Maytansinoids; Tubulin
CD138 (syndecan-1) expressed on the surface of multiple myeloma (MM) cells. [2]

HCC1954 and MDA-MB-468 cell proliferation is inhibited by SMCC-DM1, with IC50 values of 17.2 and 49.9 nM, respectively[1].

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Antibody-drug conjugates are targeted anticancer agents consisting of a cytotoxic drug covalently linked to a monoclonal antibody for tumor antigen–specific activity. Once bound to the target cell-surface antigen, the conjugate must be processed to release an active form of the drug, which can reach its intracellular target. Here, researchers used both biological and biochemical methods to better define this process for antibody-maytansinoid conjugates. In particular, researchers examined the metabolic fate in cells of huC242-maytansinoid conjugates containing either a disulfide linker (huC242-SPDB-DM4) or a thioether linker (huC242-SMCC-DM1). Using cell cycle analysis combined with lysosomal inhibitors, we showed that lysosomal processing is required for the activity of antibody-maytansinoid conjugates, irrespective of the linker. We also identified and characterized the released maytansinoid molecules from these conjugates, and measured their rate of release compared with the kinetics of cell cycle arrest. Both conjugates are efficiently degraded in lysosomes to yield metabolites consisting of the intact maytansinoid drug and linker attached to lysine. The lysine adduct is the sole metabolite from the thioether-linked conjugate. However, the lysine metabolite generated from the disulfide-linked conjugate is reduced and S-methylated to yield the lipophilic and potently cytotoxic metabolite, S-methyl-DM4. These findings provide insight into the mechanism of action of antibody-maytansinoid conjugates in general, and more specifically, identify a biochemical mechanism that may account for the significantly enhanced antitumor efficacy observed with disulfide-linked conjugates.[3]

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The immunoconjugate BT062-SMCC-DM1 demonstrated dose-dependent cytotoxicity against CD138-positive multiple myeloma cell lines (e.g., OPM1, RPMI8226). [2]
BT062-SMCC-DM1 was cytotoxic against primary tumor cells isolated from multiple myeloma patients (via negative selection), with an IC50 value of approximately 1 nmol/L at 48 hours. [2]
The cytotoxicity was specific to CD138-positive cells, as no significant cytotoxicity was observed against peripheral blood mononuclear cells from healthy volunteers at concentrations up to 12 nmol/L. The activity was also blocked by pre-incubation with non-conjugated nBT062 antibody. [2]
Treatment of OPM1 cells with BT062-SMCC-DM1 (1,000 ng/mL) induced G2-M phase cell cycle arrest after 24 hours, consistent with the antimitotic mechanism of maytansinoids. [2]
The treatment also induced apoptosis, as evidenced by a time-dependent increase in Apo 2.7-positive cells and the cleavage of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase (PARP) in OPM1 cells. This apoptotic cell death was blocked by the pan-caspase inhibitor z-VAD-fmk. [2]
The cytotoxicity of BT062-SMCC-DM1 was not blocked by interleukin-6 (IL-6) or insulin-like growth factor-I (IGF-I), cytokines known to protect MM cells from apoptosis. [2]
When MM cells were co-cultured with bone marrow stromal cells (BMSCs), which provide a protective microenvironment, the cytotoxicity of BT062-SMCC-DM1 was not inhibited, whereas dexamethasone-induced growth inhibition was significantly reduced by BMSC co-culture. [2]

The in vivo efficacy of nBT062-SPDB-DM4, nBT062-SMCC-DM1, and nBT062-SPP-DM1 was next evaluated in SCID mice bearing established CD138-positive MOLP-8 human MM cells. A single i.v. administration of the immunoconjugates caused significant dose-dependent tumor growth inhibition and tumor regression at concentrations that were well tolerated, evidenced by stable body weight. nBT062-SPDB-DM4 was the most active conjugate tested in this model. In addition, weekly dosing of the nBT062-SMCC-DM1 (six doses of 13.8 μg/kg) completely blocked tumor growth during the dosing period [2].

