Catalytic activity of anin vivo tumor targeted anti-CEA scFv::carboxypeptidase G2 fusion protein

Bhatia, J; Sharma, Surinder K.; Chester, Kerry; Pedley, R. Barbara; Boden, R.; Read, D. A.; Boxer, G. M.; Michael, N. P.; Begent, R. H. J.

doi:10.1002/(sici)1097-0215(20000215)85:4<571::aid-ijc20>3.0.co;2-1

Cited by 61 publications

(6 citation statements)

References 16 publications

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“…4 ). In fact, our results corroborated the previously reported tumor cell specificity of the anti-CEA scFv, when used to functionalize Superparamagnetic iron oxide nanoparticles [ 22 , 45 ]. In addition, we had also confirmed the CEA-targeting specificity of our system by evaluating the same NPs conditions in MSI and MSS CEA low expressing CRC cell lines.…”

Section: Targeting Capacity Of Anti-cea Scfv Functionalized Nps Towar...supporting

confidence: 90%

scFv biofunctionalized nanoparticles to effective and safe targeting of CEA-expressing colorectal cancer cells

Silveira,

Martins,

Cruz

et al. 2023

J Nanobiotechnol

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Colorectal cancer (CRC) is one of the deadliest cancers worldwide, with the 5 year survival rate in metastatic cases limited to 12%. The design of targeted and effective therapeutics remains a major unmet clinical need in CRC treatment. Carcinoembryonic antigen (CEA), a glycoprotein overexpressed in most colorectal tumors, may constitute a promising molecule for generating novel CEA-targeted therapeutic strategies for CRC treatment. Here, we developed a smart nanoplatform based on chemical conjugation of an anti-CEA single-chain variable fragment (scFv), MFE-23, with PLGA-PEG polymers to deliver the standard 5-Fluorouracil (5-FU) chemotherapy to CRC cells. We confirmed the specificity of the developed CEA-targeted NPs on the internalization by CEA-expressing CRC cells, with an enhance of threefold in the cell uptake. Additionally, CEA-targeted NPs loaded with 5-FU induced higher cytotoxicity in CEA-expressing cells, after 24 h and 48 h of treatment, reinforcing the specificity of the targeted NPs. Lastly, the safety of CEA-targeted NPs loaded with 5-FU was evaluated in donor-isolated macrophages, with no relevant impact on their metabolic activity nor polarization. Altogether, this proof of concept supports the CEA-mediated internalization of targeted NPs as a promising chemotherapeutic strategy for further investigation in different CEA-associated cancers and respective metastatic sites.Authors: Please confirm if the author names are presented accurately and in the correct sequence (given name, middle name/initial, family name). Author 1 Given name: [Maria José] Last name [Silveira]. Author 7 Given name: [Maria José] Last name [Oliveira]. Also, kindly confirm the details in the metadata are correctokAffiliations: Please check and confirm that the authors and their respective affiliations have been correctly identified and amend if necessary.ok Graphical Abstract

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Section: Targeting Capacity Of Anti-cea Scfv Functionalized Nps Towar...supporting

confidence: 90%

scFv biofunctionalized nanoparticles to effective and safe targeting of CEA-expressing colorectal cancer cells

Silveira,

Martins,

Cruz

et al. 2023

J Nanobiotechnol

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“…For Cloning of scFv-3 and scFv-7 into CPG2 fusions; first, CPG2 was cloned into the NcoI-XhoI linker region of pEHISTEV bacterial expression vector to create pJGS101; second, the scFv-3/7 was subcloned into the SalI-NotI site of pJGS101 to create pJGS201. The CPG2 was previously been used as a bioconjugate to a scFv targeting CEA[ 44 ] and was provided by Mologics (Bedford, UK). The scFv-3/7 fusion proteins were highly insoluble using the scFv lysis buffer (above) but soluble protein could be recovered in lysis buffer containing 100 mM Tris (pH 8.0), 500 mm NaCl, 10% Glycerol, 1 mM PMSF, 100 μg/ml lysozyme, 1 mM DTT, 2 mM MgCl 2 , and benzoase.…”

