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Supplementary Methods, Figures 1-5 from Preclinical Evaluation of AMG 900, a Novel Potent and Highly Selective Pan-Aurora Kinase Inhibitor with Activity in Taxane-Resistant Tumor Cell Lines

Acute Myeloid Leukemia (AML) is the most common and aggressive acute leukemia in adults, with a 25% 5-year survival rate. FLT3 is the most frequently mutated gene in AML. About 25% of AML patients harbor FLT3 internal tandem duplication (ITD) mutations, and about 8 % of patients harbor FLT3 tyrosine kinase domain (TKD) mutations. Both FLT3-ITD and FLT3-TKD mutations constitutively activate the protein, which causes poor survival. Gilteritinib is a highly potent and selective oral FLT3 inhibitor recently approved by the FDA. Although gilteritinib showed strong single agent activity in AML patients with FLT3 mutations, the development of gilteritinib resistance limits the long-term efficacy of this treatment, indicating that combination therapy may be advantageous for AML patients with FLT3 mutations. FLT3 mutations are known to be anti-apoptotic. Myeloid cell leukemia-1 (MCL-1), an anti-apoptotic protein, expressed in a large percentage of the AML patient population, plays a critical role in AML cell survival and drug resistance. AMG 176 is a potent, selective and orally bioavailable MCL-1 inhibitor, which induces rapid commitment to apoptosis in AML. Here we demonstrated that AMG 176 and gilteritinib combination treatment synergistically targeted FLT3-ITD mutated AML. A strong synergistic effect (Combination index Citation Format: Xiaoyue Chen, Sean Caenepeel, Brian Belmontes, Patricia L. McElroy, Karen Rex, Tao Osgood, Paul Hughes. Efficacy of AMG 176 in combination with gilteritinib in preclinical models of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1050.

KRAS is the most frequently mutated oncogene in cancer and encodes a key signaling protein in tumors. The p.G12C mutation of KRAS is present in approximately 13% of lung adenocarcinoma, 3% of colorectal cancer, and 2% of other solid tumors. Sotorasib (formerly known as AMG 510), the first KRASG12C inhibitor to reach clinical testing in humans, has demonstrated evidence of clinical activity as a single agent in patients with non-small cell lung (NSCLC), colorectal (CRC), endometrial, and appendiceal carcinoma. Preclinically, sotorasib has shown significant tumor growth inhibition as a single agent in multiple CDX and PDX models. The clinically-validated strategy of combining multiple inhibitors in the MAPK pathway suggests that combination strategies could yield even better outcomes for patients. Specifically, the combination of sotorasib and other inhibitors in the MAPK and AKT signaling pathways might further enhance tumor cell killing and overcome potential resistance. To test this hypothesis, in vitro combination experiments were conducted in multiple KRAS p.G12C cell lines with combination matrices of sotorasib and inhibitors of HER kinases, EGFR, SOS1, SHP2, MEK, PI3K, or mTOR, as well as an inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6). The combination of sotorasib with multiple agents resulted in robust synergistic cell killing of KRAS p.G12C tumor cells in vitro. To understand whether these observations translated in vivo, we assessed combinations of sotorasib with a SHP-2 inhibitor or a HER kinase inhibitor in pharmacodynamic assays and efficacy models in tumor xenografts. Consistent with the synergy observed in vitro, sotorasib in combination with a HER kinase inhibitor (afatinib) or a SHP2 inhibitor (RMC-4550) in vivo resulted in enhanced inhibition of MAPK signaling as measured by p-ERK in NCI-H358 tumors. In efficacy studies using the NCI-H358 xenograft model, significantly enhanced anti-tumor activity was observed with a minimally efficacious dose of sotorasib in combination with afatinib, RMC-4550, or a CDK4/6 inhibitor (palbociclib). Furthermore, enhanced anti-tumor activity was observed with sotorasib in combination with a MEK inhibitor or with the anti-EGFR monoclonal antibody panitumumab in a CRC KRAS p.G12C PDX model. Taken together, these data support the clinical evaluation of combination treatment of sotorasib with analogous agents in patients with KRAS p.G12C tumors. Citation Format: Karen Rex, Anne Y. Saiki, Tyler Holt, Alla Verlinsky, Patricia L. McElroy, Tao Osgood, Ji-Rong Sun, Marwan G. Fakih, Upendra P. Dahal, Bernd Bruenner, Victor J. Cee, Brian A. Lanman, Jude Canon, J. Russell Lipford. Combination of the KRASG12C inhibitor sotorasib with targeted agents improves anti-tumor efficacy in KRAS p.G12C cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1057.

Purpose: Despite advances in the treatment of multiple myeloma, new therapies are needed to induce more profound clinical responses. T-cell–redirected lysis triggered by bispecific antibodies recruiting T cells to cancer cells is a clinically validated mechanism of action against hematologic malignancies and CD38 is a tumor-associated antigen with near-universal expression in multiple myeloma. Thus, an anti-CD38/CD3 bispecific T-cell–recruiting antibody has the potential to be an effective new therapeutic for multiple myeloma. Experimental Design: Anti-CD38/CD3 XmAb T-cell–recruiting antibodies with different affinities for CD38 and CD3 were assessed in vitro and in vivo for their redirected T-cell lysis activity against cancer cell lines, their lower levels of cytokine release, and their potency in the presence of high levels of soluble CD38. Select candidates were further tested in cynomolgus monkeys for B-cell depletion and cytokine release properties. Results: AMG 424 was selected on the basis of its ability to kill cancer cells expressing high and low levels of CD38 in vitro and trigger T-cell proliferation, but with attenuated cytokine release. In vivo, AMG 424 induces tumor growth inhibition in bone marrow–invasive mouse cancer models and the depletion of peripheral B cells in cynomolgus monkeys, without triggering excessive cytokine release. The activity of AMG 424 against normal immune cells expressing CD38 is also presented. Conclusions: These findings support the clinical development of AMG 424, an affinity-optimized T-cell–recruiting antibody with the potential to elicit significant clinical activity in patients with multiple myeloma.

