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Introduction: Interleukin 15 (IL-15) is a potential anticancer agent and numerous engineered IL-15 agonists are currently under clinical investigation. Selective targeting of IL-15 to specific lymphocytes may enhance therapeutic effects while helping to minimize toxicities. Methods: We designed and built a heterodimeric targeted cytokine (TaCk) that consists of an anti-programmed cell death 1 receptor antibody (anti-PD-1) and an engineered IL-15. This "PD1/IL15" selectively delivers IL-15 signaling to lymphocytes expressing PD-1. We then investigated the pharmacokinetic (PK) and pharmacodynamic (PD) effects of PD1/IL15 TaCk on immune cell subsets in cynomolgus monkeys after single and repeat intravenous dose administrations. We used these results to determine the first-in-human (FIH) dose and dosing frequency for early clinical trials. Results: The PD1/IL15 TaCk exhibited a nonlinear multiphasic PK profile, while the untargeted isotype control TaCk, containing an anti-respiratory syncytial virus antibody (RSV/IL15), showed linear and dose proportional PK. The PD1/IL15 TaCk also displayed a considerably prolonged PK (half-life range â¼1.0-4.1 days) compared to wild-type IL-15 (half-life â¼1.1 h), which led to an enhanced cell expansion PD response. The PD was dose-dependent, durable, and selective for PD-1+ lymphocytes. Notably, the dose- and time-dependent PK was attributed to dynamic TMDD resulting from test article-induced lymphocyte expansion upon repeat administration. The recommended first-in-human (FIH) dose of PD1/IL15 TaCk is 0.003 mg/kg, determined based on a minimum anticipated biological effect level (MABEL) approach utilizing a combination of in vitro and preclinical in vivo data. Conclusion: This work provides insight into the complex PK/PD relationship of PD1/IL15 TaCk in monkeys and informs the recommended starting dose and dosing frequency selection to support clinical evaluation of this novel targeted cytokine.
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FLT3L-Fc is a half-life extended, effectorless Fc-fusion of the native human FLT3-ligand. In cynomolgus monkeys, treatment with FLT3L-Fc leads to a complex pharmacokinetic/pharmacodynamic (PK/PD) relationship, with observed nonlinear PK and expansion of different immune cell types across different dose levels. A minimal physiologically based PK/PD model with expansion-enhanced target-mediated drug disposition (TMDD) was developed to integrate the molecule's mechanism of action, as well as the complex preclinical and clinical PK/PD data, to support the preclinical-to-clinical translation of FLT3L-Fc. In addition to the preclinical PK data of FLT3L-Fc in cynomolgus monkeys, clinical PK and PD data from other FLT3-agonist molecules (GS-3583 and CDX-301) were used to inform the model and project the expansion profiles of conventional DC1s (cDC1s) and total DCs in peripheral blood. This work constitutes an essential part of our model-informed drug development (MIDD) strategy for clinical development of FLT3L-Fc by projecting PK/PD in healthy volunteers, determining the first-in-human (FIH) dose, and informing the efficacious dose in clinical settings. Model-generated results were incorporated in regulatory filings to support the rationale for the FIH dose selection.
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(171/200)ADCs represent a transformative class of medicine that combines the specificity of monoclonal antibodies with the potency of highly cytotoxic agents through linkers, aiming to enhance the therapeutic index of cytotoxic drugs. Given the complex molecular structures of ADCs, combining the molecular characteristics of small-molecule drugs and those of large-molecule biotherapeutics, there are several unique considerations when designing nonclinical-to-clinical PK/PD translation strategies.This complexity also demands a thorough understanding of the ADC's components-antibody, linker, and payload-to the overall toxicological, PK/PD, and efficacy profile. ADC development is a multidisciplinary endeavor requiring a strategic integration of nonclinical safety, pharmacology, and PK/PD modeling to translate from bench to bedside successfully.The ADC development underscores the necessity for a robust scientific foundation, leveraging advanced analytical and modeling tools to predict human responses and optimize therapeutic outcomes.This review aims to provide an ADC translational PK/PD framework by discussing unique aspects of ADC nonclinical to clinical PK translation, starting dose determination, and leveraging PK/PD modeling for human efficacious dose prediction and potential safety mitigation.
