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Background: T-DM1 showed remarkable activity in metastatic HER-2 positive breast cancer (mBC) and it was recently approved for clinical use in patients (pts) who previously failed Trastuzumab- and Taxanes-based therapies. Currently, little is known on the performance of T-DM1 in a “real life” scenario. Therefore, we investigated effectiveness and safety of T-DM1 in Italian daily practice. Methods: Pts baseline characteristics and clinical outcome of pts with HER-2 positive mBC treated with T-DM1 between 2013 and 2015 at 20 Italian Institutions were retrospectively collected and analyzed. Results: 300 pts were included in our analysis. Median age was 51 years (27-78); visceral metastases were present in 204 (68%) pts and brain metastases in 86 (29%). It is noteworthy that 111 (37%) pts received T-DM1 as pure second line, 83 (28%) as third line and 96 (32%) as further lines. Moreover 10 (3%) pts had T-DM1 as first line because disease recurrence occurred during or adjuvant trastuzumab of within 6 months of its completion. The overall response rate (ORR) was 40%, global disease control rate (gDCR) 64%, median progression-free survival (PFS) 7.0 months (C.I.95%: 5.6-8.4) and overall survival (OS) at 2 years 63%. Pts with 1, 2 and 3 or more metastatic site had OS at 2 years of 87%, 67% and 46%, respectively (p Conclusions: To our knowledge, this is the first real life, multicenter retrospective analysis evaluating efficacy and safety of T-DM1 in pretreated HER-2 positive mBC pts. We observed remarkable results in terms of PFS and OS, especially when T-DM1 was given early in the course of metastatic disease. Shortened PFS in patients progressing after pertuzumab suggest further analyses to better define possible molecular mechanisms of cross-resistences between two molecules. As a whole there was no evidence of significant or unexpected toxicities. Although these findings should be taken with caution due to the retrospective analysis and the different lines of previous treatment considered, we confirmed the potential therapeutic role of T-DM1 across a heterogeneous population of HER-2 positive mBC patients. The final analysis will be presented to the meeting. Citation Format: Fabi A, De Laurentiis M, Caruso M, Valle E, Moscetti L, Santini D, Cannita K, Carbognin L, Ciccarese M, Rossello R, Arpino G, Leonardi V, Montemurro F, La Verde N, Generali DG, Zambelli A, Scandurra G, Russillo M, Paris I, D'Ottavio AM, Filippelli G, Giampaglia M, Stani S, Fabbri A, Alesini D, Giannarelli D, Cognetti F. T-DM1 in HER2 positive advanced breast cancer patients: Real world practice from a multicenter observational study [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-21-11.

Thienoindoles are a new proprietary class of highly potent DNA minor groove alkylating agents. This class is characterized by a fused thiophene ring whose intrinsic electron-withdrawing character provides a nearly optimal increase in stability and potency of alkylating subunits. Furthermore, the presence of a solubilizing moiety on the minor groove portion provides compounds with physicochemical properties highly compatible with deployment as antibody payloads. Extensive optimization of this class has led to the identification of the highly potent toxin NMS-P528 with sub-nanomolar IC50 in proliferation assay across a large panel of tumor cell lines. NMS-P945, a drug-linker generated from NMS-P528, is highly suited for conjugation with targeted antibodies, consistently yielding ADCs with favorable drug-antibody ratios in the absence of significant antibody aggregation and with full maintenance of antigen binding capability. Proof of concept efficacy and mechanism of action studies were performed using NMS-P945 conjugated with trastuzumab, observing for the resulting conjugate excellent in vitro target-directed cytotoxicity in HER2-positive vs. negative cancer cell lines. In animal efficacy studies against HER2-positive human breast cancer tumors, trastuzumab-NMS-P945 ADC administration yielded complete tumor regression with no effects on body weight gain, while unarmed trastuzumab and armed control antibody showed little and no effect, respectively. PK data indicated long plasma half-life of our novel ADCs in the monkey. Promising results were also obtained with a range of different antibody/antigen systems, e.g. with an antibody directed to the ALK receptor tyrosine kinase. Extensive process research has been completed for the production of NMS-P528 and NMS-P945, resulting in an efficient stereoselective method for the preparation of GMP materials on the 100g scale and both products are proposed as a licensing opportunity for conjugation with tumor-targeting antibodies. Citation Format: Michele Caruso, Fabio Gasparri, Barbara Valsasina, Clara Albanese, Italo Beria, Ilaria Candiani, Marina Ciomei, Nicoletta Colombo, Sabrina Cribioli, Ulisse Cucchi, Eduard Felder, Ivan Fraietta, Arturo Galvani, Antonella Isacchi, Aurelio Marsiglio, Paolo Orsini, Rita Perego, Simona Rizzi, Attilio Tomasi, Sonia Troiani, Carlo Visco, Daniele Donati. Thienoindoles: New highly promising agents for antibody-drug conjugates generation [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 734.

