Nanobody Based Dual Specific CARs

Munter, Stijn De; Ingels, Joline; Goetgeluk, Glenn; Bonte, Sarah; Pille, Melissa; Weening, Karin; Kerre, Tessa; Abken, Hinrich; Vandekerckhove, Bart

doi:10.3390/ijms19020403

Cited by 95 publications

(87 citation statements)

References 48 publications

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“…The smaller size of heavy-chain-only antigen-recognition domains is also an advantage for expression from gene therapy vectors, since decreasing the size of transgenes increases the titer of viral gene therapy vectors 34 . Compared with designs that utilize two scFvs, using two heavy-chain-only antigen-recognition domains simplifies design of bispecific CAR constructs capable of recognizing two antigens 35,36 .…”

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confidence: 99%

Anti-BCMA chimeric antigen receptors with fully human heavy-chain-only antigen recognition domains

et al. 2020

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Chimeric antigen receptor (CAR)-expressing T cells targeting B-cell maturation antigen (BCMA) have activity against multiple myeloma, but improvements in anti-BCMA CARs are needed. We demonstrated recipient anti-CAR T-cell responses against a murine single-chain variable fragment (scFv) used clinically in anti-BCMA CARs. To bypass potential anti-CAR immunogenicity and to reduce CAR binding domain size, here we designed CARs with antigen-recognition domains consisting of only a fully human heavy-chain variable domain without a light-chain domain. A CAR designated FHVH33-CD8BBZ contains a fully human heavy-chain variable domain (FHVH) plus 4-1BB and CD3ζ domains. T cells expressing FHVH33-CD8BBZ exhibit similar cytokine release, degranulation, and mouse tumor eradication as a CAR that is identical except for substitution of a scFv for FHVH33. Inclusion of 4-1BB is critical for reducing activation-induced cell death and promoting survival of T cells expressing FHVH33-containing CARs. Our results indicate that heavy-chain-only anti-BCMA CARs are suitable for evaluation in a clinical trial.

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confidence: 99%

Anti-BCMA chimeric antigen receptors with fully human heavy-chain-only antigen recognition domains

et al. 2020

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“…Also mammalian cell display of nanobodies has found interesting applications, such as their use for the modification of T cells with chimeric antigen receptors (CARs) suitable for cancer immunotherapy. VHHs are preferred to scFvs because are considered less immunogenic, more stable and compact (nanoCARs) [19,100,101].…”

Section: A De Marcomentioning

confidence: 99%

“…Over the years, it emerged that VHHs are effective crystallography chaperones and molecular tools for protein structural characterization [3][4][5], convenient carriers for radioisotopes with extremely short half-life to use for in vivo PET/ SPECT imaging [6][7][8][9][10], valid immunoreagents for the controlled and oriented functionalization of nanoparticles, nanogels and biosensors [11][12][13][14] and that they can be even internalized by mammalian cells when provided with a suitable leader peptide [15]. More recently, their minimal dimension has been particularly appreciated by scientists looking for binders suitable to optimize the performance of super resolution microscopy [16][17][18] and to create tandem chimeric antigen receptors (CARs) that show higher target specificity due to the possibility of binding simultaneously multiple antigens [19]. Finally, their short sequence makes them the simplest antibody-derived candidate for rational mutagenesis and in silico improvement of biophysical features [20][21][22][23].…”

Section: Introductionmentioning

confidence: 99%

Recombinant expression of nanobodies and nanobody-derived immunoreagents

Marco

2020

Protein Expression and Purification

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A B S T R A C TAntibody fragments for which the sequence is available are suitable for straightforward engineering and expression in both eukaryotic and prokaryotic systems. When produced as fusions with convenient tags, they become reagents which pair their selective binding capacity to an orthogonal function. Several kinds of immunoreagents composed by nanobodies and either large proteins or short sequences have been designed for providing inexpensive ready-to-use biological tools. The possibility to choose among alternative expression strategies is critical because the fusion moieties might require specific conditions for correct folding or posttranslational modifications. In the case of nanobody production, the trend is towards simpler but reliable (bacterial) methods that can substitute for more cumbersome processes requiring the use of eukaryotic systems. The use of these will not disappear, but will be restricted to those cases in which the final immunoconstructs must have features that cannot be obtained in prokaryotic cells. At the same time, bacterial expression has evolved from the conventional procedure which considered exclusively the nanobody and nanobody-fusion accumulation in the periplasm. Several reports show the advantage of cytoplasmic expression, surface-display and secretion for at least some applications. Finally, there is an increasing interest to use as a model the short nanobody sequence for the development of in silico methodologies aimed at optimizing the yields, stability and affinity of recombinant antibodies.

