Structure-guided design fine-tunes pharmacokinetics, tolerability, and antitumor profile of multispecific frizzled antibodies.

Aberrant activation of Wnt/ß-catenin signaling occurs frequently in cancer. However, therapeutic targeting of this pathway is complicated by the role of Wnt in stem cell maintenance and tissue homeostasis. Here, we evaluated antibodies blocking 6 of the 10 human Wnt/Frizzled (FZD) receptors as potential therapeutics. Crystal structures revealed a common binding site for these monoclonal antibodies (mAbs) on FZD, blocking the interaction with the Wnt palmitoleic acid moiety. However, these mAbs displayed gastrointestinal toxicity or poor plasma exposure in vivo. Structure-guided engineering was used to refine the binding of each mAb for FZD receptors, resulting in antibody variants with improved in vivo tolerability and developability. Importantly, the lead variant mAb significantly inhibited tumor growth in the HPAF-II pancreatic tumor xenograft model. Taken together, our data demonstrate that anti-FZD cancer therapeutic antibodies with broad specificity can be fine-tuned to navigate in vivo exposure and tolerability while driving therapeutic efficacy.

Cellular determinants for preclinical activity of a novel CD33/CD3 bispecific T-cell engager (BiTE) antibody, AMG 330, against human AML.

CD33 is a valid target for acute myeloid leukemia (AML) but has proven challenging for antibody-drug conjugates. Herein, we investigated the cellular determinants for the activity of the novel CD33/CD3-directed bispecific T-cell engager antibody, AMG 330. In the presence of T cells, AMG 330 was highly active against human AML cell lines and primary AML cells in a dose- and effector to target cell ratio-dependent manner. Using cell lines engineered to express wild-type CD33 at increased levels, we found a quantitative relationship between AMG 330 cytotoxicity and CD33 expression; in contrast, AMG 330 cytotoxicity was neither affected by common CD33 single nucleotide polymorphisms nor expression of the adenosine triphosphate-binding cassette (ABC) transporter proteins, P-glycoprotein or breast cancer resistance protein. Unlike bivalent CD33 antibodies, AMG 330 did not reduce surface CD33 expression. The epigenetic modifier drugs, panobinostat and azacitidine, increased CD33 expression in some cell lines and augmented AMG 330-induced cytotoxicity. These findings demonstrate that AMG 330 has potent CD33-dependent cytolytic activity in vitro, which can be further enhanced with other clinically available therapeutics. As it neither modulates CD33 expression nor is affected by ABC transporter activity, AMG 330 is highly promising for clinical exploration as it may overcome some limitations of previous CD33-targeted agents.

Dose-related differences in the pharmacodynamic and toxicologic response to a novel hyperglycosylated analog of recombinant human erythropoietin in Sprague-Dawley rats with similarly high hematocrit.

We recently reported results that erythropoiesis-stimulating agent (ESA)-related thrombotic toxicities in preclinical species were not solely dependent on a high hematocrit (HCT) but also associated with increased ESA dose level, dose frequency, and dosing duration. In this article, we conclude that sequelae of an increased magnitude of ESA-stimulated erythropoiesis potentially contributed to thrombosis in the highest ESA dose groups. The results were obtained from two investigative studies we conducted in Sprague-Dawley rats administered a low (no thrombotic toxicities) or high (with thrombotic toxicities) dose level of a hyperglycosylated analog of recombinant human erythropoietin (AMG 114), 3 times weekly for up to 9 days or for 1 month. Despite similarly increased HCT at both dose levels, animals in the high-dose group had an increased magnitude of erythropoiesis measured by spleen weights, splenic erythropoiesis, and circulating reticulocytes. Resulting prothrombotic risk factors identified predominantly or uniquely in the high-dose group were higher numbers of immature reticulocytes and nucleated red blood cells in circulation, severe functional iron deficiency, and increased intravascular destruction of iron-deficient reticulocyte/red blood cells. No thrombotic events were detected in rats dosed up to 9 days suggesting a sustained high HCT is a requisite cofactor for development of ESA-related thrombotic toxicities.

Cytokines associated with increased erythropoiesis in Sprague-Dawley rats administered a novel hyperglycosylated analog of recombinant human erythropoietin.

