RESUMEN
Siglec-6 is a lectin receptor with restricted expression in the placenta, mast cells and memory B-cells. Although Siglec-6 is expressed in patients with chronic lymphocytic leukemia (CLL), its pathophysiological role has not been elucidated. We describe here a role for Siglec-6 in migration and adhesion of CLL B cells to CLL- bone marrow stromal cells (BMSCs) in vitro and compromised migration to bone marrow and spleen in vivo. Mass spectrometry analysis revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of MEC1-002 CLL cells with a Siglec-6 ligand, sTn, results in Cdc42 activation, WASP protein recruitment and F-actin polymerization, which are all associated with cell migration. Therapeutically, a Siglec-6/CD3-bispecific T-cell-recruiting antibody (T-biAb) improves overall survival in an immunocompetent mouse model and eliminates CLL cells in a patient derived xenograft model. Our findings thus reveal a migratory role for Siglec-6 in CLL, which can be therapeutically targeted using a Siglec-6 specific T-biAb.
Asunto(s)
Adhesión Celular , Movimiento Celular , Lectinas , Leucemia Linfocítica Crónica de Células B , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Humanos , Animales , Lectinas/metabolismo , Ratones , Antígenos CD/metabolismo , Antígenos CD/genética , Femenino , Linfocitos B/metabolismo , Linfocitos B/inmunología , Antígenos de Diferenciación Mielomonocítica/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Línea Celular Tumoral , Células Madre Mesenquimatosas/metabolismo , Masculino , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
On-target off-tumour toxicity limits the anticancer applicability of chimaeric antigen receptor (CAR) T cells. Here we show that the tumour-targeting specificity and activity of T cells with a CAR consisting of an antibody with a lysine residue that catalytically forms a reversible covalent bond with a 1,3-diketone hapten can be regulated by the concentration of a small-molecule adapter. This adapter selectively binds to the hapten and to a chosen tumour antigen via a small-molecule binder identified via a DNA-encoded library. The adapter therefore controls the formation of a covalent bond between the catalytic antibody and the hapten, as well as the tethering of the CAR T cells to the tumour cells, and hence the cytotoxicity and specificity of the cytotoxic T cells, as we show in vitro and in mice with prostate cancer xenografts. Such small-molecule switches of T-cell cytotoxicity and specificity via an antigen-independent 'universal' CAR may enhance the control and safety profile of CAR-based cellular immunotherapies.
RESUMEN
In vitro display technologies have been successfully utilized for the discovery and evolution of monoclonal antibodies (mAbs) for diagnostic and therapeutic applications, with phage display and yeast display being the most commonly used platforms due to their simplicity and high efficiency. As their prokaryotic or lower eukaryotic host organisms typically have no or different post-translational modifications, several mammalian cell-based display and screening technologies for isolation and optimization of mAbs have emerged and are being developed. We report here a novel and useful mammalian cell display platform based on the PiggyBac transposon system to display mAbs in a single-chain Fab (scFab) format on the surface of HEK293F cells. Immune rabbit antibody libraries encompassing ~7 × 107 independent clones were generated in an all-in-one transposon vector, stably delivered into HEK293F cells and displayed as an scFab with rabbit variable and human constant domains. After one round of magnetic activated cell sorting and two rounds of fluorescence activated cell sorting, mAbs with high affinity in the subnanomolar range and cross-reactivity to the corresponding human and mouse antigens were identified, demonstrating the power of this platform for antibody discovery. We developed a highly efficient mammalian cell display platform based on the PiggyBac transposon system for antibody discovery, which could be further utilized for humanization as well as affinity and specificity maturation.
