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The commentary by Colombo and Rich recently published in Cancer Cell provides a timely and comprehensive review of the clinical maximum tolerated doses (MTDs) of antibody-drug conjugates (ADCs) and their corresponding small molecules/chemotherapies. The authors identified similarities between their MTDs and therefore question the historic assumptions made for ADCs, namely, that they increase the MTDs of their corresponding cytotoxic molecules. However, the authors did not address the superior anti-tumor responses of ADCs compared to their corresponding chemotherapies, as reported in clinical trials. In this point of view, we propose a revised model wherein the anti-tumor activities of ADCs and consequently their therapeutic indexes (TIs) are not solely associated with changes not only in their MTDs but also in their minimal effective doses (MEDs). In addition, when using an exposure-based TI calculation method, the superior anti-tumor activities of ADCs relative to their corresponding chemotherapy can readily be explained. We discussed the clinical and preclinical data in support of lower MEDs of ADCs and generated a revised graph illustrating the TI improvements of ADCs vs chemotherapy more accurately. We believe that our revised model can provide a blueprint for future improvements in protein engineering and chemical engineering of toxins to further advance ADC research and development.

pHLA complexes represent the largest class of cell surface markers on cancer cells, making them attractive for targeted cancer therapies. Adoptive cell therapies expressing TCRs that recognize tumor specific pHLAs take advantage of the unique selectivity and avidity of TCR: pHLA interactions. More recently, additional protein binding domains binding to pHLAs, known as TCR mimics (TCRm), were developed for tumor targeting of high potency therapeutic modalities, including bispecifics, ADCs, CAR T and -NK cells. TCRm compounds take advantage of the exquisite tumor specificity of certain pHLA targets, including cell lineage commitment markers and cancer testis antigens (CTAs). To achieve meaningful anti-tumor responses, it is critical that TCRm compounds integrate both, high target binding affinities and a high degree of target specificity. In this review, we describe the most advanced approaches to achieve both criteria, including affinity- and specificity engineering of TCRs, antibodies and alternative protein scaffolds. We also discuss the status of current TCRm based therapeutics developed in the clinic, key challenges, and emerging trends to improve treatment options for cancer patients treated with TCRm based therapeutics in Oncology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gerber and Presta.)

Extra domain B splice variant of fibronectin (EDB+FN) is an extracellular matrix protein (ECM) deposited by tumor-associated fibroblasts, and is associated with tumor growth, angiogenesis, and invasion. We hypothesized that EDB+FN is a safe and abundant target for therapeutic intervention with an antibody-drug conjugate (ADC). We describe the generation, pharmacology, mechanism of action, and safety profile of an ADC specific for EDB+FN (EDB-ADC). EDB+FN is broadly expressed in the stroma of pancreatic, non-small cell lung (NSCLC), breast, ovarian, head and neck cancers, whereas restricted in normal tissues. In patient-derived xenograft (PDX), cell-line xenograft (CLX), and mouse syngeneic tumor models, EDB-ADC, conjugated to auristatin Aur0101 through site-specific technology, demonstrated potent antitumor growth inhibition. Increased phospho-histone H3, a pharmacodynamic biomarker of response, was observed in tumor cells distal to the target site of tumor ECM after EDB-ADC treatment. EDB-ADC potentiated infiltration of immune cells, including CD3+ T lymphocytes into the tumor, providing rationale for the combination of EDB-ADC with immune checkpoint therapy. EDB-ADC and anti-PD-L1 combination in a syngeneic breast tumor model led to enhanced antitumor activity with sustained tumor regressions. In nonclinical safety studies in nonhuman primates, EDB-ADC had a well-tolerated safety profile without signs of either on-target toxicity or the off-target effects typically observed with ADCs that are conjugated through conventional conjugation methods. These data highlight the potential for EDB-ADC to specifically target the tumor microenvironment, provide robust therapeutic benefits against multiple tumor types, and enhance activity antitumor in combination with checkpoint blockade., (©2022 The Authors; Published by the American Association for Cancer Research.)

Aberrant NOTCH3 signaling and overexpression is oncogenic, associated with cancer stem cells and drug resistance, yet therapeutic targeting remains elusive. Here, we develop NOTCH3-targeted antibody drug conjugates (NOTCH3-ADCs) by bioconjugation of an auristatin microtubule inhibitor through a protease cleavable linker to two antibodies with differential abilities to inhibit signaling. The signaling inhibitory antibody rapidly induces ligand-independent receptor clustering and internalization through both caveolin and clathrin-mediated pathways. The non-inhibitory antibody also efficiently endocytoses via clathrin without inducing receptor clustering but with slower lysosomal co-localization kinetics. In addition, DLL4 ligand binding to the NOTCH3 receptor mediates transendocytosis of NOTCH3-ADCs into ligand-expressing cells. NOTCH3-ADCs internalize into receptor and ligand cells independent of signaling and induce cell death in both cell types representing an atypical mechanism of ADC cytotoxicity. Treatment of xenografts with NOTCH3-ADCs leads to sustained tumor regressions, outperforms standard-of-care chemotherapy, and allows targeting of tumors that overexpress NOTCH3 independent of signaling inhibition., Competing Interests: All authors are/were employees and shareholders of Pfizer. US patents associated with the development of NOTCH3-ADCs include 8,828,401 and 9,433,687., (© 2021 The Authors.)

Purpose: Mortality due to acute myeloid leukemia (AML) remains high, and the management of relapsed or refractory AML continues to be therapeutically challenging. The reapproval of Mylotarg, an anti-CD33-calicheamicin antibody-drug conjugate (ADC), has provided a proof of concept for an ADC-based therapeutic for AML. Several other ADCs have since entered clinical development of AML, but have met with limited success. We sought to develop a next-generation ADC for AML with a wide therapeutic index (TI) that overcomes the shortcomings of previous generations of ADCs., Experimental Design: We compared the TI of our novel CD33-targeted ADC platform with other currently available CD33-targeted ADCs in preclinical models of AML. Next, using this next-generation ADC platform, we performed a head-to-head comparison of two attractive AML antigens, CD33 and CD123., Results: Our novel ADC platform offered improved safety and TI when compared with certain currently available ADC platforms in preclinical models of AML. Differentiation between the CD33- and CD123-targeted ADCs was observed in safety studies conducted in cynomolgus monkeys. The CD33-targeted ADC produced severe hematologic toxicity, whereas minimal hematologic toxicity was observed with the CD123-targeted ADC at the same doses and exposures. The improved toxicity profile of an ADC targeting CD123 over CD33 was consistent with the more restricted expression of CD123 in normal tissues., Conclusions: We optimized all components of ADC design (i.e., leukemia antigen, antibody, and linker-payload) to develop an ADC that has the potential to translate into an effective new therapy against AML., (©2020 American Association for Cancer Research.)

