PD-1-GITR-L bispecific agonist for cancer immunotherapy
An anti-PD-1-GITR-L bispecific agonist induces GITR clustering-mediated T cell activation for cancer immunotherapyChan S et al. Nature Cancer 2022 March
In the past 20 years, oncology has been revolutionized by immune checkpoint blockade therapies, although they have shown variable efficiency in some indications. Another approach to immunotherapy is to boost T cell activation using agonists of costimulatory receptors, instead of blocking immune checkpoints. One target of choice for such approach is the glucocorticoid-induced tumor necrosis factor receptor-related protein, GITR. Although preclinical studies using GITR agonists showed promising results, early-stage clinical trials only demonstrated limited success for these compounds.1 This limitation is believed to be due to the lack of potency of the compounds to induce receptor clustering, suggested to be a critical mediator of T cell activation.2A novel bispecific agonist
A recent study,3 published in Nature Cancer in March 2022, tried an alternative approach by developing and testing a bispecific agonist, associating an anti-PD1 antibody with the agonist GITR-L, hence producing a PD1-directed GITR-L. This design should allow the specific and efficient targeting of PD1+GITR+ double-positive T cells.Efficient anti-tumor efficacy
The authors first performed in vitro studies confirming that GITR clustering is critical for optimal signaling. A murine surrogate was then tested in syngeneic models, and efficiently inhibited growth of both CT26 and EMT6 tumors. Treatment with the murine surrogate increased activated, memory and proliferating CD4+ and CD8+ T cells in the blood and tumors, while reducing Tregs and exhausted CD8+ in the tumor microenvironment. Chimeric compounds (anti-muPD-1-huGITR-L and anti-huPD-1-muGITR-L) were tested in WT and single Knockin human GITR and PD-1 homozygous genetically engineered mouse models, demonstrating the requirement of PD-1 and GITR co-engagement for efficient anti-tumor activity. The mechanism of action of the bispecific agonist was further described as CD8+ T cell dependent, not CD4+, thus differing from the mechanism of action of a GITR-L:anti-PD1 combination therapy. Last, pharmacodynamic studies in non-human primates demonstrated the compound’s efficacy in this species, suggesting good translatability.An alternative approach to immunotherapy
These data show that a novel bispecific agonist specifically developed to direct GITR-L to PD1+ cells induces clustering of GITR through a FcγR independent mechanism and is thus a true T cell agonist. In addition, it has been shown to be more efficient than monotherapies, alone or in combination. This alternative approach in cancer therapy could be used with other immunotherapies in specific indications with low lymphocyte infiltrates, i.e., cold tumors.
Of note, the human GITR and PD-1 Knockin mouse models used in this study were designed and generated by genOway, a designer and provider of numerous physiologically relevant preclinical models in multiple research areas, including immuno-oncology, metabolism, cardiovascular diseases, and neuroscience.
- Choi Y, Shi Y, Haymaker CL, Naing A, Ciliberto G, Hajjar J. T-cell agonists in cancer immunotherapy. J Immunother Cancer. 2020 Oct;8(2):e000966.
- Garber K. Immune agonist antibodies face critical test. Nat Rev Drug Discov. 2020 Jan;19(1):3-5.
- Chan S, Belmar N, Ho S, Rogers B, Stickler M, Graham M, Lee E, Tran N, Zhang D, Gupta P, Sho M, MacDonough T, Woolley A, Kim H, Zhang H, Liu W, Zheng P, Dezso Z, Halliwill K, Ceccarelli M, Rhodes S, Thakur A, Forsyth CM, Xiong M, Tan SS, Iyer R, Lake M, Digiammarino E, Zhou L, Bigelow L, Longenecker K, Judge RA, Liu C, Trumble M, Remis JP, Fox M, Cairns B, Akamatsu Y, Hollenbaugh D, Harding F, Alvarez HM. An anti-PD-1-GITR-L bispecific agonist induces GITR clustering-mediated T cell activation for cancer immunotherapy. Nat Cancer. 2022 Mar;3(3):337-354.
