Mechthild Jonas

Reducing hydrophobicity of homogeneous antibody-drug conjugates improves pharmacokinetics and therapeutic index

The in vitro potency of antibody-drug conjugates (ADCs) increases with the drug-to-antibody ratio (DAR); however, ADC plasma clearance also increases with DAR, reducing exposure and in vivo efficacy. Here we show that accelerated clearance arises from ADC hydrophobicity, which can be modulated through drug-linker design. We exemplify this using hydrophilic auristatin drug linkers and PEGylated ADCs that yield uniform, high-DAR ADCs with superior in vivo performance.

Intracellular Released Payload Influences Potency and Bystander-Killing Effects of Antibody-Drug Conjugates in Preclinical Models

Antibody-drug conjugates (ADC) comprise targeting antibodies armed with potent small-molecule payloads. ADCs demonstrate specific cell killing in clinic, but the basis of their antitumor activity is not fully understood. In this study, we investigated the degree to which payload release predicts ADC activity in vitro and in vivo ADCs were generated to target different receptors on the anaplastic large cell lymphoma line L-82, but delivered the same cytotoxic payload (monomethyl auristatin E, MMAE), and we found that the intracellular concentration of released MMAE correlated with in vitro ADC-mediated cytotoxicity independent of target expression or drug:antibody ratios. Intratumoral MMAE concentrations consistently correlated with the extent of tumor growth inhibition in tumor xenograft models. In addition, we developed a robust admixed tumor model consisting of CD30(+) and CD30(-) cancer cells to study how heterogeneity of target antigen expression, a phenomenon often observed in cancer specimens, affects the treatment response. CD30-targeting ADC delivering membrane permeable MMAE or pyrrolobenzodiazepine dimers demonstrated potent bystander killing of neighboring CD30(-) cells. In contrast, a less membrane permeable payload, MMAF, failed to mediate bystander killing in vivo, suggesting local diffusion and distribution of released payloads represents a potential mechanism of ADC-mediated bystander killing. Collectively, our findings establish that the biophysical properties and amount of released payloads are chief factors determining the overall ADC potency and bystander killing. Cancer Res; 76(9); 2710-9. ©2016 AACR.

Anti-leukemic activity of lintuzumab (SGN-33) in preclinical models of acute myeloid leukemia.

Despite therapeutic advances, the long-term survival rates for acute myeloid leukemia (AML) are estimated to be 10% or less, pointing to the need for better treatment options. AML cells express the myeloid marker CD33, making it amenable to CD33-targeted therapy. Thus, the in vitro and in vivo anti-tumor activities of lintuzumab (SGN-33), a humanized monoclonal anti-CD33 antibody undergoing clinical evaluation, were investigated. In vitro assays were used to assess the ability of lintuzumab to mediate effector functions and to decrease the production of growth factors from AML cells. SCID mice models of disseminated AML with the multi-drug resistance (MDR)-negative HL60 and the MDR+, HEL9217 and TF1-α, cell lines were developed and applied to examine the in vivo antitumor activity. In vitro, lintuzumab significantly reduced the production of TNF-α-induced pro-inflammatory cytokines and chemokines by AML cells. Lintuzumab promoted tumor cell killing through antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) activities against MDR- and MDR+ AML cell lines and primary AML patient samples. At doses from 3 to 30 mg/kg, lintuzumab significantly enhanced survival and reduced tumor burden in vivo, regardless of MDR status. Survival of the mice was dependent upon the activity of resident macrophages and neutrophils. The results suggest that lintuzumab may exert its therapeutic effects by modulating the cytokine milieu in the tumor microenvironment and through effector mediated cell killing. Given that lintuzumab induced meaningful responses in a phase 1 clinical trial, the preclinical antitumor activities defined in this study may underlie its observed therapeutic efficacy in AML patients.

