ABSTRACT
T-cell-dependent bispecific antibodies (TDB) have been a major advancement in the treatment of cancer, allowing for improved targeting and efficacy for large molecule therapeutics. TDBs are comprised of one arm targeting a surface antigen on a cancer cell and another targeting an engaging surface antigen on a cytotoxic T cell. To impart this function, the antibody must be in a bispecific format as opposed to the more conventional bivalent format. Through in vitro and in vivo studies, we sought to determine the impact of changing antibody valency on solid tumor distribution and catabolism. A bivalent anti-HER2 antibody exhibited higher catabolism than its full-length monovalent binding counterpart in vivo by both invasive tissue harvesting and noninvasive single photon emission computed tomography/X-ray computed tomography imaging despite similar systemic exposures for the two molecules. To determine what molecular factors drove in vivo distribution and uptake, we developed a mechanistic model for binding and catabolism of monovalent and bivalent HER2 antibodies in KPL4 cells. This model suggests that observed differences in cellular uptake of monovalent and bivalent antibodies are caused by the change in apparent affinity conferred by avidity as well as differences in internalization and degradation rates of receptor bound antibodies. To our knowledge, this is the first study to directly compare the targeting abilities of monovalent and bivalent full-length antibodies. These findings may inform diverse antibody therapeutic modalities, including T-cell-redirecting therapies and drug delivery strategies relying upon receptor internalization.
Subject(s)
Antibodies, Bispecific/pharmacology , Antibodies, Bispecific/pharmacokinetics , Antibody Affinity , Breast Neoplasms/drug therapy , Receptor, ErbB-2/antagonists & inhibitors , T-Lymphocytes, Cytotoxic/immunology , Animals , Antibodies, Bispecific/immunology , Apoptosis , Breast Neoplasms/immunology , Breast Neoplasms/pathology , Cell Proliferation , Female , Humans , Mice , Mice, SCID , Receptor, ErbB-2/immunology , Tissue Distribution , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Calicheamicin antibody-drug conjugates (ADCs) are effective therapeutics for leukemias with two recently approved in the United States: Mylotarg (gemtuzumab ozogamicin) targeting CD33 for acute myeloid leukemia and Besponsa (inotuzumab ozogamicin) targeting CD22 for acute lymphocytic leukemia. Both of these calicheamicin ADCs are heterogeneous, aggregation-prone, and have a shortened half-life due to the instability of the acid-sensitive hydrazone linker in circulation. We hypothesized that we could improve upon the heterogeneity, aggregation, and circulation stability of calicheamicin ADCs by directly attaching the thiol of a reduced calicheamicin to an engineered cysteine on the antibody via a disulfide bond to generate a linkerless and traceless conjugate. We report herein that the resulting homogeneous conjugates possess minimal aggregation and display high in vivo stability with 50% of the drug remaining conjugated to the antibody after 21 days. Furthermore, these calicheamicin ADCs are highly efficacious in mouse models of both solid tumor (HER2+ breast cancer) and hematologic malignancies (CD22+ non-Hodgkin lymphoma). Safety studies in rats with this novel calicheamicin ADC revealed an increased tolerability compared with that reported for Mylotarg. Overall, we demonstrate that applying novel linker chemistry with site-specific conjugation affords an improved, next-generation calicheamicin ADC.
