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These are figures associated with a second manuscript that has been submitted for consideration for co-publication. It describes the platform and hopefully will facilitate the reviewers' assessment of this manuscript on XMT-1536. we are also happy to make this available as supplementary data for this publication if desired.

These are figures associated with a second manuscript that has been submitted for consideration for co-publication. It describes the platform and hopefully will facilitate the reviewers' assessment of this manuscript on XMT-1536. we are happy to make this available as supplementary data for publication in any format if desired.

Supplementary methods, data and Figures

Supplementary Figures

After significant effort over the last 30 years, antibody–drug conjugates (ADC) have recently gained momentum as a therapeutic modality, and nine ADCs have been approved by the FDA to date, with additional ADCs in late stages of development. Here, we introduce dolaflexin, a novel ADC technology that overcomes key limitations of the most common ADC platforms with two key features: a higher drug-to-antibody ratio and a novel auristatin with a controlled bystander effect. The novel, cell permeable payload, auristatin F-hydroxypropylamide, undergoes metabolic conversion to the highly potent, but less cell permeable auristatin F to balance the bystander effect through drug trapping within target cells. We conducted studies in mice, rats, and cynomolgus monkeys to complement in vitro characterization and contrasted the performance of dolaflexin with regard to antitumor activity, pharmacokinetic properties, and safety in comparison with the ADC platform utilized in the approved ADC ado-trastuzumab emtansine (T-DM1). A HER2-targeted dolaflexin ADC was shown to have a much lower threshold of antigen expression for potent cell killing in vitro, was effective in vivo in tumors with low HER2 expression, and induced tumor regressions in a xenograft model that is resistant to T-DM1.

Pyrrolobenzodiazepine (PBD) dimers are well-known highly potent antibody drug conjugate (ADC) payloads. The corresponding PBD monomers, in contrast, have received much less attention from the ADC community. We prepared several novel polyamide-linked PBD monomers and evaluated their utility as ADC payloads. The unconjugated polyamide-PBD hybrids exhibited potent antiproliferative activity (IC50 range: 10

Target selection for antibody–drug conjugates (ADC) frequently focuses on identifying antigens with differential expression in tumor and normal tissue, to mitigate the risk of on-target toxicity. However, this strategy restricts the possible target space. SLC34A2/NaPi2b is a sodium phosphate transporter expressed in a variety of human tumors including lung and ovarian carcinoma, as well as the normal tissues from which these tumors arise. Previous clinical trials with a NaPi2b targeting MMAE-ADCs have shown objective durable responses. However, the protein-based biomarker assay developed for use in that study was unable to discern a statistically significant relationship between NaPi2b protein expression and the probability of response. XMT-1536 is a NaPi2b targeting ADC comprised of a unique humanized antibody conjugated with 10–15 auristatin F- hydroxypropylamide (AF-HPA) payload molecules via the Dolaflexin platform. AF-HPA is a cell-permeable, antimitotic compound that is slowly metabolized intratumorally to an active, very low-permeable metabolite, auristatin F (AF), resulting in controlled bystander killing. We describe the preclinical in vitro and in vivo antitumor effects of XMT-1536 in models of ovarian and lung adenocarcinoma. Pharmacokinetic analysis showed approximately proportional increases in exposure in rat and monkey. Systemic free AF-HPA and AF concentrations were observed to be low in all animal species. Finally, we describe a unique IHC reagent, generated from a chimeric construct of the therapeutic antibody, that was used to derive a target expression and efficacy relationship in a series of ovarian primary xenograft cancer models.

After significant effort over the last 30 years, antibody–drug conjugates (ADC) have recently gained momentum as a therapeutic modality, and nine ADCs have been approved by the FDA to date, with additional ADCs in late stages of development. Here, we introduce dolaflexin, a novel ADC technology that overcomes key limitations of the most common ADC platforms with two key features: a higher drug-to-antibody ratio and a novel auristatin with a controlled bystander effect. The novel, cell permeable payload, auristatin F-hydroxypropylamide, undergoes metabolic conversion to the highly potent, but less cell permeable auristatin F to balance the bystander effect through drug trapping within target cells. We conducted studies in mice, rats, and cynomolgus monkeys to complement in vitro characterization and contrasted the performance of dolaflexin with regard to antitumor activity, pharmacokinetic properties, and safety in comparison with the ADC platform utilized in the approved ADC ado-trastuzumab emtansine (T-DM1). A HER2-targeted dolaflexin ADC was shown to have a much lower threshold of antigen expression for potent cell killing in vitro, was effective in vivo in tumors with low HER2 expression, and induced tumor regressions in a xenograft model that is resistant to T-DM1.

