Tubulin; microtubule; Auristatin

MMAF prevents the growth of anaplastic large cell lymphoma. In vitro cytotoxicity assays yielded IC50 values of 119, 105, 257, and 200 nM for Karpas 299, breast carcinoma H3396, renal cell carcinoma 786-O, and Caki-1 cells[4].

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In Vitro Growth Inhibition. cAC10−L1−MMAF4 was tested on a panel of both CD30 positive and negative cell lines as shown in Table 2. Cells were exposed to the ADCs continuously for 96 h, and the cytotoxic effects, as determined by Alamar Blue conversion, were compared to those of free MMAF. On a molar basis, the cAC10−L1−MMAF4 was an average of >2200-fold more potent than free MMAF and was active on all the CD30-positive cell lines tested. The effects were immunologically specific, since WSU-NHL cells, which do not express detectable levels of the CD30 antigen, were unaffected by the conjugate. Limited studies were also undertaken with the cAC10−L1−MMAF-OMe4-based conjugates, and it was found that they displayed IC50 values on Karpas 299 (0.04 nM) and L540cy (0.11 nM) cells that were comparable to those of cAC10−L1−MMAF4 (Table 2). This is consistent with the assumption stated earlier that the active form of MMAF-OMe is the free acid MMAF[4].

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Further studies were undertaken with MMAF and the cBR96−L1−MMAF8 conjugate on a LewisY positive human small cell lung carcinoma cell line (H69) and its P-glycoprotein-overexpressing counterpart H69/LX4. The results were compared to doxorubicin and cBR96−L1−doxorubicin8, a previously described conjugate with eight doxorubicin molecules/mAb. H69/LX-4 cells were approximately 100-times less sensitive to doxorubicin compared to the parental cell (Figure 2A), while there was only a 3-fold difference with MMAF (Figure 2B). This trend extended to the conjugates, in that the drug resistant cell line was refractory to BR96−L1−doxorubicin8 (Figure 2C), but quite sensitive to the corresponding MMAF conjugate (Figure 2D). These results have been confirmed on several MDR positive cell lines (data not shown), in which it has been found that MMAF and MMAF-based immunoconjugates circumvent common forms of the MDR phenotype [4].

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To evaluate the effect of the binding affinity on the cytotoxic activity of RDCs, we synthesized three different drug conjugates using repebodies with different binding affinities for EGFR, and examined their cytotoxicity towards EGFR-overexpressing HCC827 cells (Figure S9 and Table S1). The rEgH9–MMAF conjugates exhibited the highest cytotoxicity compared with the other two low-affinity conjugates. This result indicates that the binding affinity of a repebody for EGFR is critical to the cytotoxic activity of the RDCs, and optimizing the binding affinity can enhance the cytotoxic activity of drug conjugates. To investigate the relationship between the cell-surface expression level of EGFR and the cytotoxicity of the RDCs, we incubated three cancer cell lines expressing different levels of EGFR (Figure 3 a) with various concentrations of the rEgH9–MMAF conjugates for three days (Figure 3 b–d). The repebody–MMAF conjugates showed strong cytotoxic effects towards A431 and HCC827 cells in a dose-dependent manner, resulting in effective half-maximal concentrations (EC50) of 1.4 nM and 0.072 nM, respectively (Table 1). This result indicates that the repebody–MMAF conjugates have a much higher cytotoxicity than free, cell-impermeable MMAF (EC50=117.9 nM) and naked repebodies (rEgH9: EC50=17.2 nM) towards HCC827 cells. Interestingly, the repebody–MMAF conjugates showed much higher potency in HCC827 cells than in A431 cells. This result seems to be due to the fact that HCC827 cells express constitutively internalized oncogenic EGFRs and consequently have an increased uptake of repebody–MMAF conjugates.15 The cytotoxicity of the repebody–MMAF conjugates towards MCF7 cells was shown to be negligible even at a high dose (>200 nM; Table 1). Our results demonstrate that RDCs can efficiently deliver a potent anticancer drug to the target cells in a receptor-specific manner, minimizing off-target effects.

