Targeting primary acute myeloid leukemia with a new CXCR4 antagonist IgG1 antibody (PF-06747143).

The chemokine receptor CXCR4 mediates cell anchorage in the bone marrow (BM) microenvironment and is overexpressed in 25-30% of patients with acute myeloid leukemia (AML). Here we have shown that a new CXCR4 receptor antagonist IgG1 antibody (PF-06747143) binds strongly to AML cell lines and to AML primary cells inhibiting their chemotaxis in response to CXCL12. PF-06747143 also induced cytotoxicity in AML cells via Fc-effector function. To characterize the effects of PF-06747143 on leukemia progression, we used two different patient-derived xenograft (PDX) models: Patient 17CXCR4-low and P15CXCR4-high models, characterized by relatively low and high CXCR4 expression, respectively. Weekly administration of PF-06747143 to leukemic mice significantly reduced leukemia development in both models. Secondary transplantation of BM cells from PF-06747143-treated or IgG1 control-treated animals showed that leukemic progenitors were also targeted by PF-06747143. Administration of a single dose of PF-06747143 to PDX models induced rapid malignant cell mobilization into the peripheral blood (PB). These findings support evaluation of this antibody in AML therapy, with particular appeal to patients resistant to chemotherapy and to unfit patients, unable to tolerate intensive chemotherapy.

Targeting the CXCR4 pathway using a novel anti-CXCR4 IgG1 antibody (PF-06747143) in chronic lymphocytic leukemia.

BACKGROUND: The CXCR4-CXCL12 axis plays an important role in the chronic lymphocytic leukemia (CLL)-microenvironment interaction. Overexpression of CXCR4 has been reported in different hematological malignancies including CLL. Binding of the pro-survival chemokine CXCL12 with its cognate receptor CXCR4 induces cell migration. CXCL12/CXCR4 signaling axis promotes cell survival and proliferation and may contribute to the tropism of leukemia cells towards lymphoid tissues and bone marrow. Therefore, we hypothesized that targeting CXCR4 with an IgG1 antibody, PF-06747143, may constitute an effective therapeutic approach for CLL. METHODS: Patient-derived primary CLL-B cells were assessed for cytotoxicity in an in vitro model of CLL microenvironment. PF-06747143 was analyzed for cell death induction and for its potential to interfere with the chemokine CXCL12-induced mechanisms, including migration and F-actin polymerization. PF-06747143 in vivo efficacy was determined in a CLL murine xenograft tumor model. RESULTS: PF-06747143, a novel-humanized IgG1 CXCR4 antagonist antibody, induced cell death of patient-derived primary CLL-B cells, in presence or absence of stromal cells. Moreover, cell death induction by the antibody was independent of CLL high-risk prognostic markers. The cell death mechanism was dependent on CXCR4 expression, required antibody bivalency, involved reactive oxygen species production, and did not require caspase activation, all characteristics reminiscent of programmed cell death (PCD). PF-06747143 also induced potent B-CLL cytotoxicity via Fc-driven antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity activity (CDC). PF-06747143 had significant combinatorial effect with standard of care (SOC) agents in B-CLL treatment, including rituximab, fludarabine (F-ara-A), ibrutinib, and bendamustine. In a CLL xenograft model, PF-06747143 decreased tumor burden and improved survival as a monotherapy, and in combination with bendamustine. CONCLUSIONS: We show evidence that PF-06747143 has biological activity in CLL primary cells, supporting a rationale for evaluation of PF-06747143 for the treatment of CLL patients.

A novel CXCR4 antagonist IgG1 antibody (PF-06747143) for the treatment of hematologic malignancies.

The chemokine receptor CXCR4 is highly expressed and associated with poor prognosis in multiple malignancies. Upon engagement by its ligand, CXCL12, CXCR4 triggers intracellular signaling pathways that control trafficking of cells to tissues where the ligand is expressed, such as the bone marrow (BM). In hematologic cancers, CXCR4-driven homing of malignant cells to the BM protective niche is a key mechanism driving disease and therapy resistance. We developed a humanized CXCR4 immunoglobulin G1 (IgG1) antibody (Ab), PF-06747143, which binds to CXCR4 and inhibits CXCL12-mediated signaling pathways, as well as cell migration. In in vivo preclinical studies, PF-06747143 monotherapy rapidly and transiently mobilized cells from the BM into the peripheral blood. In addition, PF-06747143 effectively induced tumor cell death via its Fc constant region-mediated effector function. This Fc-mediated cell killing mechanism not only enhanced antitumor efficacy, but also played a role in reducing the duration of cell mobilization, when compared with an IgG4 version of the Ab, which does not have Fc-effector function. PF-06747143 treatment showed strong antitumor effect in multiple hematologic tumor models including non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), and multiple myeloma (MM). Importantly, PF-06747143 synergized with standard-of-care agents in a chemoresistant AML patient-derived xenograft model and in an MM model. These findings suggest that PF-06747143 is a potential best-in-class anti-CXCR4 antagonist for the treatment of hematologic malignancies, including in the resistant setting. PF-06747143 is currently in phase 1 clinical trial evaluation (registered at www.clinicaltrials.gov as #NCT02954653).

