Dale L. Ludwig

Inhibition of human leukemia in an animal model with human antibodies directed against vascular endothelial growth factor receptor 2. Correlation between antibody affinity and biological activity

Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. We recently showed that certain ‘liquid’ tumors such as leukemia not only produce VEGF, but also express functional VEGFR, resulting in an autocrine loop for tumor growth and propagation. A chimeric anti-VEGFR2 (or kinase insert domain-containing receptor, KDR) antibody, IMC-1C11, was shown to be able to inhibit VEGF-induced proliferation of human leukemia cells in vitro, and to prolong survival of nonobese diabetic-severe combined immune deficient (NOD-SCID) mice inoculated with human leukemia cells. Here we produced two fully human anti-KDR antibodies (IgG1), IMC-2C6 and IMC-1121, from Fab fragments originally isolated from a large antibody phage display library. These antibodies bind specifically to KDR with high affinities: 50 and 200 pM for IMC-1121 and IMC-2C6, respectively, as compared to 270 pM for IMC-1C11. Like IMC-1C11, both human antibodies block VEGF/KDR interaction with an IC50 of approximately 1 nM, but IMC-1121 is a more potent inhibitor to VEGF-stimulated proliferation of human endothelial cells. These anti-KDR antibodies strongly inhibited VEGF-induced migration of human leukemia cells in vitro, and when administered in vivo, significantly prolonged survival of NOD-SCID mice inoculated with human leukemia cells. It is noteworthy that the mice treated with antibody of the highest affinity, IMC-1121, survived the longest period of time, followed by mice treated with IMC-2C6 and IMC-1C11. Taken together, our data suggest that anti-KDR antibodies may have broad applications in the treatment of both solid tumors and leukemia. It further underscores the efforts to identify antibodies of high affinity for enhanced antiangiogenic and antitumor activities.

FLT3 ligand causes autocrine signaling in acute myeloid leukemia cells

The FLT3 receptor tyrosine kinase is highly expressed in most acute leukemias and frequently mutated in acute myeloid leukemia (AML). The mutated form of the receptor is constitutively activated and known to play an important role in AML, but the activation state of the overexpressed wild-type (wt) receptor is, at present, unknown. In this study, we examined the activation state of the wild-type receptor in AML. We found that the wild-type receptor was constitutively phosphorylated/activated in 8 of 12 primary AML samples and 4 of 13 leukemia cell lines. To explain why wtFLT3 is often activated, we investigated the expression of its ligand, FL, by these same cells. Coexpression of FL with FLT3 was a universal finding in both primary AML samples and leukemic-derived cell lines. To further prove that autocrine signaling was accounting for the activation, we showed that conditioned media but not fresh media was able to activate FLT3. In addition, an antibody that blocks binding of ligand to the receptor blocks FLT3 activation. Finally, depletion of FL from conditioned media is able to block the activation of FLT3. Taken together, these findings represent strong evidence that wtFLT3 is often constitutively activated in AML and thus, like its mutated form, might contribute to the altered signaling that characterizes leukemogenesis.

Simultaneous Blockade of Both the Epidermal Growth Factor Receptor and the Insulin-like Growth Factor Receptor Signaling Pathways in Cancer Cells with a Fully Human Recombinant Bispecific Antibody

Both the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR) have been implicated in the tumorigenesis of a variety of human cancers. Effective tumor inhibition has been achieved both experimentally and clinically with a number of strategies that antagonize either receptor activity. Here we constructed and produced two fully human recombinant bispecific antibodies (BsAb) that target both EGFR and IGFR, using two neutralizing human antibodies originally isolated from a phage display library. The BsAb not only retained the antigen binding capacity of each of the parent antibodies, but also were capable of binding to both targets simultaneously as demonstrated by a cross-linking enzyme-linked immunosorbent assay. Furthermore, the BsAb effectively blocked both ligands, EGF and IGF, from binding to their respective receptors, and inhibited tumor cell proliferation as potently as a combination of both the parent antibodies. More importantly, the BsAb were able to completely block activation of several major signal transduction molecules, including Akt and p44/p42 MAP kinases, by both EGF and IGF, whereas each individual parent antibody was only effective in inhibiting those signal molecules activated by the relevant single growth factor. The BsAb molecules retained good antigen binding activity after incubation with mouse serum at 37 °C for up to 6 days. Taken together, our results underscore the benefits of simultaneous targeting multiple growth factor receptor pathways for more efficacious cancer treatment. This report describes the first time use of a recombinant BsAb for targeting two tumor-associated molecules on either a single or adjacent tumor cells for enhanced antitumor activity.

Therapeutic implications of a human neutralizing antibody to the macrophage-stimulating protein receptor tyrosine kinase (RON), a c-MET family member.

