ABSTRACT
Triplex gene editing relies on binding a stable peptide nucleic acid (PNA) sequence to a chromosomal target, which alters the helical structure of DNA to stimulate site-specific recombination with a single-strand DNA (ssDNA) donor template and elicits gene correction. Here, we assessed whether the codelivery of PNA and donor template encapsulated in Poly Lactic-co-Glycolic Acid (PLGA)-based nanoparticles can correct sickle cell disease and x-linked severe combined immunodeficiency. However, through this process we have identified a false-positive PCR artifact due to the intrinsic capability of PNAs to aggregate with ssDNA donor templates. Here, we show that the combination of PNA and donor templates but not either agent alone results in different degrees of aggregation that result in varying but highly reproducible levels of false-positive signal. We have identified this phenomenon in vitro and confirmed that the PNA sequences producing the highest supposed correction in vitro are not active in vivo in both disease models, which highlights the importance of interrogating and eliminating carryover of ssDNA donor templates in assessing various gene editing technologies such as PNA-mediated gene editing.
Subject(s)
Gene Editing/methods , Anemia, Sickle Cell/genetics , Animals , False Positive Reactions , Interleukin Receptor Common gamma Subunit/genetics , Mice, SCID , Molecular Probe Techniques , Peptide Nucleic Acids , Polylactic Acid-Polyglycolic Acid CopolymerABSTRACT
Pim kinase inhibitors are promising cancer therapeutics. Pim-2, among the three Pim isoforms, plays a critical role in multiple myeloma yet inhibition of Pim-2 is challenging due to its high affinity for ATP. A co-crystal structure of a screening hit 1 bound to Pim-1 kinase revealed the key binding interactions of its indazole core within the ATP binding site. Screening of analogous core fragments afforded 1H-pyrazolo[3,4-c]pyridine (6-azaindazole) as a core for the development of pan-Pim inhibitors. Fragment and structure based drug design led to identification of the series with picomolar biochemical potency against all three Pim isoforms. Desirable cellular potency was also achieved.
Subject(s)
Antineoplastic Agents/pharmacology , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Pyrazoles/pharmacology , Pyridines/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Crystallography, X-Ray , Humans , Indazoles/chemical synthesis , Indazoles/chemistry , Indazoles/pharmacology , Mice , Models, Molecular , Proto-Oncogene Proteins c-pim-1/chemistry , Pyrazoles/chemical synthesis , Pyrazoles/chemistry , Pyridines/chemical synthesis , Pyridines/chemistry , Structure-Activity RelationshipABSTRACT
BACKGROUND: The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in multiple myeloma (MM), a blood cancer associated with uncontrolled proliferation of bone marrow plasma cells. This study aimed to develop a robust clinical pharmacodynamic (PD) assay to measure the on-target PD effects of the selective PI3K inhibitor GDC-0941 in MM patients. METHODS: We conducted an in vitro drug wash-out study to evaluate the feasibility of biochemical approaches in measuring the phosphorylation of S6 ribosomal protein (S6), one of the commonly used PD markers for PI3K pathway inhibition. We then developed a 7-color phospho-specific flow cytometry assay, or phospho flow assay, to measure the phosphorylation state of intracellular S6 in bone marrow aspirate (BMA) and peripheral blood (PB). Integrated mean fluorescence intensity (iMFI) was used to calculate fold changes of phosphorylation. Assay sensitivity was evaluated by comparing phospho flow with Meso Scale Discovery (MSD) and immunohistochemistry (IHC) assays. Finally, a sample handling method was developed to maintain the integrity of phospho signal during sample shipping and storage to ensure clinical application. RESULTS: The phospho flow assay provided single-cell PD monitoring of S6 phosphorylation in tumor and surrogate cells using fixed BMA and PB, assessing pathway modulation in response to GDC-0941 with sensitivity similar to that of MSD assay. The one-shot sample fixation and handling protocol herein demonstrated exceptional preservation of protein phosphorylation. In contrast, the IHC assay was less sensitive in terms of signal quantification while the biochemical approach (MSD) was less suitable to assess PD activities due to the undesirable impact associated with cell isolation on the protein phosphorylation in tumor cells. CONCLUSIONS: