RESUMO
Natural killer (NK) cells mediate antibody dependent cytotoxic killing of cancer cells via cross-linking FcγR on NK cells with IgG-Fc. Studies have shown that the single-hinge cleaved IgGs (scIgGs) have dysfunctional Fc and failed engagement with FcγRs on immune cells. However, little is known about how scIgGs impact on antitumor immunity in the tumor microenvironment. In this study, we revealed a significant association of tumor scIgGs with tumor progression and poor outcomes of breast cancer patients (n = 547). Using multiple mouse tumor models, we demonstrated that tumor scIgGs reduced NK cell cytotoxic activities and resulted in aggressive tumor progression. We further showed that an anti-hinge specific monoclonal antibody (AHA) rescued the dysfunctional Fc in scIgGs by providing a functional Fc and restored NK cell cytotoxic activity. These findings point to a novel immunotherapeutic strategy to enhance Fc engagement with FcγRs for activation of anticancer immunity.
Assuntos
Antineoplásicos , Neoplasias , Animais , Imunoglobulina G , Células Matadoras Naturais , Camundongos , Processos Neoplásicos , Microambiente TumoralRESUMO
The host immune system adopts multiple mechanisms involving antibodies to confront cancer cells. Accordingly, anti-tumor mAbs have become mainstays in cancer treatment. However, neither host immunity nor mAb therapies appear capable of controlling tumor growth in all cases. Structural instability of IgG was overlooked as a factor contributing to immunosuppression in the tumor microenvironment. Recently, physiological proteinases were identified that disable IgG immune effector functions. Evidence shows that these proteinases cause localized IgG impairment by selective cleavage of a single IgG peptide bond in the hinge-region. The recognition of IgG cleavage in the tumor microenvironment provides alternatives for tumor immunotherapy.
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BACKGROUND: Proteolytic impairment of the Fc effector functions of therapeutic monoclonal antibodies (mAbs) can compromise their antitumor efficacy in the tumor microenvironment and may represent an unappreciated mechanism of host immune evasion. Pertuzumab is a human epidermal growth factor receptor 2 (HER2)-targeting antibody and has been widely used in the clinic in combination with trastuzumab for treatment of HER2-overexpressing breast cancer. Pertuzumab susceptibility to proteolytic hinge cleavage and its impact on the drug's efficacy has not been previously studied. METHODS: Pertuzumab was incubated with high and low HER2-expressing cancer cells and proteolytic cleavage in the lower hinge region was detected by western blotting. The single hinge cleaved pertuzumab (scIgG-P) was purified and evaluated for its ability to mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro and anti-tumor efficacy in vivo. To assess the cleavage of trastuzumab (IgG-T) and pertuzumab (IgG-P) when simultaneously bound to the same cancer cell surface, F(ab')2 fragments of IgG-T or IgG-P were combined with the intact IgG-P and IgG-T, respectively, to detect scIgG generation by western blotting. RESULTS: Pertuzumab hinge cleavage occurred when the mAb was incubated with high HER2-expressing cancer cells. The hinge cleavage of pertuzumab caused a substantial loss of ADCC in vitro and reduced antitumor efficacy in vivo. The reduced ADCC function of scIgG-P was restored by an anti-hinge mAb specific for a cleavage site neoepitope. In addition, we constructed a protease-resistant version of the anti-hinge mAb that restored ADCC and the cell-killing functions of pertuzumab when cancer cells exressed a potent IgG hinge-cleaving protease. We also observed increased hinge cleavage of pertuzumab when combined with trastuzumab. CONCLUSION: The reduced Fc effector function of single hinge-cleaved pertuzumab can be restored by an anti-hinge mAb. The restoration effect indicated that immune function could be readily augmented when the damaged primary antibodies were bound to cancer cell surfaces. The anti-hinge mAb also restored Fc effector function to the mixture of proteolytically disabled trastuzumab and pertuzumab, suggesting a general therapeutic strategy to restore the immune effector function to protease-inactivated anticancer antibodies in the tumor microenvironment. The findings point to a novel tactic for developing breast cancer immunotherapy.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Evasão Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Animais , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados/administração & dosagem , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Fragmentos Fc das Imunoglobulinas/efeitos adversos , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Camundongos , Proteólise/efeitos dos fármacos , Receptor ErbB-2/imunologia , Evasão Tumoral/imunologia , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Immune suppression is recognized as a hallmark of cancer and this notion is largely based on studies on cellular immunity. Our recent studies have demonstrated a potential new mechanism of cancer suppression of immunity by impairment of antibody effector function mediated by proteolytic enzymes in the tumor microenvironment.
