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1.
PLoS One ; 15(7): e0236374, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735564

RESUMO

We recently reported the development of a fully-human, CD3-binding bispecific antibody for immunotherapy of malignant glioma. To translate this therapeutic (hEGFRvIII-CD3- bi-scFv) to clinical trials and to help further the translation of other similar CD3-binding therapeutics, some of which are associated with neurologic toxicities, we performed a good laboratory practice (GLP) toxicity study to assess for potential behavioral, chemical, hematologic, and pathologic toxicities including evaluation for experimental autoimmune encephalomyelitis (EAE). To perform this study, male and female C57/BL6 mice heterozygous for the human CD3 transgene (20/sex) were allocated to one of four designated groups. All animals were administered one dose level of hEGFRvIII-CD3 bi-scFv or vehicle control. Test groups were monitored for feed consumption, changes in body weight, and behavioral disturbances including signs of EAE. Urinalysis, hematologic, and clinical chemistry analysis were also performed. Vehicle and test chemical-treated groups were humanely euthanized 48 hours or 14 days following dose administration. Complete gross necropsy of all tissues was performed, and selected tissues plus all observed gross lesions were collected and evaluated for microscopic changes. This included hematoxylin-eosin histopathological evaluation and Fe-ECR staining for myelin sheath enumeration. There were no abnormal clinical observations or signs of EAE noted during the study. There were no statistical changes in food consumption, body weight gain, or final body weight among groups exposed to hEGFRvIII-CD3 bi-scFv compared to the control groups for the 2- and 14-day timepoints. There were statistical differences in some clinical chemistry, hematologic and urinalysis endpoints, primarily in the females at the 14-day timepoint (hematocrit, calcium, phosphorous, and total protein). No pathological findings related to hEGFRvIII-CD3 bi-scFv administration were observed. A number of gross and microscopic observations were noted but all were considered to be incidental background findings. The results of this study allow for further translation of this and other important CD3 modulating bispecific antibodies.


Assuntos
Anticorpos Biespecíficos/farmacologia , Complexo CD3/imunologia , Receptores ErbB/imunologia , Glioma/imunologia , Animais , Anticorpos Biespecíficos/imunologia , Peso Corporal/efeitos dos fármacos , Peso Corporal/imunologia , Complexo CD3/farmacologia , Modelos Animais de Doenças , Receptores ErbB/farmacologia , Feminino , Glioma/patologia , Glioma/terapia , Humanos , Imunoterapia/efeitos adversos , Masculino , Camundongos , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
2.
Cancer Immunol Immunother ; 69(11): 2291-2303, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32504247

RESUMO

Target expression heterogeneity and the presence of an immunosuppressive microenvironment can hamper severely the efficiency of immunotherapeutic approaches. We have analyzed the potential to encounter and overcome such conditions by a combinatory two-target approach involving a bispecific antibody retargeting T cells to tumor cells and tumor-directed antibody-fusion proteins with costimulatory members of the B7 and TNF superfamily. Targeting the tumor-associated antigens EpCAM and EGFR with the bispecific antibody and costimulatory fusion proteins, respectively, we analyzed the impact of target expression and the influence of the immunosuppressive factors IDO, IL-10, TGF-ß, PD-1 and CTLA-4 on the targeting-mediated stimulation of T cells. Here, suboptimal activity of the bispecific antibody at diverse EpCAM expression levels could be effectively enhanced by targeting-mediated costimulation by B7.1, 4-1BBL and OX40L in a broad range of EGFR expression levels. Furthermore, the benefit of combined costimulation by B7.1/4-1BBL and 4-1BBL/OX40L was demonstrated. In addition, the expression of immunosuppressive factors was shown in all co-culture settings, where blocking of prominent factors led to synergistic effects with combined costimulation. Thus, targeting-mediated costimulation showed general promise for a broad application covering diverse target expression levels, with the option for further selective enhancement by the identification and blockade of main immunosuppressive factors of the particular tumor environment.


