A Multispecific Anti-CD40 DARPin Construct Induces Tumor-Selective CD40 Activation and Tumor Regression.

The CD40 receptor is an attractive target for cancer immunotherapy. Although a modest pharmacodynamic effect is seen in patients following administration of CD40-targeting monoclonal antibodies (mAb), the doses that could be safely administered do not result in a meaningful clinical response, most likely due to the limited therapeutic window associated with systemic CD40 activation. To overcome this issue, we developed a multispecific DARPin construct, α-FAPxCD40, which has conditional activity at the site of disease. α-FAPxCD40 activation of CD40 depends on binding to fibroblast activation protein (FAP), a cell-surface protease overexpressed in the stroma of solid tumors. In vitro studies demonstrated that α-FAPxCD40 potently activates human antigen-presenting cells in the presence, but not in the absence, of FAP-positive cells. After intravenous injection, a murine surrogate construct (α-mFAPxCD40) accumulated in FAP-positive tumors, elicited rejection of 88% of these tumors, and induced memory antitumor immunity. Importantly, in contrast to the mouse anti-CD40 tested in parallel, the in vivo antitumor activity of α-mFAPxCD40 was associated neither with elevated blood cytokines nor with hepatotoxicity, both of which contribute to the clinical dose-limiting toxicities of several CD40 mAb. This study demonstrates that α-(m)FAPxCD40 engages CD40 in an FAP-restricted manner, leading to tumor eradication without signs of peripheral toxicity. This distinct preclinical profile suggests that a favorable therapeutic index may be achieved in humans. It further supports the development of α-FAPxCD40, currently tested in a first-in-human clinical study in patients with solid tumors (NCT05098405).

Antibody drug conjugates for treatment of breast cancer: Novel targets and diverse approaches in ADC design.

Breast cancer is a heterogeneous group of malignancies with a spectrum of molecular subtypes, pathologies and outcomes that together comprise the most common non-cutaneous cancer in women. Currently, over 80% of breast cancer patients are diagnosed at relatively early stages of disease where there are encouraging data on outcomes and long term survival. However, there is currently no curative option for those patients with metastatic disease and there is a substantial medical need to identify effective and safe treatment options for these patients. One approach to improve cancer therapy is by designing therapeutics directed against targets with differential levels of expression on malignant versus normal cells with the goal of improving tumor selectivity and reducing damage to normal tissues. Antibody drug conjugates (ADCs) are a rapidly evolving therapeutic class that exploits the target-selectivity of monoclonal antibodies (MAbs) to deliver cytotoxic drugs to antigen-expressing cells (Lambert & Morris, 2017; Senter, 2009; Thomas, Teicher, & Hassan, 2016; Trail, 2013). The regulatory approval of ADCs for both hematologic malignancies (brentuximab vedotin) (Younes et al., 2010) and solid tumors (ado-trastuzumab emtansine) (Amiri-Kordestani et al., 2014; Verma et al., 2012) clearly demonstrates the clinical potential of ADCs. This review will focus on targets under consideration for breast cancer directed ADCs and on the technology modifications being considered to improve ADC efficacy and safety.

Preclinical Activity of the Novel Anti-Prolactin Receptor (PRLR) Antibody-Drug Conjugate REGN2878-DM1 in PRLR-Positive Breast Cancers.

The Prolactin Receptor (PRLR) is a type 1 cytokine receptor that is expressed in a subset of breast cancers and may contribute to its pathogenesis. It is relatively overexpressed in approximately 25% of human breast tumors while expressed at low levels in some normal human tissues including the mammary gland. We developed an anti-PRLR antibody-drug conjugate (ADC), to target PRLR-positive breast cancer. REGN2878-DM1 is comprised of a fully human high-affinity function-blocking anti-PRLR IgG1 antibody (REGN2878) conjugated via a noncleavable SMCC linker to the cytotoxic maytansine derivative DM1. Both unconjugated REGN2878 and conjugated REGN2878-DM1 block PRL-mediated activation in vitro and are rapidly internalized into lysosomes. REGN2878-DM1 induces potent cell-cycle arrest and cytotoxicity in PRLR-expressing tumor cell lines. In vivo, REGN2878-DM1 demonstrated significant antigen-specific antitumor activity against breast cancer xenograft models. In addition, REGN2878-DM1 showed additive activity when combined with the antiestrogen agent fulvestrant. These results illustrate promising antitumor activity against PRLR-positive breast cancer xenografts and support the evaluation of anti-PRLR ADCs as potential therapeutic agents in breast cancer. Mol Cancer Ther; 16(7); 1299-311. ©2017 AACR.

