RESUMEN
BACKGROUND: Despite clinical success with T cell engagers (TCEs) targeting hematological malignancies, achieving a safe and efficacious dose in patients with solid tumors remains challenging. Due to potency, low levels of target antigen expression on normal tissues may not be tolerated. To overcome this, we engineered a novel conditionally active TCE design called COBRA (Conditional Bispecific Redirected Activation). Administered as prodrugs, COBRAs bind to cell surface antigens on both normal and tumor tissues but are preferentially activated within the tumor microenvironment. METHODS: A COBRA was engineered to target EGFR, TAK-186. The potency of precleaved TAK-186 relative to a non-cleavable control was assessed in vitro. Mice bearing established solid tumors expressing a range of EGFR levels were administered a single bolus of human T cells, and concurrently treated with TAK-186 and associated controls intravenously. We assessed the plasma and tumor exposure of intact and cleaved TAK-186. RESULTS: TAK-186 shows potent redirected T cell killing of antigen expressing tumor cells. In vivo efficacy studies demonstrate regressions of established solid tumors, dependent on intratumoral COBRA cleavage. Pharmacokinetic studies reveal TAK-186 is stable in circulation, but once activated is rapidly cleared due to loss of its albumin-binding half-life extension domain. CONCLUSIONS: The studies shown support the advancement of TAK-186, and the pursuit of additional COBRA TCEs for the treatment of solid tumors.
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Receptores ErbB , Neoplasias , Linfocitos T , Animales , Receptores ErbB/inmunología , Receptores ErbB/metabolismo , Humanos , Inmunoterapia , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/inmunología , Linfocitos T/inmunología , Microambiente TumoralRESUMEN
BACKGROUND: COBRA™ (COnditional Bispecific Redirected Activation) T-cell engagers are designed to target solid tumors as a single polypeptide chain prodrug that becomes activated by proteolysis in the tumor microenvironment. One COBRA molecule comprises seven Ig domains: three single-domain antibodies (sdAbs) recognizing a tumor target or human serum albumin (HSA), and CD3ε-binding variable fragment heavy chain (VH) and variable fragment light chain (VL) and their inactivated counterparts, VHi and VLi. Pairing of VH and VL, and VLi and VHi into single-chain variable fragments (Fv) is prevented by shortened inter-domain linkers. Instead, VH and VL are expected to interact with VLi and VHi, respectively, thus making a diabody whose binding to CD3ε on the T-cells is impaired. METHODS: We analyzed the structure of an epidermal growth factor receptor (EGFR) COBRA in solution using negative stain electron microscopy (EM) and small-angle X-ray scattering (SAXS). RESULTS: We found that this EGFR COBRA forms stable monomers with a very dynamic interdomain arrangement. At most, only five domains at a time appeared ordered, and only one VH-VL pair was found in the Fv orientation. Nonenzymatic posttranslational modifications suggest that the CDR3 loops in the VL-VHi pair are exposed but are buried in the VH-VLi pair. The MMP9 cleavage rate of the prodrug when bound to recombinant EGFR or HSA is not affected, indicating positioning of the MMP9-cleavable linker away from the EGFR and HSA binding sites. CONCLUSION: Here, we propose a model for EGFR COBRA where VH and VLi form an Fv, and VL and VHi do not, possibly interacting with other Ig domains. SAXS and MMP9 cleavage analyses suggest that all COBRA molecules tested have a similar structural architecture.
RESUMEN
Conditionally active COBRA™ (COnditional Bispecific Redirected Activation) T cell engagers are engineered to overcome the limitations of inherently active first-generation T cell engagers, which are unable to discern between tumor and healthy tissues. Designed to be administered as prodrugs, COBRAs target cell surface antigens upon administration, but engage T cells only after they are activated within the tumor microenvironment (TME). This allows COBRAs to be preferentially turned on in tumors while safely remaining inactive in healthy tissue. Here, we describe the development of the COBRA design and the characterization of these conditionally active T cell engagers. Upon administration COBRAs are engineered to bind to tumor-associated antigens (TAAs) and serum albumin (to extend their half-life in circulation), but are inhibited from interacting with the T cell receptor complex signaling molecule CD3. In the TME, a matrix metalloproteinase (MMP)-mediated linker cleavage event occurs within the COBRA construct, which rearranges the molecule, allowing it to co-engage TAAs and CD3, thereby activating T cells against the tumor. COBRAs are conditionally activated through cleavage with MMP9, and once active are highly potent, displaying sub-pM EC50s in T cell killing assays. Studies in tumor-bearing mice demonstrate COBRA administration completely regresses established solid tumor xenografts. These results strongly support the further characterization of the novel COBRA design in preclinical development studies.
