Robert J. Lutz

Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate.

HER2 is a validated target in breast cancer therapy. Two drugs are currently approved for HER2-positive breast cancer: trastuzumab (Herceptin), introduced in 1998, and lapatinib (Tykerb), in 2007. Despite these advances, some patients progress through therapy and succumb to their disease. A variation on antibody-targeted therapy is utilization of antibodies to deliver cytotoxic agents specifically to antigen-expressing tumors. We determined in vitro and in vivo efficacy, pharmacokinetics, and toxicity of trastuzumab-maytansinoid (microtubule-depolymerizing agents) conjugates using disulfide and thioether linkers. Antiproliferative effects of trastuzumab-maytansinoid conjugates were evaluated on cultured normal and tumor cells. In vivo activity was determined in mouse breast cancer models, and toxicity was assessed in rats as measured by body weight loss. Surprisingly, trastuzumab linked to DM1 through a nonreducible thioether linkage (SMCC), displayed superior activity compared with unconjugated trastuzumab or trastuzumab linked to other maytansinoids through disulfide linkers. Serum concentrations of trastuzumab-MCC-DM1 remained elevated compared with other conjugates, and toxicity in rats was negligible compared with free DM1 or trastuzumab linked to DM1 through a reducible linker. Potent activity was observed on all HER2-overexpressing tumor cells, whereas nontransformed cells and tumor cell lines with normal HER2 expression were unaffected. In addition, trastuzumab-DM1 was active on HER2-overexpressing, trastuzumab-refractory tumors. In summary, trastuzumab-DM1 shows greater activity compared with nonconjugated trastuzumab while maintaining selectivity for HER2-overexpressing tumor cells. Because trastuzumab linked to DM1 through a nonreducible linker offers improved efficacy and pharmacokinetics and reduced toxicity over the reducible disulfide linkers evaluated, trastuzumab-MCC-DM1 was selected for clinical development.

Expression of bbc3, a pro-apoptotic BH3-only gene, is regulated by diverse cell death and survival signals

BH3-only proteins function at a proximal point in a conserved cell death pathway by binding, through their BH3 domains, to other Bcl-2 family members and triggering mitochondrial events associated with apoptosis. Here, we describe a strongly pro-apoptotic BH3-only protein, designated Bbc3, whose expression increases in response to diverse apoptotic stimuli. bbc3 mRNA levels were induced by exposure to DNA-damaging agents and by wild-type p53, which mediates DNA damage-induced apoptosis. p53 transactivated bbc3 through consensus p53 binding sites within the bbc3 promoter region, indicating that bbc3 is a direct target of p53. Additionally, bbc3 mRNA was induced by p53-independent apoptotic stimuli, including dexamethasone treatment of thymocytes, and serum deprivation of tumor cells. Insulin-like growth factor-1 and epidermal growth factor, growth factors with broad anti-apoptotic activity, were each sufficient to suppress Bbc3 expression in serum-starved tumor cells. These results suggest that the transcriptional regulation of bbc3 contributes to the transduction of diverse cell death and survival signals.

BAK BH3 PEPTIDES ANTAGONIZE BCL-XL FUNCTION AND INDUCE APOPTOSIS THROUGH CYTOCHROME C-INDEPENDENT ACTIVATION OF CASPASES

The Bcl-2 homology 3 (BH3) domain is crucial for the death-inducing and dimerization properties of pro-apoptotic members of the Bcl-2 protein family, including Bak, Bax, and Bad. Here we report that synthetic peptides corresponding to the BH3 domain of Bak bind to Bcl-xL, antagonize its anti-apoptotic function, and rapidly induce apoptosis when delivered into intact cells via fusion to the Antennapedia homeoprotein internalization domain. Treatment of HeLa cells with the Antennapedia-BH3 fusion peptide resulted in peptide internalization and induction of apoptosis within 2–3 h, as indicated by caspase activation and subsequent poly(ADP-ribose) polymerase cleavage, as well as morphological characteristics of apoptosis. A point mutation within the BH3 peptide that blocks its ability to bind to Bcl-xL abolished its apoptotic activity, suggesting that interaction of the BH3 peptide with Bcl-2-related death suppressors, such as Bcl-xL, may be critical for its activity in cells. While overexpression of Bcl-xL can block BH3-induced apoptosis, treatment with BH3 peptides resensitized Bcl-xL-expressing cells to Fas-mediated apoptosis. BH3-induced apoptosis was blocked by caspase inhibitors, demonstrating a dependence on caspase activation, but was not accompanied by a dramatic early loss of mitochondrial membrane potential or detectable translocation of cytochrome c from mitochondria to cytosol. These findings demonstrate that the BH3 domain itself is capable of inducing apoptosis in whole cells, possibly by antagonizing the function of Bcl-2-related death suppressors.

