RESUMO
An increasing number of monoclonal antibodies and derivatives such as antibody-drug conjugates (ADC) are of the IgG1 and IgG4 isotype with distinct structural and functional properties. In cases where antibody-mediated cytotoxicity is not desired, IgG4 is often used, as its Fc region is relatively poor at inducing antibody-dependent cell-mediated or complement-dependent cytotoxicity. IgG4 ADCs with highly cytotoxic drugs against proliferating target cells but which lack or have diminished antibody effector functions against quiescent cells may have a favorable safety profile compared to IgG1. Another unique property of the IgG4 subclass is the capability to exchange half antibodies in vivo creating randomly bispecific antibodies. To investigate the functional properties of process-derived antibody species, and determine the influence of shuffling on the therapeutic efficacy, several model antibodies on the basis of the anti-CD138 antibody-drug conjugate BT062 (Indatuximab ravtansine) were generated: (I) A wild type nBT062, (II) a stable nBT062 comprising mutations to prevent half-antibody exchange, (III) a half nBT062 lacking covalent binding between two heavy chains and (IV) a stabilized, bispecific nBT062-natalizumab antibody with a second, monovalent specificity against CD49d. All nBT062 model variants were capable of CD138-specific binding and antigen-mediated internalization into cells. Furthermore, all nBT062 models inhibited tumor growth in vitro after conjugation with the maytansinoid DM4. The in vivo effects of the different molecular variants were assessed in the MAXF1322 xenograft model. The bispecific nBT062-natalizumab-DM4 demonstrated the least efficacy and was only moderately active even without the co-administration of a human IgG preparation. Wild type, stable and half nBT062-DM4 models demonstrated great anti-tumor activities. The efficacy of wild type and half nBT062-DM4 was reduced in the presence of IgG, while stable nBT062-DM4 was only marginally influenced. These pre-clinical data demonstrate the advantage of introducing half-antibody exchange-preventing mutations into therapeutic IgG4-based antibody drug-conjugates.
Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais/farmacologia , Imunoconjugados/farmacologia , Imunoglobulina G/farmacologia , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Variação Genética , Humanos , Imunoconjugados/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Camundongos , Mutação , Ligação Proteica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Triple-negative breast cancer (TNBC) is related with a poor prognosis as patients do hardly benefit from approved therapies. CD138 (Syndecan-1) is upregulated on human breast cancers. Indatuximab ravtansine (BT062) is an antibody-drug-conjugate that specifically targets CD138-expressing cells and has previously shown clinical activity in multiple myeloma. Here we show indatuximab ravtansine as a potential mono- and combination therapy for TNBC. METHODS: The effects of indatuximab ravtansine were assessed in vitro in SK-BR-3 and T47D breast cancer cell lines. The in vivo effects of indatuximab ravtansine alone and in combination with docetaxel or paclitaxel were assessed in MAXF401, MAXF1384 and MAXF1322 xenograft TNBC models. RESULTS: CD138+ SK-BR-3 and T47D cells were highly sensitive to indatuximab ravtansine. The high CD138-expressing MAXF401 xenograft model demonstrated strong inhibition of tumor growth with 4 mg/kg indatuximab ravtansine. High doses of indatuximab ravtansine (8 mg/kg), docetaxel and the combination of both led to complete remission. In the low CD138-expressing MAXF1384 xenograft model, only combination of indatuximab ravtansine and docetaxel demonstrated a significant efficacy. In the MAXF1322 xenograft model, indatuximab ravtansine alone and in combination with paclitaxel elicited complete remission. CONCLUSIONS: These data demonstrate potential use of indatuximab ravtansine in combination with docetaxel or paclitaxel for CD138-positive TNBC.