The rapidly growing field of Antibody-Drug Conjugates (ADCs) has recently spurred the study of novel drug payloads. In particular, much interest has been paid to the identification of novel cytotoxic agents, which differ in mechanism from anti-tubulin agents, currently the most commonly employed class of drug for ADC coupling. Novel drug payloads for ADC-based therapy should thus ideally possess highly potent cytotoxic activity with a diverse mechanism from tubulin agents, as well as physicochemical properties, which facilitate coupling to antibodies. Duocarmycins are a widely-described class of DNA minor groove binding alkylating agents, examples of which can exhibit picomolar activity against human tumor cell lines, as well as high activity in preclinical tumor models. The clinical development of duocarmycins as drugs in their own right has however been hampered by an extremely limited therapeutic window. Duocarmycins also typically exhibit poor physicochemical profiles, most importantly generally possessing limited aqueous solubility and a propensity to induce protein aggregation, which renders these molecules poorly tractable as optimal ADC payloads. Based on the premise that opportune modification of the duocarmycin scaffold could lead to the discovery of compounds better suited to antibody conjugation, we describe the identification and related proof of concept studies for a novel chemical series based on a proprietary thienoindole scaffold, characterized both by potent antitumor activity and by physicochemical properties that are highly compatible with deployment as antibody payloads. Extensive in vitro profiling within the thienoindole series led to selection of potent cytotoxic compounds suitable for test conjugation to humanized monoclonal antibodies, which were then submitted to in vitro/in vivo profiling. Typically, average drug/antibody ratios (DARs) of 4 could readily be achieved without induction of antibody aggregation/precipitation and without affecting natural antibody-ligand affinity. Efficacy and mechanism of action studies were carried out for thienoindole-ADCs prepared with trastuzumab and with rituximab showing target driven effects in Her2 positive vs. negative breast cancer cell lines and against CD20 positive vs. negative hematological cell lines. PK data indicated long, essentially unaffected plasma half life of our novel ADCs in the mouse. In vivo efficacy studies in the Her2-positive HCC1954 human breast cancer model showed complete tumor regression in mice treated with trastuzumab-ADC with no effects on body weight gain, while unarmed trastuzumab and armed control antibody had little and no effect, respectively. Citation Format: Barbara Valsasina, Fabio Gasparri, Italo Beria, Nicoletta Colombo, Paolo Orsini, Rita Perego, Simona Rizzi, Ulisse Cucchi, Clara Albanese, Aurelio Marsiglio, Ivan Fraietta, Marina Ciomei, Sabrina Cribioli, Carlo Visco, Eduard R. Felder, Antonella Isacchi, Enrico A. Pesenti, Arturo Galvani, Daniele Donati, Michele Caruso. Thienoindoles, a novel class of DNA minor groove alkylating agents highly suited for the generation of novel antibody drug conjugates (ADCs). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 822. doi:10.1158/1538-7445.AM2014-822

Anthracyclines are one of the most widely used classes of chemotherapy. With current success of antibody drug conjugates in the clinic, we were interested in anthracycline-based antibody-drug conjugates (ADCs). Anthracyclines currently used in systemic chemotherapy were ineffective as the ADC drug probably due to low potency of the drug. PNU-159682 was identified as a metabolic product of nemorubicin that was 700 to 2400 more potent in in vivo cytotoxicity assays than nemorubicin. We were interested to see if we could develop an effective anthracycline-based ADC using PNU-159682. For these proof of concept studies we selected the clinically validated linker maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB) used in the approved ADC brentuximab vedotin [anti-CD30-MC-vc-PAB-monomethyl auristatin E (MMAE)] and attached it to the primary alcohol of PNU-159682 to make NMS-249. For antibodies we selected the anti-CD22 and anti-CD79b antibodies that have been shown to be effective as MC-vc-PAB-MMAE ADCs in phase 1 clinical trials for the treatment of NHL. In xenograft models of NHL anti-CD22-NMS249 was as effective or more effective as anti-CD22-MC-vcPAB-MMAE. In particular WSU-DLCL2, where anti-CD22-MC-vc-PAB-MMAE was least effective, show the greatest difference between the two ADCs. To further explore if these the anti-CD22-NMS249 could be used for patients that had progressed on auristatin-based ADC, we developed two cell lines that were resistant to anti-CD22-MC-vc-PAB-MMAE by consistently treating xenograft models with increase amounts of ADC. After removing the resistant tumors and culturing the cells in vitro the cell lines retained their resistance in vivo to anti-CD22-MC-vc-PAB-MMAE but were also resistance to anti-CD79b-MC-vc-PAB-MMAE. This suggests the resistance was not target related and we found that CD22 expression was maintained in the resistant cell lines. Anti-CD22-NMS249 maintained its efficacy in the resistant cell lines. Microarray and FACs showed that the resistant cells lines were up-regulated in MDR1 (PgP). We found that in vivo, unlike other anthacyclines, PNU-159682 was not a PgP substrate this may explain the ability of the anti-CD22-NMS249 to overcome the resistance to anti-CD22-MC-vc-PAB-MMAE. These studies provide poof of concept for an anthracycline ADC that could be use in patients that have failed auristatin-based therapies. Citation Format: Andrew G. Polson, Bing Zheng, MaryAnn Go, Jeffery Lau, Shang-Fan Yu, Susan Spencer, Robert Cohen, Michele Caruso, John Flygare, Paul Polakis. Anthracycline based antibody-drug conjugates (ADCs) for the treatment of non-Hodgkin's lymphoma are effective in cell lines resistant to auristatin based ADCs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4634. doi:10.1158/1538-7445.AM2013-4634