RESUMEN
A phagemid is a plasmid that contains the origin of replication and packaging signal of a filamentous phage. Following bacterial transformation, a phagemid can be replicated and amplified as a plasmid, using a double-stranded DNA origin of replication, or it can be replicated as single-stranded DNA for packaging into filamentous phage particles. The use of phagemids enables phage display of large proteins, such as antibody fragments. Phagemid pComb3 was among the first phage display vectors used for the generation and selection of antibody libraries in the 50-kDa Fab format, a monovalent proxy of natural antibodies. Affording a robust and versatile tool for more than three decades, phage display vectors of the pComb3 phagemid family have been widely used for the discovery, affinity maturation, and humanization of antibodies in Fab, scFv, and single-domain formats from naive, immune, and synthetic antibody repertoires. In addition, they have been used for broadening phage display to the mining of nonimmunoglobulin repertoires. This review examines conceptual, functional, and molecular features of the first-generation phage display vector pComb3 and its successors, pComb3H, pComb3X, and pC3C.
RESUMEN
Monoclonal antibody (mAb)-based biologics are well established treatments of cancer. Antibody discovery campaigns are typically directed at a single target of interest, which inherently limits the possibility of uncovering novel antibody specificities or functionalities. Here, we present a target-unbiased approach for antibody discovery that relies on generating mAbs against native target cell surfaces via phage display. This method combines a previously reported method for improved whole-cell phage display selections with next-generation sequencing analysis to efficiently identify mAbs with the desired target cell reactivity. Applying this method to multiple myeloma cells yielded a panel of >50 mAbs with unique sequences and diverse reactivities. To uncover the identities of the cognate antigens recognized by this panel, representative mAbs from each unique reactivity cluster were used in a multi-omic target deconvolution approach. From this, we identified and validated three cell surface antigens: PTPRG, ICAM1, and CADM1. PTPRG and CADM1 remain largely unstudied in the context of multiple myeloma, which could warrant further investigation into their potential as therapeutic targets. These results highlight the utility of optimized whole-cell phage display selection methods and could motivate further interest in target-unbiased antibody discovery workflows.
Asunto(s)
Anticuerpos Monoclonales , Antígenos , Biblioteca de Péptidos , Humanos , Especificidad de Anticuerpos , Molécula 1 de Adhesión Celular , Técnicas de Visualización de Superficie Celular/métodos , Multiómica , Mieloma Múltiple/genéticaRESUMEN
Antibody-drug conjugates (ADCs) are cancer chemotherapeutics that utilize a monoclonal antibody (mAb)-based delivery system, a cytotoxic payload, and a chemical linker. ADC payloads must be strategically functionalized to allow linker attachment without perturbing the potency required for ADC efficacy. We previously developed a biocatalytic system for the precise functionalization of tiancimycin (TNM)-based payloads. The TNMs are anthraquinone-fused enediynes (AFEs) and have yet to be translated into the clinic. Herein, we report the translation of biocatalytically functionalized TNMs into ADCs in combination with the dual-variable domain (DVD)-mAb platform. The DVD enables both site-specific conjugation and a plug-and-play modularity for antigen-targeting specificity. We evaluated three linker chemistries in terms of TNM-based ADC potency and antigen selectivity, demonstrating a trade-off between potency and selectivity. This represents the first application of AFE-based payloads to DVDs for ADC development, a workflow that is generalizable to further advance AFE-based ADCs for multiple cancer types.