RESUMO
Pathogens that induce acute and chronic infections, as well as certain cancers, employ numerous strategies to thwart host cellular and humoral immune defenses. One proposed evasion mechanism against humoral immunity is a localized expression of extracellular proteases that cleave the IgG hinge and disable host IgG functions. Host immunity appears to be prepared to counter such a proteolytic tactic by providing a group of autoantibodies, denoted anti-hinge antibodies that specifically bind to cleaved IgGs and provide compensating functional restoration in vitro. These respective counter-measures highlight the complex interrelationships among pathogens and host immunity and suggested to us a possible means for therapeutic intervention. In this study, we combined an investigation of pathogen-mediated proteolysis of host IgGs with an immunization strategy to boost host anti-hinge antibodies. In a Staphylococcus aureus infection model using an artificial tissue cage (wiffle ball) implanted into rabbits, cleaved rabbit IgGs were detected in abundance in the abscesses of untreated animals early after infection. However, in animals previously immunized with peptide analogs of the cleaved IgG hinge to generate substantial anti-hinge antibody titers, S. aureus colony formation was markedly reduced compared to control animals or those similarly immunized with a scrambled peptide sequence. The results of this study demonstrate that extensive local proteolysis of IgGs occurs in a test abscess setting and that immunization to increase host anti-hinge antibodies provided substantial acute protection against bacterial growth.
Assuntos
Abscesso/imunologia , Imunoglobulina G/imunologia , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Animais , Carga Bacteriana , Modelos Animais de Doenças , Combinação de Medicamentos , Adjuvante de Freund/imunologia , Hemocianinas/genética , Humanos , Evasão da Resposta Imune , Imunidade Humoral , Imunização , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Extratos Vegetais , Proteólise , CoelhosRESUMO
BACKGROUND & AIMS: Many patients with inflammatory bowel disease (IBD) fail to respond to anti-tumor necrosis factor (TNF) agents such as infliximab and adalimumab, and etanercept is not effective for treatment of Crohn's disease. Activated matrix metalloproteinase 3 (MMP3) and MMP12, which are increased in inflamed mucosa of patients with IBD, have a wide range of substrates, including IgG1. TNF-neutralizing agents act in inflamed tissues; we investigated the effects of MMP3, MMP12, and mucosal proteins from IBD patients on these drugs. METHODS: Biopsy specimens from inflamed colon of 8 patients with Crohn's disease and 8 patients with ulcerative colitis, and from normal colon of 8 healthy individuals (controls), were analyzed histologically, or homogenized and proteins were extracted. We also analyzed sera from 29 patients with active Crohn's disease and 33 patients with active ulcerative colitis who were candidates to receive infliximab treatment. Infliximab, adalimumab, and etanercept were incubated with mucosal homogenates from patients with IBD or activated recombinant human MMP3 or MMP12 and analyzed on immunoblots or in luciferase reporter assays designed to measure TNF activity. IgG cleaved by MMP3 or MMP12 and antihinge autoantibodies against neo-epitopes on cleaved IgG were measured in sera from IBD patients who subsequently responded (clinical remission and complete mucosal healing) or did not respond to infliximab. RESULTS: MMP3 and MMP12 cleaved infliximab, adalimumab, and etanercept, releasing a 32-kilodalton Fc monomer. After MMP degradation, infliximab and adalimumab functioned as F(ab')2 fragments, whereas cleaved etanercept lost its ability to neutralize TNF. Proteins from the mucosa of patients with IBD reduced the integrity and function of infliximab, adalimumab, and etanercept. TNF-neutralizing function was restored after incubation of the drugs with MMP inhibitors. Serum levels of endogenous IgG cleaved by MMP3 and MMP12, and antihinge autoantibodies against neo-epitopes of cleaved IgG, were higher in patients who did not respond to treatment vs responders. CONCLUSIONS: Proteolytic degradation may contribute to the nonresponsiveness of patients with IBD to anti-TNF agents.