Targeting of humanized antibody D93 to sites of angiogenesis and tumor growth by binding to multiple epitopes on denatured collagens.

A humanized, affinity-matured IgG1 antibody, called D93, and its parental murine IgM HUI77 have been shown to specifically bind denatured collagens and thereby inhibit angiogenesis and tumor growth in various animal models. In this study, we have identified epitopes for both HUI77 and D93 on human collagen type IV. Several tryptic D93-binding peptides were identified by Western blot analysis and protein sequencing. Epitopes for D93 were ultimately identified by screening a synthetic 16-mer peptide array spanning immunoreactive tryptic peptides. D93 reacted with a peptide corresponding to alpha1(IV) P(1337)-Y(1352) that could inhibit binding of both D93 and HUI77 to denatured collagen IV in a concentration-dependent manner. A 9-mer peptide corresponding to alpha1(IV) G(1344)-Y(1352) showed maximum inhibition of D93 and HUI77 antibody binding to denatured collagen IV, and was critically dependent on the presence of hydroxyproline. D93 bound with similar affinity to denatured collagen IV and synthetic peptides with a K(D) of 1-10 microM for monovalent and of 30-63 nM for bivalent binding. Potential epitopes for D93 are highly repeated in multiple collagen types of diverse vertebrate species explaining reactivity of D93 with denatured collagens types I-V from chicken to man. Our data suggest that D93 inhibits angiogenesis and tumor growth by blockade of cryptic bioactive signals on proteolyzed collagens with importance for growth of tumors and new blood vessels.

Novel anti-denatured collagen humanized antibody D93 inhibits angiogenesis and tumor growth: An extracellular matrix-based therapeutic approach.

Matrix metalloproteases (MMPs) secreted by both tumor and endothelial cells proteolytically degrade collagen during tumor growth and neo-vascularization. This exposes cryptic binding sites on collagen with functional relevance for angiogenesis. In this report, we characterized a novel humanized monoclonal IgG1 antibody, D93. After humanization, the antibody retained the binding specificity of the parental murine IgM antibody for denatured (dn) collagen. D93 recognized dn-collagen but not native (nat) collagen of different species, including mouse, chicken, and human, indicating that its cryptic binding site(s) is conserved across species. In immunohistochemistry (IHC) studies, D93 stained the basement membrane of blood vessels in several xenograft human tumors or in surgically removed tumor tissues from patients with different types of malignancies. D93 staining was rarely or not present in normal blood vessels of healthy tissues. In in vivo experiments, D93 significantly inhibited basic fibroblast growth factor (bFGF)-induced angiogenesis in chick embryo chorioallantoic membrane (CAM) and the tumor growth of pre-established orthotopic human breast (MDA-MB-435) tumors in mice. D93 i.v. administered in mice was subsequently detected in the subendothelial basement membrane of tumor blood vessels but not blood vessels of normal tissues. Inhibition of growth of pre-established orthotopic human breast MDA-MB-435 tumors was more effective when D93 was combined with Taxol, than either treatment alone. In addition, tumors from animals treated with D93 and/or Taxol showed significantly reduced levels of the endothelial cell-marker CD31. Our data suggest that blockade of cryptic epitopes exposed on collagen IV during angiogenesis and tumor growth by a monoclonal antibody may provide a novel therapeutic modality for treatment of cancer and pathogenic neo-vascularization.

Pituitary tumorigenesis targeted by the ovine follicle-stimulating hormone beta-subunit gene regulatory region in transgenic mice.

