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1.
Nature ; 622(7984): 850-862, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37794185

RESUMO

Immune checkpoint blockade is effective for some patients with cancer, but most are refractory to current immunotherapies and new approaches are needed to overcome resistance1,2. The protein tyrosine phosphatases PTPN2 and PTPN1 are central regulators of inflammation, and their genetic deletion in either tumour cells or immune cells promotes anti-tumour immunity3-6. However, phosphatases are challenging drug targets; in particular, the active site has been considered undruggable. Here we present the discovery and characterization of ABBV-CLS-484 (AC484), a first-in-class, orally bioavailable, potent PTPN2 and PTPN1 active-site inhibitor. AC484 treatment in vitro amplifies the response to interferon and promotes the activation and function of several immune cell subsets. In mouse models of cancer resistant to PD-1 blockade, AC484 monotherapy generates potent anti-tumour immunity. We show that AC484 inflames the tumour microenvironment and promotes natural killer cell and CD8+ T cell function by enhancing JAK-STAT signalling and reducing T cell dysfunction. Inhibitors of PTPN2 and PTPN1 offer a promising new strategy for cancer immunotherapy and are currently being evaluated in patients with advanced solid tumours (ClinicalTrials.gov identifier NCT04777994 ). More broadly, our study shows that small-molecule inhibitors of key intracellular immune regulators can achieve efficacy comparable to or exceeding that of antibody-based immune checkpoint blockade in preclinical models. Finally, to our knowledge, AC484 represents the first active-site phosphatase inhibitor to enter clinical evaluation for cancer immunotherapy and may pave the way for additional therapeutics that target this important class of enzymes.


Assuntos
Imunoterapia , Neoplasias , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Proteína Tirosina Fosfatase não Receptora Tipo 2 , Animais , Humanos , Camundongos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Inibidores de Checkpoint Imunológico , Imunoterapia/métodos , Interferons/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 2/antagonistas & inibidores , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia
2.
Blood ; 127(10): 1346-54, 2016 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-26668132

RESUMO

Evidence shows that factor VIII (FVIII) ectopically expressed in platelets (2bF8) is therapeutic in FVIII(null) mice even with anti-FVIII inhibitory antibodies (inhibitors). If current efforts to generate platelets in vitro succeed, genetically manipulated platelets containing FVIII may be used therapeutically in hemophilia A patients with inhibitors. One important concern is the immunogenicity of platelet-derived FVIII. To address this concern, we infused 2bF8 transgenic (2bF8(Tg)) platelets into naïve FVIII(null) mice weekly for 8 weeks. No anti-FVIII antibodies were detected in the infused animals during the study course. We then explored whether platelet-derived FVIII is immunogenic in FVIII(null) mice with inhibitors. The 2bF8(Tg) platelets were transfused into rhF8-primed FVIII(null) mice, resulting in no augmentation of anti-FVIII antibodies. To investigate whether preconditioning affects the immune response, animals were sublethally irradiated and subsequently transfused with 2bF8(Tg) platelets. No anti-FVIII antibodies were detected in the recipients after platelet infusions. Following further challenge with rhF8, the inhibitor titer in this group was significantly lower than in naïve FVIII(null) mice utilizing the same immunization protocol. Thus, our data demonstrate that infusion of platelets containing FVIII triggers neither primary nor memory anti-FVIII immune response in FVIII(null) mice and that sublethal irradiation plus 2bF8(Tg) platelet infusion suppresses anti-FVIII immune response in FVIII(null) mice.


Assuntos
Autoanticorpos/imunologia , Inibidores dos Fatores de Coagulação Sanguínea/imunologia , Plaquetas/imunologia , Fator VIII/imunologia , Hemofilia A/imunologia , Transfusão de Plaquetas , Animais , Fator VIII/genética , Hemofilia A/genética , Camundongos , Camundongos Mutantes
3.
J Immunol ; 189(5): 2309-17, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22844120