Assuntos
Anticorpos Biespecíficos/imunologia , Antígenos de Neoplasias/imunologia , Molécula de Adesão da Célula Epitelial/imunologia , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/imunologia , Anticorpos Biespecíficos/farmacologia , Linhagem Celular Tumoral , Receptores ErbB/imunologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Linfócitos T/efeitos dos fármacos , Evasão Tumoral/efeitos dos fármacos , Evasão Tumoral/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia
3.
Cancer Immunol Immunother ; 69(10): 2125-2137, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32451681

RESUMO

CD27 is a costimulatory molecule that provides a complementary target to the PD-1/PD-L1 checkpoint axis on T cells. Combining a CD27 agonist antibody with PD-1/PD-L1 blockade has shown synergistic antitumor activity in preclinical models, which led to clinical studies of the combination in cancer patients. We theorized that coupling CD27 costimulation with PD-1/PD-L1 blockade in a bispecific antibody (BsAb) may provide greater immune activating properties than combining the individual mAbs due to enhanced CD27 activation by cross-linking through PD-L1 and Fc receptors. To test this approach, we developed CDX-527, a tetravalent PD-L1xCD27 IgG1-scFv BsAb. CDX-527 potently inhibits PD-1 signaling and induces CD27-mediated T cell costimulation through PD-L1 cross-linking. In mixed lymphocyte reaction assays, CDX-527 is more potent than the combination of the parental antibodies, suggesting that cross-linking through both Fc receptors and PD-L1 results in enhanced CD27 agonist activity. CDX-527 was shown to mediate effector function against tumor cells overexpressing either CD27 or PD-L1. In human CD27 transgenic mice, we observed that antigen-specific T cell responses to a vaccine are greatly enhanced with a surrogate PD-L1xCD27 BsAb. Furthermore, the BsAb exhibits greater antitumor activity than the combination of the parental antibodies in a syngeneic lymphoma model. A pilot study of CDX-527 in cynomolgus macaques confirmed a mAb-like pharmacokinetic profile without noted toxicities. These studies demonstrate that CDX-527 effectively combines PD-1 blockade and CD27 costimulation into one molecule that is more potent than combination of the parental antibodies providing the rationale to advance this BsAb toward clinical studies in cancer patients.


Assuntos
Anticorpos Biespecíficos/farmacologia , Formação de Anticorpos , Imunoterapia/métodos , Linfoma de Células B/terapia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/antagonistas & inibidores , Animais , Anticorpos Biespecíficos/química , Humanos , Linfoma de Células B/imunologia , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Macaca fascicularis , Masculino , Camundongos , Camundongos Transgênicos
4.
Curr Med Sci ; 40(1): 28-34, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32166662

RESUMO

Selecting an ideal molecular format from diverse structures is a major challenge in developing a bispecific antibody (BsAb). To choose an ideal format of anti-CD3 × anti-transferrin receptor (TfR) bispecific antibodies for clinical application, we constructed TfR bispecific T-cell engager (BiTE) in two extensively applied formats, including single-chain tandem single-chain variable fragments (scFvs) and double-chain diabodies, and evaluated their functional characterizations in vitro. Results demonstrated that TfR-BiTE in both formats directed potent killing of TfR+ HepG2 cells. However, compared to two-chain diabodies, scFvs were more efficient in antigen binding and TfR+ target killing. Furthermore, different domain orders in scFvs would also be evaluated because single-TfR-CD3-His was preferable to single-CD3-TfR-His in immunotherapeutic strategies. Thus, the single-chain tandem TfR-CD3 format was favored for further investigation in cancer therapy.


Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Complexo CD3/imunologia , Receptores da Transferrina/imunologia , Células A549 , Anticorpos Biespecíficos/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células HEK293 , Células Hep G2 , Humanos , Anticorpos de Cadeia Única/imunologia
5.
Eur J Cancer ; 128: 107-118, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32037061

RESUMO

More than 25 therapeutic monoclonal antibodies (mAbs) used in oncology have been approved since 1997. Their nature has been largely modified through the last 20 years, from the chimeric IgG1 rituximab with pharmacokinetic parameters specific of murin or chimeric mAbs to humanized or human mAbs. Doses and administration frequency have been chosen based on this nature. More recently, the developed and registered mAbs are mostly IgG1, IgG2, IgG3 or IgG4 humanized or 100% human. Therefore, their behavior is different from the first mAbs authorized leading to lower systemic clearance and shorter half-life due to higher cellular uptake balanced by FcRn recognition with recirculation. The complexity of the pharmacokinetics and the pharmacokinetics/pharmacodynamics relation are increased for antibody-drug conjugates or bispecific T-cell engagers. However, significant number of studies reported pharmacokinetics/pharmacodynamics relations, with positive exposure-response link justifying the exploration of the pharmacokinetics in routine clinical practice of these therapeutic mAbs to prevent treatment failures and to limit their toxicities.


Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos Imunológicos/farmacologia , Imunoconjugados/farmacologia , Neoplasias/tratamento farmacológico , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Relação Dose-Resposta a Droga , Esquema de Medicação , Monitoramento de Medicamentos , Meia-Vida , Humanos , Imunoconjugados/uso terapêutico , Taxa de Depuração Metabólica , Neoplasias/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
6.
EBioMedicine ; 52: 102625, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31981978

RESUMO

BACKGROUND: DuoBody®-CD3xCD20 (GEN3013) is a full-length human IgG1 bispecific antibody (bsAb) recognizing CD3 and CD20, generated by controlled Fab-arm exchange. Its Fc domain was silenced by introduction of mutations L234F L235E D265A. METHODS: T-cell activation and T-cell-mediated cytotoxicity were measured by flow cytometry following co-culture with tumour cells. Anti-tumour activity of DuoBody-CD3xCD20 was assessed in humanized mouse models in vivo. Non-clinical safety studies were performed in cynomolgus monkeys. FINDINGS: DuoBody-CD3xCD20 induced highly potent T-cell activation and T-cell-mediated cytotoxicity towards malignant B cells in vitro. Comparison of DuoBody-CD3xCD20 to CD3 bsAb targeting alternative B-cell antigens, or to CD3xCD20 bsAb generated using alternative CD20 Ab, emphasized its exceptional potency. In vitro comparison with other CD3xCD20 bsAb in clinical development showed that DuoBody-CD3xCD20 was significantly more potent than three other bsAb with single CD3 and CD20 binding regions and equally potent as a bsAb with a single CD3 and two CD20 binding regions. DuoBody-CD3xCD20 showed promising anti-tumour activity in vivo, also in the presence of excess levels of a CD20 Ab that competes for binding. In cynomolgus monkeys, DuoBody-CD3xCD20 demonstrated profound and long-lasting B-cell depletion from peripheral blood and lymphoid organs, which was comparable after subcutaneous and intravenous administration. Peak plasma levels of DuoBody-CD3xCD20 were lower and delayed after subcutaneous administration, which was associated with a reduction in plasma cytokine levels compared to intravenous administration, while bioavailability was comparable. INTERPRETATION: Based on these preclinical studies, a clinical trial was initiated to assess the clinical safety of subcutaneous DuoBody-CD3xCD20 in patients with B-cell malignancies. FUNDING: Genmab.


Assuntos
Anticorpos Biespecíficos/imunologia , Antígenos CD20/metabolismo , Complexo CD3/metabolismo , Citotoxicidade Imunológica , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/farmacologia , Especificidade de Anticorpos/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Humanos , Leucemia de Células B/tratamento farmacológico , Leucemia de Células B/etiologia , Leucemia de Células B/patologia , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/etiologia , Linfoma de Células B/patologia , Macaca fascicularis , Camundongos , Mutação , Proteínas Recombinantes , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Recent Results Cancer Res ; 214: 71-91, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31473849

RESUMO

Bispecific T cell engagers are antibody constructs directed to a tumor-specific target on the one hand and to CD3-positive T cells on the other hand. Blinatumomab is a compound with specificity for the pan-B cell marker CD19. Clinical activity was tested in relapsed and refractory (R/R) non-Hodgkin's Lymphoma (NHL), R/R acute lymphoblastic leukemia (ALL), and ALL patients with minimal residual disease. Trials have already been started in de novo ALL. The most clinically relevant toxicities are neurologic events and cytokine release syndrome as with other T cell-activating therapies. The mechanisms of resistance are not fully understood. Higher leukemia load and later stage disease represent unfavorable factors. Besides, an upregulation of regulatory T cells and inhibitory molecules like PD-1/PD-L1 may have a role as the loss of target by several mechanisms. The future will show whether the use of bispecifics in ALL can change the standard treatment algorithms and whether bispecific T cell engagers will also be successfully used in other malignant entities.


