Douglas Armellino

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.

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.

Antibody-targeted chemotherapy of B-cell lymphoma using calicheamicin conjugated to murine or humanized antibody against CD22

Antibody-targeted chemotherapy with immunoconjugates of calicheamicin is a clinically validated strategy in cancer therapy. This study describes the selection of a murine anti-CD22 mAb, m5/44, as a targeting agent, its conjugation to a derivative of calicheamicin (CalichDM) via either acid-labile or acid-stable linkers, the antitumor activity of CalichDM conjugated to m5/44, and its subsequent humanization by CDR grafting. Murine IgG1 mAb m5/44 was selected based on its subnanomolar affinity for CD22 and ability to be internalized into B cells. CalichDM conjugated to m5/44 caused potent growth inhibition of CD22+ human B-cell lymphomas (BCLs) in vitro. The conjugate of m5/44 with an acid-labile linker was more potent than an acid-stable conjugate, a nonbinding conjugate with a similar acid-labile linker, or unconjugated CalichDMH in inhibiting BCL growth. CalichDM conjugated to m5/44 caused regression of established BCL xenografts in nude mice. In contrast, both unconjugated m5/44 and a nonbinding conjugate were ineffective against these xenografts. Based on the potent antitumor activity of m5/44-CalichDM conjugates, m5/44 was humanized by CDR grafting to create g5/44, an IgG4 anti-CD22 antibody. Both m5/44 and g5/44 bound CD22 with subnanomolar affinity. Competitive blocking with previously characterized murine anti-CD22 mAbs suggested that g5/44 recognizes epitope A located within the first N-terminal Ig-like domain of human CD22. Antitumor efficacy of CalichDM conjugated to g5/44 against BCL xenografts was more potent than its murine counterpart. Based on these results, a calicheamicin conjugate of g5/44, CMC-544, was selected for further development as a targeted chemotherapeutic agent for the treatment of B-cell malignancies.

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.

Comprehensive interrogation of a minimalist synthetic CDR-H3 library and its ability to generate antibodies with therapeutic potential.

We have generated large libraries of single-chain Fv antibody fragments (>10(10) transformants) containing unbiased amino acid diversity that is restricted to the central combining site of the stable, well-expressed DP47 and DPK22 germline V-genes. Library WySH2A was constructed to examine the potential for synthetic complementarity-determining region (CDR)-H3 diversity to act as the lone source of binding specificity. Library WySH2B was constructed to assess the necessity for diversification in both the H3 and L3. Both libraries provided diverse, specific antibodies, yielding a total of 243 unique hits against 7 different targets, but WySH2B produced fewer hits than WySH2A when selected in parallel. WySH2A also consistently produced hits of similar quality to WySH2B, demonstrating that the diversification of the CDR-L3 reduces library fitness. Despite the absence of deliberate bias in the library design, CDR length was strongly associated with the number of hits produced, leading to a functional loop length distribution profile that mimics the biases observed in the natural repertoire. A similar trend was also observed for the CDR-L3. After target selections, several key amino acids were enriched in the CDR-H3 (e.g., small and aromatic residues) while others were reduced (e.g., strongly charged residues) in a manner that was specific to position, preferentially occurred in CDR-H3 stem positions, and tended towards residues associated with loop stabilization. As proof of principle for the WySH2 libraries to produce viable lead candidate antibodies, 114 unique hits were produced against Delta-like ligand 4 (DLL4). Leads exhibited nanomolar binding affinities, highly specific staining of DLL4+ cells, and biochemical neutralization of DLL4-NOTCH1 interaction.

Breast cancer cells respond differentially to modulation of TGFβ2 signaling after exposure to chemotherapy or hypoxia.

Intratumoral heterogeneity helps drive the selection for diverse therapy-resistant cell populations. In this study, we demonstrate the coexistence of two therapy-resistant populations with distinct properties that are reproducibly enriched under conditions that characterize tumor pathophysiology. Breast cancer cells that survived chemotherapy or hypoxia were enriched for cells expressing the major hyaluronic acid receptor CD44. However, only CD44(hi) cells that survived chemotherapy exhibited cancer stem cell (CSC) phenotypes based on growth potential and gene expression signatures that represent oncogenic signaling and metastatic prowess. Strikingly, we identified TGFβ2 as a key growth promoter of CD44(hi) cells that survived chemotherapy but also as a growth inhibitor of cells that survived hypoxia. Expression of the TGFβ receptor TGFβR1 and its effector molecule SMAD4 was required for enrichment of CD44(hi) cells exposed to the chemotherapeutic drug epirubicin, which suggests a feed-forward loop to enrich for and enhance the function of surviving CSCs. Our results reveal context-dependent effects of TGFβ2 signaling in the same tumor at the same time. The emergence of distinct resistant tumor cell populations as a consequence of prior therapeutic intervention or microenvironmental cues has significant implications for the responsiveness of recurring tumors to therapy.

