Tunable Affinity Research Articles

Reversibly Reactive Affinity Selection-Mass Spectrometry Enables Identification of Covalent Peptide Binders.

Covalent peptide binders have found applications as activity-based probes and as irreversible therapeutic inhibitors. Currently, there is no rapid, label-free, and tunable affinity selection platform to enrich covalent reactive peptide binders from synthetic libraries. We address this challenge by developing a reversibly reactive affinity selection platform termed ReAct-ASMS enabled by tandem high-resolution mass spectrometry (MS/MS) to identify covalent peptide binders to native protein targets. It uses mixed disulfide-containing peptides to build reversible peptide-protein conjugates that can enrich for covalent variants, which can be sequenced by MS/MS after reduction. Using this platform, we identified covalent peptide binders against two oncoproteins, human papillomavirus 16 early protein 6 (HPV16 E6) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 protein (Pin1). The resulting peptide binders efficiently and selectively cross-link Cys58 of E6 at 37 °C and Cys113 of Pin1 at room temperature, respectively. ReAct-ASMS enables the identification of highly selective covalent peptide binders for diverse molecular targets, introducing an applicable platform to assist preclinical therapeutic development pipelines.

Abstract 1327: Affinity tuning anti-CD123 chimeric antigen receptor natural killer cell therapy to treat acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is a highly prevalent blood and bone marrow cancer characterized by the uncontrolled growth of abnormal myeloblasts. Current treatments for AML often result in systemic toxicities for patients, many of whom will still experience relapse. There is thus an unmet need to develop safer and more effective therapies targeting AML. Chimeric antigen receptor (CAR) T cells, which are engineered to express a cancer-targeting extracellular antibody single-chain variable fragment (scFv) linked to an intracellular T cell-activating domain, have shown promise in treating B cell leukemias, but their success has been limited in myeloid leukemias. This is due in part to the lack of AML-specific target antigens. Moreover, impaired T cell function, a hallmark of AML, is also problematic when relying on autologous T cell activation upon reinfusion. We overcame the limitations of CAR T therapy in AML by developing CAR-expressing natural killer (NK) cells targeting CD123. While present on both healthy and AML cells, CD123 is expressed at >10-fold higher levels on malignant cells. Furthermore, use of NK allows use of fully functional allogeneic cells from healthy donors, since NK cells do not induce graft-versus-host-disease. To optimize the tumor cell selectivity of CAR NK cells, we employed the yeast display directed evolution platform to isolate anti-CD123 scFvs with varying affinities and are identifying and characterizing clones that maximize the interaction with CD123high AML cells over healthy CD123low cells, as determined by binding, cytotoxicity, cytokine secretion, and intracellular signaling studies. Collectively, our efforts introduce a new paradigm for engineered cell therapeutics with significant promise for AML treatment. Citation Format: Monika Kizerwetter, Huilin Yang, Ruyan Rahnama, Challice Bonifant, Jamie Spangler. Affinity tuning anti-CD123 chimeric antigen receptor natural killer cell therapy to treat acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1327.

Abstract 1919: Activity and affinity tuning next-generation immunotoxins for targeted therapy

Abstract Classic cancer-associated receptors, such as EGFR and Her2, are often overexpressed on malignant cells relative to healthy cells. Numerous molecules have been developed to leverage this differential expression with the goal of developing specific and safe targeted therapies, including antibody-drug-conjugates and immunotoxins. The general schematic of these molecules is to fuse toxic payloads -either small molecules or proteins - with a targeting moiety that binds to the cancer-specific receptor. Despite the general success of this approach, even low levels of these receptors on healthy tissues result in on-target/off-tumor toxicity thus limiting the dose of therapeutic molecules available to tumors. Targeted therapies with greater on-tumor efficacy and reduced toxicity are urgently needed in order to further advance therapeutic outcomes. To achieve this, we set out to exploit the unique features of bacterial toxins as highly modular protein delivery systems to design next-generation targeted therapies. We demonstrate the capacity of the immunotoxin platform to deliver various therapeutic proteins, with distinct activities from inhibiting protein synthesis to degrading oncogenic signaling pathways, such as the RAS/MAPK pathway. Further, we show how modifying the affinity, valency, and specificity of the receptor-binding moieties, as well as tuning the enzymatic activity of the toxic enzyme payloads interact to affect the on-target efficacy and off-target toxicity of the targeted molecules. Altogether, these data demonstrate the potential of immunotoxins as highly flexible and tunable platforms for the development of a new class of anti-cancer therapeutics. Citation Format: Huazhu (Peter) Liang, Greg L. Beilhartz, Shi Bo Cao, Roman A. Melnyk. Activity and affinity tuning next-generation immunotoxins for targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1919.

