Maureen Dougher

A general approach to site-specific antibody drug conjugates

Significance Here we demonstrate the ability to genetically incorporate nonnative amino acids into proteins in mammalian cells using both transient and stable platform expression systems that provide yields and fidelities compatible with commercial applications. To illustrate the utility of this methodology we have generated chemically homogeneous antibody drug conjugates (NDCs) with precise control over the site and stoichiometry of drug conjugation. In rodent xenograft models these NDCs display improved properties, including half-life, efficacy and safety, relative to conventional heterogeneous ADCs. These advances allow the generation of therapeutic antibody drug conjugates with medicinal chemistry like control over structure, which should greatly facilitate the optimization of their pharmacological activities. Using an expanded genetic code, antibodies with site-specifically incorporated nonnative amino acids were produced in stable cell lines derived from a CHO cell line with titers over 1 g/L. Using anti-5T4 and anti-Her2 antibodies as model systems, site-specific antibody drug conjugates (NDCs) were produced, via oxime bond formation between ketones on the side chain of the incorporated nonnative amino acid and hydroxylamine functionalized monomethyl auristatin D with either protease-cleavable or noncleavable linkers. When noncleavable linkers were used, these conjugates were highly stable and displayed improved in vitro efficacy as well as in vivo efficacy and pharmacokinetic stability in rodent models relative to conventional antibody drug conjugates conjugated through either engineered surface-exposed or reduced interchain disulfide bond cysteine residues. The advantages of the oxime-bonded, site-specific NDCs were even more apparent when low–antigen-expressing (2+) target cell lines were used in the comparative studies. NDCs generated with protease-cleavable linkers demonstrated that the site of conjugation had a significant impact on the stability of these rationally designed prodrug linkers. In a single-dose rat toxicology study, a site-specific anti-Her2 NDC was well tolerated at dose levels up to 90 mg/kg. These experiments support the notion that chemically defined antibody conjugates can be synthesized in commercially relevant yields and can lead to antibody drug conjugates with improved properties relative to the heterogeneous conjugates formed by nonspecific chemical modification.

Antitumor Efficacy of a Combination of CMC-544 (Inotuzumab Ozogamicin), a CD22-Targeted Cytotoxic Immunoconjugate of Calicheamicin, and Rituximab against Non-Hodgkin's B-Cell Lymphoma

Purpose: CMC-544 is a CD22-targeted cytotoxic immunoconjugate, currently being evaluated in B-cell non-Hodgkins lymphoma (B-NHL) patients. Rituximab is a CD20-targeted antibody commonly used in B-NHL therapy. Here, we describe antitumor efficacy of a combination of CMC-544 and rituximab against B-cell lymphoma (BCL) in preclinical models. Experimental Design: BCLs were cultured in vitro with CMC-544, rituximab, or their combination. BCLs were injected either s.c. or i.v. to establish localized s.c. BCL in nude mice or disseminated BCL in severe combined immunodeficient mice, respectively. I.p. treatment with CMC-544 or rituximab was initiated at various times either alone or in combination and its effect on s.c. BCL growth or survival of mice with disseminated BCL was monitored. Results:In vitro growth-inhibitory activity of CMC-544 combined with rituximab was additive. Rituximab but not CMC-544 exhibited effector functions, such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Rituximab was less effective in inhibiting growth of established BCL xenografts than developing xenografts. In contrast, CMC-544 was equally effective against both developing and established BCL xenografts. Although CMC-544 and rituximab individually caused partial inhibition of the growth of BCL xenografts at suboptimal doses examined, their combination suppressed xenograft growth by >90%. In a disseminated BCL model, 60% of CMC-544-treated mice and 20% of rituximab-treated mice survived for 125 days. In contrast, 90% of mice treated with the combination of CMC-544 and rituximab survived for longer than 125 days. Conclusion: The demonstration of superior antitumor activity of a combination of CMC-544 and rituximab described here provides the preclinical basis for its clinical evaluation as a treatment option for B-NHL.

Potent and Specific Antitumor Efficacy of CMC-544, a CD22-Targeted Immunoconjugate of Calicheamicin, against Systemically Disseminated B-Cell Lymphoma