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“…The presence of ASCT-2 expression in PC-3 and F98 cell lines was verified with flow cytometry. The analysis was performed using the LSR II (BD Biosciences) as described by De Munter et al 35 . Both cell lines (100,000 cells/ 100 µL) were stained at their surface with a primary (rabbit ab;…”

Section: N5-(4'-fluoro-[11'-biphenyl]-4-yl)glutamine (6)mentioning

confidence: 99%

Radiosynthesis,in vitroand preliminaryin vivoevaluation of the novel glutamine derived PET tracers [¹⁸F]fluorophenylglutamine and [¹⁸F]fluorobiphenylglutamine

Baguet

Verhoeven

Pauwelyn

et al. 2020

Preprint

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AbstractIntroductionGlucose has been deemed the driving force of tumor growth for decades. However, research has shown that several tumors metabolically shift towards glutaminolysis. The development of radiolabeled glutamine derivatives could be a useful molecular imaging tool for visualizing these tumors. We elaborated on the glutamine-derived PET tracers by developing two novel probes, namely [18F]fluorophenylglutamine and [18F]fluorobiphenylglutamineMaterials and methodsBoth tracers were labelled with fluorine-18 using our recently reported ruthenium-based direct aromatic fluorination method. Their affinity was evaluated with a [3H]glutamine inhibition experiment in a human PC-3 and a rat F98 cell line. The imaging potential of [18F]fluorophenylglutamine and [18F]fluorobiphenylglutamine was tested using a mouse PC-3 and a rat F98 tumor model.ResultsThe radiosynthesis of both tracers was successful with overall non-decay corrected yields of 18.46 ± 4.18 % (n=10) ([18F]fluorophenylglutamine) and 8.05 ± 3.25 % (n=5) ([18F]fluorobiphenylglutamine). In vitro inhibition experiments showed a moderate and low affinity of fluorophenylglutamine and fluorobiphenylglutamine, respectively, towards the human ASCT-2 transporter. Both compounds had a low affinity towards the rat ASCT-2 transporter. These results were endorsed by the in vivo experiments with low uptake of both tracers in the F98 rat xenograft, low uptake of [18F]FBPG in the mice PC-3 xenograft and a moderate uptake of [18F]FPG in the PC-3 tumors.ConclusionWe investigated the imaging potential of two novel PET radiotracers [18F]FPG and [18F]FBPG. [18F]FPG is the first example of a glutamine radiotracer derivatized with a phenyl group which enables the exploration of further derivatization of the phenyl group to increase the affinity and imaging qualities. We hypothesize that increasing the affinity of [18F]FPG by optimizing the substituents of the arene ring can result in a high-quality glutamine-based PET radiotracer.Advances in Knowledge and Implications for patient careWe hereby report novel glutamine-based PET-tracers. These tracers are tagged on the arene group with fluorine-18, hereby preventing in vivo defluorination, which can occur with alkyl labelled tracers (e.g. (2S,4R)4-[18F]fluoroglutamine). [18F]FPG shows clear tumor uptake in vivo, has no in vivo defluorination and has a straightforward production. We believe this tracer is a good starting point for the development of a high-quality tracer which is useful for the clinical visualization of the glutamine transport.

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Nanobody Based Dual Specific CARs

Cited by 95 publications

References 48 publications

Anti-BCMA chimeric antigen receptors with fully human heavy-chain-only antigen recognition domains

Anti-BCMA chimeric antigen receptors with fully human heavy-chain-only antigen recognition domains

Recombinant expression of nanobodies and nanobody-derived immunoreagents

Radiosynthesis,in vitroand preliminaryin vivoevaluation of the novel glutamine derived PET tracers [¹⁸F]fluorophenylglutamine and [¹⁸F]fluorobiphenylglutamine

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Nanobody Based Dual Specific CARs

Cited by 95 publications

References 48 publications

Anti-BCMA chimeric antigen receptors with fully human heavy-chain-only antigen recognition domains

Anti-BCMA chimeric antigen receptors with fully human heavy-chain-only antigen recognition domains

Recombinant expression of nanobodies and nanobody-derived immunoreagents

Radiosynthesis,in vitroand preliminaryin vivoevaluation of the novel glutamine derived PET tracers [18F]fluorophenylglutamine and [18F]fluorobiphenylglutamine

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Radiosynthesis,in vitroand preliminaryin vivoevaluation of the novel glutamine derived PET tracers [¹⁸F]fluorophenylglutamine and [¹⁸F]fluorobiphenylglutamine