Abstract 1220: A novel PTK7-targeted antibody-drug conjugate eliminates tumor-initiating cells and induces sustained tumor regressions

Disease relapse after treatment is common in triple-negative breast cancer (TNBC), ovarian cancer and non-small cell lung cancer (NSCLC). Therapies that target tumor-initiating cells (TICs) should improve patient survival by eliminating the cells that can drive tumor regrowth and metastasis. Here we identify Protein Tyrosine Kinase 7 (PTK7), a highly conserved but catalytically inactive receptor tyrosine kinase, as an antigen that is enriched on TICs in low-passage patient-derived xenografts (PDX) of TNBC, NSCLC and other tumor types. An anti-PTK7 antibody-drug conjugate (ADC) was generated from a humanized anti-PTK7 monoclonal antibody, a cleavable valine-citrulline-based linker and the Aur0101 auristatin microtubule inhibitor. The anti-PTK7 ADC induced sustained regressions of TNBC, NSCLC and ovarian cancer PDX, with improved activity over standard-of-care chemotherapy, and reduced the frequency of TICs as determined by serial transplantation experiments. Moreover, the ADC may have additional mechanisms of action, including an anti-angiogenic effect, that promote anti-tumor immune responses. Together these preclinical results indicate the potential of the anti-PTK7 ADC to improve the long-term survival of cancer patients. The ADC is currently being tested in a Phase 1 clinical trial, from which interim results will be presented. Citation Format: Marc Isaac Damelin, Alex Bankovich, Jeff Bernstein, Justin Lucas, Liang Chen, Sam Williams, Albert Park, Jorge Aguilar, Elana Ernstoff, Manoj Charati, Russell Dushin, Amy Jackson-Fisher, Monette Aujay, Christina Lee, Hanna Ramoth, Milly Milton, Johannes Hampl, Sasha Lazetic, Virginia Pulito, Douglas Armellino, Edward Rosfjord, Magali Guffroy, Hadi Falahatpisheh, Lindsay King, Frank Barletta, Robert Stull, Marybeth Pysz, Paul Escarpe, David Liu, Orit Foord, Brenda Gibson, Eric Powell, Christopher O’Donnell, Xiaohua Xin, Hans Peter Gerber, Puja Sapra, Scott Dylla. A novel PTK7-targeted antibody-drug conjugate eliminates tumor-initiating cells and induces sustained tumor regressions. [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 1220.

Abstract 1968: Breast cancer cells escape from chemotherapy and hypoxia by distinct mechanisms

Cancer relapse following treatment remains a significant barrier to achieving cures for many patients. An emerging framework for addressing this problem focuses on cancer stem cells (CSCs). Clinical and preclinical data suggest that CSCs survive chemotherapy and antiangiogenic therapy. In breast cancer, CSCs are marked by the cell surface expression of CD44, the major receptor for hyaluronic acid. The SKBR3 cell line was previously shown to be a clinically relevant model of breast CSCs in vivo, and we have further developed this model to enable mechanistic studies of tumor relapse in vitro. We found that chemotherapy and hypoxia both enriched for CD44 hi populations in SKBR3, but surprisingly, the populations were phenotypically distinct. CD44 hi cells from chemotherapy but not hypoxia exhibited increased tumor cell growth and increased sensitivity to the CSC-specific inhibitor salinomycin, compared to CD44 lo cells. To examine these CD44 hi and CD44 lo populations further, we performed transcriptional profiling with sorted cells. We found that the growth factor TGF beta-2 was upregulated in chemotherapy-treated CD44 hi cells and also enhanced the growth of these cells, which suggests that TGF beta-2 autocrine/paracrine signaling can promote the growth of surviving cells. We also observed an increase in xCT (SLC7A11) expression upon chemotherapy treatment and identified the CD44v8-10 variant expressed in the CD44 hi SKBR3. CD44v could therefore stabilize xCT and promote survival by lowering intracellular reactive oxygen species (ROS). We have also established a functional role for CD44 in SKBR3 cell growth: CD44 knockdown prevented colony formation, and conversely the CD44 ligand hyaluronic acid enhanced colony formation. These findings indicate that CD44 not only marks CSC populations that arise in response to chemotherapy but also functions in their survival. Our work suggests that mechanisms of tumor relapse vary based on the particular therapy, and defining these mechanisms will allow for the development of novel therapeutic strategies to enable long-term responses in the clinic. Citation Format: Siobhan O9Brien, Liang Chen, Wenyan Zhong, Douglas Armellino, Maximillian Follettie, Marc Damelin. Breast cancer cells escape from chemotherapy and hypoxia by distinct mechanisms. [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 1968. doi:10.1158/1538-7445.AM2014-1968

Abstract 475: 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) are refractory to chemotherapy and associated with short survival time. Here we integrate multiple experimental models with clinicopathological analysis of patient tumors to delineate a cellular hierarchy in NSCLC. We demonstrate 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 was 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. 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 475. doi:10.1158/1538-7445.AM2011-475