Growth Factors Inactivate the Cell Death Promoter BAD by Phosphorylation of Its BH3 Domain on Ser155

The Bcl-2 family protein BAD promotes apoptosis by binding through its BH3 domain to Bcl-xL and related cell death suppressors. When BAD is phosphorylated on either Ser112 or Ser136, it forms a complex with 14-3-3 in the cytosol and no longer interacts with Bcl-xLat the mitochondria. Here we show that phosphorylation of a distinct site Ser155, which is at the center of the BAD BH3 domain, directly suppressed the pro-apoptotic function of BAD by eliminating its affinity for Bcl-xL. Protein kinase A functioned as a BAD Ser155 kinase both in vitro and in cells. BAD Ser155 was found to be a major site of phosphorylation induced following stimulation by growth factors and prevented by protein kinase A inhibitors but not by inhibitors of the phosphatidylinositol 3-kinase/Akt pathway. Growth factors inhibited BAD-induced apoptosis in both a Ser112/Ser136- and a Ser155-dependent fashion. Thus, growth factors engage an anti-apoptotic signaling pathway that inactivates BAD by direct modification of its BH3 cell death effector domain.

Antibody-Maytansinoid Conjugates Designed to Bypass Multidrug Resistance

Conjugation of cytotoxic compounds to antibodies that bind to cancer-specific antigens makes these drugs selective in killing cancer cells. However, many of the compounds used in such antibody-drug conjugates (ADC) are substrates for the multidrug transporter MDR1. To evade the MDR1-mediated resistance, we conjugated the highly cytotoxic maytansinoid DM1 to antibodies via the maleimidyl-based hydrophilic linker PEG(4)Mal. Following uptake into target cells, conjugates made with the PEG(4)Mal linker were processed to a cytotoxic metabolite that was retained by MDR1-expressing cells better than a metabolite of similar conjugates prepared with the nonpolar linker N-succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). In accord, PEG(4)Mal-linked conjugates were more potent in killing MDR1-expressing cells in culture. In addition, PEG(4)Mal-linked conjugates were markedly more effective in eradicating MDR1-expressing human xenograft tumors than SMCC-linked conjugates while being tolerated similarly, thus showing an improved therapeutic index. This study points the way to the development of ADCs that bypass multidrug resistance.

Disulfide-Linked Antibody−Maytansinoid Conjugates: Optimization of In Vivo Activity by Varying the Steric Hindrance at Carbon Atoms Adjacent to the Disulfide Linkage

In this report, we describe the synthesis of a panel of disulfide-linked huC242 (anti-CanAg) antibody maytansinoid conjugates (AMCs), which have varying levels of steric hindrance around the disulfide bond, in order to investigate the relationship between stability to reduction of the disulfide linker and antitumor activity of the conjugate in vivo. The conjugates were first tested for stability to reduction by dithiothreitol in vitro and for plasma stability in CD1 mice. It was found that the conjugates having the more sterically hindered disulfide linkages were more stable to reductive cleavage of the maytansinoid in both settings. When the panel of conjugates was tested for in vivo efficacy in two human colon cancer xenograft models in SCID mice, it was found that the conjugate with intermediate disulfide bond stability having two methyl groups on the maytansinoid side of the disulfide bond and no methyl groups on the linker side of the disulfide bond (huC242-SPDB-DM4) displayed the best efficacy. The ranking of in vivo efficacies of the conjugates was not predicted by their in vitro potencies, since all conjugates were highly active in vitro, including a huC242-SMCC-DM1 conjugate with a noncleavable linkage which showed only marginal activity in vivo. These data suggest that factors in addition to intrinsic conjugate potency and conjugate half-life in plasma influence the magnitude of antitumor activity observed for an AMC in vivo. We provide evidence that bystander killing of neighboring nontargeted tumor cells by diffusible cytotoxic metabolites produced from target cell processing of disulfide-linked antibody-maytansinoid conjugates may be one additional factor contributing to the activity of these conjugates in vivo.