Plug-and-Play Module for Reversible and Continuous Control of DNA Strand Displacement Kinetics.

Regulatory modules for controlling the kinetics of toehold-mediated strand displacement (TMSD) play critical roles in designing dynamic and dissipative DNA chemical reaction networks (CRNs) but are hardwired into sequence designs. Herein, we introduce antitoehold (At), a plug-and-play module for reversible and continuous tuning of TMSD kinetics by temporarily occupying the toehold domain via a metastable duplex and base stacking. We demonstrate that kinetic control can be readily activated or deactivated in real time for any TMSD by simply adding At or anti-At. Continuous tuning of TMSD kinetics can also be achieved by altering the concentration of At. Moreover, the simple addition of At could readily reprogram existing TMSDs into a pulse-generation DNA CRN with continuous tunability. Our At approach also offers a new way for engineering continuously tunable DNA hybridization probes, which may find practical uses for discriminating clinically important mutations. Because of the simplicity, we anticipate that At will find wide applications for engineering DNA CRNs with diverse dynamic and dissipative behaviors, and DNA hybridization probes with tunable affinity and selectivity.

Using protein geometry to optimize cytotoxicity and the cytokine window of a ROR1 specific T cell engager.

T cell engaging bispecific antibodies have shown clinical proof of concept for hematologic malignancies. Still, cytokine release syndrome, neurotoxicity, and on-target-off-tumor toxicity, especially in the solid tumor setting, represent major obstacles. Second generation TCEs have been described that decouple cytotoxicity from cytokine release by reducing the apparent binding affinity for CD3 and/or the TAA but the results of such engineering have generally led only to reduced maximum induction of cytokine release and often at the expense of maximum cytotoxicity. Using ROR1 as our model TAA and highly modular camelid nanobodies, we describe the engineering of a next generation decoupled TCE that incorporates a "cytokine window" defined as a dose range in which maximal killing is reached but cytokine release may be modulated from very low for safety to nearly that induced by first generation TCEs. This latter attribute supports pro-inflammatory anti-tumor activity including bystander killing and can potentially be used by clinicians to safely titrate patient dose to that which mediates maximum efficacy that is postulated as greater than that possible using standard second generation approaches. We used a combined method of optimizing TCE mediated synaptic distance and apparent affinity tuning of the TAA binding arms to generate a relatively long but persistent synapse that supports a wide cytokine window, potent killing and a reduced propensity towards immune exhaustion. Importantly, this next generation TCE induced significant tumor growth inhibition in vivo but unlike a first-generation non-decoupled benchmark TCE that induced lethal CRS, no signs of adverse events were observed.

Cancer Cell Targeting Via Selective Transferrin Receptor Labeling Using Protein-Derived Carbon Dots.

Carbon dot (CD) nanoparticles offer tremendous advantages as fluorescent probes in bioimaging and biosensing; however, they lack specific affinity for biomolecules, limiting their practical applications in selective targeting. Nanoparticles with intrinsic affinity for a target have applications in imaging, cytometry, therapeutics, etc. Toward that end, we report the transferrin receptor (CD71) targeting CDs, synthesized for the first time. The formation of these particles is truly groundbreaking, as direct tuning of nanoparticle affinity was achieved by simple and careful precursor selection of a protein, which has the targeting characteristic of interest. We hypothesized that the retention of the original protein's peptides on the nanoparticle surface provides the CDs with some of the function of the precursor protein, enabling selective binding to the protein's receptor. This was confirmed with FTIR (Fourier transform infrared) data and subsequent affinity-based cell assays. These transferrin (Tf)-derived CDs have been shown to possess an affinity for CD71, a cancer biomarker that is ubiquitously expressed in nearly every cancer cell line due to its central role mediating the uptake of cellular iron. The CDs were tested using the human leukemia cell line HL60 and demonstrated the selective targeting of CD71 and specific triggering of transferrin-mediated endocytosis via clathrin-coated pits. The particle characterization results reflect a carbon-based nanoparticle with bright violet fluorescence and 7.9% quantum yield in aqueous solution. These unpresented CDs proved to retain the functional properties of the precursor protein. Indicating that this process can be repeated for other disease biomarkers for applications ranging from biosensing and diagnostic bioimaging to targeted therapeutics.