Assuntos
Fatores Biológicos/metabolismo , Imunoglobulina G/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismo , Proteólise/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Adalimumab/metabolismo , Anticorpos Monoclonais Humanizados/metabolismo , Fatores Biológicos/farmacologia , Biópsia , Estudos de Casos e Controles , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/imunologia , Colite Ulcerativa/metabolismo , Colo/imunologia , Colo/metabolismo , Colo/patologia , Doença de Crohn/tratamento farmacológico , Doença de Crohn/imunologia , Doença de Crohn/metabolismo , Epitopos/metabolismo , Etanercepte/metabolismo , Feminino , Humanos , Immunoblotting/métodos , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/imunologia , Infliximab/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Masculino , Metaloproteinase 12 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Pessoa de Meia-IdadeRESUMO
Cytotoxic therapeutic monoclonal antibodies (mAbs) often mediate target cell-killing by eliciting immune effector functions via Fc region interactions with cellular and humoral components of the immune system. Key functions include antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). However, there has been increased appreciation that along with cell-killing functions, the induction of antibody-dependent cytokine release (ADCR) can also influence disease microenvironments and therapeutic outcomes. Historically, most Fc engineering approaches have been aimed toward modulating ADCC, ADCP, or CDC. In the present study, we describe an Fc engineering approach that, while not resulting in impaired ADCC or ADCP, profoundly affects ADCR. As such, when peripheral blood mononuclear cells are used as effector cells against mAb-opsonized tumor cells, the described mAb variants elicit a similar profile and quantity of cytokines as IgG1. In contrast, although the variants elicit similar levels of tumor cell-killing as IgG1 with macrophage effector cells, the variants do not elicit macrophage-mediated ADCR against mAb-opsonized tumor cells. This study demonstrates that Fc engineering approaches can be employed to uncouple macrophage-mediated phagocytic and subsequent cell-killing functions from cytokine release.
Assuntos
Anticorpos Monoclonais , Anticorpos Antineoplásicos , Citotoxicidade Celular Dependente de Anticorpos , Citocinas/imunologia , Fragmentos Fc das Imunoglobulinas , Macrófagos/imunologia , Neoplasias/tratamento farmacológico , Engenharia de Proteínas , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Antineoplásicos/genética , Anticorpos Antineoplásicos/imunologia , Anticorpos Antineoplásicos/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/genética , Linhagem Celular Tumoral , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/farmacologia , Neoplasias/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
Trastuzumab has been used for the treatment of HER2-overexpressing breast cancer for more than a decade, but the mechanisms of action for the therapy are still being actively investigated. Ab-dependent cell-mediated cytotoxicity mediated by NK cells is well recognized as one of the key mechanisms of action for trastuzumab, but trastuzumab-mediated Ab-dependent cellular phagocytosis (ADCP) has not been established. In this study, we demonstrate that macrophages, by way of phagocytic engulfment, can mediate ADCP and cancer cell killing in the presence of trastuzumab. Increased infiltration of macrophages in the tumor tissue was associated with enhanced efficacy of trastuzumab whereas depletion of macrophages resulted in reduced antitumor efficacy in mouse xenograft tumor models. Among the four mouse FcγRs, FcγRIV exhibits the strongest binding affinity to trastuzumab. Knockdown of FcγRIV in mouse macrophages reduced cancer cell killing and ADCP activity triggered by trastuzumab. Consistently, an upregulation of FcγRIV expression by IFN-γ triggered an increased ADCP activity by trastuzumab. In an analogous fashion, IFN-γ priming of human macrophages increased the expression of FcγRIII, the ortholog of murine FcγRIV, and increased trastuzumab-mediated cancer cell killing. Thus, in two independent systems, the results indicated that activation of macrophages in combination with trastuzumab can serve as a therapeutic strategy for treating high HER2 breast cancer by boosting ADCP killing of cancer cells.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Macrófagos/imunologia , Macrófagos/metabolismo , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Receptor ErbB-2/metabolismo , Receptores de IgG/metabolismo , Animais , Citotoxicidade Celular Dependente de Anticorpos , Linhagem Celular Tumoral , Citotoxicidade Imunológica/imunologia , Modelos Animais de Doenças , Expressão Gênica , Xenoenxertos , Humanos , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Receptor ErbB-2/genética , TrastuzumabRESUMO
Primary and acquired resistance to anticancer antibody immunotherapies presents significant clinical challenges. Here, we demonstrate that proteolytic inactivation of cancer-targeting antibodies is an unappreciated contributor to cancer immune evasion, and the finding presents novel opportunities for therapeutic intervention. A single peptide bond cleavage in the IgG1 hinge impairs cancer cell killing due to structural derangement of the Fc region. Hinge-cleaved trastuzumab gradually accumulated on the surfaces of HER2-expressing cancer cell lines in vitro, and was greatly accelerated when the cells were engineered to express the potent bacterial IgG-degrading proteinase (IdeS). Similar to cancer-related matrix metalloproteinases (MMP), IdeS exposes a hinge neoepitope that we have developed an antibody, mAb2095-2, to specifically target the epitope. In in vitro studies, mAb2095-2 restored the lost antibody-dependent cell-mediated cytotoxicity functionality of cell-bound single-cleaved trastuzumab (scIgG-T). In vivo, mAb2095-2 rescued the impaired Fc-dependent tumor-suppressive activity of scIgG-T in a xenograft tumor model and restored the recruitment of immune effector cells into the tumor microenvironment. More importantly, an Fc-engineered proteinase-resistant version of mAb2095-2 rescued trastuzumab antitumor efficacy in a mouse tumor model with human cancer cells secreting IdeS, whereas trastuzumab alone showed significantly reduced antitumor activity in the same model. Consistently, an Fc-engineered proteinase-resistant version of trastuzumab also greatly improved antitumor efficacy in the xenograft tumor model. Taken together, these findings point to a novel cancer therapeutic strategy to rescue proteolytic damage of antibody effector function by an Fc-engineered mAb against the hinge neoepitope and to overcome cancer evasion of antibody immunity.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Proteólise/efeitos dos fármacos , Animais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Humanos , Imunoglobulina G/imunologia , Células MCF-7 , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Nus , Receptor ErbB-2/metabolismo , Trastuzumab/farmacologiaRESUMO
