Tag Archives: literature

The Needle Issue #25

14 Apr
Juan-Carlos-Lopez
Juan Carlos Lopez		 Andy-Marshall
Andy Marshall

The approval of multiple anti-amyloid monoclonal antibodies (mAbs) — aducanumab (Aduhelm; now withdrawn), lecanemab (Leqembi) and donanemab (Kisunla) — over the past five years has opened the era of disease-modifying Alzheimer’s drugs, albeit with only modest benefits in addressing cognitive decline (30% slowing) and associated serious safety risks, such as CNS inflammation and cerebral hemorrhages, which has limited clinical uptake. While many drug development programs target biological processes other than amyloid formation (e.g., tau and tangles, neurotransmitter receptors, neuroinflammation, autophagy, and mitochondrial or metabolic dysfunction), companies continue to optimize anti-amyloid monoclonals, but also look for alternative ways to therapeutically target Aβ.

One alternative therapeutic modality to antibodies is chimeric antigen receptor (CAR) immune cell therapy. In recent weeks, we have been thinking a lot about in vivo chimeric antigen receptor (CAR)-T therapies, which were one of the dealmaking trends in 2025, and we recommend readers check out an excellent summary of trends in the area from the consultancy firm Scitaris (you don’t even have to give them your details to download the report).

CAR-T treatments have established their clinical niche as last-ditch treatments for B-cell malignancies, with some remarkable outcomes for late-stage patients. In some cases, they have been shown to be at least twice as effective as T-cell engager bispecific antibodies in clinical studies. But they remain rather blunt instruments.

Despite advances in the clinical management of cytokine-release syndrome and immune effector cell neurotoxicity syndrome (ICANS), CAR-T treatments continue to be associated with serious risks. And while there have been advances in managing these adverse events, atypical non-ICANS neurotoxicities (NINTs) can also create serious clinical management issues, with risk factors predisposing patients to development still only poorly understood.

That said, over the past year, we have seen an increasing trend for the use of CAR-T treatments outside oncology. They have started to be applied with promising efficacy in various areas of autoimmunity (systemic lupus erythrematosus, lupus nephritis, systemic sclerosis, Sjögren’s syndrome, antisynthetase syndrome, myasthenia gravis and idiopathic inflammatory myopathies) and neuroinflammatory conditions (multiple sclerosis). In this respect, a recent paper in Science caught our attention. In it, Marco Colonna and his colleagues at Washington University in St. Louis harness astrocytes to clear amyloid plaques by promoting their ability to phagocytize Aβ.

To that end, they used in vivo gene therapy to generate astrocytes carrying chimeric antigen receptors (“CAR-As”), a strategy not unlike the one used in cancer immunotherapy. Although both macrophages (CAR-Ms) and conventional CAR-Ts have been tested in preclinical models of Alzheimer’s disease with limited success, this study reports the first attempt to directly engineer astrocytes in the body to generate CAR-As.

In broad terms, the construct used to generate CAR-As consisted of an Aβ-binding domain and the phagocytic signaling protein MEGF10 (multiple epidermal growth factor-like domains protein 10). The team examined a variety of constructs and chose two for in vivo testing. One of them combined a fragment from the Aβ-binding antibody crenezumab and MEGF10, which is primarily expressed in astrocytes. The second construct combined a fragment of aducanumab with the phagocytosis receptor Dectin-1, which is primarily expressed in microglia.

The authors packaged the constructs in an adeno-associated viral (AAV) vector under the control of an astrocyte-specific promoter and injected them intravenously into 5xFAD mice (which carry five familial Alzheimer’s disease (FAD) mutations, driving rapid Aβ plaque formation, synaptic loss, and cognitive decline starting around 2–4 months). Both CAR-As reduced amyloid burden and neuritic dystrophy, and the treatment worked both in the prophylactic and therapeutic settings.

Single-nucleus RNA sequencing and immunostaining showed that the CAR-As adopted the transcriptomic profile of activated astrocytes and readily clustered around amyloid plaques. Microglial cells, in turn, also responded to the treatment by showing a reduction of the disease-associated transcriptomic profile that is often seen after administration of monoclonal anti-Aβ antibodies. This is of interest because this disease profile of microglial cells has been suggested to contribute to the inflammatory reaction sometimes seen after Alzheimer’s immunotherapy.