Peptide ligands targeting the vesicular stomatitis virus G (VSV-G) protein for the affinity purification of lentivirus particles.

The recent uptick in the approval of ex vivo cell therapies highlights the relevance of lentivirus (LV) as an enabling viral vector of modern medicine. As labile biologics, however, LVs pose critical challenges to industrial biomanufacturing. In particular, LV purification-currently reliant on filtration and anion-exchange or size-exclusion chromatography-suffers from long process times and low yield of transducing particles, which translate into high waiting time and cost to patients. Seeking to improve LV downstream processing, this study introduces peptides targeting the enveloped protein Vesicular stomatitis virus G (VSV-G) to serve as affinity ligands for the chromatographic purification of LV particles. An ensemble of candidate ligands was initially discovered by implementing a dual-fluorescence screening technology and a targeted in silico approach designed to identify sequences with high selectivity and tunable affinity. The selected peptides were conjugated on Poros resin and their LV binding-and-release performance was optimized by adjusting the flow rate, composition, and pH of the chromatographic buffers. Ligands GKEAAFAA and SRAFVGDADRD were selected for their high product yield (50%-60% of viral genomes; 40%-50% of HT1080 cell-transducing particles) upon elution in PIPES buffer with 0.65 M NaCl at pH 7.4. The peptide-based adsorbents also presented remarkable values of binding capacity (up to 3·109 TU per mL of resin, or 5·1011 vp per mL of resin, at the residence time of 1 min) and clearance of host cell proteins (up to a 220-fold reduction of HEK293 HCPs). Additionally, GKEAAFAA demonstrated high resistance to caustic cleaning-in-place (0.5 M NaOH, 30 min) with no observable loss in product yield and quality.

Heteromultivalency enables enhanced detection of nucleic acid mutations.

Detecting genetic mutations such as single nucleotide polymorphisms (SNPs) is necessary to prescribe effective cancer therapies, perform genetic analyses and distinguish similar viral strains. Traditionally, SNP sensing uses short oligonucleotide probes that differentially bind the SNP and wild-type targets. However, DNA hybridization-based techniques require precise tuning of the probe's binding affinity to manage the inherent trade-off between specificity and sensitivity. As conventional hybridization offers limited control over binding affinity, here we generate heteromultivalent DNA-functionalized particles and demonstrate optimized hybridization specificity for targets containing one or two mutations. By investigating the role of oligo lengths, spacer lengths and binding orientation, we reveal that heteromultivalent hybridization enables fine-tuned specificity for a single SNP and dramatic enhancements in specificity for two non-proximal SNPs empowered by highly cooperative binding. Capitalizing on these abilities, we demonstrate straightforward discrimination between heterozygous cis and trans mutations and between different strains of the SARS-CoV-2 virus. Our findings indicate that heteromultivalent hybridization offers substantial improvements over conventional monovalent hybridization-based methods.

Electrically Controlled Adsorptive Membranes with Tunable Affinity for Selective Chromium (VI) Separation from Water.

Ionic contaminants such as Cr(VI) pose a challenge for water purification using membrane-based processes. However, existing membranes have low permeability and selectivity for Cr(VI). Therefore, in this study, we prepared an electrically controlled adsorptive membrane (ECAM-L) by coating a loose Cl--doped polypyrrole layer on a carbon nanotube substrate, and we evaluated the performance of ECAM-L for Cr(VI) separation from water. We also used electrochemical quartz crystal microbalance measurements and molecular dynamics and density functional theory calculations to investigate the separation mechanisms. The adsorption and desorption of Cr(VI) could be modulated by varying the electrostatic interactions between ECAM-L and Cr(VI) via potential control, enabling the cyclic use of the ECAM-L without additional additives. Consequently, the oxidized ECAM-L showed high Cr(VI) removal performance (<50 μg/L) and treatment capacity (>3500 L/m2) at a high water flux (283 L/m2/h), as well as reusability after the application of a potential. Our study demonstrates an efficient membrane design for water decontamination that can selectively separate Cr(VI) through a short electric stimulus.