Abstract 1195: SGN-CD352A: A novel humanized anti-CD352 antibody-drug conjugate for the treatment of multiple myeloma

Multiple myeloma (MM) is a hematologic malignancy of transformed plasma cells. In spite of recent advances, MM remains an incurable disease, underscoring the need to develop new targeted biological therapeutics to augment existing treatments. In this study we describe SGN-CD352A, a potent new CD352-targeting antibody-drug conjugate (ADC) under development for the treatment of MM. CD352, or SLAMF6 (Signaling Lymphocyte Activation Molecule family member 6), is a type 1 membrane protein in the SLAM family of immunoreceptors. Like other SLAM family members, CD352 is a positive regulator of natural killer (NK) cell functions. CD352 is also a tumor antigen expressed on B cell malignancies such as MM, chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL). We observed CD352 expression on the surface of malignant plasma cells in 87% (13/15) of human multiple myeloma patient samples examined by flow cytometry. Monoclonal antibodies (mAbs) specific for human CD352 were produced and a lead antibody was selected based on affinity, endocytic internalization rate, and tumor cell cytotoxic activity as an ADC. SGN-CD352A is a humanized anti-CD352 engineered cysteine (ec) mAb (h20F3ec) to which two molecules of pyrrolobenzodiazepine (PBD) dimer, a potent DNA damaging cytotoxic drug, have been conjugated. Upon binding CD352 at the MM cell surface, SGN-CD352A undergoes rapid clathrin-dependent endocytosis ( Citation Format: Tim Lewis, Devra J. Olson, Kristine A. Gordon, Sharsti L. Sandall, Jamie Miyamoto, Lori Westendorf, Germein Linares, Chris Leiske, Heather Kostner, Ivan Stone, Martha Anderson, Albina Nesterova, Mechthild Jonas, Che-Leung Law. SGN-CD352A: A novel humanized anti-CD352 antibody-drug conjugate for the treatment of multiple myeloma. [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 1195.

Characterization of SGN-CD123A, A Potent CD123-Directed Antibody–Drug Conjugate for Acute Myeloid Leukemia

Treatment choices for acute myelogenous leukemia (AML) patients resistant to conventional chemotherapies are limited and novel therapeutic agents are needed. IL3 receptor alpha (IL3Rα, or CD123) is expressed on the majority of AML blasts, and there is evidence that its expression is increased on leukemic relative to normal hematopoietic stem cells, which makes it an attractive target for antibody-based therapy. Here, we report the generation and preclinical characterization of SGN-CD123A, an antibody–drug conjugate using the pyrrolobenzodiazepine dimer (PBD) linker and a humanized CD123 antibody with engineered cysteines for site-specific conjugation. Mechanistically, SGN-CD123A induces activation of DNA damage response pathways, cell-cycle changes, and apoptosis in AML cells. In vitro, SGN-CD123A–mediated potent cytotoxicity of 11/12 CD123+ AML cell lines and 20/23 primary samples from AML patients, including those with unfavorable cytogenetic profiles or FLT3 mutations. In vivo, SGN-CD123A treatment led to AML eradication in a disseminated disease model, remission in a subcutaneous xenograft model, and significant growth delay in a multidrug resistance xenograft model. Moreover, SGN-CD123A also resulted in durable complete remission of a patient-derived xenograft AML model. When combined with a FLT3 inhibitor quizartinib, SGN-CD123A enhanced the activity of quizartinib against two FLT3-mutated xenograft models. Overall, these data demonstrate that SGN-CD123A is a potent antileukemic agent, supporting an ongoing trial to evaluate its safety and efficacy in AML patients (NCT02848248). Mol Cancer Ther; 17(2); 554–64. ©2017 AACR.

Abstract 2647: SGN-CD70A, a novel and highly potent anti-CD70 ADC, induces double-strand DNA breaks and is active in models of MDR+ renal cell carcinoma (RCC) and non-Hodgkin lymphoma (NHL)