XMT-1522 is an anti-HER2 antibody-drug conjugate (ADC) comprised of a novel anti-HER2 antibody (HT-19) and the Dolaflexin ADC platform, which allows for conjugation of 12-15 proprietary auristatin drug payload molecules per antibody without aggregation or detrimental impact on pharmacokinetics. The HT-19 antibody binds to a novel HER2 extracellular domain epitope and does not compete for HER2 binding with trastuzumab or pertuzumab. In vitro, XMT-1522 has sub-nanomolar potency in cell lines expressing as few as 25,000 HER2 antigens per cell, and is ∼100X more potent than ado-trastuzumab emtansine (T-DM1) across a panel of 25 cell lines representing a range of tumor indications and HER2 expression levels. In the BT-474 HER2-positive breast cancer model, treatment with the HT-19 antibody alone at a single 5 mg/kg dose is inactive, while XMT-1522 ADC at a single 2 mg/kg or 5 mg/kg dose induces durable complete tumor regression, indicating that the primary mechanism of XMT-1522 is cytotoxic payload delivery, not inhibition of HER2 signaling. T-DM1 at a single 5 mg/kg dose in the same model is inactive, consistent with the significant improvement in potency of XMT-1522 compared to T-DM1. Biodistribution studies using a HER2-targeted Dolaflexin ADC in the BT-474 model demonstrate accumulation of intact ADC and released active drug payload in tumor at levels significantly higher than normal tissues. In a HER2-positive patient-derived xenograft (PDX) model, XMT-1522 induces durable complete tumor regression after a single 1 mg/kg dose, while T-DM1 at a 10 mg/kg dose achieves tumor growth delay without regression. In a HER2 1+ PDX model without HER2 gene amplification, a single 3 mg/kg dose of XMT-1522 achieves partial tumor regression where a 10 mg/kg dose of T-DM1 is inactive. Combination of XMT-1522 with trastuzumab does not block the ability of the ADC to bind HER2 or efficiently internalize, and in vivo the combination of low dose XMT-1522 with full dose trastuzumab and pertuzumab is synergistic in a HER2-driven model. The exposure achieved with XMT-1522 at well-tolerated doses in cynomolgus monkey is several fold higher than the exposure needed in mice to achieve complete tumor regressions across models representing a range of HER2 expression and tumor indications. Based on these data, XMT-1522 will soon enter clinical testing in breast cancer patients with both HER2-positive tumors and HER2 low-expressing (IHC 1+ and 2+/FISH-) tumors. Citation Format: Bergstrom DA, Bodyak N, Park PU, Yurkovetskiy A, DeVit M, Yin M, Poling L, Thomas JD, Gumerov D, Xiao D, Ter-Ovanesyan E, Qin L, Uttard A, Johnson A, Lowinger TB. XMT-1522 induces tumor regressions in pre-clinical models representing HER2-positive and HER2 low-expressing breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-14-28.

Antibody drug conjugates (ADCs) for oncology applications are chemotherapeutic agents designed to selectively deliver cytotoxic drug payloads to neoplastic tissue. This book chapter reviews the latest approaches for high drug loaded ADCs. The primary focus of this review is related to ADC drug payload and antibody-drug bioconjugation linker selection strategies resulting in biotherapeutics with improved physicochemical properties, efficacy, and pharmacokinetics. A separate section of this chapter gives a brief overview of antibody targeted nanotherapeutics, a growing and diverse class of anti-cancer agents specifically designed for delivery of significant amounts of drug payload. New strategies to design the highly potent antibody targeted agents discussed in this chapter provide the opportunity to expand the list of drug payloads suitable for ADC applications and introduce agents with new mechanisms of action, which in turn may potentially lead to improvement in therapeutic index of the ADCs for the treatment of cancer.

Antibody–drug conjugates (ADC) are an emerging drug class that uses antibodies to improve cytotoxic drug targeting for cancer treatment. ADCs in current clinical trials achieve a compromise between potency and physicochemical/pharmacokinetic properties by conjugating potent cytotoxins directly to an antibody at a 4:1 or less stoichiometric ratio. Herein, we report a novel, polyacetal polymer-based platform for creating ADC that use poly-1-hydroxymethylethylene hydroxymethyl-formal (PHF), also known as Fleximer. The high hydrophilicity and polyvalency properties of the Fleximer polymer can be used to produce ADC with high drug loading without compromising physicochemical and pharmacokinetic properties. Using trastuzumab and a vinca drug derivative to demonstrate the utility of this platform, a novel Fleximer-based ADC was prepared and characterized in vivo. The ADC prepared had a vinca-antibody ratio of 20:1. It exhibited a high antigen-binding affinity, an excellent pharmacokinetic profile and antigen-dependent efficacy, and tumor accumulation in multiple tumor xenograft models. Our findings illustrate the robust utility of the Fleximer platform as a highly differentiated alternative to the conjugation platforms used to create ADC currently in clinical development. Cancer Res; 75(16); 3365–72. ©2015 AACR.

