RESUMO
To address the key challenges in the development of next-generation drug delivery systems (DDS) with desired physicochemical properties to overcome limitations regarding safety, in vivo efficacy, and solid tumor penetration, an ultrasmall folate receptor alpha (FRα) targeted silica nanoparticle (C'Dot) drug conjugate (CDC; or folic acid CDC) was developed. A broad array of methods was employed to screen a panel of CDCs and identify a lead folic acid CDC for clinical development. These included comparing the performance against antibody-drug conjugates (ADCs) in three-dimensional tumor spheroid penetration ability, assessing in vitro/ex vivo cytotoxic efficacy, as well as in vivo therapeutic outcome in multiple cell-line-derived and patient-derived xenograft models. An ultrasmall folic acid CDC, EC112002, was identified as the lead candidate out of >500 folic acid CDC formulations evaluated. Systematic studies demonstrated that the lead formulation, EC112002, exhibited highly specific FRα targeting, multivalent binding properties that would mediate the ability to outcompete endogenous folate in vivo, enzymatic responsive payload cleavage, stability in human plasma, rapid in vivo clearance, and minimal normal organ retention organ distribution in non-tumor-bearing mice. When compared with an anti-FRα-DM4 ADC, EC112002 demonstrated deeper penetration into 3D cell-line-derived tumor spheroids and superior specific cytotoxicity in a panel of 3D patient-derived tumor spheroids, as well as enhanced efficacy in cell-line-derived and patient-derived in vivo tumor xenograft models expressing a range of low to high levels of FRα. With the growing interest in developing clinically translatable, safe, and efficacious DDSs, EC112002 has the potential to address some of the critical limitations of the current systemic drug delivery for cancer management.
Assuntos
Receptor 1 de Folato , Sistemas de Liberação de Fármacos por Nanopartículas , Neoplasias , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Receptor 1 de Folato/metabolismo , Receptor 1 de Folato/uso terapêutico , Ácido Fólico/química , Nanopartículas/uso terapêutico , Neoplasias/tratamento farmacológico , Dióxido de Silício/uso terapêuticoRESUMO
The prognosis of patients diagnosed with advanced ovarian or endometrial cancer remains poor, and effective therapeutic strategies are limited. The Müllerian inhibiting substance type 2 receptor (MISIIR) is a transforming growth factor ß (TGF-ß) receptor family member, overexpressed by most ovarian and endometrial cancers while absent in most normal tissues. Restricted tissue expression, coupled with an understanding that MISIIR ligation transmits apoptotic signals to cancer cells, makes MISIIR an attractive target for tumor-directed therapeutics. However, the development of clinical MISIIR-targeted agents has been challenging. Prompted by the responses achieved in patients with blood malignancies using chimeric antigen receptor (CAR) T cell therapy, we hypothesized that MISIIR targeting may be achieved using a CAR T cell approach. Herein, we describe the development and evaluation of a CAR that targets MISIIR. T cells expressing the MISIIR-specific CAR demonstrated antigen-specific reactivity in vitro and eliminated MISIIR-overexpressing tumors in vivo. MISIIR CAR T cells also recognized a panel of human ovarian and endometrial cancer cell lines, and they lysed a battery of patient-derived tumor specimens in vitro, without mediating cytotoxicity of a panel of normal primary human cells. In conclusion, these results indicate that MISIIR targeting for the treatment of ovarian cancer and other gynecologic malignancies is achievable using CAR technology.
Assuntos
Neoplasias dos Genitais Femininos/imunologia , Imunoterapia Adotiva , Neoplasias Ovarianas/imunologia , Receptores de Antígenos Quiméricos/imunologia , Receptores de Peptídeos/imunologia , Receptores de Fatores de Crescimento Transformadores beta/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Epitopos/genética , Epitopos/imunologia , Feminino , Neoplasias dos Genitais Femininos/terapia , Humanos , Camundongos , Neoplasias Ovarianas/terapia , Receptores de Antígenos Quiméricos/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Competitive radiolabeled antibody imaging can determine the unlabeled intact antibody dose that fully blocks target binding but may be confounded by heterogeneous tumor penetration. We evaluated the hypothesis that smaller radiolabeled constructs can be used to more accurately evaluate tumor expressed receptors. PROCEDURES: The Krogh cylinder distributed model, including bivalent binding and variable intervessel distances, simulated distribution of smaller constructs in the presence of increasing doses of labeled antibody forms. RESULTS: Smaller constructs <25 kDa accessed binding sites more uniformly at large distances from blood vessels compared with larger constructs and intact antibody. These observations were consistent for different affinity and internalization characteristics of constructs. As predicted, a higher dose of unlabeled intact antibody was required to block binding to these distant receptor sites. CONCLUSIONS: Small radiolabeled constructs provide more accurate information on total receptor expression in tumors and reveal the need for higher antibody doses for target receptor blockade.