CD70 is a member of the tumor necrosis factor superfamily that is aberrantly expressed in several solid tumors and hematologic malignancies, including clear cell and papillary renal cell carcinoma (RCC) and non-Hodgkin lymphoma (NHL). Normal expression of CD70 is limited to stromal cells of the thymic medulla, mature dendritic cells, and activated B and T lymphocytes. Thus, CD70 is an attractive target for antibody-drug conjugate (ADC) based therapy. Using a panel of CD70 positive RCC and lymphoma cell lines and xenograft models, we have previously demonstrated the antitumor activity of SGN-CD70A, a novel ADC that combines a CD70-directed engineered cysteine monoclonal antibody (h1F6ec) with a highly potent, synthetic DNA cross-linking molecule, pyrrolobenzodiazepine (PBD) dimer. The strength of these results led us to develop SGN-CD70A for clinical evaluation in RCC and lymphoma. In this report, we examine the mechanism of action for SGN-CD70A and demonstrate that the formation of double strand breaks (DSB) is an early event that precedes onset of cytotoxicity in RCC and NHL cell lines. SGN-CD70A is more potent than auristatin-based CD70 ADCs in vitro and in xenograft models, including those that are MDR positive, suggesting that the PBD chemotype may overcome common resistance mechanisms. To define the mechanism(s) of targeted cytotoxicity, we examined DNA damage pathways in Caki-1, 786-0 and UM-RC-3 (RCC, MDR+) and Raji and MHH-PREB-1 (NHL) cell lines. We utilized an immunofluorescence assay to monitor DNA damage foci using antibodies specific to the tumor suppressor p53-binding protein 1 (53BP1), Meiotic recombination 11 homolog (Mre11), and Rad50. Increased amounts of foci were observed within 6 hours of treatment with 2nM PBD to levels observed in cells exposed to 10Gy of ionizing radiation. Similarly, foci were found in SGN-CD70A-treated cells. Further evidence of damage was the co-localization of phosphorylated histone H2A.X (Ser139) to the damage foci and an increase in levels of both phosphorylated Chk1 (Ser317/345) and Chk2 (Thr68) within 4 hours of treatment. The levels of both pChk1 and pChk2 continue to increase after treatment, with peaks at 24-48 hours for pChk1 and 48-72 hours for pChk2. Concomitant, we also observed an increase in both phosphorylated ATM and phosphorylated BRCA1, confirming that SGN-CD70A in vitro activates double strand break response pathways. Ongoing research is examining DNA damage pathway activation in the corresponding xenograft models to confirm our in vitro findings. Furthermore, we are developing assays to examine pH2A.X, pChk1, and pChk2 as potential biomarkers for clinical studies with SGN-CD70A. Citation Format: Sharsti Sandall, Martha Anderson, Mechthild Jonas, Albina Nesterova, Jamie Miyamoto, Ivan J. Stone, Weiping Zeng, Che-Leung Law, Timothy S. Lewis. SGN-CD70A, a novel and highly potent anti-CD70 ADC, induces double-strand DNA breaks and is active in models of MDR+ renal cell carcinoma (RCC) and non-Hodgkin lymphoma (NHL). [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 2647. doi:10.1158/1538-7445.AM2014-2647

Abstract 3694: Relationship between in vivo antitumor activity of ADC and payload release in preclinical models

Antibody-drug conjugates (ADC) deliver cytotoxic payloads to target cells via receptor-mediated internalization. We have demonstrated that SGN-35 (cAC10-vcMMAE) binds to CD30 on target cells and releases intracellular monomethyl auristatin E (MMAE) in vitro, contributing to potent killing of CD30+ cells. Using a set of auristatin-based ADCs, we evaluated whether the intratumoral concentration of the released payload is a predictive indicator of antitumor activity in preclinical models. We first treated CD30+, anaplastic large cell lymphoma (ALCL) Karpas 299 tumors with SGN-35 or a non-binding control ADC (IgG-vcMMAE). The plasma ADC and MMAE PK measurements were similar in SGN-35 and IgG-vcMMAE treated tumor-bearing animals. In contrast, SGN-35 generated 5-fold greater intratumoral MMAE exposure than IgG-vcMMAE, which correlated with tumor regression observed in the SGN-35- treated group. We then examined the relationship between released MMAE and in vitro cytotoxicity by targeting different receptors expressed on the same cells. The lymphoma cell line L82 expresses CD30, CD70, and CD71 receptors. Although auristatin-based ADCs targeting each of these targets has a different IC50, the intracellular MMAE was similar when 50% growth inhibition was achieved (approximately 200 nM). In vivo, CD30, CD70, or CD71-directed ADCs demonstrated similar antitumor activity for a given dose level (0.5, 1, or 3 mg/kg). Concordantly, equal intratumoral MMAE concentrations were observed for a given dose level, regardless of the antigen target. The cell permeable MMAE has been shown to mediate bystander killing in vitro. To evaluate whether intratumoral MMAE mediates bystander killing in vivo, an admixed tumor model consisting CD30+ and CD30- Karpas 299 cells was treated with SGN-35. As predicted, both CD30+ and CD30- tumor cells were killed, presumably due to intratumoral diffusion of MMAE across cell membranes. In contrast, a conjugate with a highly polar form of auristatin (MMAF) was unable to kill the CD30-negative cell population of the admixed tumors. These preclinical results provide evidence that the concentration of released MMAE within the tumor correlates with the ADC antitumor activity in vitro and in vivo. Moreover, the in vivo bystander activity of MMAE may improve utility of this chemotype in tumors with heterogeneous antigen expression. Citation Format: Fu Li, Xinqun Zhang, Kim Emmerton, Mechthild Jonas, Jocelyn Setter, Bill Arthur, Nicole Okeley, Robert Lyon, Dennis Benjamin, Che-Leung Law. Relationship between in vivo antitumor activity of ADC and payload release in preclinical models. [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 3694. doi:10.1158/1538-7445.AM2014-3694

