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1.
Cancer Immunol Immunother ; 72(6): 1661-1672, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-36602564

RESUMO

T cell function is central to immune reconstitution and control of residual chronic myeloid leukemia (CML) cells after treatment initiation and is associated with achieving deep molecular response as a prerequisite for treatment-free remission, the ultimate therapeutic goal in CML. ATP-pocket-binding tyrosine kinase inhibitors (TKIs) like imatinib, dasatinib, and nilotinib are widely used for treating CML, but they have shown to inhibit T cell function as an "off-target" effect. Therefore, we tested asciminib, the first-in-class BCR::ABL1 fusion protein inhibitor specifically targeting the ABL myristoyl pocket (STAMP) and compared its effects on T cell function with imatinib, dasatinib, and nilotinib. Whereas all four TKIs inhibited the expression of the co-stimulatory protein CD28, the amino acid transporter CD98, proliferation, and secretion of pro-inflammatory cytokines IFNγ, IL-6, and IL-17A upon T cell stimulation, asciminib had less impact on PD-1, activation markers, and IL-2 secretion. T cells treated with asciminib and the other TKIs maintained their ability to mobilize their respiratory capacity and glycolytic reserve, which is an important surrogate for metabolic fitness and flexibility. Overall, we found milder inhibitory effects of asciminib on T cell activation, which might be beneficial for the immunological control of residual CML cells.


Assuntos
Antineoplásicos , Leucemia Mielogênica Crônica BCR-ABL Positiva , Humanos , Dasatinibe/farmacologia , Dasatinibe/uso terapêutico , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Proteínas Tirosina Quinases , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Proteínas de Fusão bcr-abl , Resistencia a Medicamentos Antineoplásicos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico
2.
J Immunother Cancer ; 9(4)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33931470

RESUMO

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Emerging data suggest that CLL-cells efficiently evade immunosurveillance. T-cell deficiencies in CLL include immuno(metabolic) exhaustion that is achieved by inhibitory molecules, with programmed cell death 1/programmed cell death ligand 1 (PD-L1) signaling emerging as a major underlying mechanism. Moreover, CLL-cells are characterized by a close and recurrent interaction with their stromal niches in the bone marrow and lymph nodes. Here, they receive nurturing signals within a well-protected environment. We could previously show that the interaction of CLL-cells with stroma leads to c-Myc activation that is followed by metabolic adaptations. Recent data indicate that c-Myc also controls expression of the immune checkpoint molecule PD-L1. Therefore, we sought out to determine the role of stromal contact for the CLL-cells' PD-L1 expression and thus their immuno-evasive phenotype.To do so, we analyzed PD-L1 expression on CLL cell (subsets) in untreated patients and on healthy donor-derived B-cells. Impact of stromal contact on PD-L1 expression on CLL-cells and the underlying signaling pathways were assessed in well-established in vitro niche models. Ex vivo and in vitro findings were validated in the Eµ-TCL1 transgenic CLL mouse model.We found increased PD-L1 expression on CLL-cells as compared with B-cells that was further enhanced in a cell-to-cell contact-dependent manner by stromal cells. In fact, circulating recent stromal-niche emigrants displayed higher PD-L1 levels than long-time circulating CLL-cells. Using our in vitro niche model, we show that a novel Notch-c-Myc-enhancer of zeste homolog 2 (EZH2) signaling axis controls PD-L1 upregulation. Ultimately, elevated PD-L1 levels conferred increased resistance towards activated autologous T-cells.In summary, our findings support the notion that the CLL microenvironment contributes to immune escape variants. In addition, several targetable molecules (eg, Notch or EZH2) could be exploited in view of improving immune responses in patients with CLL, which warrants further in-depth investigation.