Assuntos
Anticorpos Monoclonais/metabolismo , Simulação por Computador , Imagem Molecular/métodos , Neoplasias/metabolismo , Receptores de Superfície Celular/metabolismo , Humanos , Peso MolecularRESUMO
Antibody drug conjugates (ADC), comprised of highly potent small molecule payloads chemically conjugated to a full-length antibody, represent a growing class of therapeutic agents. The targeting of cytotoxic payloads via the specificity and selectivity of the antibody has led to substantial clinical benefits. However, ADC potency can be altered by mechanisms of resistance such as overexpression of efflux pumps or anti-apoptotic proteins. DeBouganin is a de-immunized variant of bouganin, a ribosome-inactivating protein (RIP) that blocks protein synthesis, thereby leading to apoptosis. When conjugated to trastuzumab (T-deB), deBouganin was more potent than ado-trastuzumab-emtansine (T-DM1) and unaffected by resistance mechanisms to which DM1 is susceptible. To further highlight the differentiating mechanism of action of deBouganin, HCC1419 and BT-474 tumor cells that survived T-DM1 or trastuzumab-MMAE (T-MMAE) treatment were treated with an anti-HER2 C6.5 diabody-deBouganin fusion protein or T-deB. C6.5 diabody-deBouganin and T-deB were potent against HCC1419 and BT-474 cells that were resistant to T-DM1 or T-MMAE killing. The resistant phenotype involved MDR pumps, Bcl-2 family members, and the presence of additional unknown pathways. Overall, the data suggest that deBouganin is effective against tumor cell resistance mechanisms selected in response to ADCs composed of anti-microtubule payloads.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Imunoconjugados/farmacologia , Microtúbulos/efeitos dos fármacos , Antineoplásicos/química , Antineoplásicos/toxicidade , Neoplasias da Mama/tratamento farmacológico , Proteínas de Transporte/química , Linhagem Celular Tumoral , Proliferação de Células , Cromatografia Líquida de Alta Pressão , Escherichia coli , Feminino , Humanos , Imunoconjugados/química , Imunoconjugados/toxicidade , Peptídeos e Proteínas de Sinalização Intracelular , Células MCF-7 , Proteínas de Membrana/química , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/toxicidade , TrastuzumabRESUMO
Antibody Engineering & Therapeutics, the largest meeting devoted to antibody science and technology and the annual meeting of The Antibody Society, will be held in San Diego, CA on December 11-15, 2016. Each of 14 sessions will include six presentations by leading industry and academic experts. In this meeting preview, the session chairs discuss the relevance of their topics to current and future antibody therapeutics development. Session topics include bispecifics and designer polyclonal antibodies; antibodies for neurodegenerative diseases; the interface between passive and active immunotherapy; antibodies for non-cancer indications; novel antibody display, selection and screening technologies; novel checkpoint modulators / immuno-oncology; engineering antibodies for T-cell therapy; novel engineering strategies to enhance antibody functions; and the biological Impact of Fc receptor engagement. The meeting will open with keynote speakers Dennis R. Burton (The Scripps Research Institute), who will review progress toward a neutralizing antibody-based HIV vaccine; Olivera J. Finn, (University of Pittsburgh School of Medicine), who will discuss prophylactic cancer vaccines as a source of therapeutic antibodies; and Paul Richardson (Dana-Farber Cancer Institute), who will provide a clinical update on daratumumab for multiple myeloma. In a featured presentation, a representative of the World Health Organization's INN expert group will provide a perspective on antibody naming. "Antibodies to watch in 2017" and progress on The Antibody Society's 2016 initiatives will be presented during the Society's special session. In addition, two pre-conference workshops covering ways to accelerate antibody drugs to the clinic and the applications of next-generation sequencing in antibody discovery and engineering will be held on Sunday December 11, 2016.
