RESUMO
Conventional antibody-drug conjugates (ADCs) are heterogeneous mixtures of chemically distinct molecules that vary in both drugs/antibody (DAR) and conjugation sites. Suboptimal properties of heterogeneous ADCs have led to new site-specific conjugation methods for improving ADC homogeneity. Most site-specific methods require extensive antibody engineering to identify optimal conjugation sites and introduce unique functional groups for conjugation with appropriately modified linkers. Alternative nonrecombinant methods have emerged in which bifunctional linkers are utilized to cross-link antibody interchain cysteines and afford ADCs containing four drugs/antibody. Although these methods have been shown to improve ADC homogeneity and stability in vitro, their effect on the pharmacological properties of ADCs in vivo is unknown. In order to determine the relative impact of interchain cysteine cross-linking on the therapeutic window and other properties of ADCs in vivo, we synthesized a derivative of the known ADC payload, MC-MMAF, that contains a bifunctional dibromomaleimide (DBM) linker instead of a conventional maleimide (MC) linker. The DBM-MMAF derivative was conjugated to trastuzumab and a novel anti-CD98 antibody to afford ADCs containing predominantly four drugs/antibody. The pharmacological properties of the resulting cross-linked ADCs were compared with analogous heterogeneous ADCs derived from conventional linkers. The results demonstrate that DBM linkers can be applied directly to native antibodies, without antibody engineering, to yield highly homogeneous ADCs via cysteine cross-linking. The resulting ADCs demonstrate improved pharmacokinetics, superior efficacy, and reduced toxicity in vivo compared to analogous conventional heterogeneous ADCs.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Cisteína/química , Imunoconjugados/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Trastuzumab/farmacologia , Animais , Anticorpos Monoclonais/química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Reagentes de Ligações Cruzadas , Feminino , Citometria de Fluxo , Imunofluorescência , Proteína-1 Reguladora de Fusão/imunologia , Humanos , Imunoconjugados/química , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Ratos , Ratos Sprague-Dawley , Receptor ErbB-2/antagonistas & inibidores , Trastuzumab/química , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The fields of nanotechnology and medicine have merged in the development of new imaging and drug delivery agents based on nanoparticle platforms. As one example, a mutant of bacteriophage MS2 can be differentially modified on the exterior and interior surfaces for the concurrent display of targeting functionalities and payloads, respectively. In order to realize their potential for use in in vivo applications, the biodistribution and circulation properties of this class of agents must first be investigated. A means of modulating and potentially improving the characteristics of nanoparticle agents is the appendage of PEG chains. Both MS2 and MS2-PEG capsids possessing interior DOTA chelators were labeled with (64)Cu and injected intravenously into mice possessing tumor xenografts. Dynamic imaging of the agents was performed using PET-CT on a single animal per sample, and the biodistribution at the terminal time point (24 h) was assessed by gamma counting of the organs ex vivo for 3 animals per agent. Compared to other viral capsids of similar size, the MS2 agents showed longer circulation times. Both MS2 and MS2-PEG bacteriophage behaved similarly, although the latter agent showed significantly less uptake in the spleen. This effect may be attributed to the ability of the PEG chains to mask the capsid charge. Although the tumor uptake of the agents may result from the enhanced permeation and retention (EPR) effect, selective tumor imaging may be achieved in the future by using exterior targeting groups.
Assuntos
Levivirus/química , Levivirus/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Animais , Capsídeo/metabolismo , Linhagem Celular Tumoral , Radioisótopos de Cobre/administração & dosagem , Radioisótopos de Cobre/química , Feminino , Células MCF-7 , Camundongos , Camundongos Nus , Polietilenoglicóis/química , Distribuição TecidualRESUMO
A highly efficient protein bioconjugation method is described involving addition of anilines to o-aminophenols in the presence of sodium periodate. The reaction takes place in aqueous buffer at pH 6.5 and can reach high conversion in 2-5 min. The major product was characterized using X-ray crystallography, which revealed that an unprecedented oxidative ring contraction occurs after the coupling step. The compatibility of the reaction with protein substrates has been demonstrated through attachment of small molecules, polymer chains, and peptides to p-aminophenylalanine residues introduced into viral capsids through amber stop codon suppression. Coupling of anilines to o-aminophenol groups derived from tyrosine residues is also described. The compatibility of this method with thiol modification chemistry is shown through attachment of a near-IR fluorescent chromophore to cysteine residues inside the viral capsid shells, followed by attachment of integrin-targeting RGD peptides to anilines on the exterior surface.