Subject(s)
Antibiotics, Antineoplastic/therapeutic use , Calicheamicins/therapeutic use , Immunoconjugates/therapeutic use , Animals , Antibiotics, Antineoplastic/pharmacology , Calicheamicins/pharmacology , Disease Models, Animal , Humans , Immunoconjugates/pharmacology , MiceABSTRACT
BACKGROUND AND PURPOSE: Polatuzumab vedotin is an antibody-drug conjugate (ADC) being developed for non-Hodgkin's lymphoma. It contains a humanized anti-CD79b IgG1 monoclonal antibody linked to monomethyl auristatin E (MMAE), an anti-mitotic agent. Polatuzumab vedotin binds to human CD79b only. Therefore, a surrogate ADC that binds to cynomolgus monkey CD79b was used to determine CD79b-mediated pharmacological effects in the monkey and to enable first-in-human clinical trials. EXPERIMENTAL APPROACH: Polatuzumab vedotin, the surrogate ADC, and the corresponding antibodies were evaluated in different assays in vitro and in animals. In vitro assessments included binding to peripheral blood mononuclear cells from different species, binding to a human and monkey CD79b-expressing cell line, binding to human Fcγ receptors, and stability in plasma across species. In vivo, ADCs were assessed for anti-tumour activity in mice, pharmacokinetics/pharmacodynamics in monkeys, and toxicity in rats and monkeys. KEY RESULTS: Polatuzumab vedotin and surrogate ADC bind with similar affinity to human and cynomolgus monkey B cells, respectively. Comparable in vitro plasma stability, in vivo anti-tumour activity, and mouse pharmacokinetics were also observed between the surrogate ADC and polatuzumab vedotin. In monkeys, only the surrogate ADC showed B-cell depletion and B-cell-mediated drug disposition, but both ADCs showed similar MMAE-driven myelotoxicity, as expected. CONCLUSIONS AND IMPLICATIONS: The suitability of the surrogate ADC for evaluation of CD79b-dependent pharmacology was demonstrated, and anti-tumour activity, pharmacokinetics/pharmacodynamics, and toxicity data with both ADCs supported the entry of polatuzumab vedotin into clinical trials.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Burkitt Lymphoma/drug therapy , CD79 Antigens/antagonists & inhibitors , Immunoconjugates/pharmacology , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal, Humanized , Antineoplastic Agents/chemistry , Antineoplastic Agents/immunology , Binding Sites/drug effects , Burkitt Lymphoma/pathology , CD79 Antigens/immunology , Cell Line , Dose-Response Relationship, Drug , Female , Humans , Immunoconjugates/chemistry , Immunoconjugates/immunology , Macaca fascicularis , Male , Mice , Mice, SCID , Molecular Conformation , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/pathology , Rats , Rats, Sprague-Dawley , Receptors, IgG , Structure-Activity RelationshipABSTRACT
Luminal A (hormone receptor-positive) breast cancer constitutes 70% of total breast cancer patients. In an attempt to develop a targeted therapeutic for this cancer indication, we have identified and characterized Glial cell line-Derived Neurotrophic Factor (GDNF) Family Receptor Alpha 1 (GFRA1) antibody-drug conjugates (ADC) using a cleavable valine-citrulline-MMAE (vcMMAE) linker-payload. RNAseq and IHC analysis confirmed the abundant expression of GFRA1 in luminal A breast cancer tissues, whereas minimal or no expression was observed in most normal tissues. Anti-GFRA-vcMMAE ADC internalized to the lysosomes and exhibited target-dependent killing of GFRA1-expressing cells both in vitro and in vivo The ADCs using humanized anti-GFRA1 antibodies displayed robust therapeutic activity in clinically relevant cell line-derived (MCF7 and KPL-1) tumor xenograft models. The lead anti-GFRA1 ADC cross-reacts with rodent and cynomolgus monkey GFRA1 antigen and showed optimal pharmacokinetic properties in both species. These properties subsequently enabled a target-dependent toxicity study in rats. Anti-GFRA1 ADC is well tolerated in rats, as seen with other vcMMAE linker-payload based ADCs. Overall, these data suggest that anti-GFRA1-vcMMAE ADC may provide a targeted therapeutic opportunity for luminal A breast cancer patients. Mol Cancer Ther; 17(3); 638-49. ©2017 AACR.
Subject(s)
Breast Neoplasms/drug therapy , Glial Cell Line-Derived Neurotrophic Factor Receptors/antagonists & inhibitors , Immunoconjugates/pharmacology , Xenograft Model Antitumor Assays , Animals , Antibodies/chemistry , Antibodies/immunology , Antibodies/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Female , Gene Expression Regulation, Neoplastic/drug effects , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Glial Cell Line-Derived Neurotrophic Factor Receptors/immunology , HEK293 Cells , Humans , Immunoconjugates/immunology , Immunoconjugates/pharmacokinetics , MCF-7 Cells , Macaca fascicularis , Mice, Nude , Mice, SCID , Rats, Sprague-Dawley , Receptors, Steroid/metabolism , Tumor Burden/drug effects , Tumor Burden/geneticsABSTRACT
A novel disulfide linker was designed to enable a direct connection between cytotoxic pyrrolobenzodiazepine (PBD) drugs and the cysteine on a targeting antibody for use in antibody-drug conjugates (ADCs). ADCs composed of a cysteine-engineered antibody were armed with a PBD using a self-immolative disulfide linker. Both the chemical linker and the antibody site were optimized for this new bioconjugation strategy to provide a highly stable and efficacious ADC. This novel disulfide ADC was compared with a conjugate containing the same PBD drug, but attached to the antibody via a peptide linker. Both ADCs had similar efficacy in mice bearing human tumor xenografts. Safety studies in rats revealed that the disulfide-linked ADC had a higher MTD than the peptide-linked ADC. Overall, these data suggest that the novel self-immolative disulfide linker represents a valuable way to construct ADCs with equivalent efficacy and improved safety. Mol Cancer Ther; 16(5); 871-8. ©2017 AACR.