We developed a robust PD biomarker assay for the clinical evaluation of PI3K inhibitors in MM, allowing one to decipher the PD response in a relevant cell population. To our knowledge, this is the first report of an easily implemented clinical PD assay that incorporates an unbiased one-shot sample handling protocol, all (staining)-in-one (tube) phospho flow staining protocol, and an integrated modified data analysis for PD monitoring of kinase inhibitors in relevant cell populations in BMA and PB. The methods described here ensure a real-time, reliable and reproducible PD readout, which can provide information for dose selection as well as help to identify optimal combinations of targeted agents in early clinical trials.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Screening Assays, Antitumor/methods , Enzyme Inhibitors/pharmacology , Flow Cytometry/methods , Multiple Myeloma/drug therapy , Phosphoinositide-3 Kinase Inhibitors , Biomarkers, Tumor/metabolism , Bone Marrow/metabolism , Bone Marrow Cells/drug effects , Cell Proliferation , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Indazoles/pharmacology , Phosphoproteins/metabolism , Phosphorylation , Sulfonamides/pharmacologyABSTRACT
Pim kinases are promising targets for the development of cancer therapeutics. Among the three Pim isoforms, Pim-2 is particularly important in multiple myeloma, yet is the most difficult to inhibit due to its high affinity for ATP. We identified compound 1 via high throughput screening. Using property-based drug design and co-crystal structures with Pim-1 kinase to guide analog design, we were able to improve potency against all three Pim isoforms including a significant 10,000-fold gain against Pim-2. Compound 17 is a novel lead with low picomolar potency on all three Pim kinase isoforms.
Subject(s)
Drug Design , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Pyrazoles/chemistry , Pyrimidines/chemistry , Animals , Binding Sites , Cell Line , Cell Survival/drug effects , Crystallography, X-Ray , Drug Evaluation, Preclinical , Humans , Kinetics , Mice , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/metabolism , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Protein Structure, Tertiary , Proto-Oncogene Proteins c-pim-1/metabolism , Pyrazoles/chemical synthesis , Pyrazoles/pharmacology , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
CD79 Antigens/immunology , Lymphoma, Non-Hodgkin/drug therapy , Oligopeptides/therapeutic use , Animals , Antibodies, Anti-Idiotypic/therapeutic use , Antineoplastic Agents/therapeutic use , Drug Resistance, Neoplasm/drug effects , Female , Humans , Immunoconjugates/therapeutic use , Lymphoma, Non-Hodgkin/pathology , Mice , Mice, Inbred ICR , Mice, SCID , Oligopeptides/chemistry , Treatment Outcome , Tumor Burden , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Multiple myeloma remains an incurable disease, and continued efforts are required to develop novel agents and novel drug combinations with more effective anti-myeloma activity. Here, we show that the pan-PIM kinase inhibitors SGI1776 and CX6258 exhibit significant anti-myeloma activity and that combining a pan-PIM kinase inhibitor with the immunomodulatory agent lenalidomide in an in vivo myeloma xenograft mouse model resulted in synergistic myeloma cell killing without additional hematologic or hepatic toxicities. Further investigations indicated that treatment with a pan-PIM kinase inhibitor promoted increased ubiquitination and subsequent degradation of IKZF1 and IKZF3, two transcription factors crucial for survival of myeloma cells. Combining a pan-PIM kinase inhibitor with lenalidomide led to more effective degradation of IKZF1 and IKZF3 in multiple myeloma cell lines as well as xenografts of myeloma tumors. We also demonstrated that treatment with a pan-PIM kinase inhibitor resulted in increased expression of cereblon, and that knockdown of cereblon via a shRNA lentivirus abolished the effects of PIM kinase inhibition on the degradation of IKZF1 and IKZF3 and myeloma cell apoptosis, demonstrating a central role of cereblon in pan-PIM kinase inhibitor-mediated down-regulation of IKZF1 and IKZF3 and myeloma cell killing. These data elucidate the mechanism of pan-PIM kinase inhibitor mediated anti-myeloma effect and the rationale for the synergy observed with lenalidomide co-treatment, and provide justification for a clinical trial of the combination of pan-PIM kinase inhibitors and lenalidomide for the treatment of multiple myeloma.
