A bi-functional antibody-receptor domain fusion protein simultaneously targeting IGF-IR and VEGF for degradation.

Bi-specific antibodies (BsAbs), which can simultaneously block 2 tumor targets, have emerged as promising therapeutic alternatives to combinations of individual monoclonal antibodies. Here, we describe the engineering and development of a novel, human bi-functional antibody-receptor domain fusion molecule with ligand capture (bi-AbCap) through the fusion of the domain 2 of human vascular endothelial growth factor receptor 1 (VEGFR1) to an antibody directed against insulin-like growth factor - type I receptor (IGF-IR). The bi-AbCap possesses excellent stability and developability, and is the result of minimal engineering. Beyond potent neutralizing activities against IGF-IR and VEGF, the bi-AbCap is capable of cross-linking VEGF to IGF-IR, leading to co-internalization and degradation of both targets by tumor cells. In multiple mouse xenograft tumor models, the bi-AbCap improves anti-tumor activity over individual monotherapies. More importantly, it exhibits superior inhibition of tumor growth, compared with the combination of anti-IGF-IR and anti-VEGF therapies, via powerful blockade of both direct tumor cell growth and tumor angiogenesis. The unique "capture-for-degradation" mechanism of the bi-AbCap is informative for the design of next-generation bi-functional anti-cancer therapies directed against independent signaling pathways. The bi-AbCap design represents an alternative approach to the creation of dual-targeting antibody fusion molecules by taking advantage of natural receptor-ligand interactions.

A human monoclonal antibody targeting the stem cell factor receptor (c-Kit) blocks tumor cell signaling and inhibits tumor growth.

Stem cell factor receptor (c-Kit) exerts multiple biological effects on target cells upon binding its ligand stem cell factor (SCF). Aberrant activation of c-Kit results in dysregulated signaling and is implicated in the pathogenesis of numerous cancers. The development of more specific and effective c-Kit therapies is warranted given its essential role in tumorigenesis. In this study, we describe the biological properties of CK6, a fully human IgG1 monoclonal antibody against the extracellular region of human c-Kit. CK6 specifically binds c-Kit receptor with high affinity (EC 50 = 0.06 nM) and strongly blocks its interaction with SCF (IC 50 = 0.41 nM) in solid phase assays. Flow cytometry shows CK6 binding to c-Kit on the cell surface of human small cell lung carcinoma (SCLC), melanoma, and leukemia tumor cell lines. Furthermore, exposure to CK6 inhibits SCF stimulation of c-Kit tyrosine kinase activity and downstream signaling pathways such as mitogen-activated protein kinase (MAPK) and protein kinase B (AKT), in addition to reducing tumor cell line growth in vitro. CK6 treatment significantly decreases human xenograft tumor growth in NCI-H526 SCLC (T/C% = 57) and Malme-3M melanoma (T/C% = 58) models in vivo. The combination of CK6 with standard of care chemotherapy agents, cisplatin and etoposide for SCLC or dacarbazine for melanoma, more potently reduces tumor growth (SCLC T/C% = 24, melanoma T/C% = 38) compared with CK6 or chemotherapy alone. In summary, our results demonstrate that CK6 is a c-Kit antagonist antibody with tumor growth neutralizing properties and are highly suggestive of potential therapeutic application in treating human malignancies harboring c-Kit receptor.

Backbone flexibility of CDR3 and immune recognition of antigens.

Conformational entropy is an important component of protein-protein interactions; however, there is no reliable method for computing this parameter. We have developed a statistical measure of residual backbone entropy in folded proteins by using the ϕ-ψ distributions of the 20 amino acids in common secondary structures. The backbone entropy patterns of amino acids within helix, sheet or coil form clusters that recapitulate the branching and hydrogen bonding properties of the side chains in the secondary structure type. The same types of residues in coil and sheet have identical backbone entropies, while helix residues have much smaller conformational entropies. We estimated the backbone entropy change for immunoglobulin complementarity-determining regions (CDRs) from the crystal structures of 34 low-affinity T-cell receptors and 40 high-affinity Fabs as a result of the formation of protein complexes. Surprisingly, we discovered that the computed backbone entropy loss of only the CDR3, but not all CDRs, correlated significantly with the kinetic and affinity constants of the 74 selected complexes. Consequently, we propose a simple algorithm to introduce proline mutations that restrict the conformational flexibility of CDRs and enhance the kinetics and affinity of immunoglobulin interactions. Combining the proline mutations with rationally designed mutants from a previous study led to 2400-fold increase in the affinity of the A6 T-cell receptor for Tax-HLAA2. However, this mutational scheme failed to induce significant binding changes in the already-high-affinity C225-Fab/huEGFR interface. Our results will serve as a roadmap to formulate more effective target functions to design immune complexes with improved biological functions.

