Calicheamicins; DNA synthesis; antitumor antibiotic
DNA minor groove. Calicheamicin binds sequence-specifically to the minor groove of DNA, followed by oxidative DNA strand cleavage through enediyne-mediated activation. This results in double-strand DNA breaks and inhibition of DNA synthesis. The compound is a highly potent antitumor antibiotic.

With an average drug-to-antibody ratio (DAR) of 4.6, PF-06647263 (anti-EFNA4-ADC) is produced by conjugating hE22 lysine residues to the AcButDMH-N-Ac-calicheamicin-γ1 linker-payload. Antigen- and concentration-dependent cytotoxicity is elicited by PF-06647263, as exposure to the compound for 96 hours causes cell death (EC50 = approximately 1 ng/mL)[1]. In vitro treatment of pediatric primary B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells is facilitated by CMC-544, which consists of a humanized CD22 Ab conjugated to calicheamicin. With IC50 values ranging from 0.15 to 4.9 ng/mL, CMC-544 causes dose- and time-dependent cell death in a variety of ALL cell lines. In primary BCP-ALL cells, CMC-544 (10 ng/mL) exhibits efficacy and specificity [2]. Compared to G5/44-treated cells, the level of CD22 in CMC-544-treated cells has dropped and has remained low[3].
Calicheamicin binds sequence-specifically to the minor groove of DNA. Upon enediyne-mediated activation, it causes oxidative DNA strand cleavage, leading to double-strand DNA breaks. This inhibits DNA synthesis and induces cell death. The compound is a highly potent cytotoxic agent widely used as an ADC payload for targeted cancer therapy.

In both TNBC and ovarian cancer PDX, an ADC containing a humanized anti-EFNA4 monoclonal antibody coupled to the DNA-damaging agent calicheamicin results in long-lasting tumor regressions in vivo. In TNBC xenografts, PF-06647263 (0.27, 0.36 mg/kg) causes notable tumor regressions[1].
In vivo, calicheamicin is used as a payload in antibody-drug conjugates for targeted cancer therapy. The compound's potent cytotoxicity and ability to cause double-strand DNA breaks make it effective against cancer cells. It also has strong anti-Gram-positive bacteria activity.

In vitro assays for calicheamicin involve measuring its DNA cleavage activity and cytotoxicity. DNA cleavage is assessed by incubating calicheamicin with DNA and analyzing fragmentation by gel electrophoresis. Cytotoxicity is measured using cell viability assays in various cancer cell lines. The compound causes double-strand DNA breaks and inhibits DNA synthesis.

In this study, researchers investigated whether the newly developed antibody (Ab) -targeted therapy inotuzumab ozogamicin (CMC-544), consisting of a humanized CD22 Ab linked to calicheamicin, is effective in pediatric primary B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells in vitro, and analyzed which parameters determine its efficacy. CMC-544 induced dose-dependent cell kill in the majority of BCP-ALL cells, although IC(50) values varied substantially (median 4.8 ng/ml, range 0.1-1000 ng/ml at 48 h). The efficacy of CMC-544 was highly dependent on calicheamicin sensitivity and CD22/CMC-544 internalization capacity of BCP-ALL cells, but hardly on basal and renewed CD22 expression. Although CD22 expression was essential for uptake of CMC-544, a repetitive loop of CD22 saturation, CD22/CMC-544 internalization and renewed CD22 expression was not required to achieve intracellular threshold levels of calicheamicin sufficient for efficient CMC-544-induced apoptosis in BCP-ALL cells. This is in contrast to studies with the comparable CD33 immunotoxin gemtuzumab ozogamicin (Mylotarg) in acute myeloid leukemia (AML) patients, in which complete and prolonged CD33 saturation was required for apoptosis induction. These data suggest that CMC-544 treatment may result in higher response rates in ALL compared with response rates obtained in AML with Mylotarg, and that therefore clinical studies in ALL, preferably with multiple low CMC-544 dosages, are warranted.[2]