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In a severe combined immunodeficient (SCID) mouse model bearing subcutaneous MOLP-8 human multiple myeloma xenografts, a single intravenous administration of BT062-SMCC-DM1 (concentration not specified for single dose) caused significant tumor growth inhibition. Its antitumor activity was less potent compared to the conjugate BT062-SPDB-DM4 in this model. [2]
In the same MOLP-8 xenograft model, weekly dosing of BT062-SMCC-DM1 (six doses of 13.8 µg/kg) completely blocked tumor growth during the treatment period. [2]
Minimal antitumor activity was observed with free DM1 or the unconjugated nBT062 antibody, confirming that the in vivo efficacy depends on the specific targeting by the antibody-drug conjugate. [2]

Kadcyla® (T-DM1), an antibody-drug conjugates (ADCs) for HER2+ breast cancer treatment, has been approved by the Food and Drug Administration (FDA) in 2013. An ADC of random lysine conjugation, it has difficulties in DAR control and unsatisfactory PK due to uneven DAR distribution. It also gives rise to aggregation during conjugation because of the hydrophobicity nature of the cytotoxin, DM1. The linker-drug in T-DM1, SMCC-DM1 is hydrophobic and requires certain percentage of organic solvent such as DMA in the conjugation solution, limiting the manufacturing process in an organic-solvent-compatible device and adding extra costs. To address these problems, a site-specific conjugation method was developed involving full reduction of antibody and full conjugation with the bridge-like conjugator-drug, based on the work of Caddick and co-workers, to obtain a site-directed antibody-drug conjugate with DAR 4. The bridge-like conjugator was assembled with SMCC-DM1 and different lengths of hydrophilic polyethylene glycol (PEG) moiety. By applying PEG moiety in the side chain of the linker-drug, the organic solvent used in the conjugation can be reduced. When the PEG length is about 26 units, organic solvent is no longer needed in the conjugation. Reducing the amount of organic solvent in conjugation could also diminish the aggregation occurrence during the conjugation. Moreover, the conjugation configuration with the designed conjugator was also discussed in the article. The binding affinity of the resulting ADCs did not show significant decrease and the cell based assay and animal study have shown the comparable results with T-DM1.[1]

Growth inhibition assay and proliferation assay. The growth inhibitory effect of nBT062-SMCC-DM1, nBT062-SPDB-DM4, nBT062-SPP-DM1, and dexamethasone on growth of MM cell lines, PBMCs, and BM stromal cells (BMSC) was assessed by measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye absorbance, as previously described (16). One antibody molecule has attached to it ∼3.5 molecules DM4. The molecular weight of the antibody is not significantly increased by the attachment of the DM4 molecules.[2]
The ability for nBT062-SPDB-DM4 to mediate antigen-dependent bystander killing of proximal CD138-negative cells was evaluated. CD138-positive MM OPM2 cells (1 × 10-4 per well) and CD138-negative Namalwa cells (3 × 10-3 per well) were plated separately or mixed in 96-well round-bottomed plates and exposed to nBT062-SPDB-DM4 for 120 h. Cell viability was then assessed using WST-8 reagent. To evaluate growth inhibitory effects of immunoconjugates against MM cells in the BM milieu, MM cells (2 × 104 per well) were cultured for 48 h in BMSC (1 × 104 per well) coated 96-well plates (Costar) in the presence or absence of the drugs. DNA synthesis was measured by [3H]thymidine uptake, with [3H]thymidine (0.5 μCi/well) added during the last 8 h of 48-h cultures. All experiments were done in quadruplicate.[2]
Cell cycle analysis. MM cells (1 × 106) were incubated with or without agents, washed with PBS, permeabilized by a 30-min exposure to 70% ethanol at −20°C, incubated with propidium iodide (50 μg/mL) in 0.5 mL PBS containing 20 units/mL RNase A for 30 min at room temperature, and analyzed for DNA content by using flow cytometry.