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Background: Monomethyl auristatin E (MMAE) has been used as a payload for several Food and Drug Administration (FDA) approved antibody-drug conjugates (ADCs). It is known that MMAE is released from the ADC following binding, internalization and proteolytic degradation in target tissues. A striking discrepancy in systemic MMAE levels has been observed across species with 50-fold higher MMAE levels in human than that in rodents when normalized by ADC dose with unknown mechanism.Hypothesis and purpose: Multiple factors could affect systemic MMAE levels such as production and elimination of unconjugated MMAE following ADC dosing. In this study, we have explored whether MMAE displays differential red blood cell (RBC) partitioning across species that may contribute to the different MMAE levels seen between human and animals.Experiments: To determine MMAE RBC partitioning, tritium labeled MMAE ([3H]-MMAE) was incubated in whole blood from mice, rats, monkeys and humans in vitro, then RBC partitioning was determined and compared across species. To test whether MMAE released from the ADC would show any difference in RBC partitioning, pinatuzumab vedotin or polatuzumab vedotin was administered to mice, rats, and monkeys. MMAE levels were measured in both blood and plasma, and the ratios of MMAE levels were calculated as blood-to-plasma ratio (in vivo RBC partitioning).Results: Our in vitro data showed that unconjugated MMAE has a species-dependent RBC partitioning with strong RBC partitioning in mouse, rat, followed by monkey blood, whereas minimal RBC partitioning was seen in human blood. Incubation of 2 nM of MMAE in mouse blood resulted in a blood-to-plasma ratio of 11.8 ± 0.291, followed by rat, monkey, and human at 2.36 ± 0.0825, 1.57 ± 0.0250, and 0.976 ± 0.0620, respectively. MMAE RBC partitioning is also concentration-dependent, with an inverse relationship between RBC partitioning and MMAE concentration (higher RBC partitioning at lower concentration). In vivo dosing of pinatuzumab vedotin in mouse displayed systemic MMAE at about a 5-fold higher blood concentration compared to plasma concentration once MMAE reached a pseudo-equilibrium, while systemic MMAE from blood and plasma concentration showed a 1.65-fold difference in rat.Implication and conclusion: These data demonstrated that MMAE has a distinct RBC partitioning across different species, which may contribute to, at least in part, to the differential in the systemic MMAE levels observed in vivo between preclinical and clinical studies. These findings highlight the importance of fully characterizing the ADME properties of both the ADC and its payload, to enable better translation from animals to human for ADC development.
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Inhibiting MerTK on macrophages is a promising therapeutic strategy for augmenting anti-tumor immunity. However, blocking MerTK on retinal pigment epithelial cells (RPEs) results in retinal toxicity. Bispecific antibodies (bsAbs) containing an anti-MerTK therapeutic and anti-PD-L1 targeting arm were developed to reduce drug binding to MerTK on RPEs, since PD-L1 is overexpressed on macrophages but not RPEs. In this study, we present a modeling framework using in vitro receptor occupancy (RO) and pharmacokinetics (PK) data to predict efficacy, toxicity, and therapeutic index (TI) of anti-MerTK bsAbs. We first used simulations and in vitro RO data of anti-MerTK monospecific antibody (msAb) to estimate the required MerTK RO for in vivo efficacy and toxicity. Using these estimated RO thresholds, we employed our model to predict the efficacious and toxic doses for anti-MerTK bsAbs with varying affinities for MerTK. Our model predicted the highest TI for the anti-MerTK/PD-L1 bsAb with an attenuated MerTK binding arm, which was consistent with in vivo efficacy and toxicity observations. Subsequently, we used the model, in combination with sensitivity analysis and parameter scans, to suggest an optimal molecular design of anti-MerTK bsAb with the highest predicted TI in humans. Our prediction revealed that this optimized anti-MerTK bsAb should contain a MerTK therapeutic arm with relatively low affinity, along with a high affinity targeting arm that can bind to a low abundance target with slow turnover rate. Overall, these results demonstrated that our modeling framework can guide the rational design of bsAbs.