RESUMO

Diverse Ag-specific memory TCR repertoires are essential for protection against pathogens. Subunit vaccines that combine peptide or protein Ags with TLR agonists are very potent at inducing T cell immune responses, but their capacity to elicit stable and diverse memory CD4 T cell repertoires has not been evaluated. In this study, we examined the evolution of a complex Ag-specific population during the transition from primary effectors to memory T cells after peptide or protein vaccination. Both vaccination regimens induced equally diverse effector CD4 TCR repertoires, but peptide vaccines skewed the memory CD4 TCR repertoire toward high-affinity clonotypes whereas protein vaccines maintained low-affinity clonotypes in the memory compartment. CD27-mediated signaling was essential for the maintenance of low-affinity clonotypes after protein vaccination but was not sufficient to promote their survival following peptide vaccination. The rapid culling of the TCR repertoire in peptide-immunized mice coincided with a prolonged proliferation phase during which low-affinity clonotypes disappeared despite exhibiting no sign of enhanced apoptosis. Our study reveals a novel affinity threshold for memory CD4 T cell differentiation following vaccination and suggests a role for nonapoptotic cell death in the regulation of CD4 T cell clonal selection.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/imunologia , Grupo dos Citocromos c/imunologia , Memória Imunológica , Receptores de Antígenos de Linfócitos T/metabolismo , Vacinas de Subunidades Antigênicas/imunologia , Animais , Apoptose/imunologia , Linfócitos T CD4-Positivos/citologia , Proliferação de Células , Células Clonais , Grupo dos Citocromos c/administração & dosagem , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mariposas , Ligação Proteica/imunologia , Receptores de Antígenos de Linfócitos T/fisiologia , Vacinas de Subunidades Antigênicas/administração & dosagem
4.
SLAS Discov ; 29(2): 100135, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38101572

RESUMO

The cellular thermal shift assay (CETSA®) is a target engagement method widely used for preclinical characterization of small molecule compounds. CETSA® has been used for semi-quantitative readouts in whole blood with PBMC isolation, and quantitative, plate-based readouts using cell lines. However, there has been no quantitative evaluation of CETSA® in unprocessed human whole blood, which is preferred for clinical applications. Here we report two separate assay formats - Alpha CETSA® and MSD CETSA® - that require less than 100 µL of whole blood per sample without PBMC isolation. We chose RIPK1 as a proof-of-concept target and, by measuring engagement of seven different inhibitors, demonstrate high assay sensitivity and robustness. These quantitative CETSA® platforms enable possible applications in preclinical pharmacokinetic-pharmacodynamic studies, and direct target engagement with small molecules in clinical trials.


Assuntos
Bioensaio , Leucócitos Mononucleares , Humanos , Linhagem Celular Tumoral , Células HT29 , Bioensaio/métodos , Projetos de Pesquisa , Proteína Serina-Treonina Quinases de Interação com Receptores
5.
Commun Chem ; 7(1): 183, 2024 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-39152201

RESUMO

PTPN2 (protein tyrosine phosphatase non-receptor type 2, or TC-PTP) and PTPN1 are attractive immuno-oncology targets, with the deletion of Ptpn1 and Ptpn2 improving response to immunotherapy in disease models. Targeted protein degradation has emerged as a promising approach to drug challenging targets including phosphatases. We developed potent PTPN2/N1 dual heterobifunctional degraders (Cmpd-1 and Cmpd-2) which facilitate efficient complex assembly with E3 ubiquitin ligase CRL4CRBN, and mediate potent PTPN2/N1 degradation in cells and mice. To provide mechanistic insights into the cooperative complex formation introduced by degraders, we employed a combination of structural approaches. Our crystal structure reveals how PTPN2 is recognized by the tri-substituted thiophene moiety of the degrader. We further determined a high-resolution structure of DDB1-CRBN/Cmpd-1/PTPN2 using single-particle cryo-electron microscopy (cryo-EM). This structure reveals that the degrader induces proximity between CRBN and PTPN2, albeit the large conformational heterogeneity of this ternary complex. The molecular dynamic (MD)-simulations constructed based on the cryo-EM structure exhibited a large rigid body movement of PTPN2 and illustrated the dynamic interactions between PTPN2 and CRBN. Together, our study demonstrates the development of PTPN2/N1 heterobifunctional degraders with potential applications in cancer immunotherapy. Furthermore, the developed structural workflow could help to understand the dynamic nature of degrader-induced cooperative ternary complexes.