The approval of ado-trastuzumab emtansine (T-DM1) in HER2 + metastatic breast cancer validated HER2 as a target for HER2-specific antibody-drug conjugates (ADC). Despite its demonstrated clinical efficacy, certain inherent properties within T-DM1 hamper this compound from achieving the full potential of targeting HER2-expressing solid tumors with ADCs. Here, we detail the discovery of PF-06804103, an anti-HER2 ADC designed to have a widened therapeutic window compared with T-DM1. We utilized an empirical conjugation site screening campaign to identify the engineered ĸkK183C and K290C residues as those that maximized in vivo ADC stability, efficacy, and safety for a four drug-antibody ratio (DAR) ADC with this linker-payload combination. PF-06804103 incorporates the following novel design elements: (i) a new auristatin payload with optimized pharmacodynamic properties, (ii) a cleavable linker for optimized payload release and enhanced antitumor efficacy, and (iii) an engineered cysteine site-specific conjugation approach that overcomes the traditional safety liabilities of conventional conjugates and generates a homogenous drug product with a DAR of 4. PF-06804103 shows (i) an enhanced efficacy against low HER2-expressing breast, gastric, and lung tumor models, (ii) overcomes in vitro - and in vivo -acquired T-DM1 resistance, and (iii) an improved safety profile by enhancing ADC stability, pharmacokinetic parameters, and reducing off-target toxicities. Herein, we showcase our platform approach in optimizing ADC design, resulting in the generation of the anti-HER2 ADC, PF-06804103. The design elements of identifying novel sites of conjugation employed in this study serve as a platform for developing optimized ADCs against other tumor-specific targets., (©2020 American Association for Cancer Research.)

The ideal cancer target antigen (Ag) is expressed at high copy numbers on neoplastic cells, absent on normal tissues, and contributes to the survival of cancer cells. Despite significant investments in the identification of cell surface Ags, there is a paucity of targets that meet such ideal cancer target criteria. Recent clinical trials in patients with cancer treated with immune checkpoint inhibitors (ICIs) indicate that cluster of differentiation (CD)8 + T cells, by means of their T cell receptors (TCRs) recognizing intracellular targets presented as peptides in the context of human leukocyte antigen (peptide-human leukocyte antigen complex; pHLA) molecules on tumor cells, can mediate deep and long-lasting antitumor responses in patients with solid tumors. Therefore, pHLA-target Ags may represent the long sought-after, ideal targets for solid tumor targeting by high-potency oncology compounds., (Copyright © 2020 Elsevier Inc. All rights reserved.)

High-potency oncology compounds such as antibody- drug conjugates, T cell redirecting, and CAR-T cell therapies have provided transformational responses in patients with liquid tumors. However, they delivered only limited benefit to solid tumor patients due to the frequent onset of dose limiting toxicities in normal tissues. Such on-target, off-tumor toxicities are caused by recognition of targets present at low-levels on normal tissues. The apparent imbalance between the rapid development of high-potency therapeutic modalities and the slow progress in identification of cleaner targets is illustrated by the fact that most high-potency compounds currently developed in the clinic target cell surface antigens identified over 20 years ago. Therefore, identification of novel, truly tumor-specific targets is critical for the future success of high-potency oncology compounds in solid tumors. One of the most promising approaches to overcome the limitations of targeting cell surface antigens are intracellular targets. The renewed interest in this class of targets is due to the success of immune checkpoint inhibitors, which mediate their anti-tumor responses by activation of cytotoxic T cells recognizing peptide fragments of intracellular targets presented by human leukocyte antigens (HLAs) on the surface of tumor cells. Importantly, many intracellular targets belong to the class of tumor specific antigens (TSAs), which lack presentation on normal tissues. In this report we review the main classes of tumor specific antigens, including viral, neoantigens and shared self-antigens as well as tumor associated antigens (TAAs) and their relevance for therapeutic targeting of solid tumors by high-potency therapeutic modalities., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Strong evidence exists supporting the important role T cells play in the immune response against tumors. Still, the ability to initiate tumor-specific immune responses remains a challenge. Recent clinical trials suggest that bispecific antibody-mediated retargeted T cells are a promising therapeutic approach to eliminate hematopoietic tumors. However, this approach has not been validated in solid tumors. PF-06671008 is a dual-affinity retargeting (DART ® )-bispecific protein engineered with enhanced pharmacokinetic properties to extend in vivo half-life, and designed to engage and activate endogenous polyclonal T cell populations via the CD3 complex in the presence of solid tumors expressing P-cadherin. This bispecific molecule elicited potent P-cadherin expression-dependent cytotoxic T cell activity across a range of tumor indications in vitro, and in vivo in tumor-bearing mice. Regression of established tumors in vivo was observed in both cell line and patient-derived xenograft models engrafted with circulating human T lymphocytes. Measurement of in vivo pharmacodynamic markers demonstrates PF-06671008-mediated T cell activation, infiltration and killing as the mechanism of tumor inhibition.

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) that has demonstrated clinical benefit for patients with HER2 + metastatic breast cancer; however, its clinical activity is limited by inherent or acquired drug resistance. The molecular mechanisms that drive clinical resistance to T-DM1, especially in HER2 + tumors, are not well understood. We used HER2 + cell lines to develop models of T-DM1 resistance using a cyclical dosing schema in which cells received T-DM1 in an "on-off" routine until a T-DM1-resistant population was generated. T-DM1-resistant N87 cells (N87-TM) were cross-resistant to a panel of trastuzumab-ADCs (T-ADCs) with non-cleavable-linked auristatins. N87-TM cells do not have a decrease in HER2 protein levels or an increase in drug transporter protein (e.g., MDR1) expression compared with parental N87 cells. Intriguingly, T-ADCs using auristatin payloads attached via an enzymatically cleavable linker overcome T-DM1 resistance in N87-TM cells. Importantly, N87-TM cells implanted into athymic mice formed T-DM1 refractory tumors that remain sensitive to T-ADCs with cleavable-linked auristatin payloads. Comparative proteomic profiling suggested enrichment in proteins that mediate caveolae formation and endocytosis in the N87-TM cells. Indeed, N87-TM cells internalize T-ADCs into intracellular caveolin-1 (CAV1)-positive puncta and alter their trafficking to the lysosome compared with N87 cells. T-DM1 colocalization into intracellular CAV1-positive puncta correlated with reduced response to T-DM1 in a panel of HER2 + cell lines. Together, these data suggest that caveolae-mediated endocytosis of T-DM1 may serve as a novel predictive biomarker for patient response to T-DM1. Mol Cancer Ther; 17(1); 243-53. ©2017 AACR ., (©2017 American Association for Cancer Research.)