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Anticorpos Biespecíficos , Humanos , Antígeno B7-H1 , c-Mer Tirosina QuinaseRESUMO
Approved antibody-drug conjugates (ADCs) for HER2-positive breast cancer include trastuzumab emtansine and trastuzumab deruxtecan. To develop a differentiated HER2 ADC, we chose an antibody that does not compete with trastuzumab or pertuzumab for binding, conjugated to a reduced potency PBD (pyrrolobenzodiazepine) dimer payload. PBDs are potent cytotoxic agents that alkylate and cross-link DNA. In our study, the PBD dimer is modified to alkylate, but not cross-link DNA. This HER2 ADC, DHES0815A, demonstrates in vivo efficacy in models of HER2-positive and HER2-low cancers and is well-tolerated in cynomolgus monkey safety studies. Mechanisms of action include induction of DNA damage and apoptosis, activity in non-dividing cells, and bystander activity. A dose-escalation study (ClinicalTrials.gov: NCT03451162) in patients with HER2-positive metastatic breast cancer, with the primary objective of evaluating the safety and tolerability of DHES0815A and secondary objectives of characterizing the pharmacokinetics, objective response rate, duration of response, and formation of anti-DHES0815A antibodies, is reported herein. Despite early signs of anti-tumor activity, patients at higher doses develop persistent, non-resolvable dermal, ocular, and pulmonary toxicities, which led to early termination of the phase 1 trial.
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Anticorpos Monoclonais Humanizados , Antineoplásicos , Benzodiazepinas , Neoplasias da Mama , Imunoconjugados , Humanos , Animais , Feminino , Neoplasias da Mama/genética , Macaca fascicularis/genética , Receptor ErbB-2/metabolismo , Trastuzumab/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , DNARESUMO
MTBT1466A is a high-affinity TGFß3-specific humanized IgG1 monoclonal antibody with reduced Fc effector function, currently under investigation in clinical trials as a potential anti-fibrotic therapy. Here, we characterized the pharmacokinetics (PK) and pharmacodynamics (PD) of MTBT1466A in mice and monkeys and predicted the PK/PD of MTBT1466A in humans to guide the selection of the first-in-human (FIH) starting dose. MTBT1466A demonstrated a typical IgG1-like biphasic PK profile in monkeys, and the predicted human clearance of 2.69 mL/day/kg and t1/2 of 20.4 days are consistent with those expected for a human IgG1 antibody. In a mouse model of bleomycin-induced lung fibrosis, changes in expression of TGFß3-related genes, serpine1, fibronectin-1, and collagen 1A1 were used as PD biomarkers to determine the minimum pharmacologically active dose of 1 mg/kg. Unlike in the fibrosis mouse model, evidence of target engagement in healthy monkeys was only observed at higher doses. Using a PKPD-guided approach, the recommended FIH dose of 50 mg, IV, provided exposures that were shown to be safe and well tolerated in healthy volunteers. MTBT1466A PK in healthy volunteers was predicted reasonably well using a PK model with allometric scaling of PK parameters from monkey data. Taken together, this work provides insights into the PK/PD behavior of MTBT1466A in preclinical species, and supports the translatability of the preclinical data into the clinic.
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Monoclonal antibodies (mAbs) deliver great benefits to patients with chronic and/or severe diseases thanks to their strong specificity to the therapeutic target. As a result of this specificity, non-human primates (NHP) are often the only preclinical species in which therapeutic antibodies cross-react with the target. Here, we highlight the value and limitations that NHP studies bring to the design of safe and efficient early clinical trials. Indeed, data generated in NHPs are integrated with in vitro information to predict the concentration/effect relationship in human, and therefore the doses to be tested in first-in-human trials. The similarities and differences in the systems defining the pharmacokinetics and pharmacodynamics (PKPD) of mAbs in NHP and human define the nature and the potential of the preclinical investigations performed in NHPs. Examples have been collated where the use of NHP was either pivotal to the design of the first-in-human trial or, inversely, led to the termination of a project prior to clinical development. The potential impact of immunogenicity on the results generated in NHPs is discussed. Strategies to optimize the use of NHPs for PKPD purposes include the addition of PD endpoints in safety assessment studies and the potential re-use of NHPs after non-terminal studies or cassette dosing several therapeutic agents of interest. Efforts are also made to reduce the use of NHPs in the industry through the use of in vitro systems, alternative in vivo models, and in silico approaches. In the case of prediction of ocular PK, the body of evidence gathered over the last two decades renders the use of NHPs obsolete. Expert perspectives, advantages, and pitfalls with these alternative approaches are shared in this review.
