Toshihiko Takeuchi

A plasma kallikrein-dependent plasminogen cascade required for adipocyte differentiation

Here we show that plasma kallikrein (PKal) mediates a plasminogen (Plg) cascade in adipocyte differentiation. Ecotin, an inhibitor of serine proteases, inhibits cell-shape change, adipocyte-specific gene expression, and lipid accumulation during adipogenesis in culture. Deficiency of Plg, but not of urokinase or tissue-type plasminogen activator, suppresses adipogenesis during differentiation of 3T3-L1 cells and mammary-gland involution. PKal, which is inhibited by ecotin, is required for adipose conversion, Plg activation and 3T3-L1 differentiation. Human plasma lacking PKal does not support differentiation of 3T3-L1 cells. PKal is therefore a physiological regulator that acts in the Plg cascade during adipogenesis. We propose that the Plg cascade fosters adipocyte differentiation by degradation of the fibronectin-rich preadipocyte stromal matrix.

Enhancement of antibody fragment secretion into the Escherichia coli periplasm by co-expression with the peptidyl prolyl isomerase, FkpA, in the cytoplasm

Improper protein folding or aggregation can frequently be responsible for low expression and poor functional activity of antibody fragments secreted into the Escherichia coli periplasm. Expression issues also can affect selection of antibody candidates from phage libraries, since antibody fragments displayed on phage also are secreted into the E. coli periplasm. To improve secretion of properly folded antibody fragments into the periplasm, we have developed a novel approach that involves co-expressing the antibody fragments with the peptidyl prolyl cis-trans isomerase, FkpA, lacking its signal sequence (cytFkpA) which consequently is expressed in the E. coli cytosol. Cytoplasmic expression of cytFkpA improved secretion of functional Fab fragments into the periplasm, exceeding even the benefits from co-expressing Fab fragments with native, FkpA localized in the periplasm. In addition, panning and subsequent screening of large Fab and scFv naïve phage libraries in the presence of cytFkpA significantly increased the number of unique clones selected, as well as their functional expression levels and diversity.

Engineering Ecotin for Identifying Proteins with a Trypsin Fold

Ecotin is a bidentate, fold-specific inhibitor of mammalian serine-proteases produced by Escherichia coli. This molecule may be engineered to increase and/or change its affinity and specificity providing significant biotechnological potential. Since ecotin binds tightly to serine proteases of the trypsin fold, it may help to identify the role of these enzymes in different biological processes. In this work, we tested ecotin variants as an affinity purification reagent for identifying enzymes in samples of tumor progression and mammary gland involution. Initially, we used a commercial source of urokinase-type plasminogen activator (u-PA) that remained fully active after elution from an affinity column of the ecotin variant (M84R, M85R). We then successfully identified u-PA from more complex mixtures including lysates from a prostate cancer cell line and involuting mouse mammary glands. Interestingly, a membrane-type serine protease 1 was isolated from the Triton X-100-solubilized PC-3 cell lysates, and surprisingly, haptoglobin, a serine-protease homolog protein, was also identified in mammary gland lysates and in blood. Haptoglobin does not prevent ecotin inhibition of u-PA, but it may act as a carrier within blood when ecotin is used in vivo. Finally, this affinity purification matrix was also able to identify a thrombin-like enzyme from snake venom using an ecotin variant directed against thrombin. Overall, the ecotin variants acted as robust tools for the isolation and characterization of proteins with a trypsin fold. Thus, they may assist in the understanding of the role of these serine proteases and homologous proteins in different biological processes.

Development and characterization of human monoclonal antibodies that neutralize multiple TGFβ isoforms