Section: Methodsmentioning

confidence: 99%

The Development of a Recombinant scFv Monoclonal Antibody Targeting Canine CD20 for Use in Comparative Medicine

et al. 2016

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Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2) that binds both human and canine CD20. NCD1.2 has a sub-nanomolar Kd as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The VH and VL genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv) into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7) isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical VH genes but different VL genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a recombinant anti-CD20 scFv that might form a useful tool for evaluation in bioconjugate-directed anti-CD20 immunotherapies in comparative medicine.

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“…Plate blocking (2% BSA in PBS), wash (1% Tween 20 in PBS) and O-phenylenediamine dihydrochloride (0.25 mg/mL in citrate phosphate/sodium perborate buffer, all reagents from Sigma-Aldrich, Poole, UK) detection stages were as described by Bhatia et al [18]. ELISA plates (Maxisorb, Nalge Nunc, Rochester, NY, USA) were coated overnight at 47C with 0.5 mg CPG2, 0.02 mg V6 or 0.02 mg wild-type CPG2 per well diluted in PBS.…”

Section: Elisasmentioning

confidence: 99%

“…The MFE-23 part of the fusion protein has already been used in the clinic [16,17] and shows greatly reduced immunogenicity compared to the original anti-CEA antibody A5B7. The MFE-CP fusion protein has superior tumor targeting properties [18,19] and promises efficacy in the clinic. Furthermore, it is readily genetically manipulated to execute changes predicted to reduce immunogenicity or generate a functional mutant that could be given after generation of antibody to the wild-type enzyme.…”

Section: Introductionmentioning

confidence: 99%

A strategy for mapping and neutralizing conformational immunogenic sites on protein therapeutics

Spencer

Robson

Purdy³

et al. 2002

Proteomics

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Antibodies are highly specific recognition molecules which are increasingly being applied to target therapy in patients. One type of developmental antibody-based therapy is antibody directed enzyme prodrug therapy (ADEPT) for the treatment of cancer. In ADEPT, an antibody specific to a tumor marker protein delivers a drug-activating enzyme to the cancer. Subsequent intravenous administration of an inactive prodrug results in drug activation and cytotoxicity only within the locale of the tumor. Pilot clinical trials with chemical conjugates of the prodrug activating enzyme carboxypeptidase G2 (CPG2) chemically conjugated with an antibody to and carcinoembryonic antigen (CEA), have shown that CPG2-mediated ADEPT is effective but limited by formation of human antibodies to CPG2 (HACA). We have developed a recombinant fusion protein (termed MFE-CP) of CPG2 with an anti-CEA single chain Fv antibody fragment and we have developed methods to address the immunogenicity of this therapeutic. A HACA-reactive discontinuous epitope on MFE-CP was identified using the crystal structure of CPG2, filamentous phage technology and surface enhanced laser desorption/ionization affinity mass spectrometry. This information was used to create a functional mutant of MFE-CP with a significant reduction (range 19.2 to 62.5%, median 38.5%) in reactivity with the sera of 11 patients with post-therapy HACA. The techniques described here are valuable tools for identifying and adapting undesirable immunogenic sites on protein therapeutics.

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Catalytic activity of anin vivo tumor targeted anti-CEA scFv::carboxypeptidase G2 fusion protein

Cited by 61 publications

References 16 publications

scFv biofunctionalized nanoparticles to effective and safe targeting of CEA-expressing colorectal cancer cells

scFv biofunctionalized nanoparticles to effective and safe targeting of CEA-expressing colorectal cancer cells

The Development of a Recombinant scFv Monoclonal Antibody Targeting Canine CD20 for Use in Comparative Medicine

A strategy for mapping and neutralizing conformational immunogenic sites on protein therapeutics

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