Search

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.

Sensorgrams of trastuzumab-PHF-vinca binding the hErbB2 surfaces, ~420 RU (top) and ~1320 RU (bottom).

Sensorgrams of trastuzumab binding the hErbB2 surfaces, ~420 RU (top) and ~1320 RU (bottom).

Synthesis of HPV-Ala-NH2.

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

STING pathway agonism has emerged as a potential therapeutic strategy to stimulate anti-tumor immune responses. We have previously shown that tumor cell-targeted antibody-drug conjugates (ADCs) carrying a novel STING agonist induce anti-tumor activity without causing substantial elevations in systemic cytokine levels, thus suggesting a therapeutic advantage of STING agonist ADCs relative to unconjugated agonists. ADCs constitute a proven therapeutic modality that is ideally suited to enable systemic administration and delivery of the conjugated drug to desired cell types within the tumor microenvironment. In addition to delivering STING agonist into the antigen-expressing tumor cells, antigen-bound ADCs deliver STING agonist to tumor-resident myeloid cells through Fcγ receptor (FcγR)-mediated internalization. In this study we investigated the mechanism of FcγR-mediated internalization of the tumor cell-targeted STING-agonist ADCs into myeloid cells and the nature of the subsequent STING pathway activation. We developed flow cytometry-based assays to determine the changes in FcγRI, FcγRII, and FcγRIII cell surface detection levels in the presence of ADCs specifically designed to be either proficient or deficient in FcγR-binding. Combined with functional assays based on co-culture of cancer cells and FcγRI knock out myeloid cells, we identified FcγRI as the major Fcγ receptor that mediates target-bound ADC internalization into myeloid cells in vitro. Even though FcγRI is expressed only by a subset of CD11b+ myeloid cells, tumor cell-targeted ADCs induce greater production of interferons and other cytokines and more potent cancer cell killing than CD11b-targeted-ADCs, which deliver STING agonist into FcγRI- (non-productive) as well as FcγRI+ (productive) myeloid cells. Finally, we demonstrate that myeloid cells within dissociated primary human tumors from multiple donors express FcγRI and are capable of tumor cell killing in response to tumor cell-targeted STING agonist ADCs in vitro. In summary, our data indicate that the ADC-mediated delivery of a STING agonist specifically into FcγRI-expressing myeloid cells efficiently activates innate immune responses in the most relevant immune cell types within the tumor microenvironment. Citation Format: Naniye Malli Cetinbas, Kalli C. Catcott, Travis Monnell, Jahna Soomer-James, Keith Bentley, Susan Clardy, Bingfan Du, Eoin Kelleher, Marina Protopopova, Cheri Stevenson, Joshua D. Thomas, Alex Uttard, Dorin Toader, Jeremy Duvall, Raghida Bukhalid, Marc Damelin, Timothy B. Lowinger. Tumor cell-targeted STING-agonist antibody-drug conjugates achieve potent anti-tumor activity by delivering STING agonist specifically to tumor cells andFcγRI-expressing subset of myeloid cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2114.

We present here a novel therapeutic agent, XMT-2056, that results in robust anti-tumor activity mediated by an immune response through targeted delivery of a STING agonist to the tumor microenvironment. By leveraging an antibody-drug conjugate (ADC) strategy, systemic administration of a STING agonist with tumor-targeted delivery can be achieved, potentially overcoming limitations of either intratumoral or intravenous administrations of unconjugated, small molecule STING agonists. XMT-2056 was generated through conjugation of Immunosynthen, a platform that employs a novel STING agonist payload specifically designed for ADCs, to HT-19, a HER2-targeting antibody which binds to a novel epitope and does not compete for binding with either trastuzumab or pertuzumab. Initial results showed XMT-2056 has target-dependent anti-tumor activity in vivo and is well tolerated in non-human primates at significantly higher exposure levels than those required for anti-tumor activity. To evaluate the impact of HER2 expression level on the activity of XMT-2056, in vivo studies in gastric and breast cancer models with varying HER2 expression levels were conducted, and XMT-2056 showed potent anti-tumor activity in a dose dependent and target dependent manner including in models with very low expression of HER2. Because the antibody employed in XMT-2056 does not compete for binding with trastuzumab or pertuzumab, we hypothesized that there could be benefit in combining with such approved HER2-targeted therapies. This advantage was demonstrated in vivo as the combination of XMT-2056 and trastuzumab or pertuzumab showed greater anti-tumor activity compared to the administration of either agent alone. Further efforts to elucidate the mechanism(s) of the observed benefit of these combinations will be discussed. Given the innate immune activation by XMT-2056, there is also a strong rationale for combination with immune checkpoint inhibitors. To this end, administration of an XMT-2056 surrogate ADC in combination with an anti-PD1 agent improved anti-tumor activity in a ratHER2-engineered EMT-6 syngeneic mouse model. Together these data support the potential of XMT-2056 both as a monotherapy and in combination with other HER2 targeted agents as well as checkpoint inhibitors. Citation Format: Jeremy R. Duvall, Raghida A. Bukhalid, Naniye M. Cetinbas, Kalli C. Catcott, Kelly Lancaster, Keith W. Bentley, Suzanna Clark, Susan Clardy, Scott D. Collins, Anouk Dirksen, Elizabeth Ditty, Bingfan Du, Eugene W. Kelleher, Travis Monnell, Marina Protopopova, Caitlin Routhier, Cheri Stevenson, Elena Ter-Ovanesyan, Joshua D. Thomas, Alex Uttard, Jason Wang, Phonphimon Wongthida, Ling Xu, Annika Yau, Jeffrey Zurita, Dorin Toader, Marc Damelin, Timothy B. Lowinger. XMT-2056, a HER2-targeted Immunosynthen STING-agonist antibody-drug conjugate, binds a novel epitope of HER2 and shows increased anti-tumor activity in combination with trastuzumab and pertuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3503.

