastuzumab conjugated with the SiPc showed light- and antigen-dependent cytotoxicity in HER2-overexpressed cancer cell lines.

scholarly journals Systematic identification of engineered methionines and oxaziridines for efficient, stable, and site-specific antibody bioconjugation

2019 ◽  
Author(s):  
Susanna K. Elledge ◽  
Hai L. Tran ◽  
Alec H. Christian ◽  
Veronica Steri ◽  
Byron Hann ◽  
...  
Keyword(s):  
Specific Antibody ◽  
Xenograft Model ◽  
Antibody Engineering ◽  
Antibody Drug Conjugate ◽  
Site Specific ◽  
Drug Conjugates ◽  
Mouse Xenograft Model ◽  
Random Modification ◽  
The Stability ◽  
Antibody Drug

AbstractChemical modification of antibodies is one of the most important bioconjugations utilized by biologists and biotechnology. To date, the field has been dominated by random modification of lysines or more site-specific labeling of cysteines, each with attendant challenges. Recently we have developed oxaziridine chemistry for highly selective and efficient sulfimide modification of methionine called redox-activated chemical tagging (ReACT). Here, we systematically scanned methionines throughout one of the most popular antibody scaffolds, trastuzumab, for antibody engineering and drug conjugation. We tested the expression, reactivities, and stabilities of 123 single engineered methionines distributed over the surface of the antibody when reacted with oxaziridine. We found uniformly high expression for these mutants and generally good reaction efficiencies with the panel of oxaziridines. Remarkably, the stability to hydrolysis of the sulfimide varied more than ten-fold depending on temperature and the site of the engineered methionine. Interestingly, the most stable and reactive sites were those that were partially buried, likely because of their reduced access to water. There was also a ten-fold variation in stability depending on the nature of the oxaziridine, which we determined was inversely correlated with the electrophilic nature of the sulfimide. Importantly, the stabilities of the best analogs and antibody drug conjugate potencies were comparable to those reported for cysteine-maleimide modifications of trastuzumab. We also found our antibody drug conjugates to be potent in a breast cancer mouse xenograft model. These studies provide a roadmap for broad application of ReACT for efficient, stable, and site-specific antibody and protein bioconjugation.

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