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1.
Int J Mol Sci ; 25(11)2024 Jun 03.
Article in English | MEDLINE | ID: mdl-38892347

ABSTRACT

V-set immunoglobulin domain-containing 4 (VSIG4) is a B7 family protein with known roles as a C3 fragment complement receptor involved in pathogen clearance and a negative regulator of T cell activation by an undetermined mechanism. VSIG4 expression is specific for tumor-associated and select tissue-resident macrophages. Increased expression of VSIG4 has been associated with worse survival in multiple cancer indications. Based upon computational analysis of transcript data across thousands of tumor and normal tissue samples, we hypothesized that VSIG4 has an important role in promoting M2-like immune suppressive macrophages and that targeting VSIG4 could relieve VSIG4-mediated macrophage suppression by repolarizing tumor-associated macrophages (TAMs) to an inflammatory phenotype. We have also observed a cancer-specific pattern of VSIG4 isoform distribution, implying a change in the functional regulation in cancer. Through a series of in vitro, in vivo, and ex vivo assays we demonstrate that anti-VSIG4 antibodies repolarize M2 macrophages and induce an immune response culminating in T cell activation. Anti-VSIG4 antibodies induce pro-inflammatory cytokines in M-CSF plus IL-10-driven human monocyte-derived M2c macrophages. Across patient-derived tumor samples from multiple tumor types, anti-VSIG4 treatment resulted in the upregulation of cytokines associated with TAM repolarization and T cell activation and chemokines involved in immune cell recruitment. VSIG4 blockade is also efficacious in a syngeneic mouse model as monotherapy as it enhances efficacy in combination with anti-PD-1, and the effect is dependent on the systemic availability of CD8+ T cells. Thus, VSIG4 represents a promising new target capable of triggering an anti-cancer response via multiple key immune mechanisms.


Subject(s)
Neoplasms , Tumor-Associated Macrophages , Animals , Humans , Mice , Tumor-Associated Macrophages/immunology , Tumor-Associated Macrophages/metabolism , Neoplasms/immunology , Neoplasms/metabolism , Cell Line, Tumor , Lymphocyte Activation/immunology , Mice, Inbred C57BL , Cytokines/metabolism , Female , Receptors, Complement
2.
MAbs ; 14(1): 2083466, 2022.
Article in English | MEDLINE | ID: mdl-35708974

ABSTRACT

Antibody-directed nanotherapeutics (ADNs) represent a promising delivery platform for selective delivery of an encapsulated drug payload to the site of disease that improves the therapeutic index. Although both single-chain Fv (scFv) and Fab antibody fragments have been used for targeting, no platform approach applicable to any target has emerged. scFv can suffer from intrinsic instability, and the Fabs are challenging to use due to native disulfide over-reduction and resulting impurities at the end of the conjugation process. This occurs because of the close proximity of the disulfide bond connecting the heavy and light chain to the free cysteine at the C-terminus, which is commonly used as the conjugation site. Here we show that by engineering an alternative heavy chain-light chain disulfide within the Fab, we can maintain efficient conjugation while eliminating the process impurities and retaining stability. We have demonstrated the utility of this technology for efficient ADN delivery and internalization for a series of targets, including EphA2, EGFR, and ErbB2. We expect that this technology will be broadly applicable for targeting of nanoparticle encapsulated payloads, including DNA, mRNA, and small molecules.


Subject(s)
Nanoparticles , Single-Chain Antibodies , Disulfides/chemistry , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/genetics , Nanoparticles/chemistry
3.
Sci Transl Med ; 11(512)2019 10 02.
Article in English | MEDLINE | ID: mdl-31578241

ABSTRACT

Tumor necrosis factor receptor 2 (TNFR2) is the alternate receptor for TNF and can mediate both pro- and anti-inflammatory activities of T cells. Although TNFR2 has been linked to enhanced suppressive activity of regulatory T cells (Tregs) in autoimmune diseases, the viability of TNFR2 as a target for cancer immunotherapy has been underappreciated. Here, we show that new murine monoclonal anti-TNFR2 antibodies yield robust antitumor activity and durable protective memory in multiple mouse cancer cell line models. The antibodies mediate potent Fc-dependent T cell costimulation and do not result in significant depletion of Tregs Corresponding human agonistic monoclonal anti-TNFR2 antibodies were identified and also had antitumor effects in humanized mouse models. Anti-TNFR2 antibodies could be developed as a novel treatment option for patients with cancer.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/metabolism , Receptors, Tumor Necrosis Factor, Type II/antagonists & inhibitors , Receptors, Tumor Necrosis Factor, Type II/immunology , Animals , Colonic Neoplasms/immunology , Colonic Neoplasms/metabolism , Colonic Neoplasms/therapy , Disease Models, Animal , Female , Humans , Lymphocyte Activation/drug effects , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL
4.
Proc Natl Acad Sci U S A ; 116(15): 7533-7542, 2019 04 09.
Article in English | MEDLINE | ID: mdl-30898885