Antibody-drug conjugates (ADCs) are designed to bind tumor-associated antigens and deliver conjugated cytotoxic payloads to antigen-positive cells. Some ADCs also kill neighboring cells (including antigen-negative cells) by a mechanism referred to as the bystander effect. This effect can be beneficial when the antigen has heterogeneous expression among cells in a solid tumor, but it can also increase off-target toxicity of ADCs. Herein, we report on a unique pharmacologic property of the Dolaflexin platform, which provides a controlled bystander effect that retains the benefits of the bystander effect with respect to antitumor cytotoxicity but reduces the off-target toxicity. The controlled bystander effect, termed “Dolalock,” was achieved through design of a payload, auristatin F-hydroxypropylamide (AF-HPA), that is membrane-permeable and capable of bystander killing but is further catabolized to membrane-impermeable auristatin F (AF). This catabolism of the payload “locks” the highly potent AF in the cell. Using Dolaflexin-based ADCs, we investigated the extent of intracellular AF-HPA and AF release, tumor cell retention and bystander activities in vitro and in vivo. We observed both auristatin species within cells. Co-culture assays with HER2-positive and HER2-negative cells confirmed the cell permeability and bystander-killing capabilities of AF-HPA released from a Dolaflexin-based ADC. Biodistribution studies of Dolaflexin-based ADCs revealed time-dependent concentrations of AF-HPA and AF as well as significant accumulation of AF in xenografted tumor cells, consistent with the Dolalock mechanism. An additional benefit of AF formation was seen in multidrug-resistant transporter studies, which demonstrate that AF, in contrast to AF-HPA, is not a P-glycoprotein 1 (Pgp) substrate. This property may offer additional benefit in Pgp-expressing tumors. In summary, we have shown that the novel AF-HPA payload used in the Dolaflexin platform allows for a controlled bystander effect that likely contributes to the enhanced efficacy and lack of neutropenia we have observed with Dolaflexin-based ADCs. Citation Format: Susan M. Clardy, Alex Yurkovetskiy, Mao Yin, Dmitry Gumerov, Ling Xu, Elena Ter-Ovanesyan, Charlie Bu, Alex Johnson, Marina Protopopova, Qingxiu Zhang, Natalya Bodyak, Marc Damelin, David H. Lee, Donald Bergstrom, Timothy B. Lowinger. Unique pharmacologic properties of Dolaflexin-based ADCs—a controlled bystander effect [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 754.

The ADC XMT-1522 consists of a novel human IgG1 anti-HER2 monoclonal antibody and a novel, auristatin-based cytotoxic payload (Auristatin F-hydroxypropylamide, AF-HPA). An average DAR of 12 AF-HPA molecules is achieved via a biodegradable polymer conjugation platform. The non-clinical DMPK properties of XMT-1522 have been characterized in vitro in plasma and microsomal stability studies, and in vivo in plasma and tissue disposition and excretion studies. Sample analysis for total AF-HPA drug payload and released (free) AF-HPA and its metabolites was performed by ESI+ LC/MS/MS; total antibody was determined by ELISA. The half-life for AF-HPA release in plasma was found to be greater than 120 hours in all species tested. Microsomal stability studies showed that AF-HPA was further converted to other metabolites including the carboxylic acid auristatin F (AF), as well as monomethyl auristatin F-HPA (MMAF-HPA) and MMAF. The pharmacokinetic profiles of XMT-1522 were evaluated in mouse, rat and cynomolgus monkey. The antibody of XMT-1522 is cross-reactive with monkey, but not rodent, HER2. In mouse and rat, XMT-1522 exposure was dose-proportional; exposure was slightly greater than dose-proportional in monkey consistent with saturation of target-mediated clearance. All species showed extended exposure to total AF-HPA drug payload, with measured clearance and volume of distribution similar for total AF-HPA and the antibody component of XMT-1522. Exposure to free AF-HPA and AF was less than 1/1000th the exposure of total AF-HPA. These data indicate the vast majority of AF-HPA in plasma is antibody-conjugated, indicating high stability of the ADC in systemic circulation. XMT-1522 tissue disposition was studied in NCI-N87 HER2-positive gastric cancer xenograft tumor bearing mice. After a single 3 mg/kg dose of XMT-1522, free AF-HPA and its metabolite AF were measurable in tumor tissue until the last time point measured (2 weeks). Total AF-HPA and free AF-HPA achieved peak tumor concentrations 48 hours after dosing. In contrast, AF achieved peak tumor concentration 7 days after dosing and showed only a slight decline in tumor concentration at 14 days, consistent with intracellular trapping of this poorly cell-permeable metabolite. Exposure to free AF-HPA or AF in other tissues was at least an order of magnitude lower than in tumor; in tissues with measurable free drug, AF was the predominant species. XMT-1522 excretion studies, conducted in rat, indicated that the AF-HPA payload was mainly excreted by the gastrointestinal route. In the first 96 hours after administration 33% of the AF-HPA dose was excreted in feces, compared to 3% excreted in urine. The major contributing metabolites both in feces and urine were conjugated AF-HPA, AF, and free AF-HPA. In conclusion, the plasma kinetics, tissue distribution and excretion profile of XMT-1522 are acceptable for clinical evaluation in cancer patients. Citation Format: Alex Yurkovetskiy, Dmitry Gumerov, Elena Ter-Ovanesyan, Patrick Conlon, Michael Devit, Charlie Bu, Natalya Bodyak, Timothy Lowinger, Donald Bergstrom. Non-clinical pharmacokinetics of XMT-1522, a HER2 targeting auristatin-based antibody drug conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 48. doi:10.1158/1538-7445.AM2017-48