We report a chimeric monoclonal antibody (mAb) directed to a neo-epitope that is exposed in the IgG lower hinge following proteolytic cleavage. The mAb, designated 2095-2, displays specificity for IdeS-generated F(ab')2 fragments, but not for full-length IgG or for closely-related F(ab')2 fragments generated with other proteases. A critical component of the specificity is provided by the C-terminal amino acid of the epitope corresponding to gly-236 in the IgG1 (also IgG4) hinge. By its ability to bind to IdeS-cleaved anti-CD20 mAb, mAb 2095-2 fully restored antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against WIL2-S cells to the otherwise inactive anti-CD20 IgG1 F(ab')2 fragment. Similarly, 2095-2 reinstated ADCC against MDA-MB-231 cells to an anti-CD142 IgG1 F(ab')2 fragment. mAb 2095-2 was also capable of eliciting both CDC and ADCC to IgG4 F(ab')2 fragments, an IgG subclass that has weaker ADCC and CDC when intact relative to intact IgG1. The in vitro cell-based efficacy of 2095-2 was extended to the in vivo setting using platelets as a cell clearance surrogate. In a canine model, the co-administration of 2095-2 together with IdeS-generated, platelet-targeting anti-CD41/61 F(ab')2 fragment not only restored platelet clearance, but did so at a rate and extent of clearance that exceeded that of intact anti-CD41/61 IgG at comparable concentrations. To further explore this unexpected amplification effect, we conducted a rat study in which 2095-2 was administered at a series of doses in combination with a fixed dose of anti-CD41/61 F(ab')2 fragments. Again, the combination, at ratios as low as 1:10 (w/w) 2095-2 to F(ab')2, proved more effective than the anti-CD41/61 IgG1 alone. These findings suggest a novel mechanism for enhancing antibody-mediated cell-killing effector functions with potential applications in pathologic settings such as tumors and acute infections where protease activity is abundant.
Assuntos
Anticorpos Monoclonais/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/metabolismo , Anticorpos Monoclonais Murinos/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Proteínas de Bactérias/metabolismo , Plaquetas/imunologia , Plaquetas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Cisteína Endopeptidases/metabolismo , Cães , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Epitopos/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos Fab das Imunoglobulinas/farmacologia , Imunoglobulina G/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Contagem de Plaquetas , Proteólise , Ratos , RituximabRESUMO
The functional role of human antihinge (HAH) autoantibodies in normal health and disease remains elusive, but recent evidence supports their role in the host response to IgG cleavage by proteases that are prevalent in certain disorders. Characterization and potential exploitation of these HAH antibodies has been hindered by the absence of monoclonal reagents. 2095-2 is a rabbit monoclonal antibody targeting the IdeS-cleaved hinge of human IgG1. We have determined the crystal structure of the Fab of 2095-2 and its complex with a hinge analog peptide. The antibody is selective for the C-terminally cleaved hinge ending in G236 and this interaction involves an uncommon disulfide in VL CDR3. We probed the importance of the disulfide in VL CDR3 through engineering variants. We identified one variant, QAA, which does not require the disulfide for biological activity or peptide binding. The structure of this variant offers a starting point for further engineering of 2095-2 with the same specificity, but lacking the potential manufacturing liability of an additional disulfide. Proteins 2014; 82:1656-1667. © 2014 Wiley Periodicals, Inc.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Imunoglobulina G/imunologia , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Conformação Proteica , Proteólise , CoelhosRESUMO
Molecularly engineered antibodies with fit-for-purpose properties will differentiate next generation antibody therapeutics from traditional IgG1 scaffolds. One requirement for engineering the most appropriate properties for a particular therapeutic area is an understanding of the intricacies of the target microenvironment in which the antibody is expected to function. Our group and others have demonstrated that proteases secreted by invasive tumors and pathological microorganisms are capable of cleaving human IgG1, the most commonly adopted isotype among monoclonal antibody therapeutics. Specific cleavage in the lower hinge of IgG1 results in a loss of Fc-mediated cell-killing functions without a concomitant loss of antigen binding capability or circulating antibody half-life. Proteolytic cleavage in the hinge region by tumor-associated or microbial proteases is postulated as a means of evading host immune responses, and antibodies engineered with potent cell-killing functions that are also resistant to hinge proteolysis are of interest. Mutation of the lower hinge region of an IgG1 resulted in protease resistance but also resulted in a profound loss of Fc-mediated cell-killing functions. In the present study, we demonstrate that specific mutations of the CH2 domain in conjunction with lower hinge mutations can restore and sometimes enhance cell-killing functions while still retaining protease resistance. By identifying mutations that can restore either complement- or Fcγ receptor-mediated functions on a protease-resistant scaffold, we were able to generate a novel protease-resistant platform with selective cell-killing functionality.