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In this study, researchers studied the effect of CMC-544, the calicheamicin-conjugated anti-CD22 monoclonal antibody, used alone and in combination with rituximab, analyzing the quantitative alteration of target molecules, that is, CD20, CD22, CD55 and CD59, in Daudi and Raji cells as well as in cells obtained from patients with B-cell malignancies (BCM). Antibody inducing direct antiproliferative and apoptotic effect, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) were tested separately. In Daudi and Raji cells, the CDC effect of rituximab significantly increased within 12 h following incubation with CMC-544. The levels of CD22 and CD55 were significantly reduced (P<0.001 in both cells) after incubation with CMC-544, but CD20 level remained constant or increased for 12 h. Similar results were obtained in cells from 12 patients with BCM. The antiproliferative and apoptotic effect of CMC-544 were greater than that of rituximab. The ADCC of rituximab was not enhanced by CMC-544. Thus, the combination of CMC-544 and rituximab increased the in vitro cytotoxic effect in BCM cells, and sequential administration for 12 h proceeded by CMC-544 was more effective. The reduction of CD55 and the preservation of CD20 after incubation with CMC-544 support the rationale for the combined use of CMC-544 and rituximab.
In vitro cellular assays for calicheamicin involve treating cancer cells with the compound or calicheamicin-containing ADCs and measuring cell viability, DNA damage, and apoptosis. Cells are treated with various concentrations of calicheamicin, and cell viability is assessed by MTT or other metabolic assays. DNA damage is measured by comet assay or γ-H2AX foci formation.

A panel of well-annotated patient-derived xenografts (PDX) was established, and surface markers that enriched for TIC in specific tumor subtypes were empirically determined. The TICs were queried for overexpressed antigens, one of which was selected to be the target of an antibody-drug conjugate (ADC). The efficacy of the ADC was evaluated in 15 PDX models to generate hypotheses for patient stratification.
Results: We herein identified E-cadherin (CD324) as a surface antigen able to reproducibly enrich for TIC in well-annotated, low-passage TNBC and ovarian cancer PDXs. Gene expression analysis of TIC led to the identification of Ephrin-A4 (EFNA4) as a prospective therapeutic target. An ADC comprising a humanized anti-EFNA4 monoclonal antibody conjugated to the DNA-damaging agent calicheamicin achieved sustained tumor regressions in both TNBC and ovarian cancer PDX in vivo. Non-claudin low TNBC tumors exhibited higher expression and more robust responses than other breast cancer subtypes, suggesting a specific translational application for tumor subclassification.
Conclusions: These findings demonstrate the potential of PF-06647263 (anti-EFNA4-ADC) as a first-in-class compound designed to eradicate TIC. The use of well-annotated PDX for drug discovery enabled the identification of a novel TIC target, pharmacologic evaluation of the compound, and translational studies to inform clinical development.[1]

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In vivo efficacy studies[1]
Cohorts of tumor-bearing mice (140–180 mm3) were randomized into study groups of 6 to 10 based on the number of available mice. The IDBS electronic notebook statistical package, Biobook, was used for automated animal randomization. Animals were dosed by intraperitoneal injection (or intravenously for 144580) twice a week for 4 cycles with ADC, or once a week for 2 cycles with 1.5 mg/kg doxorubicin for breast PDX tumors or 5 mg/kg Cisplatin for ovarian PDX. Study groups were followed until either individual mice or entire cohort measurements reached 1,200 mm3, at which point sacrifice was indicated in accordance with approved IACUC protocols. Tumor regression was defined as a reduction in mean tumor volume after dosing. In cases where tumors regressed, time to progression (TTP) was determined to be the number of days between the first dose and the time at which mean tumor volume significantly increased (regrew) after regression.

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TIC frequency assay[1]
PDX tumor–bearing mice were treated with PF-06647263 or control ADC, and tumors were harvested at day 21 (BR13) or day 12 (BR22) after the first dose, based on when tumors were starting to regress. Tumors were harvested, dissociated, and stained as described above. Three tumors per treatment group were pooled, and live human tumor cells (murine Lineage− ESA+) were isolated by FACS, counted, and implanted into naïve animals in limiting dilution (8–10 animals per group). Mice bearing tumors that exceeded 200 mm3 were scored as positive. Poisson distribution statistics were generated by L-Calc software.
In vivo animal studies for calicheamicin typically involve administration of calicheamicin-containing ADCs to xenograft models of cancer. Efficacy is assessed by measuring tumor growth inhibition, survival, and toxicity. Calicheamicin is used as a payload in ADCs for targeted cancer therapy.

Pharmacokinetic studies of calicheamicin focus on its properties as an ADC payload. The compound has a molecular weight of 1368.35 and a molecular formula of C55H74IN3O21S4. It contains an alkyne group for click chemistry applications. The compound is a highly potent enediyne antitumor antibiotic.

Calicheamicin is a highly potent cytotoxic agent that causes double-strand DNA breaks. Its use is primarily in ADCs for targeted cancer therapy. Toxicity is related to its mechanism of action—DNA damage and cell death. The compound should be handled with extreme caution due to its high potency.