Nature Communications – Dec 2021 – 3Rs: Generate single-sex litters
Major technological breakthroughs have allowed the significant improvement of in vitro models’ physiological relevance,1 with organoids and other 3D culture methods, for example. However, in vivo studies, and thus the use of laboratory animals, are still very much needed and performed. Legislations are also evolving across the world to more tightly adjust and limit the use of laboratory animals, with the now global “Replacement, Reduction and Refinement” (3Rs) guidelines. A 2010 EU directive sets its ultimate goal to the full replacement of the use of animals for scientific purposes, while improving the welfare of the animals still needed in the meantime.2
One technical shortcoming in the 3Rs guidelines appears in research areas that focus on sex-specific traits or pathologies, such as gametogenesis, placental biology, or sex-specific cancers like prostate cancer. In these cases, research is performed on males or females only. In this context, being able to control offspring sex could significantly reduce the use of laboratory animals. As of today, the means of controlling the sex of offspring are neither effective or humane, as they mostly consist in culling the unwanted animals.
A recent study published in Nature Communications proposed a new genetic system to tackle this issue.3 In their paper, Douglas and colleagues used CRISPR/Cas9 technology to facilitate the generation of single-sex litters. Their system is built on the known lethal phenotype observed in embryos invalidated for the Topoisomerase 1 DNA replication and repair factor. Using H11Top1 females (expressing a Top1-targeting sgRNA from the H11 neutral locus on an autosome), and males expressing Cas9 from either the X (XCas9Y) or the Y (XYCas9) chromosome, they showed that co-inheritance of Cas9 and the Top1sgRNA induced sex-specific embryonic lethality with 100% efficiency. Indeed, crossing H11Top1 females with XCas9Y males produced only male descendants, whereas H11Top1 females mated with XYCas9 males only had female offspring. Interestingly, a phenomenon of litter size’s compensation occurred in these cases, as the size of these same-sex litters was superior to 50% of a control litter size. The authors also described that this system could be used to induce sex-specific postnatal phenotypes.
Several novel genetically engineered mouse models were developed for this study, including one designed and generated by genOway, a designer and provider of numerous physiologically relevant preclinical models in multiple research areas.
- Horvath P, Aulner N, Bickle M, Davies AM, Nery ED, Ebner D, Montoya MC, Östling P, Pietiäinen V, Price LS, Shorte SL, Turcatti G, von Schantz C, Carragher NO. Screening out irrelevant cell-based models of disease. Nat Rev Drug Discov. 2016 Nov;15(11):751-769.
- Douglas C, Maciulyte V, Zohren J, Snell DM, Mahadevaiah SK, Ojarikre OA, Ellis PJI, Turner JMA. CRISPR-Cas9 effectors facilitate generation of single-sex litters and sex-specific phenotypes. Nat Commun. 2021 Dec 3;12(1):6926.