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Produtos Biológicos , Animais , Humanos , Produtos Biológicos/farmacologia , Primatas , Anticorpos MonoclonaisRESUMO
RO7297089, an anti-B-cell maturation antigen (BCMA)/CD16A bispecific tetravalent antibody, is being developed as a multiple myeloma (MM) therapeutic. This study characterized nonclinical pharmacokinetics (PK), pharmacodynamics (PD), soluble BCMA (sBCMA), and soluble CD16 (sCD16) changes following administration of RO7297089 to support clinical trials. Unbound and total RO7297089 concentrations were measured in cynomolgus monkeys. RO7297089 exhibited a bi-phasic systemic concentration-time profile, similar to a typical human immunoglobulin 1 antibody. Target engagement by RO7297089 led to a robust increase (~100-fold) in total systemic sBCMA levels and relatively mild increase (~2-fold) in total sCD16 levels. To describe the relationship of nonclinical PK/PD data, we developed a target-mediated drug disposition (TMDD) model that includes the systemic target engagement of membrane BCMA (mBCMA), sBCMA, membrane CD16 (mCD16), and sCD16. We then used this model to simulate the PK/PD relationship of RO7297089 in MM patients by translating relevant PK parameters and target levels, based on the literature and newly generated data such as baseline sCD16A levels. Our model suggested that the impact of TMDD on RO7297089 exposure may be more significant in MM patients due to significantly higher expression levels of both mBCMA and sBCMA compared to healthy cynomolgus monkeys. Based on model simulations, we propose more frequent dosing of RO7297089 compared to regular monthly frequency in the clinic at the beginning of treatment to ensure sustained target engagement. This study demonstrates a translational research strategy for collecting relevant nonclinical data, establishing a TMDD model, and using simulations from this model to inform clinical dose regimens.
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Mieloma Múltiplo , Animais , Humanos , Imunoterapia , Macaca fascicularis , Mieloma Múltiplo/tratamento farmacológicoRESUMO
Advances in antibody engineering have enabled the construction of novel molecular formats in diverse shapes and sizes, providing new opportunities for biologic therapies and expanding the need to understand how various structural aspects affect their distribution properties. To assess the effect of antibody size on systemic pharmacokinetics (PK) and tissue distribution with or without neonatal Fc receptor (FcRn) binding, we evaluated a series of non-mouse-binding anti-glycoprotein D monoclonal antibody formats, including IgG [~150 kDa], one-armed IgG [~100 kDa], IgG-HAHQ (attenuated FcRn binding) [~150 kDa], F(ab')2 [~100 kDa], and F(ab) [~50 kDa]. Tissue-specific concentration-time profiles were corrected for blood content based on vascular volumes and normalized based on interstitial volumes to allow estimation of interstitial concentrations and interstitial:serum concentration ratios. Blood correction demonstrated that the contribution of circulating antibody on total uptake was greatest at early time points and for highly vascularized tissues. Tissue interstitial PK largely mirrored serum exposure profiles. Similar interstitial:serum ratios were obtained for the two FcRn-binding molecules, IgG and one-armed IgG, which reached pseudo-steady-state kinetics in most tissues. For non-FcRn-binding molecules, interstitial:serum ratios changed over time, suggesting that these molecules did not reach steady-state kinetics during the study. Furthermore, concentration-time profiles of both intact and catabolized molecule were measured by a dual tracer approach, enabling quantification of tissue catabolism and demonstrating that catabolism levels were highest for IgG-HAHQ. Overall, these data sets provide insight into factors affecting preclinical distribution and may be useful in estimating interstitial concentrations and/or catabolism in human tissues.
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Anticorpos Monoclonais , Imunoglobulina G , Antígenos de Histocompatibilidade Classe I , Humanos , Recém-Nascido , Cinética , Receptores Fc , Distribuição TecidualRESUMO
The T cell-dependent bispecific (TDB) antibody, anti-CD79b/CD3, targets CD79b and CD3 cell-surface receptors expressed on B cells and T cells, respectively. Since the anti-CD79b arm of this TDB binds only to human CD79b, a surrogate TDB that binds to cynomolgus monkey CD79b (cyCD79b) was used for preclinical characterization. To evaluate the impact of CD3 binding affinity on the TDB pharmacokinetics (PK), we utilized non-tumor-targeting bispecific anti-gD/CD3 antibodies composed of a low/high CD3 affinity arm along with a monospecific anti-gD arm as controls in monkeys and mice. An integrated PKPD model was developed to characterize PK and pharmacodynamics (PD). This study revealed the impact of CD3 binding affinity on anti-cyCD79b/CD3 PK. The surrogate anti-cyCD79b/CD3 TDB was highly effective in killing CD79b-expressing B cells and exhibited nonlinear PK in monkeys, consistent with target-mediated clearance. A dose-dependent decrease in B cell counts in peripheral blood was observed, as expected. Modeling indicated that anti-cyCD79b/CD3 TDB's rapid and target-mediated clearance may be attributed to faster internalization of CD79b, in addition to enhanced CD3 binding. The model yielded unbiased and precise curve fits. These findings highlight the complex interaction between TDBs and their targets and may be applicable to the development of other biotherapeutics.