ABSTRACT

Activation of the Met receptor tyrosine kinase, either by its ligand, hepatocyte growth factor (HGF), or via ligand-independent mechanisms, such as MET amplification or receptor overexpression, has been implicated in driving tumor proliferation, metastasis, and resistance to therapy. Clinical development of Met-targeted antibodies has been challenging, however, as bivalent antibodies exhibit agonistic properties, whereas monovalent antibodies lack potency and the capacity to down-regulate Met. Through computational modeling, we found that the potency of a monovalent antibody targeting Met could be dramatically improved by introducing a second binding site that recognizes an unrelated, highly expressed antigen on the tumor cell surface. Guided by this prediction, we engineered MM-131, a bispecific antibody that is monovalent for both Met and epithelial cell adhesion molecule (EpCAM). MM-131 is a purely antagonistic antibody that blocks ligand-dependent and ligand-independent Met signaling by inhibiting HGF binding to Met and inducing receptor down-regulation. Together, these mechanisms lead to inhibition of proliferation in Met-driven cancer cells, inhibition of HGF-mediated cancer cell migration, and inhibition of tumor growth in HGF-dependent and -independent mouse xenograft models. Consistent with its design, MM-131 is more potent in EpCAM-high cells than in EpCAM-low cells, and its potency decreases when EpCAM levels are reduced by RNAi. Evaluation of Met, EpCAM, and HGF levels in human tumor samples reveals that EpCAM is expressed at high levels in a wide range of Met-positive tumor types, suggesting a broad opportunity for clinical development of MM-131.


Subject(s)
Antibodies, Bispecific/pharmacology , Antineoplastic Agents, Immunological/pharmacology , Epithelial Cell Adhesion Molecule/antagonists & inhibitors , Hepatocyte Growth Factor/metabolism , Neoplasms, Experimental/drug therapy , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Signal Transduction/drug effects , Animals , Cell Line, Tumor , Epithelial Cell Adhesion Molecule/metabolism , Humans , Mice , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Proto-Oncogene Proteins c-met/metabolism , Xenograft Model Antitumor Assays
5.
MAbs ; 9(1): 58-67, 2017 01.
Article in English | MEDLINE | ID: mdl-27854147

ABSTRACT

Antibody-targeted nanoparticles have great promise as anti-cancer drugs; however, substantial developmental challenges of antibody modules prevent many candidates from reaching the clinic. Here, we describe a robust strategy for developing an EphA2-targeting antibody fragment for immunoliposomal drug delivery. A highly bioactive single-chain variable fragment (scFv) was engineered to overcome developmental liabilities, including low thermostability and weak binding to affinity purification resins. Improved thermostability was achieved by modifying the framework of the scFv, and complementarity-determining region (CDR)-H2 was modified to increase binding to protein A resins. The results of our engineering campaigns demonstrate that it is possible, using focused design strategies, to rapidly improve the stability and manufacturing characteristics of an antibody fragment for use as a component of a novel therapeutic construct.


Subject(s)
Drug Delivery Systems/methods , Ephrin-A2/immunology , Immunoconjugates/immunology , Nanoparticles , Single-Chain Antibodies/immunology , Animals , Humans , Immunoglobulin Variable Region/immunology , Protein Engineering/methods , Protein Stability , Receptor, EphA2 , Single-Chain Antibodies/biosynthesis
6.
MAbs ; 7(4): 752-8, 2015.
Article in English | MEDLINE | ID: mdl-25961854

ABSTRACT

Monoclonal antibodies and antibody-like molecules represent a fast-growing class of bio-therapeutics that has rapidly transformed patient care in a variety of disease indications. The discovery of antibodies that bind to particular targets with high affinity is now a routine exercise and a variety of in vitro and in vivo techniques are available for this purpose. However, it is still challenging to identify antibodies that, in addition to having the desired biological effect, also express well, remain soluble at different pH levels, remain stable at high concentrations, can withstand high shear stress, and have minimal non-specific interactions. Many promising antibody programs have ultimately failed in development due to the problems associated with one of these factors. Here, we present a simple high-performance liquid chromatography (HPLC)-based screening method to assess these developability factors earlier in discovery process. This method is robust and requires only microgram quantities of proteins. Briefly, we show that for antibodies injected on a commercially available pre-packed Zenix HPLC column, the retention times are inversely related to their colloidal stability with antibodies prone to precipitation or aggregation retained longer on the column with broader peaks. By simply varying the salt content of running buffer, we were also able to estimate the nature of interactions between the antibodies and the column. We believe this approach should generally be applicable to assessment of the developability of other classes of bio-therapeutic molecules, and that the addition of this simple tool early in the discovery process will lead to selection of molecules with improved developability characteristics.


Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/genetics , Cell Line , Chromatography, High Pressure Liquid , Humans , Hydrogen-Ion Concentration , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics
7.
Mol Cancer Ther ; 13(2): 410-25, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24282274

ABSTRACT

Although inhibition of the insulin-like growth factor (IGF) signaling pathway was expected to eliminate a key resistance mechanism for EGF receptor (EGFR)-driven cancers, the effectiveness of IGF-I receptor (IGF-IR) inhibitors in clinical trials has been limited. A multiplicity of survival mechanisms are available to cancer cells. Both IGF-IR and the ErbB3 receptor activate the PI3K/AKT/mTOR axis, but ErbB3 has only recently been pursued as a therapeutic target. We show that coactivation of the ErbB3 pathway is prevalent in a majority of cell lines responsive to IGF ligands and antagonizes IGF-IR-mediated growth inhibition. Blockade of the redundant IGF-IR and ErbB3 survival pathways and downstream resistance mechanisms was achieved with MM-141, a tetravalent bispecific antibody antagonist of IGF-IR and ErbB3. MM-141 potency was superior to monospecific and combination antibody therapies and was insensitive to variation in the ratio of IGF-IR and ErbB3 receptors. MM-141 enhanced the biologic impact of receptor inhibition in vivo as a monotherapy and in combination with the mTOR inhibitor everolimus, gemcitabine, or docetaxel, through blockade of IGF-IR and ErbB3 signaling and prevention of PI3K/AKT/mTOR network adaptation.


Subject(s)
Antibodies, Bispecific/pharmacology , Cell Proliferation/drug effects , Receptor, ErbB-3/antagonists & inhibitors , Receptor, IGF Type 1/antagonists & inhibitors , Signal Transduction/drug effects , Animals , Antibodies, Bispecific/administration & dosage , Antibodies, Bispecific/immunology , Antibodies, Monoclonal, Humanized , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Blotting, Western , Cell Line, Tumor , Deoxycytidine/administration & dosage , Deoxycytidine/analogs & derivatives , Docetaxel , Everolimus , Female , Humans , Mice, Inbred NOD , Mice, Nude , Mice, SCID , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Receptor, ErbB-3/immunology , Receptor, IGF Type 1/immunology , Sirolimus/administration & dosage , Sirolimus/analogs & derivatives , TOR Serine-Threonine Kinases/metabolism , Taxoids/administration & dosage , Tumor Burden/drug effects , Xenograft Model Antitumor Assays , Gemcitabine
8.
MAbs ; 5(2): 237-54, 2013.
Article in English | MEDLINE | ID: mdl-23392215

ABSTRACT

Multispecific antibody-like molecules have the potential to advance the standard-of-care in many human diseases. The design of therapeutic molecules in this class, however, has proven to be difficult and, despite significant successes in preclinical research, only one trivalent antibody, catumaxomab, has demonstrated clinical utility. The challenge originates from the complexity of the design space where multiple parameters such as affinity, avidity, effector functions, and pharmaceutical properties need to be engineered in concurrent fashion to achieve the desired therapeutic efficacy. Here, we present a rapid prototyping approach that allows us to successfully optimize these parameters within one campaign cycle that includes modular design, yeast display of structure focused antibody libraries and high throughput biophysical profiling. We delineate this approach by presenting a design case study of MM-141, a tetravalent bispecific antibody targeting two compensatory signaling growth factor receptors: insulin-like growth factor 1 receptor (IGF-1R) and v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ErbB3). A MM-141 proof-of-concept (POC) parent molecule did not meet initial design criteria due to modest bioactivity and poor stability properties. Using a combination of yeast display, structured-guided antibody design and library-scale thermal challenge assay, we discovered a diverse set of stable and active anti-IGF-1R and anti-ErbB3 single-chain variable fragments (scFvs). These optimized modules were reformatted to create a diverse set of full-length tetravalent bispecific antibodies. These re-engineered molecules achieved complete blockade of growth factor induced pro-survival signaling, were stable in serum, and had adequate activity and pharmaceutical properties for clinical development. We believe this approach can be readily applied to the optimization of other classes of bispecific or even multispecific antibody-like molecules.


Subject(s)
Antibodies, Bispecific , Drug Design , Protein Engineering/methods , Receptor, ErbB-3/immunology , Receptor, IGF Type 1/immunology , Single-Chain Antibodies , Animals , Antibodies, Bispecific/chemistry , Antibodies, Bispecific/genetics , Antibodies, Bispecific/immunology , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , CHO Cells , Cricetulus , Gene Library , HEK293 Cells , High-Throughput Screening Assays , Humans , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/genetics , Single-Chain Antibodies/immunology , Single-Chain Antibodies/therapeutic use
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