Cell – Sep 2021 – New Doors to Potential Novel Treatments for Obesity
Lipolysis drives expression of the constitutively active receptor GPR3 to induce adipose thermogenesis
Commented by Alessia Armezzani, PhD (Sep 2021)
Brown adipose tissue (BAT) functions as a biological furnace that warms the body by consuming circulating glucose and lipids. To our placental ancestors, this thermogenic (i.e., heat-producing) process provided a selective advantage to leave the dark underground and enter surface habitats; for modern humans, however, it may represent a real panacea to two major causes of death: overweight and obesity.1
Several studies have indeed shown that BAT plays a key role in energy metabolism by increasing lipolysis, decreasing blood glucose levels, and secreting adipokines (i.e., cytokines secreted by adipose tissue) involved in inflammation regulation, insulin action, and glucose metabolism.2 As such, BAT has become a potential therapeutic target against obesity and its associated complications.3
BAT thermogenic catabolism is activated by G protein-coupled receptors (GPCRs), a large group of cell surface receptors that responds to a variety of external signals such as peptides, carbohydrates, and lipids.4 For many years, it was believed that GPCRs could modulate intracellular signaling only upon binding to one of such molecules: a recent study conducted by a group of scientists at the Novo Nordisk Foundation has proven this belief wrong.5–7
In their paper, recently published in Cell, Olivia Sveidahl Johansen and colleagues unveiled what is indeed a new mechanism to activate the G protein-coupled receptor 3: GPR3. More specifically, they found that GPR3 possesses an innate signaling capacity (i.e., it does not require any exogenous ligand), it is constitutively active, and its expression is increased by cold exposure. Importantly, the authors also showed that GPR3 activation in BAT counteracts metabolic disease: this was elegantly demonstrated by using genetically engineered mice overexpressing GPR3 in thermogenic adipocytes, continuously fed at high caloric diet. Surprisingly, the authors found that increasing GPR3 expression in BAT is alone sufficient to drive energy expenditure and counteract metabolic disease in mice, opening new doors to potential novel treatments against obesity. Of note, the genetically engineered mice used in this study were generated by genOway, designer and provider of multiple preclinical models in several research areas, including immuno-oncology, metabolism, cardiovascular diseases, and neuroscience.
Alessia is scientific communication manager at genOway
- Obesity and overweight. https://www.who.int/news-room/fact-sheets/detail/obesity-and-overweight (2021).
- Balistreri, C. R., Caruso, C. & Candore, G. The Role of Adipose Tissue and Adipokines in Obesity-Related Inflammatory Diseases. Mediators of Inflammation 2010, 1–19 (2010).
- Razzoli, M. et al. Stress-induced activation of brown adipose tissue prevents obesity in conditions of low adaptive thermogenesis. Molecular Metabolism 5, 19–33 (2016).
- Kobilka, B. K. G protein coupled receptor structure and activation. Biochimica et Biophysica Acta (BBA) - Biomembranes 1768, 794–807 (2007).
- Wettschureck, N. & Offermanns, S. Mammalian G Proteins and Their Cell Type Specific Functions. Physiological Reviews 85, 1159–1204 (2005).
- Sveidahl Johansen, O. et al. Lipolysis drives expression of the constitutively active receptor GPR3 to induce adipose thermogenesis. Cell 184, 3502-3518.e33 (2021).
Nature – June 2021 – NOTUM as alternative therapeutic approach to prevent clonal fixation and mutant expansion in patients predisposed to colorectal cancer.
NOTUM from Apc-mutant cells biases clonal competition to initiate cancer
Commented by Amélie Rezza, PhD (Nov 2021)
Colorectal tumorigenesis has been a major focus of cancer research for decades. In 1990, Fearon and Vogelstein hypothesized a specific sequence of mutations, now called Adenoma Carcinoma Sequence, occurring during intestinal cancer development.1 Later studies confirmed that loss-of-function mutations of the tumor suppression gene APC are an early, if not initiating, event in this sequence.2 Indeed, APC is a negative regulator of WNT signaling, known as a key regulator of intestinal stem cells’ (ISC) homeostasis, intestinal epithelial cells’ proliferation, differentiation and regeneration, basically of intestinal epithelium physiopathology.3
Many labs have studied the early molecular and cellular mechanisms of colorectal cancer initiation, i.e. how Apc-mutant cells take over in the epithelium. It has been showed that Apc-mutant ISCs need to outcompete wild-type (WT) ISCs for an adenoma to develop. This phenomenon is called “fixation” and is possible thanks to the clonal advantage that Apc loss confers to mutated ISCs.4
Two recent papers published in Nature identified the secreted WNT deacylase Notum as a major effector of early stage mutation fixation, that is, intestinal cancer development.5,6 In Flanagan et al., the authors showed that Notum-upregulated expression in Apc-mutant ISCs favors their maintenance through active inhibition of WNT signaling in neighboring WT ISCs.5 This paracrine effect induces WT ISC differentiation allowing Apc-mutant cells to “win” and fix the epithelium with mutant progeny.