Assuntos
Anticorpos , Engenharia de Proteínas/métodos , Animais , Anticorpos/uso terapêutico , HumanosRESUMO
The development of antibody drug conjugates has provided enhanced potency to tumor-targeting antibodies by the addition of highly potent payloads. In the case of trastuzumab-DM1 (T-DM1), approved for the treatment of metastatic breast cancer, the addition of mertansine (DM1) to trastuzumab substantially increased progression-free survival. Despite these improvements, most patients eventually relapse due to complex mechanisms of resistance often associated with small molecule chemotherapeutics. Therefore, identifying payloads with different mechanisms of action (MOA) is critical for increasing the efficacy of targeted therapeutics and ultimately improving patient outcomes. To evaluate payloads with different MOA, deBouganin, a deimmunized plant toxin that inhibits protein synthesis, was conjugated to trastuzumab and compared with T-DM1 both in vitro and in vivo. The trastuzumab-deBouganin conjugate (T-deB) demonstrated greater potency in vitro against most cells lines with high levels of Her2 expression. In addition, T-deB, unlike T-DM1, was unaffected by inhibitors of multidrug resistance, Bcl-2-mediated resistance, or Her2-Her3 dimerization. Contrary to T-DM1 that showed only minimal cytotoxicity, T-deB was highly potent in vitro against tumor cells with cancer stem cell properties. Overall, the results demonstrate the potency and efficacy of deBouganin and emphasize the importance of using payloads with different MOAs. The data suggest that deBouganin could be a highly effective against tumor cell phenotypes not being addressed by current antibody drug conjugate formats and thereby provide prolonged clinical benefit.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Imunoconjugados/farmacologia , Maitansina/análogos & derivados , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Ado-Trastuzumab Emtansina , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Modelos Animais de Doenças , Feminino , Expressão Gênica , Genes bcl-2 , Genes erbB-2 , Humanos , Maitansina/farmacologia , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Trastuzumab , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
T cells recognize cancer cells via HLA/peptide complexes, and when disease overtakes these immune mechanisms, immunotherapy can exogenously target these same HLA/peptide surface markers. We previously identified an HLA-A2-presented peptide derived from macrophage migration inhibitory factor (MIF) and generated antibody RL21A against this HLA-A2/MIF complex. The objective of the current study was to assess the potential for targeting the HLA-A2/MIF complex in ovarian cancer. First, MIF peptide FLSELTQQL was eluted from the HLA-A2 of the human cancerous ovarian cell lines SKOV3, A2780, OV90, and FHIOSE118hi and detected by mass spectrometry. By flow cytometry, RL21A was shown to specifically stain these four cell lines in the context of HLA-A2. Next, partially matched HLA-A*02:01+ ovarian cancer (n = 27) and normal fallopian tube (n = 24) tissues were stained with RL21A by immunohistochemistry to assess differential HLA-A2/MIF complex expression. Ovarian tumor tissues revealed significantly increased RL21A staining compared with normal fallopian tube epithelium (P < 0.0001), with minimal staining of normal stroma and blood vessels (P < 0.0001 and P < 0.001 compared with tumor cells) suggesting a therapeutic window. We then demonstrated the anticancer activity of toxin-bound RL21A via the dose-dependent killing of ovarian cancer cells. In summary, MIF-derived peptide FLSELTQQL is HLA-A2-presented and recognized by RL21A on ovarian cancer cell lines and patient tumor tissues, and targeting of this HLA-A2/MIF complex with toxin-bound RL21A can induce ovarian cancer cell death. These results suggest that the HLA-A2/MIF complex should be further explored as a cell-surface target for ovarian cancer immunotherapy.
