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1.
Bioconjug Chem ; 32(4): 746-754, 2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33689309

RESUMO

Although peptide motifs represent the majority of cleavable linkers used in clinical-stage antibody-drug conjugates (ADCs), the sequences are often sensitive to cleavage by extracellular enzymes, such as elastase, which leads to systemic release of the cytotoxic payload. This action reduces the therapeutic index by causing off-target toxicities that can be dose-limiting. For example, a common side-effect of ADCs made using peptide-cleavable linkers is myelosuppression, including neutropenia. Only a few reports describe methods for optimizing peptide linkers to maintain efficient and potent tumor payload delivery while enhancing circulating stability. Herein, we address these critical limitations through the development of a tandem-cleavage linker strategy, where two sequential enzymatic cleavage events mediate payload release. We prepared dipeptides that are protected from degradation in the circulation by a sterically encumbering glucuronide moiety. Upon ADC internalization and lysosomal degradation, the monosaccharide is removed and the exposed dipeptide is degraded, which liberates the attached payload inside the target cell. We used CD79b-targeted monomethyl auristatin E (MMAE) conjugates as our model system and compared the stability, efficacy, and tolerability of ADCs made with tandem-cleavage linkers to ADCs made using standard technology with the vedotin linker. The results, where rat studies showed dramatically improved tolerability in the hematopoietic compartment, highlight the role that linker stability plays in efficacy and tolerability and also offer a means of improving an ADC's therapeutic index for improved patient outcomes.


Assuntos
Antineoplásicos/toxicidade , Antígenos CD79/toxicidade , Imunoconjugados/toxicidade , Animais , Antineoplásicos/química , Antígenos CD79/química , Endocitose , Feminino , Hidrólise , Imunoconjugados/química , Imunoconjugados/farmacocinética , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Ratos , Ratos Sprague-Dawley , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Protein Expr Purif ; 149: 75-83, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29655788

RESUMO

Expression variation among antibodies produced by stably transfected Chinese Hamster Ovary (CHO) cells is well established. While developing CHO-K1 cell lines, we encountered a human monoclonal antibody, mAb B-c, with severe manufacturability issues, including very poor expression and high levels of heavy chain (HC) dimer and high molecular weight aggregates. Using transient expression in CHO-K1 cells, we identified light chain (LC) as the source of the manufacturability issues for this antibody. While other antibodies achieved optimal expression at 1:1 or 2:1 LC to HC ratios, mAb B-c required up to a 6:1 LC:HC for maximal expression, which was still significantly lower than that for other tested antibodies. To overcome the manufacturability issues, LC shuffling was performed with the original HC to select antibodies with unique LCs which retained acceptable binding affinities. Transient CHO-K1 expression evaluation of the new LCs co-expressed with the original HC identified antibodies with high expression at a 1:1 or 2:1 LC:HC; the expression levels of these new antibodies were comparable to those of other well-expressed antibodies. Expression of these new antibodies in stably transfected CHO-K1 cells confirmed these results. In addition, antibodies containing the new LCs had very low levels of high molecular weight aggregates and HC dimer. These results demonstrate that certain antibody manufacturability issues can be attributed to LC and that engineering antibodies by pairing HCs with alternate LCs can improve antibody expression and product quality while maintaining or improving affinity.


Assuntos
Anticorpos Monoclonais Humanizados/biossíntese , Engenharia de Proteínas/métodos , Animais , Anticorpos Monoclonais Humanizados/genética , Células CHO , Cricetulus , Expressão Gênica , Vetores Genéticos , Células HEK293 , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/biossíntese , Cadeias Leves de Imunoglobulina/genética , Conformação Proteica , Multimerização Proteica , Transfecção
3.
Mol Cancer Ther ; 19(9): 1866-1874, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32651200

RESUMO

Trastuzumab and the related ADC, ado-trastuzumab emtansine (T-DM1), both target HER2-overexpressing cells. Together, these drugs have treatment indications in both early-stage and metastatic settings for HER2+ breast cancer. T-DM1 retains the antibody functionalities of trastuzumab and adds the potency of a cytotoxic maytansine payload. Interestingly, in the clinic, T-DM1 cannot always replace the use of trastuzumab plus chemotherapy administered together as single agents. We hypothesize that this failure may be due, in part, to the limited systemic exposure achieved by T-DM1 relative to trastuzumab because of toxicity-related dosing constraints on the ADC. We have developed a trastuzumab-based ADC site specifically conjugated to maytansine through a noncleavable linker. This construct, termed CAT-01-106, has a drug-to-antibody ratio (DAR) of 1.8, approximately half the average DAR of T-DM1, which comprises a mixture of antibodies variously conjugated with DARs ranging from 0 to 8. The high DAR species present in T-DM1 contribute to its toxicity and limit its clinical dose. CAT-01-106 showed superior in vivo efficacy compared with T-DM1 at equal payload dosing and was equally or better tolerated compared with T-DM1 at equal payload dosing up to 120 mg/kg in Sprague-Dawley rats and 60 mg/kg in cynomolgus monkeys. CAT-01-106 also showed improved pharmacokinetics in rats relative to T-DM1, with 40% higher ADC exposure levels. Together, the data suggest that CAT-01-106 may be sufficiently tolerable to enable clinical dosing at trastuzumab-equivalent exposure levels, combining the functions of both the antibody and the payload in one drug and potentially improving patient outcomes.