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.

Comparability studies used to assess a proposed manufacturing change for a biological product include sensitive analytical studies to confirm there are no significant differences in structural or functional attributes that may contribute to clinically meaningful changes in efficacy or safety. When a proposed change is relatively complex or when clinically relevant differences between the product before and after the change cannot be ruled out based on analytical studies, nonclinical and clinical bridging studies are generally required to confirm overall comparability. In this study, we report findings from a comparability assessment of epoetin alfa before and after a proposed manufacturing process change. Although differences in glycosylation attributes were observed, these were initially believed to be irrelevant to the product's pharmacology. This assumption was initially supported via nonclinical and clinical pharmacology studies, but a clinically meaningful difference in potency was ultimately observed in a phase 3 clinical study conducted in a sensitive patient population using a sensitive study design. These results indicate that the nonclinical assessments of structure-function relationships were insufficiently sensitive to identify clinically relevant differences resulting from differences in the glycosylation profile. This case study highlights important findings that may be relevant in the development of biosimilar epoetin alfa products.

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.

In mammalian cells, the aurora kinases (aurora-A, -B, and -C) play essential roles in regulating cell division. The expression of aurora-A and -B is elevated in a variety of human cancers and is associated with high proliferation rates and poor prognosis, making them attractive targets for anticancer therapy. AMG 900 is an orally bioavailable, potent, and highly selective pan-aurora kinase inhibitor that is active in taxane-resistant tumor cell lines. In tumor cells, AMG 900 inhibited autophosphorylation of aurora-A and -B as well as phosphorylation of histone H3 on Ser10, a proximal substrate of aurora-B. The predominant cellular response of tumor cells to AMG 900 treatment was aborted cell division without a prolonged mitotic arrest, which ultimately resulted in cell death. AMG 900 inhibited the proliferation of 26 tumor cell lines, including cell lines resistant to the antimitotic drug paclitaxel and to other aurora kinase inhibitors (AZD1152, MK-0457, and PHA-739358), at low nanomolar concentrations. Furthermore, AMG 900 was active in an AZD1152-resistant HCT116 variant cell line that harbors an aurora-B mutation (W221L). Oral administration of AMG 900 blocked the phosphorylation of histone H3 in a dose-dependent manner and significantly inhibited the growth of HCT116 tumor xenografts. Importantly, AMG 900 was broadly active in multiple xenograft models, including 3 multidrug-resistant xenograft models, representing 5 tumor types. AMG 900 has entered clinical evaluation in adult patients with advanced cancers and has the potential to treat tumors refractory to anticancer drugs such as the taxanes. Cancer Res; 70(23); 9846–54. ©2010 AACR.

The unit of erythropoietic activity has long been the standard by which erythropoietic agents are judged, but the development of long-acting agents such as darbepoetin alfa has highlighted the shortcomings of this approach. To this point, we compared the in vivo activity of Epoetin alfa and darbepoetin alfa per microgram of protein core. Using the established mass-to-unit conversion for Epoetin alfa (1 microg congruent with 200 U), we then calculated darbepoetin alfa activity in units. Activity varied with treatment regimen (1 microg darbepoetin alfa congruent with 800 U for 3 times weekly dosing to 8,000 U for a single injection). This analysis reveals the inadequacy of evaluating darbepoetin alfa activity in terms of standard erythropoietic units. We therefore propose that for molecules with heightened biological activity, a more legitimate basis for comparison is the protein mass.

Summary. Despite its frequency and impact on clinical outcomes, anaemia in cancer patients remains poorly understood and suboptimally treated. The definition of optimum treatment schedules with erythropoietic agents requires a suitable model of chemotherapy-induced progressive anaemia. This study investigated novel strategies such as once-per-chemotherapy-cycle dosing, synchronization between erythroid supportive care and chemotherapy, and definition of the optimum timing of erythroid support. A murine model of carboplatin chemotherapy/radiotherapy (CRT)-induced anaemia was used, which caused progressive anaemia across multiple cycles. Weekly administration of recombinant human erythropoietin (rHuEPO) was effective, but the longer-acting darbepoetin alpha resulted in superior responses. In all animals, anaemia became progressive and more refractory across cycles because of accumulated bone marrow damage. Exploiting a specific enzyme-linked immunosorbent assay, which could distinguish between darbepoetin alpha and endogenous erythropoietin, the effect of CRT upon the pharmacokinetics of darbepoetin alpha showed that clearance of darbepoetin alpha, and presumably erythropoietin, was at least partially dependent on a chemotherapy-sensitive pathway. Scheduling data suggested that administration of erythropoietic agents prior to chemotherapy was more effective than administration after chemotherapy. There was no evidence that erythropoietic agents exacerbated anaemia, even when administered immediately prior to CRT in an attempt to ‘prime’ erythroid cells for the effects of CRT.

For targets that are homogenously expressed, such as CD19 on cells of the B lymphocyte lineage, immunotherapies can be highly effective. Targeting CD19 with blinatumomab, a CD19/CD3 bispecific antibody construct (BiTE®), or with chimeric antigen receptor T cells (CAR-T) has shown great promise for treating certain CD19-positive hematological malignancies. In contrast, solid tumors with heterogeneous expression of the tumor-associated antigen (TAA) may present a challenge for targeted therapies. To prevent escape of TAA-negative cancer cells, immunotherapies with a local bystander effect would be beneficial. As a model to investigate BiTE®-mediated bystander killing in the solid tumor setting, we used epidermal growth factor receptor (EGFR) as a target. We measured lysis of EGFR-negative populations in vitro and in vivo when co-cultured with EGFR-positive cells, human T cells and an EGFR/CD3 BiTE® antibody construct. Bystander EGFR-negative cells were efficiently lysed by BiTE®-activated T cells only when proximal to EGFR-positive cells. Our mechanistic analysis suggests that cytokines released by BiTE®-activated T-cells induced upregulation of ICAM-1 and FAS on EGFR-negative bystander cells, contributing to T cell-induced bystander cell lysis.