A caveat of the study is that the authos saw no improvements in cognition following therapy, albeit behavioral results in mouse models have been notoriously poor at predicting outcomes in humans. However, the translational questions don’t stop there.

If in clinical practice the CAR-A approach would require an AAV vector, then immunogenicity of the treatment is going to be an issue. Pre-exposure to AAV is often a problem for gene-therapy programs, where patients are much younger. Given that Alzheimer’s is a disease associated with an elderly population, immunogenicity is likely to be exacerbated. Similarly, the delivery of 1013–1014 viral genomes to elderly patients living with Alzheimer’s—many of whom will already have a brain prone to neuroinflammation—makes the specter of unwanted side effects a major concern. In this respect, finding Alzheimer’s patients whose disease stage and age would be appropriate for a therapy with potentially highly toxic consequences for fragile recipients is also difficult to gauge.

That is not to say that CAR-immune cell therapy may not have a place in CNS disease. It just seems like neurological conditions, such as multiple sclerosis where patients are younger and potentially less fragile, are the place where much of the translational groundwork and clinical management for CAR-A or CAR-T therapies must be worked out before moving into neurodegenerative disease for elderly and cognitively compromised patients.

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The Needle Issue #24

24 Feb
Juan-Carlos-Lopez
Juan Carlos Lopez		 Andy-Marshall
Andy Marshall

X-ray crystallography has long been the go-to workhorse for providing atomic structures of drugs interacting with their protein targets. Increasingly, those static snapshots are being complemented by readouts from experimental analytical tools based on nucleic magnetic resonance (NMR) spectroscopy and cryoelectron microscopy (cryo-EM), offering drug developers a broader window into proteins as dynamic, breathing molecules. This is spurring a raft of new service provider startups, including AIffinity (Brno-Medlánky, Czech Republic), NexMR (Zürich, Switzlerand), CryoCloud (Utrecht), and Intellicule (West Lafayette, IN), all of which aim to supply drug-discovery teams with state-of-the-art platforms providing structural data with rapid turnaround times and low cost.

As many of the most compelling ‘undruggable’ targets are renowned shape shifters — aggregation-prone proteins like Tau, amyloid precursor protein (APP) or huntingtin in neurodegenerative diseases, or transcription factors like P53, KRAS and c-MYC in oncology — a lot of therapeutic startup activity has recently focused around so-called ‘intrinsically disordered proteins’ (IDPs). The ability to attain markedly different conformations under different conditions allows IDPs not only to play moonlighting roles or serve as hubs in signaling networks, but also to localize into liquid- phase condensates (or membrane-less organelles — attributes that make them acutely sensitive to mutations that can compromise specificity and lead to nonspecific binding, resulting in toxicity and disease.

As IDPs frequently resist attack by conventional drug discovery approaches, a slew of startups has sprung up to try to go after this target class, many using new structural techniques. These include Peptone (London, UK), Dewpoint Therapeutics (Boston, MA), brainQR Therapeutics (Göttingen, Germany), and Kodiform Therapeutics (Oxford, UK). Just last month, Topos Bio secured a $10.5 million seed round to “tackle ‘undruggable’ proteins driving Alzheimer’s and cancer”. Dewpoint also just announced it has dosed its first patient in a phase 1/2a trial of its lead beta-catenin program in gastric cancer and elected its MYC development candidate to take forward.

An important postscript to the startup activity targeting undruggable IDPs is that more conventional ‘druggable’ target classes, like tyrosine kinases, may also represent a fruitful hunting ground for dynamic conformational states that may have been missed by traditional crystallographic approaches. Given that conventional drug targets have relatively well-trodden clinical and commercial development paths, they may also represent simpler starting points and testing grounds for commercial programs aiming to apply the new analytical approaches to support medicinal chemistry programs around validated targets.

In a paper recently published in Science, the team of Charalampos (Babis) Kalodimos at St. Jude Children’s Research Hospital use high-resolution NMR spectroscopy to gain structural insight into how SRC family tyrosine kinases (Src, Hck, and Lck) achieve processive phosphorylation of multisite substrates.