MMAF has a much higher maximum tolerated dose in mice (>16 mg/kg) than MMAE (1 mg/kg). The MTD of cAC10-L1-MMAF₄ is 15 mg/kg in rats and 50 mg/kg in mice. With MTDs in mice and rats of >150 mg/kg and 90 mg/kg in rats, respectively, the corresponding cAC10-L4-MMAF₄ ADC was significantly less toxic[4].

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In Vivo Therapy Studies. [4]
The therapeutic effects of the ADCs were determined in nude mice with subcutaneous or disseminated Karpas 299 tumors. In animals with subcutaneous tumors, significant antitumor effects were obtained with a single injection of either cAC10−L1−MMAF4 or cAC10−L4−MMAF4, which were indistinguishably potent and active (Figure 5A). Nearly all animals that received single ADC injections at 2 mg antibody component/kg body weight were cured. In lowering the dose to 1 mg/kg, efficacy for both ADCs, dropped off in an apparently equal manner. At this dose, there were still two of six and three of six animals cured with the cAC10−L1−MMAF4 and cAC10−L4−MMAF4 ADCs, respectively.

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Immunohistochemical Analyses. [4]
Studies were undertaken to determine if cAC10−L1−MMAF4 localized into subcutaneous Karpas 299 tumors in nude mice. Animals were injected intravenously with cAC10−L1−MMAF4 or cBR96−L1−MMAF4 at 10 mg antibody component/kg body weight. Tumors were removed 24 h later, and frozen sections were evaluated for the presence of the mAb and drug components using immunohistochemistry with biotinylated anti-human-Fc and anti−MMAF mAbs as secondary antibodies, respectively. Figures 4A and 4C show that cAC10−L1−MMAF4 accumulated much more efficiently within Karpas 299 tumors compared to the cBR96 nonbinding control ADC (Figure 4B and 4D). Since both the mAb (Figure 4A) and drug (Figure 4C) moieties were detected throughout the tumor, we conclude that the ADC is delivered as an intact molecule.

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Researchers also evaluated the antitumor activity of the rEgH9–MMAF conjugates in xenograft mice using HCC827 cells. When the tumor volume reached 110 to 130 mm3, the mice were subjected to daily intravenous injections (10 mg kg−1) of the repebody–MMAF conjugates or naked repebody (rEgH9) for six days (Figure 4 a). As a positive control, cetuximab was used. The repebody–MMAF conjugates showed a significant tumor regression response (33.4 % residual tumor on day 20, ***P<0.001) compared with the naked repebody (83.4 % residual tumor on day 20, P>0.05) and cetuximab. No significant adverse effects were detected in the treated mice on day 20 (P>0.05), with the exception of transient weight loss (Figure 4 b).

Released Drug Identification. [4]
Lysosomal extracts of L540cy cells were prepared by swelling 2.4 × 108 cells in 9 mL of 0.25 M sucrose, 1 mM EDTA, and 10 mM HEPES (4-(2-hydroxyethyl)piperazine-1-ethansulfonic acid), pH 7.4. After 30 min on ice, cells were Dounce homogenized until >95% were broken as measured by Trypan Blue dye exclusion. Homogenates were centrifuged (3000g, 10 min, 4 °C) to pellet cellular debris, and the supernatant (4 × 107 cell equivalents per tube) was transferred to polyallomar ultracentrifuge tubes (13 × 51 mm) and centrifuged (17000g, 15 min, 4 °C) in a TLA100.3 rotor to isolate the lysosome-containing light mitochondrial pellet. Pellets were stored at −80 °C. The pellets were thawed and resuspended in 500 μL of 50 mM sodium acetate pH 5.0 and 2 mM DTT. cAC10−L4-[12C]MMAF4, cAC10−L4-[13C]MMAF4, and cAC10−L4-[12/13C]MMAF4 (50 μg/mL) were independently added to one pellet. After three freeze/thaw cycles to break open lysosomes, samples were incubated for 24 h at 37 °C. Cold methanol (2 vol) was added to precipitate protein, the samples were centrifuged at 14000g to pellet debris, and 100 μL of supernatant was analyzed by low resolution mass spectrometry as described above. Authentic cysteine−L4−MMAF was prepared by treating 100 μM L4−MMAF with 1 mM cysteine in PBS at room temperature for 10 min.