Subject(s)
Antibodies/administration & dosage , Benzodiazepines/administration & dosage , Immunoconjugates/administration & dosage , Neoplasms/drug therapy , Pyrroles/administration & dosage , Animals , Antibodies/chemistry , Antibodies/immunology , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Antineoplastic Agents/immunology , Benzodiazepines/chemistry , Benzodiazepines/immunology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Disulfides/chemistry , Disulfides/immunology , Humans , Immunoconjugates/chemistry , Mice , Neoplasms/immunology , Neoplasms/pathology , Pyrroles/chemistry , Pyrroles/immunology , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: Little is known concerning the onset, duration, and magnitude of direct therapeutic effects of anti-vascular endothelial growth factor (VEGF) therapies. Such knowledge would help guide the rational development of targeted therapeutics from bench to bedside and optimize use of imaging technologies that quantify tumor function in early-phase clinical trials. EXPERIMENTAL DESIGN: Preclinical studies were done using ex vivo microcomputed tomography and in vivo ultrasound imaging to characterize tumor vasculature in a human HM-7 colorectal xenograft model treated with the anti-VEGF antibody G6-31. Clinical evaluation was by quantitative magnetic resonance imaging in 10 patients with metastatic colorectal cancer treated with bevacizumab. RESULTS: Microcomputed tomography experiments showed reduction in perfused vessels within 24 to 48 h of G6-31 drug administration (P Subject(s)
Angiogenesis Inhibitors/therapeutic use
, Antibodies, Monoclonal/therapeutic use
, Colorectal Neoplasms/blood supply
, Colorectal Neoplasms/drug therapy
, Diagnostic Imaging
, Vascular Endothelial Growth Factor A/immunology
, Adolescent
, Animals
, Antibodies, Monoclonal/pharmacology
, Antibodies, Monoclonal, Humanized
, Bevacizumab
, Cell Line, Tumor
, Drug Delivery Systems
, Female
, Humans
, Mice
, Mice, Nude
, Neovascularization, Pathologic/drug therapy
, Xenograft Model Antitumor Assays
ABSTRACT
Here we describe the generation of an antibody-drug conjugate (ADC) consisting of a humanized anti-CD79b antibody that is conjugated to monomethylauristatin E (MMAE) through engineered cysteines (THIOMABs) by a protease cleavable linker. By using flow cytometry, we detected the surface expression of CD79b in almost all non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia patients, suggesting that anti-CD79b-vcMMAE could be widely used in these malignancies. By using NHL cell lines to simulate a patient population we discovered that a minimal cell-surface expression level of CD79b was required for in vitro activity. Within the subpopulation of cell lines above this minimal threshold, we found that sensitivity to free MMAE, mutation of cancer genes, and cell doubling time were poorly correlated with in vitro activity; however, the expression level of BCL-XL was correlated with reduced sensitivity to anti-CD79b-vcMMAE. This observation was supported by in vivo data showing that a Bcl-2 family inhibitor, ABT-263, strikingly enhanced the activity of anti-CD79b-vcMMAE. Furthermore, anti-CD79b-vcMMAE was significantly more effective than a standard-of-care regimen, R-CHOP (ie, rituximab with a single intravenous injection of 30 mg/kg cyclophosphamide, 2.475 mg/kg doxorubicin, 0.375 mg/kg vincristine, and oral dosing of 0.15 mg/kg prednisone once a day for 5 days), in 3 xenograft models of NHL. Together, these data suggest that anti-CD79b-vcMMAE could be broadly efficacious for the treatment of NHL.