Subject(s)
Ikaros Transcription Factor/metabolism , Multiple Myeloma/drug therapy , Multiple Myeloma/metabolism , Peptide Hydrolases/metabolism , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Adaptor Proteins, Signal Transducing , Animals , Down-Regulation/drug effects , Humans , Imidazoles/pharmacology , Lenalidomide/pharmacology , Mice , Mice, Inbred C57BL , Multiple Myeloma/enzymology , Peptide Hydrolases/biosynthesis , Proteasome Endopeptidase Complex/metabolism , Proto-Oncogene Proteins c-pim-1/metabolism , Pyridazines/pharmacology , Signal Transduction/drug effects , Ubiquitin-Protein Ligases , Xenograft Model Antitumor AssaysABSTRACT
Pim kinases have been targets of interest for a number of therapeutic areas. Evidence of durable single-agent efficacy in human clinical trials validated Pim kinase inhibition as a promising therapeutic approach for multiple myeloma patients. Here, we report the compound optimization leading to GDC-0339 (16), a potent, orally bioavailable, and well tolerated pan-Pim kinase inhibitor that proved efficacious in RPMI8226 and MM.1S human multiple myeloma xenograft mouse models and has been evaluated as an early development candidate.
Subject(s)
Antineoplastic Agents/therapeutic use , Multiple Myeloma/drug therapy , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Pyrazoles/therapeutic use , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/metabolism , Cell Line, Tumor , Dogs , Female , Humans , Macaca fascicularis , Madin Darby Canine Kidney Cells , Male , Mice, SCID , Molecular Structure , Protein Binding , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/metabolism , Proto-Oncogene Proteins c-pim-1/metabolism , Pyrazoles/chemical synthesis , Pyrazoles/metabolism , Rats, Sprague-Dawley , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
CD22 represents a promising target for antibody-drug conjugate therapy in the context of B cell malignancies since it rapidly internalizes, importing specifically bound antibodies with it. To determine the pharmacokinetic parameters of anti-CD22-MCC-DM1 and MC-MMAF conjugates, various approaches to quantifying total and conjugated antibody were investigated. Although the total antibody assay formats gave similar results for both conjugates, the mouse pharmacokinetic profile for the anti-CD22-MCC-DM1 and MC-MMAF appeared significantly different depending on the conjugated antibody assay format. Since these differences significantly impacted the PK parameters determination, we investigated the effect of the drug/antibody ratio on the total and conjugated antibody quantification using multiple assay formats. Our investigations revealed the limitations of some assay formats to quantify anti-CD22-MCC-DM1 and MC-MMAF with different drug load and in the context of a heterogeneous ADC population highlight the need to carefully plan the assay strategy for the total and conjugated antibody quantification in order to accurately determine the ADC PK parameters.
Subject(s)
Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/pharmacokinetics , Immunoconjugates/metabolism , Immunoconjugates/pharmacokinetics , Maleimides/metabolism , Maytansine/analogs & derivatives , Maytansine/metabolism , Oligopeptides/metabolism , Sialic Acid Binding Ig-like Lectin 2/immunology , Animals , Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/immunology , Humans , Immunoconjugates/analysis , Immunoconjugates/immunology , Mice , Sensitivity and SpecificityABSTRACT
Pim kinases have been identified as promising therapeutic targets for hematologic-oncology indications, including multiple myeloma and certain leukemia. Here, we describe our continued efforts in optimizing a lead series by improving bioavailability while maintaining high inhibitory potency against all three Pim kinase isoforms. The discovery of extensive intestinal metabolism and major metabolites helped refine our design strategy, and we observed that optimizing the pharmacokinetic properties first and potency second was a more successful approach than the reverse. In the resulting work, novel analogs such as 20 (GNE-955) were discovered bearing 5-azaindazole core with noncanonical hydrogen bonding to the hinge.
