Paul Balderes

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.

Anti-Vascular Endothelial Growth Factor Receptor-1 Antagonist Antibody as a Therapeutic Agent for Cancer

Purpose: Vascular endothelial growth factor receptor-1 (VEGFR-1) plays important roles in promotion of tumor growth by mediating cellular functions in tumor vascular endothelium and cancer cells. Blockade of VEGFR-1 activation has been shown to inhibit pathologic angiogenesis and tumor growth, implicating VEGFR-1 as a potential therapeutic target for the treatment of cancer. We have thus developed a VEGFR-1 antagonist human monoclonal antibody designated as IMC-18F1 and evaluated its antitumor activity in preclinical experimental models to show the therapeutic potential of the antibody for cancer treatment in clinic. Experimental Design: Human IgG transgenic mice were used for generation of anti-VEGFR-1 antibodies. Anti-VEGFR-1-specific blocking antibodies were identified using solid-phase binding and blocking assays. Inhibitory antitumor cell activity of IMC-18F1 was assessed in cell-based kinase and growth assays. Pharmacokinetic/pharmacodynamic studies were done to determine the association of antibody blood level with antitumor efficacy of the antibody in vivo. Antitumor efficacy of the anti-VEGFR-1 antibodies as monotherapy and in combination with cytotoxic agents was evaluated in human breast cancer xenograft models. Results: A fully human neutralizing antibody, IMC-18F1, was shown to be a high-affinity (KD = 54 pmol) inhibitor of VEGFR-1 ligand binding (VEGF-A, VEGF-B, and placental growth factor). IMC-18F1 inhibited ligand-induced intracellular activation of VEGFR-1 and mitogen-activated protein kinase signaling and prevented ligand-stimulated in vitro growth of breast cancer cells. In vivo, IMC-18F1 suppressed the growth of human breast tumor xenografts in association with reduced mitogen-activated protein kinase and Akt activation, reduced tumor cell proliferation, and increased tumor cell apoptosis. Pharmacokinetic/pharmacodynamic studies established a plasma elimination half-life of 5 days for IMC-18F1 and a steady-state trough plasma therapeutic threshold of 88 μg/mL. Importantly, inhibition of mouse and human VEGFR-1 with MF1 and IMC-18F1, respectively, enhanced the antitumor efficacy of cytotoxic agents commonly used to treat breast cancer. Conclusions: Based on preclinical validation studies, IMC-18F1 anti-VEGFR-1 has potential to provide clinical benefit to cancer patients.

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.

Targeting the platelet-derived growth factor receptor α with a neutralizing human monoclonal antibody inhibits the growth of tumor xenografts: Implications as a potential therapeutic target

Platelet-derived growth factor receptor α (PDGFRα) is a type III receptor tyrosine kinase that is expressed on a variety of tumor types. A neutralizing monoclonal antibody to human PDGFRα, which did not cross-react with the β form of the receptor, was generated. The fully human antibody, termed 3G3, has a Kd of 40 pmol/L and blocks both PDGF-AA and PDGF-BB ligands from binding to PDGFRα. In addition to blocking ligand-induced cell mitogenesis and receptor autophosphorylation, 3G3 inhibited phosphorylation of the downstream signaling molecules Akt and mitogen-activated protein kinase. This inhibition was seen in both transfected and tumor cell lines expressing PDGFRα. The in vivo antitumor activity of 3G3 was tested in human glioblastoma (U118) and leiomyosarcoma (SKLMS-1) xenograft tumor models in athymic nude mice. Antibody 3G3 significantly inhibited the growth of U118 (P = 0.0004) and SKLMS-1 (P < 0.0001) tumors relative to control. These data suggest that 3G3 may be useful for the treatment of tumors that express PDGFRα.

