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| Targets |
sulfo-SPDB targets the conjugation sites on antibodies (via NHS ester reaction with lysine amines) and thiol-containing cytotoxic payloads (via pyridyldithio reaction with sulfhydryls). The disulfide bond is cleaved in the reducing environment of the target cell.
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| ln Vitro |
ADC cytotoxins are connected to antibodies through an ADC connector to form ADCs [1].
sulfo-SPDB is a cleavable ADC linker that joins cytotoxic drugs (such as DM4) to antibodies, enabling precise delivery to target cells. The cleavable disulfide bond ensures controlled drug release in the reducing environment of the target cell cytoplasm (due to high intracellular glutathione concentrations), which is a key feature for optimizing ADC effectiveness. The presence of the sulfonate group improves the hydrophilic character of the linker, which helps to reduce aggregation of the ADC, a common challenge with hydrophobic payloads, and allows for higher drug-to-antibody ratios (DARs) without compromising stability. The NHS ester group reacts with primary amines on antibody lysine residues, while the pyridyldithio group reacts with free thiols on the cytotoxic payload (e.g., DM4). The linker itself has no direct cytotoxic activity; its function is to ensure stable circulation in the bloodstream and efficient payload release upon internalization into the target cell. |
| ln Vivo |
sulfo-SPDB is not designed to have direct in vivo activity; it is a building block for ADCs. The in vivo efficacy of an ADC incorporating sulfo-SPDB (e.g., IMGN853, mirvetuximab soravtansine) has been demonstrated in preclinical tumor xenograft models and in clinical trials. For example, ADCs using this linker have shown potent anti-tumor activity against folate receptor alpha (FRalpha)-positive tumors. The disulfide bond of sulfo-SPDB is stable in circulation (where glutathione levels are low) but undergoes efficient reductive cleavage once the ADC is internalized into the acidic and reducing environment of the lysosome/cytoplasm of the target cancer cell. This selective release mechanism minimizes off-target toxicity. The hydrophilic sulfo-SPDB linker has been shown to achieve higher drug-to-antibody ratios (DARs) with reduced aggregation compared to non-sulfonated SPDB, contributing to improved pharmacokinetics and therapeutic index.
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| Enzyme Assay |
sulfo-SPDB is a chemical reagent, not a biologically active compound, and is characterized chemically rather than in enzyme/receptor assays. The purity (>95%) is confirmed by HPLC. The structure is verified by ¹H NMR spectroscopy, which shows characteristic signals for the NHS ester (delta ~2.8 ppm), the pyridyldithio group (delta ~7.2-8.5 ppm for pyridine aromatic protons), and the sulfonate group (delta ~3.0-4.0 ppm). The molecular weight (406.44) is confirmed by mass spectrometry (MS). The reactivity and specificity of the NHS ester group are tested by reaction with a model primary amine (e.g., benzylamine) in a buffer (e.g., PBS, pH 7.4) and monitoring the disappearance of the starting material by LC-MS over time; the half-life of the NHS ester in aqueous buffer is typically 1-2 hours. The pyridyldithio group content is quantified by a thiol release assay: the compound is treated with a reducing agent (e.g., dithiothreitol, DTT), which reduces the disulfide bond, releasing 2-thiopyridone that absorbs at 343 nm; the concentration is calculated using the extinction coefficient (ε343 = 8,080 M-¹cm-¹). For antibody conjugation, a model ADC is prepared by first reacting the NHS ester with antibody lysines at pH 8.0, followed by reduction of antibody interchain disulfides to generate free thiols, and then reaction with the pyridyldithio group. Conjugation efficiency is assessed by SEC-HPLC, and DAR is determined by hydrophobic interaction chromatography (HIC-HPLC) or LC-MS.
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| Cell Assay |
sulfo-SPDB is not used directly in cell-based assays as it is a linker, not a therapeutic agent. Its activity is assessed as part of a complete ADC. A typical cell-based assay for an ADC built with sulfo-SPDB involves testing the ADC on antigen-positive and antigen-negative tumor cell lines. Target cells (e.g., FRalpha-positive KB or IGROV-1 cells) are seeded in 96-well plates at 5×103 cells per well in medium containing 10% FBS. The ADC (antibody conjugated to cytotoxic payload via sulfo-SPDB) is added at varying concentrations (typically 0.0001-100 nM based on antibody concentration) and incubated for 72-120 hours. Cell viability is measured by CellTiter-Glo or MTT assays. The IC50 for antigen-positive cells is calculated. For specificity assessment, the same ADC is tested on antigen-negative cells (e.g., FRalpha-negative A549 cells), which should have minimal cytotoxicity. To confirm that the mechanism of action requires linker cleavage, the assay can be performed in the presence of a reducing agent (e.g., 10 mM glutathione) that should enhance cytotoxicity, or in the presence of an endocytosis inhibitor (e.g., dynasore) that should block activity, demonstrating that ADC internalization and intracellular cleavage are required for cytotoxicity.