6.
Cancer Cell ; 42(1): 16-34, 2024 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-38157864

RESUMO

Over the last decade, the composition of the gut microbiota has been found to correlate with the outcomes of cancer patients treated with immunotherapy. Accumulating evidence points to the various mechanisms by which intestinal bacteria act on distal tumors and how to harness this complex ecosystem to circumvent primary resistance to immune checkpoint inhibitors. Here, we review the state of the microbiota field in the context of melanoma, the recent breakthroughs in defining microbial modes of action, and how to modulate the microbiota to enhance response to cancer immunotherapy. The host-microbe interaction may be deciphered by the use of "omics" technologies, and will guide patient stratification and the development of microbiota-centered interventions. Efforts needed to advance the field and current gaps of knowledge are also discussed.


Assuntos
Microbioma Gastrointestinal , Melanoma , Microbiota , Neoplasias , Humanos , Melanoma/terapia , Neoplasias/terapia , Imunoterapia , Interações entre Hospedeiro e Microrganismos
7.
Blood ; 117(1): 259-67, 2011 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-20889922

RESUMO

Factor VIII (FVIII)-specific memory B cells are essential components for regulating anamnestic antibody responses against FVIII in hemophilia A with FVIII inhibitors. We asked how stimulation and inhibition of FVIII-specific memory B cells by low and high concentrations of FVIII, respectively, are affected by concurrent activation of the innate immune system. Using CD138(-) spleen cells from hemophilic mice treated with FVIII to study restimulation and differentiation of memory B cells in vitro, we tested modulating activities of agonists for Toll-like receptors (TLRs) 2, 3, 4, 5, 7, and 9. Ligands for TLR7 and 9 were most effective. They not only amplified FVIII-specific memory responses in the presence of stimulating concentrations of FVIII, but also countered inhibition in the presence of inhibitory concentrations of FVIII. Notably, CpG oligodeoxynucleotide (CpG-ODN), a ligand for TLR9, expressed biphasic effects. It amplified memory responses at low concentrations and inhibited memory responses at high concentrations, both in vitro and in vivo. Both stimulatory and inhibitory activities of CpG-ODN resulted from specific interactions with TLR9. Despite their strong immunomodulatory effects in the presence of FVIII, ligands for TLR induced negligible restimulation in the absence of FVIII in vitro and no restimulation in the absence of FVIII in vivo.


Assuntos
Linfócitos B/imunologia , Fator VIII/imunologia , Hemofilia A/imunologia , Memória Imunológica/imunologia , Oligodesoxirribonucleotídeos/farmacologia , Receptor Toll-Like 9/imunologia , Animais , Linfócitos B/metabolismo , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Fator VIII/administração & dosagem , Fator VIII/metabolismo , Hemofilia A/metabolismo , Humanos , Ligantes , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Baço/citologia , Baço/imunologia , Baço/metabolismo , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/metabolismo
8.
Blood ; 118(11): 3154-62, 2011 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-21788339

RESUMO

Memory B cells are involved in long-term maintenance of antibody-dependent immunologic disorders. Therefore, it is essential to understand how the restimulation of FVIII-specific memory B cells in hemophilia A with FVIII inhibitors is regulated. We asked whether concurrent activation of the innate immune system by an agonist for toll-like receptor (TLR) 7 is able to facilitate the differentiation of FVIII-specific memory B cells in the absence of T-cell help. TLR7 recognizes single-stranded RNA as contained in RNA viruses such as influenza, Sendai, and Coxsackie B viruses. Our results indicate that highly purified murine memory B cells do not differentiate into FVIII-specific antibody-secreting cells in the presence of FVIII and the TLR7 agonist when cultured in the absence of CD4(+) T cells. However, CD11c(+) dendritic cells facilitate the T cell-independent differentiation of FVIII-specific memory B cells but only in the presence of FVIII and the TLR7 agonist. In contrast to T cell-dependent restimulation, the antibody response after T cell-independent restimulation of FVIII-specific memory B cells is skewed toward IgG2a, an antibody subclass that is efficient in activating the complement system and in inducing Fc-receptor-mediated effector functions, both are required for effective immune responses against pathogens.