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The antibody-drug conjugate (ADC) is a well-validated modality for the cell-specific delivery of small molecules with impact expanding rapidly beyond their originally-intended purpose of treating cancer. However, antibody-mediated delivery (AMD) remains inefficient, limiting its applicability to targeting highly potent payloads to cells with high antigen expression. Maximizing the number of payloads delivered per antibody is one key way in which delivery efficiency can be improved, although this has been challenging to carry out; with few exceptions, increasing the drug-to-antibody ratio (DAR) above â¼4 typically destroys the biophysical properties and in vivo efficacy for ADCs. Herein, we describe the development of a novel bioconjugation platform combining cysteine-engineered (THIOMAB) antibodies and recombinant XTEN polypeptides for the unprecedented generation of homogeneous, stable "TXCs" with DAR of up to 18. Across three different bioactive payloads, we demonstrated improved AMD to tumors and Staphylococcus aureus bacteria for high-DAR TXCs relative to conventional low-DAR ADCs.
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Invasive Staphylococcus aureus (S. aureus) infections are a leading cause of death and not effectively treated with prolonged standard of care antibiotics. A novel THIOMAB™ antibody antibiotic conjugate (TAC) was developed that uses a bacterial-wall specific antibody to deliver the antibiotic (dmDNA31, a rifamycin analogue) to bacteria to minimize toxicities typically seen with prolonged use of traditional antibiotics. The TAC nonclinical toxicology package included repeat dose rat and cynomolgus monkey toxicology studies for 8 weekly intravenous (IV) doses, a 7-day daily repeat dose IV toxicology study of dmDNA31 and an assessment of genotoxicity, cardiovascular toxicity, neurotoxicity and sperm parameters. TAC and dmDNA31 were well tolerated in rats and monkeys, and there was no evidence of genotoxicity, cardiovascular toxicity or neurotoxicity. Non-adverse findings were observed and included blue discoloration in skin, blood, etc. due to the blue color of dmDNA31, increased globulin due to the high doses of antibodies, and abnormal sperm morphology of small heads in male rats with no histopathology correlate in testis. This is an example of antibody-mediated delivery of an antibiotic that has the potential to offer a more effective way of eradicating infection while providing a better safety profile compared to traditional antibiotics.
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Imunotoxinas/toxicidade , Imunotoxinas/uso terapêutico , Infecções Estafilocócicas/tratamento farmacológico , Administração Intravenosa , Animais , Doenças Cardiovasculares/induzido quimicamente , Parede Celular/química , Sistemas de Liberação de Medicamentos , Feminino , Globulinas/metabolismo , Macaca fascicularis , Masculino , Testes de Mutagenicidade , Doenças do Sistema Nervoso/induzido quimicamente , Ratos , Ratos Sprague-Dawley , Espermatozoides/efeitos dos fármacos , Infecções Estafilocócicas/microbiologia , Testículo/patologiaRESUMO
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.