Interestingly, different NOTUM inhibitors are currently under development for the treatment of osteoporosis and neurodegenerative disorders.7 These new data suggest that the inhibition of NOTUM could also represent an alternative therapeutic approach to prevent clonal fixation and mutant expansion in patients predisposed to colorectal cancer.
Of note, the authors of this study used a newly developed conditional Notum allele allowing more robust recombination at the targeted locus than an existing model. This new mouse line was generated by genOway, a designer and provider of multiple preclinical models in several research areas including oncology, immuno-oncology, metabolism, cardiovascular diseases, and neuroscience.
Amélie Rezza is Innovation Project Manager at genOway
- Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell. 1990 Jun 1;61(5):759-67.
- Powell SM, Zilz N, Beazer-Barclay Y, Bryan TM, Hamilton SR, Thibodeau SN, Vogelstein B, Kinzler KW. APC mutations occur early during colorectal tumorigenesis. Nature. 1992 Sep 17;359(6392):235-7.
- Clevers H, Loh KM, Nusse R. Stem cell signaling. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control. Science. 2014 Oct 3;346(6205):1248012.
- Vermeulen L, Morrissey E, van der Heijden M, Nicholson AM, Sottoriva A, Buczacki S, Kemp R, Tavaré S, Winton DJ. Defining stem cell dynamics in models of intestinal tumor initiation. Science. 2013 Nov 22;342(6161):995-8.
- Flanagan DJ, Pentinmikko N, Luopajärvi K, Willis NJ, Gilroy K, Raven AP, Mcgarry L, Englund JI, Webb AT, Scharaw S, Nasreddin N, Hodder MC, Ridgway RA, Minnee E, Sphyris N, Gilchrist E, Najumudeen AK, Romagnolo B, Perret C, Williams AC, Clevers H, Nummela P, Lähde M, Alitalo K, Hietakangas V, Hedley A, Clark W, Nixon C, Kirschner K, Jones EY, Ristimäki A, Leedham SJ, Fish PV, Vincent JP, Katajisto P, Sansom OJ. NOTUM from Apc-mutant cells biases clonal competition to initiate cancer. Nature. 2021 Jun;594(7863):430-435.
- van Neerven SM, de Groot NE, Nijman LE, Scicluna BP, van Driel MS, Lecca MC, Warmerdam DO, Kakkar V, Moreno LF, Vieira Braga FA, Sanches DR, Ramesh P, Ten Hoorn S, Aelvoet AS, van Boxel MF, Koens L, Krawczyk PM, Koster J, Dekker E, Medema JP, Winton DJ, Bijlsma MF, Morrissey E, Léveillé N, Vermeulen L. Apc-mutant cells act as supercompetitors in intestinal tumour initiation. Nature. 2021 Jun;594(7863):436-441.
- Zhao Y, Jolly S, Benvegnu S, Jones EY, Fish PV. Small-molecule inhibitors of carboxylesterase Notum. Future Med Chem. 2021 Jun;13(11):1001-1015.
Immunity – March 2020 – Knockin C3-reporter mouse model represents a valuable tool to monitor C3 levels, a biomarker of inflamed versus uninflamed tissues
Kolev M, West EE, et al. Immunity. 2020 Mar 17. Diapedesis-Induced Integrin Signaling via LFA-1 Facilitates Tissue Immunity by Inducing Intrinsic Complement C3 Expression in Immune Cells.
genOway has generated C3-reporter mice (C3IRES‑tdTomato C57BL/6 mice) for a group of researchers, led by Claudia Kemper, who recently published their findings in Immunity.
C3 is a protein that plays a critical role in activating the complement system: several studies have indeed shown that increased C3 levels result in autoimmunity, while decreased C3 levels give rise to higher susceptibility to bacterial and viral infections. In this study, the authors demonstrated that these defects can be corrected by restoring intracellular C3 via adenovirus- or electroporation-mediated in vivo delivery.
