Auristatin
CD30 (highly expressed on Hodgkin lymphoma and anaplastic large cell lymphoma cells)[1]

Because of its membrane permeability, monomethyl auristatin E (MMAE) can effectively be released from SGN-35 within CD30+ cancer cells and cause cytotoxicity in bystander cells[1]. Colorectal and pancreatic cancer cells were sensitized to IR by MMAE in a schedule- and dose-dependent way that was correlated with mitotic arrest. Reduced clonogenic survival and more DNA double strand breaks in exposed cells are signs of radiosensitization[2].

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VcMMAE (SGN-35) kills CD30-positive cells at low picomolar concentrations, several orders of magnitude lower than the amount required for antigen saturation.
The ADC exhibits potent cytotoxicity against CD30-positive cell lines: IC₅₀ of 1.3 ng/mL (with 8 drugs per mAb) for Karpas299, 9.9 ng/mL for L540cy, and 3300 ng/mL for CD30-negative Ramos cells.
Intracellular release of MMAE from VcMMAE in CD30-positive cells reaches concentrations >400 nmol/L within 24 hours, with intracellular MMAE half-life of 15–20 h.
Released MMAE from CD30-positive cells is able to kill cocultured CD30-negative cells (bystander effect).[1]

Tumor-targeted ACPP-cRGD-MMAE combined with IR results in a more robust and noticeably prolonged tumor regression in xenograft models. In contrast, monomethyl auristatin E (MMAE) plus IR causes a delay in tumor growth[2].

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VcMMAE treatment leads to regression and cure of established Hodgkin lymphoma xenografts in mice at doses of 1 mg/kg, which is far below the maximum tolerated dose of 120 mg/kg.[1]

MMAE is efficiently released from SGN-35 within CD30(+) cancer cells and, due to its membrane permeability, is able to exert cytotoxic activity on bystander cells. This provides mechanistic insight into the pronounced preclinical and clinical antitumor activities observed with SGN-35 [1].

Cells treated with ionizing radiation (IR) and monomethyl auristatin E (MMAE, 5 nM) are collected and lysed in RIPA buffer containing inhibitors of phosphatase and protease. 30μg of the lysate are electrophoresed on 4-12% Bis-Tris gels, then they are moved to PVDF membranes and the relevant primary antibodies are added. ECL is used to develop blots.

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Cells were treated with VcMMAE at ~200 ng/mL (6 nmol/L drug equivalent) under constant exposure for up to 72 h.
Intracellular and extracellular drug concentrations were measured using radiometric and LC/MS methods.
For bystander activity assays, cocultures of CD30-positive (Karpas299 or L540cy) and CD30-negative (Ramos) cells were treated with 1 µg/mL VcMMAE or control ADC for 120 h, followed by flow cytometry analysis using anti-CD30 and anti-CD19 antibodies.[1]

Female athymic nu/nu mice aged 6-8 weeks are given a 1:1 Matrigel and PBS solution subcutaneously injected into their thighs, containing 5×106 HCT-116 or PANC-1 cells. After administering ACPP-cRGD-MMAE intravenously (IV) or orally (IR) (6 nmoles/day, totaling 18 nmoles), the mice are given the treatment. Tumor tissue is then removed, paraffin embedded, formalin fixed, and stained with the appropriate antibodies. Using the UltraMap system, DAB is used as a chromagen and the primary antibody is used at a 1:250 dilution for visualization.

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Tumor Xenograft Model Establishment: Female athymic nu/nu mice (6-8 weeks old) were injected subcutaneously into the thigh region with 5×10^6 HCT-116 or PANC-1 cells suspended in a 1:1 mixture of Matrigel and PBS. Tumors were allowed to grow until the average volume exceeded 200 mm³ before randomization into treatment groups. Tumor volume was calculated using the formula: Volume = 1/2 Length Width². [2]
Focal Tumor Irradiation: When tumors reached appropriate size, the mouse was positioned so that the tumor on the right hindlimb could be focally irradiated. The rest of the mouse, including the contralateral (left) tumor, was shielded with custom lead blocks that blocked >95% of the radiation dose, as verified by dosimeters. Radiation doses varied by experiment (e.g., 3 Gy/day, 6 Gy single dose). [2]
Drug Administration (Free MMAE): Free MMAE was dissolved in DMSO and further diluted for injection. It was administered via intravenous (IV) injection on specified days. Doses were equimolar to the MMAE payload delivered by the targeted conjugate (e.g., 6 nmoles of MMAE per injection). [2]
Drug Administration (Targeted Conjugate): ACPP-cRGD-MMAE was administered via IV injection. In efficacy studies, it was typically injected at doses containing 6 or 7.5 nmoles of MMAE per injection. The timing relative to irradiation varied: in some studies, the first dose was given one day before the first radiation fraction; in others, it was given 6 hours after irradiation on the same day. [2]
Tissue Analysis (Zymography): Tumor xenografts were excised, homogenized in Tris-SDS buffer (9 µL per mg tissue), and centrifuged. The supernatant was diluted with PBS, mixed with sample buffer, and run on gelatin zymography gels. Gels were renatured and then incubated in developing buffer to detect MMP (gelatinase) activity as clear bands against a blue background. [2]
In Vivo Optical Imaging: Tumor-bearing mice were anesthetized and injected IV with fluorescently labeled probes (e.g., ratiometric ACPP or Cy5-labeled ACPP-cRGD-MMAE). Animals were imaged 2-6 hours later using a small animal imager with appropriate excitation and emission filters. Imaging was performed both with skin on and after skin removal to reduce autofluorescence. [2]
Immunohistochemistry: Tumor tissues were harvested, formalin-fixed, and paraffin-embedded. Sections were stained with specific primary antibodies (e.g., against β3 integrin, phospho-S10 Histone H3) using an automated staining system. The primary antibody was visualized using DAB chromogen. [2]