Assuntos
Anticorpos Biespecíficos/farmacologia , Linfoma não Hodgkin/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Linfócitos T/citologia , Antígenos CD19 , Ensaios Clínicos como Assunto , Humanos , Neoplasia Residual/terapia
8.
J Exp Clin Cancer Res ; 38(1): 355, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31412896

RESUMO

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) is overexpressed in multiple cancers, which is associated with poor prognosis. Herceptin and other agents targeting HER2 have potent antitumor efficacy in patients with HER2-positive cancers. However, the development of drug resistance adversely impacts the efficacy of these treatments. It is therefore urgent to develop new HER2-targeted therapies. Bispecific antibodies (BsAbs) could guide immune cells toward tumor cells, and produced remarkable effects in some cancers. METHODS: A BsAb named M802 that targets HER2 and CD3 was produced by introducing a salt bridge and knobs-into-holes (KIHs) packing into the structure. Flow cytometry was performed to determine its binding activity and cytotoxicity. CCK-8, Annexin V/PI staining, western blotting, and ELISA were utilized to study its effect on cell proliferation, apoptosis, the signaling pathways of tumor cells, and the secretion of cytokines by immune cells. Subcutaneous tumor mouse models were used to analyze the in vivo antitumor effects of M802. RESULTS: We generated a new format of BsAb, M802, consisting of a monovalent unit against HER2 and a single chain unit against CD3. Our in vitro and in vivo experiments indicated that M802 recruited CD3-positive immune cells and was more cytotoxic than Herceptin in cells with high expression of HER2, low expression of HER2, and Herceptin resistance. Although M802 showed weaker effects than Herceptin on the PI3K/AKT and MAPK pathways, it was more cytotoxic due to its specific recognition of HER2 and its ability to recruit effector cells via its anti-CD3 moiety. CONCLUSIONS: Our results indicated that M802 exhibited potent antitumor efficacy in vitro and in vivo. M802 retained the function of Herceptin in antitumor signaling pathways, and also recruited CD3-positive immune cells to eliminate HER2-positive tumor cells. Therefore, M802 might be a promising HER2 targeted agent.


Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Complexo CD3/antagonistas & inibidores , Receptor ErbB-2/antagonistas & inibidores , Animais , Anticorpos Biespecíficos/química , Antineoplásicos Imunológicos/química , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Citotoxicidade Imunológica/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Melanoma Experimental , Camundongos , Peso Molecular , Transdução de Sinais/efeitos dos fármacos , Análise Espectral , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
9.
ACS Appl Mater Interfaces ; 11(32): 28720-28731, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31369234

RESUMO

In the present study, a capsule system that consists of a stealth carrier based on poly(ethylene glycol) (PEG) and functionalized with bispecific antibodies (BsAbs) is introduced to examine the influence of the capsule shape and size on cellular targeting. Hollow spherical and rod-shaped PEG capsules with tunable aspect ratios (ARs) of 1, 7, and 18 were synthesized and subsequently functionalized with BsAbs that exhibit dual specificities to PEG and epidermal growth factor receptor (EGFR). Dosimetry (variation between the concentrations of capsules present and capsules that reach the cell surface) was controlled through "dynamic" incubation (i.e., continuously mixing the incubation medium). The results obtained were compared with those obtained from the "static" incubation experiments. Regardless of the incubation method and the capsule shape and size studied, BsAb-functionalized PEG capsules showed >90% specific cellular association to EGFR-positive human breast cancer cells MDA-MB-468 and negligible association with both control cell lines (EGFR negative Chinese hamster ovary cells CHO-K1 and murine macrophages RAW 264.7) after incubation for 5 h. When dosimetry was controlled and the dose concentration was normalized to the capsule surface area, the size or shape had a minimal influence on the cell association behavior of the capsules. However, different cellular internalization behaviors were observed, and the capsules with ARs 7 and 18 were, respectively, the least and most optimal shape for achieving high cell internalization under both dynamic and static conditions. Dynamic incubation showed a greater impact on the internalization of rod-shaped capsules (∼58-67% change) than on the spherical capsules (∼24-29% change). The BsAb-functionalized PEG capsules reported provide a versatile particle platform for the evaluation and comparison of cellular targeting performance of capsules with different sizes and shapes in vitro.