ABSTRACT Transforming growth factor (TGF)β levels are elevated in, and drive the progression of, numerous disease states such as advanced metastatic cancer and systemic and ocular fibrosis. There are 3 main isoforms, TGFβ1, 2, and 3. As multiple TGFβ isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFβ isoforms. Fully human antibody phage display libraries were used to discover a number of antibodies that bind and neutralize various combinations of TGFβ1, 2 or 3. The primary panning did not yield any uniformly potent pan-isoform neutralizing antibodies; therefore, an antibody that displayed potent TGFβ 1, 2 inhibition, but more modest affinity versus TGFβ3, was affinity matured by shuffling with a light chain sub-library and further screening. This process yielded a high affinity pan-isoform neutralizing clone. Antibodies were analyzed and compared by binding affinity, as well as receptor and epitope competition by surface plasmon resonance methods. The antibodies were also shown to neutralize TGFβ effects in vitro in 3 assays: 1) interleukin (IL)-4 induced HT-2 cell proliferation; 2) TGFβ-mediated IL-11 release by A549 cells; and 3) decreasing SMAD2 phosphorylation in Detroit 562 cells. The antibodies’ potency in these in vitro assays correlated well with their isoform-specific affinities. Furthermore, the ability of the affinity-matured clone to decrease tumor burden in a Detroit 562 xenograft study was superior to that of the parent clone. This affinity-matured antibody acts as a very potent inhibitor of all 3 main isoforms of TGFβ and may have utility for therapeutic intervention in human disease.

An Allosteric Antibody to the Leptin Receptor Reduces Body Weight and Reverses the Diabetic Phenotype in the Lepob/Lepob Mouse

Leptin (LEP) deficiency results in major metabolic perturbations, including obesity, dyslipidemia, and diabetes. Although LEP deficiency can be treated with daily injections of a recombinant LEP, generation of an antibody activating the LEP receptor (LEPR) that has both an intrinsically long half‐life and low immunogenicity could be useful in the treatment of this condition.

Abstract LB-306: Impacting Humoral Hypercalcemia of Malignancy (HHM) and associated PTH1R-mediated morbidities: Characterization of an anti-PTH1R antagonist monoclonal antibody to reverse hypercalcemia

Humoral Hypercalcemia of Malignancy (HHM) occurs in 20-30 percent of advanced cancers, both solid tumors and hematologic malignancies, and is the most common life-threatening complication of cancer. HHM is due to tumors secreting abnormally high quantities of parathyroid hormone-related protein (PTHrP), a ligand for PTH1R (a family B GPCR) resulting in hypercalcemia. A potent and long acting PTH1R antagonist could reverse the hypercalcemia in HHM. Using XOMA’s fully-human antibody phage display libraries, highly potent anti-PTH1R antagonist monoclonal antibodies were discovered, and screened by FACS for binding to CHO-K1 cells over-expressing human PTH1R. Human and murine receptor antagonism was demonstrated in cAMP accumulation and calcium flux assays with the human Saos-2 and the rat UMR106 osteosarcoma cell lines, which express endogenous PTH1R. The epitope demonstrating the greatest receptor antagonism was localized to the N-terminal extracellular domain of human PTH1R. Antibodies were affinity matured via light chain shuffling, resulting in leads with sub-nanomolar binding affinities as measured by SPR and verified by FACS. Functionally, PTH1R is expressed on osteoblasts and osteocytes. Stimulation with PTHrP leads to increased expression of M-CSF, RANKL and other factors that drive the differentiation and activation of bone resorbing osteoclast cells. It was shown that the lead anti-PTH1R mAb inhibited both PTH- and PTHrP-induced osteoclast differentiation by greater than 10-fold. In vivo proof-of-concept was achieved in rodent models where hypercalcemia was established in rats by SC infusion of PTHrP via an osmotic pump. IV administration of 2 and 10 mg/kg antibody reduced serum calcium levels by a minimum of 2 mg/dL within 48 hours in a dose-dependent manner. Additionally, in a murine C26 tumor model of hypercalcemia associated with elevated PTHrP, the anti-PTH1R mAb showed a dose-dependent correction of hypercalcemia at doses as low as 2 mg/kg with a sustained duration of action. In this model, the antibody given at 10 mg/kg IV completely reversed the hypercalcemia within 24 hours, lowering serum calcium from greater than 15mg/dL to approximately 6mg/dL. Additional PK/PD parameters were assessed in mouse and rat models. Patients with HHM typically have a poor prognosis and HHM is a frequent reason that cancer patients are readmitted to hospitals or enter hospice care. Left untreated, hypercalcemia in these patients can cause coma or death. The highly potent anti-PTH1R antagonist mAb described here has the potential to become a first in class therapy for HHM, ameliorating hypercalcemia-associated morbidities, and extending the utility of other anti-cancer agents. This promising therapeutic antibody is currently in late stage preclinical development. Citation Format: Amer M. Mirza, Agnes Choppin, Daniel Bedinger, Rachel Hunt, Robyn Cotter, Elizabeth Pongo, Kiran Ahluwalia, Catarina Tran, Llewelyn Lao, Kristin Lind, Sujeewa Wijesuriya, Lynn Webster, Fangjiu Zhang, Kirk Johnson, Toshihiko Takeuchi, Raphael Levy. Impacting Humoral Hypercalcemia of Malignancy (HHM) and associated PTH1R-mediated morbidities: Characterization of an anti-PTH1R antagonist monoclonal antibody to reverse hypercalcemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-306. doi:10.1158/1538-7445.AM2017-LB-306