Purpose: CMC-544 is a CD22-targeted immunoconjugate of calicheamicin and exerts a potent cytotoxic effect against CD22+ B-cell lymphoma. This study evaluated antitumor efficacy of CMC-544 against systemically disseminated B-cell lymphoma. Experimental Design: Scid mice received i.v. injections of CD22+ Ramos B-cell lymphoma cells for their systemic dissemination. CMC-544, G5/44, CD33-targeted CMA-676 (control conjugate) or rituximab were given i.p. 3, 9, 15, or 21 days after B-cell lymphoma dissemination. Diseased mice were monitored daily for hind-limb paralysis and death. Histopathological examination of CMC-544-treated and vehicle-treated diseased mice was also performed. Results: Mice with disseminated B-cell lymphoma developed hind-limb paralysis within 35 days. When given up to 15 days after B-cell lymphoma dissemination, CMC-544 extended survival of the diseased mice to >100 days, and these mice were considered cured. CMC-544 was efficacious when given during both the early initiation phase and the late established phase of the disease. A single dose of CMC-544 was effective in delaying the occurrence of hind-limb paralysis. In contrast, neither CMA-676 nor unconjugated G5/44 was effective. Rituximab was effective when given early in the disease process but not when the disease was established. Histopathological analysis revealed B-cell lymphoma infiltration in brain, spinal cord, bone marrow, and kidney in vehicle-treated but not in CMC-544–treated diseased mice. Consistent with its efficacy against the disseminated B-cell lymphoma, CMC-544 also caused regression of established Ramos B-cell lymphoma xenografts in scid mice. Conclusions: CMC-544 confers strong therapeutic activity against systemic disseminated B-cell lymphoma and protects mice from hind-limb paralysis and death. These results support clinical evaluation of CMC-544 in the treatment of CD22+ lymphoid malignancies.

Antibody-Targeted Chemotherapy with the Calicheamicin Conjugate hu3S193-N-Acetyl γ Calicheamicin Dimethyl Hydrazide Targets Lewisy and Eliminates Lewisy-Positive Human Carcinoma Cells and Xenografts

Purpose: Linking a cytotoxic anticancer drug to an antibody that recognizes a tumor-associated antigen can improve the therapeutic index of the drug. We asked whether a conjugate of the cytotoxic antibiotic N-acetyl γ calicheamicin dimethyl hydrazide (CalichDMH) and an antibody recognizing Lewisy (Ley) antigen could eliminate carcinomas that express Ley. Because Ley is highly expressed on carcinomas of colon, breast, lung, ovary, and prostate, a CalichDMH conjugate targeting Ley could provide a treatment option for various cancers. Experimental Design: The humanized anti-Ley antibody hu3S193 was conjugated to CalichDMH via the bifunctional AcBut linker. Selectivity and avidity of the conjugate (hu3S193-CalichDMH) for Ley-BSA or Ley+ cells was tested by BIAcore or flow cytometry. Cytotoxicity of hu3S193-CalichDMH was compared with toxicity of a control conjugate on monolayers of Ley+ and Ley− carcinoma cells. Inhibition of tumor growth by hu3S193-CalichDMH was assessed on three types of s.c. xenografts. Results: Hu3S193-CalichDMH had similar selectivity as hu3S193. The conjugate had lower affinity for Ley-BSA but not for Ley+ cells. When tested on monolayers of human Ley+ carcinoma cells, hu3S193-CalichDMH was more cytotoxic than a control conjugate. This difference in efficacy was not noted on Ley− cells. Efficacy of hu3S193-CalichDMH depended on the expression of Ley and on the sensitivity of the cells to CalichDMH. In vivo, hu3S193-CalichDMH inhibited growth of xenografted human gastric (N87), colon (LOVO), and prostate carcinomas (LNCaP). When used against N87 xenografts, hu3S193-CalichDMH arrested tumor growth for at least 100 days. Conclusion: Hu3S193-CalichDMH can specifically eliminate Ley+ tumors. These results support development of this conjugate for treatment of carcinomas.

Long-term Tumor Regression Induced by an Antibody–Drug Conjugate That Targets 5T4, an Oncofetal Antigen Expressed on Tumor-Initiating Cells

Antibody–drug conjugates (ADC) represent a promising therapeutic modality for the clinical management of cancer. We sought to develop a novel ADC that targets 5T4, an oncofetal antigen expressed on tumor-initiating cells (TIC), which comprise the most aggressive cell population in the tumor. We optimized an anti-5T4 ADC (A1mcMMAF) by sulfydryl-based conjugation of the humanized A1 antibody to the tubulin inhibitor monomethylauristatin F (MMAF) via a maleimidocaproyl linker. A1mcMMAF exhibited potent in vivo antitumor activity in a variety of tumor models and induced long-term regressions for up to 100 days after the last dose. Strikingly, animals showed pathologic complete response in each model with doses as low as 3 mg antibody/kg dosed every 4 days. In a non–small cell lung cancer patient-derived xenograft model, in which 5T4 is preferentially expressed on the less differentiated tumor cells, A1mcMMAF treatment resulted in sustained tumor regressions and reduced TIC frequency. These results highlight the potential of ADCs that target the most aggressive cell populations within tumors, such as TICs. In exploratory safety studies, A1mcMMAF exhibited no overt toxicities when administered to cynomolgus monkeys at doses up to 10 mg antibody/kg/cycle × 2 and displayed a half-life of 5 days. The preclinical efficacy and safety data established a promising therapeutic index that supports clinical testing of A1mcMMAF. Mol Cancer Ther; 12(1); 38–47. ©2012 AACR.