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Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/farmacocinética , Afinidade de Anticorpos , Neoplasias da Mama/tratamento farmacológico , Receptor ErbB-2/antagonistas & inibidores , Linfócitos T Citotóxicos/imunologia , Animais , Anticorpos Biespecíficos/imunologia , Apoptose , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos SCID , Receptor ErbB-2/imunologia , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Advances in antibody engineering have enabled the construction of novel molecular formats in diverse shapes and sizes, providing new opportunities for cancer immunotherapeutic drug discovery while also revealing limitations in knowledge of structure-activity relationships. The current understanding of renal filtration originates largely from data reported for dextrans, IgG, albumin, and selected globular proteins. For a one-armed IgG-based T-cell imaging agent, we observed higher renal signal than typically observed for bivalent IgGs, prompting us to explore the factors governing renal filtration of biologics. We constructed a small representative library of IgG-like formats with varied shapes and hinge flexibilities falling broadly into two categories: branched molecules including bivalent IgG and (scFv)2Fc, and nonbranched molecules including one-armed IgG, one-armed IgG with stacked Fab, and one-armed IgG with a rigid IgA2 hinge. Transmission electron microscopy revealed Y-shaped structures for the branched molecules and pseudo-linear structures for the nonbranched molecules. Single-photon emission CT imaging, autoradiography, and tissue harvest studies demonstrated higher renal uptake and catabolism for nonbranched molecules relative to branched molecules. Among the nonbranched molecules, the one-armed IgG with rigid IgA2 hinge molecule demonstrated higher kidney uptake and decreased systemic exposure relative to molecules with a more flexible hinge. Our results show that differences in shape and hinge flexibility drive the increased glomerular filtration of one-armed relative to bivalent antibodies and highlight the practical advantages of using imaging to assess renal filtration properties. These findings are particularly relevant for T-cell-dependent bispecific molecules, many of which have nonstandard antibody structures.
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Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Barreira de Filtração Glomerular/metabolismo , Imunoglobulina G/imunologia , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único/métodos , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Feminino , Barreira de Filtração Glomerular/efeitos dos fármacos , Humanos , Imunoglobulina G/classificação , Camundongos SCIDRESUMO
Protein therapeutics have witnessed tremendous use and application in recent years in treatment of various diseases. Predicting efficacy and safety during drug discovery and translational development is a key factor for successful clinical development of these therapies. In general, drug related toxicities are predominantly driven by pharmacokinetic (PK) exposure at off-target sites. This work explores the ocular PK of intravenously administered protein therapeutics to understand impact of antibody format on off-site exposure. Species matched non-binding rabbit antibody proteins (rabFab and rabIgG) were intravenously administered to male New Zealand White rabbits at a single 1 mg bolus dose and exposure was measured up to 3 weeks. As anticipated based on absence of FcRn recycling, rabFab has relatively fast systemic PK (CL-943 mL/day and t1/2-1.93 days) compared to rabIgG (CL-18.5 mL/day and t1/2-8.93 days). Similarly, rabFab has lower absolute ocular exposure in ocular compartments (e.g., vitreous and aqueous humor) compared to rabIgG, despite higher relative exposures (measured as percent tissue partition in ocular tissues relative to serum, based on Cmax and AUC). In general, percent tissue partition based on AUC (in aqueous and vitreous humor) relative to serum exposure were 10.4 and 8.62 for rabFab respectively and 1.11 and 0.64 for rabIgG respectively. This work emphasizes size and format based ocular exposure of intravenously administered protein therapeutics. Findings from this work enable prediction of format based ocular exposure for systemically administered antibody based therapeutics and aid in selection of molecule format for clinical candidate to minimize ocular exposure.
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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.
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Antibióticos Antineoplásicos/uso terapêutico , Calicheamicinas/uso terapêutico , Imunoconjugados/uso terapêutico , Animais , Antibióticos Antineoplásicos/farmacologia , Calicheamicinas/farmacologia , Modelos Animais de Doenças , Humanos , Imunoconjugados/farmacologia , CamundongosRESUMO
Anti-Ly6E-seco-cyclopropabenzindol-4-one dimer antibody-drug conjugate (ADC) has been reported to form an adduct with α1-microglobulin (A1M) in animal plasma, but with unknown impact on ADC PK and tissue distribution. In this study, we compared the PK and tissue distribution of anti-Ly6E ADC with unconjugated anti-Ly6E mAb in rodents and monkeys. For PK studies, animals received an intravenous administration of anti-Ly6E ADC or unconjugated anti-Ly6E mAb. Plasma samples were analyzed for total antibody (Tab) levels and A1M adduct formation. PK parameters were generated from dose-normalized plasma concentrations. Tissue distribution was determined in tumor-bearing mice after a single intravenous dosing of radiolabeled ADC or mAb. Tissue radioactivity levels were analyzed using a gamma counter. The impact of A1M adduct formation on target cell binding was assessed in an in vitro cell binding assay. The results show that ADC Tab clearance was slower than that of mAb in mice and rats but faster than mAb in monkeys. Correspondingly, the formation of A1M adduct appeared to be faster and higher in mice, followed by rats, and slowest in monkeys. Although ADC tended to show an overall lower distribution to normal tissues, it had a strikingly reduced distribution to tumors compared with mAb, likely due to A1M adduct formation interfering with target binding, as demonstrated by the in vitro cell binding assay. Together, these data 1) demonstrate that anti-Ly6E ADC that forms A1M adduct had slower systemic clearance with strikingly reduced tumor distribution and 2) highlight the importance of selecting an appropriate linker-drug for successful ADC development. SIGNIFICANCE STATEMENT: Anti-lymphocyte antigen 6 complex, locus E, ADC with seco-cyclopropabenzindol-4-one-dimer payload formed adduct with A1M, which led to a decrease in systemic clearance but also attenuated tumor distribution. These findings demonstrate the importance of selecting an appropriate linker-drug for ADC development and also highlight the value of a mechanistic understanding of ADC biotransformation, which could provide insight into ADC molecule design, optimization, and selection.