The type II sodium-dependent potassium transporter NaPi2b (SLC34A2) is highly expressed in non-squamous NSCLC and non-mucinous ovarian cancer (OC) with restricted normal tissue expression, suggesting it may be a suitable ADC target for these indications. XMT-1536 is a novel, highly potent anti-NaPi2b ADC comprised of an average of 15 auristatin molecules conjugated to XMT-1535, a novel humanized anti-NaPi2b antibody, via the Dolaflexin ADC platform. The auristatin payload is enzymatically cleaved upon ADC trafficking to the endosome/lysosome compartment, releasing a cytotoxic auristatin derivative that is capable of bystander effect killing. In cell binding assays, XMT-1535 antibody binds to OC cells with low nanomolar affinity, which is unaffected by conjugation of the Dolaflexin drug conjugate. In vitro cytotoxicity assays show picomolar potency of XMT-1536 in OVCAR3 (OC; 32,000 NaPi2b molecules/cell; IC50 2 pM), TOV21G (OC; 10,000 NaPi2b molecules/cell; IC50 40 pM), and HCC-4006 (NSCLC; 52,000 NaPi2b molecules/cell; IC50 130 pM). In each cell line, XMT-1536 is 1-2 logs more potent than a non-binding Dolaflexin ADC control, consistent with target-dependent cytotoxic effect. XMT-1536 was tested in mouse xenograft models of OC and NSCLC. In the OVCAR3 OC model, XMT-1536 induced partial tumor regressions after a single dose of 3 mg/kg (0.21 mg/kg payload equivalent dose), and complete tumor regressions after a single dose of 5 mg/kg (0.36 mg/kg payload dose) or 3 weekly doses of 3 mg/kg. In contrast, a non-binding Dolaflexin ADC with comparable drug loading was inactive after 3 weekly administrations of 3 mg/kg, consistent with the anti-tumor activity of XMT-1536 being mediated through binding to the NaPi2b target. XMT-1536 was also tested in a patient-derived model of KRAS mutant NSCLC, where 3 weekly doses of 3 mg/kg led to significant tumor growth delay and regressions in some animals. Evaluation of XMT-1536 in additional patient derived xenograft models is on-going and will be updated at the meeting. XMT-1535 is cross-reactive with cynomolgous monkey NaPi2b, allowing an informative evaluation of whether XMT-1536 retains good tolerability in non-human primate. XMT-1536 was administered to cynomolgous monkeys in an exploratory single dose study up to 5 mg/kg ADC (4294 μg/m2 auristatin payload equivalents), with no observed target-mediated toxicity and limited adverse findings. Of note, there was no evidence of bone marrow toxicity, which has been observed generally for cleavable auristatin ADCs, and specifically for a recently published auristatin-based NaPi2b ADC (Lin et al., Clinical Cancer Research, 2015). Based on these data XMT-1536 is advancing to early clinical development for the treatment of NaPi2b-expressing tumors. Citation Format: Natalya Bodyak, Alex Yurkovetskiy, Mao Yin, Dmitry Gumerov, Reddy Bollu, Patrick Conlon, Venu R. Gurijala, Dennis McGillicuddy, Cheri Stevenson, Elena Ter-Ovanesyan, Peter U. Park, Laura Poling, Winnie Lee, Michael DeVit, Dongmei Xiao, LiuLiang Qin, Timothy B. Lowinger, Donald A. Bergstrom. Discovery and preclinical development of a highly potent NaPi2b-targeted antibody-drug conjugate (ADC) with significant activity in patient-derived non-small cell lung cancer (NSCLC) xenograft models. [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 1194.

