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1.
Breast Cancer Res Treat ; 191(2): 303-317, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34708303

RESUMO

PURPOSE: Assessment of non-clinical safety signals relies on understanding species selectivity of antibodies. This is particularly important with antibody-drug conjugates, where it is key to determine target-dependent versus target-independent toxicity. Although it appears to be widely accepted that trastuzumab does not bind mouse or rat HER2/ErbB2/neu, numerous investigators continue to use mouse models to investigate safety signals of trastuzumab and trastuzumab emtansine (T-DM1). We, therefore, conducted a broad array of both binding and biologic studies to demonstrate selectivity of trastuzumab for human HER2 versus mouse/rat neu. METHODS: Binding of anti-neu and anti-HER2 antibodies was assessed by ELISA, FACS, IHC, Scatchard, and immunoblot methods in human, rat, and mouse cell lines. In human hepatocytes, T-DM1 uptake and catabolism were measured by LC-MS/MS; cell viability changes were determined using CellTiter-Glo. RESULTS: Our data demonstrate, using different binding methods, lack of trastuzumab binding to rat or mouse neu. Structural studies show important amino acid differences in the trastuzumab-HER2 binding interface between mouse/rat and human HER2 ECD. Substitution of these rodent amino acid residues into human HER2 abolish binding of trastuzumab. Cell viability changes, uptake, and catabolism of T-DM1 versus a DM1 non-targeted control ADC were comparable, indicating target-independent effects of the DM1-containing ADCs. Moreover, trastuzumab binding to human or mouse hepatocytes was not detected. CONCLUSIONS: These data, in total, demonstrate that trastuzumab, and by extension T-DM1, do not bind rat or mouse neu, underscoring the importance of species selection for safety studies investigating trastuzumab or trastuzumab-based therapeutics.


Assuntos
Neoplasias da Mama , Maitansina , Animais , Anticorpos Monoclonais Humanizados , Cromatografia Líquida , Feminino , Humanos , Maitansina/efeitos adversos , Camundongos , Ratos , Receptor ErbB-2/genética , Espectrometria de Massas em Tandem , Trastuzumab/efeitos adversos
2.
Bioorg Med Chem Lett ; 30(4): 126907, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31902710

RESUMO

Chimeric molecules which effect intracellular degradation of target proteins via E3 ligase-mediated ubiquitination (e.g., PROTACs) are currently of high interest in medicinal chemistry. However, these entities are relatively large compounds that often possess molecular characteristics which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. Accordingly, we explored whether conjugation of chimeric degraders to monoclonal antibodies using technologies originally developed for cytotoxic payloads might provide alternate delivery options for these novel agents. In this report we describe the construction of several degrader-antibody conjugates comprised of two distinct ERα-targeting degrader entities and three independent ADC linker modalities. We subsequently demonstrate the antigen-dependent delivery to MCF7-neo/HER2 cells of the degrader payloads that are incorporated into these conjugates. We also provide evidence for efficient intracellular degrader release from one of the employed linkers. In addition, preliminary data are described which suggest that reasonably favorable in vivo stability properties are associated with the linkers utilized to construct the degrader conjugates.


Assuntos
Anticorpos Monoclonais/imunologia , Portadores de Fármacos/química , Receptor alfa de Estrogênio/imunologia , Anticorpos Monoclonais/química , Antineoplásicos/química , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Desenho de Fármacos , Receptor alfa de Estrogênio/metabolismo , Humanos , Imunoconjugados/química , Imunoconjugados/imunologia , Imunoconjugados/farmacologia , Células MCF-7 , Proteólise/efeitos dos fármacos , Receptor ErbB-2/metabolismo
3.
Bioconjug Chem ; 29(2): 267-274, 2018 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-29369629