A Phase I First-in-Human Study of Nesvacumab (REGN910), a Fully Human Anti-Angiopoietin-2 (Ang2) Monoclonal Antibody, in Patients with Advanced Solid Tumors.

PURPOSE: Nesvacumab (REGN910) is a fully human immunoglobulin G1 (IgG1) monoclonal antibody that specifically binds and inactivates the Tie2 receptor ligand Ang2 with high affinity, but shows no binding to Ang1. The main objectives of this trial were to determine the safety, tolerability, dose-limiting toxicities (DLT), and recommended phase II dose (RP2D) of nesvacumab. EXPERIMENTAL DESIGN: Nesvacumab was administered intravenously every two weeks with dose escalations from 1 to 20 mg/kg in patients with advanced solid tumors. RESULTS: A total of 47 patients were treated with nesvacumab. No patients in the dose escalation phase experienced DLTs, therefore a maximum tolerated dose (MTD) was not reached. The most common nesvacumab-related adverse events were fatigue (23.4%), peripheral edema (21.3%), decreased appetite, and diarrhea (each 10.6%; all grade ≤ 2). Nesvacumab was characterized by linear kinetics and had a terminal half-life of 6.35 to 9.66 days in a dose-independent manner. Best response by RECIST 1.1 in 43 evaluable patients included 1 partial response (adrenocortical carcinoma) of 24 weeks duration. Two patients with hepatocellular carcinoma had stable disease (SD) > 16 weeks, with tumor regression and >50% decrease in α-fetoprotein. Analyses of putative angiogenesis biomarkers in serum and tumor biopsies were uninformative for treatment duration. CONCLUSIONS: Nesvacumab safety profile was acceptable at all dose levels tested. Preliminary antitumor activity was observed in patients with treatment-refractory advanced solid tumors. On the basis of cumulative safety, antitumor activity, pharmacokinetic and pharmacodynamic data, the 20 mg/kg dose was determined to be the RP2D.

Therapeutic mechanism and efficacy of the antibody-drug conjugate BAY 79-4620 targeting human carbonic anhydrase 9.

Carbonic anhydrase IX (CAIX) is a cell surface glycoprotein that is expressed in many different tumors and yet restricted in normal tissues to the gastrointestinal tract. It is upregulated by hypoxia and correlates with tumor grade and poor survival in several tumor indications. Monoclonal antibodies (mAb) with single digit nanomolar binding affinity for CAIX were derived by panning with the recombinant ectodomain of CAIX against the MorphoSys HUCAL Gold library of human Fabs. Highest affinity Fabs were converted to full-length IgGs and subjected to further characterization based upon their avidity and selectivity for CAIX, their capacity to undergo internalization in CAIX-expressing cell lines, and their selective localization to CAIX-positive human xenografted tumors when administered to mice as fluorescent conjugates. Through this selection process, the 3ee9 mAb was identified, which upon conjugation to monomethyl auristatin E through a self-immolative enzyme-cleavable linker yielded the potent and selective CAIX antibody-drug conjugate CAIX-ADC (BAY 79-4620). In preclinical human xenograft models in mice representing several tumor indications, BAY 79-4620 showed potent antitumor efficacy and in some models showed partial and complete tumor shrinkage even following a single dose. The mechanism of action was shown by histology to involve the sequelae of events typical of antitubulin agents. Efficacy in murine preclinical models correlated semiquantitatively, with CAIX expression levels as determined by immunohistochemistry and ELISA. These preclinical data collectively support the development of BAY 79-4620 for the treatment of cancer patients with CAIX overexpressing tumors.

Dual IGF-I/II-neutralizing antibody MEDI-573 potently inhibits IGF signaling and tumor growth.

Insulin-like growth factors (IGF), IGF-I and IGF-II, are small polypeptides involved in regulating cell proliferation, survival, differentiation, and transformation. IGF activities are mediated through binding and activation of IGF-1R or insulin receptor isoform A (IR-A). The role of the IGF-1R pathway in promoting tumor growth and survival is well documented. Overexpression of IGF-II and IR-A is reported in multiple types of cancer and is proposed as a potential mechanism for cancer cells to develop resistance to IGF-1R-targeting therapy. MEDI-573 is a fully human antibody that neutralizes both IGF-I and IGF-II and inhibits IGF signaling through both the IGF-1R and IR-A pathways. Here, we show that MEDI-573 blocks the binding of IGF-I and IGF-II to IGF-1R or IR-A, leading to the inhibition of IGF-induced signaling pathways and cell proliferation. MEDI-573 significantly inhibited the in vivo growth of IGF-I- or IGF-II-driven tumors. Pharmacodynamic analysis demonstrated inhibition of IGF-1R phosphorylation in tumors in mice dosed with MEDI-573, indicating that the antitumor activity is mediated via inhibition of IGF-1R signaling pathways. Finally, MEDI-573 significantly decreased (18)F-fluorodeoxyglucose ((18)F-FDG) uptake in IGF-driven tumor models, highlighting the potential utility of (18)F-FDG-PET as a noninvasive pharmacodynamic readout for evaluating the use of MEDI-573 in the clinic. Taken together, these results demonstrate that the inhibition of IGF-I and IGF-II ligands by MEDI-573 results in potent antitumor activity and offers an effective approach to selectively target both the IGF-1R and IR-A signaling pathways.