Therapeutic use of recombinant human cytokines in humans can result in the generation of drug-specific antibodies. To predetermine the maximum potential effects of a granulocyte colony-stimulating factor (G-CSF) neutralizing auto-immunoglobulin G (auto-IgG) response during recombinant human G-CSF therapy, we developed a mouse model of mouse G-CSF (mG-CSF) neutralizing auto-IgG response. Mice were immunized and boosted with mG-CSF chemically conjugated to either keyhole limpet hemocyanin or ovalbumin on an alternating schedule. Sera were analyzed for mG-CSF-specific titers and full blood counts were performed on a Technicon H-1E. On day 252, tissues were collected for histology. IgG was protein A affinity purified from pooled mG-CSF autoimmune sera. Mice immunized with mG-CSF conjugates produced mG-CSF-specific auto-IgG responses that lasted for the length of the study. Significant neutropenia (p(max) < 0.004) was concurrent with the rise in mG-CSF-specific IgG titers. However, neutrophil counts remained at approximately 20% of preimmunization levels through day 252. Endogenous mG-CSF neutralizing auto-IgG had no significant effect on hemoglobin, erythrocyte, lymphocyte, eosinophil, basophil, and platelet counts, and had minor, transient, or no effects on monocyte counts. Bone marrow colony assays from mG-CSF autoimmune mice demonstrated no significant effect of G-CSF neutralization on the numbers or proliferative capacity of preneutrophil lineage progenitors. Purified IgG from mG-CSF autoimmune mice neutralized mG-CSF in vitro. High-titer G-CSF neutralizing auto-IgG in adult mice partially inhibited steady-state granulopoiesis and had little or no effect on steady-state levels of other hematopoietic cells.

Granulocyte colony-stimulating factor (G-CSF) has proven effective in the prophylaxis of chemotherapy-induced neutropenia and as a mobilizer of peripheral blood progenitor cells. The longevity of G-CSF action is limited by its removal from the body by two mechanisms. The first is thought to be mediated via receptors (receptor mediated clearance [RMC]) predominantly on neutrophils, the second process is likely the result of renal clearance. With the intention of developing a novel form of Filgrastim (r-met HuG-CSF) with a sustained duration of action in vivo, a new derivative named SD/01 has been made by association of Filgrastim with poly(ethylene glycol). The desired properties of this new agent would include a prolonged duration of action sufficient to cover a complete single course of chemotherapy. SD/01 is shown here to sustain significantly elevated neutrophil counts in hematopoietically normal mice for 5 days. In neutropenic mice effects were noted for at least 9 days, accompanying a significant reduction in the duration of chemotherapy induced neutropenia. Normal human volunteers showed higher than baseline ANC for around 9 to 10 days after a single injection of SD/01. Data from these normal volunteers also indicate that mobilization of CD34+ cells and progenitors may occur in a more timely manner and to around the same absolute numbers as with repeated daily injections of unmodified Filgrastim. These data indicate that SD/01 represents an efficacious novel form of Filgrastim with actions sustained for between one and two weeks from a single injection.

The effect of chronic expression of flt3 ligand (FL) on in vivo hematopoiesis was studied. Retroviral vector-mediated gene transfer was used in a mouse model of bone marrow transplantation to enforce expression of mouse FL cDNA in hematopoietic tissues. As early as 2 weeks posttransplantation, peripheral blood white blood cell counts in FL-overexpressing recipients were significantly elevated compared with controls. With the exception of eosinophils, all nucleated cell lineages studied were similarly affected in these animals. Experimental animals also exhibited severe anemia and progressive loss of marrow-derived erythropoiesis. All of the FL-overexpressing animals, but none of the controls, died between 10 and 13 weeks posttransplantation. Upon histological examination, severe splenomegaly was noted, with progressive fibrosis and infiltration by abnormal lymphoreticular cells. Abnormal cell infiltration also occurred in other organ systems, including bone marrow and liver. In situ immunocytochemistry on liver sections showed that the cellular infiltrate was CD3+/NLDC145+/CD11c+, but B220− and F4/80−, suggestive of a mixed infiltrate of dendritic cells and activated T lymphocytes. Infiltration of splenic blood vessel perivascular spaces resulted in vascular compression and eventual occlusion, leading to splenic necrosis consistent with infarction. These results show that FL can affect both myeloid and lymphoid cell lineages in vivo and further demonstrate the potential toxicity of in vivo treatment with FL.

We have studied the potential of combination growth factor treatment with GM-CSF and megakaryocyte growth and development factor (MGDF) to stimulate hematopoietic recovery in mice following bone marrow transplantation. More rapid recovery of neutrophils occurred in mice treated with recombinant murine (rm)GM-CSF plus pegylated recombinant human (PEG-rHu)MGDF than carrier treated controls, however this recovery was equivalent to the effect of treatment with rmGM-CSF alone. PEG-rHuMGDF stimulated a more rapid recovery of platelets with no effect on neutrophil recovery. At the two tested doses of rmGM-CSF (72 and 200 μg/kg/day) the platelet recovery was inferior to that in carrier treated mice. Also, the addition of rmGM-CSF to PEG-rHuMGDF had a dose-related negative impact on platelet recovery compared to PEG-rHuMGDF alone. These data suggest that the use of combination therapy in some clinical indications may lead to unexpected results. Furthermore, careful dosage studies may be necessary to identify the full potential of combined growth factors to obtain additive or synergistic effects on multilineage hematopoietic reconstitution in vivo.

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.