Development of a novel and efficient cell culture flocculation process using a stimulus responsive polymer to streamline antibody purification processes.

Recent advances in mammalian cell culture processes have significantly increased product titers, but have also resulted in substantial increases in cell density and cellular debris as well as process and product related impurities. As such, with improvements in titer, corresponding improvements in downstream processing are essential. In this study we have developed an alternative antibody harvest process that incorporates flocculation using a novel stimulus responsive polymer, benzylated poly(allylamine), followed by depth filtration. As tested on multiple antibodies, this process demonstrates high process yield, improved clearance of cells and cell debris, and efficient reduction of aggregates, host cell proteins (HCP) and DNA. A wide operating window was established for this novel flocculation process through design of experiments condition screening and optimization. Residual levels of impurities in the Protein A eluate were achieved that potentially meet requirements of drug substance and thus alleviate the burden for further impurities removal in subsequent chromatography steps. In addition, efficient clearance of residual polymer was demonstrated using a fluorescence tagged polymer in the presence of a stimulus reagent. The mechanism of HCP and aggregates removal during flocculation was also explored. This novel and efficient process can be easily integrated into current mAb purification platforms, and may overcome downstream processing challenges.

Removal of a C-terminal serine residue proximal to the inter-chain disulfide bond of a human IgG1 lambda light chain mediates enhanced antibody stability and antibody dependent cell-mediated cytotoxicity.

Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgG1λ antibodies.

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.

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.

Anti-transforming growth factor beta receptor II antibody has therapeutic efficacy against primary tumor growth and metastasis through multieffects on cancer, stroma, and immune cells.

PURPOSE: Transforming growth factor beta (TGFbeta) is a pleiotropic cytokine that affects tumor growth, metastasis, stroma, and immune response. We investigated the therapeutic efficacy of anti-TGFbeta receptor II (TGFbeta 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 TGFbeta 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-TGFbeta RII antibody blocked TGFbeta RII binding to TGFbeta 1, 2, and 3, and attenuated the TGFbeta-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-TGFbeta RII antibody as a therapeutic agent for TGFbeta RII-dependent cancer with metastatic capacity.

Development of a fully human anti-PDGFRbeta antibody that suppresses growth of human tumor xenografts and enhances antitumor activity of an anti-VEGFR2 antibody.

Platelet-derived growth factor receptor beta (PDGFRbeta) is upregulated in most of solid tumors. It is expressed by pericytes/smooth muscle cells, fibroblast, macrophage, and certain tumor cells. Several PDGF receptor-related antagonists are being developed as potential antitumor agents and have demonstrated promising antitumor activity in both preclinical and clinical settings. Here, we produced a fully human neutralizing antibody, IMC-2C5, directed against PDGFRbeta from an antibody phage display library. IMC-2C5 binds to both human and mouse PDGFRbeta and blocks PDGF-B from binding to the receptor. IMC-2C5 also blocks ligand-stimulated activation of PDGFRbeta and downstream signaling molecules in tumor cells. In animal studies, IMC-2C5 significantly delayed the growth of OVCAR-8 and NCI-H460 human tumor xenografts in nude mice but failed to show antitumor activities in OVCAR-5 and Caki-1 xenografts. Our results indicate that the antitumor efficacy of IMC-2C5 is primarily due to its effects on tumor stroma, rather than on tumor cells directly. Combination of IMC-2C5 and DC101, an anti-mouse vascular endothelial growth factor receptor 2 antibody, resulted in significantly enhanced antitumor activity in BxPC-3, NCI-H460, and HCT-116 xenografts, compared with DC101 alone, and the trend of additive effects to DC101 treatment in several other tumor models. ELISA analysis of NCI-H460 tumor homogenates showed that IMC-2C5 attenuated protein level of vascular endothelial growth factor and basic fibroblast growth factor elevated by DC101 treatment. Finally, IMC-2C5 showed a trend of additive effects when combined with DC101/chemotherapy in MIA-PaCa-2 and NCI-H460 models. Taken together, these results lend great support to the use of PDGFRbeta antagonists in combination with other antiangiogenic agents in the treatment of a broad range of human cancers.

Vascular endothelial growth factor receptor 3 is involved in tumor angiogenesis and growth.