Asunto(s)
Antineoplásicos , Inmunoconjugados , Neoplasias , Humanos , Inmunoconjugados/química , Anticuerpos Monoclonales/química , Antineoplásicos/farmacología , Antineoplásicos/química , Relación Estructura-ActividadRESUMEN
Site-specifically modified radioimmunoconjugates exhibit superior in vitro and in vivo behavior compared to analogues synthesized via traditional stochastic methods. However, the development of approaches to site-specific bioconjugation that combine high levels of selectivity, simple reaction conditions, and clinical translatability remains a challenge. Herein, we describe a novel solution to this problem: the use of dual-variable domain immunoglobulins (DVD-IgG). More specifically, we report the synthesis, in vitro evaluation, and in vivo validation of a 177Lu-labeled radioimmunoconjugate based on HER2DVD, a DVD-IgG containing the HER2-targeting variable domains of trastuzumab and the catalytic variable domains of IgG h38C2. To this end, we first modified HER2DVD with a phenyloxadiazolyl methlysulfone-modified variant of the chelator CHX-Aâ³-DTPA (PODS-CHX-A''-DTPA) and verified the site-specificity of the conjugation for the reactive lysines within the catalytic domains via chemical assay, MALDI-ToF mass spectrometry, and SDS-PAGE. The chelator-bearing immunoconjugate was subsequently labeled with [177Lu]Lu3+ to produce the completed radioimmunoconjugate, [177Lu]Lu-CHX-Aâ³-DTPAPODS-HER2DVD, in >80% radiochemical conversion and a specific activity of 29.5 ± 7.1 GBq/µmol. [177Lu]Lu-CHX-Aâ³-DTPAPODS-HER2DVD did not form aggregates upon prolonged incubation in human serum, displayed 87% stability to demetalation over a 7 days of incubation in serum, and exhibited an immunoreactive fraction of 0.95 with HER2-coated beads. Finally, we compared the pharmacokinetic profile of [177Lu]Lu-CHX-Aâ³-DTPAPODS-HER2DVD to that of a 177Lu-labeled variant of trastuzumab in mice bearing subcutaneous HER2-expressing BT-474 human breast cancer xenografts. The in vivo performance of [177Lu]Lu-CHX-Aâ³-DTPAPODS-HER2DVD matched that of 177Lu-labeled trastuzumab, with the former producing a tumoral activity concentration of 34.1 ± 12.1 %ID/g at 168 h and tumor-to-blood, tumor-to-liver, and tumor-to-kidney activity concentration ratios of 10.5, 9.6, and 21.8, respectively, at the same time point. Importantly, the DVD-IgG did not exhibit a substantially longer serum half-life than the traditional IgG despite its significantly larger size (202 kDa for the former vs 148 kDa for the latter). Taken together, these data suggest that DVD-IgGs represent a viable platform for the future development of highly effective site-specifically labeled radioimmunoconjugates for diagnostic imaging, theranostic imaging, and radioimmunotherapy.
Asunto(s)
Neoplasias de la Mama , Inmunoconjugados , Humanos , Animales , Ratones , Femenino , Inmunoconjugados/uso terapéutico , Línea Celular Tumoral , Trastuzumab/uso terapéutico , Trastuzumab/farmacocinética , Quelantes/química , Neoplasias de la Mama/tratamiento farmacológico , Ácido Pentético/química , Inmunoglobulina G/uso terapéuticoRESUMEN
BACKGROUND: Despite numerous therapeutic options, safe and curative therapy is unavailable for most patients with chronic lymphocytic leukemia (CLL). A drawback of current therapies such as the anti-CD20 monoclonal antibody (mAb) rituximab is the elimination of all healthy B cells, resulting in impaired humoral immunity. We previously reported the identification of a patient-derived, CLL-binding mAb, JML-1, and identified sialic acid-binding immunoglobulin-like lectin-6 (Siglec-6) as the target of JML-1. Although little is known about Siglec-6, it appears to be an attractive target for cancer immunotherapy due to its absence on most healthy cells and tissues. METHODS: We used a target-specific approach to mine for additional patient-derived anti-Siglec-6 mAbs. To assess the therapeutic utility of targeting Siglec-6 in the context of CLL, T cell-recruiting bispecific antibodies (T-biAbs) that bind to Siglec-6 and CD3 were engineered into single-chain variable fragment-Fc and dual-affinity retargeting (DART)-Fc constructs. T-biAbs were evaluated for their activity in vitro, ex vivo, and in vivo. RESULTS: We discovered the anti-Siglec-6 mAbs RC-1 and RC-2, which bind with higher affinity than JML-1 yet maintain similar specificity. Both JML-1 and RC-1 T-biAbs were effective at activating T cells and killing Siglec-6+ target cells. The RC-1 clone in the DART-Fc