We have investigated the potential of PEGylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a molecule related to thrombopoietin (mpl ligand or TPO) in minimizing the thrombocytopenia associated with hematopoietic ablation and peripheral blood progenitor cell (PBPC) transplant. Irradiated mice that received PBPC mobilized by PEG-rHuMGDF or granulocyte colony-stimulating factor (G-CSF) had a reduced number of thrombocytopenic days with platelets below 100 x 10(6) per mL of blood. Recipients of unmobilized PBPC had a 9 day thrombocytopenic phase which was shortened to 7 days if they were given granulocyte-macrophage colony-stimulating factor (GM-CSF)- mobilized PBPC. This was further reduced to 2 or 3 days of thrombocytopenia in recipients of G-CSF- or PEG-MGDF-mobilized PBPC. Despite our observation that PEG-rHuMGDF is a relatively modest stimulator of the mobilization of myeloid progenitors to the blood, MGDF-mobilized PBPC do effect accelerated recovery of platelets after transplantation. However, the most effective use of PEG-rHuMGDF is when it is given during the recovery phase after PBPC transplantation to hematopoietically ablated mice. Posttransplant treatment with PEG- rHuMGDF reduces thrombocytopenia to a single day or less, in recipients of most types of PBPC. Mice that were treated during the first 2 weeks after PBPC transplant with PEG-rHuMGDF had 1 thrombocytopenic day compared to 9 days in carrier-treated recipients of unmobilized PBPC and 2 to 3 days in carrier-treated recipients of the optimally mobilized PBPC from G-CSF or G-CSF/PEG-rHuMGDF treated donors. In groups where PEG-rHuMGDF was included in the mobilization protocol and used to treat recipients as well thrombocytopenia was effectively eliminated. These data show that PEG-rHuMGDF is a highly effective agent in eliminating the thrombocytopenia associated with PBPC transplantation.

Megakaryocyte growth and development factor (MGDF) has recently been identified as a ligand for the c-mpl receptor. Using retroviral- mediated gene transfer, MGDF has been overexpressed in mice to evaluate the systematic effects due to chronic exposure to this growth factor. MGDF overexpressing mice had more rapid platelet recovery than control mice after transplantation. Following this recovery, the platelet levels continued increasing to fourfold to eightfold above normal baseline levels and remained elevated (five-fold above control mice) in these animals, which are alive and well at more than 4 months posttransplantation. Increased megakaryocyte numbers were detected in a number of organs in these mice including bone marrow, spleen, liver, and lymph nodes. Prolonged overexpression of MGDF led to decreased marrow hematopoiesis, especially erythropoiesis, with a shift to extramedullary hematopoiesis in the spleen and liver. All the MGDF overexpressing mice analyzed to date developed myelofibrosis and osteosclerosis, possibly induced by megakaryocyte and platelet produced cytokines. No significant effect on other hematopoietic lineages was seen in the MGDF overexpressing mice, showing that the stimulatory effect of MGDF in vivo is restricted to the megakaryocyte lineage.

BiTE® therapeutics are single chain antibody constructs harboring two binding moieties: one directed at a tumor associated antigen and one directed at the CD3e protein, which trigger T cell dependent cellular cytotoxicity (TDCC) against targeted cancer cells. Here, we evaluated the suitability of ENPP3 as a potential BiTE® target. The ENPP3 mRNA is highly differentially expressed in clear cell Renal Cell Carcinoma (ccRCC) and the ENPP3 protein is detected with high uniformity and intensity in ccRCC tumor samples by immunohistochemistry. We demonstrated the surface expression of this protein in Renal Cancer cell lines and confirmed that the ENPP3 protein was highly restricted to the luminal side of normal epithelial layers in which it was detected (proximal renal tubules, bronchial epithelium, salivary glands). We developed highly potent anti-ENPP3 BiTE® molecules and demonstrated the in vitro TDCC activity of these molecules. Two anti-ENPP3 BiTE® molecules further demonstrated tumor formation inhibition activity in a human T cell / human target cell admixture mouse xenograft model. Citation Format: Olivier Nolan-Stevaux, Flordeliza Fajardo, Lily Liu, Suzanne Coberly, Patricia McElroy, Aaron Nazarian, Franziska Bott, Elisabeth Nahrwold, Tobias Raum, Roman Kischel, Ralf Lutterbuese, Patrick Hoffmann, Peter Kufer. Assessing ENPP3 as a renal cancer target for bispecific T-cell engager (BiTE) therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 585.

Mobilized peripheral blood progenitor cells (PBPC) have been shown to provide rapid engraftment in patients given high-dose chemotherapy. PBPC contain cells with long-term engraftment potential as shown in animal models. In this study we have further analyzed mobilized PBPC for their ability to support serial transplantation of irradiated mice. Transplantation of recombinant human granulocyte colony-stimulating factor (rhG-CSF) plus recombinant rat stem cell factor (rrSCF) mobilized PBPC resulted in 98% donor engraftment of primary recipients at 12 to 14 months post-transplantation. Bone marrow (BM) cells from these primary recipients were harvested and transplanted into secondary recipients. At 6 months posttransplantation, all surviving secondary recipients had donor engraftment. Polymerase chain reaction (PCR) analysis showed greater than 90% male cells in spleens, thymuses, and lymph nodes. Myeloid colonies from BM cells of secondary recipients demonstrated granulocyte/macrophage colony-forming cells (GM-CFC) of male origin in all animals. In comparison, transplantation of rhG-CSF mobilized PBPC resulted in decreased male engraftment in secondary recipients. BM cells from secondary recipients, who originally received PBPC mobilized by the combination of rrSCF and rhG-CSF, were further passaged to tertiary female recipients. At 6 months posttransplantation, 90% of animals had male-derived hematopoiesis by whole-blood PCR analysis. These data showed that PBPC mobilized with rhG-CSF plus rrSCF contained cells that are transplantable and able to maintain hematopoiesis for more than 26 months, suggesting that the mobilized long-term reconstituting stem cells (LTRC) have extensive proliferative potential and resemble those that reside in the BM. In addition, the data demonstrated increased mobilization of LTRC with rhG-CSF plus rrSCF compared to rhG-CSF alone.