Concurrent Design of Alloy Compositions of CZTSSe and CdZnS Using SCAPS Simulation: Potential Routes to Overcoming VOC Deficit

Solar energy is the most used renewable energy source. CZTSSe uses earth-abundant elements and has promising optoelectronic properties, resulting in becoming a viable alternative to thin film PV. This work provides design guidelines for CZTSSe-based solar cells, where CZTSSe has a tunable affinity and energy gap. The analysis is based on incorporating a ternary compound material to serve as an electron transport material (ETM). In this regard, CdZnS is a potential candidate that can be utilized as an electron transport layer whose affinity and energy gap can be tuned to adjust the band alignment at the ETL/CZTSSe interface. In order to design a high-efficiency solar cell, one has to tune both the ETL and absorber layers to have a suitable conduction band offset (CBO), thereby minimizing the non-radiative recombination which, in turn, boosts the power conversion efficiency (PCE). Thus, in our presented simulation study, we provide a codesign of alloy compositions of both the CZTSSe photoactive layer and the CdZnS ETL using SCAPS-1D simulation. It is found that using the codesign of alloy compositions of the ternary compound ETL and the absorber enhances the PCE by about 2% and, more importantly, overcomes the main issue in CZTSSe which is its open-circuit voltage (VOC) deficit. Furthermore, upon optimizing the thickness and doping of both the ETL and absorber layer, as well as the bulk defect of the absorber layer, a PCE of 17.16% is attained in this study, while the calibrated PCE based on a previously published experimental work was 12.30%.

Systematic single amino acid affinity tuning of CD229 CAR T cells retains efficacy against multiple myeloma and eliminates on-target off-tumor toxicity.

T cells expressing chimeric antigen receptors (CARs) have shown remarkable therapeutic activity against different types of cancer. However, the wider use of CAR T cells has been hindered by the potential for life-threatening toxicities due to on-target off-tumor killing of cells expressing low amounts of the target antigen. CD229, a signaling lymphocyte-activation molecule (SLAM) family member, has previously been identified as a target for CAR T cell-mediated treatment of multiple myeloma (MM) due to its high expression on the surfaces of MM cells. CD229 CAR T cells have shown effective clearance of MM cells in vitro and in vivo. However, healthy lymphocytes also express CD229, albeit at lower amounts than MM cells, causing their unintended targeting by CD229 CAR T cells. To increase the selectivity of CD229 CAR T cells for MM cells, we used a single amino acid substitution approach of the CAR binding domain to reduce CAR affinity. To identify CARs with increased selectivity, we screened variant binding domains using solid-phase binding assays and biolayer interferometry and determined the cytotoxic activity of variant CAR T cells against MM cells and healthy lymphocytes. We identified a CD229 CAR binding domain with micromolar affinity that, when combined with overexpression of c-Jun, confers antitumor activity comparable to parental CD229 CAR T cells but lacks the parental cells' cytotoxic activity toward healthy lymphocytes in vitro and in vivo. The results represent a promising strategy to improve the efficacy and safety of CAR T cell therapy that requires clinical validation.

Light-Up Aptameric Sensor of Serotonin for Point-of-Care Use.

Serotonin is a vital neurotransmitter for regulating organism functions, and its abnormal level indicates multiple diseases. Aptamer has emerged as an innovative tool for serotonin analysis very recently; however, the current aptameric sensing platform lacks design flexibility and portability. Here, we introduce a light-up aptameric sensor using designer DNA molecules with tunable affinity and dynamic response and achieve mobile phone-based detection for point-of-care use. We develop a type of allosteric DNA sensor through flanking the serotonin recognition domain with split fluorogenic sequences, where both linker lengths and split sites of the aptamer affect its function. In addition, we design a series of molecular constructs that contain nucleotide mutations and systematically investigate the structure folding and ligand binding of the aptameric molecules. The results show distinct effects of variant mutation sites on conformation change and sensing responses. Notably, the variable aptameric molecules allow affinity and dynamic response regulation, which are adaptable to diverse sensing applications that require different threshold levels. Furthermore, we demonstrate a simple surface-based assay that can use smartphone imaging to visualize results for diagnosis. In a portable and simple manner, highly sensitive and selective serotonin assay is achieved in different biofluids, with detection limits in the low nanomolar range. This study offers an alternative approach for serotonin assay using engineered aptameric molecular probes. We expect that the practical utility may make the method promising in resource-limited settings.