We previously reported an increased incidence of thrombotic toxicities in Sprague-Dawley rats administered the highest dose level of a hyperglycosylated analog of recombinant human erythropoietin (AMG 114) for 1 month as not solely dependent on high hematocrit (HCT). Thereafter, we identified increased erythropoiesis as a prothrombotic risk factor increased in the AMG 114 high-dose group with thrombotic toxicities, compared to a low-dose group with no toxicities but similar HCT. Here, we identified pleiotropic cytokines as prothrombotic factors associated with AMG 114 dose level. Before a high HCT was achieved, rats in the AMG 114 high, but not the low-dose group, had imbalanced hemostasis (increased von Willebrand factor and prothrombin time, decreased antithrombin III) coexistent with cytokines implicated in thrombosis: monocyte chemotactic protein 1 (MCP-1), MCP-3, tissue inhibitor of metalloproteinases 1, macrophage inhibitory protein-2, oncostatin M, T-cell-specific protein, stem cell factor, vascular endothelial growth factor, and interleukin-11. While no unique pathway to erythropoiesis stimulating agent-related thrombosis was identified, cytokines associated with increased erythropoiesis contributed to a prothrombotic intravascular environment in the AMG 114 high-dose group, but not in lower dose groups with a similar high HCT.

Functional erythropoietin receptor is undetectable in endothelial, cardiac, neuronal, and renal cells.

Erythropoiesis stimulating agents (ESAs) have been reported to activate erythropoietin receptors (EpoR) on cell types, including endothelial, neuronal, renal tubule, and cardiac cells. ESAs have also been reported to promote angiogenesis. However, those findings are controversial and confounded by methodologic issues. We show that EpoR mRNA was detected in essentially all cell types examined, including primary human endothelial, renal, cardiac, and neuronal cells but 10- to 100-fold lower than Epo-responsive cells using quantitative reverse-transcribed polymerase chain reaction. Total endothelial EpoR protein examined using a new monoclonal antibody was low to undetectable. Surface EpoR on endothelial cells was not detected using [(125)I]-rHuEpo surface-binding studies. There was no evidence of ESA-induced intracellular signaling in endothelial cells. There was a similar lack of EpoR expression and signaling in other cell types examined. Experiments were performed examining ESA function on these cells. An in vivo rat corneal angiogenesis assay demonstrated neo-vessel formation in response to recombinant human vascular endothelial growth factor (rHuVEGF). However, recombinant mouse Epo did not induce vessel formation. Similarly, ESAs did not reproducibly provide cytoprotection to neuronal, renal, or cardiac cells. Taken together, our data challenge the notion of presence or function of EpoR on nonhematopoietic cells, and call into question the preclinical basis for clinical studies exploring direct, "pleiotropic" actions of ESAs.

Absence of functional EpoR expression in human tumor cell lines.

Certain oncology trials showed worse clinical outcomes in the erythropoiesis-stimulating agent (ESA) arm. A potential explanation was that ESA-activated erythropoietin (Epo) receptors (EpoRs) promoted tumor cell growth. Although there were supportive data from preclinical studies, those findings often used invalidated reagents and methodologies and were in conflict with other studies. Here, we further investigate the expression and function of EpoR in tumor cell lines. EpoR mRNA levels in 209 human cell lines representing 16 tumor types were low compared with ESA-responsive positive controls. EpoR protein production was evaluated in a subset of 66 cell lines using a novel anti-EpoR antibody. EpoR(+) control cells had an estimated 10 000 to 100 000 EpoR dimers/cell. In contrast, 54 of 61 lines had EpoR protein levels lower than 100 dimers/cell. Cell lines with the highest EpoR protein levels (400-3200 dimers/cell) were studied further, and, although one line, NCI-H661, bound detectable levels of [(125)I]-recombinant human Epo (rHuEpo), none showed evidence of ESA-induced EpoR activation. There was no increased phosphorylation of STAT5, AKT, ERK, or S6RP with rHuEpo. In addition, EpoR knockdown with siRNAs did not affect viability in 2 cell lines previously reported to express functional EpoR (A2780 and SK-OV-3). These results conflict with the hypothesis that EpoR is functionally expressed in tumors.

