B cells contribute to multiple aspects of autoimmune disorders through autoantibody production, antigen presentation, cytokine secretion, and formation of tertiary lymphoid structures. B cell targeting therapies have been developed and approved for treatment of autoimmune diseases. Many approved B cell targeted therapies deplete B cells through targeting CD20 (rituximab, ocrelizumab, etc). However, CD20-mediated B cell depletion has limitations including incomplete depletion of tissue resident pathogenic B cells, inability to target CD20-negative B cells (plasmablasts), and a long recovery time for B cells. Thus, developing therapies with more efficacy on tissue resident B cells and a non-depleting mechanism of action is highly desirable.
Here we describe a novel, non-depleting, B cell inhibiting, anti-human CD19 antibody LY3541860 which binds human and cynomolgus monkey (cyno) CD19 with high affinity, does not induce ADCC or CDC, and does not cause B cell apoptosis. LY3541860 inhibited B cell activation, proliferation, and differentiation of primary human and cyno B cells ex vivo. LY3541860 also inhibited human B cell activation in vivo in humanized mice. Administration of anti-CD19 antibodies in cyno did not cause reduction of B cells and resulted in complete receptor occupancy.
We also developed a non-depleting anti-mouse CD19 antibody with similar characteristics as the anti-human CD19 antibody. The anti-mouse CD19 antibody was used to compare effects of B cell inhibition with B cell depletion in multiple mouse models of autoimmunity. Our data demonstrate that treatment with the anti-mouse CD19 antibody ameliorated clinical symptoms in multiple mouse autoimmune models including collagen-induced arthritis (CIA), non-obese diabetic (NOD) and experimental autoimmune encephalomyelitis (EAE). Moreover, this anti-mouse CD19 antibody demonstrated improved efficacy in all tested mouse autoimmune models compared with an anti-CD20 depleting antibody. No B cell depletion was noted in any of the in vivo models tested while B cell depletion was evident with the anti-CD20 antibody. Improved efficacy seen with the non-depleting CD19 antibody may be due to targeting CD20-/CD19+ plasmablasts, as well as bypassing limited B cell depletion in tissues.
Taken together, these data suggest that a non-depleting anti-CD19 antibody may demonstrate superior efficacy in treatment of autoimmune conditions compared with B cell depleting therapies.

Neural mechanism underlying the anti-nausea effect of GIP
Minrong Ai1*, Brandy Snider1, Alessia Costa2, Richard Cosgrove1, Ricardo Samms1, Paul Emmerson1, Giuseppe D’Agostino2,3, Simon M Luckman2

1Lilly Research Laboratories, Eli Lilly & Company, Indianapolis, Indiana, United States
2Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK
3The Rowett Institute, University of Aberdeen, Aberdeen, UK
*Presenter

Abstract
The success of the dual-incretin receptor agonist, Tirzepatide, in the clinic raises great interest in understanding the mechanism of action of this molecule. It is well established that GLP-1 acts on the CNS to reduce food intake and body weight, and induces nausea, while relatively little is known about GIP’s action on the brain. We have published evidence from preclinical models including rats and musk shrews which suggests that GIP has an anti-nausea effect1. Using mouse model we now show that GIP suppresses nausea-like aversive behaviors induced by either GLP-1 or PYY, two agents known to cause nausea and vomiting. To understand a possible mechanism underlying this phenomenon, we identified GIP receptor (GIPR) expressing neurons in the area postrema (AP) of rodent and non-human primate brains. These GIPR+ cells are GABAergic inhibitory neurons. Administration (SC) of GIP specifically activated these GIPR-expressing neurons in mouse brain, while GLP-1 administration (SC) activated a separate population of neurons expressing GLP1R within AP. Importantly, activation of the GABAergic GIPR+ neurons in AP suppressed the activity of the neighboring GLP1R+ neurons, suggesting a local inhibitory network. By contrast, GIPR and PYY receptor, Y2R, are expressed in the same neuronal population in AP. Activation of Y2R, a Gi coupled receptor, by peripheral PYY administration decreased cFos induction in these neurons by peripheral GIP treatment. Furthermore, we discovered that GIP and PYY co-administration led to a decreased neural activity in the dorsal parabrachial nucleus, a brain region downstream of AP that mediates aversive behaviors, when compared to PYY administration alone. Together, our results demonstrated an anti-nausea effect of GIP and elucidated a potential underlying neural basis.


Reference:
1. Borner T. et al., Diabetes 2021 Nov; 70(11): 2545-2553.