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.

Key defining attributes of an antibody-drug conjugate (ADC) include the choice of targeting antibody, linker, and the drug-to-antibody ratio (DAR). The choice of DAR, within the constraints of acceptable physicochemical properties for the given platform, is a function of balancing delivery of sufficient payload to targeted cells with the ability to achieve sustained in vivo exposures. Previous reports have described lower DAR mc-VC-MMAE conjugates, DAR = 1-2, that demonstrated higher in vivo exposure and lower clearance when compared to higher DAR (e.g. 4-8) counterparts. In theory, high DAR conjugates may be especially desirable when targeting low antigen expressing tumors or when lower potency payloads are used, as each binding and internalization event results in greater payload delivery. Here we report a systematic exploration of DAR across a much wider range than has been previously reported, by combining THIOMAB® protein engineering technology with the Dolasynthen platform. Homogeneous, site-specific ADCs spanning a discrete range of DARs – 2, 4, 6, 12, and 18 – were made by conjugation of Trastuzumab IgG1 THIOMAB constructs with 1, 2, or 3 engineered cysteines to monomeric or trimeric Dolasynthen. The cytotoxicity of the resulting well-defined ADCs was assessed in vitro in cell lines with high or low expression of HER2 antigen. Pharmacokinetic data for all test articles in mice were generated in tumor bearing mice. In high HER2 expressing cell lines, in vitro cytotoxicity by payload was comparable across DARs. In a lower HER2 expressing system, the higher DAR ADCs performed better. In vivo, our data demonstrated comparable pharmacokinetics for the Dolasynthen conjugates across all DARs. These results illustrate the utility of a DAR ranging platform, such as Dolasynthen when evaluating ADCs as it enables the interrogation of a range of antibody and payload dosing regimens. Citation Format: Kalli C. Catcott, Susan Clardy, Jack Sadowsky, Rebecca K. Rowntree, Naniye Malli Centibas, Ling Xu, Andy Polson, Kenneth Avocetien, Tyler Carter, Mark Nazzaro, Dokyong "DK" Kim, Thomas H. Pillow, Neelie Zacharias, Cong Wu, Jeffrey Zurita, Elizabeth Ditty, Stephen Bradley, Alex Uttard, Bingfan Du, William S. Sawyer, Doug Leipold, Gail Lewis Phillips, LiuLiang Qin, Kelly Slocum, Geoffrey Del Rosario, Ginny Li, Shang-Fan Yu, David Lee, Radha Iyengar, Marc Damelin, Dorin Toader, Timothy B. Lowinger. Site-specific Dolasynthen ADCs demonstrate consistent exposure across a wide range of drug-to-antibody ratios [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P167.