Assuntos
Aminofenóis/química , Compostos de Anilina/química , Capsídeo/química , Aminofenóis/metabolismo , Compostos de Anilina/metabolismo , Capsídeo/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Estrutura Molecular , OxirreduçãoRESUMO
Identification of tumor-specific cell surface antigens has proven challenging, as the vast majority of tumor-associated antigens are also expressed in normal tissues. In mesothelioma, we identified a highly specific tumor cell surface antigen that can be targeted for therapy development. Mesothelioma is caused by malignant transformation of the mesothelium, is incurable, and can be categorized into three histologic subtypes: epithelioid, biphasic, and sarcomatoid. To identity novel mesothelioma cell surface antigens with broad subtype coverage and high tissue specificity, we have previously selected phage antibody display libraries on live mesothelioma cells and tissues following counterselection on normal cells and identified a panel of human antibodies that bind all subtypes of mesothelioma, but not normal mesothelium. One of the antibodies, M25, showed high specificity against an antigen we identify here as ALPPL2. IHC on normal human tissues found that ALPPL2 is expressed only on placental trophoblasts, but not on any other normal tissues. This significant tissue specificity and broad tumor type coverage suggest that ALPPL2 could be an excellent cell surface target for therapeutic development against mesothelioma. To evaluate therapeutic potential of ALPPL2 targeting, an ALPPL2-targeted antibody-drug conjugate was developed and demonstrated potent and specific tumor killing in vitro and in vivo against both epithelioid and sarcomatoid mesothelioma. Thus, ALPPL2 belongs to a rare class of cell surface antigens classified as truly tumor specific and is well suited for therapy development against ALPPL2-expressing tumors. SIGNIFICANCE: These findings identify ALPP2 as a true tumor-specific cell surface antigen whose tissue specificity enables the development of novel therapies.
Assuntos
Fosfatase Alcalina/metabolismo , Antígenos de Superfície/metabolismo , Mesotelioma Maligno/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/imunologia , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Antineoplásicos Imunológicos/farmacologia , Células CHO , Linhagem Celular Tumoral , Cricetulus , Epitopos , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Imunoconjugados/farmacologia , Imunoglobulina G/imunologia , Masculino , Mesotelioma Maligno/tratamento farmacológico , Mesotelioma Maligno/patologia , Camundongos Endogâmicos NOD , Terapia de Alvo Molecular , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: New therapies have changed the outlook for patients with multiple myeloma, but novel agents are needed for patients who are refractory or relapsed on currently approved drug classes. Novel targets other than CD38 and BCMA are needed for new immunotherapy development, as resistance to daratumumab and emerging anti-BCMA approaches appears inevitable. One potential target of interest in myeloma is ICAM1. Naked anti-ICAM1 antibodies were active in preclinical models of myeloma and safe in patients, but showed limited clinical efficacy. Here, we sought to achieve improved targeting of multiple myeloma with an anti-ICAM1 antibody-drug conjugate (ADC). EXPERIMENTAL DESIGN: Our anti-ICAM1 human mAb was conjugated to an auristatin derivative, and tested against multiple myeloma cell lines in vitro, orthotopic xenografts in vivo, and patient samples ex vivo. The expression of ICAM1 was also measured by quantitative flow cytometry in patients spanning from diagnosis to the daratumumab-refractory state. RESULTS: The anti-ICAM1 ADC displayed potent anti-myeloma cytotoxicity in vitro and in vivo. In addition, we have verified that ICAM1 is highly expressed on myeloma cells and shown that its expression is further accentuated by the presence of bone marrow microenvironmental factors. In primary samples, ICAM1 is differentially overexpressed on multiple myeloma cells compared with normal cells, including daratumumab-refractory patients with decreased CD38. In addition, ICAM1-ADC showed selective cytotoxicity in multiple myeloma primary samples. CONCLUSIONS: We propose that anti-ICAM1 ADC should be further studied for toxicity, and if safe, tested for clinical efficacy in patients with relapsed or refractory multiple myeloma.