format was the most potent T-biAb tested and was the only anti-Siglec-6 T-biAb that eliminated Siglec-6+ primary CLL cells via autologous T cells at pathological T-to-CLL cell ratios. Tested at healthy T-to-B cell ratios, it also eliminated a Siglec-6+ fraction of primary B cells from healthy donors. The subpicomolar potency of the DART-Fc format was attributed to the reduction in the length and flexibility of the cytolytic synapse. Furthermore, the RC-1 T-biAb was effective at clearing MEC1 CLL cells in vivo and demonstrated a circulatory half-life of over 7 days. CONCLUSION: Siglec-6-targeting T-biAbs are highly potent and specific for eliminating Siglec-6+ leukemic and healthy B cells while sparing Siglec-6- healthy B cells, suggesting a unique treatment strategy for CLL with diminished suppression of humoral immunity. Our data corroborate reports that T-biAb efficacy is dependent on synapse geometry and reveal that synapse architecture can be tuned via antibody engineering. Our fully human anti-Siglec-6 antibodies and T-biAbs have potential for cancer immunotherapy. TRIAL REGISTRATION NUMBER: NCT00923507.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Linfocitos T , Linfocitos B , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , InmunoterapiaRESUMEN
The success of chimeric antigen receptor T cell (CAR-T) therapy in the treatment of hematologic malignancies has prompted the development of numerous CAR-T technologies, including switchable CAR-T (sCAR-T) systems that combine a universal CAR-T with bispecific adapter proteins. Owing to their controllability and versatility, sCAR-Ts have received considerable attention. To explore the therapeutic utility of sCAR-Ts targeting the receptor tyrosine kinase ROR1, which is expressed in hematologic and solid malignancies, and to identify bispecific adaptor proteins that efficiently mediate universal CAR-T engagement, a panel of switches based on ROR1-targeting Fabs with different epitopes and affinities was compared in in vitro and in vivo models of ROR1-expressing cancers. For switches targeting overlapping or identical epitopes, potency correlated with affinity. Surprisingly, however, we identified a switch targeting a unique epitope with low affinity but mediating potent and selective antitumor activity in vitro and in vivo. Converted to a conventional CAR-T, the same anti-ROR1 mAb (324) outperformed a clinically investigated conventional CAR-T that is based on an anti-ROR1 mAb (R12) with ~200-fold higher affinity. Thus, demonstrating therapeutic utility on their own, sCAR-Ts also facilitate higher throughput screening for the identification of conventional CAR-T candidates for preclinical and clinical studies.
Asunto(s)
Neoplasias , Receptores Quiméricos de Antígenos , Línea Celular Tumoral , Epítopos , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/terapia , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T/metabolismoAsunto(s)
Linfoma de Burkitt , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Glicoles de Propileno , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Esfingosina/análogos & derivados , Translocación GenéticaRESUMEN
Catalytic antibody 38C2 and its humanized version h38C2 harbor a uniquely reactive lysine at the bottom of a 11 Å deep pocket that permits site-specific conjugation of ß-diketone-, ß-lactam-, and heteroaryl methylsulfonyl-functionalized small and large molecules. Various dual variable domain formats pair a tumor-targeting antibody with h38C2 to enable precise, fast, and stable assembly of antibody-drug conjugates (ADCs). Here, we expand the scope of this ADC assembly strategy by mutating h38C2's reactive lysine to a cysteine. X-ray crystallography of this point mutant, h38C2_K99C, confirmed a deeply buried unpaired cysteine. Probing h38C2_K99C with maleimide, monobromomaleimide, and dibromomaleimide derivatives of a fluorophore revealed highly disparate conjugation efficiencies and stabilities. Dibromomaleimide emerged as a suitable electrophile for the precise, fast, efficient, and stable assembly of ADCs with the h38C2_K99C module. Mass spectrometry indicated the presence of a thio-monobromomaleimide linkage which was further supported by in silico docking studies. Using a dibromomaleimide derivative of the highly potent tubulin polymerization inhibitor monomethyl auristatin F, h38C2_K99C-based ADCs were found to be as potent as h38C2-based ADCs and afford a new assembly route for ADCs with single and dual payloads.