Assuntos
Biomarcadores Tumorais/metabolismo , Antígeno HLA-A2/metabolismo , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Neoplasias Ovarianas/metabolismo , Peptídeos/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Afinidade de Anticorpos , Especificidade de Anticorpos , Linhagem Celular Tumoral , Feminino , Antígeno HLA-A2/imunologia , Humanos , Neoplasias Ovarianas/patologiaRESUMO
Antibody Engineering & Therapeutics, the annual meeting of The Antibody Society, will be held in San Diego, CA in early December 2015. In this meeting preview, the chairs provide their thoughts on the importance of their session topics, which include antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, overcoming resistance to clinical immunotherapy, and building comprehensive IGVH-gene repertoires through discovering, confirming and cataloging new germline IGVH genes. The Antibody Society's special session will focus on "Antibodies to watch" in 2016, which are a subset of the nearly 50 antibodies currently in Phase 3 clinical studies. Featuring over 100 speakers in total, the meeting will commence with keynote presentations by Erica Ollmann Saphire (The Scripps Research Institute), Wayne A. Marasco (Dana-Farber Cancer Institute/Harvard Medical School), Joe W. Gray (Oregon Health & Science University), and Anna M. Wu (University of California Los Angeles), and it will conclude with workshops on the promise and challenges of using next-generation sequencing for antibody discovery and engineering from synthetic and in vivo libraries and on computational antibody design.
Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Imunoconjugados/imunologia , Engenharia de Proteínas/métodos , Anticorpos Monoclonais/uso terapêutico , Descoberta de Drogas/métodos , Humanos , Imunoconjugados/uso terapêutico , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Imunoterapia/métodos , Pesquisa Translacional Biomédica/métodosRESUMO
The 25th anniversary of the Antibody Engineering & Therapeutics Conference, the Annual Meeting of The Antibody Society, will be held in Huntington Beach, CA, December 7-11, 2014. Organized by IBC Life Sciences, the event will celebrate past successes, educate participants on current activities and offer a vision of future progress in the field. Keynote addresses will be given by academic and industry experts Douglas Lauffenburger (Massachusetts Institute of Technology), Ira Pastan (National Cancer Institute), James Wells (University of California, San Francisco), Ian Tomlinson (GlaxoSmithKline) and Anthony Rees (Rees Consulting AB and Emeritus Professor, University of Bath). These speakers will provide updates of their work, placed in the context of the substantial growth of the industry over the past 25 years.
Assuntos
Anticorpos/imunologia , Anticorpos/uso terapêutico , Engenharia de Proteínas/métodos , Anticorpos/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , HumanosRESUMO
Interferon (IFN)-γ-like the well-known antitumor biotherapeutic IFN-α-is a powerful antiproliferative and immune modulatory cytokine, but mixed results from clinical trials, together with issues of systemic toxicity, have dampened enthusiasm for its use in the treatment of cancer. We suggest that at least 2 factors reduce the antitumor efficacy of IFN-γ: (1) poorly understood survival mechanisms that protect most tumor cells from IFN-γ-induced direct cytotoxicity, and (2) the short half-life of IFN-γ in serum. In this review, we outline avenues to overcome both these limitations. First, we have identified the transcription factor nuclear factor-kappa B (NF-κB) as a protective mechanism against IFN-γ-induced necrosis, and disabling NF-κB allows IFN-γ to trigger RIP1 kinase-dependent programmed necrosis (or necroptosis) in otherwise resistant cells. Second, we propose that fusing IFN-γ to tumor-specific antibodies will stabilize IFN-γ in serum and target this cytokine to tumor cells. We expect that such IFN-γ-antibody chimeras (called immunocytokines), when combined with agents that neutralize tumor-intrinsic survival signals such as NF-κB, will exert potent tumoricidal activity with minimized systemic side effects. Although this review will focus on exploiting IFN-γ-induced necrosis for treatment of renal cell carcinoma, these approaches are also directly applicable to several human cancers in which IFNs have shown therapeutic potential.