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Imunoconjugados/farmacologia , Sindecana-1/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Mama/patologia , Linhagem Celular Tumoral , Docetaxel/farmacologia , Docetaxel/uso terapêutico , Feminino , Humanos , Imunoconjugados/uso terapêutico , Maitansina/análogos & derivados , Maitansina/farmacologia , Maitansina/uso terapêutico , Camundongos , Camundongos Nus , Paclitaxel/uso terapêutico , Sindecana-1/metabolismo , Resultado do Tratamento , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Indatuximab ravtansine is a monoclonal antibody-linked cytotoxic agent that specifically targets CD138-expressing cells. Monotherapy has been shown to significantly inhibit multiple myeloma tumour growth in vivo and improve host survival. Here, we show that in most cell lines tested, indatuximab ravtansine acts additively or even synergistically with clinically approved therapies for treatment of multiple myeloma. In addition, in vivo mouse xenograft models confirmed the activity of indatuximab ravtansine in combination with lenalidamide and lenalidomide/dexamethasone. Indatuximab ravtansine may therefore be a suitable combination partner for multiple myeloma, and a clinical study is ongoing.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Imunoconjugados/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Animais , Dexametasona/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Humanos , Lenalidomida , Maitansina/análogos & derivados , Maitansina/uso terapêutico , Camundongos , Sindecana-1/antagonistas & inibidores , Sindecana-1/imunologia , Talidomida/análogos & derivados , Talidomida/uso terapêuticoRESUMO
BACKGROUND: Glioblastoma (GBM) is the most common and malignant intracranial tumor in adults and currently incurable. To specifically target natural killer (NK) cell activity to GBM, we employed NK-92/5.28.z cells that are continuously expanding human NK cells expressing an ErbB2-specific chimeric antigen receptor (CAR). METHODS: ErbB2 expression in 56 primary tumors, four primary cell cultures, and seven established cell lines was assessed by immunohistochemistry and flow cytometry. Cell killing activity of NK-92/5.28.z cells was analyzed in in vitro cytotoxicity assays. In vivo antitumor activity was evaluated in NOD-SCID IL2Rγ(null) (NSG) mice carrying orthotopic human GBM xenografts (6 to 11 mice per group) and C57BL/6 mice carrying subcutaneous and orthotopic ErbB2-expressing murine GBM tumors (5 to 8 mice per group). Statistical tests were two-sided. RESULTS: We found elevated ErbB2 protein expression in 41% of primary GBM samples and in the majority of GBM cell lines investigated. In in vitro assays, NK-92/5.28.z in contrast to untargeted NK-92 cells lysed all ErbB2-positive established and primary GBM cells analyzed. Potent in vivo antitumor activity of NK-92/5.28.z was observed in orthotopic GBM xenograft models in NSG mice, leading to a marked extension of symptom-free survival upon repeated stereotactic injection of CAR NK cells into the tumor area (median survival of 200.5 days upon treatment with NK-92/5.28.z vs 73 days upon treatment with parental NK-92 cells, P < .001). In immunocompetent mice, local therapy with NK-92/5.28.z cells resulted in cures of transplanted syngeneic GBM in four of five mice carrying subcutaneous tumors and five of eight mice carrying intracranial tumors, induction of endogenous antitumor immunity, and long-term protection against tumor rechallenge at distant sites. CONCLUSIONS: Our data demonstrate the potential of ErbB2-specific NK-92/5.28.z cells for adoptive immunotherapy of glioblastoma, justifying evaluation of this approach for the treatment of ErbB2-positive GBM in clinical studies.
Assuntos
Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Imunoterapia Adotiva/métodos , Células Matadoras Naturais , Terapia de Alvo Molecular/métodos , Receptor ErbB-2/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Neoplasias Encefálicas/imunologia , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/imunologia , Humanos , Imuno-Histoquímica , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Receptores de Antígenos de Linfócitos T/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3ζ signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. γ-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy.