RESUMO

The valine-citrulline (Val-Cit) dipeptide and p-aminobenzyl (PAB) spacer have been commonly used as a cleavable self-immolating linker in ADC design including in the clinically approved ADC, brentuximab vedotin (Adcetris). When the same linker was used to connect to the phenol of the cyclopropabenzindolone (CBI) (P1), the resulting ADC1 showed loss of potency in CD22 target-expressing cancer cell lines (e.g., BJAB, WSU-DLCL2). In comparison, the conjugate (ADC2) of a cyclopropapyrroloindolone (CPI) (P2) was potent despite the two corresponding free drugs having similar picomolar cell-killing activity. Although the corresponding spirocyclization products of P1 and P2, responsible for DNA alkylation, are a prominent component in buffer, the linker immolation was slow when the PAB was connected as an ether (PABE) to the phenol in P1 compared to that in P2. Additional immolation studies with two other PABE-linked substituted phenol compounds showed that electron-withdrawing groups accelerated the immolation to release an acidic phenol-containing payload (to delocalize the negative charge on the anticipated anionic phenol oxygen during immolation). In contrast, efficient immolation of LD4 did not result in an active ADC4 because the payload (P4) had a low potency to kill cells. In addition, nonimmolation of LD5 did not affect the cell-killing potency of its ADC5 since immolation is not required for DNA alkylation by the center-linked pyrrolobenzodiazepine. Therefore, careful evaluation needs to be conducted when the Val-Cit-PAB linker is used to connect antibodies to a phenol-containing drug as the linker immolation, as well as payload potency and stability, affects the cell-killing activity of an ADC.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Imunoconjugados/química , Imunoconjugados/farmacologia , Fenol/química , Fenol/farmacologia , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/farmacologia , Brentuximab Vedotin , Linhagem Celular Tumoral , Ciclopropanos/química , Ciclopropanos/farmacologia , Humanos , Neoplasias/tratamento farmacológico
4.
Chemistry ; 24(19): 4830-4834, 2018 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-29493023

RESUMO

A novel strategy to attach indole-containing payloads to antibodies through a carbamate moiety and a self-immolating, disulfide-based linker is described. This new strategy was employed to connect a selective estrogen receptor down-regulator (SERD) to various antibodies in a site-selective manner. The resulting conjugates displayed potent, antigen-dependent down-regulation of estrogen receptor levels in MCF7-neo/HER2 and MCF7-hB7H4 cells. They also exhibited similar antigen-dependent modulation of the estrogen receptor in tumors when administered intravenously to mice bearing MCF7-neo/HER2 tumor xenografts. The indole-carbamate moiety present in the new linker was stable in whole blood from various species and also exhibited good in vivo stability properties in mice.


Assuntos
Indóis/química , Animais , Anticorpos Monoclonais/química , Linhagem Celular Tumoral , Humanos , Imunoconjugados/administração & dosagem , Células MCF-7 , Camundongos
5.
Mol Pharm ; 15(9): 3979-3996, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30040421

RESUMO

A number of cytotoxic pyrrolobenzodiazepine (PBD) monomers containing various disulfide-based prodrugs were evaluated for their ability to undergo activation (disulfide cleavage) in vitro in the presence of either glutathione (GSH) or cysteine (Cys). A good correlation was observed between in vitro GSH stability and in vitro cytotoxicity toward tumor cell lines. The prodrug-containing compounds were typically more potent against cells with relatively high intracellular GSH levels (e.g., KPL-4 cells). Several antibody-drug conjugates (ADCs) were subsequently constructed from PBD dimers that incorporated selected disulfide-based prodrugs. Such HER2 conjugates exhibited potent antiproliferation activity against KPL-4 cells in vitro in an antigen-dependent manner. However, the disulfide prodrugs contained in the majority of such entities were surprisingly unstable toward whole blood from various species. One HER2-targeting conjugate that contained a thiophenol-derived disulfide prodrug was an exception to this stability trend. It exhibited potent activity in a KPL-4 in vivo efficacy model that was approximately three-fold weaker than that displayed by the corresponding parent ADC. The same prodrug-containing conjugate demonstrated a three-fold improvement in mouse tolerability properties in vivo relative to the parent ADC, which did not contain the prodrug.