Antibody-drug conjugates (ADCs) represent a promising drug class that relies on monoclonal antibody recognition of specific cancer-associated antigens for targeted delivery of chemotherapeutic agents. Fleximer® ADCs utilize a polymer-based conjugation platform to enable high drug-antibody ratios (DAR) and significantly greater anti-tumor potency compared to ADCs with DAR 3-4. We previously showed that trastuzumab-dolaflexin (T-dolaflexin), a HER2-targeted ADC comprised of the antibody trastuzumab conjugated to ∼15 proprietary auristatin molecules via Fleximer, has excellent pharmacokinetics and efficacy in mouse models (AACR 2014 Abstract #2645). T-dolaflexin is efficacious at a single dose of 0.67 mg/kg in mouse xenograft models, and achieves prolonged tumor-free survival after a single 2 mg/kg dose in a low HER2-expressing model that is insensitive to ado-trastuzumab emtansine (T-DM1). We sought to test the tolerability of T-dolaflexin in exploratory mouse, rat and non-human primate toxicology studies. Cohorts of 6 mice were treated with a single dose of vehicle or T-dolaflexin at 20, 30 or 40 mg/kg and followed for 21 days. The 20 and 30 mg/kg doses were well-tolerated based on body weight loss and mortality and achieved a therapeutic index (TI) in mouse >40X. Cohorts of 4 female cynomolgus monkeys were treated with a single dose of vehicle or T-dolaflexin at 0.67, 1.34 or 2.68 mg/kg (payload doses 600, 1200 and 2400 μg/m2, respectively). Day 3 necropsy was performed on 2 animals per cohort, with recovery necropsy on Day 22 in the remaining 2 animals. All animals survived until scheduled necropsy with limited body weight loss. There were no test-article related findings on gross pathology. Most notable clinical pathology findings were transaminase elevations (primarily AST), and decreased platelet counts at Day 8. One high dose animal demonstrated laboratory findings consistent with hepatic injury with full recovery by Day 22 and no findings on gross or microscopic pathology at necropsy. There was no evidence of myelosuppression. Microscopic pathology findings were limited, with no test-article related findings in HER2-expressing organs including heart, lungs and GI tract. All doses were considered well-tolerated. Toxicokinetics demonstrated good stability of drug conjugate in plasma with t1/2 ∼5 days (comparable to antibody t1/2) and minimal exposure to free payload. Plasma exposure at the 2.68 mg/kg dose in cyno was several fold higher than mouse exposure at the minimally efficacious dose of 0.67 mg/kg, and was comparable to the mouse exposure associated with prolonged tumor free survival after a single 2mg/kg dose. Trastuzumab-dolaflexin, a highly potent Fleximer-based ADC, demonstrates a favorable TI in tumor models with low HER2 expression where current HER2-directed therapies are inactive. Citation Format: Natalya Bodyak, Alex Yurkovetskiy, Peter U. Park, Dmitry R. Gumerov, Michael DeVit, Mao Yin, Joshua D. Thomas, LiuLiang Qin, Timothy B. Lowinger, Donald A. Bergstrom. Trastuzumab-dolaflexin, a highly potent Fleximer-based antibody-drug conjugate, demonstrates a favorable therapeutic index in exploratory toxicology studies in multiple species. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 641. doi:10.1158/1538-7445.AM2015-641

ASN004 is an Antibody Drug Conjugate (ADC) that targets the 5T4 oncofetal antigen (trophoblast glycoprotein), which is highly expressed in a wide range of malignant tumors, while having very limited expression in normal tissues. ASN004 incorporates a novel single-chain homo-dimer antibody, Fleximer® linker technology (Mersana Therapeutics), and several cytotoxic dolastatin (auristatin) analog warheads per ADC molecule (drug/antibody ratio ∼15). ASN004 shows high affinity for the 5T4 antigen and for 5T4-expressing tumor cells. As well, ASN004 shows potent cytotoxicity that is selective for 5T4-expressing tumor cells. ASN004 provides strong tumor regression and tumor-free survivors in multiple tumor xenograft models, at well-tolerated doses as low as 0.3 mg/kg iv. Furthermore, ASN004 causes tumor regression when administered to xenografts bearing more advanced (500 mm3) tumors. Robust, potent efficacy for ASN004 has also been demonstrated in head-to-head comparison studies with relevant reference ADCs. A favorable pharmacokinetics profile was observed in rodents and primates. Dose-range finding exploratory toxicology studies have been completed in both pharmacological and non-pharmacological non-clinical species. Efforts toward IND-enabling safety studies with this promising new agent are in progress. Citation Format: Roger A. Smith, Nitin K. Damle, Sanjeeva P. Reddy, Alex Yurkovetskiy, Natalya Bodyak, Mao Yin, Dmitry Gumerov, Elena Ter-Ovanesyan, Liu Qin, Peter U. Park, Timothy B. Lowinger, Sandeep Gupta. ASN004, a novel 5T4-targeted Dolaflexin™ antibody drug conjugate, causes complete regression in multiple solid tumor models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1693. doi:10.1158/1538-7445.AM2015-1693