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma de Células Renais/tratamento farmacológico , Interferon gama/uso terapêutico , Neoplasias Renais/tratamento farmacológico , Anticorpos Monoclonais/uso terapêutico , Carcinoma de Células Renais/patologia , Humanos , Neoplasias Renais/patologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Necrose , Complexo de Proteínas Formadoras de Poros Nucleares , Proteínas de Ligação a RNA , Proteínas Recombinantes de Fusão/uso terapêuticoRESUMO
Metastatic renal cell carcinoma (RCC) is an incurable disease in clear need of new therapeutic interventions. In early-phase clinical trials, the cytokine IFN-γ showed promise as a biotherapeutic for advanced RCC, but subsequent trials were less promising. These trials, however, focused on the indirect immunomodulatory properties of IFN-γ, and its direct anti-tumor effects, including its ability to kill tumor cells, remains mostly unexploited. We have previously shown that IFN-γ induces RIP1 kinase-dependent necrosis in cells lacking NF-κB survival signaling. RCC cells display basally-elevated NF-κB activity, and inhibiting NF-κB in these cells, for example by using the small-molecule proteasome blocker bortezomib, sensitizes them to RIP1-dependent necrotic death following exposure to IFN-γ. While these observations suggest that IFN-γ-mediated direct tumoricidal activity will have therapeutic benefit in RCC, they cannot be effectively exploited unless IFN-γ is targeted to tumor cells in vivo. Here, we describe the generation and characterization of two novel 'immunocytokine' chimeric proteins, in which either human or murine IFN-γ is fused to an antibody targeting the putative metastatic RCC biomarker CD70. These immunocytokines display high levels of species-specific IFN-γ activity and selective binding to CD70 on human RCC cells. Importantly, the IFN-γ immunocytokines function as well as native IFN-γ in inducing RIP1-dependent necrosis in RCC cells, when deployed in the presence of bortezomib. These results provide a foundation for the in vivo exploitation of IFN-γ-driven tumoricidal activity in RCC.
Assuntos
Ligante CD27/antagonistas & inibidores , Carcinoma de Células Renais/patologia , Interferon gama/farmacologia , Neoplasias Renais/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Animais , Antivirais/farmacologia , Ácidos Borônicos/farmacologia , Bortezomib , Ligante CD27/metabolismo , Carcinoma de Células Renais/enzimologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Neoplasias Renais/enzimologia , Camundongos , Necrose , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Pirazinas/farmacologia , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Especificidade da EspécieRESUMO
UNLABELLED: Persistent signaling by the oncogenic EGF receptor (EGFR) is a major source of cancer resistance to EGFR targeting. We established that inactivation of 2 sterol biosynthesis pathway genes, SC4MOL (sterol C4-methyl oxidase-like) and its partner, NSDHL (NADP-dependent steroid dehydrogenase-like), sensitized tumor cells to EGFR inhibitors. Bioinformatics modeling of interactions for the sterol pathway genes in eukaryotes allowed us to hypothesize and then extensively validate an unexpected role for SC4MOL and NSDHL in controlling the signaling, vesicular trafficking, and degradation of EGFR and its dimerization partners, ERBB2 and ERBB3. Metabolic block upstream of SC4MOL with ketoconazole or CYP51A1 siRNA rescued cancer cell viability and EGFR degradation. Inactivation of SC4MOL markedly sensitized A431 xenografts to cetuximab, a therapeutic anti-EGFR antibody. Analysis of Nsdhl-deficient Bpa(1H/+) mice confirmed dramatic and selective loss of internalized platelet-derived growth factor receptor in fibroblasts, and reduced activation of EGFR and its effectors in regions of skin lacking NSDHL. SIGNIFICANCE: This work identifies a critical role for SC4MOL and NSDHL in the regulation of EGFR signaling and endocytic trafficking and suggests novel strategies to increase the potency of EGFR antagonists in tumors.