The C3-reporter mice are therefore invaluable tools to monitor the intracellular expression of C3 in vivo, and distinguish inflamed versus uninflamed tissues.
Commented by Alessia Armezzani, PhD (Apr 2020)
For more information on genOway’s customized mouse models, please follow the link:
Nature Neuroscience – March 2020 – Cxcr4CreER(T2)-IRES-eGFP Knockin mouse model as a universal tool to study...
The Cxcr4CreER(T2)-IRES-eGFP Knockin mouse model as a universal tool to study functions of hematopoietic stem cell (HSC)-derived cells
Werner Y, et al. Cxcr4 distinguishes HSC‑derived monocytes from microglia and reveals monocyte immune responses to experimental stroke. Nat Neurosci. 2020
Commented by Alessia Armezzani, PhD (Mar 2020)
The Cxcr4 Knockin mouse model was designed and generated by genOway for a group of researchers, led by Prof. Ralf Stumm, who recently published their work in Nature Neuroscience.
By taking advantage of the CreER(T2)-IRES-eGFP design, the authors could distinguish HSC‑derived monocytes from tissue-resident microglia and macrophages, and demonstrate that Cxcr4 promotes initial monocyte infiltration and subsequent territorial restriction of monocyte-derived macrophages to infarct tissue.
This model is a powerful tool to:
- Trace the lineage of HSC-derived cells such as monocytes and microglia, due to eGFP expression under the control of the Cxcr4 promoter
- Track monocyte fate and function in injured brain tissues (e.g., experimental stroke), due to the inducible expression of Cre upon tamoxifen induction
Figure: Immunofluorescences for GFP, Iba1 and Tmem119 reveals the presence of Cxcr4+ cells after stroke induction in coronal brain sections. Interestingly, Cxcr4 is expressed in infiltrating monocytes, but not in reactive microglia.
Alessia is scientific communication manager at genOway
For more information on genOway’s customized mouse models:
Science – Feb 2020 – VISTA is a checkpoint regulator for naïve T cell quiescence and peripheral tolerance.
Humanized Immune Checkpoint VISTA mouse model contributes to research published in Science
ElTanbouly MA, Zhao Y, et al. Science. 2020
VISTA is a checkpoint regulator for naïve T cell quiescence and peripheral tolerance.
Commented by Alessia Armezzani, PhD (Feb 2020)
A humanized VISTA mouse developed by genOway has proved to be an excellent tool to unveil a new role for this immune checkpoint inhibitor, already known to play a key role in suppressing T cell activation.
In this study, freshly published in Science, a team of researchers at the Geisel School of Medicine at Dartmouth and ImmuNext performed in vivo analyses, demonstrating that VISTA is critical in maintaining T cell self-tolerance. This was elegantly shown by targeting VISTA with agonist and antagonist antibodies, whose specificity was assessed in the humanized VISTA mouse model as well as in other human and murine samples.
Alessia is scientific communication manager at genOway
For more information on genOway’s humanized immune checkpoint catalog, please follow the link:
Cell – Jan 2020 – A finely tuned crosstalk between enteric neurons and muscularis macrophages: evidence from a conditional Knockout...
Matheis F, Muller PA, Graves CL, Gabanyi I, Kerner ZJ, Costa-Borges D, Ahrends T, Rosenstiel P, Mucida D.
Adrenergic Signaling in Muscularis Macrophages Limits Infection-Induced Neuronal Loss.
Cell. 2020 Jan.
Commentary by Alessia Armezzani, PhD (Jan 2020)
A recent study, freshly published in Cell by a group of researchers at Rockefeller University, demonstrates that enteric infections cause rapid neuronal loss via the two non-canonical inflammasomes Nlrp6 and Casp11. This was elegantly demonstrated by infecting conditional Knockout mice with an attenuated strain of Salmonella enterica and, subsequently, quantifying the numbers of enteric neurons.