Depending on the cell line, cells are treated with serial dilutions of test molecules and incubated for four to six days. The Alamar Blue dye reduction assay is used to evaluate cellular growth and reduce data in order to produce IC50 values[1].

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In Vitro Growth Inhibition. [4]
Log phase cultures of cells were collected and cells plated at seeding densities ranging from 500 to 10000 cells/well according to predetermined conditions. After incubating 24 h to allow surface protein reconstitution, serial dilutions of test molecules were added and cultures incubated a further 4−6 days depending on cell line. Assessment of cellular growth and data reduction to generate IC50 values was done using Alamar Blue dye reduction assay as previously described, according to previously published methods. Briefly, a 40% solution (wt/vol) of Alamar Blue was freshly prepared in complete media just before cultures were added. Ninety-two hours after drug exposure, Alamar Blue solution was added to cells to constitute 10% culture volume. Cells were incubated for 4 h, and dye reduction was measured on a Fusion HT fluorescent plate reader.

Mice: The size of the subcutaneous Karpas 299 tumor is 300 mm³, and three animals per group are given one intravenous injection of either cAC10-L1-MMAF₄ or cBR96-L1-MMAF₄ (10 mg antibody component/kg body weight). Immunohistochemistry evaluation is used to stain 5 μm-thin frozen tissue sections after the tumors have been removed and placed in an optimal cutting temperature compound[1].

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In Vivo Therapy Experiments. [4]
For the localized, subcutaneous disease model of anaplastic large cell lymphoma, 5 × 106 Karpas 299 cells were implanted into the right flanks of C.B.-17 SCID mice. Therapy was initiated when the tumor size in each group of six animals averaged approximately 100 mm3. Treatments consisted of a single injection of solutions of the conjugates or controls in PBS intravenously (tail vein). Tumor volume was determined using the formula (L × W2)/2. For the disseminated ALCL model, 1 × 106 Karpas 299 were injected in the tail vein into C. B.-17 SCID mice. Single dose injection treatment was performed at 9 days after tumor injection.

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In Vivo Localization of Antibodies via Immunohistochemistry. [4]
When subcutaneous Karpas 299 tumor size reached 300 mm3, three animals per group received one injection of 10 mg antibody component/kg body weight of either cAC10−L1−MMAF4 or cBR96−L1−MMAF4 intravenously. Tumors were then removed and placed in optimal cutting temperature (OCT) compound, and 5 μm-thin frozen tissue sections were stained using immunohistochemistry evaluation. Briefly, frozen tissues on the slides were air-dried then fixed in 4% paraformaldehyde for 15 min at room temperature. Endogenous peroxidase activity was blocked using 0.6% H2O2 for 15 min. Additional blocking for endogenous biotin was done using the Avidin−Biotin Blocking kit. Biotinylated-anti-human-Fc and biotinylated-anti-drug antibodies were incubated on tissues at 2 μg/mL concentration for 1 h at room temperature. Following incubation of slides with avidin conjugated to HRP, 3,3‘-diabenzidine (DAB) was used as a substrate for HRP. Tissues were counterstained using hematoxylin, slides were dehydrated, and slips were applied. Images were taken using the Zeiss Axiovert light microscope.

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In vivo antitumor activity of the RDCs.[2]
a) Nude xenograft mice (HCC827) were administered with the rEgH9–MMAF conjugates, naked repebodies, or cetuximab (10 mg kg−1) intravenously every day for six days after tumor establishment. The tumor size was measured every third day for 20 days (mean±SD; n=6). b) Changes in the mouse body weight. After administration, the body weights of each mouse were measured every third day.