Sorafenib (BAY 43-9006) inhibits tumor growth and vascularization and induces tumor apoptosis and hypoxia in RCC xenograft models.

PURPOSE: New research findings have revealed a key role for vascular endothelial growth factor (VEGF) in the stimulation of angiogenesis in clear cell renal carcinoma (RCC) which is a highly vascularized and treatment-resistant tumor. Sorafenib (BAY 43-9006, Nexavar) is a multi-kinase inhibitor which targets receptor tyrosine and serine/threonine kinases involved in tumor progression and tumor angiogenesis. The effect of sorafenib on tumor growth and tumor histology was assessed in both ectopic and orthotopic mouse models of RCC. METHODS: Sorafenib was administered orally to mice bearing subcutaneous (SC, ectopic) or sub-renal capsule (SRC, orthotopic) tumors of murine (Renca) or human (786-O) RCC. Treatment efficacy was determined by measurements of tumor volume and tumor growth delay. In mechanism of action studies, using the 786-O and Renca RCC tumor models, the effect of sorafenib was assessed after dosing for 3 or 5 days in the SC models and 21 days in the SRC models. Inhibition of tumor angiogenesis was assessed by measuring level of CD31 and alpha-smooth muscle actin (alphaSMA) staining by immunohistochemistry (IHC). The effect of sorafenib on MAPK signaling, cell cycle progression and cell proliferation was also assessed by IHC by measuring levels of phospho-ERK, phospho-histone H3 and Ki-67 staining, respectively. The extent of tumor apoptosis was measured by terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assays. Finally, the effects of sorafenib on tumor hypoxia was assessed in 786-O SC model by injecting mice intravenously with pimonidazole hydrochloride 1 h before tumor collection and tumor sections were stained with a FITC-conjugated Hypoxyprobe antibody. RESULTS: Sorafenib produced significant tumor growth inhibition (TGI) and a reduction in tumor vasculature of both ectopic and orthotopic Renca and 786-O tumors, at a dose as low as 15 mg/kg when administered daily. Inhibition of tumor vasculature was observed as early as 3 days post-treatment, and this inhibition of angiogenesis correlated with increased level of tumor apoptosis (TUNEL-positive) and central necrosis. Consistent with these results, a significant increase in tumor hypoxia was also observed 3 days post-treatment in 786-O SC model. However, no significant effect of sorafenib on phospho-ERK, phospho-histone H3 or Ki-67 levels in either RCC tumor model was observed. CONCLUSION: Our results show the ability of sorafenib to potently inhibit the growth of both ectopically- and orthotopically-implanted Renca and 786-O tumors. The observed tumor growth inhibition and tumor stasis or stabilization correlated strongly with decreased tumor angiogenesis, which was due, at least in part, to inhibition of VEGF and PDGF-mediated endothelial cell and pericyte survival. Finally, sorafenib-mediated inhibition of tumor growth and angiogenesis occurred at concentrations equivalent to those achieved in patients in the clinic.

Sorafenib (BAY 43-9006, Nexavar), a dual-action inhibitor that targets RAF/MEK/ERK pathway in tumor cells and tyrosine kinases VEGFR/PDGFR in tumor vasculature.