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| Animal Protocol |
sulfo-SPDB is not administered to animals directly; it is used to synthesize ADCs, which are then tested in vivo. A typical in vivo protocol for an ADC built with sulfo-SPDB involves a murine xenograft model. Female athymic nude mice (6-8 weeks old, 18-22 g) are injected subcutaneously in the flank with 5-10×10⁶ target antigen-positive tumor cells (e.g., FRalpha-positive KB or IGROV-1 cells) in 100 microL of PBS mixed 1:1 with Matrigel. When tumors reach 100-200 mm3, mice are randomized into treatment groups (n=8-10). The ADC (e.g., mirvetuximab soravtansine) is formulated in PBS or a suitable vehicle and administered intravenously (i.v.) via tail vein injection at doses of 1-10 mg/kg (based on antibody content). Control groups receive vehicle alone, non-targeting ADC (isotype control), or unconjugated antibody. Tumor volumes are measured with digital calipers every 3-4 days (volume = length × width2 × 0.5). Body weights are recorded as a general indicator of toxicity. Treatment is typically administered as a single dose or weekly for 2-4 cycles. At the end of the study (typically after 21-28 days or when tumor volumes reach ethical limits), mice are euthanized. Tumors are excised, weighed, and processed for histology (Ki-67 staining for proliferation, cleaved caspase-3 for apoptosis) and for measurement of payload concentrations by LC-MS.
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| ADME/Pharmacokinetics |
sulfo-SPDB is a cleavable ADC linker, and its pharmacokinetics are studied as part of the complete ADC conjugate. The linker itself is not administered as a standalone entity. For an ADC, the PK is characterized by the antibody portion, with the linker affecting the stability and release of the payload. ADC PK is typically biphasic, with a distribution phase (alpha) and a terminal elimination phase (beta) with a half-life of several days (e.g., 4-7 days for IgG-based ADCs). The stability of the disulfide bond in circulation is assessed by measuring the concentration of intact ADC (conjugated antibody) versus the concentration of unconjugated payload released in plasma over time. In vitro plasma stability assays: the ADC is spiked into human or mouse plasma at 37degC for up to 14 days, and samples are taken at multiple time points to quantify payload release by LC-MS. The sulfo-SPDB linker is designed to be stable in plasma (low glutathione) and cleavable in the reducing intracellular environment. The volume of distribution for the ADC is typically low (Vd ~ plasma volume) due to the large size of the antibody, limiting extravascular distribution.sulfo-SPDB is a chemical linker used for ADC synthesis, not a therapeutic agent itself.
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| Toxicity/Toxicokinetics |
The toxicity profile is evaluated for the complete ADC, not for the linker alone. In preclinical toxicology studies (rat and cynomolgus monkey) for ADCs incorporating sulfo-SPDB (e.g., mirvetuximab soravtansine), the primary dose-limiting toxicities are typically on-target effects related to the microtubule inhibitor payload (e.g., neutropenia, peripheral neuropathy, thrombocytopenia). The linker's disulfide bond contributes to the safety profile by ensuring that the payload is released primarily inside target cells. Premature cleavage of the disulfide bond in circulation could lead to systemic release of the cytotoxic payload and increased off-target toxicity. The sulfonate group on sulfo-SPDB increases water solubility compared to non-sulfonated SPDB, reducing the risk of ADC aggregation, which can lead to immunogenicity and rapid clearance. Genotoxicity, cardiotoxicity (hERG), and hepatotoxicity are not associated with the linker itself. For laboratory handling, sulfo-SPDB is handled as a chemical: wear gloves, lab coat, and eye protection. The compound should be stored at -20degC under argon to prevent hydrolysis of the NHS ester and oxidation of the disulfide bond.
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| References | |
| Additional Infomation |
sulfo-SPDB is a cleavable linker used in the synthesis of antibody-drug conjugates (ADCs). It is the linker used in the clinically approved ADC mirvetuximab soravtansine (Elahere™) for the treatment of folate receptor alpha (FRalpha)-positive, platinum-resistant ovarian cancer. The linker's name "SPDB" stands for "N-succinimidyl 4-(2-pyridyldithio)butanoate," and the "sulfo" prefix indicates the presence of the sulfonate group that enhances solubility. sulfo-SPDB enables high drug-to-antibody ratios (DAR) of up to 3-4 without significant aggregation. The linker attaches to antibody lysine residues (via NHS ester) and to the reduced thiol of the maytansinoid payload DM4. The pyridyldithio group acts as a protected thiol that is activated upon reduction, allowing conjugation to the payload. The disulfide bond is cleaved in the reducing environment of the lysosome/cytoplasm. This product is for research use only and is not for human use.
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| Molecular Formula |
C13H14N2O7S3
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|---|---|
| Molecular Weight |
406.45
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| Exact Mass |
405.996
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| CAS # |
1193111-39-5
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| PubChem CID |
53248044
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| Appearance |
Off-white to yellow solid powder
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| LogP |
0
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
25
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| Complexity |
604
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=S(C(CCSSC1=NC=CC=C1)C(ON2C(CCC2=O)=O)=O)(O)=O
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| InChi Key |
FUHCFUVCWLZEDQ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C13H14N2O7S3/c16-11-4-5-12(17)15(11)22-13(18)9(25(19,20)21)6-8-23-24-10-3-1-2-7-14-10/h1-3,7,9H,4-6,8H2,(H,19,20,21)
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| Chemical Name |
1-(2,5-dioxopyrrolidin-1-yl)oxy-1-oxo-4-(pyridin-2-yldisulfanyl)butane-2-sulfonic acid
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| Synonyms |
sulfoSPDB sulfo SPDB
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage. (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ≥ 125 mg/mL (~307.54 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4603 mL | 12.3016 mL | 24.6033 mL | |
| 5 mM | 0.4921 mL | 2.4603 mL | 4.9207 mL | |
| 10 mM | 0.2460 mL | 1.2302 mL | 2.4603 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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