The retention half-life of intracellular MMAE released by VcMMAE in CD30-positive cells is 15-20 hours. The released MMAE can diffuse out of the cells and accumulate in the culture medium, but at concentrations far below intracellular levels. [1]

The maximum tolerated dose of VcMMAE in mice was 120 mg/kg. [1]

[1]. Intracellular Activation of SGN-35, a Potent Anti-CD30 Antibody-Drug Conjugate. Clinical Cancer Research (2010), 16(3), 888-897.

[2]. Tumor radiosensitization by monomethyl auristatin E: mechanism of action and targeted delivery. Cancer Res. 2015 Apr 1;75(7):1376-87.

[3]. Jianmin Fang, et al. Anti-her2 antibody and conjugate thereof. US 20160304621 A1.

VcMMAE is an antibody-drug conjugate composed of an anti-CD30 monoclonal antibody cAC10 linked to monomethylaurestatin E (MMAE) via a protease-cleavable dipeptide linker (mc-valine-citrulline-PABC).
This antibody-drug conjugate enters the cell via CD30-mediated endocytosis, and is subsequently cleaved by lysosomal proteolysis to release free MMAE.
The released MMAE can cross the cell membrane and exert cytotoxic effects on neighboring antigen-negative cells (bystander effect), which is of great significance for the treatment of heterogeneous tumors. [1]

C68H105N11O15

1316.6260

1315.779

C, 62.03; H, 8.04; N, 11.70; O, 18.23

646502-53-6

VcMMAE;646502-53-6

46944733

White to off-white solid powder

1.2±0.1 g/cm3

1347.6±65.0 °C at 760 mmHg

768.8±34.3 °C

0.0±0.3 mmHg at 25°C

1.556

6.04

8

15

39

94

2520

12

O(C([H])([H])[H])[C@]([H])([C@]([H])(C(N([H])[C@]([H])(C([H])([H])[H])[C@]([H])(C1C([H])=C([H])C([H])=C([H])C=1[H])O[H])=O)C([H])([H])[H])[C@]1([H])C([H])([H])C([H])([H])C([H])([H])N1C(C([H])([H])[C@]([H])([C@]([H])([C@@]([H])(C([H])([H])[H])C([H])([H])C([H])([H])[H])N(C([H])([H])[H])C([C@]([H])(C([H])(C([H])([H])[H])C([H])([H])[H])N([H])C([C@]([H])(C([H])(C([H])([H])[H])C([H])([H])[H])N(C(=O)OC([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])N([H])C([C@]([H])(C([H])([H])C([H])([H])C([H])([H])N([H])C(N([H])[H])=O)N([H])C([C@]([H])(C([H])(C([H])([H])[H])C([H])([H])[H])N([H])C(C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])N1C(C([H])=C([H])C1=O)=O)=O)=O)=O)C([H])([H])[H])=O)=O)OC([H])([H])[H])=O

NLMBVBUNULOTNS-HOKPPMCLSA-N

InChI=1S/C68H105N11O15/c1-15-43(8)59(51(92-13)38-55(83)78-37-23-27-50(78)61(93-14)44(9)62(85)71-45(10)60(84)47-24-18-16-19-25-47)76(11)66(89)57(41(4)5)75-65(88)58(42(6)7)77(12)68(91)94-39-46-29-31-48(32-30-46)72-63(86)49(26-22-35-70-67(69)90)73-64(87)56(40(2)3)74-52(80)28-20-17-21-36-79-53(81)33-34-54(79)82/h16,18-19,24-25,29-34,40-45,49-51,56-61,84H,15,17,20-23,26-28,35-39H2,1-14H3,(H,71,85)(H,72,86)(H,73,87)(H,74,80)(H,75,88)(H3,69,70,90)/t43-,44+,45+,49-,50-,51+,56-,57-,58-,59-,60+,61+/m0/s1

4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate

mc-vc-PAB-MMAE; VcMMAE; Vc-MMAE; MC-VC-PAB-MMAE; MMAE Vc linker, MMAE antibody conjugate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: 1) This product is not stable in solution, please use freshly prepared working solution for optimal results; 2) Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 54 mg/mL (~41.0 mM)
H2O : < 0.1 mg/mL
Ethanol: ~50 mg/mL (38 mM)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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