The fetal oncogene 5T4 is a cell surface protein, with overexpression observed in a variety of cancers as compared to normal adult tissue. The ability to select patients with tumors that express high levels of 5T4 may enrich a clinical trial cohort with patients most likely to respond to 5T4 targeted therapy. To that end, we developed assays to measure 5T4 in both tumors and in circulating tumor cells (CTCs). We identified the presence of 5T4 in both adenocarcinoma and squamous cell carcinoma of lung, in all clinical stages and grades of disease. CTCs were identified in peripheral blood from the majority of patients with NSCLC, and 5T4 was detectable in most samples. Although 5T4 was present in both CTCs and tumors in most patients, there was no concordance between relative amount in either sample type. Clinical response rates of patients treated with the therapies directed against 5T4 in early stage clinical trials, as determined by these assays, may provide important insights into the biology of 5T4 in tumors and the mechanisms of action of 5T4-targeting therapy.

As the antibody drug conjugate (ADC) community continues to shift towards site-specific conjugation technology, there is a growing need to understand how the site of conjugation impacts the biophysical and biological properties of an ADC. In order to address this need, we prepared a carefully selected series of engineered cysteine ADCs and proceeded to systematically evaluate their potency, stability, and PK exposure. The site of conjugation did not have a significant influence on the thermal stability and in vitro cytotoxicity of the ADCs. However, we demonstrate that the rate of cathepsin-mediated linker cleavage is heavily dependent upon site and is closely correlated with ADC hydrophobicity, thus confirming other recent reports of this phenomenon. Interestingly, conjugates with high rates of cathepsin-mediated linker cleavage did not exhibit decreased plasma stability. In fact, the major source of plasma instability was shown to be retro-Michael mediated deconjugation. This process is known to be impeded by succinimide hydrolysis, and thus, we undertook a series of mutational experiments demonstrating that basic residues located nearby the site of conjugation can be a significant driver of succinimide ring opening. Finally, we show that total antibody PK exposure in rat was loosely correlated with ADC hydrophobicity. It is our hope that these observations will help the ADC community to build "design rules" that will enable more efficient prosecution of next-generation ADC discovery programs.

Purpose: Adverse reactions reported in patients treated with antibody-calicheamicin conjugates such as gemtuzumab ozogamicin (Mylotarg) and inotuzumab ozogamicin include thrombocytopenia and sinusoidal obstruction syndrome (SOS). The objective of this experimental work was to investigate the mechanism for thrombocytopenia, characterize the liver injury, and identify potential safety biomarkers. Experimental Design: Cynomolgus monkeys were dosed intravenously at 6 mg/m 2 /dose once every 3 weeks with a nonbinding antibody-calicheamicin conjugate (PF-0259) containing the same linker-payload as gemtuzumab ozogamicin and inotuzumab ozogamicin. Monkeys were necropsied 48 hours after the first administration (day 3) or 3 weeks after the third administration (day 63). Results: PF-0259 induced acute thrombocytopenia (up to 86% platelet reduction) with nadirs on days 3 to 4. There was no indication of effects on megakaryocytes in bone marrow or activation of platelets in peripheral blood. Microscopic evaluation of liver from animals necropsied on day 3 demonstrated midzonal degeneration and loss of sinusoidal endothelial cells (SECs) associated with marked platelet accumulation in sinusoids. Liver histopathology on day 63 showed variable endothelial recovery and progression to a combination of sinusoidal capillarization and sinusoidal dilation/hepatocellular atrophy, consistent with early SOS. Among biomarkers evaluated, there were early and sustained increases in serum hyaluronic acid (HA) that correlated well with serum aspartate aminotransferase and liver microscopic changes, suggesting that HA may be a sensitive diagnostic marker of the liver microvascular injury. Conclusions: These data support the conclusion that target-independent damage to liver SECs may be responsible for acute thrombocytopenia (through platelet sequestration in liver sinusoids) and development of SOS. Clin Cancer Res; 23(7); 1760-70. ©2016 AACR ., (©2016 American Association for Cancer Research.)

Disease relapse after treatment is common in triple-negative breast cancer (TNBC), ovarian cancer (OVCA), 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 recurrence and metastasis. We demonstrate that protein tyrosine kinase 7 (PTK7), a highly conserved but catalytically inactive receptor tyrosine kinase in the Wnt signaling pathway, is enriched on TICs in low-passage TNBC, OVCA, and NSCLC patient-derived xenografts (PDXs). To deliver a potent anticancer drug to PTK7-expressing TICs, we generated a targeted antibody-drug conjugate (ADC) composed of a humanized anti-PTK7 monoclonal antibody, a cleavable valine-citrulline-based linker, and Aur0101, an auristatin microtubule inhibitor. The PTK7-targeted ADC induced sustained tumor regressions and outperformed standard-of-care chemotherapy. Moreover, the ADC specifically reduced the frequency of TICs, as determined by serial transplantation experiments. In addition to reducing the TIC frequency, the PTK7-targeted ADC may have additional antitumor mechanisms of action, including the inhibition of angiogenesis and the stimulation of immune cells. Together, these preclinical data demonstrate the potential for the PTK7-targeted ADC to improve the long-term survival of cancer patients., (Copyright © 2017, American Association for the Advancement of Science.)

Drug resistance limits the effectiveness of cancer therapies. Despite attempts to develop curative anticancer treatments, tumors evolve evasive mechanisms limiting durable responses. Hence, diverse therapies are used to attack cancer, including cytotoxic and targeted agents. Antibody-drug conjugates (ADC) are biotherapeutics designed to deliver potent cytotoxins to cancer cells via tumor-specific antigens. Little is known about the clinical manifestations of drug resistance to this class of therapy; however, recent preclinical studies reveal potential mechanisms of resistance. Because ADCs are a combination of antibody and small molecule cytotoxin, multifactorial modes of resistance are emerging that are inherent to the structure and function of the ADC. Decreased cell-surface antigen reduces antibody binding, whereas elevated drug transporters such as MDR1 and MRP1 reduce effectiveness of the payload. Inherent to the uniqueness of the ADC, other novel resistance mechanisms are emerging, including altered antibody trafficking, ADC processing, and intracellular drug release. Most importantly, the modular nature of the ADC allows components to be switched and replaced, enabling development of second-generation ADCs that overcome acquired resistance. This review is intended to highlight recent progress in our understanding of ADC resistance, including approaches to create preclinical ADC-refractory models and to characterize their emerging mechanisms of resistance. Mol Cancer Ther; 15(12); 2825-34. ©2016 AACR., (©2016 American Association for Cancer Research.)

There is a considerable ongoing work to identify new cytotoxic payloads that are appropriate for antibody-based delivery, acting via mechanisms beyond DNA damage and microtubule disruption, highlighting their importance to the field of cancer therapeutics. New modes of action will allow a more diverse set of tumor types to be targeted and will allow for possible mechanisms to evade the drug resistance that will invariably develop to existing payloads. Spliceosome inhibitors are known to be potent antiproliferative agents capable of targeting both actively dividing and quiescent cells. A series of thailanstatin-antibody conjugates were prepared in order to evaluate their potential utility in the treatment of cancer. After exploring a variety of linkers, we found that the most potent antibody-drug conjugates (ADCs) were derived from direct conjugation of the carboxylic acid-containing payload to surface lysines of the antibody (a "linker-less" conjugate). Activity of these lysine conjugates was correlated to drug-loading, a feature not typically observed for other payload classes. The thailanstatin-conjugates were potent in high target expressing cells, including multidrug-resistant lines, and inactive in nontarget expressing cells. Moreover, these ADCs were shown to promote altered splicing products in N87 cells in vitro, consistent with their putative mechanism of action. In addition, the exposure of the ADCs was sufficient to result in excellent potency in a gastric cancer xenograft model at doses as low as 1.5 mg/kg that was superior to the clinically approved ADC T-DM1. The results presented herein therefore open the door to further exploring splicing inhibition as a potential new mode-of-action for novel ADCs.