Maximum tolerated dose. [4] The maximum tolerated dose (MTD) of cAC10-MMAF conjugates was determined in BALB/c mice and Sprague-Dawley rats and defined as the highest dose that did not cause more than 20% weight loss, distress, or significant toxicity in either animal. This dose was typically within 20% of the dose that caused the aforementioned events. The MTD of cAC10-L1-MMAF4 in mice was 50 mg/kg and in rats it was 15 mg/kg. The corresponding cAC10-L4-MMAF4 ADCs were much less toxic, with MTDs of >150 mg/kg (the highest dose tested, which did not result in significant toxicity) in mice and 90 mg/kg in rats. This clearly demonstrates that the way a drug is linked to a mAb has a significant impact on the tolerability of an ADC, and that ADCs lacking a peptide spacer in the linker are much less toxic than peptide-based ADCs.

[1]. EphA2 targeted chemotherapy using an antibody drug conjugate in endometrial carcinoma. Clin Cancer Res. 2010 May 1;16(9):2562-70.

[2]. Enzymatic prenylation and oxime ligation for the synthesis of stable and homogeneous protein-drug conjugates for targeted therapy. Angew Chem Int Ed Engl. 2015 Oct 5;54(41):12020-4.

[3]. Strategies and Advancement in Antibody-Drug Conjugate Optimization for Targeted CancerTherapeutics. Biomol Ther (Seoul). 2015 Nov;23(6):493-509.

[4]. Enhanced activity of monomethylauristatin F through monoclonal antibody delivery: effects of linker technology on efficacy and toxicity. Bioconjug Chem. 2006 Jan-Feb;17(1):114-24.

Targeted therapy based on protein-drug conjugates has attracted much attention due to its high efficiency and low side effects. However, how to efficiently and stably conjugate drugs to protein binders remains a challenge. This article proposes a chemical enzymatic method for the efficient preparation of highly stable and pure drug conjugates. The method involves inserting a CaaX sequence into the C-terminus of the protein binder, isoprene modification using farnesyltransferase, and drug conjugation via oxime linkage reaction. MMAF and EGFR specific repetitive antibodies were used as antitumor drugs and protein binders, respectively. This method can precisely control the synthesis of high-yield and high-purity repetitive antibody-drug conjugates. The practicality of this method is reflected in the significant stability of the repetitive antibody-drug conjugates in human plasma, negligible off-target effects, and significant antitumor activity in vivo. This method can be widely used to prepare highly homogeneous and stable PDCs for targeted therapy. [2] Antibody-drug conjugates utilize antibodies as delivery carriers to deliver highly efficient cytotoxic molecules against tumor-associated antigens for cancer treatment. Key parameters for the successful development of antibody-drug conjugates (ADCs) for clinical use include: the selection of the tumor target antigen, the antibody targeting that target, the cytotoxic molecule, the linker connecting the cytotoxic molecule and the antibody, and the conjugation chemistry method for linking the cytotoxic molecule to the antibody. The recent approvals of Adectris® (anti-CD30 ADC) and Kadcyla® (anti-HER2 ADC) reflect advancements in these core ADC technologies. The potential approval of the anti-CD22 conjugate and encouraging new clinical data for the anti-CD19 and anti-CD33 conjugates represent further progress in this field. Enriching antibody-drug conjugates (ADCs) using newly developed potent cytotoxic molecules and linkers is also a research direction targeting multiple tumor targets. However, the complexity of ADC components, the complexity of conjugation methods, and off-target toxicity remain major challenges in ADC strategy design to maximize their therapeutic potential. This article will explore the emergence of clinical ADCs, the latest trends in optimization strategies, and the research results of ADCs applying the latest optimized strategies. Furthermore, this article will discuss how to make ADCs more suitable for a wider range of disease indications in the future, as well as the challenges and prospects. [3] We have previously demonstrated that ADCs linked to cAC10 (anti-CD30) and the anti-mitotic agent monomethylaurestatin E (MMAE) exhibit significant activity against antigen-positive tumor models both in vitro and in vivo. MMAF is a novel anti-mitotic aurestatin derivative with a charged phenylalanine residue at its C-terminus, which weakens its cytotoxic activity compared to its uncharged counterpart, MMAE, likely due to impaired intracellular transport. In vitro cytotoxicity studies showed that mAb-maleimide hexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl-MMAF (mAb-L1-MMAF) conjugates were more than 2200 times more potent than free MMAF in various CD30-positive hematopoietic cell lines. Similar to cAC10-L1-MMAE, the corresponding MMAF antibody-drug conjugates (ADCs) can cure or regress established xenograft tumors at tolerable doses. To further optimize ADCs, researchers constructed several novel linkers in which various components of the L1 linker were altered or deleted. One of the most promising linkers incorporates a non-cleavable maleimide hexanoyl (L4) spacer between the drug and the monoclonal antibody. cAC10-L4-MMAF exhibited comparable in vitro activity against multiple cell lines to cAC10-L1-MMAF, and similar in vivo activity. Importantly, the tolerable dose of cAC10-L4-MMAF was more than three times that of cAC10-L1-MMAF. Liquid chromatography-mass spectrometry (LCMS) studies showed that the drug released from cAC10-L4-MMAF is a cysteine-L4-MMAF adduct, likely due to the degradation of the monoclonal antibody within the target cell lysosome. This novel linker technology appears well-suited for drugs with relatively low cell permeability and tolerance to amino acid substitutions. Therefore, linker modifications can significantly impact toxicity and could lead to the development of novel antibody-drug conjugates (ADCs) with dramatically improved therapeutic indices. [4]