Assuntos
3-Hidroxiesteroide Desidrogenases/genética , Receptores ErbB/metabolismo , Oxigenases de Função Mista/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Cetuximab , Colesterol/metabolismo , Endocitose , Receptores ErbB/antagonistas & inibidores , Humanos , Masculino , Camundongos , Camundongos SCID , Camundongos Transgênicos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Transporte ProteicoRESUMO
Fibroblast activation protein (FAP) is a serine protease selectively expressed on tumor stromal fibroblasts in epithelial carcinomas and is important in cancer growth, adhesion, and metastases. As FAP enzymatic activity is a potent therapeutic target, we aimed to identify inhibitory antibodies. Using a competitive inhibition strategy, we used phage display techniques to identify 53 single-chain variable fragments (scFvs) after three rounds of panning against FAP. These scFvs were expressed and characterized for binding to FAP by surface plasmon resonance and flow cytometry. Functional assessment of these antibodies yielded an inhibitory scFv antibody, named E3, which could attenuate 35% of FAP cleavage of the fluorescent substrate Ala-Pro-7-amido-4-trifluoromethylcoumarin compared with nonfunctional scFv control. Furthermore, a mutant E3 scFv was identified by yeast affinity maturation. It had higher affinity (4-fold) and enhanced inhibitory effect on FAP enzyme activity (3-fold) than E3. The application of both inhibitory anti-FAP scFvs significantly affected the formation of 3-dimensional FAP-positive cell matrix, as demonstrated by reducing the fibronectin fiber orientation from 41.18% (negative antibody control) to 34.06% (E3) and 36.15% (mutant E3), respectively. Thus, we have identified and affinity-maturated the first scFv antibody capable of inhibiting FAP function. This scFv antibody has the potential to disrupt the role of FAP in tumor invasion and metastasis.
Assuntos
Gelatinases/antagonistas & inibidores , Gelatinases/imunologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/imunologia , Serina Endopeptidases/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Afinidade de Anticorpos , Endopeptidases , Citometria de Fluxo , Gelatinases/genética , Gelatinases/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Terapia de Alvo Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Biblioteca de Peptídeos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
Aberrant expression and activation of EGF receptor (EGFR) has been implicated in the development and progression of many human cancers. As such, targeted therapeutic inhibition of EGFR, for example by antibodies, is a promising anticancer strategy. The overall efficacy of antibody therapies results from the complex interplay between affinity, valence, tumor penetration and retention, and signaling inhibition. To gain better insight into this relationship, we studied a panel of EGFR single-chain Fv (scFv) antibodies that recognize an identical epitope on EGFR but bind with intrinsic monovalent affinities varying by 280-fold. The scFv were converted to Fab and IgG formats, and investigated for their ability to bind EGFR, compete with EGF binding, and inhibit EGF-mediated downstream signaling and proliferation. We observed that the apparent EGFR-binding affinity for bivalent IgG plateaus at intermediate values of intrinsic affinity of the cognate Fab, leading to a biphasic curve describing the ratio of IgG to Fab affinity. Mathematical modeling of antibody-receptor binding indicated that the biphasic effect results from nonequilibrium assay limitations. This was confirmed by further observation that the potency of EGF competition for antibody binding to EGFR improved with both intrinsic affinity and antibody valence. Similarly, both higher intrinsic affinity and bivalent binding improved the potency of antibodies in blocking cellular signaling and proliferation. Overall, our work indicates that higher intrinsic affinity combined with bivalent binding can achieve avidity that leads to greater in vitro potency of antibodies, which may translate into greater therapeutic efficacy.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos/imunologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/imunologia , Anticorpos Monoclonais/farmacologia , Afinidade de Anticorpos/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/genética , Expressão Gênica , Humanos , Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/imunologiaRESUMO
Recombinant immunotoxins, consisting of single-chain variable fragments (scFv) genetically fused to polypeptide toxins, represent potentially effective candidates for cancer therapeutics. We evaluated the affinity of various anti-Her2/neu scFv fused to recombinant gelonin (rGel) and its effect on antitumor efficacy and off-target toxicity. A series of rGel-based immunotoxins were created from the human anti-Her2/neu scFv C6.5 and various affinity mutants (designated ML3-9, MH3-B1, and B1D3) with affinities ranging from 10(-8) to 10(-11) mol/L. Against Her2/neu-overexpressing tumor cells, immunotoxins with increasing affinity displayed improved internalization and enhanced autophagic cytotoxicity. Targeting indices were highest for the highest affinity B1D3/rGel construct. However, the addition of free Her2/neu extracellular domain (ECD) significantly reduced the cytotoxicity of B1D3/rGel because of immune complex formation. In contrast, ECD addition had little impact on the lower affinity constructs in vitro. In vivo studies against established BT474 M1 xenografts showed growth suppression by all immunotoxins. Surprisingly, therapy with the B1D3-rGel induced significant liver toxicity because of immune complex formation with shed Her2/neu antigen in circulation. The MH3-B1/rGel construct with intermediate affinity showed effective tumor growth inhibition without inducing hepatotoxicity or complex formation. These findings show that while high-affinity constructs can be potent antitumor agents, they may also be associated with mistargeting through the facile formation of complexes with soluble antigen leading to significant off-target toxicity. Constructs composed of intermediate-affinity antibodies are also potent agents that are more resistant to immune complex formation. Therefore, affinity is an exceptionally important consideration when evaluating the design and efficacy of targeted therapeutics.