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Growth Inhibition Assay: MM cells, peripheral blood mononuclear cells, or bone marrow stromal cells were cultured in appropriate media. Cells were treated with serial dilutions of BT062-SMCC-DM1 (or other agents/controls) for specified durations (e.g., 48-72 hours). Cell viability/proliferation was assessed by measuring the absorbance of a formazan dye (MTT assay) or by quantifying DNA synthesis via [³H]thymidine incorporation during the final hours of culture. [2]
Cell Cycle Analysis: MM cells were incubated with or without the test agent. After treatment, cells were washed, fixed/permeabilized with ethanol, and stained with propidium iodide solution containing RNase. The DNA content was then analyzed by flow cytometry to determine cell cycle distribution. [2]
Apoptosis Detection (Apo 2.7 staining): Treated MM cells were washed, stained with a PE-conjugated Apo 2.7 antibody, and analyzed by flow cytometry to quantify the percentage of apoptotic cells. [2]
Western Blotting: MM cells treated with BT062-SMCC-DM1 were harvested, washed, and lysed. Whole-cell lysates were separated by SDS-PAGE, transferred to membranes, and probed with specific antibodies against proteins like PARP, caspase-3, caspase-8, caspase-9, and α-tubulin (loading control) to detect cleavage products indicative of apoptosis. [2]
Cytokine/Bone Marrow Stromal Cell Co-culture Assay: To assess the impact of the microenvironment, MM cells were cultured in the presence of recombinant cytokines (IL-6, IGF-I) or on a pre-established layer of bone marrow stromal cells. The cells were then treated with BT062-SMCC-DM1 or control agents. Cell proliferation/viability was subsequently measured via [³H]thymidine incorporation or MTT assay to determine if the protective effects were overcome. [2]

Green fluorescent protein–positive human MM xenograft mouse model and SCID-hu mouse model.[2]
OPM1 cells were transfected with green fluorescent protein (OPM1GFP+) using a lentiviral vector, as previously described. CB17 SCID mice (48-54 days old) were purchased from Charles River Laboratories. All animal studies were conducted according to protocols approved by the Animal Ethics Committee of the Dana-Farber Cancer Institute. Mice were inoculated s.c. with 5 × 106 OPM1GFP+ MM cells in 100 μL RPMI 1640. When tumors became palpable, mice were assigned into the treatment group receiving 200 μg conjugate per mouse via tail vein injection weekly or the control group receiving vehicle alone. Caliper measurements of the longest perpendicular tumor diameters were done every alternate day to estimate the tumor volume using the following formula representing the three-dimensional volume of an ellipse: 4 / 3 × (width / 2)2 × (length / 2). Animals were sacrificed when tumors reached 2 cm or when moribund. Survival was evaluated from the first day of treatment until death. Tumor growth was evaluated using caliper measurements from the first day of treatment until day of sacrifice, day 10 for control, and day 21 for the nBT062-SPDB-DM4 treatment group. Mice were monitored by whole-body fluorescence imaging using Illumatool Bright Light System LT-9900 (Lightools Research) after shaving the tumor area. The images were captured with a Canon IXY digital 700 camera. Ex vivo analysis of tumor image was captured with a LEICA DM IL microscope connected to the LEICA DFC300 FX camera at 40 units/0.60.
Human fetal long bones were implanted into CB17 SCID mice (SCID-hu), as previously described (18). Briefly, 4 wk after bone implantation, 2.5 × 106 INA-6 cells in a final volume of 100 μL of RPMI 1640 were injected directly into the human BM cavity in the SCID-hu mice. An increase in the levels of soluble human IL-6 receptor (shuIL-6R), which is released by INA-6 cells, was used as a parameter of MM cell growth and burden of disease in SCID-hu mice. Mice developed measurable serum shuIL-6R ∼4 wk after INA-6 cell injection and then received 0.176 mg conjugate or vehicle control via tail vein injection weekly for 7 wk. After treatments, blood samples were collected and assayed for shuIL-6R levels by an ELISA.

