Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies

Rickert, Keith; Grinberg, Luba; Woods, R. Claude; Wilson, Susan; Bowen, Michael A.; Baca, Manuel

doi:10.1080/19420862.2016.1145865

Cited by 17 publications

(14 citation statements)

References 17 publications

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“…Meca32 is a high-affinity rat anti-mouse antibody targeting PV1 commonly used for IHC. We determined the sequence of Meca32 via de novo protein sequencing 10 . Identification of Meca32 protein sequence allowed for generation of novel constructs including bispecific antibodies.…”

Section: Resultsmentioning

confidence: 99%

Targeted drug delivery via caveolae-associated protein PV1 improves lung fibrosis

Marchetti¹,

Burwell²,

Peterson

et al. 2019

Commun Biol

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Systemic administration of bio-therapeutics can result in only a fraction of drug reaching targeted tissues, with the majority of drug being distributed to tissues irrelevant to the drug’s site of action. Targeted delivery to specific organs may allow for greater accumulation, better efficacy, and improved safety. We investigated how targeting plasmalemma vesicle-associated protein (PV1), a protein found in the endothelial caveolae of lungs and kidneys, can promote accumulation in these organs. Using ex vivo fluorescence imaging, we show that intravenously administered αPV1 antibodies localize to mouse lungs and kidneys. In a bleomycin-induced idiopathic pulmonary fibrosis (IPF) mouse model, αPV1 conjugated to Prostaglandin E 2 (PGE 2 ), a known anti-fibrotic agent, significantly reduced collagen content and fibrosis whereas a non-targeted PGE 2 antibody conjugate failed to slow fibrosis progression. Our results demonstrate that PV1 targeting can be utilized to deliver therapeutics to lungs and this approach is potentially applicable for various lung diseases.

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Section: Resultsmentioning

confidence: 99%

Targeted drug delivery via caveolae-associated protein PV1 improves lung fibrosis

Marchetti¹,

Burwell²,

Peterson

et al. 2019

Commun Biol

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“…The diversity created by gene recombination and somatic hypermutation makes protein sequencing of monoclonal antibodies a challenge [34]. Mass spectrometry-based sequencing will provide a single unambiguous sequence for variable domains, which can result in the need for empirical testing of candidate sequences, sometimes iteratively, to identity one that can replicate the activity of the parental antibody [35]. Here, we describe an approach to antibody protein sequencing using the phage display technology and a combinatorial library of sequences followed by mass spectrometry, aiming to investigate the native targets probable to express B10 and C01 mimotopes in Leishmania .…”

Section: Discussionmentioning

confidence: 99%

Leishmania infantum β-Tubulin Identified by Reverse Engineering Technology through Phage Display Applied as Theranostic Marker for Human Visceral Leishmaniasis

Costa

Alves

Carneiro

et al. 2019

IJMS

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Two Leishmania infantum mimotopes (B10 and C01) identified by phage display showed to be antigenic and immunogenic for visceral (VL) and tegumentary (TL) leishmaniasis; however, their biological targets in the parasites have not been identified. The aim of the present study was to investigate the native antigens expressing both mimotopes, and to use them in distinct immunological assays. For this, a subtractive phage display technology was used, where a combinatorial library of single-chain variable fragments (scFv) was employed and the most reactive monoclonal antibodies for each target were captured, being the target antigens identified by mass spectrometry. Results in immunoblotting and immunoprecipitation assays showed that both monoclonal scFvs antibodies identified the β-tubulin protein as the target antigen in L. infantum. To validate these findings, the recombinant protein was cloned, purified and tested for the serodiagnosis of human leishmaniasis, and its immunogenicity was evaluated in PBMC derived from healthy subjects and treated or untreated VL patients. Results showed high diagnostic efficacy, as well as the development of a specific Th1 immune response in the cell cultures, since higher IFN-γ and lower IL-10 production was found.

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“…For LOB12.3 and 3H3-derived Abs, the VL and VH of LOB12.3 and 3H3 were synthesized according to published amino acid sequence 38 . Using indicated VH and mouse IgG1 and IgG2a constant region as template, the full length of heavy chain was obtained by overlapping PCR.…”

Section: Methodsmentioning

confidence: 99%

Optimization of 4-1BB antibody for cancer immunotherapy by balancing agonistic strength with FcγR affinity

Wu³

et al. 2019

Nat Commun

107

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Costimulation of T cell responses with monoclonal antibody agonists (mAb-AG) targeting 4-1BB showed robust anti-tumor activity in preclinical models, but their clinical development was hampered by low efficacy (Utomilumab) or severe liver toxicity (Urelumab). Here we show that isotype and intrinsic agonistic strength co-determine the efficacy and toxicity of anti-4-1BB mAb-AG. While intrinsically strong agonistic anti-4-1BB can activate 4-1BB in the absence of FcγRs, weak agonistic antibodies rely on FcγRs to activate 4-1BB. All FcγRs can crosslink anti-41BB antibodies to strengthen co-stimulation, but activating FcγR-induced antibody-dependent cell-mediated cytotoxicity compromises anti-tumor immunity by deleting 4-1BB + cells. This suggests balancing agonistic activity with the strength of FcγR interaction as a strategy to engineer 4-1BB mAb-AG with optimal therapeutic performance. As a proof of this concept, we have developed LVGN6051, a humanized 4-1BB mAb-AG that shows high anti-tumor efficacy in the absence of liver toxicity in a mouse model of cancer immunotherapy.

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Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies

Cited by 17 publications

References 17 publications

Targeted drug delivery via caveolae-associated protein PV1 improves lung fibrosis

Targeted drug delivery via caveolae-associated protein PV1 improves lung fibrosis

Leishmania infantum β-Tubulin Identified by Reverse Engineering Technology through Phage Display Applied as Theranostic Marker for Human Visceral Leishmaniasis

Optimization of 4-1BB antibody for cancer immunotherapy by balancing agonistic strength with FcγR affinity

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