Assuntos
Anticorpos Monoclonais , Citotoxicidade Celular Dependente de Anticorpos , Sítios de Ligação de Anticorpos , Engenharia de Proteínas , Proteólise , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/genética , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Sítios de Ligação de Anticorpos/genética , Sítios de Ligação de Anticorpos/imunologia , Linhagem Celular , Humanos , Imunoglobulina G , Receptores de IgG/genética , Receptores de IgG/imunologiaRESUMO
Tumor-associated macrophages (TAMs) have been shown to promote tumor progression, and increased TAM infiltration often correlates with poor prognosis. However, questions remain regarding the phenotype of macrophages within the tumor and their role in mAb-dependent cytotoxicity. This study demonstrates that whereas TAMs have protumor properties, they maintain Fc-dependent anti-tumor function. CD11b(+)CD14(+) TAMs isolated from primary human breast tumors expressed activating FcγRs. To model breast cancer TAMs in vitro, conditioned medium from breast cancer cells was used to drive human peripheral monocyte differentiation into macrophages. Tumor-conditioned macrophages were compared with in vitro derived M1 and M2a macrophages and were found to promote tumor cell invasion and express M2a markers, confirming their protumor potential. However, unlike M2a macrophages, tumor-conditioned macrophages expressed FcγRs and phagocytosed tumor cells in the presence of a tumor Ag-targeting mAb, unmasking an underappreciated tumoricidal capacity of TAMs. In vivo macrophage depletion reduced the efficacy of anti-CD142 against MDA-MB-231 xenograft growth and metastasis in SCID/beige mice, implicating a critical role for macrophages in Fc-dependent cell killing. M-CSF was identified in tumor-conditioned media and shown to be capable of differentiating macrophages with both pro- and anti-tumor properties. These results highlight the plasticity of TAMs, which are capable of promoting tumor progression and invasion while still retaining tumoricidal function in the presence of tumor-targeting mAbs.
Assuntos
Anticorpos Antineoplásicos/imunologia , Antígenos de Neoplasias/imunologia , Neoplasias da Mama/imunologia , Macrófagos/imunologia , Fagocitose , Receptores de IgG/imunologia , Animais , Neoplasias da Mama/patologia , Antígeno CD11b/imunologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Meios de Cultivo Condicionados/farmacologia , Progressão da Doença , Feminino , Humanos , Imunofenotipagem , Receptores de Lipopolissacarídeos/imunologia , Fator Estimulador de Colônias de Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos , Camundongos SCID , Invasividade Neoplásica/imunologia , Transplante de Neoplasias , Cultura Primária de CélulasRESUMO
INTRODUCTION: Recent studies reported that human IgG antibodies are susceptible to specific proteolytic cleavage in their lower hinge region, and the hinge cleavage results in a loss of Fc-mediated effector functions. Trastuzumab is a humanized IgG1 therapeutic monoclonal antibody for the treatment of HER2-overexpressing breast cancers, and its mechanisms of action consist of inhibition of HER2 signaling and Fc-mediated antibody-dependent cellular cytotoxicity (ADCC). The objective of this study is to investigate the potential effect of proteinase hinge cleavage on the efficacy of trastuzumab using both a breast cancer cell culture method and an in vivo mouse xenograft tumor model. METHODS: Trastuzumab antibody was incubated with a panel of human matrix metalloproteinases, and proteolytic cleavage in the lower hinge region was detected using both western blotting and mass spectrometry. Single hinge cleaved trastuzumab (scIgG-T) was purified and evaluated for its ability to mediate ADCC and inhibition of breast cancer cell proliferation in vitro as well as anti-tumor efficacy in the mouse xenograft tumor model. Infiltrated immune cells were detected in tumor tissues by immunohistochemistry. RESULTS: scIgG-T retains HER2 antigen binding activity and inhibits HER2-mediated downstream signaling and cell proliferation in vitro when compared with the intact trastuzumab. However, scIgG-T lost Fc-mediated ADCC activity in vitro, and had significantly reduced anti-tumor efficacy in a mouse xenograft tumor model. Immunohistochemistry showed reduced immune cell infiltration in tumor tissues treated with scIgG-T when compared with those treated with the intact trastuzumab, which is consistent with the decreased ADCC mediated by scIgG-T in vitro. CONCLUSION: Trastuzumab can be cleaved by matrix metalloproteinases within the lower hinge. scIgG-T exhibited a significantly reduced anti-tumor efficacy in vivo due to the weakened immune effector function such as ADCC. The results suggest that the lower hinge cleavage of trastuzumab can occur in the tumor microenvironment where matrix metalloproteinases often have high levels of expression and scIgG-T might compromise its anti-tumor efficacy in the clinic. However, further studies are needed to validate these hypotheses in the clinical setting.