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MOLP-8 Xenograft Model (SCID Mice): Severe combined immunodeficient mice were inoculated subcutaneously with MOLP-8 human multiple myeloma cells suspended in a mixture of serum-free media and Matrigel. When tumors reached approximately 100 mm³, mice were randomized into treatment groups. BT062-SMCC-DM1 was administered as a bolus intravenous injection, either as a single dose or weekly (e.g., six doses of 13.8 µg/kg). Tumor dimensions were measured regularly with calipers, and tumor volume was calculated. Mouse body weight was monitored as an indicator of toxicity. [2]
SCID-hu Mouse Model: Human fetal long bones were implanted into CB17 SCID mice. Four weeks later, IL-6-dependent INA-6 multiple myeloma cells were injected directly into the implanted human bone marrow cavity. Tumor burden was monitored by measuring serum levels of soluble human IL-6 receptor released by the INA-6 cells. Mice with established tumors were treated via tail vein injection with BT062-SMCC-DM1 or vehicle control weekly for 7 weeks. Blood samples were collected periodically to assay soluble IL-6 receptor levels. [2]

In the MOLP-8 xenograft model, treatment with an effective dose of BT062-SMCC-DM1 was well tolerated, and the body weight of mice in the treatment group remained stable. [2]

[1]. Site-specific and hydrophilic ADCs through disulfide-bridged linker and branched PEG. Bioorg Med Chem Lett. 2018 May 1;28(8):1363-1370.

[2]. The monoclonal antibody nBT062 conjugated to cytotoxic Maytansinoids has selective cytotoxicity against CD138-positive multiple myeloma cells in vitro and in vivo. Clin Cancer Res. 2009 Jun 15;15(12):4028-37. doi: 10.1158/1078-0432.CCR-08-2867.

[3]. Antibody-maytansinoid conjugates are activated in targeted cancer cells by lysosomal degradation and linker-dependent intracellular processing. Cancer Res. 2006 Apr 15;66(8):4426-33.

Objective: We investigated the antitumor effects of the mouse/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with a highly cytotoxic maytansin derivative on multiple myeloma (MM) cells in vitro and in vivo. Experimental Design: We examined the growth inhibitory effects of BT062-SPDB-DM4, BT062-SMCC-DM1, and BT062-SPP-DM1 on MM cell lines and primary tumor cells from MM patients. We also examined the in vivo activity of these drugs in a human MM cell xenograft mouse model and a severe combined immunodeficiency (SCID) mouse model (SCID-hu model), in which mice were implanted with bone fragments injected with human MM cells. Results: The anti-CD138 immunoconjugates significantly inhibited the growth of MM cell lines and primary tumor cells from MM patients, and showed no cytotoxicity to peripheral blood mononuclear cells from healthy volunteers. In multiple myeloma (MM) cells, these compounds induced G2/M phase cell cycle arrest, followed by apoptosis associated with caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase (PARP) cleavage. Unconjugated nBT062 completely blocked the cytotoxicity induced by the nBT062-maytansin conjugate, confirming that specific binding is necessary for cytotoxicity induction. Furthermore, the nBT062-maytansin conjugate blocked the adhesion of MM cells to bone marrow stromal cells. Co-culture of MM cells with bone marrow stromal cells protected cells from dexamethasone-induced death but had no effect on the cytotoxicity of the immunoconjugate. Importantly, nBT062-SPDB-DM4 and nBT062-SPP-DM1 significantly inhibited MM tumor growth in vivo and prolonged host survival in both human MM xenograft mouse models and SCID-hu mouse models. Conclusion: These results provide a preclinical framework for evaluating nBT062-matansine derivatives in clinical trials to improve the prognosis of MM patients. [2]