Antibody-drug conjugates are effective in the treatment of HER2-amplified breast cancer and Hodgkin's lymphoma, but current ADC technologies have faced limitations expanding the addressable patient population and target space. Ado-trastuzumab emtansine (T-DM1) is an ADC with 3-4 cytotoxic drugs per antibody that was recently approved for HER2 IHC 3+ or HER2-amplified breast cancer. Even within this high HER2-expressing population, several studies have now shown greater T-DM1 benefit in patients with HER2 mRNA expression above the median. These data suggest the need for more potent anti-HER2 ADCs to maximize benefit for HER2 IHC 3+ or amplified patients, and to extend HER2 ADC therapy to low HER2-expressing patients (HER2 IHC 1+/2+). XMT-1522 is an anti-HER2 ADC that uses a novel, human anti-HER2 antibody optimized for cytotoxic payload delivery, and is non-competitive with trastuzumab or pertuzumab for HER2 binding. Each antibody is conjugated to ∼15 proprietary auristatin molecules using Fleximer, a biodegradable hydrophilic polymer. XMT-1522 shows nanomolar potency in cultured tumor cells with HER2 receptor densities as low as 10,000 per cell, and is typically 1-3 logs more potent than T-DM1 across a panel of 25 tumor cell lines. In mouse xenograft studies XMT-1522 has excellent pharmacokinetic properties and achieves complete tumor regressions at well-tolerated doses. In the high HER2-expressing N87 gastric cancer model (800,000 HER2 receptors/cell), complete regressions are achieved with a single 1 mg/kg dose of XMT-1522, while 10 mg/kg T-DM1 is required for comparable activity. In the same model, the XMT-1522/trastuzumab/pertuzumab triple combination results in tumor regressions where the same doses of XMT-1522 alone or the trastuzumab/pertuzumab doublet result in tumor stasis. In the low HER2-expressing JIMT-1 breast cancer (79,000 HER2/cell) and SNU5 gastric cancer (22,000 HER2/cell) models, complete regressions are achieved with single 1 mg/kg or 0.67 mg/kg doses of XMT-1522, respectively, while T-DM1 is inactive at doses ≥10 mg/kg. In non-human primates XMT-1522 demonstrates good stability of drug conjugate in plasma with t1/2 ∼5 days (comparable to antibody t1/2) and minimal exposure to free payload. Despite the high potency of XMT-1522 in low HER2 tumor models, there is no XMT-1522-related toxicity observed in critical HER2-expressing tissues including heart and lung. The preclinical data support testing XMT-1522 as a single agent in tumors with low HER2 expression where current HER2-directed therapies are not indicated. Furthermore, combination of XMT-1522 with trastuzumab and/or pertuzumab achieves efficient cytotoxic payload delivery while retaining the potential for full inhibition of HER2 signaling, which may be necessary to improve on current regimens in HER2-driven tumors. Citation Format: Donald A. Bergstrom, Natalya Bodyak, Alex Yurkovetskiy, Peter U. Park, Michael DeVit, Mao Yin, Laura Poling, Joshua D. Thomas, Dmitry Gumerov, Dongmei Xiao, Elena Ter-Ovanesyan, LiuLiang Qin, Alex Uttard, Alex Johnson, Timothy B. Lowinger. A novel, highly potent HER2-targeted antibody-drug conjugate (ADC) for the treatment of low HER2-expressing tumors and combination with trastuzumab-based regimens in HER2-driven tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-231. doi:10.1158/1538-7445.AM2015-LB-231

The application of biodegradable polymers to antibody-drug conjugate (ADC) design can provide numerous advantages, including significantly higher drug-antibody ratios, the use of alternative payloads with potencies considered insufficient for direct conjugation, the improvement of ADC physico-chemical properties, especially for highly hydrophobic payloads, and the potential expansion of protein recognition scaffolds beyond the commonly used IgGs. The basis of the novel polymer-based conjugation system described herein is a hydrophilic, fully biodegradable polyacetal carrier, (poly(1-hydroxymethylethylene hydroxymethylformal) or PHF) modified with chemically orthogonal linkers. A bioconjugation linker is used for efficient covalent attachment of a targeting moiety to the PHF scaffold, while a second, chemically distinct linker is used to attach multiple copies of a drug payload to the polymer to control the mechanism and rate of drug release. Utilizing multiple copies of a proprietary dolastatin derivative chemically conjugated to PHF, we have developed a potent and effective drug conjugation platform for ADC application, which has been named Dolaflexin™. Here, we report the preparation and characterization of a novel trastuzumab DolaflexinTM ADC, employing a maleimide-based bioconjugation approach. The resulting ADC, with a drug-antibody ratio of 20, exhibits enhanced stability and improved pharmacokinetics, with a prolonged plasma half-life and tumor-specific accumulation. Active drug release and accumulation in tumor tissue was also confirmed by LC/MS/MS methods. The activity of this novel trastuzumab-dolaflexin ADC was evaluated in multiple tumor xenograft models with significant variations in target antigen expression levels. Models including BT474 breast cancer, NCI-N87 gastric cancer, and JIMT1 breast cancer models were utilized, and comparisons to a variety of controls and ADC reference standards were made. Significant advantages of the polyacetal polymer-based ADCs in comparison to conventional ADCs, particularly in models with low target antigen expression, were observed. Details of these studies and potential applications for the development of new ADC therapeutics based on this approach will be presented. Citation Format: Alex Yurkovetskiy, Natalya Bodyak, Mao Yin, Joshua D. Thomas, Patrick Conlon, Cheri A. Stevenson, Alex Uttard, LiuLiang Qin, Dmitry R. Gumerov, Elena Ter-Ovaneysan, Venu R. Gurijala, Dennis McGillicuddy, Roberta E. Glynn, Michael DeVit, Laura L. Poling, Peter U. Park, Timothy B. Lowinger. Advantages of polyacetal polymer-based ADCs: Application to low expression targets. [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 2645. doi:10.1158/1538-7445.AM2014-2645

The proceptive and investigative behavior of estrous female golden hamsters was studied by introducing females into an arena with pairs of males in mesh containers (intact golden hamster - intact Turkish hamster; intact - Harderianectomized (HX) golden hamster; HX golden hamster - golden hamster smeared by male fresh Harderian gland homogenate (HXHG). During 10-min tests the number and duration of lordosis reactions, the time spent in each half of arena, the total time and the number of sniffing bouts were recorded. When one male of the pair was a conspecific and another one was a Turkish hamster all measures were significantly higher towards the conspecific male. Females also preferred an intact male over an HX male. In the HX male - HXHG male comparison, females showed greater proceptive and investigative interest in the HXHG males. Thus, the degree of expression of female proceptive behavior increased in the following order: heterospecific male, HX conspecific male, HX conspecific male plus male Harderian gland homogenate, intact conspecific male. We conclude that the Harderian gland of the male golden hamster contains a signal which can provoke sexual response of the female.