Assuntos
Benzodiazepinas/química , Dissulfetos/química , Imunoconjugados/química , Pró-Fármacos/química , Pirróis/química , Linhagem Celular Tumoral , Cisteína/metabolismo , Glutationa/metabolismo , Humanos , Imunoconjugados/metabolismo , Estrutura Molecular
6.
Breast Cancer Res Treat ; 164(3): 581-591, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28493046

RESUMO

PURPOSE: The extent to which efficacy of the HER2 antibody Trastuzumab in brain metastases is limited by access of antibody to brain lesions remains a question of significant clinical importance. We investigated the uptake and distribution of trastuzumab in brain and mammary fat pad grafts of HER2-positive breast cancer to evaluate the relationship of these parameters to the anti-tumor activity of trastuzumab and trastuzumab emtansine (T-DM1). METHODS: Mouse transgenic breast tumor cells expressing human HER2 (Fo2-1282 or Fo5) were used to establish intracranial and orthotopic tumors. Tumor uptake and tissue distribution of systemically administered 89Zr-trastuzumab or muMAb 4D5 (murine parent of trastuzumab) were measured by PET and ELISA. Efficacy of muMAb 4D5, the PI3K/mTOR inhibitor GNE-317, and T-DM1 was also assessed. RESULTS: 89Zr-trastuzumab and muMAb 4D5 exhibited robust uptake into Fo2-1282 brain tumors, but not normal brains. Uptake into brain grafts was similar to mammary grafts. Despite this, muMAb 4D5 was less efficacious in brain grafts. Co-administration of muMAb 4D5 and GNE-317, a brain-penetrant PI3K/mTOR inhibitor, provided longer survival in mice with brain lesions than either agent alone. Moreover, T-DM1 increased survival in the Fo5 brain metastasis model. CONCLUSIONS: In models of HER2-positive breast cancer brain metastasis, trastuzumab efficacy does not appear to be limited by access to intracranial tumors. Anti-tumor activity improved with the addition of a brain-penetrant PI3K/mTOR inhibitor, suggesting that combining targeted therapies is a more effective strategy for treating HER2-positive breast cancer brain metastases. Survival was also extended in mice with Fo5 brain lesions treated with T-DM1.


Assuntos
Antineoplásicos Imunológicos/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/secundário , Neoplasias da Mama/tratamento farmacológico , Pirimidinas/administração & dosagem , Receptor ErbB-2/genética , Tiofenos/administração & dosagem , Trastuzumab/administração & dosagem , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/farmacocinética , Antineoplásicos Imunológicos/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Pirimidinas/farmacocinética , Receptor ErbB-2/metabolismo , Análise de Sobrevida , Tiofenos/farmacocinética , Distribuição Tecidual , Trastuzumab/farmacocinética , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Bioconjug Chem ; 28(8): 2086-2098, 2017 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-28636382

RESUMO

Conjugation of small molecule payloads to cysteine residues on proteins via a disulfide bond represents an attractive strategy to generate redox-sensitive bioconjugates, which have value as potential diagnostic reagents or therapeutics. Advancement of such "direct-disulfide" bioconjugates to the clinic necessitates chemical methods to form disulfide connections efficiently, without byproducts. The disulfide connection must also be resistant to premature cleavage by thiols prior to arrival at the targeted tissue. We show here that commonly employed methods to generate direct disulfide-linked bioconjugates are inadequate for addressing these challenges. We describe our efforts to optimize direct-disulfide conjugation chemistry, focusing on the generation of conjugates between cytotoxic payloads and cysteine-engineered antibodies (i.e., THIOMAB antibody-drug conjugates, or TDCs). This work culminates in the development of novel, high-yielding conjugation chemistry for creating direct payload disulfide connections to any of several Cys mutation sites in THIOMAB antibodies or to Cys sites in other biomolecules (e.g., human serum albumin and cell-penetrating peptides). We conclude by demonstrating that hindered direct disulfide TDCs with two methyl groups adjacent to the disulfide, which have heretofore not been described for any bioconjugate, are more stable and more efficacious in mouse tumor xenograft studies than less hindered analogs.