GITR/TNFRSF18 is a member of the TNF-receptor superfamily preferentially expressed on regulatory T cells (Tregs) and activated T effector cells. Antibody agonists to GITR claim two distinct mechanisms to overcome the repressive tumor microenvironment and drive anti-tumor efficacy in vivo: receptor agonism (forward signaling) on T effector cells and FcγR-mediated Treg depletion. We sought to better understand the contribution of these two mechanisms using pharmacodynamic readouts relating target coverage, Treg depletion and efficacy using isotypic variants of a surrogate antibody against mouse GITR, DTA-1. First, target coverage was determined in spleen, tumor and draining lymph node following treatment with a single dose of mouse IgG2a DTA-1. In this study, efficacy correlated with doses that covered >90% GITR-expressing intratumoral leukocytes and depleted >90% intratumoral Tregs at 24 hours post-dose. Though displaying equivalent agonistic activity in vitro and achieving a similar level of target coverage, the mouse IgG1 N297A variant of DTA-1 neither depleted Tregs nor displayed anti-tumor activity in vivo, in confirmation of recent literature. To further explore the influence of Fc engagement, additional DTA-1 isotypic variants were generated and tested in vivo. In this study, we confirmed that preferential engagement of Fcγ receptors was necessary for optimal activity, as the mouse IgG1 DTA-1 variant failed to regress tumors. Additionally, we identified a variant with enhanced Treg depletion properties, however, the enhanced depletion did not translate to improved anti-tumor efficacy. Lastly, we sought to understand if mouse IgG2a DTA-1-would enhance the effect of PD-1 / PD-L1 blockade in vivo. Using the MC38 tumor model, we observed synergistic tumor regression in the combination group versus either monotherapy. Given the likely non-overlapping mechanism of the antibodies, the results suggest that an ADCC-enabled agonistic GITR antibody could provide benefit to human cancer patients in combination with, or refractory to, PD-1/PD-L1 inhibitors

Recent clinical data demonstrate the significance of T cells in anti-tumor activity. For instance, the CD19/CD3 bispecific T cell engager (BiTE) blinatumomab is a proven means of harnessing T cells for cancer treatment. BiTE antibody constructs comprise an anti-CD3 scFv (single chain variable fragment) linked to an scFv binding a tumor-associated antigen (TAA). One potential challenge for TAA-targeted therapeutics is that treatment may only eliminate TAA-expressing tumor cells and heterogeneity of TAA expression becomes a potential means of resistance. To prevent escape of TAA-negative tumor cells, a treatment modality with a bystander effect on TAA-negative cells may be desirable. To evaluate the potential of BiTE antibodies to mediate bystander cell killing, mixtures of TAA-positive and -negative (bystander) cells were co-cultured with human T cells and the effect of BiTE antibodies tested. Lysis of TAA-expressing and bystander cells was evaluated using both imaging and viability assays. For this study, we used BiTE antibodies recognizing either epidermal growth factor receptor (EGFR) or CD33. In the presence of TAA-positive cells, T cells were activated and bystander cells lysed. In the absence of TAA-positive cells, bystander cells were not killed. Bystander cell lysis was also observed in a xenograft mouse model with subcutaneous tumors comprising EGFR-positive and -negative cancer cells, and human T-cells. The mechanism of BiTE-mediated bystander killing was further investigated. In the presence of TAA-positive cells, T cells released many cytokines, including IFN-γ and TNFα. However, exposure of bystander cells to just the soluble factors released by T cells did not induce their lysis, suggesting that a direct interaction between BiTE-activated T cells and bystander cells was required. BiTE treatment induced the expression on bystander cells of intercellular adhesion molecule 1 (ICAM-1), a protein involved in formation of cytolytic T cell synapses with target cells. ICAM-1 upregulation on bystander cells was also observed following exposure to recombinant IFN-γ and TNFα. These findings suggest that exposure of bystander cells to cytokines secreted by BiTE-activated T cells caused ICAM-1 expression on bystander cells leading to their improved attachment and cytolytic synapse formation. Blockade of ICAM-1 by an antibody partially protected bystander cells from lysis. Our data suggest a model where BiTE-activated T cells secrete cytokines that cause upregulation of ICAM-1 on TAA-negative cells. This can then lead to T cell binding and T cell-induced bystander cell lysis. This mechanism is not expected to cause systemic cell death because only those cells proximal to the activated T cell in the tumor environment would be exposed to sufficiently high concentrations of ICAM-1-inducing cytokines. However, this locally confined bystander cell lysis may be sufficient to enable effective treatment of tumors that are heterogeneous for TAA expression. Citation Format: Sandra L. Ross, Marika Mulen, Patricia L. McElroy, Julie Lofgren, Gordon Moody, Patrick A. Baeuerle, Angela Coxon, Tara L. Arvedson. BiTE antibody constructs can mediate bystander tumor cell killing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-232. doi:10.1158/1538-7445.AM2015-LB-232

Despite its frequency and impact on clinical outcomes, anaemia in cancer patients remains poorly understood and suboptimally treated. The definition of optimum treatment schedules with erythropoietic agents requires a suitable model of chemotherapy-induced progressive anaemia. This study investigated novel strategies such as once-per-chemotherapy-cycle dosing, synchronization between erythroid supportive care and chemotherapy, and definition of the optimum timing of erythroid support. A murine model of carboplatin chemotherapy/radiotherapy (CRT)-induced anaemia was used, which caused progressive anaemia across multiple cycles. Weekly administration of recombinant human erythropoietin (rHuEPO) was effective, but the longer-acting darbepoetin alpha resulted in superior responses. In all animals, anaemia became progressive and more refractory across cycles because of accumulated bone marrow damage. Exploiting a specific enzyme-linked immunosorbent assay, which could distinguish between darbepoetin alpha and endogenous erythropoietin, the effect of CRT upon the pharmacokinetics of darbepoetin alpha showed that clearance of darbepoetin alpha, and presumably erythropoietin, was at least partially dependent on a chemotherapy-sensitive pathway. Scheduling data suggested that administration of erythropoietic agents prior to chemotherapy was more effective than administration after chemotherapy. There was no evidence that erythropoietic agents exacerbated anaemia, even when administered immediately prior to CRT in an attempt to "prime" erythroid cells for the effects of CRT.