Assuntos
Anticorpos Monoclonais/farmacologia , Imunoconjugados/farmacologia , Molécula 1 de Adesão Intercelular/genética , Mieloma Múltiplo/tratamento farmacológico , ADP-Ribosil Ciclase 1/antagonistas & inibidores , ADP-Ribosil Ciclase 1/imunologia , Adulto , Idoso , Animais , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Anti-Idiotípicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Citometria de Fluxo , Xenoenxertos , Humanos , Imunoconjugados/imunologia , Molécula 1 de Adesão Intercelular/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologiaRESUMO
Although initially responsive to androgen signaling inhibitors (ASIs), metastatic castration-resistant prostate cancer (mCRPC) inevitably develops and is incurable. In addition to adenocarcinoma (adeno), neuroendocrine prostate cancer (NEPC) emerges to confer ASI resistance. We have previously combined laser capture microdissection and phage antibody display library selection on human cancer specimens and identified novel internalizing antibodies binding to tumor cells residing in their tissue microenvironment. We identified the target antigen for one of these antibodies as CD46, a multifunctional protein that is best known for negatively regulating the innate immune system. CD46 is overexpressed in primary tumor tissue and CRPC (localized and metastatic; adeno and NEPC), but expressed at low levels on normal tissues except for placental trophoblasts and prostate epithelium. Abiraterone- and enzalutamide-treated mCRPC cells upregulate cell surface CD46 expression. Genomic analysis showed that the CD46 gene is gained in 45% abiraterone-resistant mCRPC patients. We conjugated a tubulin inhibitor to our macropinocytosing anti-CD46 antibody and showed that the resulting antibody-drug conjugate (ADC) potently and selectively kills both adeno and NEPC cell lines in vitro (sub-nM EC50) but not normal cells. CD46 ADC regressed and eliminated an mCRPC cell line xenograft in vivo in both subcutaneous and intrafemoral models. Exploratory toxicology studies of the CD46 ADC in non-human primates demonstrated an acceptable safety profile. Thus, CD46 is an excellent target for antibody-based therapy development, which has potential to be applicable to both adenocarcinoma and neuroendocrine types of mCRPC that are resistant to current treatment.
Assuntos
Adenocarcinoma/metabolismo , Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias/metabolismo , Proteína Cofatora de Membrana/metabolismo , Tumores Neuroendócrinos/metabolismo , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/imunologia , Androstenos/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/imunologia , Anticorpos Antineoplásicos/farmacologia , Afinidade de Anticorpos , Antígenos de Neoplasias/imunologia , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Benzamidas , Linhagem Celular Tumoral , Feminino , Humanos , Macaca fascicularis , Masculino , Proteína Cofatora de Membrana/genética , Proteína Cofatora de Membrana/imunologia , Tumores Neuroendócrinos/tratamento farmacológico , Tumores Neuroendócrinos/imunologia , Nitrilas , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Proteínas Recombinantes de Fusão , Transdução de Sinais/efeitos dos fármacos , Terapêutica , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Multiple myeloma is incurable by standard approaches because of inevitable relapse and development of treatment resistance in all patients. In our prior work, we identified a panel of macropinocytosing human monoclonal antibodies against CD46, a negative regulator of the innate immune system, and constructed antibody-drug conjugates (ADCs). In this report, we show that an anti-CD46 ADC (CD46-ADC) potently inhibited proliferation in myeloma cell lines with little effect on normal cells. CD46-ADC also potently eliminated myeloma growth in orthometastatic xenograft models. In primary myeloma cells derived from bone marrow aspirates, CD46-ADC induced apoptosis and cell death, but did not affect the viability of nontumor mononuclear cells. It is of clinical interest that the CD46 gene resides on chromosome 1q, which undergoes genomic amplification in the majority of relapsed myeloma patients. We found that the cell surface expression level of CD46 was markedly higher in patient myeloma cells with 1q gain than in those with normal 1q copy number. Thus, genomic amplification of CD46 may serve as a surrogate for target amplification that could allow patient stratification for tailored CD46-targeted therapy. Overall, these findings indicate that CD46 is a promising target for antibody-based treatment of multiple myeloma, especially in patients with gain of chromosome 1q.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Antineoplásicos/farmacologia , Imunoconjugados/farmacologia , Proteína Cofatora de Membrana/antagonistas & inibidores , Mieloma Múltiplo/tratamento farmacológico , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/imunologia , Linhagem Celular Tumoral , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 1/imunologia , Dosagem de Genes/imunologia , Humanos , Imunoconjugados/imunologia , Proteína Cofatora de Membrana/genética , Proteína Cofatora de Membrana/imunologia , Camundongos , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