Assuntos
Cisteína , Dissulfetos/química , Imunoconjugados/química , Peptídeos/química , Engenharia de Proteínas , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Humanos , Imunoconjugados/genética , Camundongos
8.
Bioorg Med Chem ; 24(22): 6075-6081, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27745990

RESUMO

A Pd-catalysed amination method is used to convert seco-CBI, a synthetic analogue of the alkylating subunit of the duocarmycin natural products, from the phenol to amino form. This allows efficient enantioselective access to the more potent S enantiomer of aminoCBI and its incorporation into analogues of DNA minor groove cross-linking agents. Evaluation in a panel of nine human tumour cell lines shows that the bifunctional agents containing aminoCBI are generally less cytotoxic than their phenolCBI analogues and more susceptible to P-glycoprotein-mediated resistance. However, all bifunctional agents are potent cytotoxins, some in the sub-pM IC50 range, with in vitro properties that compare favourably with established microtubule-targeted ADC payloads.


Assuntos
Anticorpos/farmacologia , Antineoplásicos/farmacologia , Reagentes de Ligações Cruzadas/farmacologia , Indóis/farmacologia , Anticorpos/química , Antineoplásicos/síntese química , Antineoplásicos/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Reagentes de Ligações Cruzadas/síntese química , Reagentes de Ligações Cruzadas/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Duocarmicinas , Humanos , Indóis/química , Estrutura Molecular , Pirrolidinonas/química , Pirrolidinonas/farmacologia , Relação Estrutura-Atividade
9.
Cancer Discov ; 12(1): 204-219, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34544753

RESUMO

PIK3CA is one of the most frequently mutated oncogenes; the p110a protein it encodes plays a central role in tumor cell proliferation. Small-molecule inhibitors targeting the PI3K p110a catalytic subunit have entered clinical trials, with early-phase GDC-0077 studies showing antitumor activity and a manageable safety profile in patients with PIK3CA-mutant breast cancer. However, preclinical studies have shown that PI3K pathway inhibition releases negative feedback and activates receptor tyrosine kinase signaling, reengaging the pathway and attenuating drug activity. Here we discover that GDC-0077 and taselisib more potently inhibit mutant PI3K pathway signaling and cell viability through unique HER2-dependent mutant p110a degradation. Both are more effective than other PI3K inhibitors at maintaining prolonged pathway suppression. This study establishes a new strategy for identifying inhibitors that specifically target mutant tumors by selective degradation of the mutant oncoprotein and provide a strong rationale for pursuing PI3Kα degraders in patients with HER2-positive breast cancer. SIGNIFICANCE: The PI3K inhibitors GDC-0077 and taselisib have a unique mechanism of action; both inhibitors lead to degradation of mutant p110a protein. The inhibitors that have the ability to trigger specific degradation of mutant p110a without significant change in wild-type p110a protein may result in improved therapeutic index in PIK3CA-mutant tumors.See related commentary by Vanhaesebroeck et al., p. 20.This article is highlighted in the In This Issue feature, p. 1.


Assuntos
Antineoplásicos , Neoplasias da Mama , Classe I de Fosfatidilinositol 3-Quinases , Imidazóis , Oxazepinas , Inibidores de Fosfoinositídeo-3 Quinase , Receptor ErbB-2 , Feminino , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases/genética , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Oxazepinas/farmacologia , Oxazepinas/uso terapêutico , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase/uso terapêutico , Receptor ErbB-2/genética
10.
J Med Chem ; 63(17): 9603-9622, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32787101

RESUMO

Cytotoxic pyrrolobenzodiazepine (PBD)-dimer molecules are frequently utilized as payloads for antibody-drug conjugates (ADCs), and many examples are currently in clinical development. In order to further explore this ADC payload class, the physicochemical properties of various PBD-dimer molecules were modified by the systematic introduction of acidic and basic moieties into their chemical structures. The impact of these changes on DNA binding, cell membrane permeability, and in vitro antiproliferation potency was, respectively, determined using a DNA alkylation assay, PAMPA assessments, and cell-based cytotoxicity measurements conducted with a variety of cancer lines. The modified PBD-dimer compounds were subsequently incorporated into CD22-targeting ADCs, and these entities were profiled in a variety of in vitro and in vivo experiments. The introduction of a strongly basic moiety into the PBD-dimer scaffold afforded a conjugate with dramatically worsened mouse tolerability properties relative to ADCs derived from related payloads, which lacked the basic group.