Delineation of a Cellular Hierarchy in Lung Cancer Reveals an Oncofetal Antigen Expressed on Tumor-Initiating Cells

Poorly differentiated tumors in non-small cell lung cancer (NSCLC) have been associated with shorter patient survival and shorter time to recurrence following treatment. Here, we integrate multiple experimental models with clinicopathologic analysis of patient tumors to delineate a cellular hierarchy in NSCLC. We show that the oncofetal protein 5T4 is expressed on tumor-initiating cells and associated with worse clinical outcome in NSCLC. Coexpression of 5T4 and factors involved in the epithelial-to-mesenchymal transition were observed in undifferentiated but not in differentiated tumor cells. Despite heterogeneous expression of 5T4 in NSCLC patient-derived xenografts, treatment with an anti-5T4 antibody-drug conjugate resulted in complete and sustained tumor regression. Thus, the aggressive growth of heterogeneous solid tumors can be blocked by therapeutic agents that target a subpopulation of cells near the top of the cellular hierarchy.

CD20-specific antibody-targeted chemotherapy of non-Hodgkin’s B-cell lymphoma using calicheamicin-conjugated rituximab

Tumor-targeted delivery of a potent cytotoxic agent, calicheamicin, using its immunoconjugates is a clinically validated therapeutic strategy. Rituximab is a human CD20-specific chimeric antibody extensively used in B-NHL therapy. We investigated whether conjugation to calicheamicin can improve the anti-tumor activity of rituximab against human B-cell lymphoma (BCL) xenografts in preclinical models. BCL cells were cultured with rituximab or its calicheamicin conjugates and their in vitro growth was monitored. BCL cells were injected s.c. to establish localized xenografts in nude mice or i.v. to establish disseminated BCL in severe combined immunodeficient (scid) mice. I.p. treatment with rituximab or its calicheamicin conjugates was initiated and its effect on s.c. BCL growth or survival of mice with disseminated BCL was monitored. Conjugation of calicheamicin to rituximab vastly enhanced its growth inhibitory activity against BCL in vitro. Conjugation to calicheamicin had no deleterious effect on the effector functional activity of rituximab. Calicheamicin conjugated to rituximab with an acid-labile linker exhibited greater anti-tumor activity against s.c. BCL xenografts and improved survival of mice with disseminated BCL over that of unconjugated rituximab. Anti-tumor activities of rituximab conjugated to calicheamicin via an acid-stable linker were similar to that of unconjugated rituximab. Superior anti-tumor efficacy exhibited by a calicheamicin immunoconjugate of rituximab with an acid-labile linker over that of rituximab demonstrates the therapeutic potential of CD20-specific antibody-targeted chemotherapy strategy in the treatment of B-NHL.

Enhanced Antitumor Activity of an Anti-5T4 Antibody–Drug Conjugate in Combination with PI3K/mTOR inhibitors or Taxanes

Purpose: Targeted treatment of solid or liquid tumors with antibody–drug conjugates (ADCs) can lead to promising clinical benefit. The aim of the study is to investigate combination regimens of auristatin-based ADCs in preclinical models of cancer. Experimental Design: An auristatin-based anti-5T4 antibody conjugate (5T4-ADC) and auristatin payloads were combined with the dual PI3K/mTOR catalytic site inhibitor PF-05212384 (PF-384) or taxanes in a panel of tumor cell lines. Drug interactions in vitro were evaluated using cell viability assays, apoptosis induction, immunofluorescence, mitotic index, and immunoblotting. Breast cancer cells treated with auristatin analogue or 5T4-ADC were profiled by total- and phospho-proteomics. Antitumor efficacy of selected combinations was evaluated in 5T4-positive human breast or lung tumor xenografts in vivo. Results: In vitro, auristatin-based agents displayed strong synergistic or additive activity when combined with PF-384 or taxanes, respectively. Further, treatment of 5T4-ADC plus PF-384 resulted in stronger induction of apoptosis and cell line–specific attenuation of pAKT and pGSK. Interestingly, proteomic analysis revealed unique effects of auristatins on multiple components of mRNA translation. Addition of PF-384 further amplified effects of 5T4-ADC on translational components, providing a potential mechanism of synergy between these drugs. In human tumor xenografts, dual targeting with 5T4-ADC/PF-384 or 5T4-ADC/paclitaxel produced substantially greater antitumor effects with longer average survival as compared with monotherapy treatments. Conclusions: Our results provide a biologic rationale for combining 5T4-ADC with either PI3K/mTOR pathway inhibitors or taxanes and suggest that mechanisms underlying the synergy may be attributed to cellular effects of the auristatin payload. Clin Cancer Res; 22(2); 383–94. ©2015 AACR.