Activating mutations in Ras and B-RAF were identified in several human cancers. In addition, several receptor tyrosine kinases, acting upstream of Ras, were found either mutated or overexpressed in human tumors. Because oncogenic activation of the Ras/RAF pathway may lead to a sustained proliferative signal resulting in tumor growth and progression, inhibition of this pathway represents an attractive approach for cancer drug discovery. A novel class of biaryl urea that inhibits C-RAF kinase was discovered using a combination of medicinal and combinatorial chemistry approaches. This effort culminated in the identification of the clinical candidate BAY 43-9006 (Sorafenib, Nexavar), which has recently been approved by the FDA for advanced renal cell carcinoma in phase III clinical trials. Sorafenib inhibited the kinase activity of both C-RAF and B-RAF (wild type and V600E mutant). It inhibited MEK and ERK phosphorylation in various cancer cell lines and tumor xenografts and exhibited potent oral antitumor activity in a broad spectrum of human tumor xenograft models. Further characterization of sorafenib revealed that this molecule was a multikinase inhibitor that targeted the vascular endothelial growth factor receptor family (VEGFR-2 and VEGFR-3) and platelet-derived growth factor receptor family (PDGFR-beta and Kit), which play key roles in tumor progression and angiogenesis. Thus, sorafenib may inhibit tumor growth by a dual mechanism, acting either directly on the tumor (through inhibition of Raf and Kit signaling) and/or on tumor angiogenesis (through inhibition of VEGFR and PDGFR signaling). In phase I and phase II clinical trials, sorafenib showed limited side effects and, more importantly, disease stabilization. This agent is currently being evaluated in phase III clinical trials in renal cell and hepatocellular carcinomas.

BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis.

The RAS/RAF signaling pathway is an important mediator of tumor cell proliferation and angiogenesis. The novel bi-aryl urea BAY 43-9006 is a potent inhibitor of Raf-1, a member of the RAF/MEK/ERK signaling pathway. Additional characterization showed that BAY 43-9006 suppresses both wild-type and V599E mutant BRAF activity in vitro. In addition, BAY 43-9006 demonstrated significant activity against several receptor tyrosine kinases involved in neovascularization and tumor progression, including vascular endothelial growth factor receptor (VEGFR)-2, VEGFR-3, platelet-derived growth factor receptor beta, Flt-3, and c-KIT. In cellular mechanistic assays, BAY 43-9006 demonstrated inhibition of the mitogen-activated protein kinase pathway in colon, pancreatic, and breast tumor cell lines expressing mutant KRAS or wild-type or mutant BRAF, whereas non-small-cell lung cancer cell lines expressing mutant KRAS were insensitive to inhibition of the mitogen-activated protein kinase pathway by BAY 43-9006. Potent inhibition of VEGFR-2, platelet-derived growth factor receptor beta, and VEGFR-3 cellular receptor autophosphorylation was also observed for BAY 43-9006. Once daily oral dosing of BAY 43-9006 demonstrated broad-spectrum antitumor activity in colon, breast, and non-small-cell lung cancer xenograft models. Immunohistochemistry demonstrated a close association between inhibition of tumor growth and inhibition of the extracellular signal-regulated kinases (ERKs) 1/2 phosphorylation in two of three xenograft models examined, consistent with inhibition of the RAF/MEK/ERK pathway in some but not all models. Additional analyses of microvessel density and microvessel area in the same tumor sections using antimurine CD31 antibodies demonstrated significant inhibition of neovascularization in all three of the xenograft models. These data demonstrate that BAY 43-9006 is a novel dual action RAF kinase and VEGFR inhibitor that targets tumor cell proliferation and tumor angiogenesis.

Monoclonal antibody mediated intracellular targeting of tallysomycin S(10b).

The potency of tallysomycin S(10b) (TLM S(10b)) an analogue bleomycin was enhanced by up to 875-fold when it was conjugated to the internalizing antibody BR96. Attachment to the antibody is achieved via a Cathepsin B cleavable linker. The enhancement in potency is believed to be a result of cellular uptake of the conjugate upon antigen binding followed by rapid release of the drug inside the lysosome. This method provides a novel approach for increasing the potency and therapeutic index of nominally moderately-active cytotoxic agents.

BR96 conjugates of highly potent anthracyclines.

The 6-maleimidocaproylhydrazone derivatives of highly potent antitumor agents 5-Diacetoxypentyldoxorubicin and Morpholinodoxorubicin were synthesized and conjugated to monoclonal antibody BR96 and control IgG. Immunoconjugate molar ratios were generally 7.5-8.5, and dimer aggregate levels were low. The linkers released parent drug at lysosomal pH 5, while they remained stable at neutral pH. BR96 conjugates were highly potent and antigen specific in vitro. The BR96-DAPDOX conjugate demonstrated an IC(50) of 0.03 micrometer and was at least 300-fold more potent than a non-binding IgG-DAPDOX control conjugate.

Monoclonal antibody drug immunoconjugates for targeted treatment of cancer.