Anti–Transforming Growth Factor β Receptor II Antibody Has Therapeutic Efficacy against Primary Tumor Growth and Metastasis through Multieffects on Cancer, Stroma, and Immune Cells

Purpose: Transforming growth factor β (TGFβ) is a pleiotropic cytokine that affects tumor growth, metastasis, stroma, and immune response. We investigated the therapeutic efficacy of anti–TGFβ receptor II (TGFβ RII) antibody in controlling metastasis and tumor growth as well as enhancing antitumor immunity in preclinical tumor models. Experimental Design: We generated neutralizing antibodies to TGFβ RII and assessed the antibody effects on cancer, stroma, and immune cells in vitro. The efficacy and mechanism of action of the antibody as monotherapy and in combination with chemotherapy in suppression of primary tumor growth and metastasis were evaluated in several tumor models. Results: Anti–TGFβ RII antibody blocked TGFβ RII binding to TGFβ 1, 2, and 3, and attenuated the TGFβ-mediated activation of downstream Smad2 kinase, invasion of cancer cells, motility of endothelial and fibroblast cells, and induction of immunosuppressive cells. Treatment with the antibody significantly suppressed primary tumor growth and metastasis and enhanced natural killer and CTL activity in tumor-bearing mice. Immunohistochemistry analysis showed cancer cell apoptosis and massive necrosis, and increased tumor-infiltrating T effector cells and decreased tumor-infiltrating Gr-1+ myeloid cells in the antibody-treated tumors. Fluorescence-activated cell sorting analysis indicated the significant reduction of peripheral Gr-1+/CD11b+ myeloid cells in treated animals. Concomitant treatment with the cytotoxic agent cyclophosphamide resulted in a significantly increased antitumor efficacy against primary tumor growth and metastasis. Conclusions: These preclinical data provide a foundation to support using anti–TGFβ RII antibody as a therapeutic agent for TGFβ RII–dependent cancer with metastatic capacity. Clin Cancer Res; 16(4); 1191–205

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.

The Structural Basis for the Function of Two Anti-VEGF Receptor 2 Antibodies

The anti-VEGF receptor 2 antibody IMC-1121B is a promising antiangiogenic drug being tested for treatment of breast and gastric cancer. We have determined the structure of the 1121B Fab fragment in complex with domain 3 of VEGFR2, as well as the structure of a different neutralizing anti-VEGFR2 antibody, 6.64, also in complex with VEGFR2 domain 3. The two Fab fragments bind at opposite ends of VEGFR2 domain 3; 1121B directly blocks VEGF binding, whereas 6.64 may prevent receptor dimerization by perturbing the domain 3:domain 4 interface. Mutagenesis reveals that residues essential for VEGF, 1121B, and 6.64 binding are nonoverlapping among the three contact patches.

Blood-Brain-Barrier-Penetrating 6-Halogenopurines Suitable as Pro-Probes for Positron Emission Tomography are Substrates for Human Glutathione Transferases

2015 For ‘acidic’ (pI < 7.0) or ‘neutral’ antibodies (pI: 7.0–8.0), it is challenging to operate traditional anion exchange chromatography in a product flow-through mode to achieve adequate clearance of HCP, DNA, leached ProA, HMW and viruses while maintaining high process yield. In this study, the authors developed a scalable mAb polishing step using a new salt tolerant chromatographic resin. Utilizing a combination of high-throughput condition screening in 96-well plates and optimization in smallscale column models, a polishing step was developed that demonstrated high process yield and efficient clearance of impurities for multiple acidic or neutral antibodies. Pilot scale production demonstrated scalability of the step. This polishing step can be easily integrated into most current Protein A/AEX two-column antibody purification platforms.

Abstract 3539: Anti-CSF-1R antibodies reduce tumor-associated macrophages and inhibit tumor growth in preclinical models