Multimeric Anti-DR5 IgM Agonist Antibody IGM-8444 Is a Potent Inducer of Cancer Cell Apoptosis and Synergizes with Chemotherapy and BCL-2 Inhibitor ABT-199.

Death receptor 5 (DR5) is an attractive target for cancer therapy due to its broad upregulated expression in multiple cancers and ability to directly induce apoptosis. Though anti-DR5 IgG antibodies have been evaluated in clinical trials, limited efficacy has been attributed to insufficient receptor crosslinking. IGM-8444 is an engineered, multivalent agonistic IgM antibody with 10 binding sites to DR5 that induces cancer cell apoptosis through efficient DR5 multimerization. IGM-8444 bound to DR5 with high avidity and was substantially more potent than an IgG with the same binding domains. IGM-8444 induced cytotoxicity in a broad panel of solid and hematologic cancer cell lines but did not kill primary human hepatocytes in vitro, a potential toxicity of DR5 agonists. In multiple xenograft tumor models, IGM-8444 monotherapy inhibited tumor growth, with strong and sustained tumor regression observed in a gastric PDX model. When combined with chemotherapy or the BCL-2 inhibitor ABT-199, IGM-8444 exhibited synergistic in vitro tumor cytotoxicity and enhanced in vivo efficacy, without augmenting in vitro hepatotoxicity. These results support the clinical development of IGM-8444 in solid and hematologic malignancies as a monotherapy and in combination with chemotherapy or BCL-2 inhibition.

Structure, Function, and Therapeutic Use of IgM Antibodies.

Natural immunoglobulin M (IgM) antibodies are pentameric or hexameric macro-immunoglobulins and have been highly conserved during evolution. IgMs are initially expressed during B cell ontogeny and are the first antibodies secreted following exposure to foreign antigens. The IgM multimer has either 10 (pentamer) or 12 (hexamer) antigen binding domains consisting of paired µ heavy chains with four constant domains, each with a single variable domain, paired with a corresponding light chain. Although the antigen binding affinities of natural IgM antibodies are typically lower than IgG, their polyvalency allows for high avidity binding and efficient engagement of complement to induce complement-dependent cell lysis. The high avidity of IgM antibodies renders them particularly efficient at binding antigens present at low levels, and non-protein antigens, for example, carbohydrates or lipids present on microbial surfaces. Pentameric IgM antibodies also contain a joining (J) chain that stabilizes the pentameric structure and enables binding to several receptors. One such receptor, the polymeric immunoglobulin receptor (pIgR), is responsible for transcytosis from the vasculature to the mucosal surfaces of the lung and gastrointestinal tract. Several naturally occurring IgM antibodies have been explored as therapeutics in clinical trials, and a new class of molecules, engineered IgM antibodies with enhanced binding and/or additional functional properties are being evaluated in humans. Here, we review the considerable progress that has been made regarding the understanding of biology, structure, function, manufacturing, and therapeutic potential of IgM antibodies since their discovery more than 80 years ago.

Mutation of hepatocyte nuclear factor-1beta inhibits Pkhd1 gene expression and produces renal cysts in mice.

Hepatocyte nuclear factor-1beta (HNF-1beta) is a Pit-1, Oct-1/2, UNC-86 (POU)/homeodomain-containing transcription factor that regulates tissue-specific gene expression in the liver, kidney, and other organs. Humans with autosomal dominant mutations of HNF-1beta develop maturity-onset diabetes of the young type 5 (MODY5) and congenital cystic abnormalities of the kidney. Autosomal recessive polycystic kidney disease (ARPKD) is an inherited cystic disorder that produces renal failure in infants and children and is caused by mutations of PKHD1. The proximal promoter of the mouse Pkhd1 gene contains an evolutionarily conserved HNF-1-binding site that is located near a region of deoxyribonuclease hypersensitivity. HNF-1beta and the structurally related HNF-1alpha bind specifically to the Pkhd1 promoter and stimulate gene transcription. Mutations of the HNF-1 site or expression of a dominant-negative HNF-1beta mutant inhibit Pkhd1 promoter activity in transfected cells. Transgenic mice expressing a dominant-negative HNF-1beta mutant under the control of a kidney-specific promoter develop renal cysts, similarly to humans with MODY5. Pkhd1 transcripts are absent in the cells lining the cysts but are present in morphologically normal surrounding tubules. These studies identify a link between two cystic disease genes, HNF1beta (MODY5) and PKHD1 (ARPKD). HNF-1beta directly regulates the transcription of Pkhd1, and inhibition of PKHD1 gene expression may contribute to the formation of renal cysts in humans with MODY5.