Targeted tumorigenesis in transgenic mice has been a powerful tool for the study of gene expression and oncogenesis, as well as for the production of differentiated immortal cell lines from rare cell types. Follicle-stimulating hormone (FSH) is secreted by the gonadotrope cells of the anterior pituitary gland and plays a pivotal role in mammalian reproduction. Here we have used the regulatory region of the ovine FSH beta gene to direct expression of the SV40 T antigen oncogene to gonadotrope cells in the pituitary of transgenic mice. Two of five transgenic mouse lines bearing this fusion gene rapidly developed pituitary tumors, with appearance of adenomatous foci as early as 6 weeks of age, resulting in death by 12 weeks of age in both genders. Histologic examination of tumor development over time revealed that increases in cell proliferation and dysplasia were accompanied by decreases in synthesis of pituitary hormones, indicating dedifferentiation of the pituitary cells. Histological features observed in these tumors were in agreement with this rapid transformation of cell phenotype. Tumors were multifocal in origin, and the most highly transformed cell types observed consisted of giant pale basophilic cells with enormous hyperploid nuclei associated with infiltrating neuronal-like cells, which were very abundant at later stages of tumor development. Mitotic indices were much higher in transgenic than wild-type pituitaries, as expected. Morphologic analysis of the gonads of these transgenic mice showed no major developmental differences, as compared to wild-type littermates, however the length of the seminiferous tubules in transgenic males was greater than age-matched wild-type animals. Despite this phenotype difference, both genders were fertile, with normal sperm development observed in males and normal estrous cycle stages in females. Moreover, while 8 -- 10-week-old transgenic males had much lower blood levels of FSH than littermates, transgenic female FSH levels were the same as those of wild-type females. These animals offer a unique and potentially useful model of organ-specific tumorigenesis, where a multistage pathway of tumor development is evident, both histologically and temporally. Study of such models will advance our knowledge on the physiological and molecular mechanisms involved in gene expression as well as tumor formation.

A unique case of combined pituitary hormone deficiency caused by a PROP1 gene mutation (R120C) associated with normal height and absent puberty.

We report a 28-year-old-female who presented with primary amenorrhoea, absence of puberty, obesity and normal stature. The subject was clearly short as a child, with a height more than 2 SD below normal until the age of 15 years. The pubertal growth spurt failed to develop. She continued growing at a prepubertal rate until growth ceased at the age of 20 years, reaching her final adult height of 157 cm (SDS -0.86) without hormonal treatment. A combined pituitary hormone stimulation test of anterior pituitary function showed deficiencies of GH, LH and FSH, and low normal serum levels of TSH and PRL. Magnetic resonance imaging revealed a hypoplastic pituitary with markedly reduced pituitary height. In addition, a whole body dual energy X-ray absorptiometry scan showed high levels of body fat (54%). Combined pituitary hormone deficiencies with a hypoplastic pituitary suggested the diagnosis of a Prophet of Pit-1 (PROP1) gene mutation. Normal stature in this case, however, confounded this diagnosis. Sequencing of PROP1 revealed homozygosity for a single base-pair substitution (C to T), resulting in the replacement of an Arg by a Cys at codon 120 (R120C) in the third helix of the homeodomain of the Prop-1 protein. To our knowledge, this is the first report of a patient with a mutation in the PROP1 gene that attained normal height without hormonal treatment, indicating a new variability in the PROP1 phenotype, with important implications for the diagnosis of these patients. We suggest that this can be explained by (i) the presence of low levels of GH in the circulation during childhood and adolescence; (ii) the lack of circulating oestrogen delaying epiphyseal fusion, resulting in growth beyond the period of normal growth; and (iii) fusion of the epiphyseal plates, possibly as a result of circulating oestrogens originating from peripheral conversion of androgens by adipose tissue.

Transcriptional activation of the ovine follicle-stimulating hormone-beta gene by gonadotropin-releasing hormone involves multiple signal transduction pathways.

GnRH regulates gonadotrope cells through GnRH receptor activation of the PKC-, MAPK-, and calcium-activated signaling cascades. Due to the paucity of homologous model systems expressing FSHbeta, little is known about the specific mechanisms involved in transcriptional regulation of this gene by GnRH. Previous studies from our laboratory demonstrated that the gonadotrope-derived LbetaT2 cell line expresses FSHbeta mRNA. In the present study we characterized the mechanisms involved in GnRH regulation of the FSHbeta promoter using this cell model. Using transfection assays, we show that GnRH regulation of the ovine FSHbeta promoter involves at least two elements, present between -4152/-2878 and -2550/-1089 bp, in association with one or several elements within the proximal region of the promoter. Surprisingly, the two activating protein-1 sites previously shown to be involved in the FSHbeta response to GnRH in heterologous cells do not play a role in GnRH responsiveness in the gonadotrope cell model. Here we demonstrate that calcium influx itself is not sufficient to confer the response, but it is necessary for both 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and GnRH induction of the FSHbeta gene. Moreover, we show that GnRH regulation of FSHbeta gene expression is mediated by PKC and establish the presence of multiple PKC isozymes in LbetaT2 cells. Interestingly, GnRH and TPA induce activity of the FSHbeta promoter through different, although possibly overlapping, pools of PKC isoforms. This is further supported by the use of a MAPK inhibitor, which abolishes the induction of FSHbeta by GnRH, but not by TPA. In conclusion, we have demonstrated that calcium, PKC, and MAPK signaling systems are all involved in the induction of FSHbeta gene expression by GnRH in the LbetaT2 mouse gonadotrope cell model.