Bispecific antibodies offer a promising approach for the treatment of cancer but can be challenging to engineer and manufacture. Here we report the development of PF-06671008, an extended-half-life dual-affinity re-targeting (DART ® ) bispecific molecule against P-cadherin and CD3 that demonstrates antibody-like properties. Using phage display, we identified anti-P-cadherin single chain Fv (scFv) that were subsequently affinity-optimized to picomolar affinity using stringent phage selection strategies, resulting in low picomolar potency in cytotoxic T lymphocyte (CTL) killing assays in the DART format. The crystal structure of this disulfide-constrained diabody shows that it forms a novel compact structure with the two antigen binding sites separated from each other by approximately 30 Å and facing approximately 90° apart. We show here that introduction of the human Fc domain in PF-06671008 has produced a molecule with an extended half-life (-4.4 days in human FcRn knock-in mice), high stability (T m 1 > 68 °C), high expression (>1 g/L), and robust purification properties (highly pure heterodimer), all with minimal impact on potency. Finally, we demonstrate in vivo anti-tumor efficacy in a human colorectal/human peripheral blood mononuclear cell (PBMC) co-mix xenograft mouse model. These results suggest PF-06671008 is a promising new bispecific for the treatment of patients with solid tumors expressing P-cadherin.

Blockade of immune-checkpoints has emerged as one of the most promising approaches to improve the durability of anti-tumor responses in cancer patients. However, the fraction of patients experiencing durable responses to single agent immune checkpoint inhibitor treatment remains limited. Recent clinical reports suggest that patients responding best to checkpoint blockade therapies display higher levels of CD8(+) T-cells in the tumor prior to treatment. Therefore, combination treatments of immune-checkpoint inhibitors with compounds that increase the number of tumor infiltrating CD8(+) T cells may expand the therapeutic benefit of immuno-oncology (IO) drugs. Immunogenic cell death (ICD) of tumor cells is induced by certain classes of cytotoxic compounds and represents a potent stimulator of effector T-cell recruitment to tumors. In addition, several cytotoxics directly stimulate dendritic cell activation and maturation, resulting in improved anti-tumor immune responses when combined with IO compounds. Among them, several cytotoxic agents are currently utilized as payloads for antibody-drug conjugates (ADCs). Therefore, identification of optimal combination regimens between ADC- and IO compounds holds strong promise to overcome the current limitations of immune checkpoint inhibitors, by increasing the recruitment of CD8(+) effector T-cells to the tumor core. Here we review the emerging field of ADC/IO combination research, with a focus on how to optimally combine both modalities. The answer to this question may have a broader impact on oncology drug development, as synergistic activities between IO compounds and ADCs may increase the formation of tumor specific immunological memory, ultimately leading to durable responses in a larger fraction of cancer patients., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Calicheamicin is a DNA-damaging agent that, following intracellular activation, binds to DNA in the minor groove and introduces double-strand DNA breaks, leading to G2/M arrest and subsequent cell death. Importantly, the mechanism of action of calicheamicin is fundamentally different from the tubulin-binding class of cytotoxics targeting the mitotic spindle, which represent the most common class of payloads for antibody-drug conjugates (ADCs) currently undergoing clinical development. Spindle poisons that target tubulin, including auristatins and maytansines, are most effective against rapidly proliferating cells. In contrast, calicheamicin induces DNA double-strand breaks and apoptosis independent of cell cycle progression. Such properties may be advantageous when targeting malignant cells that are not markedly different in their proliferation status compared to normal cells. Here we review calicheamicin conjugates, with a particular focus on the preclinical- and clinical development of inotuzumab ozogamicin, targeting the CD22 antigen expressed on a large variety of hematologic malignancies. In pre-clinical experiments, inotuzumab ozogamicin potently induced tumor regressions in models of non-Hodgkin's lymphoma (NHL), either alone or in combination with the anti-CD20 antibody Rituximab. Promising anti-tumor responses were observed in early stage clinical trials, where inotuzumab ozogamicin was administered either as single agent or in combination with Rituximab. Consistent with the cell cycle independent mechanism of action of the calicheamicin payload, high rates of complete responses were observed in less aggressive forms of lymphomas, including follicular lymphoma (FL) and relapsed, diffuse large B-cell lymphoma (DLBCL). Inotuzumab ozogamicin is currently being tested in phase III clinical trials in acute lymphocytic leukemia (ALL). Particular focus is dedicated to reviewing the pre-clinical and clinical data generated with this compound in NHL and to outline future focus areas for pre-clinical- and clinical research of inotuzumab ozogamicin, and the calicheamicin class of antibody-drug conjugates more generally., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Purpose: Triple-negative breast cancer (TNBC) and ovarian cancer each comprise heterogeneous tumors, for which current therapies have little clinical benefit. Novel therapies that target and eradicate tumor-initiating cells (TIC) are needed to significantly improve survival., Experimental Design: A panel of well-annotated patient-derived xenografts (PDX) was established, and surface markers that enriched for TIC in specific tumor subtypes were empirically determined. The TICs were queried for overexpressed antigens, one of which was selected to be the target of an antibody-drug conjugate (ADC). The efficacy of the ADC was evaluated in 15 PDX models to generate hypotheses for patient stratification., Results: We herein identified E-cadherin (CD324) as a surface antigen able to reproducibly enrich for TIC in well-annotated, low-passage TNBC and ovarian cancer PDXs. Gene expression analysis of TIC led to the identification of Ephrin-A4 (EFNA4) as a prospective therapeutic target. An ADC comprising a humanized anti-EFNA4 monoclonal antibody conjugated to the DNA-damaging agent calicheamicin achieved sustained tumor regressions in both TNBC and ovarian cancer PDX in vivo. Non-claudin low TNBC tumors exhibited higher expression and more robust responses than other breast cancer subtypes, suggesting a specific translational application for tumor subclassification., Conclusions: These findings demonstrate the potential of PF-06647263 (anti-EFNA4-ADC) as a first-in-class compound designed to eradicate TIC. The use of well-annotated PDX for drug discovery enabled the identification of a novel TIC target, pharmacologic evaluation of the compound, and translational studies to inform clinical development., (©2015 American Association for Cancer Research.)