Assuntos
Antígeno B7-H1/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores Notch/metabolismo , Células Estromais/metabolismo , Linfócitos T/metabolismo , Animais , Antígeno B7-H1/genética , Estudos de Casos e Controles , Linhagem Celular , Técnicas de Cocultura , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Ativação Linfocitária , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Comunicação Parácrina , Transdução de Sinais , Células Estromais/imunologia , Linfócitos T/imunologia , Células Tumorais Cultivadas , Evasão Tumoral , Microambiente Tumoral
3.
Oncoimmunology ; 7(7): e1445454, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29900057

RESUMO

D-2-hydroxyglutarate (D-2HG) is released by various types of malignant cells including acute myeloid leukemia (AML) blasts carrying isocitrate dehydrogenase (IDH) gain-of-function mutations. D-2HG acting as an oncometabolite promotes proliferation, anoikis, and differentiation block of hematopoietic cells in an autocrine fashion. However, prognostic impact of IDH mutations and high D-2HG levels remains controversial and might depend on the overall mutational context. An increasing number of studies focus on the permissive environment created by AML blasts to promote immune evasion. Impact of D-2HG on immune cells remains incompletely understood. Here, we sought out to investigate the effects of D-2HG on T-cells as key mediators of anti-AML immunity. D-2HG was efficiently taken up by T-cells in vitro, which is in line with high 2-HG levels measured in T-cells isolated from AML patients carrying IDH mutations. T-cell activation was slightly impacted by D-2HG. However, D-2HG triggered HIF-1a protein destabilization resulting in metabolic skewing towards oxidative phosphorylation, increased regulatory T-cell (Treg) frequency, and reduced T helper 17 (Th17) polarization. Our data suggest for the first time that D-2HG might contribute to fine tuning of immune responses.

4.
Cancer Lett ; 303(2): 128-39, 2011 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-21339041

RESUMO

Bispecific scFv antibody-derivatives (bsscFvs) recruiting natural killer (NK) cells for the lysis of malignant cells have therapeutic potential. However, a bsscFv specific for the B-lymphoid tumor antigen CD19 and the trigger molecule CD16 on NK cells had similar affinities for both antigens (42 and 58nM, respectively) and was not optimal for cytotoxicity. Therefore, a bispecific tribody (bsTb) was constructed with two binding sites for CD19 and one for CD16. This bsTb contained a CD19-specific Fab fragment carrying a CD16-specific scFv fused to its light chain and a CD19-specific scFv fused to its heavy chain. The bsTb was compared with a bispecific bibody (bsBb) lacking the CD19-specific scFv. The bsTb had 3-fold greater avidity for CD19 than the bsBb (8 and 24nM, respectively), while both had equal affinity for CD16 (56nM). Both molecules mediated antibody-dependent cellular cytotoxicity (ADCC) of leukemia-derived SEM cells and primary cells from leukemia patients. The bsTb showed half-maximum effective concentrations (EC(50)) of 55pM and promoted equal lysis as the bsBb and the bsscFv at 6- and 12-fold lower concentrations, respectively. Among these three molecules the bsTb showed the most promising in vitro properties which are anticipated to be displayed also in vivo.


Assuntos
Anticorpos Biespecíficos/química , Antígenos CD19/química , Linfoma de Células B/imunologia , Receptores de IgG/química , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Linhagem Celular Tumoral , Dimerização , Citometria de Fluxo/métodos , Humanos , Fragmentos de Imunoglobulinas , Imunoterapia/métodos , Cinética , Leucócitos Mononucleares/citologia , Linfoma de Células B/terapia , Ligação Proteica , Proteínas Recombinantes de Fusão/química
5.
MAbs ; 3(1): 21-30, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21081841

RESUMO

A single-chain triplebody (sctb) 33-ds16-ds19 comprising two distal single-chain Fv fragments (scFvs) specific for the lymphoid antigen CD19 and the myeloid antigen CD33 flanking a central scFv specific for CD16, which is the low affinity Fc-receptor (FcγRIII) present on natural killer cells and macrophages, was produced and its properties were investigated. CD33 and CD19 in combination are present on acute leukemiablasts with mixed lineage phenotype, but not on normal human hematopoietic cells. For comparison, two bispecific scFvs (bsscFvs), ds19-ds16 and 33-ds16, with monovalent binding to CD19 and CD33, respectively, were also studied. The sctb 33-ds16-ds19 specifically interacted with all 3 antigens. On the antigen double-positive cell line BV-173, the sctb bound with 2-fold greater avidity than bsscFv ds19-ds16 (KD = 21 vs. 42 nM) and with 1.4-fold greater avidity than bsscFv 33-ds16 (KD = 29 nM). All 3 fusion proteins had similar affinity for CD16 and sufficient thermic stability in human serum. In antibody-dependent cellular cytotoxicity (ADCC) reactions with human mononuclear cells as effectors, the sctb promoted lysis of BV-173 cells at 23-fold lower concentrations than bsscFv ds19-ds16 and at 1.4-fold lower concentrations than bsscFv 33-ds16. The sctb also mediated potent ADCC of the antigen double-positive mixed lineage leukemia cell line SEM, and the half-maximal concentration EC50 for BV-173 cells was 7 pM. Therefore, CD19 and CD33 are present on the surface of these leukemic cell lines such that they can be connected by a single sctb molecule, permitting the recruitment of NK cells via CD16 and tumor cell lysis.