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL In cancer, increased infiltration of macrophages within and surrounding the tumor mass correlates with increased tumor invasiveness and growth. In addition, presence of tumor-associated macrophages (TAMs) has been shown to correlate with poor prognosis, particularly in breast, prostate, ovarian and cervical cancer. TAM proliferation, differentiation and survival is dependent on Colony Stimulating Factor - 1 Receptor (CSF-1R) activation, a type III integral membrane tyrosine kinase receptor selectively expressed on cells of the mononuclear phagocyte lineage. Given that TAMs enhance tumor growth and that activation of the CSF-1R pathway is required for TAM function, an antibody against mouse CSF-1R was generated for proof-of-principle studies. CS7, a monoclonal anti-mouse CSF-1R antibody inhibited both CSF-1 and IL-34 binding to mouse CSF-1R, leading to inactivation of the receptor and downstream signaling molecules. In addition, CS7 prevented monocyte proliferation and macrophage differentiation with IC50s of 0.1 nM and 0.75 nM, respectively. In murine models of breast cancer using CSF-1-secreting MDA-MB-231, Hcc1954, 4T1 or EMT6 tumor cells, CS7 treatment led to a marked reduction in TAMs and an associated decrease in tumor growth. In contrast, breast tumor xenografts with CSF-1 non-secreting breast tumor cell lines JimT1 and MCF-7 had limited or no decrease in tumor volume following CS7 treatment. In prostate models, DU145 tumor cell line xenografts (CSF-1-secreting) but not PC3 (CSF-1 non-secreting) tumor growth was inhibited by CS7, recaptitulating the results seen with breast xenografts. Thus, in breast and prostate preclinical models, CSF-1 secretion by tumor cells is a prerequisite for sensitivity to anti-CSF-1R treatment. Taken together, targeting CSF-1R with a monoclonal antibody inhibits CSF-1R signaling via CSF-1 and IL-34, prevents monocytic to macrophage differentiation, and reduces tumor volume in preclinical models, validating CSF-1R as a target for therapeutic application in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3539. doi:1538-7445.AM2012-3539

Abstract A235: Anti-CSF-1R antibodies reduce tumor-associated macrophages and inhibit tumor growth in preclinical models.

In cancer, increased infiltration of macrophages within and surrounding the tumor mass correlates with increased tumor invasiveness and growth. In addition, presence of tumor-associated macrophages (TAMs) has been shown to correlate with poor prognosis, particularly in breast, prostate, ovarian, and cervical cancer. TAM proliferation, differentiation, and survival is dependent on CSF-1R activation, a type III integral membrane tyrosine kinase receptor selectively expressed on cells of the mononuclear phagocyte lineage. Given that TAMs enhance tumor growth and that activation of the CSF-1R pathway is required for TAM function, an antibody against mouse CSF-1R was generated for proof-of-principle studies. CS7, a monoclonal anti-mouse CSF-1R antibody inhibited both CSF-1 and IL-34 binding to mouse CSF-1R, leading to inactivation of the receptor and downstream signaling molecules. In addition, CS7 prevented monocyte proliferation and macrophage differentiation with IC50s of 0.1 nM and 0.75 nM, respectively. In murine models of breast cancer using CSF-1-secreting MDA-MB-231, Hcc1954, 4T1 or EMT6 tumor cells, CS7 treatment led to a marked reduction in TAMs and an associated decrease in tumor growth. In contrast, breast tumor xenografts with CSF-1 non-secreting breast tumor cell lines JimT1 and MCF-7 had limited or no decrease in tumor volume following CS7 treatment. In prostate models, DU145 tumor cell line xenografts (CSF-1-secreting) but not PC3 (CSF-1 non-secreting) tumor growth was inhibited by CS7, recaptitulating the results seen with breast xenografts. Thus, in breast and prostate preclinical models, CSF-1 secretion by tumor cells is a prerequisite for sensitivity to anti-CSF-1R treatment. Taken together, targeting CSF-1R with a monoclonal antibody inhibits CSF-1R signaling via CSF-1 and IL-34, prevents monocytic to macrophage differentiation, and reduces tumor volume in preclinical models, validating CSF-1R as a target for therapeutic application in cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A235. Citation Format: Yanxia Li, Sneha Mathew, Lan Wu, Ying Wang, Kris Persaud, Douglas Burtrum, Paul Balderes, David Surguladze, John Haurum, Dale Ludwig, Jacqueline Doody. Anti-CSF-1R antibodies reduce tumor-associated macrophages and inhibit tumor growth in preclinical models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A235.