Assuntos
Benzodiazepinas/química , Dimerização , Imunoconjugados/efeitos adversos , Imunoconjugados/química , Pirróis/química , Segurança , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fenômenos Químicos , DNA/química , DNA/metabolismo , Humanos , Imunoconjugados/metabolismo , Imunoconjugados/farmacologia , Modelos Moleculares , Conformação de Ácido Nucleico
11.
ACS Med Chem Lett ; 8(10): 1037-1041, 2017 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-29057047

RESUMO

The tubulysins are promising anticancer cytotoxic agents due to the clinical validation of their mechanism of action (microtubule inhibition) and their particular activity against multidrug-resistant tumor cells. Yet their high potency and subsequent systemic toxicity make them prime candidates for targeted therapy, particularly in the form of antibody-drug conjugates (ADCs). Here we report a strategy to prepare stable and bioreversible conjugates of tubulysins to antibodies without loss of activity. A peptide trigger along with a quaternary ammonium salt linker connection to the tertiary amine of tubulysin provided ADCs that were potent in vitro. However, we observed metabolism of a critical acetate ester of the drug in vivo, resulting in diminished conjugate activity. We were able to circumvent this metabolic liability with the judicious choice of a propyl ether replacement. This modified tubulysin ADC was stable and effective against multidrug-resistant lymphoma cell lines and tumors.

12.
J Med Chem ; 60(23): 9490-9507, 2017 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-29112410

RESUMO

Three rationally designed pyrrolobenzodiazepine (PBD) drug-linkers have been synthesized via intermediate 19 for use in antibody-drug conjugates (ADCs). They lack a cleavable trigger in the linker and consist of a maleimide for cysteine antibody conjugation, a hydrophilic spacer, and either an alkyne (6), triazole (7), or piperazine (8) link to the PBD. In vitro IC50 values were 11-48 ng/mL in HER2 3+ SK-BR-3 and KPL-4 (7 inactive) for the anti-HER2 ADCs (HER2 0 MCF7, all inactive) and 0.10-1.73 µg/mL (7 inactive) in CD22 3+ BJAB and WSU-DLCL2 for anti-CD22 ADCs (CD22 0 Jurkat, all inactive at low doses). In vivo antitumor efficacy for the anti-HER2 ADCs in Founder 5 was observed with tumor stasis at 0.5-1 mg/kg, 1 mg/kg, and 3-6 mg/kg for 6, 8, and 7, respectively. Tumor stasis at 2 mg/kg was observed for anti-CD22 6 in WSU-DLCL2. In summary, noncleavable PBD-ADCs exhibit potent activity, particularly in HER2 models.


Assuntos
Antineoplásicos/química , Antineoplásicos/uso terapêutico , Benzodiazepinas/química , Benzodiazepinas/uso terapêutico , Imunoconjugados/química , Imunoconjugados/uso terapêutico , Neoplasias/tratamento farmacológico , Pirróis/química , Pirróis/uso terapêutico , Animais , Antineoplásicos/farmacologia , Benzodiazepinas/farmacologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dimerização , Feminino , Humanos , Imunoconjugados/farmacologia , Camundongos , Modelos Moleculares , Pirróis/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/antagonistas & inibidores
13.
Clin Cancer Res ; 22(15): 3755-63, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-26920887

RESUMO

PURPOSE: HER2-positive breast cancer is heterogeneous. Some tumors express mutations, like activating PIK3CA mutations or reduced PTEN expression, that negatively correlate with response to HER2-targeted therapies. In this exploratory analysis, we investigated whether the efficacy of trastuzumab emtansine (T-DM1), an antibody-drug conjugate comprised of the cytotoxic agent DM1 linked to the HER2-targeted antibody trastuzumab, was correlated with the expression of specific biomarkers in the phase III EMILIA study. EXPERIMENTAL DESIGN: Tumors were evaluated for HER2 (n = 866), EGFR (n = 832), and HER3 (n = 860) mRNA expression by quantitative reverse transcriptase PCR; for PTEN protein expression (n = 271) by IHC; and for PIK3CA mutations (n = 259) using a mutation detection kit. Survival outcomes were analyzed by biomarker subgroups. T-DM1 was also tested on cell lines and in breast cancer xenograft models containing PIK3CA mutations. RESULTS: Longer progression-free survival (PFS) and overall survival (OS) were observed with T-DM1 compared with capecitabine plus lapatinib in all biomarker subgroups. PIK3CA mutations were associated with shorter median PFS (mutant vs. wild type: 4.3 vs. 6.4 months) and OS (17.3 vs. 27.8 months) in capecitabine plus lapatinib-treated patients, but not in T-DM1-treated patients (PFS, 10.9 vs. 9.8 months; OS, not reached in mutant or wild type). T-DM1 showed potent activity in cell lines and xenograft models with PIK3CA mutations. CONCLUSIONS: Although other standard HER2-directed therapies are less effective in tumors with PI3KCA mutations, T-DM1 appears to be effective in both PI3KCA-mutated and wild-type tumors. Clin Cancer Res; 22(15); 3755-63. ©2016 AACR.