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Anticuerpos Biespecíficos , Antígenos de Neoplasias , Antineoplásicos Inmunológicos , Inmunoterapia , Activación de Linfocitos , Neoplasias Experimentales/terapia , Linfocitos T/inmunología , Animales , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Antineoplásicos Inmunológicos/química , Antineoplásicos Inmunológicos/inmunología , Antineoplásicos Inmunológicos/farmacología , Células HT29 , Humanos , Células Jurkat , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias Experimentales/inmunología , Ingeniería de Proteínas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Amassin-1 mediates a rapid cell adhesion that tightly adheres sea urchin coelomocytes (body cavity immunocytes) together. Three major structural regions exist in amassin-1: a short beta region, 3 coiled coils, and an olfactomedin domain. Amassin-1 contains 8 disulfide-bonded cysteines that, upon reduction, render it inactive. Truncated forms of recombinant amassin-1 were expressed and purified from Pichia pastoris and their disulfide bonding and biological activities investigated. Expressed alone, the olfactomedin domain contained 2 intramolecular disulfide bonds, existed in a monomeric state, and inhibited amassin-1-mediated clotting of coelomocytes by a calcium-dependent cell-binding activity. The N-terminal beta region, containing 3 cysteines, was not required for clotting activity. The coiled coils may dimerize amassin-1 in a parallel orientation through a homodimerizing disulfide bond. Neither amassin-1 fragments that were disulfide-linked as dimers or that were engineered to exist as dimers induced coelomocytes clotting. Clotting required higher multimeric states of amassin-1, possibly tetramers, which occurred through the N-terminal beta region and (or) the first segment of coiled coils.
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Moléculas de Adhesión Celular/química , Proteínas de la Matriz Extracelular/química , Glicoproteínas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Calcio/metabolismo , Moléculas de Adhesión Celular/metabolismo , Dimerización , Disulfuros/metabolismo , Glicoproteínas/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Pichia/genética , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Olfactomedin (OLF) domain proteins maintain extracellular protein-protein interactions in diverse phyla. Only one OLF family member, amassin-1, has been described from the sea urchin Strongylocentrotus purpuratus, a basal invertebrate deuterostome. Amassin-1 mediates intercellular adhesion of coelomocytes (immunocytes). Here we describe the protein structural features of four additional OLF proteins, the total for the genome being five. Phylogenetically, four of these proteins (the amassins) form a subgroup among previously identified OLF proteins. The fifth OLF protein is within the colmedin subfamily and contains a type II transmembrane domain, collagen repeats, and an OLF domain. Sea urchin OLF proteins represent an intermediate diversification between protostomes and vertebrates. Transcripts of all five OLF family members are in coelomocytes and adult radial nerve tissue. Transcripts for some OLF proteins increase during late larval stages. Transcript levels for amassin-1 increase 1,000,000-fold, coinciding with formation of the adult urchin rudiment within the larval body.
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Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Strongylocentrotus purpuratus/genética , Strongylocentrotus purpuratus/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Secuencia Conservada , Cartilla de ADN/genética , Proteínas de la Matriz Extracelular/química , Regulación del Desarrollo de la Expresión Génica , Variación Genética , Glicoproteínas/química , Larva/crecimiento & desarrollo , Larva/metabolismo , Datos de Secuencia Molecular , Filogenia , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Strongylocentrotus purpuratus/crecimiento & desarrollo , Distribución TisularRESUMEN
A family of animal proteins is emerging which contain a conserved protein motif known as an olfactomedin (OLF) domain. Novel extracellular protein-protein interactions occur through this domain. The OLF-family member amassin, from the sea urchin Strongylocentrotus purpuratus, has previously been identified to mediate a rapid cell-adhesion event resulting in a large aggregation of coelomocytes, the circulating immune cells. In this work, heterologous expression and purification of the OLF domain from amassin was carried out and initial crystallization trials were performed. A native data set has been collected, extending to 2.7 A under preliminary cryoconditions, using an in-house generator. This work leads the way to the determination of the first structure of an OLF domain.
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Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/aislamiento & purificación , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/aislamiento & purificación , Glicoproteínas/química , Glicoproteínas/aislamiento & purificación , Animales , Moléculas de Adhesión Celular/genética , Cristalización , Cristalografía por Rayos X , Escherichia coli/genética , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/genética , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Strongylocentrotus purpuratusRESUMEN
Sea urchins have a fluid-filled body cavity, the coelom, containing four types of immunocytes called coelomocytes. Within minutes after coelomic fluid is removed from the body cavity, a massive cell-cell adhesion of coelomocytes occurs. This event is referred to as clotting. Clotting is thought to be a defense mechanism against loss of coelomic fluid if the body wall is punctured, and it may also function in the cellular encapsulation of foreign material and microbes. Here we show that this intercoelomocyte adhesion is mediated by amassin, a coelomic plasma protein with a relative molecular mass (Mr) of 75 kD. Amassin forms large disulfide-bonded aggregates that adhere coelomocytes to each other. One half of the amassin protein comprises an olfactomedin (OLF) domain. Structural predictions show that amassin and other OLF domain-containing vertebrate proteins share a common architecture. This suggests that other proteins of the OLF family may function in intercellular adhesion. These findings are the first to demonstrate a function for a protein of the OLF family.