Avidity‐Based Selection of Tissue‐Specific CAR‐T Cells from a Combinatorial Cellular Library of CARs

Ma, Peixiang; Ren, Ping; Zhang, Chuyue; Tang, Jiaxing; Zheng, Yu; Zhu, Xuekai; Fan, Kun; Li, Guanglei; Zhu, Wei; Sang, Wei; Min, Chenyu; Chen, Wenzhang; Huang, Xingxu; Yang, Guang; Lerner, Richard A.

doi:10.1002/advs.202003091

Cited by 11 publications

(9 citation statements)

References 54 publications

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“…While this provides useful information regarding the scFv itself, this analysis does not consider the geography of chimeric antigen receptor or target antigen expression within the cell membrane, nor the influence of secondary binding interactions mediated by other pro-adhesive molecules. Accordingly, some studies of scFv affinity have proven poorly predictive of CAR function ( 36 ). To measure the global strength of interaction between our panel of CD19-specific CAR T-cells and a target cell that expresses this antigen, we undertook z-Movi avidity testing.…”

Section: Discussionmentioning

confidence: 99%

Engineering of an Avidity-Optimized CD19-Specific Parallel Chimeric Antigen Receptor That Delivers Dual CD28 and 4-1BB Co-Stimulation

Halim

Das²,

Larcombe-Young

et al. 2022

Front. Immunol.

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Co-stimulation is critical to the function of chimeric antigen receptor (CAR) T-cells. Previously, we demonstrated that dual co-stimulation can be effectively harnessed by a parallel (p)CAR architecture in which a CD28-containing second generation CAR is co-expressed with a 4-1BB containing chimeric co-stimulatory receptor (CCR). When compared to linear CARs, pCAR-engineered T-cells elicit superior anti-tumor activity in a range of pre-clinical models. Since CD19 is the best validated clinical target for cellular immunotherapy, we evaluated a panel of CD19-specific CAR and pCAR T-cells in this study. First, we generated a panel of single chain antibody fragments (scFvs) by alanine scanning mutagenesis of the CD19-specific FMC63 scFv (VH domain) and these were incorporated into second generation CD28+CD3ζ CARs. The resulting panel of CAR T-cells demonstrated a broad range of CD19 binding ability and avidity for CD19-expressing tumor cells. Each scFv-modified CAR was then converted into a pCAR by co-expression of an FMC63 scFv-targeted CCR with a 4-1BB endodomain. When compared to second generation CARs that contained an unmodified or mutated FMC63 scFv, each pCAR demonstrated a significant enhancement of tumor re-stimulation potential and IL-2 release, reduced exhaustion marker expression and enhanced therapeutic efficacy in mice with established Nalm-6 leukemic xenografts. These data reinforce the evidence that the pCAR platform delivers enhanced anti-tumor activity through effective provision of dual co-stimulation. Greatest anti-tumor activity was noted for intermediate avidity CAR T-cells and derived pCARs, raising the possibility that effector to target cell avidity is an important determinant of efficacy.

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Section: Discussionmentioning

confidence: 99%

Engineering of an Avidity-Optimized CD19-Specific Parallel Chimeric Antigen Receptor That Delivers Dual CD28 and 4-1BB Co-Stimulation

Halim

Das²,

Larcombe-Young

et al. 2022

Front. Immunol.

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“…S-trimer-His and SA3-hFc were crosslinked using collision-induced dissociation (CID)cleavable cross-linker, disuccinimidyl sulfoxide (DSSO) following the described procedure [22]. Briefly, S-trimer-His and SA3-hFc were mixed in PBS and incubated for 30 min at RT.…”

Section: Chemical Crosslinking and Mass Spectrometrymentioning

confidence: 99%

Potent NTD-Targeting Neutralizing Antibodies against SARS-CoV-2 Selected from a Synthetic Immune System