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Tumorais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Maitansina/análogos & derivados , Receptor ErbB-2/metabolismo , Trastuzumab/uso terapêutico , Ado-Trastuzumab Emtansina , Animais , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Imunológicos/efeitos adversos , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Modelos Animais de Doenças , Feminino , Seguimentos , Humanos , Estimativa de Kaplan-Meier , Maitansina/administração & dosagem , Maitansina/efeitos adversos , Maitansina/uso terapêutico , Camundongos , Metástase Neoplásica , Estadiamento de Neoplasias , Fosfatidilinositol 3-Quinases , Modelos de Riscos Proporcionais , Retratamento , Trastuzumab/administração & dosagem , Trastuzumab/efeitos adversos , Ensaios Antitumorais Modelo de Xenoenxerto
14.
ACS Med Chem Lett ; 7(11): 988-993, 2016 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-27882196

RESUMO

Disulfide bonds could be valuable linkers for a variety of therapeutic applications requiring tunable cleavage between two parts of a molecule (e.g., antibody-drug conjugates). The in vitro linker immolation of ß-mercaptoethyl-carbamate disulfides and DNA alkylation properties of associated payloads were investigated to understand the determinant of cell killing potency of anti-CD22 linked pyrrolobenzodiazepine (PBD-dimer) conjugates. Efficient immolation and release of a PBD-dimer with strong DNA alkylation properties were observed following disulfide cleavage of methyl- and cyclobutyl-substituted disulfide linkers. However, the analogous cyclopropyl-containing linker did not immolate, and the associated thiol-containing product was a poor DNA alkylator. As predicted from these in vitro assessments, the related anti-CD22 ADCs showed different target-dependent cell killing activities in WSU-DLCL2 and BJAB cell lines. These results demonstrate how the in vitro immolation models can be used to help design efficacious ADCs.

15.
Clin Cancer Res ; 21(1): 123-33, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25370470

RESUMO

PURPOSE: Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate (ADC) comprising the cytotoxic agent DM1 conjugated to trastuzumab with a stable linker. Thrombocytopenia was the dose-limiting toxicity in the phase I study, and grade ≥3 thrombocytopenia occurred in up to 13% of patients receiving T-DM1 in phase III studies. We investigated the mechanism of T-DM1-induced thrombocytopenia. EXPERIMENTAL DESIGN: The effect of T-DM1 on platelet function was measured by aggregometry, and by flow cytometry to detect the markers of activation. The effect of T-DM1 on differentiation and maturation of megakaryocytes (MK) from human hematopoietic stem cells was assessed by flow cytometry and microscopy. Binding, uptake, and catabolism of T-DM1 in MKs, were assessed by various techniques including fluorescence microscopy, scintigraphy to detect T-[H(3)]-DM1 and (125)I-T-DM1, and mass spectrometry. The role of FcγRIIa was assessed using blocking antibodies and mutant constructs of trastuzumab that do not bind FcγR. RESULTS: T-DM1 had no direct effect on platelet activation and aggregation, but it did markedly inhibit MK differentiation via a cytotoxic effect. Inhibition occurred with DM1-containing ADCs but not with trastuzumab demonstrating a role for DM1. MKs internalized these ADCs in a HER2-independent, FcγRIIa-dependent manner, resulting in intracellular release of DM1. Binding and internalization of T-DM1 diminished as MKs matured; however, prolonged exposure of mature MKs to T-DM1 resulted in a disrupted cytoskeletal structure. CONCLUSIONS: These data support the hypothesis that T-DM1-induced thrombocytopenia is mediated in large part by DM1-induced impairment of MK differentiation, with a less pronounced effect on mature MKs.