Assuntos
Expressão Gênica , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptor ErbB-2/imunologia , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Proteínas Recombinantes de Fusão/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Evolução Clonal , Citotoxicidade Imunológica , Modelos Animais de Doenças , Epitopos de Linfócito T/imunologia , Feminino , Vetores Genéticos/genética , Humanos , Imunofenotipagem , Imunoterapia , Lentivirus/genética , Teste de Cultura Mista de Linfócitos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapia , Fenótipo , Transdução Genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Targeted T cells are emerging as effective non-toxic therapies for cancer. Multiple elements, however, contribute to the overall pathogenesis of cancer through both distinct and redundant mechanisms. Hence, targeting multiple cancer-specific markers simultaneously could result in better therapeutic efficacy. We created a functional chimeric antigen receptor-the TanCAR, a novel artificial molecule that mediates bispecific activation and targeting of T cells. We demonstrate the feasibility of cumulative integration of structure and docking simulation data using computational tools to interrogate the design and predict the functionality of such a complex bispecific molecule. Our prototype TanCAR induced distinct T cell reactivity against each of two tumor restricted antigens, and produced synergistic enhancement of effector functions when both antigens were simultaneously encountered. Furthermore, the TanCAR preserved the cytolytic ability of T cells upon loss of one of the target molecules and better controlled established experimental tumors by recognition of both targets in an animal disease model. This proof-of-concept approach can be used to increase the specificity of effector cells for malignant versus normal target cells, to offset antigen escape or to allow for targeting the tumor and its microenvironment.Molecular Therapy-Nucleic Acids (2013) 2, e105; doi:10.1038/mtna.2013.32; published online 9 July 2013.
RESUMO
Transfer of tumor-specific T-cell receptor (TCR) genes into patient T cells is a promising strategy in cancer immunotherapy. We describe here a novel vector (CD8-LV) derived from lentivirus, which delivers genes exclusively and specifically to CD8(+) cells. CD8-LV mediated stable in vitro and in vivo reporter gene transfer as well as efficient transfer of genes encoding TCRs recognizing the melanoma antigen tyrosinase. Strikingly, T cells genetically modified with CD8-LV killed melanoma cells reproducibly more efficiently than CD8(+) cells transduced with a conventional lentiviral vector. Neither TCR expression levels, nor the rate of activation-induced death of transduced cells differed between both vector types. Instead, CD8-LV transduced cells showed increased granzyme B and perforin levels as well as an up-regulation of CD8 surface expression in a small subpopulation of cells. Thus, a possible mechanism for CD8-LV enhanced tumor cell killing may be based on activation of the effector functions of CD8(+) T cells by the vector particle displaying OKT8-derived CD8-scFv and an increase of the surface density of CD8, which functions as coreceptor for tumor-cell recognition. CD8-LV represents a powerful novel vector for TCR gene therapy and other applications in immunotherapy and basic research requiring CD8(+) cell-specific gene delivery.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Citotoxicidade Imunológica/genética , Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T/genética , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/fisiologia , Células Cultivadas , Técnicas de Transferência de Genes , Terapia Genética/métodos , Células HEK293 , Humanos , Imunoterapia Adotiva/métodos , Células Jurkat , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Biológicos , Neoplasias/genética , Especificidade de Órgãos/genética , Regulação para Cima/genética , Regulação para Cima/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Natural killer (NK) cells hold promise for adoptive cancer immunotherapy but are dependent on cytokines such as interleukin (IL)-2 for growth and cytotoxicity. Here, we investigated the consequences of ectopic expression of IL-15 in human NK cells. IL-2 and IL-15 belong to the common γ chain family of cytokines and have overlapping activities. Transduction of clinically applicable NK-92 cells with lentiviral vectors encoding human IL-15 resulted in predominantly intracellular expression of the cytokine, and STAT5 activation, proliferation and cytotoxicity of the producer cells in the absence of IL-2. Growth of non-transduced bystander cells was not supported, allowing rapid enrichment of gene-modified cells solely by IL-2 withdrawal. This was also the case upon transduction of NK-92 and NKL cells with a bicistronic lentiviral vector encoding IL-15 and a chimeric antigen receptor (CAR) targeting the pancarcinoma antigen EpCAM. Effector cells co-expressing CAR and IL-15 continued to proliferate in the absence of exogenous cytokines and displayed high and selective cell-killing activity against EpCAM-expressing breast carcinoma cells that were resistant to the natural cytotoxicity of unmodified NK cells. This strategy facilitates rapid isolation and continuous expansion of retargeted NK cells and may extend their potential clinical utility.