[1]. Anti-EFNA4 Calicheamicin Conjugates Effectively Target Triple-Negative Breast and Ovarian Tumor-Initiating Cells to Result in Sustained Tumor Regressions. Clin Cancer Res. 2015 Sep 15;21(18):4165-73

[2].The novel calicheamicin-conjugated CD22 antibody inotuzumab ozogamicin (CMC-544) effectively kills primary pediatric acute lymphoblastic leukemia cells. Leukemia. 2012 Feb;26(2):255-64

[3]. CMC-544 (inotuzumab ozogamicin), an anti-CD22 immuno-conjugate of calicheamicin, alters the levels of target molecules of malignant B-cells. Leukemia. 2009 Jul;23(7):1329-36.

Calicheamicin is a highly potent, 10-membered enediyne antitumor antibiotic isolated from Micromonospora echinospora. It binds sequence-specifically to the DNA minor groove, causing double-strand DNA breaks and inhibiting DNA synthesis. Calicheamicin is widely used as a payload in antibody-drug conjugates for targeted cancer therapy. It has a molecular formula of C55H74IN3O21S4 and a molecular weight of 1368.35.

C55H74IN3O21S4

1368.3415

1367.274

C, 48.28; H, 5.45; I, 9.27; N, 3.07; O, 24.55; S, 9.37

108212-75-5

N-Acetyl-Calicheamicin;108212-76-6

10953353

White to yellow solid powder

1.6±0.1 g/cm3

1.662

11.89

8

27

24

84

2500

19

IC1C(C([H])([H])[H])=C(C(=C(C=1O[C@@]1([H])[C@@]([H])([C@@]([H])([C@]([H])([C@]([H])(C([H])([H])[H])O1)O[H])OC([H])([H])[H])O[H])OC([H])([H])[H])OC([H])([H])[H])C(=O)S[C@]1([H])[C@@]([H])(C([H])([H])[H])O[C@]([H])(C([H])([H])[C@]1([H])O[H])ON([H])[C@]1([H])[C@@]([H])(C([H])([H])[H])O[C@]([H])([C@@]([H])([C@@]1([H])O[H])O[C@@]1([H])C([H])([H])[C@@]([H])([C@]([H])(C([H])([H])O1)N([H])C([H])([H])C([H])([H])[H])OC([H])([H])[H])O[C@@]1([H])C#CC([H])=C([H])C#C[C@@]2(C([H])([H])C(C(=C1/C/2=C(/[H])\C([H])([H])SSSC([H])([H])[H])N([H])C(=O)OC([H])([H])[H])=O)O[H] |t:126|

HXCHCVDVKSCDHU-IXTZGUNISA-N

InChI=1S/C55H74IN3O21S4/c1-12-57-30-24-73-35(22-34(30)68-6)78-48-43(63)40(26(3)75-53(48)77-33-17-15-13-14-16-19-55(67)23-32(61)41(58-54(66)72-10)38(33)29(55)18-20-82-84-81-11)59-80-36-21-31(60)50(28(5)74-36)83-51(65)37-25(2)39(56)46(49(71-9)45(37)69-7)79-52-44(64)47(70-8)42(62)27(4)76-52/h13-14,18,26-28,30-31,33-36,40,42-44,47-48,50,52-53,57,59-60,62-64,67H,12,20-24H2,1-11H3,(H,58,66)/b14-13-,29-18+/t26-,27+,28-,30+,31+,33+,34+,35+,36+,40-,42+,43+,44-,47-,48-,50-,52+,53?,55-/m1/s1

S-((2R,3S,4S,6S)-6-((((2R,3S,4S,5R)-5-(((2S,4S,5S)-5-(ethylamino)-4-methoxytetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-(((2S,5Z,9S,13E)-9-hydroxy-12-((methoxycarbonyl)amino)-13-(2-(methyltrisulfanyl)ethylidene)-11-oxobicyclo[7.3.1]trideca-1(12),5-dien-3,7-diyn-2-yl)oxy)-2-methyltetrahydro-2H-pyran-3-yl)amino)oxy)-4-hydroxy-2-methyltetrahydro-2H-pyran-3-yl) 4-(((2S,3R,4R,5S,6S)-3,5-dihydroxy-4-methoxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-3-iodo-5,6-dimethoxy-2-methylbenzothioate

Calicheamicin; Calicheamicin gamma(1,I); 108212-75-5; calicheamicin gamma(1)I; 113440-58-7; Calichemicin gamma1; Calicheamicin gamma(1)I.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~25 mg/mL (~18.27 mM)

Solubility in Formulation 1: ≥ 3 mg/mL (2.19 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 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (1.83 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 3: 2.5 mg/mL (1.83 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 4: 10% DMSO+90% Corn Oil: ≥ 3 mg/mL (2.19 mM)

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