Assuntos
Linfócitos B/imunologia , Células Dendríticas/fisiologia , Fator VIII/imunologia , Memória Imunológica/imunologia , Receptor 7 Toll-Like/agonistas , Animais , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/imunologia , Epitopos/efeitos dos fármacos , Epitopos/imunologia , Memória Imunológica/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/imunologia , Linfócitos T/imunologia , Linfócitos T/fisiologia
9.
J Thromb Haemost ; 21(3): 488-498, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36696197

RESUMO

BACKGROUND: We previously demonstrated that busulfan preconditioning enabled sustained therapeutic platelet-derived factor VIII (FVIII) expression in naïve FVIIInull mice transplanted with 2bF8-transduced Sca-1+ cells. However, in mice with pre-existing inhibitors, platelet-FVIII expression was lost. OBJECTIVE: In this study, we aimed to describe the mechanism of this platelet-FVIII loss. METHODS: We monitored platelet-FVIII expression in FVIIInull mice that were immunized with rhFVIII to induce inhibitors and subsequently conditioned with busulfan before whole bone marrow transplantation or Sca-1+ hematopoietic stem cell transplantation (HSCT) from 2bF8 transgenic (2bF8Tg) mice. Busulfan with or without antithymocyte globulin or anti-CD8 antibody was employed before 2bF8Tg HSCT. Interferon gamma-ELISpot assay was used to assess which subset of cells was the target in platelet-FVIII loss. B-cell-deficient homozygous mutant mice were used to determine whether platelet-FVIII loss in FVIII-primed mice was mediated by antibody-dependent cellular cytotoxicity. RESULTS: Platelet-FVIII expression was sustained in 2bF8Tg bone marrow transplantation but not in 2bF8Tg HSCT recipients. CD8 T-cell depletion in addition to busulfan preconditioning restored platelet-FVIII expression in 2bF8Tg-HSCT recipients. ELISpot analyses showed that FVIII-primed CD8 T cells were efficiently restimulated by 2bF8Tg-Sca-1+ cells and secreted interferon gamma, but were not stimulated by 2bF8Tg platelets/megakaryocytes, suggesting that 2bF8Tg-Sca-1+ cells are targets for FVIII-primed CD8 T cells. When 2bF8Tg-Sca-1+ cells were transplanted into FVIII-primed homozygous mutant mice preconditioned with busulfan, no FVIII expression was detected, suggesting that antibody-dependent cellular cytotoxicity was not the mechanism of platelet-FVIII loss in FVIII-primed mice. CONCLUSION: Pre-existng immunity can alter the engraftment of 2bF8Tg-Sca-1+ cells through the cytotoxic CD8 T-cell-mediated pathway. Sufficient eradication of FVIII-primed CD8 T cells is critical for the success of platelet gene therapy in hemophilia A with inhibitors.


Assuntos
Hemofilia A , Hemostáticos , Camundongos , Animais , Bussulfano/metabolismo , Interferon gama/metabolismo , Plaquetas/metabolismo , Camundongos Knockout , Linfócitos T CD8-Positivos
10.
J Immunol ; 184(2): 573-81, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-20007533

RESUMO

The clonal composition of the T cell response can affect its ability to mediate infection control or to induce autoimmunity, but the mechanisms regulating the responding TCR repertoire remain poorly defined. In this study, we immunized mice with wild-type or mutated peptides displaying varying binding half-lives with MHC class II molecules to measure the impact of peptide-MHC class II stability on the clonal composition of the CD4 T cell response. We found that, although all peptides elicited similar T cell response size on immunization, the clonotypic diversity of the CD4 T cell response correlated directly with the half-life of the immunizing peptide. Peptides with short half-lives focused CD4 T cell response toward high-affinity clonotypes expressing restricted public TCR, whereas peptides with longer half-lives broadened CD4 T cell response by recruiting lower-affinity clonotypes expressing more diverse TCR. Peptides with longer half-lives did not cause the elimination of high-affinity clonotypes, and at a low dose, they also skewed CD4 T cell response toward higher-affinity clonotypes. Taken collectively, our results suggest the half-life of peptide-MHC class II complexes is the primary parameter that dictates the clonotypic diversity of the responding CD4 T cell compartment.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Peptídeos/imunologia , Animais , Autoimunidade , Células Clonais/imunologia , Meia-Vida , Imunização , Camundongos , Mutação , Peptídeos/genética , Estabilidade Proteica
11.
Immunol Cell Biol ; 89(1): 54-9, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20956989