Assuntos
Imunotoxinas/farmacologia , Receptor ErbB-2/imunologia , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Anticorpos de Cadeia Única/farmacologia , Animais , Afinidade de Anticorpos , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Humanos , Imunotoxinas/química , Imunotoxinas/imunologia , Camundongos , Camundongos Nus , Neoplasias/imunologia , Proteínas Inativadoras de Ribossomos Tipo 1/imunologia , Anticorpos de Cadeia Única/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Rapid and sensitive detection of serum tumor biomarkers are needed to monitor cancer patients for disease progression. Highly sensitive piezoelectric microcantilever sensors (PEMS) offer an attractive tool for biomarker detection; however, their utility in the complex environment encountered in serum has yet to be determined. As a proof of concept, we have functionalized PEMS with antibodies that specifically bind to HER2, a biomarker (antigen) that is commonly overexpressed in the blood of breast cancer patients. The function and sensitivity of these anti-HER2 PEMS biosensors was initially assessed using recombinant HER2 spiked into human serum. Their ability to detect native HER2 present in the serum of breast cancer patients was then determined. We have found that the anti-HER2 PEMS were able to accurately detect both recombinant and naturally occurring HER2 at clinically relevant levels (>2 ng/mL). This indicates that PEMS-based biosensors provide a potentially effective tool for biomarker detection.
Assuntos
Análise Química do Sangue/métodos , Neoplasias da Mama/sangue , Eletricidade , Espaço Extracelular , Receptor ErbB-2/sangue , Receptor ErbB-2/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/química , Estudos de Casos e Controles , Humanos , Masculino , Estrutura Terciária de Proteína , Receptor ErbB-2/imunologia , TrastuzumabRESUMO
Antibody drugs are widely used in cancer therapy, but conditions to maximize tumor penetration and efficacy have yet to be fully elucidated. In this study, we investigated the impact of antibody binding affinity on tumor targeting and penetration with affinity variants that recognize the same epitope. Specifically, we compared four derivatives of the C6.5 monoclonal antibody (mAb), which recognizes the same HER2 epitope (monovalent K(D) values ranging from 270 to 0.56 nmol/L). Moderate affinity was associated with the highest tumor accumulation at 24 and 120 hours after intravenous injection, whereas high affinity was found to produce the lowest tumor accumulation. Highest affinity mAbs were confined to the perivascular space of tumors with an average penetration of 20.4 ± 7.5 µm from tumor blood vessels. Conversely, lowest affinity mAbs exhibited a broader distribution pattern with an average penetration of 84.8 ± 12.8 µm. In vitro internalization assays revealed that antibody internalization and catabolism generally increased with affinity, plateauing once the rate of HER2 internalization exceeded the rate of antibody dissociation. Effects of internalization and catabolism on tumor targeting were further examined using antibodies of moderate (C6.5) or high-affinity (trastuzumab), labeled with residualizing ((111)In-labeled) or nonresidualizing ((125)I-labeled) radioisotopes. Significant amounts of antibody of both affinities were degraded by tumors in vivo. Furthermore, moderate- to high-affinity mAbs targeting the same HER2 epitope with monovalent affinity above 23 nmol/L had equal tumor accumulation of residualizing radiolabel over 120 hours. Results indicated equal tumor exposure, suggesting that mAb penetration and retention in tumors reflected affinity-based differences in tumor catabolism. Together, these results suggest that high-density, rapidly internalizing antigens subject high-affinity antibodies to greater internalization and degradation, thereby limiting their penetration of tumors. In contrast, lower-affinity antibodies penetrate tumors more effectively when rates of antibody-antigen dissociation are higher than those of antigen internalization. Together, our findings offer insights into how to optimize the ability of therapeutic antibodies to penetrate tumors.