Abstract 625: Preclinical characterization of an auristatin-based anti-CD19 drug conjugate, SGN-19A

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL CD19 has multiple attributes that make it an attractive target for an antibody-drug conjugate (ADC). CD19 is uniformly expressed on the cell surface of almost all malignancies of B cell origin, including non-Hodgkin lymphoma (NHL), B cell precursor acute lymphoblastic leukemia (B-ALL), and chronic lymphocytic leukemia (CLL). In addition, CD19 has very limited normal tissue expression, restricted only to B lymphocytes and their precursors, and it internalizes rapidly, which are favorable characteristic for targeted drug delivery. SGN-19A is an ADC comprised of a humanized anti-CD19 mAb and the potent cytotoxic drug-linker maleimidocaproyl monomethyl auristatin F (mcMMAF). Here we demonstrate internalization and trafficking, in vivo antitumor activity, and in vivo on-target pharmacodynamic effect of B cell depletion following SGN-19A treatment. Internalization and trafficking of SGN-19A through the endosomal-lysosomal pathway resulted in potent in vitro cytotoxic activity against CD19+ cell lines derived from B-ALL, follicular lymphoma, diffused large B cell lymphoma, Burkitts lymphoma, and plasma cell leukemia. In mouse xenografts modeling different B-lineage malignancies, SGN-19A demonstrated antitumor activity including tumor regression and prolonged survival at doses well below its maximum tolerated dose. We also show that the in vivo antitumor activity is directly related to the duration of the pharmacokinetic half-life among a panel of ADCs targeting CD19. Since SGN-19A cross-reacts with its ortholog expressed in non-human primates, we examined its pharmacodynamic effect on B cell depletion in cynomolgus monkeys. Depletion of peripheral B cells was observed following SGN-19A and correlated with results from terminal immunohistochemical (IHC) analysis demonstrating the depletion of Ki67+ germinal center B cells. Furthermore, using an antibody against mcMMAF we confirmed delivery of the cytotoxic agent to the B cell areas in peripheral lymphoid tissues. Taken together, these results support the further evaluation of SGN-19A as a therapy for B-lineage derived malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 625. doi:10.1158/1538-7445.AM2011-625

Abstract 1285: Tumor associated macrophages can process antibody-drug conjugates and contribute to antitumor activity in preclinical xenograft models