Vascular endothelial growth factor receptor 3 (VEGFR-3) binds VEGF-C and VEGF-D and is essential for the development of the lymphatic vasculature. Experimental tumors that overexpress VEGFR-3 ligands induce lymphatic vessel sprouting and enlargement and show enhanced metastasis to regional lymph nodes and beyond, whereas a soluble form of VEGFR-3 that blocks receptor signaling inhibits these changes and metastasis. Because VEGFR-3 is also essential for the early blood vessel development in embryos and is up-regulated in tumor angiogenesis, we wanted to determine if an antibody targeting the receptor that interferes with VEGFR-3 ligand binding can inhibit primary tumor growth. Our results show that antibody interference with VEGFR-3 function can inhibit the growth of several human tumor xenografts in immunocompromised mice. Immunohistochemical analysis showed that the blood vessel density of anti-VEGFR-3-treated tumors was significantly decreased and hypoxic and necrotic tumor tissue was increased when compared with tumors treated with control antibody, indicating that blocking of the VEGFR-3 pathway inhibits angiogenesis in these tumors. As expected, the anti-VEGFR-3-treated tumors also lacked lymphatic vessels. These results suggest that the VEGFR-3 pathway contributes to tumor angiogenesis and that effective inhibition of tumor progression may require the inhibition of multiple angiogenic targets.

Nonvascular VEGF receptor 3 expression by corneal epithelium maintains avascularity and vision.

Transparency of the cornea, the window of the eye, is a prerequisite for vision. Angiogenesis into the normally avascular cornea is incompatible with good vision and, therefore, the cornea is one of the few tissues in the human body where avascularity is actively maintained. Here, we provide evidence for a critical mechanism contributing to corneal avascularity. VEGF receptor 3, normally present on lymphatic and proliferating blood vascular endothelium, is strongly constitutively expressed by corneal epithelium and is mechanistically responsible for suppressing inflammatory corneal angiogenesis.

Inhibition of VEGFR-3 activation with the antagonistic antibody more potently suppresses lymph node and distant metastases than inactivation of VEGFR-2.

Lymph nodes are the first site of metastases for most types of cancer, and lymph node status is a key indicator of patient prognosis. Induction of tumor lymphangiogenesis by vascular endothelial growth factor-C (VEGF-C) has been shown to play an important role in promoting tumor metastases to lymph nodes. Here, we employed receptor-specific antagonist antibodies in an orthotopic spontaneous breast cancer metastasis model to provide direct evidence for the key role of VEGFR-3 activation in metastasis. Inhibition of VEGFR-3 activation more potently suppressed regional and distant metastases than inactivation of VEGFR-2, although VEGFR-2 blockade was more effective in inhibiting angiogenesis and tumor growth. Despite prominent proliferation, metastases were not vascularized in any of the control and treatment groups, indicating that the growth of metastases was not dependent on angiogenesis at the secondary site for the duration of the experiment. Systemic treatment with either VEGFR-2 or VEGFR-3 antagonistic antibodies suppressed tumor lymphangiogenesis, indicating that VEGFR-3 signaling affects the rate of tumor cell entry into lymphatic vessels through both lymphangiogenesis-dependent and independent mechanisms. Combination treatment with the anti-VEGFR-2 and anti-VEGFR-3 antibodies more potently decreased lymph node and lung metastases than each antibody alone. These results validate the concept of targeting the lymphatic dissemination and thereby very early steps of the metastatic process for metastasis control and suggest that a combination therapy with antiangiogenic agents may be a particularly promising approach for controlling metastases.

Kaposi's sarcoma-associated herpesvirus activation of vascular endothelial growth factor receptor 3 alters endothelial function and enhances infection.

Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8) is the etiologic agent of Kaposi's sarcoma, an endothelial neoplasm. This gamma-herpesvirus encodes for several unique proteins that alter target cell function, including the virion envelope-associated glycoprotein B (gB). Glycoprotein B has an RGD (Arg-Gly-Asp) motif at the extracellular amino terminus region and binds to the alpha3beta1 surface integrin, which enhances virus entry. We now report that gB can activate the vascular endothelial growth factor receptor 3 (VEGFR-3) on the surface of microvascular endothelial cells and trigger receptor signaling, which can modulate endothelial migration and proliferation. Furthermore, we observed that VEGFR-3 expression and activation enhance KSHV infection and participate in KSHV-mediated transformation. These functional changes in the endothelium may contribute to the pathogenesis of Kaposi's sarcoma and suggest that interventions that inhibit gB activation of VEGFR-3 could be useful in the treatment of this neoplasm.

A recombinant, fully human, bispecific antibody neutralizes the biological activities mediated by both vascular endothelial growth factor receptors 2 and 3.