RON is a member of the c-MET receptor tyrosine kinase family. Like c-MET, RON is expressed by a variety of epithelial-derived tumors and cancer cell lines and it is thought to play a functional role in tumorigenesis. To date, antagonists of RON activity have not been tested in vivo to validate RON as a potential cancer target. In this report, we used an antibody phage display library to generate IMC-41A10, a human immunoglobulin G1 (IgG1) antibody that binds with high affinity (ED50 = 0.15 nmol/L) to RON and effectively blocks interaction with its ligand, macrophage-stimulating protein (MSP; IC50 = 2 nmol/L). We found IMC-41A10 to be a potent inhibitor of receptor and downstream signaling, cell migration, and tumorigenesis. It antagonized MSP-induced phosphorylation of RON, mitogen-activated protein kinase (MAPK), and AKT in several cancer cell lines. In HT-29 colon, NCI-H292 lung, and BXPC-3 pancreatic cancer xenograft tumor models, IMC-41A10 inhibited tumor growth by 50% to 60% as a single agent, and in BXPC-3 xenografts, it led to tumor regressions when combined with Erbitux. Western blot analyses of HT-29 and NCI-H292 xenograft tumors treated with IMC-41A10 revealed a decrease in MAPK phosphorylation compared with control IgG-treated tumors, suggesting that inhibition of MAPK activity may be required for the antitumor activity of IMC-41A10. To our knowledge, this is the first demonstration that a RON antagonist and specifically an inhibitory antibody of RON negatively affects tumorigenesis. Another major contribution of this report is an extensive analysis of RON expression in approximately 100 cancer cell lines and approximately 300 patient tumor samples representing 10 major cancer types. Taken together, our results highlight the potential therapeutic usefulness of RON activity inhibition in human cancers.

Insulin-like Growth Factor-I Receptor Signaling Blockade Combined with Radiation

Signaling through the insulin-like growth factor-I receptor (IGF-IR) is implicated in cellular proliferation, apoptosis, carcinogenesis, metastasis, and resistance to cytotoxic cancer therapies. Targeted disruption of IGF-IR signaling combined with cytotoxic therapy may therefore yield improved anticancer efficacy over conventional treatments alone. In this study, a fully human anti-IGF-IR monoclonal antibody A12 (ImClone Systems, Inc., New York, NY) is examined as an adjunct to radiation therapy. IGF-IR expression is shown for a diverse cohort of cell lines, whereas targeted IGF-IR blockade by A12 inhibits IGF-IR phosphorylation and activation of the downstream effectors Akt and mitogen-activated protein kinase. Anchorage-dependent proliferation and xenograft growth is inhibited by A12 in a dose-dependent manner, particularly for non-small cell lung cancer lines. Clonogenic radiation survival of H226 and H460 cells grown under anchorage-dependent conditions is impaired by A12, demonstrating a radiation dose-enhancing effect for IGF-IR blockade. Postradiation anchorage-independent colony formation is inhibited by A12 in A549 and H460 cells. In the H460 xenograft model, combining A12 and radiation significantly enhances antitumor efficacy compared with either modality alone. These effects may be mediated by promotion of radiation-induced, double-stranded DNA damage and apoptosis as observed in cell culture. In summary, these results validate IGF-IR signal transduction blockade as a promising strategy to improve radiation therapy efficacy in human tumors, forming a basis for future clinical trials.

Synergy between an IGF-1R antibody and Raf/MEK/ERK and PI3K/Akt/mTOR pathway inhibitors in suppressing IGF-1R-mediated growth in hematopoietic cells.

The Insulin-like growth factor-1 receptor (IGF-1R) is overexpressed in a variety of tumors including breast, prostate and myeloma. Thus, IGF-1R and its downstream signaling effectors are good candidates for molecular-based targeted antitumor therapies. Indeed, protein inhibitors of IGF-1R signaling and IGF-1R blocking antibodies are undergoing clinical trials. Herein, the molecular basis for antibody-mediated IGF-1R signal inhibition has been investigated in a hematopoietic cell line model, FDC-P1, that has been rendered interleukin-3 independent in a ligand-dependent manner through retroviral-mediated expression of IGF-1R (FD/IGF-1R). Furthermore, the ability of an anti-IGF-1R antibody to synergize with signal-transduction pathway inhibitors and induce apoptosis was determined. The αIGF-1R antibody, A12, was capable of arresting IGF-1 or insulin-induced FD/IGF-1R cell proliferation in the G1 phase of the cell cycle and resulted in apoptotic induction. A12 effectiveness could be potentiated through combination treatment with small molecule inhibitors of the Ras/Raf/MEK/ERK or PI3K/Akt/mTOR pathways. These results validate the use of the FD/IGF-1R cells to evaluate the effectiveness and mechanisms of targeted IGF-1R therapeutic strategies.