The SRC enzyme family is essential for rapid and coordinated signaling in processes such as cell migration and T-cell activation. In addition, SRC family kinases are frequently overexpressed in tumors, contributing to the activation not only of multiple scaffold or signaling proteins, such as receptor tyrosine kinases (e.g., EGFR, FGFR, PDGFR or IGF1R), but also of downstream effectors (e.g., MAPKs, FAK, paxillin, p130Cas, ELMO1 and RAC1). Although there are approved drugs like the multikinase inhibitor Sprycel (dasatinib) that bind the SRC active site, these drugs have such extensive off-target and adverse side effects that there is a pressing need for new paths to more-selective SRC inhibitors.

SRC enzymes share a conserved domain organization, with a disordered N-tail, a tandem SH3–SH2 module, a kinase domain, and a disordered C-tail. All can carry out processive phosphorylation — a phenomenon where the enzyme phosphorylates multiple residues in a substrate during a single encounter. Each of these catalytic cycles typically requires ATP binding, phosphate transfer and ADP release, and ADP release is often the rate-limiting step. So, a question that has long puzzled structural biologists is how ADP-release–constrained kinases achieve sufficiently rapid turnover to successfully perform their function.

Using NMR spectroscopy with cryogenic probes — which reduce electronic/thermal noise and increase sensitivity up to five-fold compared with room-temperature probes — the St. Jude team characterized the conformational ensemble of the Src kinase domain and identified three interconverting states: a predominant active state, a previously described inactive Src/CDK-like state, and a hitherto unknown low-populated intermediate state positioned linearly between the other two. Structural determination revealed that this intermediate state displays features that are distinct from the active and inactive states. Its activation loop is partially folded, the P-loop is displaced inward, and the αC helix is shifted upward. This conformation binds ADP poorly relative to the active and inactive states, suggesting that it facilitates nucleotide release.

Using mutational analyses, the researchers then confirmed the functional importance of this intermediate state. Variants that eliminated this intermediate state while stabilizing the active state showed slower ADP dissociation, reduced catalytic turnover and impaired processive phosphorylation of the multisite Src substrate p130Cas. Instead of generating a fully phosphorylated substrate in a single binding event, these mutants accumulated partially phosphorylated intermediates. Equivalent mutations in other kinases of the SRC family, Lck and Hck, similarly reduced catalytic efficiency and impaired multisite phosphorylation of their respective physiological substrates CD3ζ and ELMO1 in Jurkat cells. Furthermore, these mutations compromised cellular functions measured via in vitro assays, including T-cell activation using Lck-deficient Jurkat cells and migration of mouse embryo fibroblasts lacking Src, Yes and Fyn in the presence of fibronectin. These molecular and functional findings indicate that the intermediate state is evolutionarily conserved and essential for processive activity across the SRC family.

Mechanistically, the work establishes that rapid ADP release, enabled by transient sampling of a structurally constrained intermediate, is critical for sustaining catalytic turnover rates that exceed the speed of substrate dissociation. More broadly, it shows that kinase conformational landscapes are tuned not only for switching between active and inactive states, but also for optimizing specific kinetic steps within the catalytic cycle.

From a drug developer’s standpoint, because Sprycel and other inhibitors target the active or inactive conformations of the SRC active site, the identification of a low-populated, functionally indispensable intermediate suggests a completely new strategy to target tyrosine kinases: selectively stabilize or destabilize the intermediate state to fine-tune catalytic turnover and processivity rather than simply blocking activity. Targeting such transient conformations could enable more precise modulation of signaling output, potentially improving selectivity and reducing off-target effects in kinase-directed therapies.

We look forward to seeing how many more of these intermediate states are uncovered in other kinase targets and whether pharmacological inhibitors targeting this state have advantages over orthosteric or allosteric chemotypes that conventionally have been used to inhibit the kinase active site or lock it in an inactive conformation. What is clear is that ultrafast NMR measurements of binding and state behavior are a powerful differentiating tool for understanding kinase activity where static structures aren’t enough.

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