Abstract 3095: The development of CPI as a novel, next-generation DNA-targeting payload for ADCs

DNA targeting drugs represent one of cornerstones of anti-cancer therapy for both hematologic and solid tumor indications. Low potency anti-DNA compounds (e.g. platins, anthracyclines) are included in many standard-of-care (SOC) regimens, however their modest activity and overall toxicity profiles limit their therapeutic potential. To increase the therapeutic window for DNA-damaging agents, high potency anti-DNA compounds with enhanced anti-tumor activity have been delivered to tumors as payloads of targeting modalities such as antibody-drug conjugates (ADCs). Herein, we describe the development of a novel DNA-damaging compound comprised of a dimeric structure of cyclopropylpyrrolo[e]indolones (CPIs) that was designed to alkylate DNA and generate toxic interstrand crosslinks (ICLs). In response to the CPI-induced formation of ICLs, CPI treatment of cells primarily activates the Fancomia anemia DNA damage response pathway, whereas other successful DNA-damaging ADC payloads such as calicheamicin activate double-strand break response pathways. CPI shows ~860-fold greater potency than calicheamicin in a panel of cell lines derived from a broad spectrum of tumor indications. Importantly, this new CPI payload retains potent activity in calicheamicin- and SOC-resistant tumor models (including overcoming overexpression of drug efflux pumps). When evaluated as payloads on anti-CD33 targeting ADCs, the CPI conjugate showed dramatically improved efficacy over the corresponding calicheamicin conjugates in MDR+ tumor models. As a site-specific conjugate, the CPI ADC shows enhanced in vivo stability and possesses a wider therapeutic window than the corresponding conventional calicheamicin conjugate and other leading DNA-damaging conjugates on the CD33 platform. Citation Format: Jennifer Kahler, Maureen Dougher, Jane Xu, Matthew Doroski, Andreas Maderna, Russell Dushin, Stephane Thibault, Mauricio Leal, Madan Katragadda, Christopher J. O9Donnell, Matthew Sung, Puja Sapra. The development of CPI as a novel, next-generation DNA-targeting payload for ADCs [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 3095. doi:10.1158/1538-7445.AM2017-3095

Abstract 2530: Long-term tumor regression induced by a novel antibody drug conjugate that targets 5T4, an oncofetal antigen expressed on tumor-initiating cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Antibody-drug conjugates (ADCs) represent a promising therapeutic modality for the clinical management of cancer. We have developed a novel ADC that targets 5T4 (also known as TPBG), a tumor-associated antigen that rapidly internalizes. In preclinical models of NSCLC, 5T4 was identified as a marker of undifferentiated tumorigenic cells that express properties of tumor-initiating cells (TICs) and was associated with a highly proliferating (Ki67 positive) cell phenotype. Based on the biological properties of 5T4, we designed the ADC by conjugating an internalizing humanized anti-5T4 antibody (Ab), A1, to the tubulin inhibitor monomethylauristatin F via a maleimidocaproyl (mc) linker (A1mcMMAF) with a drug/antibody ratio of ∼ 4. The A1 Ab has a Kd of 0.9 nM against the 5T4 antigen, binds to a broad range of 5T4 expressing cell lines, and rapidly internalizes (66% internalized within 4h). As an ADC, A1mcMMAF retains similar properties to the unconjugated Ab. In vitro, A1mcMMAF exhibited potent and target-specific cytotoxic activity against a panel of 5T4+ cell lines with an IC50 of 5 ng Ab/ml against high 5T4 expressing cells. In vivo, A1mcMMAF exhibited potent anti-tumor activity in all five tumor models tested (with a broad range of 5T4 expression) and caused long-term regressions monitored up to 100 days after the last dose. Strikingly, animals were cured of disease in every model with doses as low as 3 mg Ab/kg Q4d x 4. In a lung cancer patient-derived xenograft in which 5T4 is not uniformly expressed, but instead is preferentially expressed on the less differentiated sub-population of tumor cells, A1mcMMAF treatment resulted in sustained tumor regression. This result highlights the clinical potential of ADCs that target aggressive cell subpopulations such as TICs. In exploratory safety studies, A1mcMMAF exhibited no overt toxicities when administered at 10 mg Ab/kg/cycle x 2 to cynomolgus monkeys and had a half-life of ∼5 days. The encouraging efficacy and safety data of the conjugate resulting in high therapeutic index in preclinical models warrants further clinical testing of this ADC. 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 2530. doi:1538-7445.AM2012-2530