Subject(s)
Indazoles/chemistry , Indazoles/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Animals , Biological Availability , Crystallography, X-Ray , Humans , Indazoles/metabolism , Indazoles/pharmacokinetics , Intestinal Mucosa/metabolism , Molecular Docking Simulation , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/metabolism , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacokinetics , Proto-Oncogene Proteins c-pim-1/metabolism , RatsABSTRACT
Unexpected drug activities discovered during clinical testing establish the need for better characterization of compounds in human disease-relevant conditions early in the discovery process. Here, we describe an approach to characterize drug function based on statistical analysis of protein expression datasets from multiple primary human cell-based models of inflammatory disease. This approach, termed Biologically Multiplexed Activity Profiling (BioMAP), provides rapid characterization of drug function, including mechanism of action, secondary or off-target activities, and insights into clinical phenomena. Using three model systems containing primary human endothelial cells and peripheral blood mononuclear cells in different environments relevant to vascular inflammation and immune activation, we show that BioMAP profiles detect and discriminate multiple functional drug classes, including glucocorticoids; TNF-alpha antagonists; and inhibitors of HMG-CoA reductase, calcineurin, IMPDH, PDE4, PI-3 kinase, hsp90, and p38 MAPK, among others. The ability of cholesterol lowering HMG-CoA reductase inhibitors (statins) to improve outcomes in rheumatic disease patients correlates with the activities of these compounds in our BioMAP assays. In addition, the activity profiles identified for the immunosuppressants mycophenolic acid, cyclosporin A, and FK-506 provide a potential explanation for a reduced incidence of posttransplant cardiovascular disease in patients receiving mycophenolic acid. BioMAP profiling can allow integration of meaningful human biology into drug development programs.
Subject(s)
Drug Evaluation, Preclinical/methods , Endothelial Cells/drug effects , Endothelium, Vascular/drug effects , Leukocytes, Mononuclear/drug effects , Vasculitis/drug therapy , Anti-Inflammatory Agents/pharmacology , Cells, Cultured/drug effects , Coculture Techniques , Cytokines/antagonists & inhibitors , Drug Design , Endothelium, Vascular/cytology , Enzyme Inhibitors/pharmacology , Enzyme-Linked Immunosorbent Assay , Humans , Immunosuppressive Agents/pharmacology , Models, Biological , Pharmaceutical Preparations/classification , RNA, Small Interfering/pharmacology , Transfection , Umbilical VeinsABSTRACT
Bispecific antibodies and antibody fragments in various formats have been explored as a means to recruit cytolytic T cells to kill tumor cells. Encouraging clinical data have been reported with molecules such as the anti-CD19/CD3 bispecific T cell engager (BiTE) blinatumomab. However, the clinical use of many reported T cell-recruiting bispecific modalities is limited by liabilities including unfavorable pharmacokinetics, potential immunogenicity, and manufacturing challenges. We describe a B cell-targeting anti-CD20/CD3 T cell-dependent bispecific antibody (CD20-TDB), which is a full-length, humanized immunoglobulin G1 molecule with near-native antibody architecture constructed using "knobs-into-holes" technology. CD20-TDB is highly active in killing CD20-expressing B cells, including primary patient leukemia and lymphoma cells both in vitro and in vivo. In cynomolgus monkeys, CD20-TDB potently depletes B cells in peripheral blood and lymphoid tissues at a single dose of 1 mg/kg while demonstrating pharmacokinetic properties similar to those of conventional monoclonal antibodies. CD20-TDB also exhibits activity in vitro and in vivo in the presence of competing CD20-targeting antibodies. These data provide rationale for the clinical testing of CD20-TDB for the treatment of CD20-expressing B cell malignancies.