Assuntos
Anticorpos Biespecíficos , Antineoplásicos Imunológicos , Sistemas de Liberação de Medicamentos , Polietilenoglicóis , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/farmacologia , Células CHO , Cápsulas , Cricetulus , Humanos , Camundongos , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Células RAW 264.7
10.
Haemophilia ; 25(5): 789-796, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31373431

RESUMO

INTRODUCTION: The formation of neutralizing antifactor VIII (fVIII) antibodies, called inhibitors, is the most common complication in modern haemophilia A care. Novel non-factor replacement therapies, such as emicizumab, have sought to address the limitations of bypassing agents for bleeding management in patients with inhibitors. However, immune tolerance induction (ITI) remains the primary method for eradicating inhibitors and restoring the hemostatic response to fVIII. AIM: The aim of this study was to review a case series of paediatric patients with haemophilia A and inhibitors who have received ITI for inhibitor eradication concomitantly with emicizumab prophylaxis for bleeding prevention. METHODS: Single institution retrospective chart review of paediatric patients with severe haemophilia A receiving ITI and emicizumab. RESULTS: Seven patients were included in this cohort. Six patients used three different recombinant fVIII products at 100 IU/kg three times per week, and one patient used a plasma-derived fVIII product at an initial dose of 50 IU/kg three times per week. Three patients achieved a negative inhibitor titre <0.6 Chromogenic Bethesda Units per mL (CBU/mL), and two patients achieved a normal fVIII recovery ≥66%. There were nine bleeding events in four patients, but no thrombotic events. All patients remained on ITI and emicizumab. CONCLUSION: Immune tolerance induction while on emicizumab prophylaxis is a feasible approach in paediatric haemophilia A patients with inhibitors. This is the first report of the concomitant use of ITI in patients receiving emicizumab prophylaxis described as the 'Atlanta Protocol'.


Assuntos
Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Hemofilia A/tratamento farmacológico , Tolerância Imunológica/efeitos dos fármacos , Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Estudos Retrospectivos
11.
Molecules ; 24(16)2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31394786

RESUMO

Vascular endothelial growth factor (VEGF) inhibition by the addition of bevacizumab to the chemotherapy regimen of metastatic colorectal cancer leads to an improved outcome. However, anti-angiogenic tumor therapy targeting a single factor may be limited by complementary mechanisms. Angiopoietin-2 (Ang-2, ANGPT2) is another important factor that cooperates with VEGF to drive tumor angiogenesis. It was shown that high Ang-2 levels are associated with a poor clinical outcome of colorectal cancer patients treated with bevacizumab-containing therapy. Therefore, combined inhibition of VEGF and Ang-2 was supposed to improve anti-angiogenic therapy. Here, we evaluated the efficacy of a bispecific antibody (CrossMab) co-targeting VEGF and Ang-2 in combination with chemotherapy in a chemoresistant colorectal carcinoma model. Antitumor activity was evaluated in athymic nude mice bearing subcutaneous DLD1 xenograft tumors and treated with anti-VEGF (B20), anti-Ang-2 (LC06) and anti-VEGF/Ang-2 (CrossMab) antibodies. Chemotherapy consisted of 5-FU and irinotecan. Resected tumors were analyzed immunohistochemically. First, an impact of targeting each single factor but also a clear advantage of co-targeting both factors could be demonstrated. Accordingly, tumor tissue showed strong staining for VEGF and Ang-2. Chemotherapy alone was less effective. Efficient tumor growth inhibition could be achieved by treatment with anti-VEGF/chemotherapy, single CrossMab and CrossMab/chemotherapy, which resulted in 3 out of 10, 6 out of 10 and 10 out of 10 complete responses, respectively, during seven weeks. Complete retarded tumors were characterized by massive intratumoral necrosis surrounded by layers of vital tumor cells and connective tissue with CD31-positive vessels at the periphery. In some cases, a distinct feature known as vessel co-option could be observed. In conclusion, the data from this model clearly support the strategy of co-targeting VEGF and Ang-2 and further demonstrate the beneficial impact of co-treatment with chemotherapy. The clear superiority of the CrossMab-containing regimen compared to clinical standard anti-VEGF/chemotherapy warrants further analyses in other models.