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Neoplasias/imunologia , Neoplasias Ovarianas/imunologia , Receptor ErbB-2/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacocinética , Afinidade de Anticorpos/imunologia , Antígenos de Neoplasias/metabolismo , Ligação Competitiva , Linhagem Celular Tumoral , Endocitose/imunologia , Epitopos/imunologia , Epitopos/metabolismo , Feminino , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Imuno-Histoquímica , Radioisótopos de Índio , Radioisótopos do Iodo , Camundongos , Camundongos SCID , Mutação , Transplante de Neoplasias , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Fatores de Tempo , Distribuição Tecidual , Transplante HeterólogoRESUMO
We have investigated real-time, label-free, in-situ detection of human epidermal growth factor receptor 2 (Her2) in diluted serum using the first longitudinal extension mode of a lead zirconate-lead titanate (PZT)/glass piezoelectric microcantilever sensor (PEMS) with H3 single-chain variable fragment (scFv) immobilized on the 3-mercaptopropyltrimethoxysilane (MPS) insulation layer of the PEMS surface. We showed that with the longitudinal extension mode, the PZT/glass PEMS consisting of a 1 mm long and 127 µm thick PZT layer bonded with a 75 µm thick glass layer with a 1.8 mm long glass tip could detect Her2 at a concentration of 6-60 ng/ml (or 0.06-0.6 nM) in diluted human serum, about 100 times lower than the concentration limit obtained using the lower-frequency flexural mode of a similar PZT/glass PEMS. We further showed that with the longitudinal mode, the PZT/glass PEMS determined the equilibrium H3-Her2 dissociation constant K(d) to be 3.3±0.3 × 10(-8) M consistent with the value, 3.2±0.28 ×10(-8) M deduced by the surface plasmon resonance method (BIAcore).
RESUMO
Piezoelectric microcantilever sensors (PEMS) can be sensitive tools for the detection of proteins and cells in biological fluids. However, currently available PEMS can only be used a single time or must be completely stripped and refunctionalized prior to subsequent uses. Here we report the successful use of an alternative regeneration protocol employing high salt concentrations to remove the target, leaving the functional probe immobilized on the microcantilever surface. Our model system employed the extracellular domain (ECD) of recombinant human Epidermal Growth Factor Receptor (EGFR) as the probe and anti-human EGFR polyclonal antibodies as the target. We report that high concentrations of MgCl2 dissociated polyclonal antibodies specifically bound to EGFR ECD immobilized on the sensor surface without affecting its bioactivity. This simple regeneration protocol both minimized the time required to re-conjugate the probe and preserved the density of probe immobilized on PEMS surface, yielding identical biosensor sensitivity over a series of assays.
Assuntos
Técnicas Biossensoriais/instrumentação , Anticorpos/química , Antígenos/química , Biomarcadores/análise , Técnicas Biossensoriais/métodos , Desenho de Equipamento , HumanosRESUMO
Many factors contribute to successful tumor targeting by antibodies. Besides properties of the tumor tissue and general antibody pharmacology, a relationship exists between an antibody and its antigen that can shape penetration, catabolism, specificity, and efficacy. The affinity and avidity of the binding interactions play critical roles in these dynamics. In this work, we review the principles that guide models predicting tumor penetration and cellular internalization while providing a critical overview of studies aimed at experimentally determining the specific role of affinity and avidity in these processes. One should gain the perspective that binding affinity can, in part, dictate the localization of antibodies in tumors, leading to high concentrations in the perivascular space or low concentrations diffused throughout the tumor. These patterns can be simply due to the diminution of available dose by binding antigen and are complicated by internalization and degradation stemming from slow rates of dissociation. As opposed to the trend of simply increasing affinity to increase efficacy, novel strategies that increase avidity and broaden specificity have made significant progress in tumor targeting.