The application of biodegradable, polyacetal polymers to antibody-drug conjugate (ADC) design can provide numerous advantages, including significantly higher capacity for drug payload (∼20 drugs per antibody), the use of payloads with low potency that are not suitable for direct conjugation, the improvement of physicochemical properties for ADC, especially with highly hydrophobic payloads and the use of protein recognition scaffolds beyond the commonly used IgGs. The basis of this novel polyacetal polymer-based conjugation system is a hydrophilic, fully biodegradable polyacetal carrier (PHF or poly(1-hydroxymethylethylene hydroxymethylformal, or Fleximer®) modified with chemically orthogonal linkers. One linker is used to covalently attach a targeting moiety (mAb or alternative), while a second, chemically distinct linker is used to attach a drug payload and to control the mechanism and rate of drug release. Previously, we have reported potent anti-tumor activity with trastuzumab-s-Dolaflexin™, an anti-HER2 ADC composed of trastuzumab and a proprietary dolastatin derivative coupled to a Fleximer scaffold (Dolaflexin™). In that example, Dolaflexin was conjugated to the antibody through interchain cysteine residues via a hindered disulfide linkage (AACR Annual Meeting 2013 Abstract #4331). Unlike direct drug-cysteine linked ADCs that can result in destabilization of antibody by disruption of interchain disulfide bridges, we have shown that Dolaflexin conjugation via cysteines in the antibody hinge region stabilizes the resulting ADCs through the formation of inter-chain bridge structures. Trastuzumab-s-Dolaflexin ADC exhibited a prolonged plasma half-life, tumor-specific accumulation, and potent anti-tumor activity in vivo. Here, we report a novel trastuzumab-m-Dolaflexin ADC using a maleimide linker that further enhances the pharmacokinetics of the ADC and demonstrates complete regressions of established HER2+ BT-474 xenograft tumors in SCID mice even at a single dose of 2.5 mg/kg. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C238. Citation Format: Joshua Thomas, Alex Yurkovetskiy, Natalya Bodyak, Mao Yin, Patrick Conlon, Cheri Stevenson, Alex Uttard, Liu Qin, Dmitry Gumerov, Elena Ter-Ovaneysan, Michael DeVit, Laura L. Poling, Peter U. Park, Timothy B. Lowinger. Polyacetal polymer-based anti-HER2 antibody-drug conjugate employing cysteine bioconjugation through thioether linkage allows a high drug loading of dolastatin-derived payload with excellent pharmacokinetics and potent anti-tumor activity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C238.

The application of polymers to antibody drug conjugate (ADC) design and preparation can provide numerous advantages, including 1) significantly higher capacity for drug payload; 2) utilization of alternative payloads not suitable for direct conjugation approaches; 3) improvement of physicochemical properties of resulting ADCs; 4) utilization of protein recognition scaffolds beyond the commonly used IgGs; and 5) improvements in PK and biodistribution. Herein we present results of a novel, biodegradable polyacetal polymer-based conjugation system to create next-generation ADCs. The basis of this new conjugation system is a hydrophilic, fully biodegradable polyacetal carrier (PHF or poly(1-hydroxymethylethylene hydroxymethylformal) modified with chemically orthogonal linkers. One linker is used to covalently attach a targeting moiety (mAb or alternative) via cysteine conjugation, while a second, chemically distinct linker is used to attach a drug payload and to control the mechanism and rate of drug release. Previously we have reported highly efficacious polyacetal ADCs prepared by utilizing random lysine modification. In this report we present an alternative cysteine-based bioconjugation strategy. Conventional, direct drug-cysteine linked ADCs are destabilized due to loss of inter-chain disulfide bridges. In contrast, the polyacetal polymer conjugated via cysteines in the antibody hinge region stabilizes the resulting ADCs through the formation of inter-chain bridge structures. To demonstrate the benefits of this approach, we prepared Her-2 targeted ADCs with protein recognition scaffolds ranging in size from 15 kDa to 150 kDa, all targeting the Her-2 antigen, and bearing an anti-tubulin agent as the drug payload. ADCs were highly active and selective in vitro in Her-2 expressing cell lines. These polyacetal cysteine-based ADCs exhibited prolonged plasma and tumor exposure in the Her-2 expressing BT474 mouse xenograft model and tumor-specific accumulation. The ADCs were well-tolerated, and resulted in 100% tumor-free survivors. Citation Format: Alex Yurkovetskiy, Natalya Bodyak, Mao Yin, Joshua Thomas, Patrick Conlon, Cheri Stevenson, Alex Uttard, Liu Qin, Dmitry Gumerov, Elena Ter-Ovaneysan, Michael DeVit, Timothy B. Lowinger. Advantages of polyacetal polymer-based antibody drug conjugates employing cysteine bioconjugation. [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 4331. doi:10.1158/1538-7445.AM2013-4331