Abstract LB-149: Discovery and characterization of fully human monoclonal antibodies that neutralize multiple TGFβ isoforms

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA TGFβ levels are elevated in, and can drive the progression of, numerous disease states including advanced metastatic cancer and fibrosis. The exact role of each TGFβ isoform in disease is an area of active research. To help elucidate the therapeutic importance and test the value of inhibiting multiple TGFβ isoforms, we generated antibodies with differing specificities to each of the three TGFβ isoforms. Antibodies that functionally neutralized TGFβ1/2, TGFβ2/3, and TGFβ1/2/3 were characterized in vitro, and in vivo. Two surface plasmon resonance (SPR)-based kinetic methods were used to compare the binding affinities and rate constants of the three antibodies. All three antibodies had single digit nanomolar affinities (KD) or better to the TGFβ isoforms, ranging from 1.7pM to 1400pM. The antibodies were characterized in cell based assays for their ability to effect signaling, cell proliferation, and stimulated cytokine release, and EC50 values for each were obtained. Potency in each of the cell assays directly correlated with the affinity of the antibody for the TGFβ isoform used to elicit the effect.These antibodies were then used to elucidate the role of each of the TGFβ isoforms in more complex systems in vivo. Detroit 562, a human metastatic pharyngeal carcinoma derived cell line, was shown to express all three TGFβ isoforms in vitro and in vivo. 5x106 cells were implanted subcutaneously on the flanks of athymic ( nu/nu ) mice. Tumors were established to ∼200 mm3, and treatment initiated at day 7 post implant. Groups were treated with 10mg/kg IP twice weekly. At Day 28, blocking all three TGFβ isoforms with a pan reactive TGFβ1/2/3 antibody significantly reduced mean tumor volume (219 ± 38 mm3) compared to a control IgG (1022 ± 241mm3) (n= 12 mice per group). Additionally, dose-ranging studies were performed (1, 3, or 10 mg/kg) to determine the relative potency of the antibodies. We found that tumors in mice treated with either the anti-TGFβ1/2 or the pan anti-TGFβ1/2/3 inhibitor antibody (3mg/kg twice weekly) showed notable and significant (p<0.005) differences in mean tumor volumes and mean tumor weights at day 30 compared to IgG control antibody, with average tumor volume reductions of 63%, and 64% respectively. In this model, inhibition of tumor growth by the anti-TGFβ1/2 antibody was indistinguishable from inhibition by the anti-TGFβ1/2/3 antibody, suggesting that TGFβ3 may not play a role in tumor progression. Conversely, in this model, inhibition of TGFβ2/3 alone was insufficient to significantly impact tumor growth. These data suggest that effective neutralization of TGFβ1/2 function may be sufficient to inhibit tumor growth and that neutralizing all three TGFβ isoforms is not required. Specific and targeted inhibition of select TGFβ isoforms offers a more focused therapeutic approach which has potential for improved safety. Experiments are underway to elucidate the role of the various TGFβ isoforms in key aspects of tumor biology. Citation Format: Amer Mirza, Daniel Bedinger, Llewelyn Lao, Shireen Khan, Toshihiko Takeuchi. Discovery and characterization of fully human monoclonal antibodies that neutralize multiple TGFβ isoforms. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-149. doi:10.1158/1538-7445.AM2014-LB-149