Subject(s)
Antibodies, Bispecific/therapeutic use , Antigens, CD20/immunology , CD3 Complex/immunology , Leukemia, B-Cell/therapy , T-Lymphocytes/immunology , Animals , Antibodies, Bispecific/immunology , Antibodies, Bispecific/pharmacokinetics , Humans , Leukemia, B-Cell/immunology , Macaca fascicularis , Mice , Mice, TransgenicABSTRACT
MET, the receptor for hepatocyte growth factor (HGF), plays an important role in signaling normal and tumor cell migration and invasion. Here, we describe a previously unrecognized mechanism that promotes MET expression in multiple tumor cell types. The levels of the Pim-1 protein kinase show a positive correlation with the levels of MET protein in human tumor cell lines and patient-derived tumor materials. Using small interfering RNA (siRNA), Pim knockout mice, small-molecule inhibitors, and overexpression of Pim-1, we confirmed this correlation and found that Pim-1 kinase activity regulates HGF-induced tumor cell migration, invasion, and cell scattering. The novel biochemical mechanism for these effects involves the ability of Pim-1 to control the translation of MET by regulating the phosphorylation of eukaryotic initiation factor 4B (eIF4B) on S406. This targeted phosphorylation is required for the binding of eIF4B to the eIF3 translation initiation complex. Importantly, Pim-1 action was validated by the evaluation of patient blood and bone marrow from a phase I clinical trial of a Pim kinase inhibitor, AZD1208. These results suggest that Pim inhibitors may have an important role in the treatment of patients where MET is driving tumor biology.
Subject(s)
Eukaryotic Initiation Factors/genetics , Hepatocyte Growth Factor/metabolism , Proto-Oncogene Proteins c-met/biosynthesis , Proto-Oncogene Proteins c-pim-1/biosynthesis , Animals , Biphenyl Compounds/pharmacology , Cell Line, Tumor , Cell Movement , Eukaryotic Initiation Factor-3/genetics , HeLa Cells , Humans , Mice , Mice, Knockout , Neoplasm Invasiveness , Phosphorylation , Protein Binding , Protein Biosynthesis , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-met/genetics , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Proto-Oncogene Proteins c-pim-1/genetics , RNA Interference , RNA, Small Interfering , Signal Transduction , Thiazolidines/pharmacologyABSTRACT
Clinical results from the latest strategies for T-cell activation in cancer have fired interest in combination immunotherapies that can fully engage T-cell immunity. In this study, we describe a trastuzumab-based bispecific antibody, HER2-TDB, which targets HER2 and conditionally activates T cells. HER2-TDB specifically killed HER2-expressing cancer cells at low picomolar concentrations. Because of its unique mechanism of action, which is independent of HER2 signaling or chemotherapeutic sensitivity, HER2-TDB eliminated cells refractory to currently approved HER2 therapies. HER2-TDB exhibited potent antitumor activity in four preclinical model systems, including MMTV-huHER2 and huCD3 transgenic mice. PD-L1 expression in tumors limited HER2-TDB activity, but this resistance could be reversed by anti-PD-L1 treatment. Thus, combining HER2-TDB with anti-PD-L1 yielded a combination immunotherapy that enhanced tumor growth inhibition, increasing the rates and durability of therapeutic response.
Subject(s)
Antibodies, Bispecific/immunology , Lymphocyte Activation , Receptor, ErbB-2/immunology , T-Lymphocytes/immunology , Animals , Antibodies, Bispecific/pharmacology , Antibodies, Monoclonal, Humanized/pharmacology , Breast Neoplasms/immunology , Breast Neoplasms/pathology , Cell Line, Tumor , Female , Humans , Rats , Rats, Sprague-Dawley , TrastuzumabABSTRACT
Antibody-drug conjugates (ADC), potent cytotoxic drugs linked to antibodies via chemical linkers, allow specific targeting of drugs to neoplastic cells. We have used this technology to develop the ADC DCDT2980S that targets CD22, an antigen with expression limited to B cells and the vast majority of non-Hodgkin lymphomas (NHL). DCDT2980S consists of a humanized anti-CD22 monoclonal IgG1 antibody with a potent microtubule-disrupting agent, monomethyl auristatin E (MMAE), linked to the reduced cysteines of the antibody via a protease cleavable linker, maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB). We describe the efficacy, safety, and pharmacokinetics of DCDT2980S in animal models to assess its potential as a therapeutic for the treatment of B-cell malignancies. We did not find a strong correlation between in vitro or in vivo efficacy and CD22 surface expression, nor a correlation of sensitivity to free drug and in vitro potency. We show that DCDT2980S was capable of inducing complete tumor regression in xenograft mouse models of NHL and can be more effective than rituximab plus combination chemotherapy at drug exposures that were well tolerated in cynomolgus monkeys. These results suggest that DCDT2980S has an efficacy, safety, and pharmacokinetics profile that support potential treatment of NHL.
Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Antineoplastic Agents/pharmacology , Immunoconjugates/pharmacology , Lymphoma, B-Cell/drug therapy , Lymphoma, Non-Hodgkin/drug therapy , Oligopeptides/pharmacology , Sialic Acid Binding Ig-like Lectin 2/immunology , Animals , Antibodies, Monoclonal/pharmacokinetics , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal, Humanized/pharmacokinetics , Female , Humans , Immunoconjugates/pharmacokinetics , Lymphoma, B-Cell/immunology , Lymphoma, B-Cell/metabolism , Macaca fascicularis , Mice , Mice, Inbred ICR , Mice, SCID , Random Allocation , Xenograft Model Antitumor AssaysABSTRACT
Fc receptor-like 5 (FcRL5/FcRH5/IRTA2/CD307) is a surface protein expressed selectively on B cells and plasma cells. We found that FcRL5 was expressed at elevated levels on the surface of plasma cells from the bone marrow of patients diagnosed with multiple myeloma. This prevalence in multiple myeloma and narrow pattern of normal expression indicate that FcRL5 could be a target for antibody-based therapies for multiple myeloma, particularly antibody-drug conjugates (ADC), potent cytotoxic drugs linked to antibodies via specialized chemical linkers, where limited expression on normal tissues is a key component to their safety. We found that FcRL5 is internalized upon antibody binding, indicating that ADCs to FcRL5 could be effective. Indeed, we found that FcRL5 ADCs were efficacious in vitro and in vivo but the unconjugated antibody was not. The two most effective consisted of our anti-FcRL5 antibody conjugated through cysteines to monomethylauristatin E (MMAE) by a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vcPAB) linker (anti-FcRL5-MC-vcPAB-MMAE) or conjugated via lysines to the maytansinoid DM4 through a disulfide linker (anti-FcRL5-SPDB-DM4). These two ADCs were highly effective in vivo in combination with bortezomib or lenalidomide, drugs in use for the treatment of multiple myeloma. These data show that the FcRL5 ADCs described herein show promise as an effective treatment for multiple myeloma.
Subject(s)
Antibodies, Neoplasm/immunology , Antineoplastic Agents/therapeutic use , Immunoconjugates/therapeutic use , Molecular Targeted Therapy , Multiple Myeloma/drug therapy , Multiple Myeloma/immunology , Receptors, Cell Surface/immunology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Dose-Response Relationship, Drug , Endocytosis/drug effects , Humans , Immunoconjugates/chemistry , Immunoconjugates/pharmacology , Mice , Mice, SCID , Receptors, Fc , Reproducibility of Results , Treatment OutcomeABSTRACT
The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.