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alfa-Globulinas/metabolismo , Antineoplásicos Imunológicos/farmacocinética , Imunoconjugados/farmacocinética , Neoplasias/tratamento farmacológico , Animais , Antígenos de Superfície , Antineoplásicos Imunológicos/administração & dosagem , Linhagem Celular Tumoral , Feminino , Proteínas Ligadas por GPI/antagonistas & inibidores , Humanos , Imunoconjugados/administração & dosagem , Macaca fascicularis , Taxa de Depuração Metabólica , Camundongos , Neoplasias/patologia , Ratos , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A localized positive charge on IgG (referred to as a "charge patch") shows an adverse effect on pharmacokinetics (PK), so it would seem to be best practice to avoid charge patches during the discovery stage and closely monitor charge interactions during the development process. In certain circumstances, however, charge patches are required for target binding, in which case completely removing charge patches is not feasible. Therefore, quantitative measurement of a charge patch and its impact on PK is critical to the success of therapeutic antibody development. In this article, we generated mutations of a recombinant human antibody (referred to as mAb1) with disrupted charge patches to investigate how charge patches on IgG antibodies impact both target-binding affinity and PK-related factors. We conclude that it is important to modulate the size of the charge patch in order to balance target-binding affinity and PK.
Assuntos
Regiões Determinantes de Complementaridade , Imunoglobulina G , Regiões Determinantes de Complementaridade/genética , HumanosRESUMO
Invasive Staphylococcus aureus infection is a leading cause of infectious disease-related deaths because S. aureus survives within host phagocytic cells, from which the bacteria are not adequately eliminated using current antibiotic treatments. Anti-S. aureus THIOMAB antibody-antibiotic conjugate (TAC), an anti-S. aureus antibody conjugated with antibiotic payload dmDNA31, was designed to deliver antibiotics into phagocytes, thereby killing intracellular S. aureus Herein, we present the distribution, metabolism/catabolism, and elimination properties for this modality. The tissue distribution of TAC and the release and elimination of its payload dmDNA31 were characterized in rats using multiple approaches. Intravenous injection of unconjugated [14C]dmDNA31 to rats resulted in a rapid clearance in both systemic circulation and tissues, with biliary secretion as the major route of elimination. Six major metabolites were identified. When [14C]dmDNA31 was conjugated to an antibody as TAC and administered to rat intravenously, a sustained exposure was observed in both systemic circulation and tissues. The dmDNA31 in blood and tissues mainly remained in conjugated form after administering TAC, although minimal deconjugation of dmDNA31 from TAC was also observed. Several TAC catabolites were identified, which were mainly eliminated through the biliary-fecal route, with dmDNA31 and deacetylated dmDNA31 as the most abundant catabolites. In summary, these studies provide a comprehensive characterization of the distribution, metabolism/catabolism, and elimination properties of TAC. These data fully support further clinical development of TAC for the invasive and difficult-to-treat S. aureus infection. SIGNIFICANCE STATEMENT: The present studies provide a comprehensive investigation of the absorption, distribution, metabolism/catabolism, and elimination of the first antibody-antibiotic conjugate developed for the treatment of an infectious disease. Although many antibody-drug conjugates are in development for various disease indications, only a limited amount of absorption, distribution, metabolism/catabolism, and elimination information is available in the literature. This study demonstrates the use of radiolabeling technology to delineate the absorption, distribution, metabolism/catabolism, and elimination properties of a complex modality and help address the key questions related to clinical pharmacological studies.