Target selection and clinical chimeric antigen receptor T cell activity against solid tumors

AbstractChimeric antigen receptor (CAR) T cell therapy is a relatively new form of targeted therapy that has demonstrated impressive success in treating hematological malignancies. It has been challenging to translate this success to solid tumors. Reasons for this include barriers to delivery, tumor heterogeneity, cancer cells' ability to evade the immune system as well as identifying the optimal target. Most CAR T clinical trials have targeted well‐characterized cancer targets with significant preclinical and in some cases clinical validation. Published results from some of these trials show signs of anti‐cancer activity that warrant encouragement, but also caution, given instances of unacceptable toxicity. The narrow therapeutic window is complicated by the ability of CAR T cells to expand in patients regardless of dose. Here, we review those trials showing encouraging results in the context of target selection. It is clear that more specific tumor targeting is required, either by affinity tuning to avoid low‐level target expression in healthy cells, logic gating, or the identification of new targets that are more cancer specific.

Use of aequorin-based indicators for monitoring Ca2+ in acidic organelles

Over the last years, there is accumulating evidence that acidic organelles can accumulate and release Ca2+ upon cell activation. Hence, reliable recording of Ca2+ dynamics in these compartments is essential for understanding the physiopathological aspects of acidic organelles. Genetically encoded Ca2+ indicators (GECIs) are valuable tools to monitor Ca2+ in specific locations, although their use in acidic compartments is challenging due to the pH sensitivity of most available fluorescent GECIs. By contrast, bioluminescent GECIs have a combination of features (marginal pH sensitivity, low background, no phototoxicity, no photobleaching, high dynamic range and tunable affinity) that render them advantageous to achieve an enhanced signal-to-noise ratio in acidic compartments. This article reviews the use of bioluminescent aequorin-based GECIs targeted to acidic compartments. A need for more measurements in highly acidic compartments is identified.

Abstract LB333: Cytokine affinity tuning using the AlphaSeq platform to generate targeted immuno-oncology therapeutics

Abstract Here, we demonstrate a novel approach for generating affinity detuned cytokines that can be fused to antibodies for selective immune cell activation to improve anti-tumor responses and mitigate systemic toxicity. Although cytokine therapies have demonstrated curative effects in some cancer patients, clinical use remains limited with current toxicity profiles accompanying systemic administration. Next generation cytokine approaches include conditional signaling focused to sites of interest, including the tumor microenvironment or specific immune cell populations. In this presentation, we share a novel approach for generating detuned cytokine therapeutic candidates using the AlphaSeq platform, which involves the repurposing of yeast agglutination and mating to quantitatively measure protein-protein interactions at a library-on-library scale. We show, for the first time, how AlphaSeq can be used to measure interactions between cytokines and their receptors as well as generate engineered cytokines with a broad range of affinities. A saturated mutational library was created for a cytokine of interest and subsequently screened against a second library consisting of axis receptor chains, their species orthologs and off-target receptors. Cytokine variants with lower affinity than parental were recombinantly expressed as Fc fusion proteins to orthogonally measure affinity with biolayer interferometry and characterize potency with an in vitro human PBMC phosflow assay. Finally, detuned cytokine candidates were fused to antibodies recognizing surface antigens of interest to demonstrate cell population-specific signaling. Our results show the AlphaSeq platform can accurately quantitate thousands of cytokine variant affinities simultaneously against multiple relevant receptors which translates to signaling potency in primary human cells. When engineered as antibody fusion proteins, candidate immunocytokines are identified that only induce signaling in intended cell populations. AlphaSeq’s rapid, comprehensive affinity determination can be utilized to develop a portfolio of clinically relevant therapeutic immunocytokines. Citation Format: Ryan Swanson, Davis Goodnight, Jeff Adamo, Kyle Minch, Emily Engelhart, Randolph Lopez, David Younger. Cytokine affinity tuning using the AlphaSeq platform to generate targeted immuno-oncology therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB333.

CD19 CAR antigen engagement mechanisms and affinity tuning.

Chimeric antigen receptor (CAR) T cell therapy relies on T cells that are guided by synthetic receptors to target and lyse cancer cells. CARs bind to cell surface antigens through an scFv (binder), the affinity of which is central to determining CAR T cell function and therapeutic success. CAR T cells targeting CD19 were the first to achieve marked clinical responses in patients with relapsed/refractory B cell malignancies and to be approved by the U.S. Food and Drug Administration (FDA). We report cryo-EM structures of CD19 antigen with the binder FMC63, which is used in four FDA-approved CAR T cell therapies (Kymriah, Yescarta, Tecartus, and Breyanzi), and the binder SJ25C1, which has also been used extensively in multiple clinical trials. We used these structures for molecular dynamics simulations, which guided creation of lower- or higher-affinity binders, and ultimately produced CAR T cells endowed with distinct tumor recognition sensitivities. The CAR T cells exhibited different antigen density requirements to trigger cytolysis and differed in their propensity to prompt trogocytosis upon contacting tumor cells. Our work shows how structural information can be applied to tune CAR T cell performance to specific target antigen densities.