We have evaluated the durability of engraftment and the potential of remobilization in mice reconstituted with mobilized peripheral blood progenitor cells (PBPC). Female mice which had been reconstituted with cytokine-mobilized PBPC from male donors were serially transplanted into second, third, fourth and fifth lethally irradiated female recipients at intervals of 6-10 months. Male-derived hematopoiesis was determined in recipient mice at each serial transplantation. Male-positive CFCs were detected after 5 passages for 45 months, but declined from95% at passage 1 to 74% at passage 2, 33% at passage 4, and 28% at passage 5. Long-term survival also declined from 97% at passage 2 to 53% at passage 4, and 27% at passage 5. The results demonstrated that mobilized PBPC were able to provide engraftment for more than 45 months, but the engraftment provided by mobilized PBPC decreased at each serial passage. In addition, mice reconstituted with mobilized PBPC (at 1 year post transplantation) were treated with the same cytokines as in the primary mobilization (remobilization). The remobilized PBPC were harvested and transplanted into lethally irradiated secondary recipients. Male-derived CFCs were evaluated at 20 months post transplantation. Mice transplanted with PBPC remobilized with rhG-CSF or rhG-CSF plus rrSCF-PEG showed 70% and 89% male-positive CFCs respectively, demonstrating that mice reconstituted with mobilized PBPC could be remobilized and that the remobilized PBPC were also capable of providing long-term hematopoietic reconstitution. Our studies demonstrated that mobilized PBPC have extensive proliferative or self-renewal capacity to provide durable engraftment and that marrow repopulating cells in PBPC reconstituted mice can be remobilized, suggesting that patients who relapse after PBPC transplantation may be remobilized for a second transplantation to support additional chemotherapy.

Aurora kinases A and B play essential roles in multiple stages of mitosis and are frequently overexpressed in a subset of human cancers, including acute myeloid leukemia (AML) (Ikezoe T et al, 2007). AMG 900, a potent and highly selective small molecule inhibitor of aurora kinases, is currently in Phase 1 clinical testing in adult patients with AML. In this study, we report the preclinical effects of AMG 900 in AML cell lines. We show that AMG 900 inhibits the phosphorylation of Histone H3 on serine-10 (a proximal substrate of aurora-B) leading to aborted cell division, apoptosis and/or polyploidy. We evaluated the activity of AMG 900 and two other well-characterized aurora kinase inhibitors [AZD1152-hQPA (B-selective AKI) and MLN8054 (A-selective AKI)] in a panel of AML cell lines. AMG 900 inhibited proliferation in all 10 cell lines at single-digit nanomolar concentrations. At effective concentrations, AMG 900 and AZD1152-hQPA showed similar cellular phenotypes, indicating that the activity of AMG 900 may occur through inhibition of aurora-B. A subset of cell lines sensitive to AMG 900 and MLN8054 were insensitive to AZD1152-hQPA, suggesting that AMG 900 may be less susceptible to resistance mediated by drug-efflux (Grundy M et al, 2011). Two AMG 900 oral dosing schedules are being evaluated in the ongoing AML clinical trial; patients receive either 4 or 7 consecutive daily doses followed by a drug holiday in 14-day cycles. In this study, we evaluated the in vivo anti-proliferative effects of AMG 900 using the two dose schedules in the skeleton of NOD/SCID IL2γnull mice bearing MOLM-13 (AML) cells expressing luciferase. To assess tumor cell proliferation in vivo, we used 18FLT (radioactive thymidine analog) PET/CT imaging, a technique that has been used to monitor early treatment response in the bone marrow of AML patients (Vanderhoek M et al, 2011). Mice were imaged for luciferase activity and 18FLT uptake before treatment and at multiple time-points during the drug holiday phase within the 14-day cycle. While the two AMG 900 dosing schedules resulted in a similar decrease in tumor burden across study time points (as measured by luciferase activity), they differed in the timing of skeletal 18FLT responses. Mice administered AMG 900 showed an attenuated skeletal 18FLT uptake compared with the vehicle group, followed by an 18FLT flare. This 18FLT flare event is notably higher using the AMG 900 4-day schedule, although the cumulative dose is similar for both schedules. This difference in 18FLT flaring may indicate the schedules differ in the duration and/or level of target inhibition in the skeletal tumor and bone marrow cells. Mice treated with sunitinib (positive control agent) did not show a skeletal 18FLT flare during the drug holiday, suggesting its mode of action is distinct from that of AMG 900. At the end of the study, mouse bone marrow was assessed for tumor burden by flow cytometry. Mice treated with AMG 900 showed a significant decrease in tumor burden compared with the vehicle group. Interestingly, the mice administered AMG 900 7-day schedule showed the most suppression of tumor growth compared with either AMG 900 4-day schedule or sunitinib. Together, our preclinical studies demonstrate that AMG 900 is a potent inhibitor of aurora kinases that robustly suppresses the growth of AML cells in vitro and in vivo. Furthermore, we highlight the utility of in vivo imaging to monitor AMG 900 drug action, which may help to inform future dose scheduling and drug combination studies. Disclosures: Cheung: Amgen Inc: Employment, Equity Ownership. Juan:Amgen Inc.: Employment, Equity Ownership. Wayne:Amgen Inc.: Employment, Equity Ownership. Hanestad:Amgen Inc.: Employment, Equity Ownership. Keegan:Amgen Inc.: Employment, Equity Ownership. Huard:Amgen Inc.: Employment, Equity Ownership. McElroy:Amgen Inc.: Employment, Equity Ownership. Stanton:Amgen Inc.: Employment, Equity Ownership. Bush:Amgen Inc.: Employment, Equity Ownership. Kendall:Amgen Inc.: Employment, Equity Ownership. Radinsky:Amgen Inc.: Employment, Equity Ownership. Abella:Amgen Inc. : Employment, Equity Ownership. Pieslor:Amgen Inc.: Employment, Equity Ownership. Friberg:Amgen Inc.: Employment, Equity Ownership. Coxon:Amgen Inc.: Employment, Equity Ownership. Gamelin:Amgen Inc: Employment, Equity Ownership. Payton:Amgen Inc.: Employment, Equity Ownership. Off Label Use: AMG 900 is currently in phase 1 clinical development, there is no approved label.