In addition, researchers found that resident macrophages rapidly respond to enteric pathogens by activating the β2-adrenergic receptor (β2-AR) signaling that, in turn, triggers the synthesis of neuroprotective polyamines, thereby limiting neuronal loss. These findings suggest an extremely sophisticated interplay between enteric neurons and macrophages similar to what has been already described for the central nervous system.
Alessia is scientific communication manager at genOway
Nature – Oct 2019 – VISTA is an acidic pH-selective ligand for PSGL-1.
Johnston RJ, Su LJ, Pinckney J, Critton D, Boyer E, Krishnakumar A, Corbett M, Rankin AL, Dibella R, Campbell L, Martin GH, Lemar H, Cayton T, Huang RY, Deng X, Nayeem A, Chen H, Ergel B, Rizzo JM, Yamniuk AP, Dutta S, Ngo J, Shorts AO, Ramakrishnan R, Kozhich A, Holloway J, Fang H, Wang YK, Yang Z, Thiam K, Rakestraw G, Rajpal A, Sheppard P, Quigley M, Bahjat KS, Korman AJ.
VISTA is an acidic pH-selective ligand for PSGL-1.
Nature. 2019 Oct.
Commentary by Alessia Armezzani, PhD (Oct 2019)
Immune checkpoint inhibitors play a critical role in cancer suppression.(1,2) For this reason, antibodies targeting immune checkpoint inhibitors such as CTLA-4 and PD-1 have become standard of care in an increasing number of solid tumors, including metastatic melanomas, non-small-cell lung carcinomas and liver cancer.(3) These immunotherapies offer patients a durable remission from diseases whose outcomes were previously invariably terminal; over time, however, some tumors become resistant to such molecules due to innate and acquired resistance. These limitations have pushed scientists to understand the biological mechanisms responsible for the development of such resistances, and establish new therapeutic strategies.(4)
VISTA is an immune checkpoint inhibitor that has been identified as a potential mediator of resistance to anti-CTLA-4 and anti-PD-1 immunotherapies.(5,6) However, to date, it has not been frequently used for therapeutic intervention due to a lack of understanding of its mechanism of action in immune responses.
Along these lines, a study conducted by a group of researchers at Bristol-Myers Squibb, freshly published in Nature, shed some light on VISTA and its interaction with PSGL-1.(7) In particular, Johnston and colleagues found that VISTA binds and suppresses T cells at acidic pH such as that typically measured in tumor microenvironments. Pharmacokinetic experiments conducted on our humanized VISTA mice treated with VISTA-blocking antibodies showed indeed that acidic pH-selective antibodies accumulate preferentially within tumors and exhibit longer serum mean resistance time than non-pH-selective antibodies. Moreover, the authors demonstrated that tumor growth in human VISTA Knockin mice bearing mouse colorectal carcinoma MC38 cells is significantly reduced in animals treated with combination therapy (anti-VISTA and anti-PD-1) compared to those that received monotherapy (anti-PD-1).
Alessia is scientific communication manager at genOway
- Paluch, Christopher, et al. "Immune checkpoints as therapeutic targets in autoimmunity." Front Immunol. 2018 Oct 8
- Armezzani, A. "Immune checkpoint inhibitors: A strategy to tackle cancer?" genOway, 2019 Jul
- Darvin, Pramod, et al. "Immune checkpoint inhibitors: recent progress and potential biomarkers." Exp Mol Med. 2018 Dec 13
- Wang, Jinghua, et al. "VSIG‐3 as a ligand of VISTA inhibits human T‐cell function." Immunology. 2019 Jan
- Kakavand, Hojabr, et al. "Negative immune checkpoint regulation by VISTA: a mechanism of acquired resistance to anti-PD-1 therapy in metastatic melanoma patients." Mod Pathol. 2017 Dec
- Nowak, Elizabeth C., et al. "Immunoregulatory functions of VISTA." Immunol Rev. 2017 Mar
- Johnston, Robert J., et al. "VISTA is an acidic pH-selective ligand for PSGL-1." Nature. 2019 Oct