The primary mechanism of antibody-drug conjugates (ADCs) is the targeted delivery of a cytotoxic payload via cancer antigen mediated internalization. However, stromal components in the tumor microenvironment may also play a role in ADC penetration, distribution, and processing. Here, we study the potential roles of Fc-FcγR interaction between tumor-associated macrophages (TAMs) and ADCs in the antitumor activity observed in xenograft models. In the CD30+ L428 Hodgkin lymphoma (HL) model, the anti-CD30 ADC (cAC10-vcMMAE) and a non-binding control ADC (h00-vcMMAE) showed similar antitumor activity over a two-week period post treatment initiation. The antitumor activity was correlated with payload release, as h00-vcMMAE produced intratumoral MMAE concentration comparable to cAC10-vcMMAE in this time frame. Histopathology analysis of L-428 xenografts revealed high abundance of TAMs, leading to the hypothesis that TAMs can internalize and process ADCs for payload release. Using immunohistochemistry and flow cytometry, we confirmed that h00-vcMMAE bound to macrophage cell lines and TAMs. We further examined the presence of TAMs in additional xenograft models and correlated that to the antitumor activity of non-binding h00-vcMMAE. High levels of TAMs were observed in the KM-H2 HL and BR620 breast cancer models that were sensitive to h00-vcMMAE. In contrast, xenograft models with much fewer TAMs (DOHH2, SU-DHL8, and Karpas-299) did not respond to h00-vcMMAE treatment. Interestingly, h00-vcMMAF, releasing a membrane non-permeable payload, had no activity on the L-428 tumor model. These data suggest TAM-processed drug can mediate bystander tumor killing when the released payload is membrane permeable. To evaluate whether Fc-FcγR interaction plays a role in the ADC uptake by TAMs, we mutated the Fc region of h00-vcMMAE to decrease FcγR binding affinity (E233P:L234V:L235, G1V1). h00G1V1-vcMMAE lost its cytotoxicity activity in FcγR+ THP-1 monocytes. Furthermore, h00G1V1-vcMMAE could no longer mediate tumor regression or growth delay in three xenograft models that are sensitive to h00-vcMMAE treatment. These results suggest ADC-FcγR interaction is required for ADC processing by TAMs in these xenografts. These results suggest that TAMs can contribute to ADC processing through FcγR interaction in preclinical tumor models. Moreover, TAM-processed membrane permeable payload can mediate bystander tumor cell killing and contribute to ADC activity. Although this phenomenon may be an additional mechanism of ADCs in vivo, whether TAMs play a role in patients’ response to ADCs requires further correlated studies in clinical trials. Citation Format: Fu Li, Michelle Ulrich, Mechthild Jonas, Germein Linares, Xinqun Zhang, Lori Westendorf, Dennis Benjamin, Che-Leung Law. Tumor associated macrophages can process antibody-drug conjugates and contribute to antitumor activity in preclinical 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 1285.

Abstract 351: Uptake of antibody-drug conjugates by cultured Kupffer cells can predict pharmacokinetics

Interest in antibody-drug conjugates (ADCs) has increased rapidly in the oncology field over the past several years. Recent advances in the ADC field have been achieved in part by an improved understanding of how conjugation with drug-linkers impacts the biophysical, pharmacokinetic (PK), and biodistribution properties of a monoclonal antibody. Most of these advances have been driven by in vivo preclinical observations, an inevitably low throughput endeavor. A higher throughput in vitro method that would allow some level of predictive power for ADC disposition in vivo would be highly advantageous, requiring less test article and allowing for much faster turnaround. Thus far no such methods for ADCs have been reported, in contrast to the situation for the prediction of the metabolic clearance of small molecule therapeutics, for which cultured hepatocytes are widely used as an in vitro tool. It has been generally assumed that clearance of ADCs is driven by cells of the monocyte phagocytic system, and we recently published immunohistochemistry (IHC) data implicating hepatic sinusoidal endothelium and the resident liver macrophages, Kupffer cells (KCs), in the accelerated clearance of highly-loaded ADCs. We sought to use this observation to develop a convenient assay that might correlate ADC uptake in vitro with in vivo PK. Initial fluorescent microscopy experiments revealed that cultured rat KCs stain intensely when incubated with fluorescently labeled ADCs, but much less so with unconjugated antibody, similar to the observed IHC results. In an effort to further improve the throughput of this method to allow the rapid comparison of many ADCs, we moved to a FACS-based platform to quantify the amount of KC-associated fluorescent conjugate. To quantify the effect of different drug-linkers, we prepared homogeneous ADCs with 8 drugs per antibody and observed that the KC staining intensity varied widely depending upon the characteristics of the drug-linker that was conjugated. KCs incubated with ADCs loaded with vc-PAB-MMAE had a mean fluorescent intensity (MFI) >20 fold higher than unconjugated Antibody. Additionally, ADCs loaded with MMAE that contain a polyethylene glycol (PEG) masking moiety in the linker had a much lower MFI than those without PEG. This FACS method allowed us to compare fluorescent uptake by KCs with ADC clearance determined in vivo. We have observed that ADC uptake by cultured KCs in vitro correlates well with ADC PK, allowing for more rapid assessment of the PK impact of new drug classes, drug-linker designs, and conjugation methodologies. Citation Format: David Meyer, Sara Shum, Mechthild Jonas, Martha Anderson, Joshua Hunter, Nagendra Chemuturi, Nicole Okeley, Robert Lyon. Uptake of antibody-drug conjugates by cultured Kupffer cells can predict pharmacokinetics. [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 351.