Chemotherapy-induced thrombocytopenia (CIT) is a serious complication of some cancer therapies and can necessitate treatment delay or chemotherapy dose reduction. Romiplostim is a thrombopoietin (TPO) receptor agonist approved for the treatment of immune thrombocytopenia (ITP). The ability of romiplostim to promote platelet recovery in a mouse model of multicycle chemotherapy/radiation therapy (CRT)-induced thrombocytopenia was examined. A secondary aim was to determine the optimum dose and timing of romiplostim treatment. In humans, an inverse relationship between platelet numbers and endogenous (e) TPO concentrations has been reported. Studies confirmed a similar relationship in both naïve mice and mice with CIT. Platelet numbers and eTPO concentration were measured during three 28-day cycles of CRT in mice. eTPO did not increase during the first five days after each CRT (the ‘eTPO gap’ time period, eTPO concentration of 2.8 ± 0.88 ng/mL), then increased to a peak 10 days after each CRT treatment (6.1 ± 1.5 ng/mL), as platelet numbers decreased. The window of time before eTPO concentration increased was used to design dosing paradigms that would supplement eTPO activity while concentrations were low. Since it was anticipated that patients would be unlikely to receive primary prophylaxis for first cycle CIT, a model was established in which romiplostim treatment was administered during the second or third cycle of CRT. In an attempt to ‘bridge’ the eTPO gap, mice were treated with 10 to 1000 μg/kg of romiplostim given on day 0, 1 or 2 after CRT in either cycle 2, cycle 3 or in both cycles, and platelet numbers were measured throughout that cycle. In some mice the total dose of romiplostim was divided over three days (i.e. one third on each of days 0, 1 and 2). Platelet recovery occurred faster in animals that received romiplostim in most conditions tested, with platelet counts significantly higher for groups that received CRT plus romiplostim compared to CRT alone (as determined by ANOVA with Dunnett's post-hoc test). Romiplostim also provided benefit in terms of decreasing the severity of the platelet nadir. Doses of >100 μg/kg given on day 0 significantly lessened the platelet nadir in both cycle 2 and cycle 3. Fractionating the dose and administering it over 3 days was effective but not superior to a single administration on day 0. In summary, an inverse relationship between platelet numbers and eTPO concentrations was observed during multiple cycles of CRT in mice. This decrease in eTPO created a ‘eTPO gap’ in each CRT cycle, which was effectively bridged by romiplostim treatment. These data may provide a rationale for clinical studies of romiplostim in patients undergoing myelosuppressive chemotherapy. Citation Format: Patricia L. McElroy, Keri Buck, Michael Eschenberg, Barbra J. Sasu, Graham Molineux. Romiplostim promotes platelet recovery in a mouse model of multicycle chemotherapy-induced thrombocytopenia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4109. doi:10.1158/1538-7445.AM2013-4109

Megakaryocyte growth and development factor (MGDF) is a recently characterized ligand for the cell surface receptor mpl. We have evaluated the effects of polyethylene glycollated recombinant human MGDF (PEG-rHuMGDF) on recovery of hematopoietic cells in mice following bone marrow transplantation (BMT) to support lethal irradiation. Mice treated with PEG-rHuMGDF (50 micrograms/kg/d) had accelerated recovery of platelet numbers compared with BMT mice treated with carrier or recombinant human granulocyte colony-stimulating factor (rHuG-CSF, 72 or 200 micrograms/kg/d). In contrast, PEG-rHuMGDF had no effect on white blood cell (WBC) or red blood cell (RBC) recovery. As previously reported, animals treated with rHuG-CSF had an enhanced recovery of WBC but not platelet or RBC levels. Interestingly, BMT receipient mice treated with the combination of PEG-rHuMGDF and rHuG-CSF showed simultaneous enhanced recovery of both leukocytes and platelets. PEGylated rHuMGDF was found to be considerably more potent than non- PEGylated rHuMGDF in this setting. PEG-rHuMGDF is an effective growth factor for enhancing platelet recovery in mice following BMT either alone or in combination with rHuG-CSF. It will be of interest to evaluate in a clinical setting the ratios of PEG-rHuMGDF and rHuG-CSF for simultaneous administration of these factors and accelerated recovery of both leukocytes and platelets.

Background: The aurora family of serine-threonine kinases (aurora-A, -B, -C) regulate cell-cycle progression in mammalian cells. Aurora-A and aurora-B are essential for proper chromosome congression, segregation, and cytokinesis during mitosis, whereas aurora-C function appears restricted to male meiosis. Aurora-B is responsible for the direct phosphorylation of histone H3 on serine-10. Aurora-A and aurora-B expression is elevated in a variety of human cancers and is associated with advanced clinical staging and poor prognosis. The emergence of aurora kinases as key mitotic regulators and their potential role in tumorigenesis has focused substantial interest in developing selective small molecule inhibitors for the treatment of human cancers. Objective: The aim of this study was to determine the in vitro effects of AMG 900 on a panel of tumor cell lines and profiling its antitumor activity across multiple human xenograft models. Results: AMG 900 is a novel ATP competitive small molecule inhibitor that is potent and highly selective for aurora kinases A, B, and C. In cells, AMG 900 inhibits autophosphorylation of aurora-A and aurora-B as well as the phosphorylation of histone H3. The predominant cellular response of tumor cells to AMG 900 treatment is aborted cell division, which leads to endoreduplication and cell death. The effect of AMG 900 was tested in a panel of 26 cell lines to evaluate its potential to inhibit cell proliferation across multiple tumor types. AMG 900 inhibited the proliferation of tumor cell lines at low nanomolar concentrations (EC50 values 1-6 nM), including a number of multidrug resistant (MDR) cell lines expressing ATP-binding cassette transporters (P-gp and BCRP1). In contrast, paclitaxel and three aurora kinase inhibitors (AZD1152, MK-0457, and PHA-739358) showed a loss of potency in these MDR cell lines compared to matched parental cell lines. In nude mice, oral administration of AMG 900 inhibited phosphorylation of histone H3 in tumors in a concentration- and dose-dependent manner. The effect of AMG 900 was tested in a panel of nine human xenografts representing five tumor types (breast, colon, lung, pancreatic, and uterine). Oral administration of AMG 900 at 15 mg/kg BID for two consecutive days per week or 3 mg/kg BID every day inhibited tumor growth (50-97%, P < 0.005) in all nine of the xenograft models. Importantly, three of these xenograft models are resistant to taxanes. Mice treated with efficacious doses of AMG 900 showed a transient loss of body weight and bone marrow cellularity, consistent with its on-mechanism effects on normal proliferating cells. Conclusion: Based on these preclinical activities, AMG 900 has the potential to treat advanced cancers, including tumors resistant to chemotherapeutic agents such as paclitaxel and docetaxel. AMG 900 is currently undergoing phase 1 clinical evaluation in patients with advanced solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4437.