Assuntos
Anticorpos Monoclonais Humanizados/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Linhagem Celular , Modelos Animais de Doenças , Feminino , Humanos , Metaloproteinases da Matriz/metabolismo , Camundongos , Dados de Sequência Molecular , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/patologia , Ligação Proteica , Proteólise , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Receptores de IgG/metabolismo , Trastuzumab , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
There are currently ~25 recombinant full-length IgGs (rIgGs) in the market that have been approved by regulatory agencies as biotherapeutics to treat various human diseases. Most of these are based on IgG1k framework and are either chimeric, humanized or human antibodies manufactured using either Chinese hamster ovary (CHO) cells or mouse myeloma cells as the expression system. Because CHO and mouse myeloma cells are mammalian cells, rIgGs produced in these cell lines are typically N-glycosylated at the conserved asparagine (Asn) residues in the CH2 domain of the Fc, which is also the case with serum IgGs. The Fc glycans present in these rIgGs are for the most part complex biantennary oligosaccharides with heterogeneity associated with the presence or the absence of several different terminal sugars. The major Fc glycans of rIgGs contain 0 or 1 or 2 (G0, G1 and G2, respectively) terminal galactose residues as non-reducing termini and their relative proportions may vary depending on the cell culture conditions in which they were expressed. Since glycosylation is strongly associated with antibody effector functions and terminal galactosylation may affect some of those functions, a panel of commercially available therapeutic rIgGs expressed in CHO cells and mouse myeloma cells were examined for their galactosylation patterns. The results suggest that the rIgGs expressed in CHO cells are generally less galactosylated compared to the rIgGs expressed in mouse myeloma cells. Accordingly, rIgGs produced in CHO cells tend to contain higher levels of G0 glycans compared with rIgGs produced in mouse myeloma cell lines. Despite the apparent wide variability in galactose content, adverse events or safety issues have not been associated with specific galactosylation patterns of therapeutic antibodies. Nevertheless, galactosylation may have an effect on the mechanisms of action of some therapeutic antibodies (e.g., effector pathways) and hence further studies to assess effects on product efficacy may be warranted for such antibodies. For antibodies that do not require effector functions for biological activity, however, setting a narrow specification range for galactose content may be unnecessary.
Assuntos
Anticorpos Monoclonais/metabolismo , Galactose/metabolismo , Imunoterapia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/uso terapêutico , Células CHO , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Cricetinae , Galactose/análogos & derivados , Galactose/química , Humanos , Marketing , Camundongos , Polissacarídeos/química , Processamento de Proteína Pós-TraducionalRESUMO
The mammalian antibody repertoire comprises immunoglobulin (Ig) molecules of multiple isotypes and subclasses with varying functional properties. Among the four subclasses of the human IgG isotype, we found that IgG2 exhibits a particular resistance to human and bacterial proteases that readily cleave the IgG1 hinge region in vitro. Autoantibodies (IgGs) that recognize points of proteolytic cleavage in the IgG1 hinge are widespread in the healthy human population, suggesting that IgG1 fragmentation and the generation of cryptic antigens for host immune surveillance commonly occur in vivo. We previously reported that autoantibodies to cleaved IgG1s can restore Fc-mediated effector functions that are lost following proteolytic cleavage of the hinge. In contrast, it was not possible to demonstrate an analogous cohort of autoantibodies to IgG2 hinge epitope analogs, and there appeared to be no functional component in human serum with the ability to reconstitute Fc effector functions to a cell-bound IgG2 fragment. Thus, the results indicate that among the IgG subclasses, human IgG2 is uniquely resistant to a number of known pathological proteases and that autoimmune recognition to potential cleavage points in the IgG2 hinge appears to be absent in human circulation.