Asunto(s)
Cisteína , Inmunoconjugados , Anticuerpos Monoclonales/química , Cisteína/química , Inmunoconjugados/química , Lisina/químicaRESUMEN
Comparative analyses of four anthraquinone-fused enediyne biosynthetic gene clusters (BGCs) identified YpmL as a cytochrome P450 enzyme unique to the yangpumicin (YPM) BGC. In vitro characterization of YpmL established it as a hydroxylase, catalyzing C-6 hydroxylation in YPM A biosynthesis. In vivo application of YpmL enabled engineered production of four new tiancimycin analogues (14-17). Evaluation of their cytotoxicity against selected human cancer cell lines shed new insights into the enediyne structure-activity relationship.
Asunto(s)
Antraquinonas , Proteínas Bacterianas , Sistema Enzimático del Citocromo P-450 , Antraquinonas/química , Antraquinonas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Conformación Molecular , Estereoisomerismo , Streptomyces/enzimologíaRESUMEN
Antibody-drug conjugates directed against tumor-specific targets have allowed targeted delivery of highly potent chemotherapy to malignant cells while sparing normal cells. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein with limited expression on normal adult tissues and is overexpressed on the surface of malignant cells in mantle cell lymphoma, acute lymphocytic leukemia with t(1;19)(q23;p13) translocation, and chronic lymphocytic leukemia. This differential expression makes ROR1 an attractive target for antibody-drug conjugate therapy, especially in malignancies such as mantle cell lymphoma and acute lymphocytic leukemia, in which systemic chemotherapy remains the gold standard. Several preclinical and phase 1 clinical studies have established the safety and effectiveness of anti-ROR1 monoclonal antibody-based therapies. Herein we describe a humanized, first-in-class anti-ROR1 antibody-drug conjugate, huXBR1-402-G5-PNU, which links a novel anti-ROR1 antibody (huXBR1-402) to a highly potent anthracycline derivative (PNU). We found that huXBR1-402-G5-PNU is cytotoxic to proliferating ROR1+ malignant cells in vitro and suppressed leukemia proliferation and extended survival in multiple models of mice engrafted with human ROR1+ leukemia. Lastly, we show that the B-cell lymphoma 2 (BCL2)-dependent cytotoxicity of huXBR1-402-G5-PNU can be leveraged by combined treatment strategies with the BCL2 inhibitor venetoclax. Together, our data present compelling preclinical evidence for the efficacy of huXBR1-402-G5-PNU in treating ROR1+ hematologic malignancies.
Asunto(s)
Neoplasias Hematológicas , Inmunoconjugados , Leucemia Linfocítica Crónica de Células B , Linfoma de Células del Manto , Animales , Anticuerpos Monoclonales , Linfoma de Células del Manto/tratamiento farmacológico , Linfoma de Células del Manto/genética , RatonesRESUMEN
Triple-negative breast cancer (TNBC) is a highly diverse group of malignant neoplasms which tend to have poor outcomes, and the development of new targets and strategies to treat these cancers is sorely needed. Antibody-drug conjugate (ADC) therapy has been shown to be a promising targeted therapy for treating many cancers, but has only rarely been tried in patients with TNBC. A major reason the efficacy of ADC therapy in the setting of TNBC has not been more fully investigated is the lack of appropriate target molecules. In this work we were able to identify an effective TNBC target for use in immunotherapy. We were guided by our previous observation that in some breast cancer patients the protein tropomyosin receptor kinase B cell surface protein (TrkB) had become immunogenic, suggesting that it was somehow sufficiently chemically different enough (presumably by mutation) to escaped immune tolerance. We postulated that this difference might well offer a means for selective targeting by antibodies. We engineered site-specific ADCs using a dual variable domain (DVD) format which combines anti-TrkB antibody with the h38C2 catalytic antibody. This format enables rapid, one-step, and homogeneous conjugation of ß-lactam-derivatized drugs. Following conjugation to ß-lactam-derivatized monomethyl auristatin F, the TrkB-targeting DVD-ADCs showed potency against multiple breast cancer cell lines, including TNBC cell lines. In addition, our isolation of antibody that specifically recognized the breast cancer-associated mutant form of TrkB, but not the wild type TrkB, indicates the possibility of further refining the selectivity of anti-TrkB DVD-ADCs, which should enhance their therapeutic index. These results confirmed our supposition that TrkB is a potential target for immunotherapy for TNBC, as well as for other cancers with mutated cell surface proteins.