Recently, the team at DeepMind has released a deep learning based protein folding structure prediction algorithm called Alphafold2. This algorithm has achieved remarkable scores at the CASP14 competition, and in many cases achieves remarkable accuracy. However, there are limitations to Alphafold2, and in some cases Alphafold2 staggeringly fails. Because domains are the functional unit of drug development, it would be useful to characterize the accuracy of AlphaFold2’s predictions at each domain. We performed a sequence based structure alignment between 1) the full length of the AlphaFold-predicted and protein structures, and 2) the AlphaFold-predicted and protein structures over just the specific PFAM domain. We calculated the root mean square deviation (RMSD) of the two structures over the domain and full length structures. The average RMSD value over the full length alignments were .75 Angstroms, with the median being .64 Angstroms. Importantly, this dataset includes structures proprietary to Lilly, and that were not present in the RCSB PDB that was used as AlphaFold’s training set. We found that of the 53 domains that had proteins with structures that covered at least 20% of the domain and had at least 90% sequence identity with the AlphaFold2 sequence, that all of them had RMSD values of less than 1 Angstrom, with 29 having RMSD values below .5 Angstroms, suggesting that AlphaFold is able to predict these 53 domains with very high confidence. There were an additional 305 domains that did not have sufficient data to perform this analysis, suggesting that proteins of interest containing those domains may be less reliable.

Advancing Nose-to-Brain Delivery of Nucleic Acids through Localization

Samantha M. Sarett1, Zhefeng Li1, Anisha D’Souza3, Hao-Cheng Chueh1, Christopher Wiethoff1, Peng Fang1, Erica Mondo1, Christina Zhang1, Claire Moront1, Ryan C. Hill1, Sarah Dicker1, Benjamin S. Bleier2, Mansoor Amiji3, Michelle Lynn Hall1
Eli Lilly and Company1; Massachusetts Eye and Ear Institute2; Northeastern University3

Nucleic acids like short interfering RNA (siRNA) are an emerging class of drugs with the capability of addressing previously untreatable (central nervous system) CNS diseases at the genetic level. However, broad clinical application of nucleic acid drugs has been hampered by their inherently poor delivery properties. To date, the only clinically validated option for CNS delivery of nucleic acids is a highly invasive direct injection.
Nose-to-brain delivery (N2B) is a minimally invasive approach that circumvents the BBB and could effectively deliver siRNA to the CNS. Deposition at the olfactory epithelium (located in the upper nasal cavity) allows transport of drugs alongside the olfactory and trigeminal nerves to the brain. However, the fundamental biological differences between rodent and human nasal biology have historically hindered development of drugs that leverage the N2B pathway. For example, 95% of the rodent nasal epithelial surface area allows N2B transit compared to 5 – 8 % for humans. Thus, we hypothesized that a key issue for N2B delivery could be inefficient / variable localization and retention at the target area.
In our investigation of the potential for N2B delivery of nucleic acids, we confirmed the validity of the transit pathway for siRNA therapeutics using pipet-based intranasal instillation in rats. However, absent more efficient localization of dose, magnitude of siRNA delivery was low (1x10-3 – 1x10-2 % ID/g in olfactory bulb and trigeminal nerve). Subsequently, we improved the consistency and degree of siRNA distribution to the CNS through localization by two distinct methods. First, we used catheter-based intranasal infusion in rats to localize the dose to the back of the nasal cavity. Second, we evaluated a translatable approach that both localizes and retains the dose at the rodent epithelium via implantation of a minimally invasive nasal depot (MIND). In both cases, distribution to the brain increased by >10-fold in the olfactory bulb and trigeminal nerve, and siRNA was also quantified in deeper brain regions (e.g., frontal cortex, hippocampus, striatum). RNAScope data depicting siRNA distribution to the olfactory bulb and surface layers of the cortex supported siRNA’s transit through the distinct N2B pathway. In pilot studies, N2B-delivered siRNA also achieved knockdown in regions along that pathway (trigeminal nerve (25%), brain stem (50%), and rostral frontal cortex (20%)).
This data highlights the necessity of efficient localization and retention of siRNA at the olfactory epithelium for effective N2B delivery. Our data shows that addressing this challenge (e.g., via the simple, endoscopic application of MIND in human patients) could bridge the translatability gap for the N2B route. This motivates a continued investigation of the minimally invasive N2B route for delivery of nucleic acid therapeutics to the CNS.