A novel colonic repressor element regulates intestinal gene expression by interacting with Cux/CDP.

Intestinal gene regulation involves mechanisms that direct temporal expression along the vertical and horizontal axes of the alimentary tract. Sucrase-isomaltase (SI), the product of an enterocyte-specific gene, exhibits a complex pattern of expression. Generation of transgenic mice with a mutated SI transgene showed involvement of an overlapping CDP (CCAAT displacement protein)-GATA element in colonic repression of SI throughout postnatal intestinal development. We define this element as CRESIP (colon-repressive element of the SI promoter). Cux/CDP interacts with SI and represses SI promoter activity in a CRESIP-dependent manner. Cux/CDP homozygous mutant mice displayed increased expression of SI mRNA during early postnatal development. Our results demonstrate that an intestinal gene can be repressed in the distal gut and identify Cux/CDP as a regulator of this repression during development.

Glycoengineering: the effect of glycosylation on the properties of therapeutic proteins.

Therapeutic proteins have revolutionized the treatment of many diseases but low activity or rapid clearance limits their utility. New approaches have been taken to design drugs with enhanced in vivo activity and half-life to reduce injection frequency, increase convenience, and improve patient compliance. One recently used approach is glycoengineering, changing protein-associated carbohydrate to alter pharmacokinetic properties of proteins. This technology has been applied to erythropoietin and resulted in the discovery of darbepoetin alfa (DA), a hyperglycosylated analogue of erythropoietin that contains two additional N-linked carbohydrates, a threefold increase in serum half-life and increased in vivo activity compared to recombinant human erythropoietin (rHuEPO). The increased serum half-life allows for less frequent dosing to maintain target hemoglobin levels in anemic patients. Carbohydrates on DA and other molecules can also increase molecular stability, solubility, increase in vivo biological activity, and reduce immunogenicity. These properties are discussed.

Romiplostim promotes platelet recovery in a mouse model of multicycle chemotherapy-induced thrombocytopenia.

Chemotherapy-induced thrombocytopenia can lead to chemotherapy treatment delays or dose reductions. The ability of romiplostim, a thrombopoietin (TPO) mimetic, to promote platelet recovery in a mouse model of multicycle chemotherapy/radiation therapy (CRT)-induced thrombocytopenia was examined. In humans, an inverse relationship between platelet counts and endogenous TPO (eTPO) concentration exists. In a CRT mouse model, eTPO was not elevated during the first 5 days after CRT treatment (the "eTPO gap"), then increased to a peak 10 days after each CRT treatment in an inverse relationship to platelet counts seen in humans. To bridge the eTPO gap, mice were treated with 10-1,000 µg/kg of romiplostim on day 0, 1, or 2 after CRT. In some mice, the romiplostim dose was approximately divided over 3 days. Platelet recovery occurred faster with romiplostim in most conditions tested. Romiplostim doses of ≥100 µg/kg given on day 0 significantly lessened the platelet nadir. Fractionating the dose over 3 days did not appear to confer a large advantage. These data may provide a rationale for clinical studies of romiplostim in chemotherapy-induced thrombocytopenia.

The Broad Anti-AML Activity of the CD33/CD3 BiTE Antibody Construct, AMG 330, Is Impacted by Disease Stage and Risk.