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Inmunoconjugados/uso terapéutico , Inmunoterapia/métodos , Glicoproteínas de Membrana/inmunología , Oligopéptidos/uso terapéutico , Receptor trkB/inmunología , Neoplasias de la Mama Triple Negativas/terapia , Línea Celular Tumoral , Femenino , Humanos , Proteínas de la Membrana , Terapia Molecular Dirigida , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Bruton tyrosine kinase inhibitors (BTKis) are a preferred treatment of patients with chronic lymphocytic leukemia (CLL). Indefinite therapy with BTKis, although effective, presents clinical challenges. Combination therapy can deepen responses, shorten treatment duration, and possibly prevent or overcome drug resistance. We previously reported on a CD19/CD3-bispecific antibody (bsAb) that recruits autologous T-cell cytotoxicity against CLL cells in vitro. Compared with observations with samples from treatment-naïve patients, T cells from patients being treated with ibrutinib expanded more rapidly and exerted superior cytotoxic activity in response to the bsAb. In addition to BTK, ibrutinib also inhibits interleukin-2 inducible T-cell kinase (ITK). In contrast, acalabrutinib, does not inhibit ITK. Whether ITK inhibition contributes to the observed immune effects is unknown. To better understand how BTKis modulate T-cell function and cytotoxic activity, we cultured peripheral blood mononuclear cells (PBMCs) from BTKi-naive and ibrutinib- or acalabrutinib-treated CLL patients with CD19/CD3 bsAb in vitro. T-cell expansion, activation, differentiation, and cytotoxicity were increased in PBMCs from patients on treatment with either BTKi compared with that observed for BKTi-naïve patients. BTKi therapy transcriptionally downregulated immunosuppressive effectors expressed by CLL cells, including cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and CD200. CTLA-4 blockade with ipilimumab in vitro increased the cytotoxic activity of the bsAb in BTKi-naïve but not BTKi-treated PBMCS. Taken together, BTKis enhance bsAb-induced cytotoxicity by relieving T cells of immunosuppressive restraints imposed by CLL cells. The benefit of combining bsAb immunotherapy with BTKis needs to be confirmed in clinical trials.
Asunto(s)
Adenina/análogos & derivados , Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Anticuerpos Biespecíficos/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Piperidinas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Adenina/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD19/inmunología , Benzamidas/uso terapéutico , Complejo CD3/inmunología , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Ipilimumab/uso terapéutico , Leucemia Linfocítica Crónica de Células B/inmunología , Masculino , Persona de Mediana Edad , Pirazinas/uso terapéutico , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Over the past 25 years, antibody therapeutics have emerged as clinically and commercially successful pharmaceuticals, rapidly approaching 100 Food and Drug Administration approvals with combined annual global sales exceeding $100 billion. Nearly half of the marketed antibody therapeutics are used in oncology. These antibody-based cancer therapies can be broken down into three categories based on their different mechanisms of action, i.e., (i) natural properties, (ii) engagement of cytotoxic T cells, and (iii) delivery of cytotoxic payloads. Both natural and engineered properties of the antibody molecule are founded on its highly stable and modular architecture. In this review we provide an overview and outlook of the rapidly evolving landscape of antibody-based cancer therapy.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunoterapia Activa/métodos , Inmunoterapia Adoptiva/métodos , Neoplasias/terapia , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Neoplasias/metabolismoRESUMEN
The ammosamides (AMMs) are a family of pyrroloquinoline alkaloids that exhibits a wide variety of bioactivities. A biosynthetic gene cluster (BGC) that is highly homologous in both gene content and genetic organization to the amm BGC was identified by mining the Streptomyces uncialis DCA2648 genome, leading to the discovery of a sub-family of new AMM congeners, named ammosesters (AMEs). The AMEs feature a C-4a methyl ester, differing from the C-4a amide functional group characteristic to AMMs, and exhibit modest cytotoxicity against a broad spectrum of human cancer cell lines, expanding the structure-activity relationship for the pyrroloquinoline family of natural products. Comparative analysis of the ame and amm BGCs supports the use of a scaffold peptide as an emerging paradigm for the biosynthesis of the pyrroloquinoline family of natural products. AME and AMM biosynthesis diverges from a common intermediate by evolving the pathway-specific Ame24 O-methyltransferase and Amm20 amide synthetase, respectively. These findings will surely inspire future efforts to mimic Nature's combinatorial biosynthetic strategies for natural product structural diversity.