Monoclonal antibodies (mAb) directed to tumor-associated antigens (TAA) or antigens differentially expressed on the tumor vasculature have been covalently linked to drugs that have different mechanisms of action and various levels of potency. The use of these mAb immunoconjugates to selectively deliver drugs to tumors has the potential to both improve antitumor efficacy and reduce the systemic toxicity of therapy. Several immunoconjugates, particularly those that incorporate internalizing antibodies and tumor-selective linkers, have demonstrated impressive activity in preclinical models. Immunoconjugates that deliver doxorubicin, maytansine and calicheamicin are currently being evaluated in clinical trials. The feasibility of using immunoconjugates as cancer therapeutics has been clearly demonstrated. Gemtuzumab ozogamicin, a calicheamicin conjugate that targets CD33, has recently been approved by the Food and Drug Administration (FDA) for treatment of acute myelogenous leukemia (AML). This review concentrates on the properties of the tumor and the characteristics of the mAb, linker, and drugs that influence the efficacy, potency, and selectivity of immunconjugates selected for cancer treatment.

Monoclonal antibody conjugates of doxorubicin prepared with branched peptide linkers: inhibition of aggregation by methoxytriethyleneglycol chains.

High mole ratio BR96 immunoconjugates were synthesized using branched peptide-doxorubicin linkers designed to liberate doxorubicin following antigen-specific internalization into lysosomes. However, these immunoconjugates are highly prone to noncovalent, dimeric aggregation. We hypothesize that this is due to (1) the hydrophobic nature of the peptides, (2) the loss of positive charge upon amide formation at the 3'-amino group of doxorubicin, and (3) the proximity of the peptide hydrophobic residues to form efficient intermolecular stacking interactions. By introducing a hydrophilic methoxytriethylene glycol chain onto the doxorubicin portion of the branched peptide linkers, aggregation has been eliminated or greatly reduced in the immunoconjugate products. The methoxytriethylene glycol chain was linked to the doxorubicin moiety of the linker via a hydrazone bond that is stable at pH 7 but hydrolyzes rapidly at pH 5 to release free drug. BR96 immunoconjugates synthesized from methoxytriethylene glycol-modified branched peptide-doxorubicin linkers are highly potent and immunospecific in vitro. The data suggest that the methoxytriethylene glycol chain hydrolyzes as designed upon antigen-specific internalization into tumor lysosomes in vitro, where enzymatic degradation of the peptide linker releases free doxorubicin.

Cathepsin B-labile dipeptide linkers for lysosomal release of doxorubicin from internalizing immunoconjugates: model studies of enzymatic drug release and antigen-specific in vitro anticancer activity.

The anticancer drug doxorubicin (DOX) has been linked to chimeric BR96, an internalizing monoclonal antibody that binds to a Lewis(y)-related, tumor-associated antigen, through two lysosomally cleavable dipeptides, Phe-Lys and Val-Cit, giving immunoconjugates 72 and 73. A self-immolative p-aminobenzyloxycarbonyl (PABC) spacer between the dipeptides and the DOX was required for rapid and quantitative generation of free drug. DOX release from model substrate Z-Phe-Lys-PABC-DOX 49 was 30-fold faster than from Z-Val-Cit-PABC-DOX 42 with the cysteine protease cathepsin B alone, but rates were identical in a rat liver lysosomal preparation suggesting the participation of more than one enzyme. Conjugates 72 and 73 showed rapid and near quantitative drug release with cathepsin B and in a lysosomal preparation, while demonstrating excellent stability in human plasma. Against tumor cell lines with varying levels of BR96 expression, both conjugates showed potent, antigen-specific cytotoxic activity, suggesting that they will be effective in delivering DOX selectively to antigen-expressing carcinomas.

Doxorubicin immunoconjugates containing bivalent, lysosomally-cleavable dipeptide linkages.

Bivalent doxorubicin (DOX)-dipeptides (16a-c) were prepared and conjugated to the monoclonal antibody BR96. The dipeptides are cleaved by lysosomal proteases following internalization of the resulting immunoconjugates. Conjugate 18b demonstrated antigen-specific in vitro tumor cell killing activity (IC(50)=0.2 microM) that was equipotent to DOX with a near doubling of drug molecules/MAb. Size exclusion chromatography showed 18b to be a noncovalent dimer that was formed immediately upon conjugation.

Synthesis of an immunoconjugate of camptothecin.

The first immunoconjugate of camptothecin has been synthesized wherein the drug is attached to the tumor-recognizing antibody BR96 via a Cathepsin B cleavable linker. Endocytosis of the immunoconjugate upon binding to the tumor cell followed by enzymatic cleavage of the linker inside the endosome ensures tumor-specific release of the drug. In this way, it is hoped that the dose-limiting side effects associated with camptothecin can be eliminated while the antitumor activity is preserved.