Antibody-drug conjugates (ADC) are emerging as clinically effective therapy. We hypothesized that cancers treated with ADCs would acquire resistance mechanisms unique to immunoconjugate therapy and that changing ADC components may overcome resistance. Breast cancer cell lines were exposed to multiple cycles of anti-Her2 trastuzumab-maytansinoid ADC (TM-ADC) at IC80 concentrations followed by recovery. The resistant cells, 361-TM and JIMT1-TM, were characterized by cytotoxicity, proteomic, transcriptional, and other profiling. Approximately 250-fold resistance to TM-ADC developed in 361-TM cells, and cross-resistance was observed to other non-cleavable-linked ADCs. Strikingly, these 361-TM cells retained sensitivity to ADCs containing cleavable mcValCitPABC-linked auristatins. In JIMT1-TM cells, 16-fold resistance to TM-ADC developed, with cross-resistance to other trastuzumab-ADCs. Both 361-TM and JIMT1-TM cells showed minimal resistance to unconjugated mertansine (DM1) and other chemotherapeutics. Proteomics and immunoblots detected increased ABCC1 (MRP1) drug efflux protein in 361-TM cells, and decreased Her2 (ErbB2) in JIMT1-TM cells. Proteomics also showed alterations in various pathways upon chronic exposure to the drug in both cell models. Tumors derived from 361-TM cells grew in mice and were refractory to TM-ADC compared with parental cells. Hence, acquired resistance to trastuzumab-maytansinoid ADC was generated in cultured cancer cells by chronic drug treatment, and either increased ABCC1 protein or reduced Her2 antigen were primary mediators of resistance. These ADC-resistant cell models retain sensitivity to other ADCs or standard-of-care chemotherapeutics, suggesting that alternate therapies may overcome acquired ADC resistance. Mol Cancer Ther; 14(4); 952-63. ©2015 AACR., (©2015 American Association for Cancer Research.)

Most oncology compounds entering clinical development have passed stringent preclinical pharmacology evaluation criteria. However, only a small fraction of experimental agents induce meaningful antitumor activities in the clinic. Low predictability of conventional preclinical pharmacology models is frequently cited as a main reason for the unusually high clinical attrition rates of therapeutic compounds in oncology. Therefore, improvement in the predictive values of preclinical efficacy models for clinical outcome holds great promise to reduce the clinical attrition rates of experimental compounds. Recent reports suggest that pharmacology studies conducted with patient derived xenograft (PDX) tumors are more predictive for clinical outcome compared to conventional, cell line derived xenograft (CDX) models, in particular when therapeutic compounds were tested at clinically relevant doses (CRDs). Moreover, the study of the most malignant cell types within tumors, the tumor initiating cells (TICs), relies on the availability of preclinical models that mimic the lineage hierarchy of cells within tumors. PDX models were shown to more closely recapitulate the heterogeneity of patient tumors and maintain the molecular, genetic, and histological complexity of human tumors during early stages of sequential passaging in mice, rendering them ideal tools to study the responses of TICs, tumor- and stromal cells to therapeutic intervention. In this commentary, we review the progress made in the development of PDX models in key areas of oncology research, including target identification and validation, tumor indication search and the development of a biomarker hypothesis that can be tested in the clinic to identify patients that will benefit most from therapeutic intervention., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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.

Objectives of the present investigation were: (1) to compare three literature reported tumor growth inhibition (TGI) pharmacodynamic (PD) models and propose an optimal new model that best describes the xenograft TGI data for antibody drug conjugates (ADC), (2) to translate efficacy of the ADC Trastuzumab-emtansine (T-DM1) from mice to patients using the optimized PD model, and (3) to apply the translational strategy to predict clinically efficacious concentrations of a novel in-house anti-5T4 ADC, A1mcMMAF. First, the performance of all four of the PD models (i.e. 3 literature reported + 1 proposed) was evaluated using TGI data of T-DM1 obtained from four different xenografts. Based on the estimates of the pharmacodynamic/pharmacokinetic (PK/PD) modeling, a secondary parameter representing the efficacy index of the drug was calculated, which is termed as the tumor static concentration (TSC). TSC values derived from all four of the models were compared with each other, and with literature reported values, to assess the performance of these models. Subsequently, using the optimized PK/PD model, PD parameters obtained from different cell lines, human PK, and the proposed translational strategy, clinically efficacious doses of T-DM1 were projected. The accuracy of projected efficacious dose range for T-DM1 was verified by comparison with the clinical doses. Aforementioned strategy was then applied to A1mcMMAF for projecting its efficacious concentrations in clinic. TSC values for A1mcMMAF, obtained by fitting TGI data from 4 different xenografts with the proposed PK/PD model, were estimated to range from 0.6 to 11.5 μg mL⁻¹. Accordingly, the clinically efficacious doses for A1mcMMAF were projected retrospectively. All in all, the improved PD model and proposed translational strategy presented here suggest that appropriate correction for the clinical exposure and employing the TSC criterion can help translate mouse TGI data to predict first in human doses of ADCs.

The Antibody Drug Conjugate (ADC) is a therapeutic modality consisting of a monoclonal antibody attached to a cytotoxic, small-molecule payload. The antibody portion of the ADC serves as a transport vehicle that recognizes and binds to a protein antigen expressed in tumor tissues. The localized delivery and release of the payload within or near malignant cells allows for targeted delivery of a potent cytotoxic agent to diseased tissue, while reducing damage to antigen-negative, normal tissues. Recent years have witnessed an explosive increase in ADC-based therapies, due mainly to clinical reports of activity in both hematologic and epithelial cancers. Accompanying this upsurge in ADC development is a renewed interest in natural product cytotoxins, which are typically highly potent cell-killing agents, but suffer from poor drug-like properties and narrow safety margins when systemically administered as conventional chemotherapeutics. In this review, we discuss recent advances related to the construction of ADCs, the optimization of ADC safety and efficacy, and the increasingly pivotal roles of natural product payloads in the current and future landscape of ADC therapy.

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.

