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Purpose The purpose of this study was to investigate the albumin-binding compound 111 In-C4-DTPA as an imaging agent for the detection of endogenous albumin accumulation in tumors. Methods 111 In-C4-DTPA was injected in healthy nude mice for pharmacokinetic and biodistribution studies (10 min, 1, 6, 24, and 48 h, n = 4) and subsequently in tumor-bearing mice for single-photon emission computed tomography/X-ray-computed tomography (SPECT/CT) imaging studies. Four different human tumor xenograft models (LXFL529, OVXF899, MAXFTN401, and CXF2081) were implanted subcutaneously unilaterally or bilaterally (n = 4-8). After intravenous administration of 111 In-C4-DTPA, SPECT/CT images were collected over 72 h at 4-6 time points. Additionally, gamma counting was performed for the blood, plasma, lungs, heart, liver, spleen, kidneys, muscle, and tumors at 72 h post-injection. Results 111 In-C4-DTPA bound rapidly to circulating albumin upon injection, and the radiolabeled albumin conjugate thus formed was stable in murine and human serum. SPECT/CT images demonstrated a time-dependent uptake with a maximum of 2.7-3.8% ID/cm 3 in the tumors at approximately 24 h post-injection and mean tumor/muscle ratios in the range of 3.2-6.2 between 24 and 72 h post-injection. The kidneys and bladder were the predominant elimination organs. Gamma counting at 72 h postinjection showed 1.3-2.5% ID/g in the tumors and mean tumor/muscle ratios in the range of 4.9-9.4. Conclusion 111 In-C4-DTPA bound rapidly to circulating albumin upon injection and showed time-dependent uptake in the tumors demonstrating a potential for clinical application as a companion imaging diagnostic for albumin-binding anticancer drugs.Keywords Albumin . Drug carrier . Imaging agent . SPECT/CT imaging . Tumor accumulation . 111 In S. Daum and J. P. Magnusson contributed equally to this work.

Drug delivery systems in oncology for treating cancer are based on different drug release mechanisms at the tumor site including primarily hydrolytic, reductive, enzymatic and/or acid-sensitive cleavage. Drug carrier conjugates that incorporate an acid-sensitive breaking-point exploit the extra- and intracellular acidic environment of the tumor. Important requirements for acid-sensitive bonds are high stability of the carrier-bound drug in the blood circulation and an effective or sustained release of the active drug in the acidic tumor interstitium and acidic endosomes/lysosomes of tumor cells. In addition, sufficient stability of the acid-sensitive bond is a prerequisite for galenic formulation and reconstitution. As a continuation of our development of aldoxorubicin, an acid-sensitive albumin-binding drug of doxorubicin, which is being clinically assessed in advanced clinical trials (www.cytrx.com), we have set out to design a novel acid-sensitive drug release platform that is applicable to carriers such as serum proteins and antibodies. The key components of this specific drug delivery system are linkers containing a thiol-binding maleimide group and a substituted aromatic hydrazine to form an acid-cleavable hydrazone bond with suitable carbonyl-containing drugs. For establishing and fine-tuning the pH-dependent release profiles, the aromatic moiety of the linker was substituted with a spectrum of electron-withdrawing groups, and reacted with the anthracyclines doxorubicin and nemorubicin. The resulting hydrazone derivatives were conjugated to the cysteine-34 position of albumin as a model protein. Subsequently, the drug release was determined at physiological and acidic conditions as well as in human plasma. It was discovered that by variation of the electron-withdrawing groups, the pH dependent release of the drug could be substantially varied between 1-50 h in the pH range of 4-5. The technology using these linkers was coined LADR™ (Linker Activated Drug Release) and has the additional advantage that the galenic formulation and reconstitution of LADR™-based drugs is facilitated. We have applied the LADR™ technology to several anticancer drugs such as vinblastine and gemcitabine. In summary, LADR™ is an innovative and versatile linker technology creating a platform for developing acid-sensitive drug carrier conjugates that allow the carrier-bound drug to be released in a controlled manner resulting in sustained exposure to cancer cells. Citation Format: Khalid Abu Ajaj, Stephan David Koester, Friederike Inga Nollmann, Simon Waltzer, Olga Fuchs, André Warnecke, Felix Kratz. LADR™: A novel linker activated drug release technology for drug delivery. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4858.