SAR3419: An Anti-CD19-Maytansinoid Immunoconjugate for the Treatment of B-Cell Malignancies

SAR3419 is a novel anti-CD19 humanized monoclonal antibody conjugated to a maytansine derivate through a cleavable linker for the treatment of B-cell malignancies. SAR3419 combines the strengths of a high-potency tubulin inhibitor and the exquisite B-cell selectivity of an anti-CD19 antibody. The internalization and processing of SAR3419, following its binding at the surface of CD19-positive human lymphoma cell lines and xenograft models, release active metabolites that trigger cell-cycle arrest and apoptosis, leading to cell death and tumor regression. SAR3419 has also been shown to be active in different lymphoma xenograft models, including aggressive diffuse large B-cell lymphoma, resulting in complete regressions and tumor-free survival. In these models, the activity of SAR3419 compared favorably with rituximab and lymphoma standard of care chemotherapy. Two phase I trials with 2 different schedules of SAR3419 as a single agent were conducted in refractory/relapsed B-cell non-Hodgkin lymphoma. Activity was reported in both schedules, in heavily pretreated patients of both follicular and diffuse large B-cell lymphoma subtypes, with a notable lack of significant hematological toxicity, validating SAR3419 as an effective antibody-drug conjugate and opening opportunities in the future. Numerous B-cell–specific anti-CD19 biologics are available to treat B-cell non-Hodgkin lymphoma, and early phase I results obtained with SAR3419 suggest that it is a promising candidate for further development in this disease. In addition, thanks to the broad expression of CD19, SAR3419 may provide treatment options for B-cell leukemias that are often CD20-negative. Clin Cancer Res; 17(20); 6448–58. ©2011 AACR.

αv Integrin-Targeted Immunoconjugates Regress Established Human Tumors in Xenograft Models

Purpose: Targeted delivery of cytotoxic agents to solid tumors through cell surface antigens can potentially reduce systemic toxicity and increase the efficacy of the targeted compounds. The purpose of this study was to show the feasibility of treating solid tumors by targeting αv integrins with antibody-maytansinoid conjugates and to test the relative in vivo activities of several linker-maytansinoid chemistries. Experimental Design: CNTO 364, CNTO 365, and CNTO 366 are targeted cytotoxic agents created by conjugating the CNTO 95 anti-αv integrin antibody with three distinct maytansinoid-linker structures. These structures were designed to have varying degrees of chemical substitution surrounding the disulfide bond linking the cytotoxic agent to the antibody. A model conjugate was shown to be specifically cytotoxic in vitro and highly active against established human tumor xenografts in immunocompromised rats. The in vivo antitumor activities of CNTO 364, CNTO 365, and CNTO 366 were compared in rat xenograft models. Results: CNTO 365, with a linker chemistry of expected intermediate stability, was shown to be substantially more active than the other two conjugates with lesser or greater substitution around the disulfide linkage. Conclusion: CNTO 95–maytansinoid immunoconjugates are potent antitumor agents against αv integrin–expressing human carcinomas. These studies show for the first time the feasibility of targeting αv integrins on solid tumors with tumor-activated prodrugs. The DM4 linker-maytansinoid configuration of CNTO 365 was substantially more active in the models tested here when compared with alternative configurations with greater or lesser chemical substitution surrounding the linker.