Conventional monoclonal antibody (mAb) therapeutics interfering with cellular signaling of their respective target antigens are frequently limited in their ability to induce significant anti-tumor activities when administered as single agents in patients with solid tumors. To overcome these limitations, several new technologies are being developed to empower biotherapeutics and to improve their anti-tumor activities, while maintaining their high tumor selectivity and superior safety profiles. The various efficacy enhancement technologies developed for mAbs can be divided broadly into two categories: First, technologies that improve the intrinsic anti-tumor activities of conventional immunoglobulin mAb formats, including the enhancement of effector cell functions and modulations of target binding properties, including interference with multiple signaling pathways. The second category of empowered biologics combines complementary anti-tumor modalities independent of the IgG format, including antibody drug conjugates (ADCs). In addition, bispecific compounds designed to recruit different subsets of inflammatory cells to the tumor environment, also belong to the mechanistic complementation strategy. This approach termed redirected immune cell killing, belongs to one the most promising new biotherapeutic platforms developed in oncology. Over 20 bispecific compounds are currently being developed pre-clinically, and several compounds are undergoing early stage clinical trials. In this report, we review the progress made in the development of bispecific biotherapeutics in the context of ADCs, redirected T- and B-cell killing and targeting of multiple signaling pathways. We also discuss the status of the clinical development of this class of compounds in oncology and the promises and challenges this field is currently facing., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Vascular endothelial growth factor and its receptors, FLK1/KDR and FLT1, are key regulators of angiogenesis. Unlike FLK1/KDR, the role of FLT1 has remained elusive. FLT1 is produced as soluble (sFLT1) and full-length isoforms. Here, we show that pericytes from multiple tissues produce sFLT1. To define the biologic role of sFLT1, we chose the glomerular microvasculature as a model system. Deletion of Flt1 from specialized glomerular pericytes, known as podocytes, causes reorganization of their cytoskeleton with massive proteinuria and kidney failure, characteristic features of nephrotic syndrome in humans. The kinase-deficient allele of Flt1 rescues this phenotype, demonstrating dispensability of the full-length isoform. Using cell imaging, proteomics, and lipidomics, we show that sFLT1 binds to the glycosphingolipid GM3 in lipid rafts on the surface of podocytes, promoting adhesion and rapid actin reorganization. sFLT1 also regulates pericyte function in vessels outside of the kidney. Our findings demonstrate an autocrine function for sFLT1 to control pericyte behavior., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Introduction: Despite the progress made in the past 20 years in understanding the molecular events leading to the formation of cancer, the success of targeted antitumor agents in solid tumors has lagged behind the scientific discoveries. The most difficult to treat patient segments are those with refractory solid tumors, resistant to standard chemotherapy, and novel therapeutic compounds with improved therapeutic indexes are needed. Antibody drug conjugates (ADCs) are poised to become an important class of cancer therapeutics, as evidenced by the promising objective response rates when administered as single agents to chemorefractory cancer patients., Areas Covered: The basic concept for ADCs is to combine the strengths of the two most successful classes of therapeutic compounds developed in oncology, the high selectivity of antibodies with the unrivaled potency of small molecules, with the goal to improve the therapeutic index. Currently, approximately 60 ADCs are being developed in oncology. Among them, about 20 are undergoing clinical testing, the majority of which are tubulin inhibitor-based immunoconjugates. Herein, we review ADCs targeting solid tumors, with the focus on 11 programs currently undergoing clinical development., Expert Opinion: Key challenges the ADC field is facing, including potency and safety, can be addressed effectively by introducing novel research concepts with transformational potential for ADC development.

The immunotherapy of Hodgkin's lymphoma (HL) has been particularly challenging because of the unique features of tumor intrinsic and host mediated factors, interfering with the antitumor activities of therapeutic antibodies. Despite a wide array of compounds tested successfully in preclinical studies, immunotherapy in HL patients resulted in only limited success when compared to the significant improvements in patient survival provided by chemotherapeutic agents. Antibody-drug conjugates (ADCs) may surmount the restrictions posed by the unique pathobiology of HL tumors as they combine the selective tumor targeting of monoclonal antibodies with the potent anti-neoplastic activities of cytotoxic drugs. In early clinical trials, this class of compounds induced robust antitumor effects in patients with relapsed or refractory lymphoproliferative diseases, in the absence of overt toxicities, while naked antibodies failed to induce therapeutic benefit. Here we review some of the unique features of HL tumor biology and the key advantages of ADC-based lymphoma therapies, which may ultimately account for the improved therapeutic benefit provided by ADCs compared to first generation immunotherapeutics tested in HL patients., (2010 Elsevier Inc. All rights reserved.)

Vascular endothelial growth factor A (VEGF-A) is a well-characterized regulator of physiological and pathological angiogenesis. Multiple therapeutic compounds interfering with VEGF-A-regulated signal transduction pathways are currently being developed for the treatment of neoplasias and other malignancies associated with pathological angiogenesis. A major challenge in developing anti-VEGF therapies are tumor intrinsic refractoriness and the emergence of treatment-induced resistance. A variety of molecular and cellular mechanisms contribute to tumor angiogenesis, including the recruitment of bone marrow (BM)-derived endothelial cell progenitors (EPCs) and inflammatory cells to the tumor mass. Among the latter, two types of tumor infiltrating, inflammatory cells were recently identified to mediate refractoriness to anti-VEGF treatment: CD11b + Gr1+ myeloid derived suppressor cells (MDSC) and tumor-associated macrophages (TAMs). In this chapter, we review some of the inflammatory components regulating tumor angiogenesis and their roles in mediating refractoriness toward anti-VEGF treatment. In addition, we discuss potential therapeutic strategies targeting angiogenic pathways regulated by inflammatory cells. A better understanding of the biological and molecular events involved in mediating refractoriness to anti-VEGF treatment may help to further improve therapeutic strategies targeting tumor angiogenesis.

Rheumatoid arthritis (RA) is characterized by inflammation and cellular proliferation in the synovial lining of joints that result in cartilage and bone destruction. Although the etiology of RA is unclear, activated lymphocytes and proinflammatory molecules, in particular TNF superfamily members, have been implicated in the disease pathology. A TNF superfamily member, CD70, is found on activated lymphocytes and shown to be important in memory and effector responses of lymphocytes. CD70 is expressed at high levels on chronically activated T cells in patients with autoimmune disorders, including RA. The involvement of CD70 in the progression of RA, however, remains unknown. In this study, we report effects of targeting CD70 on disease pathogenesis by using an anti-mouse CD70 Ab in a murine model of collagen-induced arthritis (CIA). In addition to blocking CD70 binding to its receptor CD27, the anti-CD70 Ab used also engages Fc-dependent effector functions including Ab-dependent cellular cytotoxicity, phagocytosis, and complement fixation. Treatment of mice with anti-CD70 Ab both before the onset or after the established disease in CIA model resulted in marked improvements in disease severity and significant reduction in the production of autoantibodies. Histopathological analyses of the joints of mice revealed a substantial reduction of inflammation, and bone and cartilage destruction in response to the anti-CD70 Ab treatment. These results uncover a novel role for CD27-CD70 interactions in the regulation of in vivo inflammatory response leading to arthritis, and provide a molecular basis to support the rationale for anti-CD70 therapy for autoimmune and inflammatory diseases.

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-alpha, cell lines were developed and applied to examine the in vivo antitumor activity. In vitro, lintuzumab significantly reduced the production of TNFalpha-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.

Reducing the blood supply of tumors is one modality to combat cancer. Monoclonal antibodies are now established as a key therapeutic approach for a range of diseases. Owing to the ability of antibodies to selectively target endothelial cells within the tumor vasculature, vascular targeting programs have become a mainstay in oncology drug development. However, the antitumor activity of single agent administration of conventional anti-angiogenic compounds is limited and the improvements in patient survival are most prominent in combinations with chemotherapy. Furthermore, prolonged treatment with conventional anti-angiogenic drugs is associated with toxicity and drug resistance. These circumstances provide a strong rationale for novel approaches to enhance the efficacy of mAbs targeting tumor vasculature such as antibody-drug conjugates (ADCs).Here, we review trends in the development of ADCs targeting tumor vasculature with the aim of informing future research and development of this class of therapeutics.