Vascular endothelial growth factors (VEGF) and their receptors (VEGFR) have been implicated to play important roles in tumor-associated angiogenesis and lymphangiogenesis, and hence in tumor growth and metastasis. We previously produced a number of fully human antibodies directed against VEGF receptor 2 (VEGFR2) and VEGF receptor 3 (VEGFR3) and showed that these antibodies are capable of inhibiting growth factor (VEGF and VEGF-C)-induced receptor activation, migration, and proliferation of human endothelial cells. In this report, we constructed and produced a bispecific antibody, a diabody, using the variable domain genes of two neutralizing antibodies, IMC-1121 to VEGFR2 and hF4-3C5 to VEGFR3. The diabody binds to both VEGFR2 and VEGFR3 in a dose-dependent manner, and blocks interaction between VEGF/VEGFR2, VEGF-C/VEGFR2, and VEGF-C/VEGFR3. In cell-based assays, the diabody neutralized both VEGF and VEGF-C-stimulated activation of VEGFR2, VEGFR3, and p44/p42 mitogen-activated protein kinase in endothelial cells. Furthermore, the diabody was able to inhibit both VEGF and VEGF-C-induced migration of endothelial cells. Taken together, our results suggest that a dual blockade of both VEGFR2 and VEGFR3 simultaneously may represent a more potent approach to effective cancer therapy.

Complete and specific inhibition of adult lymphatic regeneration by a novel VEGFR-3 neutralizing antibody.

BACKGROUND: New lymphatic growth may contribute to tumor metastasis. Activation of vascular endothelial growth factor receptor 3 (VEGFR-3) by its ligands VEGF-C and -D is necessary for embryonic and tumor lymphangiogenesis. However, the exact role of VEGFR-3 signaling in adult lymphangiogenesis and in lymphatic vessel survival and regeneration is unclear. METHODS: A novel rat monoclonal antibody to murine VEGFR-3, mF4-31C1, which potently antagonizes the binding of VEGF-C to VEGFR-3, was developed. We tested the effects of systemic mF4-31C1 administration in a mouse tail skin model of lymphatic regeneration, either with or without local overexpression of VEGF-C, and we observed lymphatic and blood vessel regeneration over time using microlymphangiography and immunostaining. RESULTS: Normal mice regenerated complete and functional lymphatic vessels within 60 days of surgery. In athymic mice implanted with VEGF-C-overexpressing human breast carcinoma cells, lymphatic regeneration took place over 25 days and resulted in hyperplastic vessels. Under either condition, no lymphatic regeneration occurred in mice receiving mF4-31C1 during the regeneration period. Blood angiogenesis and preexisting lymphatic vessels were unaffected, both in morphology and in function. CONCLUSIONS: Blocking VEGFR-3 completely and specifically prevented both physiologically normal and tumor VEGF-C-enhanced lymphangiogenesis in the adult mouse but had no effect on either blood angiogenesis or the survival or function of existing lymphatic vessels. Thus, targeting VEGFR-3 with specific inhibitors may block new lymphatic growth exclusively.

Involvement of the VEGF receptor 3 in tubular morphogenesis demonstrated with a human anti-human VEGFR-3 monoclonal antibody that antagonizes receptor activation by VEGF-C.

In this report we utilize a novel antagonist antibody to the human VEGFR-3 to elucidate the role of this receptor in in vitro tubular morphogenesis of bovine and human endothelial cells (EC cells) induced by VEGF-C. The antibody hF4-3C5 was obtained by panning a human phage display library on soluble human VEGFR-3. The binding affinity constant of hF4-3C5 significantly exceeds that of the interaction of VEGFR-3 with VEGF-C. hF4-3C5 strongly inhibits the binding of soluble VEGFR-3 to immobilized VEGF-C and abolishes the VEGF-C-mediated mitogenic response of cells that expresses a chimeric human VEGFR-3-cFMS receptor. In fluorescence experiments, hF4-3C5 reactivity is observed with human lymphatic endothelial cells (LECs) and human umbilical vein endothelial cells (HUVECs). Binding of hF4-3C5 shows that about half of bovine aortic endothelial (BAE) cells express VEGFR-3 and cells in this subpopulation are primarily responsible for the chemotactic response to the mature form of VEGF-C (VEGF-C(DeltaNDeltaC)). This response was strongly inhibited by the addition of hF4-3C5. In vitro tube formation by BAE cells induced by VEGF-C(DeltaNDeltaC) was reduced by greater than 60% by hF4-3C5 whereas the response to VEGF(165) was unaffected. Addition of hF4-3C5 together with an antagonist antibody to VEGFR-2 completely abolished the response to VEGF-C(DeltaNDeltaC). Similar results were obtained with HUVECs. Together, these findings point to a role for VEGFR-3 in vascular tubular morphogenesis and highlight the utility of hF4-3C5 as a tool for the investigation of the biology of VEGFR-3.