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Researchers reported that the huC242-matansine conjugate huC242-SMCC-DM1, which has a thioether-containing “non-cleavable” linker, was at least as effective in vitro as the selected conjugate huC242-SPDB-DM4, which has a disulfide-containing “cleavable” linker. This was surprising, as huC242-SMCC-DM1 showed significantly lower in vivo activity in various xenograft tumor models. To investigate this puzzle, we conducted a series of experiments to elucidate the cell-killing mechanisms of these conjugates. The results showed that activation of both conjugates required lysosomal degradation of the antibody components in the conjugates. However, we identified and characterized different maytansine metabolites produced during the intracellular processing of huC242-SPDB-DM4, which provides a possible mechanism for its superior antitumor efficacy. [3]

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BT062-SMCC-DM1 is an antibody-drug conjugate composed of a mouse/human chimeric anti-CD138 monoclonal antibody nBT062 linked to the cytotoxic maytansin class drug DM1 via an indestructible thioether bond (SMCC linker). [2]
This conjugate is designed to target the potent cytotoxic drug DM1 to CD138-expressing multiple myeloma cells. DM1 is an antimitotic agent that inhibits microtubule polymerization. [2]
This study shows that BT062-SMCC-DM1 can overcome traditional drug resistance mechanisms in multiple myeloma, such as the protective effect of the bone marrow stromal cell microenvironment and the effects of survival cytokines such as IL-6 and IGF-I. [2]
In the tested models, the disulfide-linked conjugate BT062-SPDB-DM4 showed higher potency in vivo than BT062-SMCC-DM1. [2]

C51H66CLN5O16S

1072.6116528511

1071.391

C, 57.11; H, 6.20; Cl, 3.30; N, 6.53; O, 23.87; S, 2.99

1228105-51-8

92131096

White to off-white solid powder

1.4±0.1 g/cm3

1.626

3.28

2

17

15

74

2250

8

ClC1C(=CC2CC(C)=CC=CC([C@]3(C[C@@H]([C@@H](C)C4[C@](C)(C(CC(N(C)C=1C=2)=O)OC([C@H](C)N(C)C(CCSC1CC(N(C1=O)CC1CCC(C(=O)ON2C(CCC2=O)=O)CC1)=O)=O)=O)O4)OC(N3)=O)O)OC)OC |t:7,9|

IADUWZMNTKHTIN-IOBAKXROSA-N

InChI=1S/C51H66ClN5O16S/c1-27-10-9-11-37(69-8)51(67)25-35(70-49(66)53-51)28(2)45-50(4,72-45)38(24-42(61)55(6)33-21-31(20-27)22-34(68-7)44(33)52)71-47(64)29(3)54(5)39(58)18-19-74-36-23-43(62)56(46(36)63)26-30-12-14-32(15-13-30)48(65)73-57-40(59)16-17-41(57)60/h9-11,21-22,28-30,32,35-38,45,67H,12-20,23-26H2,1-8H3,(H,53,66)/b11-9-,27-10+/t28-,29+,30?,32?,35+,36?,37-,38-,45?,50+,51+/m1/s1

2,5-dioxopyrrolidin-1-yl 4-((3-((3-(((2S)-1-(((14S,16S,33S,2R,4R,10E,12Z,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl)oxy)-1-oxopropan-2-yl)(methyl)amino)-3-oxopropyl)thio)-2,5-dioxopyrrolidin-1-yl)methyl)cyclohexane-1-carboxylate

DM1-SMCC; DM1 SMCC; DM1SMCC; DM1-SMCC; SCHEMBL20153009; IADUWZMNTKHTIN-MLSWMBHTSA-N; 1613362-80-3; SMCC-DM1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Ship with dry ice.

DMSO : ~16.67 mg/mL (~15.54 mM)

Solubility in Formulation 1: 2 mg/mL (1.86 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2 mg/mL (1.86 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2 mg/mL (1.86 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 10% DMSO+ 40% PEG300+ 5% Tween-80+ 45% saline: 2 mg/mL (1.86 mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)