Despite major advances in the treatment of non-Hodgkin lymphoma (NHL), including the use of chemotherapeutic agents and the anti-CD20 antibody rituximab, the majority of patients eventually relapse, and salvage treatments with non-cross-resistant compounds are needed to further improve patient survival. Here, we evaluated the antitumor effects of the microtubule destabilizing agent monomethyl auristatin E (MMAE) conjugated to the humanized anti-CD19 antibody hBU12 via a protease-sensitive valine-citrulline (vc) dipeptide linker. hBU12-vcMMAE induced potent tumor cell killing against rituximab-sensitive and -resistant NHL cell lines. CD19 can form heterodimers with CD21, and high levels of CD21 were reported to interfere negatively with the activity of CD19-targeted therapeutics. However, we observed comparable internalization, intracellular trafficking, and drug release in CD21(low) and CD21(high), rituximab-sensitive and -refractory lymphomas treated with hBU12-vcMMAE. Furthermore, high rates of durable regressions in mice implanted with these tumors were observed, suggesting that both rituximab resistance and CD21 expression levels do not impact on the activity of hBU12-vcMMAE. Combined, our data suggest that hBU12-vcMMAE may represent a promising addition to the treatment options for rituximab refractory NHL and other hematologic malignancies, including acute lymphoblastic leukemia.

Limb development constitutes a central model for the study of tissue and organ patterning; yet, the mechanisms that regulate the patterning of limb vasculature have been left understudied. Vascular patterning in the forming limb is tightly regulated in order to ensure sufficient gas exchange and nutrient supply to the developing organ. Once skeletogenesis is initiated, limb vasculature undergoes two seemingly opposing processes: vessel regression from regions that undergo mesenchymal condensation; and vessel morphogenesis. During the latter, vessels that surround the condensations undergo an extensive rearrangement, forming a stereotypical enriched network that is segregated from the skeleton. In this study, we provide evidence for the centrality of the condensing mesenchyme of the forming skeleton in regulating limb vascular patterning. Both Vegf loss- and gain-of-function experiments in limb bud mesenchyme firmly established VEGF as the signal by which the condensing mesenchyme regulates the vasculature. Normal vasculature observed in limbs where VEGF receptors Flt1, Flk1, Nrp1 and Nrp2 were blocked in limb bud mesenchyme suggested that VEGF, which is secreted by the condensing mesenchyme, regulates limb vasculature via a direct long-range mechanism. Finally, we provide evidence for the involvement of SOX9 in the regulation of Vegf expression in the condensing mesenchyme. This study establishes Vegf expression in the condensing mesenchyme as the mechanism by which the skeleton patterns limb vasculature.

A network of capillaries branches from the hyaloid vascular system and surrounds the mammalian lens throughout much of its embryonic development. These vessels are presumed to be important for the growth and maturation of the lens, although the lenses of non-mammalian vertebrates have no comparable vessels. Over expression of VEGF-A in the lens increases the extent of these capillaries, but it is not known whether VEGF-A from the lens is necessary for their formation or survival. To address this question, we deleted Vegfa in the lens. This prevented the formation of the capillary networks adjacent to the lens capsule, but did not alter nearby hyaloid vessels at the surface of the retina. Postnatal lenses lacking Vegfa were smaller than wild type and, by 1 month of age, many had mild nuclear opacities. These opacities regressed with age. The lens is hypoxic throughout most of life and VEGF-A expression is often regulated by the transcription factor, hypoxia inducible factor-1. Lenses lacking Hif1a were of apparently normal size, had markedly reduced levels of mRNA for VEGF-A and glyceraldehyde-3-phosphate dehydrogenase, but had normal-appearing capillaries covering their surface. We conclude that VEGF-A from the lens is necessary for the formation of the normal hyaloid vascular system and that lack of these capillaries was the most likely cause of growth retardation during fetal and early postnatal lens development. In the absence of HIF-1 function, sufficient VEGF-A is produced by the lens to promote capillary formation. Further study is needed to explain the formation of the mild opacities seen in some lenses lacking Vegfa and their regression later in life.

SGN-40 is a therapeutic antibody targeting CD40, which induces potent anti-lymphoma activities via direct apoptotic signalling cells and by cell-mediated cytotoxicity. Here we show antibody-dependent cellular phagocytosis (ADCP) by macrophages to contribute significantly to the therapeutic activities and that the antitumour effects of SGN-40 depend on Fc interactions.

Purpose: The antitubulin agent monomethyl auristatin F (MMAF) induces potent antitumor effects when conjugated via protease cleavable linkers to antibodies targeting internalizing, tumor-specific cell surface antigens. Humanized 1F6 (h1F6) is a humanized monoclonal antibody targeting CD70, a member of the tumor necrosis factor family that is expressed on hematologic malignancies and carcinomas. Here, we tested h1F6-maleimidocaproyl (mc) MMAF conjugates, consisting of an uncleavable mc linker, for their ability to interfere with the growth of CD70-positive carcinomas., Experimental Design: To evaluate the optimal drug per antibody ratio, we conjugated either four or eight MMAF molecules to the cysteines that comprise the interchain disulfides of h1F6 and determined antitumor activities in vitro and in xenografted mice. The tumor types tested included glioblastoma, patient-derived renal cell carcinoma (RCC) cell isolates, and standard RCC tumor cell lines., Results: All h1F6-mcMMAF conjugates potently interfered with the growth of all carcinomas in vitro and resulted in complete responses of RCC tumors implanted orthotopically or s.c. in mice. In vitro, h1F6-mcMMAF(8) was generally more potent than h1F6-mcMMAF(4). However, h1F6-mcMMAF(4) displayed equal or better efficacy than h1F6-mcMMAF(8) when administered to tumor-bearing mice., Conclusions: We showed that h1F6-mcMMAF conjugates inhibited the growth of human carcinomas and that increased drug loading, while improving potency in vitro, did not substantially affect the pharmacodynamic and pharmacokinetic properties in vivo. Based on these findings, h1F6-mcMMAF(4), designated SGN-75, has been identified as a potential antibody-drug conjugate for clinical development.