Assuntos
Anticorpos Monoclonais Humanizados/efeitos adversos , Antineoplásicos/efeitos adversos , Neoplasias da Mama/tratamento farmacológico , Maitansina/análogos & derivados , Trombocitopenia/patologia , Ado-Trastuzumab Emtansina , Anticorpos Monoclonais Humanizados/administração & dosagem , Antineoplásicos/administração & dosagem , Neoplasias da Mama/complicações , Neoplasias da Mama/patologia , Diferenciação Celular/efeitos dos fármacos , Feminino , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/efeitos adversos , Maitansina/administração & dosagem , Maitansina/efeitos adversos , Megacariócitos/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Plasma Rico em Plaquetas/efeitos dos fármacos , Receptor ErbB-2/metabolismo , Trombocitopenia/induzido quimicamente , Trombocitopenia/etiologia , Trastuzumab
16.
Cancer Chemother Pharmacol ; 74(5): 969-80, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25186956

RESUMO

PURPOSE: Trastuzumab emtansine (T-DM1), an antibody-drug conjugate (ADC) comprised of trastuzumab linked to the antimitotic agent DM1, has shown promising results in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer. Investigations of the mechanisms of the action of ADCs, including T-DM1, have been primarily descriptive or semiquantitative. However, quantitative pharmacokinetic/pharmacodynamic (PK/PD) analysis may provide insights into their complex behavior. The analyses described herein applied PK/PD modeling to nonclinical studies of maytansinoid conjugates. METHODS: The maytansinoid conjugates T-DM1 and T-SPP-DM1, with thioether and disulfide linkers, respectively, were tested in mouse efficacy, PK, and tumor uptake studies. (3)[H]DM1-bearing ADCs were used to facilitate the quantitation of the ADCs in plasma, as well as ADC and ADC catabolites in tumors. Three mechanistic PK/PD models were used to characterize plasma ADC, tumor ADC, and tumor catabolite concentrations. Tumor catabolite concentrations were used to fit tumor response. Model parameters were estimated using R software and nonlinear least squares regression. RESULTS: Plasma ADC-associated DM1 concentrations of T-DM1 decreased more slowly than those of T-SPP-DM1, likely due to slower DM1 release. A comparison of the mechanistic models found that the best model allowed catabolism and catabolite exit rates to differ between ADCs, that T-DM1 exhibited both faster tumor catabolism and catabolite exit rate from tumors than T-SPP-DM1; findings inconsistent with expected behavior based on the physicochemical nature of the respective catabolites. Tumor catabolite concentrations adequately described tumor response with both ADCs showing similar potency. CONCLUSION: Mechanistic PK/PD studies described herein provided results that confirmed and challenged current hypotheses, and suggested new areas of investigation.


Assuntos
Anticorpos Monoclonais Humanizados/farmacocinética , Maitansina/análogos & derivados , Modelos Biológicos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Ado-Trastuzumab Emtansina , Algoritmos , Animais , Anticorpos Monoclonais Humanizados/sangue , Área Sob a Curva , Linhagem Celular Tumoral , Feminino , Humanos , Maitansina/sangue , Maitansina/farmacocinética , Camundongos Nus , Trastuzumab , Resultado do Tratamento , Trítio
17.
Cancer Res ; 74(19): 5561-71, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25228655

RESUMO

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.