Assuntos
Ado-Trastuzumab Emtansina/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Imunoconjugados/administração & dosagem , Maitansina/química , Trastuzumab/química , Ado-Trastuzumab Emtansina/efeitos adversos , Ado-Trastuzumab Emtansina/farmacocinética , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Imunoconjugados/efeitos adversos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Macaca fascicularis , Dose Máxima Tolerável , Ratos , Ratos Sprague-Dawley , Receptor ErbB-2/metabolismo , Trastuzumab/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
4.
J Immunol Methods ; 391(1-2): 60-71, 2013 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-23454004

RESUMO

Phage display antibody libraries have a proven track record for the discovery of therapeutic human antibodies, increasing the demand for large and diverse phage antibody libraries for the discovery of new therapeutics. We have constructed naïve antibody phage display libraries in both Fab and scFv formats, with each library having more than 250 billion clones that encompass the human antibody repertoire. These libraries show high fidelity in open reading frame and expression percentages, and their V-gene family distribution, VH-CDR3 length and amino acid usage mirror the natural diversity of human antibodies. Both the Fab and scFv libraries show robust sequence diversity in target-specific binders and differential V-gene usage for each target tested, supporting the use of libraries that utilize multiple display formats and V-gene utilization to maximize antibody-binding diversity. For each of the targets, clones with picomolar affinities were identified from at least one of the libraries and for the two targets assessed for activity, functional antibodies were identified from both libraries.


Assuntos
Técnicas de Visualização da Superfície Celular , Fragmentos Fab das Imunoglobulinas/imunologia , Biblioteca de Peptídeos , Receptor de Insulina/imunologia , Receptor TIE-2/imunologia , Anticorpos de Cadeia Única/imunologia , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Especificidade de Anticorpos , Células CHO , Cricetinae , Cricetulus , Humanos , Fragmentos Fab das Imunoglobulinas/biossíntese , Fragmentos Fab das Imunoglobulinas/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fases de Leitura Aberta , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/genética , Receptor TIE-2/genética , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genética , Transfecção
5.
Cell ; 111(3): 357-67, 2002 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-12419246

RESUMO

The Ski family of nuclear oncoproteins represses TGF-beta signaling through interactions with the Smad proteins. The crystal structure of the Smad4 binding domain of human c-Ski in complex with the MH2 domain of Smad4 reveals specific recognition of the Smad4 L3 loop region by a highly conserved interaction loop (I loop) from Ski. The Ski binding surface on Smad4 significantly overlaps with that required for binding of the R-Smads. Indeed, Ski disrupts the formation of a functional complex between the Co- and R-Smads, explaining how it could lead to repression of TGF-beta, activin, and BMP responses. Intriguingly, the structure of the Ski fragment, stabilized by a bound zinc atom, resembles the SAND domain, in which the corresponding I loop is responsible for DNA binding.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas Nucleares/química , Proteínas Proto-Oncogênicas/química , Transdução de Sinais , Transativadores/química , Fator de Crescimento Transformador beta/metabolismo , Sequência de Aminoácidos , Linhagem Celular Transformada , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Estrutura Terciária de Proteína , Proteína Smad4 , Células Tumorais Cultivadas
6.
J Biol Chem ; 277(45): 43335-43, 2002 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-12218060

RESUMO

The endothelial cell protein C receptor (EPCR) is a type 1 transmembrane protein found primarily on endothelium that binds both protein C and activated protein C with similar affinity. EPCR augments the activation of protein C by the thrombin-thrombomodulin complex. To determine the physiological importance of EPCR, we generated EPCR-deficient mice by homologous targeting in embryonic stem cells. Genotyping of progeny obtained from EPCR(+/-) interbreeding indicated that EPCR(-/-) embryos died on or before embryonic day 10.5 (E10.5). Reverse transcriptase-PCR confirmed the absence of EPCR mRNA in EPCR(-/-) embryos. EPCR(-/-) embryos removed from extra-embryonic membranes and tissues at day E7.5 and cultured in vitro developed beyond E10.5, suggesting a role for EPCR in the normal function of the placenta and/or at the materno-embryonic interface. Immunohistochemistry revealed the lack of EPCR in trophoblast giant cells of EPCR(-/-) embryos. These cells, which normally express EPCR, are in direct contact with the maternal circulation and its clotting factors. In EPCR(-/-) embryos, greatly increased fibrin deposition was detected around these cells. To prevent this fibrin deposition, EPCR(+/-)-crossed female mice received a daily subcutaneous injection of enoxaparin through pregnancy. Although some EPCR(-/-) embryos were rescued from midgestational lethality, this regimen yielded no EPCR(-/-) pups. We conclude that EPCR is essential for normal embryonic development. Moreover, EPCR plays a key role in preventing thrombosis at the maternal-embryonic interface.


Assuntos
Fatores de Coagulação Sanguínea/genética , Morte Fetal , Doenças Placentárias/genética , Receptores de Superfície Celular/genética , Animais , Sequência de Bases , Fatores de Coagulação Sanguínea/metabolismo , Primers do DNA , Embrião de Mamíferos/fisiologia , Feminino , Genótipo , Idade Gestacional , Camundongos , Camundongos Knockout , Técnicas de Cultura de Órgãos , Doenças Placentárias/patologia , Gravidez , RNA Mensageiro/genética , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/metabolismo , Trombose
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