An anti-CD70 antibody conjugated to monomethylauristatin F (MMAF) via a valine-citrulline dipeptide containing linker has been shown previously to have potent antitumor activity in renal cell cancer xenograft studies. Here, we generated a panel of humanized anti-CD70 antibody IgG variants and conjugated them to MMAF to study the effect of isotype (IgG1, IgG2, and IgG4) and Fcgamma receptor binding on antibody-drug conjugate properties. All IgG variants bound CD70+ 786-O cells with an apparent affinity of approximately 1 nmol/L, and drug conjugation did not impair antigen binding. The parent anti-CD70 IgG1 bound to human FcgammaRI and FcgammaRIIIA V158 and mouse FcgammaRIV and this binding was not impaired by drug conjugation. In contrast, binding to these Fcgamma receptors was greatly reduced or abolished in the variant, IgG1v1, containing the previously described mutations, E233P:L234V:L235A. All conjugates had potent cytotoxic activity against six different antigen-positive cancer cell lines in vitro with IC50 values of 30 to 540 pmol/L. The IgGv1 conjugate with MMAF displayed improved antitumor activity compared with other conjugates in 786-O and UMRC3 models of renal cell cancer and in the DBTRG05-MG glioblastoma model. All conjugates were tolerated to > or =40 mg/kg in mice. Thus, the IgG1v1 MMAF conjugate has an increased therapeutic index compared with the parent IgG1 conjugate. The improved antitumor activity of the IgG1v1 auristatin conjugates may relate to increased exposure as suggested by pharmacokinetic analysis. The strategy used here for enhancing the therapeutic index of antibody-drug conjugates is independent of the antigen-binding variable domains and potentially applicable to other antibodies.

Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with cutaneous hyperreactivity to environmental stimuli, resulting in increased infiltration of inflammatory cells, IgE production and enhanced expression of various co-stimulatory molecules, Th2 cytokines and chemokines. Antigen presenting cells (APCs) are critical for AD disease pathogenesis and interaction between APCs and inflammatory T cells represents an important signal required for induction and maintenance of the inflammatory process. CD40 was shown to be upregulated on inflammatory cells in patients with atopic dermatitis; however the identity of these cells and their correlation with disease severity remained unknown. To address these questions, we determined CD40 expression in skin lesions and on peripheral blood cells from AD patients and identified a positive correlation between the numbers of CD40 positive cells and disease severity. Furthermore, we identified the nature of CD40 positive cells in skin lesions to include CD1a+ and CD11b+ APCs. Finally, a correlation between serum PARC and IgE levels and the numbers of CD40+ cells in AD skin lesions was found. Combined, these findings demonstrate that CD40 is upregulated on APCs, cell types previously shown to contribute to AD disease pathology, and suggest that therapeutic strategies targeting CD40 positive cells may provide benefit to AD patients.

Anti-CD30 diabodies were engineered with two cysteine mutations for site-specific drug conjugation in each chain of these homodimeric antibody fragments. Diabodies were conjugated with approximately 4 equivalents of the anti-tubulin drugs, monomethyl auristatin E or F, via a protease-cleavable dipeptide linker, to create the conjugates, diabody-vcE4 and diabody-vcF4, respectively. Diabody conjugation had only minor (<3-fold) effects on antigen binding. Diabody-vcF4 was potently cytotoxic against the antigen-positive cell lines, Karpas-299 (34 pmol/L IC(50)) and L540cy (22 pmol/L IC(50)), and was 8- and 21-fold more active than diabody-vcE4 against these cell lines, respectively. Clearance of diabody-vcF4 (99-134 mL/d/kg) was 5-fold slower than for the nonconjugated diabody in naive severe combined immunodeficient mice. Diabody-vcF4 had potent and dose-dependent antitumor activity against established Karpas-299 xenografts and gave durable complete responses at well-tolerated doses. Biodistribution experiments with diabody-[(3)H]-vcF4 (0.72-7.2 mg/kg) in tumor-bearing mice showed a dose-dependent increase in total auristatin accumulation in tumors (< or =520 nmol/L) and decrease in relative auristatin accumulation (< or =8.1 %ID/g), with peak localization at 4 to 24 h after dosing. Diabody-vcF4 had approximately 4-fold lower cytotoxic activity than the corresponding IgG1-vcF4 conjugate in vitro. A similar potency difference was observed in vivo despite 25- to 34-fold faster clearance of diabody-vcF4 than IgG1-vcF4. This may reflect that dose-escalated diabody-vcF4 can surpass IgG1-vcF4 in auristatin delivery to tumors, albeit with higher auristatin exposure to some organs including kidney and liver. Diabody-drug conjugates can have potent antitumor activity at well-tolerated doses and warrant further optimization for cancer therapy.

CD133/prominin-1 is a pentaspan transmembrane glycoprotein overexpressed in various solid tumours including colorectal and glioblastomas. CD133 was found here to be highly expressed in >or=50% of pancreatic, gastric and intrahepatic cholangiocarcinomas. Quantitative flow cytometric analysis showed that a panel of established hepatocellular, pancreatic and gastric cancer cell lines expressed CD133 at levels higher than normal epithelial cells or bone marrow progenitor cells. A murine anti-human CD133 antibody (AC133) conjugated to a potent cytotoxic drug, monomethyl auristatin F (MMAF), effectively inhibited the growth of Hep3B hepatocellular and KATO III gastric cancer cells in vitro with IC(50) values of 2-7 ng ml(-1). MMAF induced apoptosis in the cancer cells as measured by caspase activation. The anti-CD133-drug conjugate (AC133-vcMMAF) was shown to internalise and colocalised with the lysosomal marker CD107a in the sensitive cell lines. In contrast, in the resistant cell line Su.86.86, the conjugate internalised and colocalised with the caveolae marker, Cav-1. Addition of ammonium chloride, an inhibitor of lysosomal trafficking and processing, suppressed the cytotoxic effect of AC133-vcMMAF in both Hep3B and KATO III. Anti-CD133-drug conjugate treatment resulted in significant delay of Hep3B tumour growth in SCID mice. Anti-CD133 antibody-drug conjugates warrant further evaluation as a therapeutic strategy to eradicate CD133+ tumours.

The antibody-drug conjugate (ADC) cAC10-vcMMAE consists of the tubulin inhibitor monomethyl auristatin E (MMAE) conjugated to the chimeric anti-CD30 monoclonal antibody cAC10. This ADC potently interferes with the growth of CD30-positive haematological tumours, including Hodgkin lymphoma (HL) and anaplastic large-cell lymphoma. This study found improved antitumour activity in a preclinical model of HL when SGN-35 was combined with chemotherapeutic regimens such as ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine) or gemcitabine. Improved efficacy was also observed in high tumour burden models, indicating that combining ADCs with chemotherapeutic agents may be advantageous for the treatment of patients with relapsed or refractory HL.

The glomerular microvasculature is particularly susceptible to injury in thrombotic microangiopathy, but the mechanisms by which this occurs are unclear. We report the cases of six patients who were treated with bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), in whom glomerular disease characteristic of thrombotic microangiopathy developed. To show that local reduction of VEGF within the kidney is sufficient to trigger the pathogenesis of thrombotic microangiopathy, we used conditional gene targeting to delete VEGF from renal podocytes in adult mice; this resulted in a profound thrombotic glomerular injury. These observations provide evidence that glomerular injury in patients who are treated with bevacizumab is probably due to direct targeting of VEGF by antiangiogenic therapy., (Copyright 2008 Massachusetts Medical Society.)