ABSTRACT
Small-cell lung cancer (SCLC) is an aggressive cancer for which immune checkpoint inhibitors (ICI) have had only limited success. Bispecific T-cell engagers are promising therapeutic alternatives for ICI-resistant tumors, but not all patients with SCLC are responsive. Herein, to integrate CD137 costimulatory function into a T-cell engager format and thereby augment therapeutic efficacy, we generated a CD3/CD137 dual-specific Fab and engineered a DLL3-targeted trispecific antibody (DLL3 trispecific). The CD3/CD137 dual-specific Fab was generated to competitively bind to CD3 and CD137 to prevent DLL3-independent cross-linking of CD3 and CD137, which could lead to systemic T-cell activation. We demonstrated that DLL3 trispecific induced better tumor growth control and a marked increase in the number of intratumoral T cells compared with a conventional DLL3-targeted bispecific T-cell engager. These findings suggest that DLL3 trispecific can exert potent efficacy by inducing concurrent CD137 costimulation and provide a promising therapeutic option for SCLC.
Subject(s)
CD3 Complex , Intracellular Signaling Peptides and Proteins , Lung Neoplasms , Membrane Proteins , Small Cell Lung Carcinoma , T-Lymphocytes , Tumor Necrosis Factor Receptor Superfamily, Member 9 , Small Cell Lung Carcinoma/immunology , Small Cell Lung Carcinoma/pathology , Small Cell Lung Carcinoma/drug therapy , Small Cell Lung Carcinoma/therapy , Small Cell Lung Carcinoma/metabolism , Humans , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Lung Neoplasms/drug therapy , Tumor Necrosis Factor Receptor Superfamily, Member 9/metabolism , CD3 Complex/immunology , Animals , Mice , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Membrane Proteins/immunology , Antibodies, Bispecific/pharmacology , Antibodies, Bispecific/therapeutic use , Cell Line, Tumor , Lymphocyte Activation/immunology , Female , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Agonistic antibodies targeting CD137 have been clinically unsuccessful due to systemic toxicity. Because conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigens, we exploited extracellular adenosine triphosphate (exATP), which is a hallmark of the tumor microenvironment and highly elevated in solid tumors, as a broadly tumor-selective switch. We generated a novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the presence of exATP. STA551 demonstrated potent and broad antitumor efficacy against all mouse and human tumors tested and a wide therapeutic window without systemic immune activation in mice. STA551 was well tolerated even at 150 mg/kg/week in cynomolgus monkeys. These results provide a strong rationale for the clinical testing of STA551 against a broad variety of cancers regardless of antigen expression, and for the further application of this novel platform to other targets in cancer therapy. SIGNIFICANCE: Reported CD137 agonists suffer from either systemic toxicity or limited efficacy against antigen-specific cancers. STA551, an antibody designed to agonize CD137 only in the presence of extracellular ATP, inhibited tumor growth in a broad variety of cancer models without any systemic toxicity or dependence on antigen expression.See related commentary by Keenan and Fong, p. 20.This article is highlighted in the In This Issue feature, p. 1.
Subject(s)
Adenosine Triphosphate , Neoplasms , Animals , Antibodies, Monoclonal/pharmacology , Antigens, Neoplasm , Immunotherapy , Mice , Neoplasms/drug therapy , Tumor Microenvironment , Tumor Necrosis Factor Receptor Superfamily, Member 9ABSTRACT
The antibody drug market is rapidly expanding, and various antibody engineering technologies are being developed to create antibodies that can provide better benefit to patients. Although bispecific antibody drugs have been researched for more than 30â¯years, currently only a limited number of bispecific antibodies have achieved regulatory approval. Of the few successful examples of industrially manufacturing a bispecific antibody, the "common light chain format" is an elegant technology that simplifies the purification of a whole IgG-type bispecific antibody. Using this IgG format, the bispecific function can be introduced while maintaining the natural molecular shape of the antibody. In this article, we will first introduce the outline, prospects, and limitations of the common light chain format. Then, we will describe the identification and optimization process for ERY974, an anti-glypican-3â¯×â¯anti-CD3ε T cell-redirecting bispecific antibody with a common light chain. This format includes one of Chugai's proprietary technologies, termed ART-Ig technology, which consists of a method to identify a common light chain, isoelectric point (pI) engineering to purify the desired bispecific IgG antibody from byproducts, and Fc heterodimerization by an electrostatic steering effect. Furthermore, we describe some tips for de-risking the antibody when engineering a T cell redirecting antibody.