Genome-wide association studies have identified genes modulating microglia and inflammation to be associated with an enhanced risk for Alzheimer’s disease (AD). Furthermore, increased microglial activation has been associated with the progression of AD. In the rTg4510 tau transgenic AD model, we consistently observed an age-dependent elevation in the microglia marker IBA-1, while gene expression profiling in rTg4510 brain tissues also revealed a robust age-dependent increase in microglia activation-related markers, and a moderate increase in microglia cell- and proliferation-related markers, in comparison to wild-type littermates. Notably, the increase in microglia markers correlated with the accumulation in tau pathology. Consistent with our observations, previous findings in the literature demonstrated that inhibition of colony-stimulating factor-1 receptor (CSF-1R) depleted microglia and reduced tau pathology in multiple tau models.
CSF-1R, a membrane-bound receptor tyrosine kinase, is known for its important role in the proliferation and survival of microglia. CSF-1R has two ligands, CSF-1 and IL-34. We became interested in IL-34, because of its selective activity in the brain. IL-34 knockout removes about half of microglia in mouse brain, while minimally affecting macrophages/monocytes in the periphery. It is unknown to what extent IL-34 inhibition will impact tau pathology. We therefore set out to test this with a high-affinity murine antibody raised against IL-34.
In a prevention paradigm, young rTg4510 mice were treated with an IgG1 control mAb (50 mg/kg), or dose-dependently with an anti-IL-34 mAb (5-, 15-, or 50- mg/kg), or a high dose of a CSF-1R inhibitor (PLX3397) as a positive control. The anti-IL-34 mAb significantly reduced microglia dose-dependently and the expression of proinflammatory cytokine markers. At the highest dose, anti-IL-34 mAb reduced microglia by ~50%, phenocopying the magnitude of microglia reduction in IL-34 knockout mice. Surprisingly, anti-IL-34 mAb demonstrated a robust reduction of tau pathology, with a magnitude comparable to PLX3397, which elicited a much stronger reduction of microglia (~95%). In addition, some brain regions showed evidence for the slowing of neurodegeneration. Our data suggested that partial reduction of microglia is sufficient to robustly reduce tau pathology in rTg4510 mice, and supported the anti-IL-34 mAb as a novel therapy to treat tauopathies and AD.

This ePanel will celebrate the inaugural winners of the Michelson Philanthropies & Science Prize for Immunology, an international prize that focuses on transformative research in human immunology, with trans-disease applications to accelerate vaccine and immunotherapeutic discovery. The prize is intended to encourage and support young investigators from a wide range of disciplines and will be awarded annually based on work done in the past three years.

This year’s recipients are:

• Grand prize winner Paul Bastard, MD, PhD, (Laboratory of Human Genetics of Infectious Diseases, Institut Imagine, INSERM & University of Paris and The Rockefeller University, New York)
  Why do people die from COVID-19?: Autoantibodies neutralizing type I interferons increase with age.
• Finalist Scott B. Biering, PhD, (Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley)
  One antibody to treat them all: Conserved flavivirus protein holds potential as target for versatile vaccines and therapies.
• Finalist Lisa Wagar, PhD, (Institute for Immunology, University of California, Irvine)
  Small centers of defense: Deciphering immune responses to viruses and vaccines using human tonsil organoids.

Following welcome remarks by Dr. Gary Michelson, Michelson Philanthropies founder and co-chair, Bill Moran, Publisher of AAAS/Science, United States Senator Alex Padilla of California, and Seth Scanlon, Editor of AAAS/Science, the recipients will present their award winning research and participate in a Q&A with the audience.

There is a need for wastewater based epidemiological (WBE) methods that integrate multiple,
variously sized surveillance sites across geographic areas. We developed a novel indexing method,
Melvin’s Index, that provides a normalized and standardized metric of wastewater pathogen load
for qPCR assays that is resilient to surveillance site variation. To demonstrate the utility of Melvin’s
Index, we used qRT-PCR to measure SARS-CoV-2 genomic RNA levels in influent wastewater from 19
municipal wastewater treatment facilities (WWTF’s) of varying sizes and served populations across the
state of Minnesota during the Summer of 2020. SARS-CoV-2 RNA was detected at each WWTF during
the 20-week sampling period at a mean concentration of 8.5 × 104
genome copies/L (range 3.2 × 102–
1.2 × 109
genome copies/L). Lag analysis of trends in Melvin’s Index values and clinical COVID-19
cases showed that increases in indexed wastewater SARS-CoV-2 levels precede new clinical cases by
15–17 days at the statewide level and by up to 25 days at the regional/county level. Melvin’s Index is
a reliable WBE method and can be applied to both WWTFs that serve a wide range of population sizes
and to large regions that are served by multiple WWTFs.