Assuntos
Anticorpos Monoclonais/imunologia , Autoanticorpos/imunologia , Éxons Codificadores da Região de Dobradiça , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Peptídeo Hidrolases/metabolismo , Peptídeos/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Citotoxicidade Celular Dependente de Anticorpos , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Humanos , Imunoglobulina G/química , Macrófagos/imunologia , Dados de Sequência Molecular , Peptídeos/química , FagocitoseRESUMO
The effective functioning of immunoglobulins and IgG mAbs in removing pathological cells requires that the antigen binding regions and the Fc (effector) domain act in concert. The hinge region that connects these domains itself presents motifs that engage Fc receptors on immune effector cells to achieve cell lysis. In addition, sequences in the lower hinge/CH2 and further down the CH2 region are involved in C1q binding and complement-mediated cell killing. Proteolytic enzymes of little relevance to human physiology were successfully used for decades to generate fragments of IgGs for reagent and therapeutic use. It was subsequently noted that tumor-related and microbial proteases also cleaved human IgG specifically in the hinge region. We have shown previously that the "nick" of just one of the lower hinge heavy chains of IgG unexpectedly prevented many effector functions without impacting antigen binding. Of interest, related single-cleaved IgG breakdown products were detected in breast carcinoma extracts. This suggested a pathway by which tumors might avoid host immune surveillance under a cloak of proteolytically-generated, dysfunctional antibodies that block competent IgG binding. The host immune system cannot be blind to this pathway since there exists a widespread, low-titer incidence of anti-hinge (cleavage-site) antibodies in the healthy population. The prevalence of anti-hinge reactivity may reflect an ongoing immune recognition of normal IgG catabolism. Tumor growth and bacterial infections potentially generate hostile proteolytic environments that may pose harsh challenges to host immunity. Recent findings involving physiologically-relevant proteases suggest that the potential loss of key effector functions of host IgGs may result from subtle and limited proteolytic cleavage of IgGs and that such events may facilitate the incursion of invasive cells in local proteolytic settings.
Assuntos
Infecções Bacterianas/enzimologia , Infecções Bacterianas/imunologia , Evasão da Resposta Imune , Imunoglobulina G/metabolismo , Neoplasias/enzimologia , Neoplasias/imunologia , Peptídeo Hidrolases/metabolismo , Evasão Tumoral , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Autoanticorpos/imunologia , Infecções Bacterianas/terapia , Proteínas de Bactérias/metabolismo , Éxons Codificadores da Região de Dobradiça/genética , Éxons Codificadores da Região de Dobradiça/imunologia , Humanos , Imunoglobulina G/imunologia , Dados de Sequência Molecular , Neoplasias/terapiaRESUMO
The successful elimination of pathogenic cells and microorganisms by the humoral immune system relies on effective interactions between host immunoglobulins and Fc gamma receptors on effector cells, in addition to the complement system. Essential Ig motifs that direct those interactions reside within the conserved IgG lower hinge/CH2 interface. We noted that a group of tumor-related and microbial proteases cleaved human IgG1s in that region, and the "nick" of just one of the heavy chains profoundly inhibited IgG1 effector functions. We focused on IgG1 monoclonal antibodies (mAbs) since IgG1 is the most abundant human subclass and demonstrates robust Fc-mediated effector functions. The loss of Fc-mediated cell killing activities was correlated with diminished binding to the Fc gamma family of receptors, but a similar decrease in affinity was not observed toward the FcRn receptor that maintains IgG in circulation. Endogenous human IgG cleavage products of comparable size to mAbs with the single cleavage were detected by Western blot analysis in synovial fluid from patients with rheumatoid arthritis and in breast carcinoma extracts. Their detection is problematic under physiological conditions, since there is no loss of structure, and antigen-binding capability is unaffected. These findings suggest that within the hostile proteolytic microenvironments associated with many diseases, key effector functions of host IgGs, or therapeutic Abs, may be compromised.