The CD33/CD3-bispecific T-cell engaging (BiTE) antibody construct, AMG 330, potently lyses CD33+ leukemic cells in vitro. Using specimens from 41 patients with acute myeloid leukemia (AML), we studied the factors that might contribute to clinical response or resistance. For this purpose, thawed aliquots of primary AML samples were immunophenotypically characterized and subjected to various doses of AMG 330 in the presence or absence of healthy donor T-cells. After 48 hours, drug-specific cytotoxicity was quantified and correlated with CD33 expression levels, amounts of T-cells present, and other disease characteristics. AMG 330 caused modest cytotoxicity that was correlated with the amount of autologous T-cells (P = 0.0001) but not CD33 expression, as AMG 330 exerted marked cytotoxic effects in several specimens with minimal CD33 expression. With healthy donor T-cells added, AMG 330 cytotoxicity depended on the drug dose and effector:target (E:T) cell ratio. High cytotoxic activity was observed even with minimal CD33 expression, and AMG 330 cytotoxicity and CD33 expression correlated only at high E:T cell ratio and high AMG 330 doses (P<0.003). AMG 330 resulted in significantly higher cytotoxicity in specimens from patients with newly diagnosed AML than those with relapsed/refractory disease despite similar levels of CD33 on myeloblasts. AMG 330 cytotoxicity also appeared greater in specimens from patients with favorable-risk disease as compared to other specimens. Together, our data demonstrate that AMG 330 is highly active in primary AML specimens across the entire disease spectrum, while suggesting the presence of yet undefined, CD33-independent, relative resistance mechanisms in specific patient subsets.

Characterization of bispecific T-cell Engager (BiTE) antibodies with a high-capacity T-cell dependent cellular cytotoxicity (TDCC) assay.

The Bispecific T-cell Engager (BiTE) antibody modality is a clinically validated immunotherapeutic approach for targeting tumors. Using T-cell dependent cellular cytotoxicity (TDCC) assays, we measure the percentage of specific cytotoxicity induced when a BiTE molecule engages T-cells, redirects T-cell mediated cytolysis, and ultimately kills target cells. We establish a novel luminescence-based TDCC assay quantified by measuring cell viability via constitutive expression of luciferase. The luciferase-based TDCC assay performance is valid and comparable to an adenosine triphosphate (ATP)-based detection method. We demonstrate that the luciferase-based TDCC assay is an efficient homogeneous assay format that is amenable to both suspension and adherent target cells. The luciferase-based TDCC assay eliminates the need for plate-washing protocols, allowing for higher-throughput screening of BiTE antibodies and better data quality. Assay capacity is also improved by performing serial dilutions of BiTE antibodies in 384-well format with an automated liquid handler. We describe here a robust, homogeneous TDCC assay platform with capacity for in vitro assessment of BiTE antibody potency and efficacy using multiple tumor cell lines and T-cell donors.

Erythropoiesis stimulating agents: approaches to modulate activity.

Recombinant human erythropoietin (rHuEPO), such as the approved agents epoetin alfa and epoetin beta, has been used successfully for over 20 years to treat anemia in millions of patients. However, due to the relatively short half-life of the molecule (approximately 8 hours), frequent dosing may be required to achieve required hemoglobin levels. Therefore, a need was identified in some anemic patient populations for erythropoiesis stimulating agents with longer half-lives that required less frequent dosing. This need led to the development of second generation molecules which are modified versions of rHuEPO with improved pharma-cokinetic and pharmacodynamic properties such as darbepoetin alfa, a hyperglycosylated analog of rHuEPO, and pegzyrepoetin, a pegylated rHuEPO. Third generation molecules, such as peginesatide, which are peptide mimetics that have no sequence homology to rHuEPO have also recently been developed. The various molecular, pharmacokinetic, and pharmacodynamic properties of these and other erythropoiesis stimulating agents will be discussed in this review.

The effect of erythropoietin on normal and neoplastic cells.