Introduction and objectives: Maytansine and its analogs (DM1 and DM4) are potent microtubule-targeting compounds that inhibit proliferation of cells during mitosis.1 Unfortunately, their narrow therapeutic window prevents a clinical application of these molecules. So far only T-DM1, an antibody-maytansinoid conjugate targeting the HER2 receptor, has been approved for the treatment of resistant breast cancer. Previous work on maytansinoids showed that their potent cytotoxic activity is related to the nature of the substituent at the C3 acyloxy side chain. In order to harness the potential of compounds of the maytansinoid family while diminishing dose-limiting side effects, we synthesized a library of novel analogs which can be attached to serum albumin in vivo through an acid-sensitive linker ensuring release of the active maytansinoid at the tumor site. All novel maytansine analogs contain a keto moiety as an attachment point for the linker and differ in their substitution pattern at C3. The analogs we studied can be classified into those containing an amino acid spacer between C3 and the keto group (maytansine-type) and those obtained through direct esterification at the C3-OH-position with various carboxylic acids (ansamitocin-type). Methods: All the newly synthesized molecules were screened for their in vitro cytotoxicity against 11 cancer cell lines. In order to explore the influence of the maytansine functional groups at C3, we designed novel analogs with different amino acid spacers at this position. We evaluated the influence of the chain length, the degree of steric hindrance, the need of the chiral center and the influence of the N-methyl on the cytotoxicity as well as stability in murine and human blood plasma. Moreover, we studied the structure-activity relationship of the ansamitocin-type of analogs including the influence of the chain length, the degree of steric hindrance and the effect of introducing different heteroatoms in the alkyl chain. Result and conclusion: A total of 32 new maytansinoid analogs were synthesized, and seven of them were found to be more potent than the parent drug maytansine in inhibiting the growth of human cancer cells in vitro. Clear SARs were identified for both classes of compounds. Based on these studies, lead compounds have been selected for creating albumin-binding derivatives and their further in vivo evaluation.2 1) W. C. Widdison et al, J. Med. Chem., 49: 4392-4408 (2006); K. A. Poon et al, Toxicology and Applied Pharmacology, 273: 298-313 (2013); H. L. Perez et al, Drug Discovery Today, 19:869-881 (2014) 2) Abstract "In vivo efficacy of novel acid-sensitive albumin-binding Maytansinoid-based prodrugs in human cancer xenograft models in nude mice" Citation Format: Friederike I. Nollmann, Patricia Perez Galan, Javier Garcia Fernandez, Heidi K. Walter, Johannes P. Magnusson, Federico Medda, Felix Kratz, Stephan D. Koester, Khalid Abu Ajaj, Lara Pes, Serghei Chercheja, Anna Warnecke. Structure-activity relationship studies and biological evaluation of novel maytansinoids, a class of highly selective tubulin inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1657.

Auristatins are highly cytotoxic antimitotic tubulin-binding peptides. Of this family, only Adcetris®, an antibody drug conjugate (ADC) derived from monomethyl auristatin E (MMAE), is approved and marketed. Other auristatins such as dolastatin 10, dolastatin 15, and auristatin PE reached phase 1 and 2 clinical trials, but due to systemic toxicity and low antitumor activity they were discontinued.1 Because of the high cytotoxicity, it is important to deliver these drugs selectively to the tumor avoiding premature release in the blood circulation. We developed an acid-sensitive drug delivery system that uses circulating endogenous serum albumin as a macromolecular carrier. The drug is rapidly bound covalently to albumin upon i.v. injection and, following accumulation in the tumor due to enhanced uptake and retention mechanisms, the highly cytotoxic agent is released in a pH-dependent manner.2 Herein we present in-vivo data of the considerably improved efficacy of two auristatin E derived prodrugs, AE-Keto-Sulf07 and AE-Ester-Sulf07, compared to the parent compound auristatin E. The acid-sensitive hydrazone prodrugs were prepared from the respective carbonyl-containing auristatin E derivatives and the water-solubilizing maleimide-bearing linker Sulf07. Female NMRI nu/nu mice were inoculated subcutaneously with patient- or cell-derived human tumor xenografts (A375, A2780, RXF631, LXFA737) and randomized (n=7-8 per group) with starting tumor volumes in the range of 140-350 mm3. AE-Keto-Sulf07 showed excellent antitumor response over a wide dose range (3.0-6.5 mg/kg twice per week over 3-4 weeks), with optimal dosage at 4.5 mg/kg twice per week over 4 weeks. AE-Ester-Sulf07 was highly efficacious between 1.9 and 2.4 mg/kg dosed twice per week over 3-4 weeks or at 3.8 mg/kg dosed once per week over 4 weeks, but caused cumulative skin lesions due to scratching and biting. In contrast, auristatin E was dosed at 0.3 mg/kg twice per week over 3-4 weeks and was only marginally active. In summary, we have shown for the first time that auristatin E derivatives, namely AE-Keto-Sulf07 and AE-Ester-Sulf07, bound to circulating albumin demonstrate promising antitumor efficacy and induce statistically significant long-term partial or complete remission in a panel of human xenograft models in mice. Citation Format: Stephan D. Koester, Lara Pes, Johannes P. Magnusson, Serghei Chercheja, Federico Medda, Friederike I. Nollmann, Patricia Perez Galan, Javier Garcia Fernandez, Heidi-Kristin Walter, Khalid Abu Ajaj, Anna Warnecke, Felix Kratz. Superior efficacy of novel albumin-binding auristatin E-based prodrugs compared to auristatin E in a panel of human xenograft models in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3703.