Wang

Liu

et al. 2023

Vaccines

Self Cite

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The majority of neutralizing antibodies (NAbs) against SARS-CoV-2 recognize the receptor-binding domain (RBD) of the spike (S) protein. As an escaping strategy, the RBD of the virus is highly variable, evolving mutations to thwart a natural immune response or vaccination. Targeting non-RBD regions of the S protein thus provides a viable alternative to generating potential, robust NAbs. Using a pre-pandemic combinatorial antibody library of 1011, through an alternate negative and positive screening strategy, 11 non-RBD-targeting antibodies are identified. Amongst one NAb that binds specifically to the N-terminal domain of the S protein, SA3, shows mutually non-exclusive binding of the angiotensin-converting enzyme 2 receptor with the S protein. SA3 appears to be insensitive to the conformational change and to interact with both the “open” and “closed” configurations of the trimeric S protein. SA3 shows compatible neutralization as S-E6, an RBD-targeting NAb, against the wild type and variant of concern (VOC) B.1.351 (Beta) of the SARS-CoV-2 pseudo virus. More importantly, the combination of SA3 with S-E6 is synergistic and recovers from the 10-fold loss in neutralization efficacy against the VOC B.1.351 pseudo virus.

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“…While simple in principle, the modular design of CARs can become quite complex, with factors such as the affinity of the scFv, the length of the extracellular hinge region, the structure of the transmembrane region, and the identities of the intracellular costimulatory domains each having a significant impact on the final CAR activity 139 . At present, it is not possible to reliably predict how a specific CAR design will function without laborious empirical testing 140 , and while large libraries (10 6 constructs) of CARs can be generated and transferred into immune cells simultaneously, only a small fraction of the total library can be analyzed 141,142 . The functional screening technologies described herein may be able to address the screening bottleneck of synthetic CAR molecules by translating functional CAR assessment into a high-throughput screening process that can analyze entire CAR libraries using functional cell sorting technologies.…”

Section: Limitation Of Current Analysismentioning

confidence: 99%

Single-cell sorting based on secreted products for functionally defined cell therapies

et al. 2022

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Cell therapies have emerged as a promising new class of “living” therapeutics over the last decade and have been particularly successful for treating hematological malignancies. Increasingly, cellular therapeutics are being developed with the aim of treating almost any disease, from solid tumors and autoimmune disorders to fibrosis, neurodegenerative disorders and even aging itself. However, their therapeutic potential has remained limited due to the fundamental differences in how molecular and cellular therapies function. While the structure of a molecular therapeutic is directly linked to biological function, cells with the same genetic blueprint can have vastly different functional properties (e.g., secretion, proliferation, cell killing, migration). Although there exists a vast array of analytical and preparative separation approaches for molecules, the functional differences among cells are exacerbated by a lack of functional potency-based sorting approaches. In this context, we describe the need for next-generation single-cell profiling microtechnologies that allow the direct evaluation and sorting of single cells based on functional properties, with a focus on secreted molecules, which are critical for the in vivo efficacy of current cell therapies. We first define three critical processes for single-cell secretion-based profiling technology: (1) partitioning individual cells into uniform compartments; (2) accumulating secretions and labeling via reporter molecules; and (3) measuring the signal associated with the reporter and, if sorting, triggering a sorting event based on these reporter signals. We summarize recent academic and commercial technologies for functional single-cell analysis in addition to sorting and industrial applications of these technologies. These approaches fall into three categories: microchamber, microfluidic droplet, and lab-on-a-particle technologies. Finally, we outline a number of unmet needs in terms of the discovery, design and manufacturing of cellular therapeutics and how the next generation of single-cell functional screening technologies could allow the realization of robust cellular therapeutics for all patients.

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Avidity‐Based Selection of Tissue‐Specific CAR‐T Cells from a Combinatorial Cellular Library of CARs

Cited by 11 publications

References 54 publications

Engineering of an Avidity-Optimized CD19-Specific Parallel Chimeric Antigen Receptor That Delivers Dual CD28 and 4-1BB Co-Stimulation

Engineering of an Avidity-Optimized CD19-Specific Parallel Chimeric Antigen Receptor That Delivers Dual CD28 and 4-1BB Co-Stimulation

Potent NTD-Targeting Neutralizing Antibodies against SARS-CoV-2 Selected from a Synthetic Immune System

Single-cell sorting based on secreted products for functionally defined cell therapies

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