Assuntos
Inibidores da Angiogênese/farmacologia , Angiopoietina-2/antagonistas & inibidores , Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Neoplasias Colorretais/patologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Angiopoietina-2/metabolismo , Animais , Biópsia , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Terapia de Alvo Molecular , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
12.
J Immunol ; 203(3): 585-592, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31332079

RESUMO

Effector lymphocytes are multifunctional cells of the immune system that promote cytolysis of pathogen-infected cells and nascent tumors. Tumors must learn to evade effectors and employ a wide variety of mechanisms to do so. Bispecific Abs (BsAbs) are an emerging cancer immunotherapy approach seeking to re-engage either T effectors or NK cells with malignant cells. Possessing specificity for effector cells on one end and a tumor Ag on the other, these molecules work by attracting effectors to the target cell to build an immunologic synapse and induce tumor cell killing. The BsAb blinatumomab, for example, has specificity for the T cell-activating cell surface protein CD3 and the B cell Ag CD19. The only BsAb with regulatory approval currently, blinatumomab is used in the treatment of relapsed or refractory B cell acute lymphoblastic leukemia. Many additional BsAbs are in preclinical development, however, targeting many different tumor types. The variety of potential effector cells and cancer Ags, along with potential combination therapies, make BsAbs an active area of drug development. In this review, we discuss cancer recognition by the immune system and structural and mechanistic aspects of BsAbs. We summarize key steps in preclinical development and subsequent translation to medical practice. Future directions for BsAbs include combinations with a wide variety of both immunologic and nonimmunologic therapies. Defining their optimum clinical use is at early stages.


Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Antineoplásicos/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Linfócitos T/imunologia , Antígenos CD19/imunologia , Antígenos de Neoplasias/imunologia , Complexo CD3/imunologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia
14.
Haemophilia ; 25(5): 731-737, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31294904

RESUMO

INTRODUCTION: Emicizumab (Hemlibra® ) recently became available and requires an adaptation for managing bleeding, suspected bleeding and emergency or scheduled invasive procedures in haemophilia A patients with inhibitor. This implicates a multidisciplinary approach and redaction of recommendations for care that must be regularly adapted to the available data. AIM: The following text aims to provide a guide for the management of people with haemophilia A with inhibitor treated with emicizumab in case of bleeding or invasives procedures. METHODS: The French network on inherited bleeding disorders (MHEMO), the French Reference Centre on Haemophilia (CRH), in collaboration with the French Working Group on Perioperative Haemostasis (GIHP) have been working together to make proposals for the management of these situations. RESULTS: Haemostatic treatment and other medications should be given stepwise, according to the severity and location of the bleeding or the risk of bleeding of the procedure as well as the haemostatic response obtained at each step in order to ensure an optimal benefit/risk ratio. CONCLUSION: The lack of data means that it is only possible to issue proposals rather than recommendations.


Assuntos
Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Hemofilia A/tratamento farmacológico , Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , França , Hemostasia , Humanos
15.
J Immunol ; 203(4): 1076-1087, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31253728

RESUMO

Elicitation of tumor cell killing by CD8+ T cells is an effective therapeutic approach for cancer. In addition to using immune checkpoint blockade to reinvigorate existing but unresponsive tumor-specific T cells, alternative therapeutic approaches have been developed, including stimulation of polyclonal T cell cytolytic activity against tumors using bispecific T cell engager (BiTE) molecules that simultaneously engage the TCR complex and a tumor-associated Ag. BiTE molecules are efficacious against hematologic tumors and are currently being explored as an immunotherapy for solid tumors. To understand mechanisms regulating BiTE molecule--mediated CD8+ T cell activity against solid tumors, we sought to define human CD8+ T cell populations that efficiently respond to BiTE molecule stimulation and identify factors regulating their cytolytic activity. We find that human CD45RA+CCR7- CD8+ T cells are highly responsive to BiTE molecule stimulation, are enriched in genes associated with cytolytic effector function, and express multiple unique inhibitory receptors, including leukocyte Ig-like receptor B1 (LILRB1). LILRB1 and programmed cell death protein 1 (PD1) were found to be expressed by distinct CD8+ T cell populations, suggesting different roles in regulating the antitumor response. Engaging LILRB1 with its ligand HLA-G on tumor cells significantly inhibited BiTE molecule-induced CD8+ T cell activation. Blockades of LILRB1 and PD1 induced greater CD8+ T cell activation than either treatment alone. Together, our data suggest that LILRB1 functions as a negative regulator of human CD8+ effector T cells and that blocking LILRB1 represents a unique strategy to enhance BiTE molecule therapeutic activity against solid tumors.