Assuntos
Citocinas/biossíntese , Imunoterapia/métodos , Interleucina-15/biossíntese , Células Matadoras Naturais/imunologia , Receptores de Antígenos/imunologia , Animais , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Linhagem Celular Tumoral , Citocinas/genética , Citocinas/imunologia , Citotoxicidade Imunológica/genética , Citotoxicidade Imunológica/imunologia , DNA Complementar/genética , Molécula de Adesão da Célula Epitelial , Humanos , Interleucina-15/genética , Interleucina-15/imunologia , Interleucina-2/imunologia , Interleucina-2/farmacologia , Células K562 , Células Matadoras Naturais/metabolismo , Leucemia Eritroblástica Aguda/imunologia , Leucemia Eritroblástica Aguda/terapia , Melanoma/imunologia , Melanoma/terapia , Camundongos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Antígenos/biossíntese , Receptores de Antígenos/genéticaRESUMO
Treatment of high-risk neuroblastoma (NB) represents a major challenge in paediatric oncology. Alternative therapeutic strategies include antibodies targeting the disialoganglioside GD(2) , which is expressed at high levels on NB cells, and infusion of donor-derived natural killer (NK) cells. To combine specific antibody-mediated recognition of NB cells with the potent cytotoxic activity of NK cells, here we generated clonal derivatives of the clinically applicable human NK cell line NK-92 that stably express a GD(2) -specific chimeric antigen receptor (CAR) comprising an anti-GD(2) ch14.18 single chain Fv antibody fusion protein with CD3-ζ chain as a signalling moiety. CAR expression by gene-modified NK cells facilitated effective recognition and elimination of established GD(2) expressing NB cells, which were resistant to parental NK-92. In the case of intrinsically NK-sensitive NB cell lines, we observed markedly increased cell killing activity of retargeted NK-92 cells. Enhanced cell killing was strictly dependent on specific recognition of the target antigen and could be blocked by GD(2) -specific antibody or anti-idiotypic antibody occupying the CAR's cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD(2) -specific NK cells was also found against primary NB cells and GD(2) expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos/imunologia , Neoplasias do Jejuno/terapia , Células Matadoras Naturais/metabolismo , Neuroblastoma/terapia , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Complexo CD3/genética , Complexo CD3/imunologia , Linhagem Celular Tumoral , Criança , Gangliosídeos/genética , Gangliosídeos/imunologia , Expressão Gênica , Engenharia Genética , Vetores Genéticos , Humanos , Imunoterapia Adotiva , Neoplasias do Jejuno/imunologia , Neoplasias do Jejuno/secundário , Jejuno/imunologia , Jejuno/patologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Neuroblastoma/imunologia , Neuroblastoma/secundário , Receptores de Antígenos/genética , Receptores de Antígenos/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Retroviridae , Transdução GenéticaRESUMO
PURPOSE: Our goal was to target melanoma antigens to the dendritic cell-specific receptor DEC-205. DEC-205 is an antigen receptor expressed on dendritic cells and has been shown to guide antigens to MHC class I and II compartments for processing and presentation to T cells. EXPERIMENTAL DESIGN: The melanoma tumor-associated antigen (TAA), gp100, was fused to the single-chain fragment variable (scFv) specific for DEC-205. The binding capacity of the scFv was tested on lymph node-isolated CD11c+ cells. Mixed lymphocyte reactions were carried out to show an increased proliferative capacity of gp100 antigen-specific CD4 and CD8 T cells. Furthermore the scFv-TAA was used in a therapeutic setting using two different melanoma mouse models. RESULTS: C57Bl/6 mice were injected with scFv-DEC-205-gp100, monoclonal antibody anti-DEC-205, or PBS. Using fluorescence-activated cell sorting, we showed that lymph node CD11c+ dendritic cells stained positive for the binding of the scFv-mDEC-205-gp100 and the anti-DEC-205 