Antibody targeting of the insulin-like growth factor I receptor enhances the anti-tumor response of multiple myeloma to chemotherapy through inhibition of tumor proliferation and angiogenesis

Although many multiple myeloma (MM) patients initially respond to cytotoxic therapy, most eventually relapse. Novel therapeutic strategies employing a combination of chemotherapy with targeted biologics may significantly enhance the response of tumor cells to treatment. We tested a fully human anti-IGF-IR antibody (A12) against MM, and showed specific inhibition of IGF-I or serum -induced IGF-IR signaling in MM cells in vitro. The A12 as a single agent was demonstrated to exert modest to significant inhibition of tumor growth in vivo in various subcutaneous xenograft MM models. The A12 was also evaluated in a disseminated xenograft MM.1S NOD/SCID model as monotherapy or in combination with other drugs (bortezomib, melphalan) currently in clinical use. The tumor burden, as determined by luciferase bioimaging, was sharply decreased, and overall survival significantly prolonged when the therapies were combined. Immunohistochemical analysis demonstrated that the A12 treated tumors had significantly decreased vascularization compared to control tumors. Furthermore, most MM lines constitutively secreted significant quantities of VEGF, and this was enhanced following IGF-I treatment. Inhibition of IGF-IR by the A12 in vitro suppressed both constitutive and IGF-I-induced secretion of VEGF, indicating that a putative anti-angiogenic mechanism associated with the A12 treatment may contribute to its anti-tumor effect.

Monoclonal antibody therapeutics and apoptosis

The potential for disease-specific targeting and low toxicity profiles have made monoclonal antibodies attractive therapeutic drug candidates. Antibody-mediated target cell killing is frequently associated with immune effector mechanisms such as antibody-directed cellular cytotoxicity, but they can also be induced by apoptotic processes. Antibody-directed mechanisms, including antigen crosslinking, activation of death receptors, and blockade of ligand-receptor growth or survival pathways, can elicit the induction of apoptosis in targeted cells. Depending on their mechanism of action, monoclonal antibodies can induce targeted cell-specific killing alone or can enhance target cell susceptibility to chemo- or radiotherapeutics by effecting the modulation of antiapoptotic pathways. This review will focus on the mechanisms by which antibodies are capable of eliciting programmed cell death either directly or indirectly within tumor cells.

IgG isotype, glycosylation, and EGFR expression determine the induction of antibody-dependent cellular cytotoxicity in vitro by cetuximab

PURPOSE To evaluate the antibody-dependent cellular cytotoxicity (ADCC) of cetuximab, an anti-epidermal growth factor receptor (EGFR) IgG1 antibody, in vitro. METHODS Binding to human Fc receptors was measured by ELISA. ADCC against a panel of tumor cell lines was evaluated using peripheral blood mononuclear cells or NK cells as effectors and lactate dehydrogenase release as a marker of cell killing. Cetuximab was compared with two glycan variants of cetuximab and with panitumumab, an anti-EGFR IgG2. RESULTS Cetuximab bound with high affinity to FcγRI (EC50 = 0.13 nM) and FcγRIIIa (EC50 = 6 nM) and effectively induced ADCC across multiple tumor cell lines. Panitumumab and aglycosylated cetuximab did not bind to FcγRI or FcγRIIIa nor have ADCC activity even at high effector-target cell ratios, even though the EGFR-binding affinity of cetuximab and panitumumab were shown to be comparable (KD = 87 pM and 83 pM, respectively). The extent of cetuximab-elicited ADCC was associated with the level of EGFR expression on tumor cells. CONCLUSIONS Cetuximab elicits effective ADCC activity against a wide range of tumor cells in vitro. This activity is dependent on antibody glycosylation and IgG1 isotype as well as tumor-cell EGFR expression. These findings suggest that ADCC may contribute to the antitumor activity of cetuximab.

DEGA/AMIGO-2, a leucine-rich repeat family member, differentially expressed in human gastric adenocarcinoma: effects on ploidy, chromosomal stability, cell adhesion/migration and tumorigenicity.

We have discovered DEGA, a novel cDNA differentially expressed in human gastric adenocarcinomas. The DEGA gene product contains a signal peptide, five leucine-rich repeat motifs and a single IgG, and transmembrane domain, suggesting its residence on the plasma membrane. Transfection of 293 cells with a DEGA-GFP fusion construct confirmed its cell surface localization. Although the cytosolic portion of the DEGA gene product does not contain known protein domains, approximately one-fifth of these residues are either a serine or a threonine, suggesting that DEGA may play a role in signal transduction. BLAST searches revealed DEGA to be an exact match to AMIGO-2, a recently identified, but functionally uncharacterized protein related to AMIGO, a leucine-rich repeat containing cell adhesion molecule implicated in axon tract development. In this report, we show that DEGA/AMIGO-2 mRNA is differentially expressed in ∼45% of tumor versus normal tissue from gastric adenocarcinoma patients. Stable expression of a DEGA/AMIGO-2 antisense construct in the gastric adenocarcinoma cell line, AGS, led to altered morphology, increased ploidy, chromosomal instability, decreased cell adhesion/migration, and a nearly complete abrogation of tumorigenicity in nude mice. These findings suggest a potential etiologic role for DEGA/AMIGO-2 in gastric adenocarcinoma.