Antibody drug conjugates (ADCs) are rapidly establishing themselves as an important class of chemotherapeutic agents, with impressive therapeutic potential both in hematological malignancies and in solid tumors, as evidenced by SGN-035 and T-DM1. Despite these impressive results, limitations in the current technologies remain. Current ADCs are typically limited to the use of full-size mAbs, providing excellent target recognition and pharmacokinetics (PK), but tolerating the conjugation of only 3-4 payload molecules. This limitation in payload capacity necessitates the use of extremely toxic drugs such as the auristatins and maytansinoids to maximize the therapeutic effect while maintaining the drug load at a low stoichiometric ratio. The vast majority of less potent but often more specific agents with proven anti-cancer activity are largely excluded from incorporation in ADCs. Similarly, a diversity of smaller (alternative) protein recognition scaffolds, such as scFvs, Fabs, diabodies, minibodies etc. are not readily utilized for ADCs (because of their smaller size, they are often associated with poor PK, and even lower capacity for direct drug conjugation. We wish to report our results with a novel, biodegradable-polymer based conjugation system, which provides several advantages for next-generation ADCs, including: 1) significant drug loading of diverse classes of anti-cancer agents; 2) excellent physicochemical and PK properties; and 3) flexibility for use with full-sized mAbs as well as mAb alternatives such as Fabs. The basis of this new conjugation system is a hydrophilic, fully biodegradable polyacetal carrier (PHF or poly(1-hydroxymethylethylene hydroxymethylformal)) covalently linked via separate, optimized linkers to a targeting moiety (mAb or alternative) and 10-40 molecules of drug payload. The optimized stability of the linker employed for conjugation of the polymer-drug conjugate to the targeting molecule ensures stability in the circulation, while the enzymatically cleavable linker utilized for drug-polymer conjugation provides a controllable, predictable pattern of intracellular drug release. Employing well characterized mAbs and mAb Fab fragments in combination with diverse cytotoxic agents as well as kinase inhibitors, we have demonstrated that this new ADC conjugation system provides several potential advantages over existing approaches. For example, trastuzumab was efficiently conjugated to a PHF-vinca polymer conjugate, with a ratio of 16-20 small molecules per antibody. Clear evidence of in vivo activity was demonstrated in multiple xenograft models. Pharmacokinetic and tissue disposition studies conducted in these models confirmed extended plasma ADC exposure (T1/2 of 3-4 days) and significant drug intratumoral accumulation, correlating well with the high ADC efficacy observed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4633. doi:1538-7445.AM2012-4633

Targeted delivery remains one of the biggest challenges in the development of RNAi-based therapeutics. Cequent has developed a proprietary delivery technique for RNA interference, transkingdom RNAi (tkRNAi), in which non-pathogenic bacteria are engineered to invade specific target cells, produce and release short hairpin RNA (shRNA). We have previously shown tkRNAi to be successful in cell culture assays, in a mouse model for human colon cancer (APCmin), and in non-human primates (cynomolgus monkeys), by suppressing the oncogene, β-catenin. β-catenin is the key oncogene implicated in Colorectal Cancer (CRC) and Familial Adenomatous Polyposis (FAP); the latter being an orphan hereditary disease resulting in the formation of hundreds of polyps in the gastrointestinal tract and ultimately leads to the development of colon cancer without surgical intervention. No pharmaceutical treatment is available for patients with FAP. The non-human primate studies designed to evaluate on-target toxicities were conducted with a precursor (CEQ501) to our clinical candidate, CEQ508. Following optimization, which most importantly included platform engineering to improve hairpin production and processing, we conducted a large GLP mouse bridging toxicity study comparing off-target effects of CEQ508 and CEQ501. No CEQ508 or CEQ501-related adverse responses were identified in the following study parameters analyzed: clinical observations, body weights, serum chemistry, hematology, cytokines, gross or histopathology. As no test article-related changes were identified under the conditions of this study at the highest dose evaluated, the No Observed Effect Level (NOEL) for daily oral administration of either CEQ508 or CEQ501 was 5×109 cfu/day, or 2×1011 cfu/kg/day. (CEQ501 in non-human primates showed a similar safety profile with the No Observed Adverse Effect Level (NOAEL) determined to be 1×1012 cfu/day, or 2×1011 cfu/kg/day). Together with multiple internally conducted pharmacology and pharmacokinetic experiments, Cequent used this data in the nonclinical section of an IND filed in late 2009. In addition to presenting detailed in vivo data from our filing, an update on further development will be presented on CEQ508 for the proposed daily dosing of FAP patients with this oral tkRNAi therapeutic targeting β-catenin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4513.