C39H66CLN5O8

768.4231

767.46

1415246-68-2

MMAF;745017-94-1;MMAF-d8 hydrochloride;MMAF sodium;1799706-65-2;MMAF-OMe;863971-12-4;MMAF-d8

78357791

White to off-white solid

5.016

5

9

21

53

1160

9

Cl[H].O(C([H])([H])[H])[C@]([H])([C@]([H])(C(N([H])[C@]([H])(C(=O)O[H])C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[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([H])C([H])([H])[H])=O)=O)OC([H])([H])[H])=O

BUPKFQQDMNUXOY-KMYLZLQDSA-N

InChI=1S/C39H65N5O8.ClH/c1-12-25(6)34(43(9)38(48)33(24(4)5)42-37(47)32(40-8)23(2)3)30(51-10)22-31(45)44-20-16-19-29(44)35(52-11)26(7)36(46)41-28(39(49)50)21-27-17-14-13-15-18-27;/h13-15,17-18,23-26,28-30,32-35,40H,12,16,19-22H2,1-11H3,(H,41,46)(H,42,47)(H,49,50);1H/t25-,26+,28-,29-,30+,32-,33-,34-,35+;/m0./s1

(2S)-2-[[(2R,3R)-3-methoxy-3-[(2S)-1-[(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl-[(2S)-3-methyl-2-[[(2S)-3-methyl-2-(methylamino)butanoyl]amino]butanoyl]amino]heptanoyl]pyrrolidin-2-yl]-2-methylpropanoyl]amino]-3-phenylpropanoic acid;hydrochloride

Monomethylauristatin F hydrochloride; MMAF Hydrochloride; MMAF (Hydrochloride); 1415246-68-2; (2S)-2-[[(2R,3R)-3-methoxy-3-[(2S)-1-[(3R,4S,5S)-3-methoxy-5-methyl-4-[methyl-[(2S)-3-methyl-2-[[(2S)-3-methyl-2-(methylamino)butanoyl]amino]butanoyl]amino]heptanoyl]pyrrolidin-2-yl]-2-methylpropanoyl]amino]-3-phenylpropanoic acid;hydrochloride; MMAFHydrochloride; Monomethylauristatin F Hydrochloride; SCHEMBL21111548; ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine hydrochloride; MMAF hydrochloride

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, avoid exposure to moisture.

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

DMSO: ~27 mg/mL (~35.1 mM)
H2O: < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.25 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 25.0 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: ≥ 2.5 mg/mL (3.25 mM) (saturation unknown) 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.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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: ≥ 2.5 mg/mL (3.25 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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