Subject(s)
Antibodies, Bispecific , Immunoglobulin G , Immunoglobulin Light Chains , Protein Engineering/methods , Animals , CD3 Complex/immunology , Glypicans/immunology , Humans , MiceABSTRACT
Monoclonal antibodies are widely used to target disease-related antigens. However, because conventional antibody binds to the antigen but cannot eliminate the antigen from plasma, and rather increases the plasma antigen concentration by reducing the clearance of the antigen, some clinically important antigens are still difficult to target with monoclonal antibodies because of the huge dosages required. While conventional antibody can only bind to the antigen, some natural endocytic receptors not only bind to the ligands but also continuously eliminate them from plasma by pH-dependent dissociation of the ligands within the acidic endosome and subsequent receptor recycling to the cell surface. Here, we demonstrate that an engineered antibody, named sweeping antibody, having both pH-dependent antigen binding (to mimic the receptor-ligand interaction) and increased binding to cell surface neonatal Fc receptor (FcRn) at neutral pH (to mimic the cell-bound form of the receptor), selectively eliminated the antigen from plasma. With this novel antigen-sweeping activity, antibody without in vitro neutralizing activity exerted in vivo efficacy by directly eliminating the antigen from plasma. Moreover, conversion of conventional antibody with in vitro neutralizing activity into sweeping antibody further potentiated the in vivo efficacy. Depending on the binding affinity to FcRn at neutral pH, sweeping antibody reduced antigen concentration 50- to 1000-fold compared to conventional antibody. Thereby, sweeping antibody antagonized excess amounts of antigen in plasma against which conventional antibody was completely ineffective, and could afford marked reduction of dosage to a level that conventional antibody can never achieve. Thus, the novel mode of action of sweeping antibody provides potential advantages over conventional antibody and may allow access to the target antigens which were previously undruggable by conventional antibody.
Subject(s)
Antibodies, Monoclonal/metabolism , Antigens/metabolism , Histocompatibility Antigens Class I/metabolism , Protein Engineering/methods , Receptors, Fc/metabolism , Animals , Antigens/blood , Humans , Hydrogen-Ion Concentration , Immunoglobulin G/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutation/genetics , Protein Binding/immunology , Signal Transduction/immunologyABSTRACT
In the course of screening for an inhibitor of ER stress-induced XBP1 activation, we isolated a new member of the triene-ansamycin group compound, trierixin, from a culture broth of Streptomyces sp. AC 654. Trierixin was purified by column chromatography on silica gel and by HPLC. The molecular formula of trierixin is C(37)H(52)N(2)O(8)S. Trierixin inhibited thapsigargin-induced XBP1-luciferase activation in HeLa/XBP1-luc cells and endogenous XBP1 splicing in HeLa cells with an IC(50) of 14 ng/ml and 19 ng/ml, respectively. Moreover, in the process of isolating trierixin, we isolated structurally related mycotrienin II and trienomycin A as inhibitors of ER stress-induced XBP1 activation from a culture broth of a trierixin-producing strain. This study provides the first observation that triene-ansamycins have a novel inhibitory effect against XBP1 activation.
Subject(s)
Anti-Bacterial Agents/pharmacology , DNA-Binding Proteins/antagonists & inhibitors , Lactams, Macrocyclic/pharmacology , Nuclear Proteins/antagonists & inhibitors , Streptomyces/chemistry , Alanine/analogs & derivatives , Alanine/isolation & purification , Anti-Bacterial Agents/classification , Anti-Bacterial Agents/isolation & purification , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Fermentation , HeLa Cells , Humans , Hydroquinones/isolation & purification , Inhibitory Concentration 50 , Lactams, Macrocyclic/classification , Lactams, Macrocyclic/isolation & purification , Luciferases/drug effects , Regulatory Factor X Transcription Factors , Reverse Transcriptase Polymerase Chain Reaction , Stress, Physiological/chemically induced , Stress, Physiological/physiopathology , Thapsigargin , Transcription Factors , X-Box Binding Protein 1ABSTRACT
Trierixin, a new member of the triene-ansamycin group, has been isolated from the fermentation broth of Streptomyces sp. AC654 as an inhibitor of ER stress-induced XBP1 activation. The structure of trierixin was determined on the basis of its spectroscopical and chemical properties. Trierixin possessed a 21-membered macrocyclic lactam, which contains a methylthio-benzenediol structure, and a cyclohexanecarbonylalanine moiety. Trierixin is thus elucidated as 21-thiomethylmycotrienin II.