With optimal target antigen selection antibody-based therapeutics can be very effective agents for hematologic malignancies, but none have yet been approved for myeloma. Rituximab and brentuximab vedotin are examples of success for the naked antibody and antibody-drug conjugate classes, respectively. Plasma cell myeloma is an attractive disease for antibody-based targeting due to target cell accessibility and the complementary mechanism of action with approved therapies. Initial antibodies tested in myeloma were disappointing. However, recent results from targeting well-characterized antigens have been more encouraging. In particular, the CD38 and CD138 targeted therapies are showing single-agent activity in early phase clinical trials. Here we will review the development pipeline for naked antibodies and antibody-drug conjugates for myeloma. There is clear clinical need for new treatments, as myeloma inevitably becomes refractory to standard agents. The full impact is yet to be established, but we are optimistic that the first FDA-approved antibody therapeutic(s) for this disease will emerge in the near future.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Imunoconjugados/uso terapêutico , Imunoterapia/métodos , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/terapia , Animais , Anticorpos Monoclonais/imunologia , Humanos , Imunoconjugados/imunologiaRESUMO
Chemical cross-linking of proteins combined with mass spectral analysis is a powerful technique that can be utilized to yield protein structural information, such as the spatial arrangement of multi-protein complexes or the folding of monomeric proteins. The succinimidyl ester cross-linking reagents are commonly used to cross-link primary amine-containing amino acids (N-terminus and lysine). However, in this study they were used to react with tyrosines as well, which allowed for the formation of cross-links between two primary amines, one primary amine and one tyrosine, or two tyrosines. This result is extremely important to the chemical cross-linking community for two reasons: (1) all possible cross-linked residues must be considered when analyzing data from these experiments to generate correct distance constraints and structural information, and (2) utilizing the versatility of these cross-linking reagents allows more information content to be generated from a single cross-linking reagent, which may increase the number of cross-links obtained in the experiment. Herein, we study the reactivity of the succinimidyl ester labeling and cross-linking reagents with angiotensin I and oxidized insulin beta-chain. Using the succinimidyl acetate labeling reagent, the reactivity of the N-terminus was found to be greater than either lysine or tyrosine. However, a selectivity of the cross-linking reagent was observed for either tyrosine or lysine depending on the pH of the reaction solution. In acidic pH, it was observed that tyrosine was more reactive, while in alkaline pH lysine was more reactive. Exploiting this selectivity predominantly N-terminus-tyrosine or tyrosine-tyrosine cross-links were favored at acidic pH, while N-terminus-tyrosine or tyrosine-lysine cross-links were favored at alkaline pH.
Assuntos
Reagentes de Ligações Cruzadas/química , Lisina/química , Tirosina/química , Angiotensina I/química , Insulina/química , Oxirredução , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Antibody drug conjugates (ADCs) are an emerging class of targeted therapeutics with the potential to improve therapeutic index over traditional chemotherapy. Drugs and linkers have been the current focus of ADC development, in addition to antibody and target selection. Recently, however,the importance of conjugate homogeneity has been realized. The current methods for drug attachment lead to a heterogeneous mixture, and some populations of that mixture have poor in vivo performance. New methods for site-specific drug attachment lead to more homogeneous conjugates and allow control of the site of drug attachment. These subtle improvements can have profound effects on in vivo efficacy and therapeutic index. This review examines current methods for site-specific drug conjugation to antibodies, and compares in vivo results with their non-specifically conjugated counterparts. The apparent improvement in pharmacokinetics and the reduced off target toxicity warrant further development of this site-specific modification approach for future ADC development.
Assuntos
Imunoconjugados/química , Imunoconjugados/uso terapêutico , Animais , HumanosRESUMO
Mycobacterium tuberculosis possesses an unusual cell wall that is replete with virulence-enhancing lipids. One cell wall molecule unique to pathogenic M. tuberculosis is polyacyltrehalose (PAT), a pentaacylated, trehalose-based glycolipid. Little is known about the biosynthesis of PAT, although its biosynthetic gene cluster has been identified and found to resemble that of the better studied M. tuberculosis cell wall component sulfolipid-1. In this study, we sought to elucidate the function of papA3, a gene from the PAT locus encoding a putative acyltransferase. To determine whether PapA3 participates in PAT assembly, we expressed the protein heterologously and evaluated its acyltransferase activity in vitro. The purified enzyme catalyzed the sequential esterification of trehalose with two palmitoyl groups, generating a diacylated product similar to the 2,3-diacyltrehalose glycolipids of M. tuberculosis. Notably, PapA3 was selective for trehalose; no activity was observed with other structurally related disaccharides. Disruption of the papA3 gene from M. tuberculosis resulted in the loss of PAT from bacterial lipid extracts. Complementation of the mutant strain restored PAT production, demonstrating that PapA3 is essential for the biosynthesis of this glycolipid in vivo. Furthermore, we determined that the PAT biosynthetic machinery has no cross-talk with that for sulfolipid-1 despite their related structures.