Assuntos
Anticorpos Biespecíficos/farmacologia , Antígenos CD/imunologia , Imunoterapia/métodos , Receptor B1 de Leucócitos Semelhante a Imunoglobulina/imunologia , Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Anticorpos Biespecíficos/imunologia , Humanos , Receptor B1 de Leucócitos Semelhante a Imunoglobulina/antagonistas & inibidores , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos T/imunologia , Células Tumorais Cultivadas
16.
Int J Hematol ; 110(4): 419-430, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31254165

RESUMO

Emicizumab is a bispecific antibody to factor (F) IX/IXa and FX/FXa, which mimics FVIIIa cofactor function. Emicizumab prophylaxis significantly decreases bleeding events for patients with hemophilia A (PwHA). However, global hemostatic monitoring in emicizumab-treated PwHA remains poorly investigated. Using rotational thromboelastometry (ROTEM), we evaluated coagulation potentials of whole blood samples from seven emicizumab-treated PwHA who participated in ACE001JP/ACE002JP studies. Dose-dependent coagulation-enhancing effects of emicizumab to whole blood from PwHA mixed with an anti-FVIII C2 antibody in vitro were evident by non-activated ROTEM analysis (NATEM). The relationship between FVIII levels and NATEM parameters in PwHA not participating in the clinical trials demonstrated that CT + CFT inversely correlated with FVIII levels. These parameters were defined as NATEM-based grading of coagulation potential; 'T1' (FVIII < 1 IU/dL), 'T2' (1 ≤ , < 12 IU/dL) and 'T3' (12 ≤ , < 60 IU/dL). Coagulation function in emicizumab-treated PwHA was determined to be 'T2' or 'T3,' and was dependent on plasma emicizumab concentration. Improvement of NATEM-based grades corresponded with significant reduction of bleeding episodes, except for target joints, and differences were due to the time to reach the coagulation-steady state in individual patients. The NATEM analysis may be useful for intra- and inter-individual evaluation of coagulation function in emicizumab-treated PwHA.


Assuntos
Anticorpos Biespecíficos/administração & dosagem , Anticorpos Monoclonais Humanizados/administração & dosagem , Coagulação Sanguínea , Hemofilia A/sangue , Hemofilia A/tratamento farmacológico , Hemorragia/prevenção & controle , Tromboelastografia/métodos , Adolescente , Adulto , Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Anticoagulantes , Criança , Relação Dose-Resposta a Droga , Feminino , Hemofilia A/complicações , Hemorragia/etiologia , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
18.
MAbs ; 11(6): 1175-1190, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31181988

RESUMO

We describe a bispecific dual-antagonist antibody against human B cell activating factor (BAFF) and interleukin 17A (IL-17). An anti-IL-17 single-chain variable fragment (scFv) derived from ixekizumab (Taltz®) was fused via a glycine-rich linker to anti-BAFF tabalumab. The IgG-scFv bound both BAFF and IL-17 simultaneously with identical stoichiometry as the parental mAbs. Stability studies of the initial IgG-scFv revealed chemical degradation and aggregation not observed in either parental antibody. The anti-IL-17 scFv showed a high melting temperature (Tm) by differential scanning calorimetry (73.1°C), but also concentration-dependent, initially reversible, protein self-association. To engineer scFv stability, three parallel approaches were taken: labile complementary-determining region (CDR) residues were replaced by stable, affinity-neutral amino acids, CDR charge distribution was balanced, and a H44-L100 interface disulfide bond was introduced. The Tm of the disulfide-stabilized scFv was largely unperturbed, yet it remained monodispersed at high protein concentration. Fluorescent dye binding titrations indicated reduced solvent exposure of hydrophobic residues and decreased proteolytic susceptibility was observed, both indicative of enhanced conformational stability. Superimposition of the H44-L100 scFv (PDB id: 6NOU) and ixekizumab antigen-binding fragment (PDB id: 6NOV) crystal structures revealed nearly identical orientation of the frameworks and CDR loops. The stabilized bispecific molecule LY3090106 (tibulizumab) potently antagonized both BAFF and IL-17 in cell-based and in vivo mouse models. In cynomolgus monkey, it suppressed B cell development and survival and remained functionally intact in circulation, with a prolonged half-life. In summary, we engineered a potent bispecific antibody targeting two key cytokines involved in human autoimmunity amenable to clinical development.