BackgroundThe tumor microenvironment is a complex, multicellular system, composed not only of malignant cancer cells but also of a diversity of stromal cells including vascular cells, immune cells, and fibroblasts that support tumorigenesis. Antigens expressed on these cells tend to be widely expressed across a range of malignancies, presenting unique opportunities for development of anti-cancer therapies.MethodsWe have previously demonstrated that STING-agonist antibody-drug conjugates (Immunosynthen ADCs) targeting tumor cell antigens induce target-dependent anti-tumor immune responses in vitro and in vivo. To that effect, we hypothesized that Immunosynthen ADCs targeting tumor-associated antigens would coordinate immune-mediated killing of cancer cells not expressing the tumor-associated antigens (antigen-negative cancer cells) and induce anti-tumor activity.ResultsHerein, we demonstrate that targeting tumor-associated antigens with STING-agonist ADCs activate the STING pathway in immune cells via Fcγ receptor-mediated uptake. In addition, due to the intrinsic ability of certain tumor-associated cells to activate the STING pathway, STING-agonist ADCs targeting those cells can induce STING signaling in both the targeted cells and the immune cells, which constitutes a therapeutic advantage of ADCs that activate the STING pathway. In triple co-cultures of antigen-positive tumor-associated cells, antigen-negative cancer cells, and immune cells, the STING-agonist ADC specifically induced potent cell killing of the antigen-negative cancer cells with minimal impact on the immune and tumor-associated cells, thus representing a non-traditional, yet highly effective mechanism of ADC targeting. In vivo efficacy studies showed that STING-agonist ADCs developed for two tumor-associated antigens induced complete, sustained tumor regressions in syngeneic tumor models and exhibited immunological memory after rechallenge. CD8+ T cells contributed to the anti-tumor activity of the STING-agonist ADCs.ConclusionsIn summary, Immunosynthen STING-agonist ADCs targeting tumor-associated antigens represent a novel approach for ADC-mediated cancer immunotherapy and enable the multifaceted activation of the STING pathway in a tumor-targeted manner beyond tumor antigens.

XMT-1660 is a novel Dolasynthen-based antibody drug conjugate carrying a DolaLock payload with controlled bystander effect and targeting B7-H4, a tumor antigen that is broadly expressed on the cell surface in breast, ovarian and endometrial cancers. B7-H4 (VTCN1) exerts immunosuppressive effects by suppression of T cell proliferation and is expressed on tumor-associated macrophages (TAMs) as well as epithelial tumor cells. XMT-1660 is comprised of an anti-B7-H4 antibody site-specifically conjugated to Dolasynthen, with a total of 6 DolaLock Auristatin F-HPA (AF-HPA) anti-tubulin payloads per antibody (DAR-6). To select the optimal ADC, three ADCs using the same antibody and DolaLock payload were compared: site-specific Dolasynthen-based DAR-2 and DAR-6 ADCs, and a stochastically conjugated Dolaflexin-based DAR-12 ADC. In vitro, no significant differences were observed among the 3 ADCs: all exhibited specific recognition of B7-H4 and elicited potent cytotoxicity against B7-H4-expressing cancer cells. In vivo, XMT-1660 consistently exhibited more anti-tumor activity than the other ADCs in TNBC models and ER+/HER2- models after single, equivalent doses based on payload. XMT-1660 demonstrated dose-dependent anti-tumor activity and induced sustained tumor regressions after a single administration. XMT-1660 and the Dolasynthen DAR-2 ADC both exhibited improved pharmacokinetics in mouse relative to the Dolaflexin DAR 12 ADC. These data indicate that XMT-1660 exhibited a superior preclinical profile to the other ADCs and more generally demonstrate the importance of DAR-ranging studies to identify the optimal antibody-drug conjugate for a given target. These results, as well as results from exploratory toxicology studies in non-human primates, strongly support the clinical development of XMT-1660. Citation Format: Shawn P. Fessler, Jason Wang, Scott D. Collins, LiuLiang Qin, Kenneth Avocetien, Ling Xu, Ronald Eydelloth, Steven Vonderfecht, Chen-Ni Chin, Steven Bradley, Susan Clardy, Anouk Dirksen, Elizabeth Ditty, Bingfan Du, Dokyong Kim, Rebecca Mosher, Elena Ter-Ovanesyen, Kelly Slocum, Alex Uttard, Phonphimon Wongthida, Jeffrey Zurita, Dorin Toader, Marc Damelin, Timothy B. Lowinger. XMT-1660, a B7-H4-targeted Dolasynthen antibody-drug conjugate for the treatment of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 907.