Background: The aurora family of serine-threonine kinases (Aurora-A, -B, -C) regulate cell-cycle progression in mammalian cells. Aurora kinases A and B are essential for proper chromosome congression, segregation, and cytokinesis during mitosis, whereas aurora kinase C function appears restricted to male meiosis. Aurora kinase B is responsible for the direct phosphorylation of histone H3 on serine-10 (p-histone H3). Aurora kinase A and B expression is elevated in a variety of human cancers and is associated with advanced clinical staging and poor prognosis. The emergence of aurora kinases as key mitotic regulators and their potential role in tumorigenesis has focused substantial interest in developing selective small molecule inhibitors for the treatment of human cancers. AMG 900 is a novel ATP competitive small molecule inhibitor that is highly potent and selective for aurora kinases A, B, and C. Objective: The aim of this study was to characterize the in vivo effects of AMG 900 by establishing a pharmacokinetic-pharmacodynamic relationship and profiling its antitumor activity across multiple human xenografts models using a variety of dosing schedules. Results: In nude mice, oral administration of AMG 900 inhibited phosphorylation of histone H3 in tumors and bone marrow in a time- and dose-dependent manner. The degree of p-histone H3 suppression in cells correlated with plasma drug concentrations. The effect of AMG 900 was tested in a panel of human tumor xenograft models. Oral administration of AMG 900 at 15 mg/kg BID for two consecutive days per week or 3 mg/kg BID every day inhibited tumor growth (50–97% inhibition; p ≤ 0.0044) in all nine of the xenograft models tested compared with the vehicle control group. Importantly, three of these xenograft models are resistant to paclitaxel, docetaxel, and doxorubicin. Mice treated with efficacious doses of AMG 900 showed a transient loss of body weight (0–10%) and bone marrow cellularity, consistent with its on-mechanism effects on normal proliferating cells. Since aurora kinase inhibitors are known to cause neutropenia, studies were performed to investigate the effects of prophylactic treatment of PEGylated-G-CSF (SD/02). In mice, administration of SD/02 shortened the duration of AMG 900-induced neutropenia. Conclusion: Based on these preclinical activities, AMG 900 has the potential to treat advanced cancers, including tumors resistant to standard-of-care chemotherapeutic agents such as paclitaxel and docetaxel. AMG 900 is currently undergoing phase 1 clinical evaluation in patients with advanced solid tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C194.

Introduction: Recombinant human erythropoietin (rHuEPO) has become a standard treatment for the alleviation of anemia resulting from chronic renal failure and chemotherapy induced anemia. Treatment with rHuEPO corrects anemia, resulting in a broad range of clinical benefits including improved quality of life. Darbepoetin alfa (DA), a super-sialylated erythropoietic stimulating protein (ESP) with a 3-fold longer serum half-life than rHuEPO, is approved for use in the treatment of anemia. Another ESP investigated was a hyperglycosylated analog of DA having a greater number of carbohydrate chains and sialic acids (AMG205). Objective: Our intention was to develop and characterize new ESPs and examine their relative activity and ability to maintain normal hemoglobin levels in a mouse model with stable anemia (hemoglobin 2g/dL below initial levels, > 98 days). Methods and Results: Clearance of ESPs was reduced and in vivo potency was increased in proportion to number of carbohydrate chains; AMG205 was cleared the slowest and was the most potent of the 3 test molecules. A regimen of repeated low dose exposure to cisplatin (CDDP) was established that resulted in a stable reduction of hemoglobin levels by 2–3 g/dL (12.23±0.75g/dL). The hemoglobin range in control animals not treated with CDDP in this experiment was (14.53±0.57g/dL). Animals made anemic by repeated administration of CDDP were treated with rHuEPO (20μg/kg), DA (10μg/kg) or AMG205 (10μg/kg). Each group consisted of 15 mice. Following the initial injection, hemoglobin was measured every 2–3 days, and subsequent injections were administered when hemoglobin values were < 13 g/dL. Animals were so treated for 98 days. In this manner the number of injections of each ESP required to maintain normal hemoglobin was determined. Mice treated for 98 days required more injections of rHuEPO to maintain normal Hb levels than DA (13 vs.7 injections), while mice treated with AMG205 required 5 injections. Conclusion: Normal hemoglobin levels were maintained over a 3 month period with repeated administration of the ESPs investigated. The ESPs with increased carbohydrate content had reduced clearance, increased in vivo potency and could maintain hemoglobin within a normal range with fewer injections than rHuEPO.

Therapeutic use of cytokines in humans can result in the generation of antibodies that bind to the drug. In order to predict the potential effects of an autoantibody response to recombinant human G-CSF therapy, we developed a novel, reproducible mouse model of anti-G-CSF specific humoral immunity. Two groups of mice were immunized and boosted with mouse G-CSF (mG-CSF) chemically conjugated to either KLH or OVA on an alternating schedule. All mice immunized with the mG-CSF conjugates produced high titer, mG-CSF specific IgG responses that lasted for the length of the study (252 days). Significant neutropenia was concurrent with the rise in anti-mG-CSF IgG titers (p