Assuntos
Anticorpos Biespecíficos , Doenças Autoimunes/tratamento farmacológico , Fator Ativador de Células B/antagonistas & inibidores , Interleucina-17/antagonistas & inibidores , Anticorpos de Cadeia Única , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Fator Ativador de Células B/imunologia , Feminino , Células HEK293 , Células HT29 , Humanos , Interleucina-17/imunologia , Macaca fascicularis , Camundongos , Camundongos Transgênicos , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacologia
19.
Mol Neurobiol ; 56(11): 7420-7432, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31041656

RESUMO

The amyloid ß (Aß) peptide, correlated with development of Alzheimer's disease (AD), is produced by sequential proteolytic cleavage of the amyloid precursor protein (APP) by ß- and γ-secretases. Alternative proteolytic cleavage of APP by α-secretase prevents formation of Aß peptide and produces a neuroprotective protein, a soluble fragment of APPα (sAPPα). We previously generated a single-chain variable domain antibody fragment (scFv) that binds APP at the ß-secretase cleavage site and blocks cleavage of APP (iBsec1), and a second scFv which has been engineered to have α-secretase-like activity that increases α-secretase cleavage of APP (Asec1a) and showed that a bispecific antibody (Diab) combining both iBsec1 and Asec1a constructs protects mammalian cells from oxidative stress. Here, we show that the diabody is an effective therapeutic agent in a mouse model of AD. An apolipoprotein B (ApoB) binding domain peptide was genetically added to the diabody to facilitate transfer across the blood-brain barrier, and a recombinant human adeno-associated virus 2/8 (rAAV2/8) was used as a vector to express the gene constructs in a APP/PS1 mouse model of AD. The diabody increased levels of sAPPα, decreased Aß deposits and levels of oligomeric Aß, increased neuronal health as indicated by MAP2 and synaptophysin staining, increased hippocampal neurogenesis, and most importantly dramatically increased survival rates compared with untreated mice or mice treated only with the ß-secretase inhibitor. These results indicate that altering APP processing to inhibit ß-site activity while simultaneously promoting α-secretase processing provides substantially increased neuronal benefits compared with inhibition of ß-secretase processing alone and represents a promising new therapeutic approach for treating AD.


Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Anticorpos Biespecíficos/farmacologia , Neurônios/metabolismo , Doença de Alzheimer/metabolismo , Amiloide/metabolismo , Animais , Encéfalo/metabolismo , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/metabolismo , Modelos Animais de Doenças , Feminino , Gliose/patologia , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Presenilina-1/metabolismo , Anticorpos de Cadeia Única/metabolismo , Solubilidade , Sinapses/metabolismo
20.
Thromb Haemost ; 119(7): 1084-1093, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31064025

RESUMO

Emicizumab bridges activated factor IX (FIX) and FX to restore the tenase function mediated by activated FVIII (FVIIIa), which is deficient in people with haemophilia A (PwHA). Unlike FVIII, emicizumab does not require activation to function; thus, in coagulation assays, the behavior of emicizumab may differ from that of FVIII. The objective of this study was to assess the effect of emicizumab on coagulation assays, including potential interference behavior that may produce inaccurate or misleading results. A variety of clotting-based, amidolytic/chromogenic, latex particle-enhanced turbidometric, and enzyme-linked immunosorbent methods were investigated. As expected based on its pharmacologic mechanism of action, emicizumab exhibited strong activity on the activated partial thromboplastin time (aPTT), which resulted in interference with several aPTT-based assays, most importantly the one-stage FVIII activity assay; these assays are not recommended for PwHA receiving emicizumab therapy. Pharmacodynamic activity of emicizumab, as measured by FVIII chromogenic assays, was species-dependent due to the binding specificity of the drug antibody. Outside of FVIII assays, emicizumab did not interfere with assays based on immunologic or chromogenic principles, nor with clotting assays based on nonintrinsic pathway activators, thus offering alternative choices where aPTT-based assays might otherwise be used. The observed interferences are in line with the unique mechanism of action of emicizumab. Potential interferences should be taken into account in the selection of coagulation assays and interpretation of coagulation assay test results for PwHA receiving emicizumab therapy.


Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Testes de Coagulação Sanguínea/métodos , Fator VIIIa/metabolismo , Hemofilia A/diagnóstico , Plasma/metabolismo , Biomimética , Coagulação Sanguínea , Fator IXa/metabolismo , Fator X/metabolismo , Hemofilia A/tratamento farmacológico , Humanos , Tempo de Tromboplastina Parcial , Ligação Proteica
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