Abstract 3719: Bicyclic peptides for PET imaging of MT1-MMP expressing tumors

The Bicycle® technology is based on repertoires of short peptides displayed on the surface of bacteriophages which can be modified with homo-trifunctional organochemical scaffolds, thus creating large diverse libraries of constrained, bicyclic peptides. These large combinatorial libraries have been extensively used for iterative selections to identify high affinity binding peptides to a wide array of targets, including receptors, interleukins and proteases. Bicyclic peptides are chemically synthesized macrocyclic entities with drug-like properties that exhibit sub-nanomolar affinities and exquisite selectivity towards targets. Unlike biologics, their synthetic nature allows facile modulation of metabolic and pharmacokinetic properties, as well as site-specific conjugation to effector molecules such as fluorophores, radionuclides, and cytotoxic drugs. In the present work, novel phage display derived bicyclic peptides were identified targeting the matrix metalloproteinase 14 (also known as MT1-MMP), a tumor associated surface protein overexpressed in a variety of cancers (i.e. lung, breast). A prototype bicyclic peptide with high affinity to MT1-MMP (Kd at ~1 nM) was identified, and confocal microscopy using fluoresceinated bicyclic peptide derivatives shows target-dependent internalisation in MT1-MMP+ cells. In the in vivo mouse, selective tumor binding in an MT1-MMP+ xenograft model is demonstrated for a DOTA conjugate loaded with Ga-68 or Lu-177. Upon proteolytic optimization of the prototype bicycle peptide, a striking enhancement in tumor signal is observed in biodistribution studies. Compared to radiolabeled antibodies directed against the same target, the lead compound showed fast background clearance ( Together, tumor targeting bicyclic peptides can, through their small size and high selectivity, facilitate efficient penetration and visualization of tumors in vivo, demonstrating their potential as diagnostic imaging agents in profiling and therapeutic management of patients. Citation Format: Daniel Teufel, Helen Harrison, Spencer Campbell, Catherine Stace, Edward Walker, Robert J. Lutz, Peter Park, Matthias Eder, Ulrike Bauder-Wust, Ursula Schierbaum, Karin Leotta, Klaus Kopka, Uwe Haberkorn. Bicyclic peptides for PET imaging of MT1-MMP expressing tumors [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 3719. doi:10.1158/1538-7445.AM2017-3719

Abstract 5144: BT1718, a novel bicyclic peptide-maytansinoid conjugate targeting MT1-MMP for the treatment of solid tumors: Design of bicyclic peptide and linker selection

Bicycles® are novel binding agents comprising small bicyclic peptides (1.5-3 KDa) constrained via a chemical scaffold, selected for high affinity and selectivity to targets of interest. MT1 (MMP14/MT1-MMP) is a membrane-associated metalloprotease overexpressed in many solid tumours and is implicated in tumor invasion and metastasis. MT1 expression positively correlates with poor prognosis. Phage libraries containing 1015 unique peptide sequences were post-translationally cyclized with thiol-reactive scaffold and used in an optimized, high-throughput selection process to identify Bicycles® to the hemopexin domain of MT1. Additional iterative rounds of directed phage based screening were used to optimize affinity and off-phage non-natural amino acids were introduced at select positions to improve plasma stability to generate the lead Bicycle binder. The lead anti-MT1 Bicycle was further modified with a sarcosyl spacer to form N241. N241 binds specifically to the hemopexin domain of MT1 with a Kd of approximately 2 nM with no binding observed to the catalytic domain of the protease nor to any of the related MMP family members tested. Importantly and in contrast to most antibodies, N241 binds with similar affinity to MT1 from multiple species including rodent, dog and non-human primate. Since the expected rapid tumor penetration and specific binding of these small peptidyl-binders makes them ideal for use in targeted delivery approaches, a series of Bicycle drug conjugates (BDCs) were prepared; N241 was conjugated to potent maytansinoid cytotoxics via linkers which varied in their cleavability. Though all the BDCs maintained high affinity for MT1, efficacy toward MT1-positive human tumor mouse xenografts varied with linker stability. BDCs with the most stable linkers were the least active suggesting that optimal tumor activation was obtained with linkers that could be cleaved more rapidly. Due to the rapid clearance and limited systemic exposure of these small-targeting BDCs, only the most labile linker showed toxicity in the mouse studies. Of the BDCs tested, BT1718, composed of N241 and DM1 conjugated via the SPP linker, demonstrated an optimal therapeutic index. Potent anti-tumor efficacy with BT1718 was observed across a panel of MT1-positive xenografts with complete tumor regressions observed in most models at doses that were well tolerated. In one example, HT-1080 fibrosarcoma subcutaneous xenografts were intraveneously treated with BT1718 when the tumor size had reached approximately 180 mm3. BT1718 given at 3 mg/kg once a week resulted in tumor stasis while BT1718 given at 10 mg/kg once a week or 3 mg/kg twice a week induced complete regression. In summary, BT1718, a highly active, targeted drug conjugate with unique pharmacological properties is a promising therapeutic candidate for the treatment of MT1-MMP-positive solid tumors. Citation Format: Helen Harrison, Gavin Bennett, Diane Blakeley, Amy Brown, Spencer Campbell, Liuhong Chen, Robert J. Lutz, Silvia Pavan, Katerine van Rietschoten, Daniel Teufel, Peter U. Park, Kevin Lee. BT1718, a novel bicyclic peptide-maytansinoid conjugate targeting MT1-MMP for the treatment of solid tumors: Design of bicyclic peptide and linker selection [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 5144. doi:10.1158/1538-7445.AM2017-5144