Assuntos
Anticorpos Biespecíficos/imunologia , Ativação Linfocitária , Receptor ErbB-2/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Ratos , Ratos Sprague-Dawley , Trastuzumab
18.
J Med Chem ; 57(19): 7890-9, 2014 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-25191794

RESUMO

Antibody-drug conjugates (ADCs) have a significant impact toward the treatment of cancer, as evidenced by the clinical activity of the recently approved ADCs, brentuximab vedotin for Hodgkin lymphoma and ado-trastuzumab emtansine (trastuzumab-MCC-DM1) for metastatic HER2+ breast cancer. DM1 is an analog of the natural product maytansine, a microtubule inhibitor that by itself has limited clinical activity and high systemic toxicity. However, by conjugation of DM1 to trastuzumab, the safety was improved and clinical activity was demonstrated. Here, we report that through chemical modification of the linker-drug and antibody engineering, the therapeutic activity of trastuzumab maytansinoid ADCs can be further improved. These improvements include eliminating DM1 release in the plasma and increasing the drug load by engineering four cysteine residues into the antibody. The chemical synthesis of highly stable linker-drugs and the modification of cysteine residues of engineered site-specific antibodies resulted in a homogeneous ADC with increased therapeutic activity compared to the clinically approved ADC, trastuzumab-MCC-DM1.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/síntese química , Imunoconjugados/farmacologia , Maitansina/análogos & derivados , Engenharia de Proteínas , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Trastuzumab
19.
Mol Cancer Ther ; 11(5): 1133-42, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22408268

RESUMO

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate consisting of the anti-HER2 antibody trastuzumab linked via a nonreducible thioether linker to the maytansinoid antitubulin agent DM1. T-DM1 has shown favorable safety and efficacy in patients with HER2-positive metastatic breast cancer. In previous animal studies, T-DM1 exhibited better pharmacokinetics (PK) and slightly more efficacy than several disulfide-linked versions. The efficacy findings are unique, as other disulfide-linked antibody-drug conjugates (ADC) have shown greater efficacy than thioether-linked designs. To explore this further, the in vitro and in vivo activity, PK, and target cell activation of T-DM1 and the disulfide-linked T-SPP-DM1 were examined. Both ADCs showed high in vitro potency, with T-DM1 displaying greater potency in two of four breast cancer cell lines. In vitro target cell processing of T-DM1 and T-SPP-DM1 produced lysine-N(ε)-MCC-DM1, and lysine-N(ε)-SPP-DM1 and DM1, respectively; in vivo studies confirmed these results. The in vitro processing rates for the two conjugate to their respective catabolites were similar. In vivo, the potencies of the conjugates were similar, and T-SPP-DM1 had a faster plasma clearance than T-DM1. Slower T-DM1 clearance translated to higher overall tumor concentrations (conjugate plus catabolites), but unexpectedly, similar levels of tumor catabolite. These results indicate that, although the ADC linker can have clear impact on the PK and the chemical nature of the catabolites formed, both linkers seem to offer the same payload delivery to the tumor.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Maitansina/análogos & derivados , Ado-Trastuzumab Emtansina , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/metabolismo , Anticorpos Monoclonais Humanizados/farmacocinética , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Maitansina/química , Maitansina/metabolismo , Maitansina/farmacocinética , Maitansina/farmacologia , Camundongos , Camundongos Nus , Receptor ErbB-2/metabolismo , Trastuzumab , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Clin Breast Cancer ; 11(5): 275-82, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21729661

RESUMO

Increased understanding of the molecular mechanisms of tumorigenesis has led to the development of novel agents that target tumor cells with minimal effects on normal cells. The success of this approach is exemplified by the development of monoclonal antibodies directed toward antigens expressed selectively by tumor cells. The conjugation of these monoclonal antibodies with potent cytotoxic drugs has the potential to further improve efficacy while retaining a favorable safety profile. Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) currently in clinical development. It combines the humanized antibody trastuzumab, which targets the human epidermal growth factor receptor 2 (HER2) receptor on cancer cells, and the potent antimicrotubule agent DM1 using a unique highly stable linker. When T-DM1 binds to HER2, a proportion of the receptors are thought to be internalized by the process of receptor endocytosis, followed by the intracellular release of an active form of DM1, which in turn kills the tumor cell. This review presents the rationale for the development of T-DM1 and summarizes the preclinical and clinical data for this novel agent for the treatment of breast cancer.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Receptor ErbB-2/metabolismo , Anticorpos Monoclonais Humanizados/administração & dosagem , Antineoplásicos/administração & dosagem , Neoplasias da Mama/metabolismo , Feminino , Humanos , Trastuzumab , Resultado do Tratamento
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