Assuntos
Antígenos CD19/imunologia , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Especificidade de Anticorpos/imunologia , Antígenos CD/metabolismo , Antígenos CD19/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Western Blotting , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Citotoxicidade Imunológica/imunologia , Citometria de Fluxo , Células HEK293 , Humanos , Cinética , Leucemia/imunologia , Leucemia/patologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ligação Proteica/imunologia , Estabilidade Proteica , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Temperatura
6.
Br J Haematol ; 150(5): 574-86, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20636437

RESUMO

Two trivalent constructs consisting of single-chain Fv antibody fragments (scFvs) specific for the interleukin-3 receptor alpha chain (CD123), CD33 and the Fcgamma-receptor III (CD16) were designed and characterized for the elimination of acute myeloid leukaemia (AML) cells. The dual targeting single-chain Fv triplebody (sctb) [123 x ds16 x 33] and the mono targeting sctb [123 x ds16 x 123] both specifically bound their respective target antigens and were stable in human serum at 37 degrees C for at least 5 d. Both constructs induced potent antibody-dependent cellular cytotoxicity (ADCC) of two different AML-derived CD33- and CD123 double-positive cell lines in the low picomolar range using isolated mononuclear cells (MNCs) as effector cells. In these experiments the dual targeting molecule produced significantly stronger lysis than the mono targeting agent. In addition, the sctbs showed a high potency in mediating ADCC of primary leukaemia cells isolated from peripheral blood or bone marrow of seven AML patients. Hence, these novel molecules displayed potent anti-leukaemic effects against AML cells in vitro and represent attractive candidates for further preclinical development.


Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Subunidade alfa de Receptor de Interleucina-3/imunologia , Leucemia Mieloide Aguda/imunologia , Anticorpos de Cadeia Única/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Proteínas Ligadas por GPI , Humanos , Fragmentos de Imunoglobulinas/imunologia , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/patologia , Receptores de IgG/imunologia , Proteínas Recombinantes/imunologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Células Tumorais Cultivadas
7.
J Immunother ; 33(6): 599-608, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20551837

RESUMO

Single-chain Fv triplebodies (sctb), consisting of a single polypeptide chain with 3 single-chain antibody variable fragments connected in tandem, were generated as antileukemic agents. A CD19-specific sctb of this format has previously been shown to be superior to a bispecific single-chain Fv antibody fragment (bsscFv) for the elimination of leukemic B-lineage cells, but corresponding targeted agents for the treatment of acute myeloid leukemia are still lacking. For this purpose, both a bsscFv and a sctb specific for CD33 and the trigger molecule CD16 (FcgammaRIII) were produced. The sctb displayed 3.5-fold greater avidity for CD33 than the bsscFv 33xds16, whereas both had close to equal affinity for CD16. In antibody-dependent cellular cytotoxicity (ADCC) reactions with human mononuclear cells as effectors, both the bsscFv 33xds16 and the sctb induced lysis of tumor cells with half maximum effective concentrations (EC50) in the low picomolar range. It is interesting to note that the sctb promoted equal lysis of human leukemia-derived cell lines at 10 to 200-fold lower concentrations than the bsscFv. Both molecules mediated ADCC of primary patient cells. In conclusion, both the bsscFv 33xds16 and the sctb 33xds16x33 eliminated acute myeloid leukemia cells in ADCC reactions, but the novel sctb format showed significantly greater specific activity.