The Dolasynthen platform incorporates the highly potent anti-mitotic agent auristatin F-HPA (AF-HPA), with its associated DolaLock mechanism of controlled bystander effect, and enables the synthesis of antibody-drug conjugates (ADCs) with precise control of the drug-to-antibody ratio (DAR) and site-specific bioconjugation. XMT-1592 is a novel ADC comprised of an anti-NaPi2b antibody and Dolasynthen, conjugated in a site-specific manner to yield DAR 6. NaPi2b, also known as SLC34A2, is a transmembrane sodium-phosphate transporter that is broadly expressed on tumor cells in ovarian carcinoma, NSCLC lung adenocarcinoma and other tumor types. Recent studies have shown that NaPi2b expression is enriched in the EGFR and KRAS mutant subtypes of lung adenocarcinoma. Binding studies showed a specific, high-affinity interaction of XMT-1592 with NaPi2b that was not affected by conjugated Dolasynthen. XMT-1592 elicited potent and specific in vitro cytotoxicity against NaPi2b-expressing ovarian carcinoma cells. XMT-1592 exhibited potent and specific in vivo activity in NaPi2b-expressing tumor xenografts derived from ovarian carcinoma or lung adenocarcinoma. Consistent with the targeted delivery benefits of the ADC approach, XMT-1592 yielded high and sustained concentrations of AF-HPA to tumors but not normal tissues. To evaluate the benefits of site-specific bioconjugation of Dolasynthen, we conducted in vitro and in vivo comparisons of XMT-1592 to a stochastically conjugated version of the ADC. XMT-1592 had improved in vivo activity, pharmacokinetics, and clinical pathology relative to its stochastic counterpart. Taken together, these results support XMT-1592 as a development candidate for the treatment of NaPi2b-expressing tumors. Citation Format: Shawn Fessler, Anouk Dirksen, Scott D. Collins, Ling Xu, Winnie Lee, Jason Wang, Ron Eydelloth, Elena Ter-Ovanesyen, Jeffrey Zurita, Elizabeth Ditty, Barrett Nehilla, Susan Clardy, Susan Clardy, Tyler Carter, Kenneth Avocetien, Mark Nazzaro, Nam Le, Kalli C. Catcott, Alex Uttard, Bingfan Du, Chen-Ni Chin, Rebecca Mosher, Kelly Slocum, Liuliang Qin, David Lee, Dorin Toader, Marc Damelin, Timothy B. Lowinger. XMT-1592, a site-specific Dolasynthen-based NaPi2b-targeted antibody-drug conjugate for the treatment of ovarian cancer and lung adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2894.

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 (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.

Dolasynthen is a novel, fully synthetic, structurally homogeneous platform that enables the construction of ADCs with tunable drug-to-antibody ratios (DAR), from a low of 2 to a high of 24. The resulting ADCs exhibit excellent physicochemical properties and fully homogeneous conjugates can be created through a variety of bioconjugation chemistries. Analogous to our first platform, Dolaflexin®, Dolasynthen is loaded with the proprietary payload Auristatin F hydroxypropylamide (AF-HPA) with precisely defined numbers of the cytotoxin per Dolasynthen scaffold.Studies that evaluate the tolerability and efficacy of Dolasynthen in preclinical models are described herein. ADCs containing a range of DAR values were generated following conjugation of Dolasynthen to two different monoclonal antibodies. The DAR of the ADCs was achieved by controlled reduction of native disulfide bonds in IgG1 antibodies, chromatographic fractionation, or through use of site-specific conjugation technologies. ADCs with both DAR6 and DAR12 were evaluated in vitro and also in vivo in the mouse, rat and monkey, for efficacy, tolerability and PK. Dolasynthen conjugates had excellent physicochemical properties and displayed the expected cell binding and in vitro cytotoxicities. In vivo pharmacology of Dolasynthen ADCs in in vivo xenograft models showed dose-dependent tumor growth inhibition at low mg/kg mAb doses. Tolerability in the rat at multiple doses was determined, including histopathological evaluation. Dolasynthen ADCs showed excellent PK characteristics in mouse, rat and NHP. Overall, the Dolasynthen platform appears to offer significant potential for clinical application. Citation Format: Dorin Toader, Marc Damelin, Anouk Dirksen, Shawn P. Fesler, Scott D. Collins, Barrett J. Nehilla, Jian Xu, Ling Xu, Kalli C. Cattcott, Alex Uttard, Winnie Lee, Susan Clardy, Cheri A. Stevenson, LiuLiang Qin, Patrick R. Conlon, Mariya V. Kozytska, Chen-Ni Chin, David H. Lee, Timothy B. Lowinger. Dolasynthen–a novel, homogeneous Auristatin F hydroxypropyl amide antibody-drug conjugate platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2687.

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 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