RESUMO

Protective immunity against a variety of infections depends on the amplification and differentiation of rare naïve antigen-specific CD4 and CD8 T cells. Recent evidence indicates that the clonotypic composition of the responding T-cell compartment has a critical role in the immune defense against pathogens. The present review compares and contrasts how naive CD4 and CD8 T cells recognize their cognate antigen, and discusses the factors that regulate the genesis and maintenance of the CD4 and CD8 T-cell receptor repertoire diversity.


Assuntos
Imunidade Adaptativa/imunologia , Antígenos/imunologia , Linfócitos T/imunologia , Animais , Apresentação de Antígeno/imunologia , Deleção Clonal/imunologia , Humanos , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/citologia
12.
Immunology ; 130(1): 16-22, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20331477

RESUMO

New vaccines based on soluble recombinant antigens (Ags) require adjuvants to elicit long-lasting protective humoral and cellular immunity. Despite the importance of CD4 T helper cells for the generation of long-lived memory B and CD8 T cells, the impact of adjuvants on CD4 T-cell responses is still poorly understood. Adjuvants are known to promote dendritic cell (DC) maturation and migration to secondary lymphoid organs where they present foreign peptides bound to class II major histocompatibility complex molecules (pMHCII) to naïve CD4 T cells. Random and imprecise rearrangements of genetic elements during thymic development ensure that a vast amount of T-cell receptors (TCRs) are present in the naïve CD4 T-cell repertoire. Ag-specific CD4 T cells are selected from this vast pre-immune repertoire based on the affinity of their TCR for pMHCII. Here, we review the evidence demonstrating a link between the adjuvant and the specificity and clonotypic diversity of the CD4 T-cell response, and consider the potential mechanisms at play.


Assuntos
Adjuvantes Imunológicos/farmacologia , Linfócitos T CD4-Positivos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Vacinas/imunologia , Animais , Apresentação de Antígeno/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos
13.
Front Immunol ; 9: 1950, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30237796

RESUMO

Delivery of gene therapy as well as of biologic therapeutics is often hampered by the immune response of the subject receiving the therapy. We have reported that effective gene therapy for hemophilia utilizing platelets as a delivery vehicle engenders profound tolerance to the therapeutic product. In this study, we investigated whether this strategy can be applied to induce immune tolerance to a non-coagulant protein and explored the fundamental mechanism of immune tolerance induced by platelet-targeted gene delivery. We used ovalbumin (OVA) as a surrogate non-coagulant protein and constructed a lentiviral vector in which OVA is driven by the platelet-specific αIIb promoter. Platelet-specific OVA expression was introduced by bone marrow transduction and transplantation. Greater than 95% of OVA was stored in platelet α-granules. Control mice immunized with OVA generated OVA-specific IgG antibodies; however, mice expressing OVA in platelets did not. Furthermore, OVA expression in platelets was sufficient to prevent the rejection of skin grafts from CAG-OVA mice, demonstrating that immune tolerance developed in platelet-specific OVA-transduced recipients. To assess the mechanism(s) involved in this tolerance we used OTII mice that express CD4+ effector T cells specific for an OVA-derived peptide. After platelet-specific OVA gene transfer, these mice showed normal thymic maturation of the T cells ruling against central tolerance. In the periphery, tolerance involved elimination of OVA-specific CD4+ effector T cells by apoptosis and expansion of an OVA-specific regulatory T cell population. These experiments reveal the existence of natural peripheral tolerance processes to platelet granule contents which can be co-opted to deliver therapeutically important products.


Assuntos
Plaquetas , Deleção Clonal/genética , Técnicas de Transferência de Genes , Terapia Genética/métodos , Tolerância Periférica/genética , Linfócitos T Reguladores/imunologia , Animais , Camundongos , Camundongos Transgênicos , Linfócitos T Reguladores/patologia
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