Assuntos
Anticorpos Biespecíficos/farmacologia , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Imunoterapia , Leucemia Mieloide Aguda/imunologia , Receptores de IgG/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/genética , Afinidade de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos CD/biossíntese , Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/biossíntese , Antígenos de Diferenciação Mielomonocítica/genética , Células CHO , Simulação por Computador , Cricetinae , Cricetulus , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Engenharia de Proteínas , Estabilidade Proteica , Receptores de IgG/genética , Receptores de IgG/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico
8.
Br J Haematol ; 148(6): 879-89, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20064159

RESUMO

Four new single-chain Fv antibody fragments (scFvs) specific for the human leucocyte surface antigen CD123 (interleukin-3 receptor alpha) were generated to achieve preferential targeting of leukaemia stem cells (LSCs) in acute myeloid leukaemia (AML). The scFvs were isolated from a phage display library generated with spleen RNA from mice, immunized with a fusion protein consisting of the extracellular domain of CD123 and the Fc domain of a human immunoglobulin G1. The scFvs displayed CD123-specific binding on tumour cells (binding constants (K(D)) 4.5-101 nmol/l). The scFv with the highest affinity was used to design two cell death-inducing molecules. First, an immunotoxin, a fusion protein with truncated Pseudomonas Exotoxin A, induced potent apoptosis of AML-derived MOLM-13 and SKNO-1 cells at nanomolar concentrations. Second, the fusion to another scFv, specific for the low affinity Fcgamma-receptor III (CD16), created a bispecific single chain Fv (bsscFv). This bsscFv [123 x ds16] mediated potent lysis of AML-derived MOLM-13, THP-1 and SKNO-1 cells in antibody-dependent cellular cytotoxicity (ADCC) reactions at picomolar concentrations. The recruitment of CD16-positive effector cells for the lysis of AML cells via CD123 represents a novel combination with attractive prospects for future clinical testing.


Assuntos
Fragmentos de Imunoglobulinas/imunologia , Subunidade alfa de Receptor de Interleucina-3/imunologia , Leucemia Mieloide Aguda/imunologia , Anticorpos de Cadeia Única/imunologia , ADP Ribose Transferases/imunologia , Animais , Citotoxicidade Celular Dependente de Anticorpos , Apoptose/imunologia , Toxinas Bacterianas/imunologia , Células CHO , Cricetinae , Cricetulus , Relação Dose-Resposta Imunológica , Epitopos , Exotoxinas/imunologia , Humanos , Imunotoxinas/imunologia , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos BALB C , Células Tumorais Cultivadas , Fatores de Virulência/imunologia , Exotoxina A de Pseudomonas aeruginosa
9.
J Immunother ; 31(9): 871-84, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18833000

RESUMO

A novel bispecific antibody-derived recombinant protein targeting leukemias and lymphomas was designed, a single-chain Fv triple body (sctb) consisting of 1 polypeptide chain with 3 scFvs connected in tandem. The distal scFvs were specific for the tumor antigen CD19, and the central scFv for the trigger molecule CD16 (FcgammaRIII) on natural killer (NK) cells and macrophages. We had previously built a disulphide stabilized (ds) bsscFv [19 x 16] with monovalent binding for CD19 from ds components. The sctb ds[19 x 16 x 19] also used ds components and displayed 3-fold greater avidity for CD19 than the bsscFv (KD = 13 vs. 42 nM), whereas both had equal affinity for CD16 (KD = 58 nM). Plasma half-lives in mice were 4 and 2 hours for the sctb and the bsscFv, respectively. In antibody-dependent cellular cytotoxicity reactions with human mononuclear cells as effectors, the sctb promoted equal lysis of leukemic cell lines and primary cells from leukemia and lymphoma patients at 10-fold to 40-fold lower concentrations than the bsscFv. This new format may also be applicable to a variety of other tumor antigens and effector molecules. With half-maximum effective concentrations (EC50) in the low picomolar range, the sctb ds[19 x 16 x 19] is an attractive candidate for further preclinical evaluation.


Assuntos
Anticorpos Biespecíficos/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD19/imunologia , Fragmentos de Imunoglobulinas/imunologia , Células Matadoras Naturais/imunologia , Leucemia/terapia , Receptores de IgG/imunologia , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/uso terapêutico , Antígenos CD19/genética , Antígenos CD19/metabolismo , Células CHO , Clonagem Molecular , Cricetinae , Cricetulus , Humanos , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/metabolismo , Fragmentos de Imunoglobulinas/uso terapêutico , Imunoterapia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Leucemia/imunologia , Leucemia/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Receptores de IgG/genética , Receptores de IgG/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico
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