Asunto(s)
Genoma Bacteriano , Pirroles/metabolismo , Quinolinas/metabolismo , Streptomyces/metabolismo , Amidas/química , Amidas/metabolismo , Productos Biológicos/química , Productos Biológicos/metabolismo , Humanos , Familia de Multigenes , Pirroles/química , Quinolinas/química , Streptomyces/química , Streptomyces/genéticaRESUMEN
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002-2003. Horseshoe bats (genus Rhinolophus) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related to SARS-CoV-2, has been identified in one horseshoe-bat species. Here we characterize the ability of the S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, pangolin coronavirus (PgCoV), RaTG13, and LyRa11, a bat virus similar to SARS-CoV-1, to bind a range of ACE2 orthologs. We observed that the PgCoV RBD bound human ACE2 at least as efficiently as the SARS-CoV-2 RBD, and that both RBDs bound pangolin ACE2 efficiently. We also observed a high level of variability in binding to closely related horseshoe-bat ACE2 orthologs consistent with the heterogeneity of their RBD-binding regions. However five consensus horseshoe-bat ACE2 residues enhanced ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 pseudoviruses by an enzymatically inactive immunoadhesin form of human ACE2 (hACE2-NN-Fc). Two of these mutations impaired neutralization of SARS-CoV-1 pseudoviruses. An hACE2-NN-Fc variant bearing all five mutations neutralized both SARS-CoV-2 pseudovirus and infectious virus more efficiently than wild-type hACE2-NN-Fc. These data suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of soluble ACE2.
Asunto(s)
Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/inmunología , COVID-19/inmunología , COVID-19/virología , Quirópteros/metabolismo , SARS-CoV-2/genética , Animales , COVID-19/genética , Quirópteros/genética , Especificidad del Huésped/genética , Especificidad del Huésped/inmunología , Humanos , Modelos Moleculares , Mutación , Unión Proteica/genética , Unión Proteica/fisiología , Receptores Virales/metabolismo , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Monoclonal antibody h38C2 is a humanized catalytic antibody that has been used to generate various immunoconjugate species such as chemically programmed antibodies, antibody-drug conjugates, and antibody-siRNA conjugates. Highly efficient and specific conjugation of h38C2 occurs at its uniquely reactive lysine (Lys) residue buried inside the antibody's catalytic pocket. We recently reported the rational mutation of this Lys residue at position 99 in the heavy chain variable domain to an arginine (Arg) residue. The Lys99Arg mutation can be site-selectively conjugated with molecules containing a hapten-like triazolyl-phenylglyoxal (TPG) unit. Here we show that this conjugation is facilitated by the unusual pH-sensitive reactivity of the Arg99 residue, consistent with an indirectly measured pKa of 5.2. The Arg99/TPG conjugation holds promise to further expand the versatility of the h38C2 conjugation platform, such as for the generation of antibody conjugates with dual payloads.