monoclonal antibody, whereas the PBS-injected animals were negative. In mixed lymphocyte reactions, bone marrow-derived dendritic cells pulsed with scFv-mDEC-205-gp100 significantly increased proliferation of gp100-specific CD8+ and CD4+ T cells beyond gp100 peptide-pulsed or nonpulsed bone marrow-derived dendritic cells. Finally, in B16/F10 and RET models, a concentration-dependent suppression of tumor growth using scFv-mDEC-205-gp100 (66% reduction of tumor volume), in comparison with gp100 peptide vaccination, was observed. CONCLUSIONS: Our results indicate that the scFv-mDEC-205-gp100 targets TAA to dendritic cells in vivo for presentation on both MHC class I and II molecules. In vivo, this leads to an improved immune response and a decrease in tumor growth rate.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos CD/imunologia , Antígenos de Neoplasias/imunologia , Células Dendríticas/imunologia , Fragmentos de Imunoglobulinas/uso terapêutico , Lectinas Tipo C/imunologia , Melanoma Experimental/terapia , Glicoproteínas de Membrana/imunologia , Receptores de Superfície Celular/imunologia , Animais , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor , Antígeno gp100 de MelanomaRESUMO
Of all human endogenous retroviruses known today, HERV-K is the only one that has been shown to produce viral particles. While the first of the approximately 30 HERV-K sequences integrated into the human genome more than 40 million years ago, evidence is accumulating that HERV-K was active more recently, provirus HERV-K113 being the youngest sequence found. However, it is unclear which HERV-K sequences code for the viral particles that are produced by human germ-cell tumours or melanomas. Here, we show that the provirus HERV-K113, cloned into a baculovirus expression vector, is capable of producing intact particles of retroviral morphology, exhibiting the typical structure of those particles that were characterized in cell lines derived from human germ-cell tumours. Thus, the HERV-K113 sequence is a candidate for particle production in vivo and for an active human endogenous retrovirus of today.
Assuntos
Retrovirus Endógenos/fisiologia , Provírus/genética , Spodoptera/metabolismo , Replicação Viral/fisiologia , Animais , Baculoviridae/genética , Células Cultivadas/ultraestrutura , DNA Viral/análise , Retrovirus Endógenos/genética , Retrovirus Endógenos/patogenicidade , Vetores Genéticos , Genoma Humano , Humanos , Provírus/classificação , Provírus/patogenicidade , Provírus/fisiologia , Vírion/genéticaRESUMO
The aim of this study was to investigate whether depletion of CD4(+)CD25(+) regulatory T cells (Treg) from melanoma patients affects immune responses against tumors. By application of recombinant IL-2-diphteria toxin fusion protein, also known as ONTAK, we were able to significantly reduce the frequency of Treg in peripheral blood, whereas other cell populations remained unaffected. The reduction of Treg started immediately after the first bolus of ONTAK with a dose of 5 microg ONTAK per kg bodyweight and lasted for 13 days with subsequent recovery thereafter. Successive ONTAK treatments further reduced the number of circulating Treg. Using the contact sensitizer DCP we show that all patients developed vast eczema after Treg depletion, whereas no or only mild eczematous reactions were detectable before ONTAK treatment. Corresponding induction of DCP-specific CD4(+) and CD8(+) T cells were detectable. Moreover, after immunization of ONTAK treated patients with tumor antigen peptides, MelanA/MART-1 and gp100, significant induction of peptide specific CD8(+) T cells could be observed in 90% of the patients treated. These cells displayed effector functions, as they were able to lyse peptide-pulsed target cells and secreted IFNgamma upon restimulation. In aggregate, our data indicate that ONTAK depletes Treg in vivo significantly, resulting in enhanced immune functions and substantial development of antigen-specific CD8(+) T cells in vaccinated individuals.