Subject(s)
Antibodies/blood , Antibodies/immunology , Binding Sites, Antibody/immunology , Immunoconjugates/chemistry , Immunoconjugates/immunology , Immunoglobulin G/chemistry , Protein Engineering , Aminobenzoates/chemistry , Aminobenzoates/immunology , Animals , Antibodies/chemistry , Antibodies, Monoclonal, Humanized/chemistry , Antibodies, Monoclonal, Humanized/immunology , Cell Line, Tumor , Cell Survival , Cysteine/chemistry , Humans , Immunoconjugates/administration & dosage , Immunoglobulin G/immunology , Macaca fascicularis , Maleimides/chemistry , Mammary Neoplasms, Experimental/drug therapy , Mammary Neoplasms, Experimental/immunology , Maytansine/chemistry , Maytansine/immunology , Mice , Mice, Nude , Models, Molecular , Oligopeptides/chemistry , Oligopeptides/immunology , Protein Conformation , Rats , Structure-Activity Relationship , Sulfhydryl Compounds/chemistry , TrastuzumabABSTRACT
The primary function of B cells, critical components of the adaptive immune response, is to produce antibodies against foreign antigens, as well as to perform isotype class switching, which changes the heavy chain of an antibody so that it can interact with different repertoires of effector cells. CD40 is a member of the tumor necrosis factor superfamily of cell surface receptors that transmits survival signals to B cells. In contrast, in B cell cancers, stimulation of CD40 signaling results in a heterogeneous response in which cells can sometimes undergo cell death in response to treatment, depending on the system studied. We found an association between sensitivity to CD40 stimulation and mutation of the tumor suppressor p53 in a panel of non-Hodgkin's lymphoma cell lines. Consistent with p53's tumor suppressor role, we found that higher levels of intrinsic DNA damage and increased proliferation rates, as well as higher levels of BCL6, a transcriptional repressor proto-oncogene, were associated with sensitivity to CD40 stimulation. In addition, CD40 treatment-resistant cell lines were sensitized to CD40 stimulation after the introduction of DNA-damaging agents. Using gene expression analysis, we also showed that resistant cell lines exhibited a preexisting activated CD40 pathway and that an mRNA expression signature comprising CD40 target genes predicted sensitivity and resistance to CD40-activating agents in cell lines and mouse xenograft models. Finally, the gene signature predicted tumor shrinkage and progression-free survival in patients with diffuse large B cell lymphoma treated with dacetuzumab, a monoclonal antibody with partial CD40 agonist activity. These data show that CD40 pathway activation status may be useful in predicting the antitumor activity of CD40-stimulating therapeutic drugs.
Subject(s)
Antibodies, Monoclonal/therapeutic use , CD40 Antigens/immunology , Immunotherapy/methods , Lymphoma, Large B-Cell, Diffuse/drug therapy , Lymphoma, Large B-Cell, Diffuse/immunology , Animals , Antibodies, Monoclonal, Humanized , B-Lymphocytes/immunology , CD40 Antigens/genetics , CD40 Ligand/genetics , CD40 Ligand/immunology , Cell Line, Tumor , Gene Expression Profiling , Humans , In Situ Hybridization, Fluorescence , Mice , Microarray Analysis , Proto-Oncogene Mas , Transplantation, Heterologous , Tumor Suppressor Protein p53/immunologyABSTRACT
Antibodies directed against B cells are in use for the treatment of non-Hodgkin's lymphoma and autoimmune disorders. The B-cell-restricted surface antigen CD79b, a signaling component of the B-cell receptor, has been shown as a promising antibody target in mouse efficacy models of systemic lupus erythematosus. Anti-CD79b antibody-drug conjugates (ADC), cytotoxic drugs linked through specialized chemical linkers to antibodies, are effective in mouse xenograft models of non-Hodgkin's lymphoma. We were interested in evaluating the systemic effects of anti-CD79b antibodies and ADCs in normal animals as a step toward the development of these molecules as therapeutics. As we were unable to identify any cell surface binding anti-human CD79b antibodies that were cross-reactive to other species, we developed an antibody to cynomolgus monkey (Macaca fascicularis) CD79b (anti-cyCD79b). The anti-cynomolgus antibody, anti-cyCD79b (10D10), and the maytansine (tubulin inhibitor)-conjugated ADC, anti-cyCD79b (10D10)-MCC-DM1, were administered to cynomolgus monkeys at approximately 30 mg/kg (6,000 microg DM1/m(2)) for two doses 3 weeks apart. Anti-cyCD79b and anti-cyCD79b-MCC-DM1 resulted in peripheral blood B-cell depletion of approximately 65% and approximately 94%, respectively. In addition, anti-cyCD79b-MCC-DM1 resulted in near-complete absence of splenic germinal centers, an observation supporting an effect on dividing B cells. Both molecules were well tolerated, with minimal findings for the antibody and findings for the ADC limited to the lymphoid and hematopoietic systems, liver, and peripheral nerves. These preclinical data suggest that targeting CD79b with antibodies or ADCs may provide safe and effective therapies for B-cell malignancies and autoimmune diseases.