Erythropoietin (Epo) is an essential hormone that binds and activates the Epo receptor (EpoR) resident on the surface of erythroid progenitor cells, thereby promoting erythropoiesis. Recombinant human erythropoietin has been used successfully for over 20 years to treat anemia in millions of patients. In addition to erythropoiesis, Epo has also been reported to have other effects, such as tissue protection and promotion of tumor cell growth or survival. This became of significant concern in 2003, when some clinical trials in cancer patients reported increased tumor progression and worse survival outcomes in patients treated with erythropoiesis-stimulating agents (ESAs). One of the potential mechanisms proffered to explain the observed safety issues was that functional EpoR was expressed in tumors and/or endothelial cells, and that ESAs directly stimulated tumor growth and/or antagonized tumor ablative therapies. Since then, numerous groups have performed further research evaluating this potential mechanism with conflicting data and conclusions. Here, we review the biology of endogenous Epo and EpoR expression and function in erythropoiesis, and evaluate the evidence pertaining to the expression of EpoR on normal nonhematopoietic and tumor cells.

Expression and function of erythropoietin receptors in tumors: implications for the use of erythropoiesis-stimulating agents in cancer patients.

Safety concerns surrounding the use of recombinant human erythropoietin (Epo) to treat anemia in cancer patients were raised after 2 recent clinical studies reported a worse survival outcome in patients who received epoetin alpha or epoetin beta compared with patients who received placebo. Although those findings contrasted with previous clinical studies, which demonstrated no difference in survival for cancer patients who received erythropoiesis-stimulating agents (ESAs), some investigators have suggested a potential role for ESAs in promoting tumor growth through 1) stimulation of Epo receptors (EpoR) expressed in tumors, 2) stimulation and formation of tumor vessels, and/or 3) enhanced tumor oxygenation. The first and second hypotheses appeared to be supported by some EpoR expression and ESA in vitro studies. However, these conclusions have been challenged because of poor specificity of EpoR-detection methodologies, conflicting data from different groups, and the lack of correlation between in vitro data and in vivo findings in animal tumor models. For this report, the authors reviewed the biology of EpoR in erythropoiesis and compared and contrasted the reported findings on the role of ESAs and EpoR in tumors.

Anti-Epo receptor antibodies do not predict Epo receptor expression.

Investigators using anti-EpoR antibodies for immunoblotting and immunostaining have reported erythropoietin receptor (EpoR) expression in nonhematopoietic tissues including human tumors. However, these antibodies detected proteins of 66 to 78 kDa, significantly larger than the predicted molecular weight of EpoR (56-57 kDa). We investigated the specificity of these antibodies and showed that they all detected non-EpoR proteins. C-20 detected 3 proteins in tumor cell lines (35, 66, and 100 kDa). Sequences obtained from preparative gels had similarity to the C-20-immunizing peptide. The 66-kDa protein was a heat shock protein (HSP70) to which antibody binding was abrogated in peptide competition experiments. Antibody M-20 readily identified a 59-kDa EpoR protein. However, neither M-20 nor C-20 was suitable for detection of EpoR using immunohistochemical methods. We concluded that these antibodies have limited utility for detecting EpoR. Thus, reports of EpoR expression in tumor cells using these antibodies should be viewed with caution.

Regulation of kidney-specific Ksp-cadherin gene promoter by hepatocyte nuclear factor-1beta.

Kidney-specific cadherin (Ksp-cadherin) is a tissue-specific member of the cadherin family that is expressed exclusively in the kidney and developing genitourinary tract. Recent studies have shown that the proximal 250 bp of the Ksp-cadherin gene promoter are sufficient to direct tissue-specific gene expression in vivo and in vitro. The proximal 120 bp of the promoter are evolutionarily conserved between mouse and human and contain a DNase I hypersensitive site that is kidney cell specific. At position -55, the promoter contains a consensus recognition site for hepatocyte nuclear factor-1 (HNF-1). Mutations of the consensus HNF-1 site and downstream GC-boxes inhibit promoter activity in transfected cells. HNF-1alpha and HNF-1beta bind specifically to the -55 site, and both proteins transactivate the promoter directly. Expression of Ksp-cadherin is not altered in the kidneys of HNF-1alpha-deficient mice. However, expression of a gain-of-function HNF-1beta mutant stimulates Ksp-cadherin promoter activity in transfected cells, whereas expression of a dominant-negative mutant inhibits activity. These studies identify Ksp-cadherin as the first kidney-specific promoter that has been shown to be regulated by HNF-1beta. Mutations of HNF-1beta, as occur in humans with inherited renal cysts and diabetes, may cause dysregulated Ksp-cadherin promoter activity.