Potential Regulation for Surface-Enhanced Raman Scattering Detection and Identification of Carotenoids.

Surface-enhanced Raman scattering (SERS) is often impaired by the limited affinity of molecules to plasmonic substrates. Here, we use carbon fiber microelectrodes modified with silver nanoparticles as a plasmonic microsubstrate with tunable affinity for enrichment and molecular identification by SERS. The silver nanoparticles self-assemble by electrostatic interaction with diamine molecules that are electrochemically grafted onto the surface of the microelectrodes. β-carotene and trans-β-Apo-8'-carotenal, producing similar resonant SERS spectra, are employed as model molecules to study the effect of electroenrichment and SERS screening for different electrode potentials. The data show that at a characteristic electrode potential, the low affinity of polyene chains without hydrophilic groups to the substrate can be overcome. Different potentials were applied to recognize the two types of carotenoids by their typical SERS signal, and the applicability of this strategy was further confirmed in the environment of a real cell culture. The results indicate that by regulating the potential, carotenoid molecules with a similar molecular structure can be selectively quantified and identified by SERS. The developed SERS-active microelectrode is expected to help the development of portable, miniaturized point-of-care diagnostic SERS sensors.

240 - Single-Chain Variable Fragment Affinity Tuning Can Optimize Anti-AML CAR-NK Cell Functionality

240 - Single-Chain Variable Fragment Affinity Tuning Can Optimize Anti-AML CAR-NK Cell Functionality

Nanoscopy of single antifreeze proteins reveals that reversible ice binding is sufficient for ice recrystallization inhibition but not thermal hysteresis

Antifreeze proteins (AFPs) bind ice to reduce freezing temperatures and arrest ice crystal ripening, making AFPs essential for the survival of many organisms in ice-laden environments and attractive as biocompatible antifreezes in many applications. While their activity was identified over 50 years ago, the physical mechanisms through which they function are still debated because experimental insights at the molecular scale remain elusive. Here, we introduce subzero nanoscopy by the design and incorporation of a freezing stage on a commercial super-resolution setup to resolve the interfacial dynamics of single AFPs with ice crystal surfaces. Using this method, we demonstrate irreversible binding and immobilization (i.e., pinning) of individual proteins to the ice/water interface. Surprisingly, pinning is lost and adsorption becomes reversible when freezing point depression activity, but not ice recrystallization inhibition, is eliminated by a single mutation in the ice-binding site of the AFP. Our results provide direct experimental evidence for the adsorption-inhibition paradigm, pivotal to all theoretical descriptions of freezing point depression activity, but also reveal that reversible binding to ice must be accounted for in an all-inclusive model for AFP activity. These mechanistic insights into the relation between interfacial interactions and activity further our understanding and may serve as leading principles in the future design of highly potent, biocompatible antifreezes with tunable affinity.

Overcoming on-target, off-tumour toxicity of CAR T cell therapy for solid tumours.

Therapies with genetically modified T cells that express chimeric antigen receptors (CARs) specific for CD19 or B cell maturation antigen (BCMA) are approved to treat certain B cell malignancies. However, translating these successes into treatments for patients with solid tumours presents various challenges, including the risk of clinically serious on-target, off-tumour toxicity (OTOT) owing to CAR T cell-mediated cytotoxicity against non-malignant tissues expressing the target antigen. Indeed, severe OTOT has been observed in various CAR T cell clinical trials involving patients with solid tumours, highlighting the importance of establishing strategies to predict, mitigate and control the onset of this effect. In this Review, we summarize current clinical evidence of OTOT with CAR T cells in the treatment of solid tumours and discuss the utility of preclinical mouse models in predicting clinical OTOT. We then describe novel strategies being developed to improve the specificity of CAR T cells in solid tumours, particularly the role of affinity tuning of target binders, logic circuits and synthetic biology. Furthermore, we highlight control strategies that can be used to mitigate clinical OTOT following cell infusion such as regulating or eliminating CAR T cell activity, exogenous control of CAR expression, and local administration of CAR T cells.