Subject(s)
Antibodies/immunology , Antineoplastic Agents/pharmacology , CD79 Antigens/immunology , Amino Acid Sequence , Animals , Antibody Formation/drug effects , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , CD79 Antigens/chemistry , Cell Membrane/drug effects , Cell Membrane/metabolism , Cross Reactions/drug effects , Flow Cytometry , Humans , Immune Tolerance/drug effects , Macaca fascicularis/blood , Macaca fascicularis/immunology , Maytansine/pharmacology , Mice , Molecular Sequence Data , Spleen/drug effects , Spleen/immunology , Spleen/pathology , Xenograft Model Antitumor AssaysABSTRACT
Antibody-drug conjugates (ADC), potent cytotoxic drugs covalently linked to antibodies via chemical linkers, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells while reducing side effects. Here, we systematically examine the potential targets and linker-drug combinations that could provide an optimal ADC for the treatment for non-Hodgkin's lymphoma. We identified seven antigens (CD19, CD20, CD21, CD22, CD72, CD79b, and CD180) for potential treatment of non-Hodgkin's lymphoma with ADCs. ADCs with cleavable linkers mediated in vivo efficacy via all these targets; ADCs with uncleavable linkers were only effective when targeted to CD22 and CD79b. In target-independent safety studies in rats, the uncleavable linker ADCs showed reduced toxicity, presumably due to the reduced release of free drug or other toxic metabolites into the circulation. Thus, our data suggest that ADCs with cleavable linkers work on a broad range of targets, and for specific targets, ADCs with uncleavable linkers provide a promising opportunity to improve the therapeutic window for ADCs in humans.
Subject(s)
Antineoplastic Agents/administration & dosage , Immunotoxins/pharmacology , Lymphoma, Non-Hodgkin/drug therapy , Maytansine/analogs & derivatives , Oligopeptides/administration & dosage , Sulfhydryl Compounds/administration & dosage , Animals , Antigens, CD/biosynthesis , Antigens, CD/immunology , Antineoplastic Agents/pharmacokinetics , B-Lymphocytes/immunology , Cross-Linking Reagents/administration & dosage , Cross-Linking Reagents/pharmacokinetics , Female , Immunotoxins/immunology , Immunotoxins/pharmacokinetics , Lymphoma, Non-Hodgkin/immunology , Maytansine/administration & dosage , Maytansine/pharmacokinetics , Mice , Mice, Inbred ICR , Mice, SCID , Oligopeptides/pharmacokinetics , Rats , Sulfhydryl Compounds/pharmacokinetics , Xenograft Model Antitumor AssaysABSTRACT
Targeting cytotoxic drugs to cancer cells using antibody-drug conjugates (ADCs), particularly those with stable linkers between the drug and the antibody, could be an effective cancer treatment with low toxicity. However, for stable-linker ADCs to be effective, they must be internalized and degraded, limiting potential targets to surface antigens that are trafficked to lysosomes. CD79a and CD79b comprise the hetrodimeric signaling component of the B-cell receptor, and are attractive targets for the use of ADCs because they are B-cell-specific, expressed in non-Hodgkin lymphomas (NHL), and are trafficked to a lysosomal-like compartment as part of antigen presentation. We show here that the stable-linker ADCs anti-CD79b-MCC-DM1 and anti-CD79b-MC-MMAF are capable of target-dependent killing of nonHodgkin lymphoma cell lines in vitro. Further, these 2 ADCs are equally effective as low doses in xenograft models of follicular, mantle cell, and Burkitt lymphomas, even though several of these cell lines express relatively low levels of CD79b in vivo. In addition, we demonstrate that anti-CD79b ADCs were more effective than anti-CD79a ADCs and that, as hypothesized, anti-CD79b antibodies downregulated surface B-cell receptor and were trafficked to the lysosomal-like major histocompatibility complex class II-positive compartment MIIC. These results suggest that anti-CD79b-MCC-DM1 and anti-CD79b-MC-MMAF are promising therapeutics for the treatment of NHL.