Assuntos
Imunoglobulina G/química , Imunoglobulina G/metabolismo , Peptídeo Hidrolases/metabolismo , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Neoplasias da Mama/enzimologia , Membrana Celular/imunologia , Feminino , Humanos , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/metabolismo , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ratos , Receptores de IgG/metabolismo , Serina Endopeptidases/metabolismo , Staphylococcus aureus/enzimologia , Streptococcus pyogenes/enzimologiaRESUMO
A comparative in vitro survey of physiologically relevant human and microbial proteinases defined a number of enzymes that induced specific hinge domain cleavage in human IgG1. Several of these proteinases have been associated with tumor growth, inflammation, and infection. A majority of the identified proteinases converted IgG to F(ab')(2), and a consistent feature of their action was a transient accumulation of a single-cleaved intermediate (scIgG). The scIgG resulted from the relatively rapid cleavage of the first hinge domain heavy chain, followed by a slower cleavage of the second chain to separate the Fc domain from F(ab')(2). Major sites of enzymatic cleavage were identified or confirmed from the mass of the F(ab')(2) or Fab fragments and/or the amino-terminal amino acid sequence of the Fc for each enzyme including human matrix metalloproteinases (MMPs) 3 and 12, human cathepsin G, human neutrophil elastase (Fab), staphylococcal glutamyl endopeptidase I and streptococcal immunoglobulin-degrading enzyme (IdeS). The cleavage sites in IgG1 by MMP-3, cathepsin G and IdeS were used to guide the synthesis of peptide analogs containing the corresponding carboxy-termini to be used as immunogens in rabbits. Rabbit antibodies were successfully generated that showed selective binding to different human F(ab')(2)s and other hinge-cleavage fragments, but not to intact IgG. In Western blotting studies of synovial fluids from individuals with rheumatoid arthritis, the rabbit antibodies yielded patterns consistent with the presence of endogenous IgG fragments including F(ab')(2) and the single-cleaved IgG intermediate. The detection in synovial fluid of IgG fragments similar to those observed in the in vitro biochemical studies suggests that proteolysis of IgG may contribute to localized immune dysfunction in inflammatory environments.
Assuntos
Artrite Reumatoide/imunologia , Bactérias/enzimologia , Fragmentos de Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Processamento de Proteína Pós-Traducional , Líquido Sinovial/imunologia , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Western Blotting , Ensaio de Imunoadsorção Enzimática , Humanos , Concentração de Íons de Hidrogênio , Fragmentos de Imunoglobulinas/isolamento & purificação , Imunoglobulina G/química , Dados de Sequência Molecular , Peptídeos/imunologia , CoelhosRESUMO
BACKGROUND: The ability of abciximab to prevent fibrinogen binding to activated platelets indicates it may also promote dissolution of platelet-rich thrombi. The present study examined the capacity of abciximab to reverse platelet aggregation in vitro. METHODS AND RESULTS: Experiments were performed on blood from healthy non-medicated donors. Platelet aggregate formation and disaggregation were monitored turbidimetrically. Platelet-bound fibrinogen was measured by flow cytometry. For disaggregation studies, platelets were first stimulated with either ADP or the 11-mer thrombin receptor activating peptide (TRAP), then varying amounts of abciximab were added at periodic intervals after agonist addition. Platelet disaggregation was detected by comparing the extent of light transmittance at 4 min after addition of either abciximab or saline to PRP. ATP release was simultaneously monitored by chemi-luminescence. When added 1 min after low concentrations of ADP, abciximab rapidly (< 1 min) dispersed platelet aggregates in a dose-dependent manner, with complete disaggregation observed with 6.25 microg/mL of the beta3 antagonist. In contrast, equivalent concentrations of abciximab did not induce appreciable disaggregation to platelets stimulated with TRAP (10 microM). Platelet counts of samples that had undergone complete disaggregation, as assessed by aggregometry, were equivalent to baseline, indicating dispersal of aggregates to single cells. Concentrations of abciximab that produced complete disaggregation induced partial displacement of platelet-bound fibrinogen (52 +/- 8% inhibition of fibrinogen binding at 12.5 microg/ml abciximab). The disaggregation effectiveness of abciximab decreased as the time between ADP and subsequent abciximab addition widened, and as the amount of both dense granule release and agonist stimulation increased. However, pre-treatment of platelets with acetylsalicylic acid (ASA) did not potentiate platelet disaggregation induced by abciximab. CONCLUSIONS: These data indicate that abciximab facilitates the dispersal of newly formed platelet aggregates in vitro, by partially displacing fibrinogen from activated GPIIb/IIIa receptors. In vivo, abciximab may destabilize coronary thrombi by preventing aggregate formation and dispersing mural thrombi.